Activation of cell-surface and intracellular receptor-mediated immunity results in rapid transcriptional reprogramming that underpins disease resistance. However, the mechanisms by which co... Show moreActivation of cell-surface and intracellular receptor-mediated immunity results in rapid transcriptional reprogramming that underpins disease resistance. However, the mechanisms by which co-activation of both immune systems lead to transcriptional changes are not clear. Here, we combine RNA-seq and ATAC-seq to define changes in gene expression and chromatin accessibility. Activation of cell-surface or intracellular receptor-mediated immunity, or both, increases chromatin accessibility at induced defence genes. Analysis of ATAC-seq and RNA-seq data combined with publicly available information on transcription factor DNA-binding motifs enabled comparison of individual gene regulatory networks activated by cell-surface or intracellular receptor-mediated immunity, or by both. These results and analyses reveal overlapping and conserved transcriptional regulatory mechanisms between the two immune systems. Show less
MotivationDeveloping a robust and performant data analysis workflow that integrates all necessary components whilst still being able to scale over multiple compute nodes is a challenging task. We... Show moreMotivationDeveloping a robust and performant data analysis workflow that integrates all necessary components whilst still being able to scale over multiple compute nodes is a challenging task. We introduce a generic method based on the microservice architecture, where software tools are encapsulated as Docker containers that can be connected into scientific workflows and executed using the Kubernetes container orchestrator.ResultsWe developed a Virtual Research Environment (VRE) which facilitates rapid integration of new tools and developing scalable and interoperable workflows for performing metabolomics data analysis. The environment can be launched on-demand on cloud resources and desktop computers. IT-expertise requirements on the user side are kept to a minimum, and workflows can be re-used effortlessly by any novice user. We validate our method in the field of metabolomics on two mass spectrometry, one nuclear magnetic resonance spectroscopy and one fluxomics study. We showed that the method scales dynamically with increasing availability of computational resources. We demonstrated that the method facilitates interoperability using integration of the major software suites resulting in a turn-key workflow encompassing all steps for mass-spectrometry-based metabolomics including preprocessing, statistics and identification. Microservices is a generic methodology that can serve any scientific discipline and opens up for new types of large-scale integrative science.Availability and implementationThe PhenoMeNal consortium maintains a web portal (https://portal.phenomenal-h2020.eu) providing a GUI for launching the Virtual Research Environment. The GitHub repository https://github.com/phnmnl/ hosts the source code of all projects. Show less
Ghotra, V.P.; He, S.; Horst, G. van der; Nijhoff, S.; Bont, H. de; Lekkerkerker, A.; ... ; Danen, E.H. 2015
Improved targeted therapies are needed to combat metastatic prostate cancer. Here, we report the identification of the spleen kinase SYK as a mediator of metastatic dissemination in zebrafish and... Show moreImproved targeted therapies are needed to combat metastatic prostate cancer. Here, we report the identification of the spleen kinase SYK as a mediator of metastatic dissemination in zebrafish and mouse xenograft models of human prostate cancer. Although SYK has not been implicated previously in this disease, we found that its expression is upregulated in human prostate cancers and associated with malignant progression. RNAi-mediated silencing prevented invasive outgrowth in vitro and bone colonization in vivo, effects that were reversed by wild-type but not kinase-dead SYK expression. In the absence of SYK expression, cell surface levels of the progression-associated adhesion receptors integrin α2β1 and CD44 were diminished. RNAi-mediated silencing of α2β1 phenocopied SYK depletion in vitro and in vivo, suggesting an effector role for α2β1 in this setting. Notably, pharmacologic inhibitors of SYK kinase currently in phase I-II trials for other indications interfered similarly with the invasive growth and dissemination of prostate cancer cells. Our findings offer a mechanistic rationale to reposition SYK kinase inhibitors for evaluation in patients with metastatic prostate cancer. Show less
Drabsch, Y.S.; He, S.; Zhang, L.; Snaar-Jagalska, B.E.; Dijke, P. ten 2013
A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in... Show moreA quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline. Show less
Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf–MEK–MAPK pathway.... Show moreAlthough the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf–MEK–MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf–MEK–MAPK pathway was also shown to be deregulated in zebrafish liver tumours. Based on the genetic conservation between zebrafish and human liver tumours, the zebrafish was used as an animal model to better understand the molecular basis of hepatocellular carcinoma. Here we establish an inducible oncogenic zebrafish cell model, in which oncogenic human Raf-1(ΔRaf1) can be post-transcriptionally activated in zebrafish liver cells by administration of 4-hydroxytamoxifen (4HT). The ΔRaf1 activation resulted in the hyperactivation of the zebrafish MEK–ERK cascade, promoted cell growth and proliferation, and inhibited apoptosis. The mitogenic transformation of the ZFL–ΔRaf1–ER cells was confirmed by in vivo allo-transplantation and in silico microarray analyses. Gene expression profiling of cells treated with 4HT and a MEK-inhibitor identified a Raf–MEK-dependent signature set. This transcriptome response was compared to zebrafish and human liver cancer transcriptomes. We identified, and validated by quantitative PCR, a set of genes transcriptionally regulated by hyperactive MAPK signalling in ZFL–ΔRaf1–ER cells, zebrafish liver tumours and human liver tumours, suggesting that the in vitro zebrafish liver cell model can be used for further study of the molecular basis of human hepatocellular carcinoma. The molecular targeting of the commonly regulated hepatocellular carcinoma genes using the ZFL–ΔRaf1–ER cell model can be applied for high-throughput preclinical target discovery. Show less
As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this... Show moreAs the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties were compared. We also developed a set of tool methods to investigate cellular signaling events in zebrafish cell lines. Our case studies illustrated that zebrafish cell lines are as reliable models as the widely used mammalian cell cultures. Taking advantage of the transparency of zebrafish embryos and cell implantation protocols, zebrafish cell lines can serve as a bridge platform between in vitro, in silico, ex vivo and in vivo studies in order to enhance our understanding of molecular mechanisms underlying disease progression. Show less
The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1... Show moreThe MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior–posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis. Show less
Krens, S.F.G.; He, S.; Spaink, H.P.; Snaar-Jagalska, B.E. 2006
