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Rimann M.,ZHAW Zurich University of Applied Sciences | Angres B.,Cellendes GmbH | Patocchi-Tenzer I.,Tecan Schweiz AG | Braum S.,Tecan Schweiz AG | Graf-Hausner U.,ZHAW Zurich University of Applied Sciences
Journal of Laboratory Automation | Year: 2014

Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development. © 2013 Society for Laboratory Automation and Screening.

Beck J.,ETH Zurich | Maerki S.,ETH Zurich | Maerki S.,Tecan Schweiz AG | Posch M.,University of Dundee | And 10 more authors.
Nature Cell Biology | Year: 2013

Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3-KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3-KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3-KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis. © 2013 Macmillan Publishers Limited. All rights reserved.

Almario J.,University Claude Bernard Lyon 1 | Almario J.,CNRS Microbial Ecology | Gobbin D.,ETH Zurich | Gobbin D.,Research and Innovation Center | And 6 more authors.
Research in Microbiology | Year: 2014

Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN+ biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN- biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems. © 2014 Institut Pasteur.

Iten M.,ZHAW Zurich University of Applied Sciences | Weibel R.,ZHAW Zurich University of Applied Sciences | Konig I.,ZHAW Zurich University of Applied Sciences | Beckbissinger R.,Hamilton Bonaduz AG | And 6 more authors.
JALA - Journal of the Association for Laboratory Automation | Year: 2010

Robotic liquid-handling systems can be equipped with disposable pipetting tips or fixed reusable pipetting tips. The use of disposable tips is perceived as the best option to avoid carry over (CO) of analyte from sample to sample. We recently developed standardized CO test procedures that allow precise and reproducible quantification of CO for fixed reusable tips. We used these test procedures to reduce CO of the analytes fluorescein, IgG, and hepatitis B surface antigen (HBsAg) to minimal levels. Variations of washing intensity, using water as a washing solution, did not lower CO below acceptable target levels. These target levels would preclude a false-positive detection of IgG and HBsAg in human serum when a negative sample is measured subsequent to a sample with a high analyte concentration. We therefore integrated a decontamination step into the washing procedure. Screening of 12 decontamination solutions showed that sodium hypochlorite solution was very efficient in reducing CO. Optimization led to a final washing routine in which tips are exposed for 0.2. s to 0.17. M NaOCl and subsequently rinsed with 2. mL of water to remove any remaining decontaminant solution. The washing procedure only takes 15. s and is thus suitable for high-throughput applications. The procedure was able to lower CO of IgG and HBsAg in human sera below relevant levels. The decontamination step with hypochlorite can easily be integrated into different liquid-handling systems and is likely to be effective against CO of most proteins and peptides. © 2010 Society for Laboratory Automation and Screening.

Weibel R.,ZHAW Zurich University of Applied Sciences | Iten M.,ZHAW Zurich University of Applied Sciences | Konig I.,ZHAW Zurich University of Applied Sciences | Beckbissinger R.,Hamilton Bonaduz AG | And 6 more authors.
JALA - Journal of the Association for Laboratory Automation | Year: 2010

Robotic liquid-handling systems using fixed reusable pipetting tips are used not only in fully integrated in vitro clinical diagnostic analyzers, but also in open platform robotic liquid-handling systems. These are not confined to particular diagnostic assays, as customers adapt a large variety of assays on these platforms. One major problem with the use of fixed, reusable tips is the carry over (CO) of analyte from sample to sample. Despite widespread use of fixed tips in open platform systems, systematic studies on procedures to quantify CO for analytes other than nucleic acids are missing. In a consortium with three liquid-handling system suppliers and one coating specialist, we developed test procedures for the quantification of CO. The procedures were standardized and tested with the analytes fluorescein, immunoglobulin G, and hepatitis B surface antigen as model substances for small organic molecules, antibodies, and complex biomolecules. The test procedures allow the reproducible quantification of the CO with intra- and interassay precisions of less than 6% coefficient of variation. They were used to investigate the effect of different tip coatings on the CO of the three analytes. Fluoropolymers, inorganic-organic nanocomposites, sodium-silicate glass, titanium dioxide, and silicone resins, which are used in special applications, showed only small differences in CO. The CO test procedures can be easily transferred to different liquid-handling systems and used with different analytes. © 2010 Society for Laboratory Automation and Screening.

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