GeneExpression Systems, Inc & Appasani Research Conferences and Educational Institue (ARCEI.org)
Jontly Presents

RNAi & Single Cell Biology-2011 Summit

April 4 - 5, 2011
Venue: Hilton Garden Inn, 420 Totten Pond Road, Waltham 02451, USA

This Year RNAi-2011-Meeting Will Be held in Conjunction with Single Cell Biology-2011-Meeting.

The Most Popular and The Best Global Event in the RNAi Field!

Click Here for Detailed Agenda

Scientific Committe:

	Krishnarao Appasani, PhD., MBA Founder & CEO GeneExpression Systems, Inc Waltham, MA USA
	Mehmet F. Yanik, PhD. Robert J. Shillman Assistant Professor of Electrical Engineering Massachusetts Institute of TechnologyCambridge, MA, USA
	Dmitry Samarsky, PhD. VP, Technology Development RXi Pharmaceuticals Worcester, MA, USA
	Joseph Loscalzo, MD., PhD.                                   Physician-in-Chief & Professor of Medicine Brigham & Women’s Hospital & Harvard Medical School Boston, MA, USA

Keynote Speakers

	Martin Chalfie, PhD.                         Inaugural Speaker William R Kenan Jr. Professor, Nobel Laureate        Chair of Biological Sciences Columbia University New York, NY, USA Title:Teasing out cell-specific effects of lethal genes using SID-1-directed feeding RNAi
	Robert A. Weinberg,Ph.D. Member, Whitehead Institute & Professor of Biology Director, MIT Ludwig Center for Molecular Oncology Massachusetts Institute of Technology Cambridge, MA, USA Title: Formation of cancer stem cells and malignant progression
	Xiaoliang Sunney Xie, PhD.                      Mallinckrodt Professor of Chemistry              Harvard University Cambridge, MA, USA Title: Life at the Single Molecule Level

RNAi Speakers		Single Cell Biology Speakers
	Rama Natarajan, PhD. Professor in Diabetes Division Beckman Research Inst. of City of Hope Duarte, CA USA Ttile: Role of MicroRNAs in Diabetic Kidney Disease		Francesco S. Pavone, PhD.               Professor & Group leader European Lab. Non-linear Spectroscopy University of Florence Sesto Fiorentino, Italy Title: Very high speed and ultraprecise manipulation and imaging of a single bio-molecule
	Stoil D. Dimitrov, MD., PhD. Research Fellow in Genetics Dana-Farber Cancer Institute, Boston, MA, USA Title: TBA		Ching-Hwa Kiang, PhD.       Assistant Professor of Physics & Astronomy Rice University, Houston, TX USA Title: single molecule analysis of viruses
	Joseph P. Mizgerd, Sc.D.                            Professor of Medicine and Microbiology Boston University School of Medicine Boston, MA, USA Title: microRNAs directed cytokine expression		Michael Strano, PhD.                       Charles and Hilda Roddey Associate Professor of Chem. Engg Massachusetts Inst. Of Technology Cambridge, MA, USA Title: Single molecule signaling from EGFR using carbon nanotubes
	Joseph Loscalzo, MD., PhD. Physician-in-Chief & Chairman of Medicine Brigham & Women’s Hospital Harvard Medical School Boston, MA, USA Title: MicroRNA-210:  An Hypoxamir that Regulates Cell Metabolism		Ola Söderberg, PhD.                        Associate Professor of Genetics and Pathology Uppsala University Uppsala, Sweden Title: Using proximity ligation assays to explore protein-interactions in clinical specimens
	Mike Blower, PhD.                                     Assistant Professor of Molecular Biology Massachusetts General Hospital Boston, MA, USA Title: single cell analysis of piwi-rnas		Somenath Roy, PhD.                        Research Scientist A Star-Inst. of Bioengineering and Nanotechnology, Singapore Title: Bridging the nano-gaps with DNA: Implication to molecular electronics and medical diagnostics
	Hao Zhu, MD.                                    Hematology/Oncology Fellow Children’s Hospital of Boston Boston, MA, USA Title: The Lin28/let-7 axis regulates glucose metabolism through the PI3K-mTOR pathway		Philip R. Dormitzer, MD, PhD.                    Head, Viral Advanced Programs Global Program Team Novartis Vaccines and Diagnostics Cambridge, MA, USA Title: Study of rotavirus particles near-atomic resolution using cryo-Electron Microscopy
	M.L (NIkki) Harter, PhD. Associate Professor Case Western Reserve University School of Medicine, Cleveland, OH, USA Title: UV-responsive microRNAs are differentially expressed in the melanocytes of a healthy person vs. that of a melanoma patient, in situ		Natasha Paul, Ph.D.                                    Scientific Investigator TriLink BioTechnologies, Inc. San Diego, CA , USA Title: Hot Start dNTPs – Novel Chemistries for Use in Advanced PCR Applications
	Oliver Steinbach, PhD. Head of Bio-Molecular Engineering Philips Research Laboratories Eindhoven, The Netherlands Title: Imag(in)e Global – Release Local Image Guided Ultrasound-Triggered Drug Delivery		David Duffy, PhD.                             Senior Director Platform Research Quanterix Corp. Cambridge, MA, USA Title: Single molecule ELISA
	Dawei Jiang, PhD.                            Fellow in the Lab of Dr. David Clapham HHMI and Children's Hospital Boston Boston, MA, USA Title: Genome-wide RNAi screen identifies the Wolf-Hirschhorn syndrome candidate gene, Letm1, as a mitochondrial Ca2+/H+ antiporter		Dr. Hiroyuki Kishi                                       Assistant Professor of Immunology University of Toyama, Toyama, Japan Title: Single cell manipulation methods
	Seyedtaghi Takyar, MD., PhD.  Assistant Professor of Medicine Yale University School of Medicine New Haven, CT, USA Title: MicroRNA (miR)-1 Inhibits Angiogenesis and Adaptive Th2 Inflammation in the Lung via a VEGF/Myeloproliferative Leukemia Virus Oncogene (Mpl)-Dependent Mechanism		Vasilis Ntziachristos, PhD.              Director Institute for Biological and Medical Imaging Technical University of Munich Munich, Germany Title: Listening to light: new cellular imaging capabilities using opto-acoustic tomography
	Francisco Navarro, Ph.D. Immune Disease Institute Harvard Medical School Boston, MA, USA Title: Analysis of the role of miR-34a in the p53 tumor suppressor network		Madhavi Krishnan, Ph. D.                Marie Curie Research Fellow Laboratory of Physical Chemistry ETH Zurich, Zurich, Switzerland Title: New methods for detection, imaging and manipulation of nano-objects: going beyond fluorsence labeling and optical tweezing
	Nathan D. Lawson, PhD.                  Associate Professor in Gene Function University of Massachusetts Medical School Worcester, MA, USA Title: The role of microRNAs in Vegf signaling, and angiogenesis
	Alexander J.R. Bishop, Ph.D.      Assistant Professor Cellular & Structural Biology University of Texas Health Sciences Center San Antonio, TX, USA Title: RNAi screening, systems biology and comparative biology used to reveal novel mechanisms to augment cisplatin effectiveness
	Joanne Kamens, PhD.          Sr. Director of Discovery RXi Pharmaceuticals, Inc. Worcester, MA, USA Title: Novel “Self-Delivering” RNAi Compounds for Therapeutic Development

Exhibitors are welcome to reserve their booth space early!

Please contact if you are interested in speaking in the scientific or Technology workshops of this meeting.

GeneExpression Systems, Inc.
P.O. Box 540170
Waltham, MA 02454 USA
Tel: (781) 891-8181
Fax: (781) 891-8234
E-mail: Genexpsys@expressgenes.com
www.expressgenes.com

ALL ABSTRACTS

RNAi & MicroRNAs Abstracts

Teasing out cell-specific effects of lethal genes using SID-1-directed feeding RNAi
Martin Chalfie, PhD., William R Kenan Jr. Professor & Chair of Biological Sciences, Columbia University, New York, USA

Easily discernable defects occur when Caenorhabditis elegans is fed bacteria making dsRNA for several of its genes.  Unfortunately, this feeding RNAi does not usually affect neurons, an inability that correlates with the absence in most neurons of SID-1, a protein needed for the transport of dsRNA into cells.  Expression of the wild-type sid-1 gene in neurons overcomes this difficulty. By using a cell-specific promoter to direct sid-1(+) and sid-1 mutant animals, we produced strains in which feeding RNAi is seen in a selective set of neurons.  We have used these strains to test the requirement of lethal genes in the touch-sensing neurons of C. elegans.

 Formation of Cancer Stem Cells and Malignant Progression
Robert Weinberg, PhD. Professor of Biology & Director of MIT Ludwig Center for Molecular Oncology, Whitehead Institute for Biomedical Research, MIT, Cambridge MA  USA

Research over the past decade has revealed a cell-biological program, termed the epithelial-mesenchymal transition (EMT) that is used in normal embryonic morphogenesis to convert epithelial cells into mesenchymal derivatives and is exploited opportunistically by carcinoma cells, which thereby gain a variety of cell-biological traits associated with high-grade malignancy; these include motility, invasiveness, and a heightened resistance to apoptosis.  Our recent work has indicated another, unexpected outcome of the EMT: epithelial cells that have passed through an EMT acquire many of the attributes of stem cells, an outcome that pertains both to normal epithelial cells and their neoplastic derivatives.  The biological and medical consequences of this will be explored.

 MicroRNA-210:  An Hypoxamir that Regulates Cell Metabolism
Joseph Loscalzo, MD., PhD., Physician-in-Chief & Chairman, Dept of Medicine & Hersey Professor of the Theory and Practice of Medicine, Brigham & Women’s Hospital & Harvard Medical School, Boston, MA, USA

A unique group of microRNAs, the ‘hypoxamirs,’ is upregulated by hypoxia in all cells.  A key hypoxamir is microRNA-210 (miR210), which is evolutionarily conserved and ubiquitously expressed in hypoxic cells via induction by hypoxia inducible factor-alpha.  A key target of miR210 is the iron-sulfur cluster assembly proteins (ISCU1/2), which facilitate the assembly of non-heme iron-sulfur clusters into apoproteins, including mitochondrial cytochromes and aconitase.  Hypoxic induction of miR210 suppresses expression of ISCU1/2, which, in turn, impairs mitochondrial electron transport and oxidative phosphorylation, shifting cellular metabolism to anaerobic glycolysis (Pasteur effect).  These observations demonstrate an important adaptive mechanism that limits reactive oxygen species generation by hypoxic mitochondrial metabolism.     

 Novel “Self-Delivering” RNAi Compounds for Therapeutic Development
Joanne Kamens, Ph.D., Senior Director Research Collaboration Management
RXi Pharmaceuticals, Worcester, MA, USA

A novel type of RNAi compound, “self-delivering” or sd-rxRNA™, has been discovered through systematic medicinal chemistry screening.  sd-rxRNA does not require a delivery vehicle for efficient cellular uptake or tissue distribution.  Local administration results in rapid and efficient cellular uptake that translates into significant silencing activity in vivo.  RXi has selected its first Development Candidate in dermal anti-scarring with RXI-109.  Preclinical efficacy data in skin and eye will be presented to support the development of sd-rxRNAs as potential therapies for the indications with a fibrosis component and with potential for treatment in many other therapeutic areas. 

 Imag(in)e Global – Release Local Image Guided Ultrasound-Triggered Drug Delivery
Oliver Steinbach, PhD., Head of Bio-Molecular Engineering, Molecular & Nuclear Medicine, Philips Research Laboratories, Eindhoven, AE, The Netherlands

Medical imaging technologies are becoming an integral part of therapeutic interventions. Ultrasound triggered drug delivery is an emerging application for which delivery vehicles and image guidance techniques are being developed.  The local delivery of therapeutic molecules mediated by ultrasound is a novel approach to address unmet clinical needs by providing a minimally-invasive platform for delivery of pharmaceuticals. In this lecture we show advances in temperature and pressure sensitive agents for ultrasound-triggered, image-guided local release. Temperature sensitive liposomes with incorporated drugs and MRI-imaging labels as well as pressure sensitive, microbubbles loaded with various drugs and specific ultrasound imaging and release characteristics are described. The use of ultrasound for the delivery of drugs has been demonstrated in particular the field of cardiology and oncology for a variety of therapeutics ranging from small drug molecules to biologics and nucleic acids. An overview is given on the potential applications of both types of delivery vehicles:
-Use of drug loaded, temperature (liposomes) and pressure sensitive (microbubbles) systems for ultrasound triggered drug delivery
-Tracking, monitoring and quantifying drug release with ultrasound or MRI through the incorporation of imaging labels in drug carriers
-Formulations and applications for different drug formats: small molecules, biologics, nucleic-acid based therapeutics
-Examples from different disease areas such as oncology, cardiology, metabolic diseases

 MicroRNA (miR)-1 Inhibits Angiogenesis and Adaptive Th2 Inflammation in the Lung via a VEGF/Myeloproliferative Leukemia Virus Oncogene (Mpl)-Dependent Mechanism   
 Seyedtaghi Takyar, MD., PhD., Assistant Professor of Medicine, Yale University School of Medicine, New Haven, CT, USA

Background: Lung endothelial cell activation and neo-angiogenesis are prominent features of severe asthma and VEGF (Vascular Endothelial Growth Factor) plays a critical role in lung angiogenesis and inflammation by inducing these responses. We have previously shown that blocking VEGF in an Ovalbumin (OVA) sensitization and challenge model inhibits aeroallergen-induced adaptive Th2 inflammation and that transgenic overexpression of VEGF in the lung leads to Th2 inflammation and neoangiogenesis (Lee et al, 2004).

Recently we reported that miR-1 is downregulated in the lung endothelial cells exposed to VEGF and that miR-1 supplementation inhibits selective angiogenic effects of VEGF in vitro and in vivo. Here we demonstrate that direct targets of microRNAs can be identified by analysis of the mRNAs recruited to RNA Induced Silencing Complex (RISC) using an Argonaute (Ago2) pull down approach. We also show that in vivo supplementation of miR-1 modulates the lung angiogenesis and Th2 inflammation through downregulation of the identified target gene.

Methods: We transfected mouse lung endothelial cells (MLECs) with miR-1 and used differential microarray analysis to identify potential miR-1 targets. In the next step, mRNAs recruited by miR-1 to the RISC in endothelial cells were characterized by Ago2 immunoprecipitation and validated in a 3’UTR-luciferase assay. The functional significance of the identified mRNA was tested in vitro by transfection of endothelial cells with the corresponding siRNA and in vivo by intranasal delivery. Mice were sensitized to ovalbumin (OVA) with alum and then challenged with OVA by aerosol. MiR-1 or scrambled control or siRNA against c-Mpl were administered intranasally at the time of challenge and cellular inflammation and cytokine levels were assessed.

Results: A comparative analysis of the VEGF-responsive genes and the mRNAs recruited to the RISC by miR-1 showed that Myeloproliferative Leukemia Virus Oncogene (Mpl) is one of the few genes that is upregulated by VEGF and directly targeted by miR-1. RNA interference against MPL inhibited VEGF-induced angiogenic responses in vitro and in vivo. Intranasal delivery of miR-1 reduced the eosinophilic inflammation and Th2 cytokine levels in the BAL and lungs of OVA sensitized and challenged mice. The level of Mpl in the lung increased after OVA challenge and VEGF overexpression and was downregulated by miR-1 supplementation. Mpl knockdown inhibited Th2 inflammation in the OVA model.

Conculsions:   These studies demonstrate that:
- MiR-1 down-regulation is an integral component of Th2 inflammation and miR-1 supplementation inhibits adaptive Th2 immune responses.
- In the lung endothelium miR-1 recruits MPL mRNA to the RISC and inhibition of MPL down regulates the angiogenic response.
- Mpl is upregulated in the lung during Th2 inflammation and directly targeted by miR-1.

Implications: Intranasal delivery of miR-1 or an siRNA against its target, Mpl, can be used as a therapeutic strategy in the management of Th2 lung disease such as asthma.

Genome-wide RNAi screen identifies the Wolf-Hirschhorn syndrome candidate gene, Letm1, as a mitochondrial Ca2+/H+ antiporter

Dawei Jiang, PhD., Postdoctoral Fellow in the Lab of Dr. David Clapham, Children's Hospital Boston & Harvard Medical School, Howard Hughes Medical Institute
Boston, MA, USA

Dawei Jiang, Linlin Zhao, and David E. Clapham§
Dept. of Cardiology, Howard Hughes Medical Institute, Children's Hospital Boston, and the Dept. of Neurobiology, Harvard Medical School. Enders Building 1309, Boston, MA USA

Mitochondria are integral components of cellular signals, regulating cytoplasmic calcium (Ca2+) levels, stimulating mitochondrial ATP production, and regulating apoptosis.  Although several transporter and ion channel mechanisms have been measured in mitochondria, the molecules that govern Ca2+ movement across the inner mitochondrial membrane are unknown.  We conducted a genome-wide, high-throughput Drosophila RNA interference (RNAi) screen to identify genes that control mitochondrial Ca2+ transport, utilizing a specific mitochondrial matrix targeted fluorescence indicator protein.  After the primary screen, comprehensive bioinformatic analyses and secondary screen, Letm1, was found to specifically mediate coupled Ca2+ / H+ exchange.  RNAi knockdown, overexpression, and liposome reconstitution of the purified Letm1 protein show that Letm1 is a mitochondrial Ca2+/H+ antiporter. Letm1 is mutant in Wolf-Hirschhorn syndrome, a condition in humans consistent with mitochondrial failure.

 Analysis of the role of miR-34a in the p53 tumor suppressor network
Francisco Navarro, Ph.D., Immune Disease Institute, Harvard Medical School, Boston, MA USA
miR-34a is a well-known tumor suppressor microRNA that is under-expressed in a wide range of cancers. miR-34a is known to directly suppress the expression of genes involved in cell cycle regulation and apoptosis. To study the role of miR-34a in p53 tumor suppressor function, we used an unbiased biochemical approach, based on the streptavidin pull-down of mRNAs associated with transfected biotin-conjugated miR-34a, to identify candidate miR-34a regulated genes. Transcripts for multiple genes known to inhibit p53 function were enriched in the biotin-miR-34a pull-down. Our data suggest a p53-miR-34a feed forward loop where p53 activates miR-34a transcription and miR-34a in turn enhances p53 function by suppressing the expression of many of its negative regulators.

 Roles of the uridyltransferase enzyme Zcchc11
Joseph P. Mizgerd, Sc.D., Professor of Medicine and Microbiology
Director of Pulmonary Center, Boston University School of Medicine, Boston, MA USA

Zcchc11 is a nucleotidyltransferase enzyme that can add uridines to the 3’ ends of miRNAs, pre-miRNAs, and mRNAs.  Uridylation of IL-6-targeting mir-26 family members relieves them of their ability to silence IL-6 expression, suggesting that Zcchc11 can enhance cytokine expression and contribute to immune function.  The uridylation of pre-miRNAs or mRNAs can decrease the stability of these RNA species.  There is also evidence for Zcchc11 functioning independent of uridyltransferase activity.  Biological roles of Zcchc11 are only beginning to be appreciated, but this protein has been implicated in inflammatory signaling, cytokine expression, stem cell maintenance, and the cell cycle.

RNAi screening, systems biology and comparative biology used to reveal novel mechanisms to augment cisplatin effectiveness
Alexander J.R. Bishop, Ph.D., Assistant Professor, University of Texas Health sciences Center, Dept. Cellular and Structural Biology, San Antonio, TX USA

Using a Drosophila tissue culture based genomic RNAi screen we have identified numerous genes and pathways conserved to mammalian cells that are necessary for surviving various DNA damages. Fusing this data with the protein interactome suggests many possible interactions between survival pathways. Exploring one such pathway interaction we suggest a novel strategy for augmenting cisplatin treatment of ovarian cancers.

 The Lin28/let-7 axis regulates glucose metabolism through the PI3K-mTOR pathway
Hao Zhu, MD., Hematology/Oncology Fellow, George Daley Lab, DFCI-Children’s Hospital, Boston, MA USA

let-7 microRNAs repress oncogene translation and promote embryonic stem cell (ESC) differentiation, while the RNA-binding proteins Lin28a and Lin28b block the processing of all let-7 familymembers to promote ESC pluripotency and tumorigenesis. In transgenic mice, overexpression of both Lin28a and LIN28B establish a metabolic state characterized by enhanced glucose uptake and insulin sensitivity. Overexpression of let-7, on the other hand, leads to impaired glucose uptake, due to let-7­-mediated repression of the PI3K-mTOR pathway. The Lin28a mediated glucose uptake and insulin-sensitive phenotype is abrogated by the mTORC1 inhibitor rapamycin. These data establish the Lin28/let-7 pathway as a regulator of mammalian glucose metabolism, with implications for cancer and stem cell metabolism.

Single Cell Biology Abstracts

Life at the Single Molecule Level
Xiaoliang Sunney Xie, PhD. Mallinckrodt Professor of Chemistry and Chemical Biology
Harvard University, Cambridge, MA USA

In a living cell, gene expression—the transcription of DNA to messenger RNA followed by translation to protein—occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved. Can one monitor these processes in a living cell in real time?  How do cells with identical genes exhibit different phenotypes?  Recent advances in single-molecule imaging in living cells allow these questions to be answered at the molecular level in a quantitative manner. It was found that low probability events of single molecules can have important biological consequences.

 Single Molecule Arrays (SiMoATM) for Life Science Research and In Vitro Diagnostics
David Duffy, PhD., Senior Director Platform Research, Quanterix Corp., Cambridge, MA USA

We will describe our Single Molecule Array (SiMoA™) technology that we are developing for applications in life science researchand in vitro diagnostics.  SiMoA is based on the isolation of individual molecules in arrays of femtoliter wells and detection by fluorescence imaging.  The digital nature of SiMoA enhances sensitivity to enzyme label by 68,000-fold over traditional analog approaches.  We have used SiMoA to develop digital ELISAs that are 1000-fold more sensitive than conventional ELISA.  We will describe the SiMoA approach and instrumentation, and provide examples of how it has been used to detect proteins that were previously undetectable. 

Very high speed and ultraprecise manipulation and imaging of a single bio-molecule
Francesco S. Pavone, PhD., Professor & Head of the Biophotonics, European Laboratory for Non-linear spectroscopy, University of Florence-Department of Physics, Sesto Fiorentino, Italy

In this contribution we will present our recent developments in terms of single molecule imaging and manipulation with high precision and high speed control of forces applied. An approach based on the use of optical tweezers with fast tracking procedures will demonstrate the possibility to visualize a single molecule at work with a nanometer accuracy and to apply and sense forces on it with a temporal resolution of 10s. Also, thanks to fast tracking procedures and the application of optical tweezers , we will show the possibility to increase the colocalisation accuracy in space of the single molecule with a scheme using trapping and fluorescence detection (FIAT). Some examples of applications will be discussed.

High-throughput vertebrate screening at single-cell resolution in vivo
Mehmet Yanik, PhD. Associate Professor, Dept of Electrical engineering, MIT, Cambridge, MA USA    

We demonstrate a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on vertebrates - zebrafish larvae. The system automatically loads zebrafish from multiwell plates, and positions and rotates them for high-speed confocal imaging and laser manipulation of deep organs. The system enables cellular-resolution large-scale in vivo studies of complex processes such as organ development, neural degeneration and regeneration, stem cell proliferation, cardiovascular, immune, endocrine and nervous system functions, pathogenesis, cancer and tissue specificity and toxicity of drugs.

Observing Dynamic States of DNA and Proteins Using Single-Molecule Manipulation
Ching-Hwa Kiang, PhD. Professor of Physics & Astronomy, Rice University, Houston, TX, USA

Single-molecule manipulation studies open a door for a close-up investigation of complex biological interactions at the molecular level.  We used an atomic force microscope to pull poly(dA) molecules and found that poly(dA) has two states when highly stretched, and the unique base interaction of poly(dA) makes it more stable at large extensions.  We have also studied the dynamic states of of von Willebrand Factor, a key protein in blood coagulation.  These results suggest that single-molecule manipulation technique has potential to be developed into a general tool for studying the dynamics of protein and nucleic acid complexes.

A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells
Dr. Hiroyuki Kishi, professor of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan

Antigen-specific human monoclonal antibodies are important candidates for therapeutic agents. Here, we present a method for detecting individual antigen-specific antibody-secreting cells (ASCs) using microwell array chips, which enables the analysis of live lymphocytes on a single-cell basis and offers a rapid, efficient, and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for virus antigens from human peripheral blood lymphocytes, and produced human monoclonal antibodies with virus-neutralizing activities within a week. Our method would open the way for the generation of therapeutic antibodies for individual patients.

Bridging the nano-gaps with DNA: Implications to molecular electronics and medical diagnostics
Somenath Roy, PhD., Research Scientist, A Star-Inst. of Bioengineering and Nanotechnology
Singapore

Charge transport through the -stacked DNA base pairs has drawn remarkable attention owing to its implications to multiple physiologic processes, biosensors and futuristic molecular electronics. We have investigated the current carrying capacity of a single DNA molecule (80 base pair) by bridging it across a nanogap between a pair of single-walled carbon nanotubes. While a hybridization-induced change in conductivity indicated its application in biosensing, the gate-voltage dependent I-V characteristics suggested the potential use of a DNA molecule as a channel in a field-effect transistor. When it comes to a full-length genomic DNA, we have designed a mass-producible sensor array, in which the termini of a target DNA molecule hybridizes with two capture probes immobilized across a vertical nanogap. The unique design of the vertical nanogap sensor, along with the two-probe approach, has enabled us to achieve a detection limit of 1.0 fM with a capability to discriminate single-base mismatch. We could also detect messenger-RNA (mRNA) on the identical platform with sub-fM sensitivity upon a slight modification in the probe design.

Listening to light: new cellular imaging capabilities using opto-acoustic tomography
Vasilis Ntziachristos, PhD., Director of the Institute for Biological and Medical Imaging,
Technical University of Munich & Helmholtz Zentrum München, Germany

Optical imaging is unequivocally the most versatile and widely used visualization modality in clinical practice and life sciences research. In recent years, advances in photonic technologies and image formation methods have received particular attention for non-invasively revealing information on the molecular basis of disease and treatment. An increasing availability of endogenous reporters such as fluorescent proteins and probes with physiological and molecular specificity enable insights to cellular and sub-cellular processes through entire small animals, embryos, fish and insects and have revolutionized the role of imaging on the laboratory bench, well beyond the depth capability of modern microscopy. This talk describes current progress with instruments and methods for in-vivo opto-acoustic tomography of whole intact animals and model biological organisms. We show how new tomographic concepts are necessary for accurate and quantitative molecular investigations in tissues and why it could be potentially a valuable tool for accelerated investigations of therapeutic efficacy and outcome. We further demonstrate that cellular function and bio-chemical changes can be detected in-vivo, through intact tissues at high sensitivity and molecular specificity. Examples of imaging enzyme up-regulation, carcinogenesis and gene-expression are given. The potential for clinical translation is further outlined.

Using proximity ligation assays to explore protein-interactions in clinical specimens
Ola Söderberg, PhD., Associate Professor of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden

Measurements in single cells are required to identify cell-to-cell variation in a heterogeneous cell population such as a tissue section. The activity status of a protein or signaling pathway can be visualized with in situ Proximity Ligation Assays (in situ PLA) using a pair of antibodies targeting the interacting proteins or PTM, using an attached DNA molecule to the antibodies to template the creation of a circular DNA molecule that is a surrogate marker for the interaction. It can then be amplified by rolling circle amplification (RCA) and be detected, with a single-molecule resolution, in fixed cells or tissues.

Near atomic resolution electron cryomicroscopy analysis of rotavirus cell entry
Philip R. Dormitzer, MD, PhD., Head, Viral Advanced Programs Global Program Team, Senior Project Leader, Viral Vaccine Research, Novartis Vaccines and Diagnostics, Cambridge, MA USA

Near atomic resolution electron cryomicroscopy has reduced the time needed to determine the structures of rigid icosahedral particles from years to months or even weeks.  We have used this technique, in combination with other structural and functional analyses, to dissect the mechanism of cell entry by rotavirus.  The studies reveal the details of how the capsid protein of this non-enveloped virus triggers un-coating in low calcium and the viral spike protein rearranges in a manner analogous to the membrane fusion proteins of enveloped viruses.  We are now exploring approaches to broaden the application of this powerful technique.

Hot Start dNTPs – Novel Chemistries for Use in Advanced PCR Applications
Natasha Paul, Ph.D., Scientific Investigator, TriLink BioTechnologies, Inc., San Diego, CA USA

Recently developed Hot Start dNTPs are a distinct approach to Hot Start activation in PCR that employs modified nucleoside triphosphates with a thermolabile protecting group at the 3-hydroxyl.  This thermolabile chemistry can be applied to dNTP analogs such as dUTP for UNG decontamination methods and 7-deaza-dGTP for GC-rich target amplification.  In addition, further studies have led to the development of 3-protecting groups that deprotect more quickly than the current 3-modification group, allowing application to fast PCR.  With the evolving chemistry of Hot Start dNTPs, the areas of application benefiting from the versatility and flexibility of this technology continue to grow.

PCR is a widely used scientific tool whose specificity can be increased by the use of Hot Start technologies. Although many Hot Start technologies exist, recently developed CleanAmpTM dNTPs are a distinct approach that employs modified nucleoside triphosphates with a thermolabile protecting group at the 3-hydroxyl.  The presence of the protecting group blocks low temperature primer extension, which can often be a significant problem in PCR.  At higher temperatures, the protecting group is released to allow for incorporation by the DNA polymerase and more specific amplification of the intended target. These modified dNTPs provide comparable performance to other Hot Start technologies and can be used with thermostable DNA polymerases to turn a reaction into a Hot Start version.  This thermolabile chemistry can be applied to dNTP analogs such as dUTP, which is used in UNG decontamination methods, and 7-deaza-dGTP, which is used to amplify difficult GC-rich targets.  In addition, further studies have led to the development of 3-protecting groups that deprotect more quickly than the current 3-modification group, allowing these modified dNTPs to be used in fast PCR.  With the evolving chemistry of CleanAmpTM dNTPs, the areas of application benefiting from the versatility and flexibility of this technology continue to grow.

 New methods for detection, imaging and manipulation of nano-objects: going beyond fluorsence labeling and optical tweezing
Madhavi Krishnan, Ph. D., Marie Curie Research Fellow in the lab of Dr. Vahid Sandoghdar, PhD., Professor of Physical Chemistry, ETH Zurich, Switzerland

The advent of fluorescence microscopy and spectroscopy in the 1990s ushered in single molecule detection as a powerful tool for a wide range of studies, ranging from biophysics to quantum optics. However, this approach has been limited to chromophores with high quantum efficiency. Over the past few years, we have developed extinction detection and spectroscopy as an alternative to fluorescence for investigating single nano-objects such as metallic nanoparticles, viruses, quantum dots, and organic molecules. After a general introduction to the operation principle of this method, I will discuss a specific application of imaging single virus motion and its interaction with receptor lipids. Furthermore, I present a new nanofluidic technique for contact-free and stable trapping of nanoparticles such as gold and polymer beads or lipid vesicles. I will end with the prospects of fluorescence-free imaging and trapping of biological entities as small as single proteins.