Sunday, November 12

5:00-6:00 pm Early Registration

Monday, November 13

7:00 am Registration and Morning Coffee

Novel Fluorescent Probes and Techniques

7:50 Chairperson’s Opening Remarks
Andrew Bradbury, Ph.D., Staff Scientist, Biosciences Division, Los Alamos National Laboratory

8:00 The Creation of a Novel Fluorescent Protein by Guided Consensus Engineering
Andrew Bradbury, Ph.D., Staff Scientist, Biosciences Division, Los Alamos National Laboratory
We have created a novel fluorescent protein based on the consensus derived from the alignment of 31 fluorescent proteins. This consensus protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to GFP. Recent data on the characterization of this novel fluorescent protein will be presented.

8:30 RNA Visualization in Live Bacterial Cells Using Fluorescent Protein Complementation
Natalia Broude, Ph.D., Research Associate Professor, Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University 
We developed a new fluorescent imaging technique with very low background signal for studying RNA dynamics in vivo. Using this technology, we observed synchronous spatial and temporal changes of fluorescence in single bacterial cells. Our approach presents a substantial advance for cellular imaging since it adds fluorescent protein complementation to the existing arsenal of methods for RNA labeling in vivo. 

9:00 Optimized GFP Fragment Complementation Assays for Protein Tagging and Detection
Geoffrey S. Waldo, Ph.D., Bioscience Division, Los Alamos National Laboratory
We describe the properties and applications of self-assembling GFP fragments for monitoring protein expression in vivo and in vitro, and for 'discovering' compact protein domains that are suitable for downstream applications. These fragments can also be used for monitoring the assembly of multi-protein complexes and for high-throughput protein expression assays.

9:30 Use of Kaede Fusions to Visualize Recycling and Degradation of G Protein - Coupled Receptors
Dr. Antje Schmidt, Staff Scientist, Leibniz Institute of Molecular Pharmacology
G protein-coupled receptors (GPCRs) represent important drug targets. Trafficking of GPCRs has been widely studied using fusions with green fluorescent protein (GFP) derivatives. For many experiments, however, marker proteins would be desirable whose fluorescence can be converted once the protein has reached a particular membrane compartment. Here we show that fusions with the Kaede protein from the coral Trachyphylla geoffroy may be useful to fill this gap. Kaede emits green fluorescence that can be irreversibly converted to red using UV light (364 nm). With the help of C-terminal Kaede fusions and confocal laser scanning microscopy, we have established a novel methodology to study GPCR recycling and degradation: Initially, receptors at the plasma membrane are internalized using agonists. The fluorescence is then switched from green to red. Thereafter, trafficking of the receptors to target compartments such as the plasma membrane or lysosomes can be easily visualised by monitoring their new fluorescence.

10:00 Networking Coffee Break 

10:30 Exploiting the Convergence of Fluorescence Imaging, Cellular Networks, and Chemical Biology 
John K. Westwick, Ph.D., President and CSO, Odyssey Thera, Inc.
Biological responses to drugs are determined by the architecture and dynamics of the cellular signal transduction network. We are using massively parallel arrays of high content, fluorescence-based assays, including Protein-fragment Complementation Assays (PCA), to perform live cell, pathway-based drug discovery and drug profiling. Our approach enables assessment of “un-drugable” targets, and identification of on-target and off-target effects of lead compounds at an early stage in the discovery process. 

11:15 Solutions Showcase 
Get informed on the newest technology and developments. 
Contact Carol Dinerstein at 781-972-5471 for sponsorship opportunities.

11:30 Novel Fluorescent Probes to Measure and Manipulate the p53 Pathway
Kenneth Giuliano, Ph.D., Principal Scientist, Research and Development, Cellumen, Inc.
The p53 tumor suppressor pathway contains potential drug targets, such as the ubiquitin ligase HDM2, that are difficult to screen with conventional high content screening (HCS) methods. We have generated tools to manipulate expression of an HDM2-GFP fusion protein with a tightly-regulated promoter system, and to measure the interaction of p53 and HDM2 with a new fluorescent-protein-based protein:protein interaction positional biosensor. Multiplexed HCS are used to simultaneously monitor several aspects of p53 pathway activity, including cell cycle regulation, organelle function, DNA damage response, and apoptosis. These tools facilitate a new systems cell biology approach to studying pathway activity that enables drug discovery for novel target classes.

12:00 Panel Discussion with Speakers

12:30 Lunch on your own (Luncheon Workshop Sponsorship Available)

Assay Development

2:00 Fluorescent Protein Assays Using 1536-Well-Plate-Based Laser-Scanning Microplate Cytometry and qHTS
Doug Auld, Ph.D., Group Leader Genomic Assay Technologies, NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health
The National Institute of Health Chemical Genomics Center (NCGC) has developed a process termed “quantitative HTS” (qHTS) where > 70,000 compounds are screened as concentration-titration series in a high-throughput mode. The process has been applied to a variety of fluorescent protein - based assays utilizing the Acumen Explorer laser plate cytometer. This talk will focus on case studies covering a wide range of biology that has been successfully applied using microplate cytometry at the NCGC. Specifically, the ability to measure cytoplasmic to nuclear translocation using cells expressing fluorescent protein fusions (BioImage cell lines, Promega’s HaloTag technology), and the use of bifurcated FPs (Odyssey Thera) will be illustrated.

2:30 A FRET Cell-Based Assay for Insulin - Receptor Activation
Shane Marine, Ph.D., Department of Automated Biotechnology, Merck & Co., Inc.

3:00 A Novel Generic High Throughput System for Kinase Assays
Jun Wu, M.D., Ph.D., Product Manager, Biochemical Assays, Nanostream Inc.
Protein kinases control many cellular activities through protein phosphorylation and dephosphorylation in response to extra cellular signals. Abnormality in the activities of the kinases in these regulation pathways have been linked to many human diseases. Classical screening platforms, e.g., radioactive methods and ELISA-based assay methods, generally require multiple labor-intensive and time-consuming steps. To address these kinase assay limitations, we have developed a generic high-throughput screening assay. In this assay, nonphosphorylated peptide was used as a substrate., The phosphorylated peptide was resolved from nonphosphorylated peptide via micro parallel chromatography. In contrast to a traditional plate reader based kinase assay, the Nanostream LD system could detect less than 10% conversion of enzymatic reactions and Z’ factor ranges from 0.6 to 0.85. In addition, this system can enable real time monitoring of kinase kinetics. From a single experiment, the optimal enzyme, ATP, and substrate concentrations can be determined. Furthermore, the collected data allows Km determination for ATP and substrate and for time course study. Compared with conventional HPLC and LC-MS, our system enables parallel chromatographic analysis of 24 samples. The system is simple to use, and the integrated system software permits fast and accurate data analysis. The system can be used to facilitate target identification and assay development in drug discovery.

3:30 Refreshment Break, Poster and Exhibit Viewing

4:15 Optical Imaging for Anti-Cancer Drug Discovery and Development
Wafik El-Deiry, M.D., Ph.D., Professor, Department of Medicine, Genetics, Pharmacology, University of Pennsylvania School of Medicine
In the last several years we have developed the infrastructure and have performed high through-put cell-based screening using optical imaging including bioluminescence as a read-out to identify candidate therapeutic agents. Target validation and anti-tumor effects have been demonstrated. We have also been developing various strategies to image drug effects in vivo including assays of apoptosis and gene expression changes using molecular beacons or Q-dot labeled probes. We are moving in the direction of using medicinal chemistry and genomic approaches to continue to discover and develop novel anti-cancer drugs as well as imaging probes. Optical imaging provides a powerful tool for screening, target validation, and in vivo studies of anti-tumor effects. The NCI-funded Network for translational research in Optical Imaging has supported these efforts.

Real-Time Imaging

4:45 Whole-Body Subcellular Imaging in the Live Mouse
Robert M. Hoffman, Ph.D., President, AntiCancer, Inc.
Dual-color cancer cells expressing GFP in the nucleus and RFP in the cytoplasm have been developed. These cells enable nuclear-cytoplasmic dynamics, cell cycle analysis, apoptosis, nuclear and cytoplasmic shape changes, and numerous other processes to be visualized in the living mouse. The Olympus IV100 whole-mouse laser-scanning microscope with ultra-thin diameter objectives enable the dual-color cells to be visualized external to the mouse. Using host mouse models expressing GFP in all cells enables for the first time the study of tumor-host interaction at the cellular level in real time. This technology will lead to the development of the new field of in vivo cell biology to study both normal and disease processes in the live animal at the subcellular level.

5:15 Panel Discussion with Speakers

5:45 Networking Reception in Exhibit Hall

6:45 End of Day One of Imaging Week

For more information about this conference, please contact:
Margit Eder, Ph.D., Conference Director, Cambridge Healthtech Institute 
Phone: 781-972-5478 – Fax: 781-972-5425 - E-mail: meder@healthtech.com

For sponsorship or exhibiting information, please contact:
Carol Dinerstein, Business Development Manager, Cambridge Healthtech Institute 
Phone: 781-972-5471 – Fax: 781-972-5425  – E-mail: dinerstein@healthtech.com