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High-throughput assays for molecular markers of cytotoxicity

English title High-throughput assays for molecular markers of cytotoxicity
Applicant Aguzzi Adriano
Number 157745
Funding scheme R'EQUIP
Research institution Institut für Neuropathologie Departement für Pathologie Universitätsspital Zürich
Institution of higher education University of Zurich - ZH
Main discipline Cellular Biology, Cytology
Start/End 01.12.2014 - 30.11.2015
Approved amount 609'632.00
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All Disciplines (2)

Discipline
Cellular Biology, Cytology
Molecular Biology

Keywords (7)

drug discovery; acoustic liquid dispensing workstation; disease-related markers; biomarkers; High throughput screening; RNAi screening; automation

Lay Summary (German)

Lead
Seit der Entzifferung des menschlichen Genoms ist es möglich, jedes einzelne Gen auf seine Relevanz zu einer spezifischen Krankheit zu prüfen. Die grosse Anzahl der Gene sowie die Notwendigkeit von Wiederholungen bedingt Millionen von Pipettierschritten für jedes Experiment. Deshalb planen wir die Etablierung einer automatisierten Workstation für akustischen Flüssigkeitstransfer. Diese Technik wird es uns ermöglichen, Genom-Abfragen in einem erschwinglichen finanziellen Rahmen durchzuführen. Geplant ist die Abfrage des gesamten menschlichen Genoms im Hinblick auf Prionenkrankheiten, Herzkreislauf-Erkrankungen, Krebsentstehung, Diabetes und Muskelkrankheiten. Die Technologie ist überdies auch geeignet, um Sammlungen von bioaktiven Substanzen auf ihre Wirksamkeit gegen pathologische Zellzustände zu testen. Der Zugang zur automatisierten Workstation und das Wissen, welches aus dieser Kooperation hervorgeht, wird auch Forschenden anderer Institutionen zugänglich gemacht werden.
Lay summary
Seit der Entzifferung des menschlichen Genoms ist es möglich, jedes einzelne Gen auf seine Relevanz zu einer spezifischen Krankheit zu prüfen. Dazu werden Techniken der Gen-Suppression (siRNA) und Gen-Ablation (CRISPR) verwendet. Die grosse Anzahl der existierenden Gene sowie die statistische Notwendigkeit von Wiederholungen bedingt Millionen von Pipettierschritten für jedes Experiment – was von konventionellen Robotern kaum bewältigt werden kann, geschweige denn von menschlichem Laborpersonal. Deshalb planen wir die Etablierung einer automatisierten Workstation für akustischen Flüssigkeitstransfer. Diese neuartige Technologie erlaubt den Transfer in Nanoliterschritten von Flüssigkeiten und Zellen mittels Schallwellen auf 384- und 1536-Well Mikrotiterplatten ohne Benutzung von Pipettenspitzen. Daraus ergeben sich entscheidende Vorteile, wie z.B. höhere Flexiblität, tiefere Betriebskosten, weniger Ausschuss, einfachere Software, Miniaturisierung der Ansätze, und sehr viel höhere Präzision. Diese Technik wird es uns erstmals ermöglichen, Genom-Abfragen in einem erschwinglichen finanziellen Rahmen durchzuführen. Die Antragsteller sind Experten auf den Gebieten der Prionenkrankheiten, der Herzkreislauf-Erkrankungen, der Krebsentstehung, des Diabetes und der Muskelkrankheiten; sie planen die Abfrage des gesamten menschlichen Genoms im Hinblick auf diese Erkrankungen. In einer späteren Phase ist die Technologie aber auch geeignet, um öffentlich zugängliche Sammlungen von bioaktiven Substanzen (sog. "compound libraries") auf ihre Wirksamkeit gegen pathologische Zellzustände zu testen. Schliesslich ist geplant, sowohl den Zugang zur automatisierten Workstation als auch das Wissen, welches aus dieser Kooperation hervorgeht, Forschenden anderer Institutionen zugänglich zu machen.

Direct link to Lay Summary Last update: 17.11.2014

Responsible applicant and co-applicants

Publications

Publication
New and emerging roles of small RNAs in neurodegeneration, muscle, cardiovascular and inflammatory diseases.
Hruska-Plochan Marian, Li Bei, Kyburz Diego, Krützfeldt Jan, Landmesser Ulf, Aguzzi Adriano, Polymenidou Magdalini (2015), New and emerging roles of small RNAs in neurodegeneration, muscle, cardiovascular and inflammatory diseases., in Swiss Medical Weekly, w14192.

Collaboration

Group / person Country
Types of collaboration
Prof. Dr. Diego Kyburz, University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Dr. Tuomas Knowles, University of Cambridge Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Dr. Philip Gribbon, ScreeningPort, Hamburg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Novartis Institutes for BioMedical Research, Inc. United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Dr. Ulf Landmesser Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Dr. Jan Krützfeldt Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Ioannis Xenarios, Swiss Institute of Bioinformatics, University of Lausanne Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Dr. Magdalini Polymenidou Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Lucas Pelkmans, Institute of Molecular Life Sciences, University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Dr. Markus Stoffel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Eli Lilly and Company United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events



Self-organised

Title Date Place
Joint Retreat 2015 30.04.2015 Richisau GL, Switzerland

Associated projects

Number Title Start Funding scheme
160672 Immunotherapy of familial prion diseases 01.02.2015 ERA-E-RARE
141761 Mechanisms of ectopic brown adipose tissue formation in normal and insulin resistant states 01.01.2013 ProDoc
144862 Deciphering the mechanisms of TDP-43 and FUS/TLS mediated neurotoxicity 01.09.2013 SNSF Professorships
141193 The prion protein in health and disease 01.06.2012 Project funding
128474 Role of microRNAs from myogenic progenitors in adult skeletal muscle function and the implications for type 2 diabetes 01.10.2010 SNSF Professorships
140336 Inflammation and acute coronary syndrome (ACS) - novel strategies for prevention and clinical management 01.02.2012 SPUM
149990 Molecular mechanisms and pathophysiological relevance of NLRP3 inflammasome activation in monocyte subpopulations from patients with coronary disease: Impact of modified lipoproteins and miRNAs 01.04.2014 Project funding
152978 Role of microRNAs from myogenic progenitors in adult skeletal muscle function and the implications for type 2 diabetes 01.10.2014 SNSF Professorships

Abstract

We are a consortium of scientists within the University Hospital and Children’s Hospital of Zurich (USZ and KISPI), including two Clinical Research Priority Programs (CRPPs, or KFSPs). We have formed a multidisciplinary team designing innovative platforms for high throughput screens (HTS). The individual research teams strive to create a synergistic HTS-based approach to new assays for protein aggregation and “protein infectivity”, RNAi-based screens for human disease, and to identify novel clinically relevant therapeutic targets. In this context, we propose to create a flexible, largely automated high-throughput platform adaptable to a broad variety of assay formats for the discovery of new molecular markers of cytoxicity and therapeutics related to a broad range of human pathologies:•A high-throughput prion quantification and replication assay, to be used for RNAi-based whole-transcriptome screens for prion modifiers (A. Aguzzi)•A high-throughput, miniaturized automated platform for low-cost genotyping of genetically modified animals (A. Aguzzi)•A high-throughput screening platform for miRNA mimics and inhibitors for mitochondrial function in human myotubes (J. Krützfeldt)•A molecular high-throughput screening assay to measure and quantify protein aggregation in ALS and FTD (M. Polymenidou) •A high-throughput synthetic lethality screening assay using RNAi technology in genetically modified endometrial carcinoma cell lines (P. Wild)•A high-throughput screening platform for the identification of novel anti-leukemic agents to treat refractory childhood acute lymphoblastic leukemia (J.P. Bourquin)•A high-throughput screening platform to identify microRNAs as targets for prevention and treatment of atherosclerotic vascular disease (U. Landmesser)Although our goals are scientifically and biologically diverse, they all rely on the same technology platforms. We therefore propose the acquisition of an automated Acoustic Liquid Dispenser Workstation, which incorporates an Acoustic Liquid Dispenser, a cell incubator, a sophisticated plate reader, and equipment for plate handling through the “R'Equip” funding instrument. The proposed Acoustic Liquid Dispensing Workstation is an innovative instrument that utilizes focused ultrasound to transfer liquids. It provides a high-throughput technology for precisely dispensing nanoliter amounts of water and DMSO-based solutions, and even cells, without pipette tips, into 384 and 1536-well destination plates and has many advantages over conventional robots (higher flexibility, much lower running costs, no pipette tips, user-friendly software, smaller volumes, and much higher precision). We expect that this advanced technology will significantly strengthen our efforts in implementing the envisaged high-throughput platforms for the screening of biological and functional markers that play important roles in neurodegenerative diseases, cancer, diabetes, and cardiovascular diseases. The automated workstation and the operational know-how will also be accessible to researchers from other institutions as a cost-effective and highly innovative way to run siRNA, microRNA, and other high-throughput screenings and assays in the future.
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