Publications

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Papers we were directly involved and/or provided the technology platform

Contents

Biotechniques: Automated feature detection and imaging for high-resolution screening of zebrafish embryos

Biotechniques. 2011 May;50(5):319-24. Peravali R, Gehrig J, Giselbrecht S, Lütjohann DS, Hadzhiev Y, Müller F, Liebel U.


Cover (magnify)

Automated high resolution microscopy (magnify)


  • Abstract: The development of automated microscopy platforms has enabled large-scale observation of biological processes, thereby complementing genome scale biochemical techniques. However, commercially available systems are restricted either by fixed-field-of-views, leading to potential omission of features of interest, or by low-resolution data of whole objects lacking cellular detail. This limits the efficiency of high-content screening assays, especially when large complex objects are used as in whole-organism screening. Here we demonstrate a toolset for automated intelligent high-content screening of whole zebrafish embryos at cellular resolution on a standard wide-field screening microscope. Using custom-developed algorithms, predefined regions of interest-such as the brain-are automatically detected. The regions of interest are subsequently imaged automatically at high magnification, enabling rapid capture of cellular resolution data. We utilize this approach for acquiring 3-D datasets of embryonic brains of transgenic zebrafish. Moreover, we report the development of a mold design for accurate orientation of zebrafish embryos for dorsal imaging, thereby facilitating standardized imaging of internal organs and cellular structures. The toolset is flexible and can be readily applied for the imaging of different specimens in various applications.
  • a detailed protocol can be found in the BioTechniques' Protocol Guide 2012 | link to protocol

Bioinformatics: "Sciencenet" - Towards a global search and share engine for all scientific knowledge

Bioinformatics, Oxford Journal. 2011 Apr 14. Lütjohann DS, Shah AH, Christen MP, Richter F, Knese K, Liebel U.


Sciencenet Search engine(magnify)

* Abstract: Modern biological experiments create vast amounts of data which are geographically distributed. These datasets consist of petabytes of raw data and billions of documents. Yet to the best of our knowledge, a search engine technology that searches and crosslinks all different data types in life sciences does not exist. We have developed a prototype distributed scientific search engine technology, "Sciencenet", which facilitates rapid searching over this large data space. By "bringing the search engine to the data" we do not require server farms. This platform also allows users to contribute to the search index and publish their large scale data to support e-Science. Furthermore, a community-driven method guarantees that only scientific content is crawled and presented. Our peer-to-peer approach is sufficiently scalable for the science web without performance or capacity tradeoff. Availability and Implementation: The free to use search portal webpage and the downloadable client are accessible here: http://sciencenet.kit.edu. The web portal for index administration is implemented in ASP.NET, the "AskMe" experiment publisher is written in Python 2.7, and the backend "YaCy" search engine is based on Java 1.6. Supplementary Material: Detailed instructions and descriptions can be found on the project

BMC Biology "A high-throughput chemically induced inflammation assay in zebrafish"

http://www.biomedcentral.com/1741-7007/8/151


Automated ChiN Assay paper (magnify)
  • Background

Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immuno-modulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high content screening.

  • Results

Here, we show that specific, non-invasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sub-lethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real-time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immuno-modulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.

  • Conclusions

This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds towards new immuno-modulatory therapies. We have called this method the Chemically-Induced Inflammation Assay, or ChIn Assay.

See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

Nature: "Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes" (Mitocheck Team EMBL)


Mitocheck Methods paper (magnify)
  • Abstract:

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.

Nature Methods: "Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos"


Zebrafish High Content Screen (magnify)
  • We analyzed 30.000 Zebrafish via High Content Screening microscopy and novel image processing methods.
  • Published online: 8 November 2009 | doi:10.1038/nmeth.1396
  • Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos
    • Jochen Gehrig1,2,5, Markus Reischl3,5, Éva Kalmár1,5, Marco Ferg1, Yavor Hadzhiev1,2, Andreas Zaucker1,2, Chengyi Song1,4, Simone Schindler1, Urban Liebel1 & Ferenc Müller1,2

Abstract: link to Nature Methods

  • Zebrafish embryos offer a unique combination of high-throughput capabilities and the complexity of the vertebrate animal for a variety of phenotypic screening applications.
  • However, there is a need for automation of imaging technologies to exploit the potential of the transparent embryo. Here we report a high-throughput pipeline for registering domain-specific reporter expression in zebrafish embryos with the aim of mapping the interactions between cis-regulatory modules and core promoters.
  • Automated microscopy coupled with custom-built embryo detection and segmentation software allowed the spatial registration of reporter activity for 202 enhancer-promoter combinations, based on images of thousands of embryos. The diversity of promoter-enhancer interaction specificities underscores the importance of the core promoter sequence in cis-regulatory interactions and provides a promoter resource for transgenic reporter studies.
  • The technology described here is also suitable for the spatial analysis of fluorescence readouts in genetic, pharmaceutical or toxicological screens.


Journal of Cell Biology: "Novel cargo-binding site in the beta and delta subunits of coatomer" (Schwappach group)


Novel Cargo paper (magnify)
  • Abstract: Arginine (R)-based ER localization signals are sorting motifs that confer transient ER localization to unassembled subunits of multimeric membrane proteins. The COPI vesicle coat binds R-based signals but the molecular details remain unknown. Here, we use reporter membrane proteins based on the proteolipid Pmp2 fused to GFP and allele swapping of COPI subunits to map the recognition site for R-based signals. We show that two highly conserved stretches--in the beta- and delta-COPI subunits--are required to maintain Pmp2GFP reporters exposing R-based signals in the ER. Combining a deletion of 21 residues in delta-COP together with the mutation of three residues in beta-COP gave rise to a COPI coat that had lost its ability to recognize R-based signals, whilst the recognition of C-terminal di-lysine signals remained unimpaired. A homology model of the COPI trunk domain illustrates the recognition of R-based signals by COPI.


Nature Methods: "High-throughput RNAi screening by time-lapse imaging of live human cells" (Mitocheck team EMBL)


Mitocheck Methods paper (magnify)


  • Abstract: RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.


Journal of Biotechnology "An RNAi screening platform to identify secretion machinery in mammalian cells"


Secretion Screen paper (magnify)


  • Abstract: Integrative approaches to study protein function in a cellular context are a vital aspect of understanding human disease. Genome sequencing projects provide the basic catalogue of information with which to unravel gene function, but more systematic applications of this resource are now necessary. Here, we describe and test a platform with which it is possible to rapidly use RNA interference in cultured mammalian cells to probe for proteins involved in constitutive protein secretion. Synthetic small interfering RNA molecules are arrayed in chambered slides, then incubated with cells and an assay for secretion performed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. In test arrays of 92 siRNA spots targeting 37 prospective membrane traffic proteins, our approach identifies 7 of these as being important for the correct delivery of a secretion marker to the cell surface. Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components.


FEBS letters: "A microscope-based screening platform for large-scale functional protein analysis in intact cells"


Screening platform development (magnify)
  • A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.


Bioinformatics: "Harvester': a fast meta search engine of human protein resources"


Bioinformatic Harvester (magnify)
  • Abstract: We have developed a Web-based tool named 'Harvester' that bulk-collects bioinformatic data on human proteins from various databases and prediction servers. The information on every single protein is assembled on a single HTML page as a combination of database screen-shots and plain text. A full text meta search engine, similar to Google trade mark, allows screening of the whole genome proteome for current protein functions and predictions in a few seconds. With Harvester it is now possible to compare and check the quality of different database entries and prediction algorithms on a single page. A feedback forum allows users to comment on Harvester and to report database inconsistencies. AVAILABILITY: The service is freely available to the academic community.


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