Grant Abstracts 2012   Broad Institute Inc. Feng Zhang Cambridge, MA $1,000,000 June 2012 Neuropsychiatric diseases arise from a combination of genetic and environmental factors that influence the molecular, morphological and physiological properties of neurons and glia in the brain.  Elucidation and treatment of these diseases will benefit from understanding how specific brain cell types connect and signal in neural circuits, and how genetic factors affect their cellular function.  Transgenic techniques have been widely used to target specific cell populations with fluorescent reporter (e.g. GFP) and modulator (e.g. channelrhodopsin-2) genes to probe their role in disease mechanisms.  However these conventional gene targeting strategies depend on the identification of unique molecular markers and promoters and are largely limited to the mouse, whereas other animal models that are commonly used for neuroscience and disease studies (e.g. rats and primates) are still mostly inaccessible.  Additionally, since many non-rodent animal models have long reproductive cycles, fundamentally new approaches for cell-type specific gene targeting are highly desirable.  The researchers propose to develop and apply a generalizable genetic circuit for detecting endogenous transcription states to enable conditional genetic manipulation of specific cell types.  The proposed technology could have broad applications for both disease research as well future therapeutic interventions.   J. David Gladstone Institutes Sheng Ding, Steve Finkbeiner, Shinya Yamanaka San Francisco, CA $1,000,000 June 2012 The human brain can carry out more operations per second than the most powerful computer.  It also has capacities no computer is likely to ever have, such as consciousness and self- awareness.  Thanks to human induced pluripotent stem cells and other induced neural precursor cells, scientists can now make, in theory, any type of brain cell from adult skin cells.  Still, only a few types of human brain cells have been made, and the process for making them remains inefficient.  The investigators propose to establish an interdisciplinary program to understand how cells that are reprogrammed to pluripotency can be instructed to generate each type of cell in the brain.  They have assembled a team that can create, in the laboratory, a number of cell types that compose the brain.  In this project they propose to make certain brain cells (e.g., von Economo neurons that are thought to confer unique abilities, such as emotional and social salience, to the human brain) that do not exist in standard laboratory animals.  This work could lay the foundation for both the development of cellular therapies to treat neurological diseases and for a greater understanding of what makes us unique as humans.   Oregon Health & Science University Joe Gray Portland, OR $1,000,000 June 2012 This project will initiate development of image-based approaches that will allow architectural analysis of the nanoscale molecular assemblies that regulate information flow in cells.  The team will coordinately develop multi-scale imaging, labeling chemistry and computer science to visualize specific molecular assemblies in cells and to localize these relative to cellular ultrastructural features using a new integrated light and electron microscope (iLEM) to analyze the signaling architecture.  The iLEM consists of a custom-designed fluorescent microscope mounted on the side port of a transmission electron microscope.  The project team will develop the technologies to enable the detection of the molecular structures that regulate information flow within cells and learn how these structures assemble, integrate signals from multiple sources, and shuttle the information to action centers in cells that control activities such as growth, death and movement.  The research team expects that their iLEM analysis approaches eventually will be broadly applicable to studies of regulatory signal networks in normal and diseased tissues.   University of California, San Diego Steven Dowdy, Yitzhak Tor San Diego, CA $1,000,000 June 2012 The discovery of RNA interference (RNAi), a natural gene silencing mechanism with exquisite selectivity for all 23,000 human mRNAs, opened the door to a global therapeutic approach that can be tailored to keep evolutionary pace with pandemic viral outbreaks or for personalized medicine.  However, use of RNAi as a therapeutic is severely hindered by the negatively charged phosphate backbone: it simply cannot enter cells on its own.  Current RNAi delivery approaches utilize nanoparticles that are 5,000-fold larger than the actual RNAi cargo, which due to this size, have inherently poor pharmacological properties.  Using an interdisciplinary chemistry and molecular approach, we seek funds to build a paradigm shifting RNAi technology that radically shrinks the delivery size to the smallest possible, self delivering monomeric RNAi-inducing molecule via synthesis of neutral, bioreversible phosphotriester RiboNucleic Neutrals.  Once inside cells, cytoplasmic enzymes unmask the phosphate groups and convert them into negatively charged, active RNAi molecules.  Although the technology has broad applicability, the team will establish efficacy of this delivery system in mouse models of cancer.   University of Wisconsin, Madison Aseem Ansari, Parameswaran Ramanathan, Jennifer Reed, David C. Schwartz Madison, WI $1,000,000 June 2012 The ability to compose or “program” genomes to perform desired functions will yield insights into how gene networks govern life and will stimulate innovation in many disciplines that interface with biology.  The current approach to synthetic genomes involves copying an existing small genome via time-intensive and cost-prohibitive methods.  The UW team of engineers, chemists and biologists seeks to create a multifaceted system – a genome foundry – that permits rapid, inexpensive fabrication of de novo designed genomes.  The interdisciplinary team, will develop an integrated system to enable design-to-fabricated genome production.  The proposed “genome foundries” will comprise an inter-locking suite of computational tools, nanofluidic instrumentation, hardware fabrication languages, and custom-designed synthetic gene switches. The new technology envisioned could enable widespread invention of genome-aided solutions to fundamental and applied problems and the ability to compose and automate the synthesis of large DNA molecules could spur innovations in DNA-based nano-device fabrication and DNA computing.   Grant Abstracts 2011   Carnegie Mellon University Adrien Treuille, Rhiju Das Pittsburgh, PA $1,000,000 December 2011 Computational models cannot yet robustly design RNAs that fold correctly in vitro, slowing the development of vital antibacterial, antitumor, and antiviral therapies.  The research team proposes a novel strategy to rapidly advance RNA bioengineering:  Internet-scale RNA design competitions rigorously scored by wet-lab feedback and organized through an online game.  Launched in early 2011, EteRNA hosts a community of over 25,000 citizen scientists who outperform existing RNA design methods.  The research team proposes to formalize this community’s insights into the first extensively validated automated RNA design algorithm, EteRNAbot.  They also propose to expand the game’s scale to thousands of hypotheses per month, enabling a new scale of public involvement in hypothesis-driven research.  Finally, they propose to extend the EteRNA interface from secondary structure design to fully 3D engineering of RNA-based biomedical sensors and scaffolds.  They will evaluate success via high-throughput experimentation; by the publication of tools and principles generated by EteRNA players; and by the widespread adoption of EteRNA’s novel high-throughput experimentation paradigm into future citizen science projects.   Columbia University Dirk Englund, Jonathan Owen, Rafael Yuste New York, NY $1,000,000 December 2011 The research team proposes to develop a novel method for direct, real-time imaging of neuronal voltage signals.  Neuroscience has traditionally relied on electrodes to measure the activity of neurons, one at a time.  It is, however, the interaction of many cells in neuronal circuits that generates behavior, and tools to monitor such ensemble activity, while still preserving single-cell resolution, are lacking.  To tackle this technical challenge, the team will use color centers in diamond nanocrystals, together with techniques derived from atomic physics, to sense electric fields with unprecedented resolution.  Diamond is well suited for use in biological preparations because it is chemically inert, cytocompatible and ideal for coupling to biological molecules.  The team proposes to harness these properties in diamond nanoprobes to measure electrical signals from neurons in real-time, in neuronal cell cultures, in neuronal circuits in brain slices and in awake mice.   Purdue University Joseph Irudayaraj, Feng Zhou, Sophie Lelièvre, Ann Kirchmaier West Lafayette, IN $1,000,000 December 2011 It is becoming increasingly clear that genetic mutations alone cannot fully account for devastating chronic adult-onset diseases, neurological disorders and cancer.  Rather, environmental assaults also contribute significantly to disease etiology and pathology.  Environmental factors act on epigenetic processes in a cell to alter gene expression without causing a genetic mutation.  This gives the cell plasticity to respond to the environment without causing permanent damage to its DNA.  Understanding how epigenetic marks govern gene expression at the single cell level will foster development of intervention strategies to change cell fate by inducing or reversing epigenetic modifications.  However, current knowledge of how these marks function is limited by existing technologies.  To break through this critical limitation, the research group will create, refine and validate a set of technologies that will enable researchers to identify, examine and even alter epigenetic marks in a locus-specific manner at the single cell level.  They will test this system in a neural stem cell differentiation model and validate it in a 3D breast epithelial tissue culture system.  This toolkit will be designed to enable users to customize strategies to identify and alter epigenetic processes at a single gene in individual cells to reset key events during neural differentiation or tumor formation.   Texas Children's Hospital Huda Zoghbi (Howard Hughes Medical Institute), Daoun Ji, Jianrong Tang, Yuri Dabaghian, Daniel Curry, Akash Patel Houston, TX $1,000,000 December 2011 Intellectual disabilities and autism spectrum disorders (lD/ASDs) represent a huge health care burden.  Dozens of genes that perform diverse molecular functions have been shown to cause these disorders when mutated, deleted or duplicated.  The challenge is to understand how such disparate molecular changes result in similar phenotypes and how we can treat these disorders given their genetic heterogeneity.  The investigators’ overarching hypothesis is that diverse molecular changes alter neural network activity and synaptic homeostasis; that such alterations in network activity lead to the overlapping cognitive and motor phenotypes; and that manipulating the aberrant activity, through modalities such as deep brain stimulation (DBS), will prove effective in treating this broad class of disorders.  They will investigate neural circuit dysfunction and treatment response in genetically-accurate models of ID/ASDs.  Specifically, they will determine the neural circuit activity in genetic models of Rett and Angelman syndromes using in vivo recordings of single units, synaptic plasticity, and network oscillations in freely-moving mice for each disease model.  They will then relate the circuit abnormalities to the cognitive deficits of these mice, and finally, will use DBS to explore possible therapeutic intervent.   University of California, Irvine H. Kumar Wickramasinghe, Kavita Arora, Rahul Warrior, Edward Nelson, Arthur Lander, Alice Yamada Irvine, CA $1,000,000 December 2011 The team proposes a unique instrument (the Single Cell Analyzer, SCA) for analysis of mRNA levels (at copy numbers of 5 to 2,000 molecules) in space and time within a living cell.  Current understanding of disease and developmental processes is largely based on biochemical or gene expression studies in the whole organ or tissue, coupled with phenotypic analysis of mutations in the context of the whole organism.  The SCA quantitatively queries gene expression at the single cell level, permitting examination of biological processes at much greater resolution.  The team has demonstrated that an active atomic force microscope (AFM) probe chemically modified with DNA primers can be inserted into a cell, without destruction, to selectively capture mRNA via an applied attractive dielectrophoretic force, and subsequently quantified.  Since mRNA levels can be assessed without affecting cell viability, the same cell can be monitored over time.  The proposed instrument will have applications in areas ranging from developmental and systems biology to personalized medicine, cancer diagnosis and stem cell research, with the potential to transform cell biology much as PCR has revolutionized molecular biology.  While transcript levels can be measured in fixed samples, no technology exists today that can dynamically quantify mRNA expression in individual living cells.   University of Florida Laura Ranum, Maurice Swanson Gainesville, FL $1,000,000 December 2011 Well-established rules of translational initiation have been used as a cornerstone in molecular biology to understand gene expression and to frame fundamental questions about what proteins a cell synthesizes, how proteins work and to predict the consequences of mutations.  For a group of neurological diseases caused by the abnormal expansion of short segments of DNA (e.g. CAG/CTG repeats), mutations within or outside of predicted coding and non-coding regions are thought to cause disease by protein gain or loss-of-function or RNA gain-of-function mechanisms.  The research team unexpectedly discovered the canonical rules of protein translation do not apply for these repeats and in the absence of an ATG initiation codon, expanded CAG and CTG often express homopolymeric proteins in all three frames (e.g. polyGln, polyAla, polySer from CAG repeats).  This Repeat Associated Non-ATG translation (RAN translation) depends on RNA structure and repeat length and occurs when expansion constructs are integrated into mammalian brain.  Additionally, they showed that RAN translation occurs in two human CAG/CTG expansion diseases: DM1 and SCA8.  The project will pursue critical questions:  How does RAN translation work?  Is RAN translation a key, previously unrecognized cellular process?  Are repetitive sequences throughout the genome translated into proteins and if so, what is their function?   Washington University in St. Louis Robi Mitra, Stephen Johnson, Mark Johnston St. Louis, MO $1,000,000 December 2011 There is still a great deal to learn about the design principles that guide vertebrate development.  Since all cells in an organism contain the same genes, transcribing different sets of genes confers a cell’s specialized role.  Which genes get turned on or off to create a particular cell type at the right time, in the right place during the development of an organism?  This is one of the pivotal questions in developmental biology.  The research team has developed a technology, Transposon “Calling Cards,” to attack this question in a novel way, and has shown that it works in yeast and mammalian cell cultures.  They propose to develop this technology, which allows them to mark every place in the genome where genes are active, for use in a living animal.  If successful, they will be able to record gene expression along different cell lineages throughout the development of a living vertebrate.  The team will be able to produce a complete record of gene activation at different stages of vertebrate development, watching as cells and their progeny specialize and form organs.  These data would contribute to the field of developmental biology by providing a blueprint for the generation of all cell types, and could ultimately guide the reprogramming of embryonic or induced pluripotent stem cells to produce specific cell types for personalized transplants, such as pancreatic beta cells to treat diabetes.   Salk Institute for Biological Studies Joseph Ecker, Terry Sejnowski, Margarita Behrens La Jolla, CA $1,000,000 June 2011 The human genome sequence lists every DNA base of the roughly 3 billion bases that makes up the genome, but it doesn’t tell us about how its function is regulated.  That job belongs to the epigenome, the layer of genetic control beyond the regulation inherent in the sequence of the genes themselves.  Methylation of DNA at the carbon 5 position of cytosines constitutes an important epigenetic layer that contributes to the definition of transcriptional and regulatory potential of the genome.  DNA methylation in cells dynamically changes during aging and is susceptible to environmental influences.  To shed light on the dynamic variation of these marks in normal and diseased cells, novel technologies must be developed for the creation of base resolution maps of DNA methylation in single cells.  The interdisciplinary team proposes to develop an approach to “read the epigenome” information from single chromosomes within single neurons.  Being able to study DNA methylation in its entirety in single cells will greatly increase our understanding of how gene expression is influenced by the environment, including the impact of diet and stress, as well as how genome function is regulated in health but dysfunctional in disease states such as schizophrenia and other mental disorders.   Thomas Jefferson University Isidore Rigoutsos Philadelphia, PA $1,000,000 June 2011 Despite very significant advances in the field of non-coding RNAs (ncRNAs), much remains to be uncovered.  This project will study a novel and currently under-characterized class of non coding RNA transcripts (ncRNAs) that are linked to pyknons.  Pyknons are a group of DNA motifs that were discovered several years ago using a data agnostic pattern discovery approach and which exhibit a number of intriguing properties.  To date the pyknon framework has correctly anticipated several discoveries in the RNA field, including: the existence of novel classes of short ncRNAs with specific lengths; the role of messenger RNAs (mRNAs) as a source of regulatory ncRNAs; and the targeting of the introns of unspliced mRNAs by exon-derived ncRNAs.  The planned work will combine computational analyses with modern experimental techniques to study samples from several diverse human conditions in order to determine the rules governing the pyknons’ biogenesis, processing and mechanisms of regulatory action.   University of California, Davis David Jay Segal, Anne Knowlton, Jan Nolta, Scott Simon, David Rocke Davis, CA $1,000,000 June 2011 One of the greatest challenges facing biomedical research since the sequencing of the human genome has been to understand the role of genetic variation in human disease.  Many genetic variants have been associated with common diseases.  However, determining the functional consequences of these variants has been difficult.  Several variants are often inherited together in tightly linked blocks, making it difficult to determine the causative variant.  Additionally, people have millions of other genetic differences, making it difficult to correlate cellular phenotypes with a particular variant.  Different gene sets are expressed in different cells, but it is difficult to extract disease-relevant cells from large numbers of patients.  Here the PIs propose a new method for the functional analysis of genetic variation, using custom nucleases to genetically modify individual variants in induced pluripotent stem cells.  They will focus on variants at the 9p2l region of the genome that have been associated with coronary artery disease.  The methods developed should provide a new way to unlock the wealth of data from genome wide association studies, and to probe the genetic architecture of common diseases.   Grant Abstracts 2010   Colorado State University Randy A. Bartels, Stuart Tobet Fort Collins, CO $1,000,000 2010 The research team of Randy Bartels and Stuart Tobet at Colorado State University proposes to develop and build a new imaging technology that will allow a direct view of the complex world of molecular communication within and between cells. Achieving the project’s goal requires a new approach to forming images of the spatio-temporal concentration of biomolecules and increases the sensitivity of unlabeled molecular detection in optical microscopy by up to six orders of magnitude. They propose to observe changes in molecule concentrations at up to picomolar levels. This sensitivity will use a new approach to capturing vibrational Raman spectral information using properties of ultrafast laser pulses interacting with coherent quantum Raman-active molecular vibrations. Images formed from this ultra sensitive Doppler shifted Raman detection will be combined with multimodal nonlinear microscope images to simultaneously capture morphology, cell translation and molecular signaling. A new view of molecular signaling leading to changes in cell mobility and cell differentiation may be revealed by this new technology.   Fred Hutchinson Cancer Research Center Harlan S. Robins, Christopher S. Carlson Seattle, WA $1,000,000 2010 FHCRC researchers led by Harlan Robins and Christopher Carlson propose to develop advanced DNA sequencing methods to measure past and present pathogenic exposure in humans. By sequencing tens of millions of antibody genes per person – over 100 times more genes than we can sequence with existing technology – they aim to identify rearrangements that are shared by individuals with the same disease. A catalogue of unique B cell DNA signatures corresponding to distinct pathogen exposures and disease states would comprise a library of associations that can be quickly and inexpensively accessed by clinicians to diagnose and predict risk of future diseases. Eventually, such a repository could be used to screen for novel correlations between environmental exposures and existing or emerging diseases.   University of California, Irvine Enrico Gratton, J. Lawrence Marsh, Michelle Digman Irvine, CA $1,000,000 2010 UCI scientists Enrico Gratton, Lawrence Marsh and Michelle Digman will develop optical microscopy technology capable of imaging biologically relevant events in live animals across cellular and subcellular scales, and in 3D at significant tissue depths. They will use their unique feedback based imaging methodology that has the capability to capture weak signals at the speeds necessary to detect fast biochemical reactions (~ms), and at the resolution needed to allow visualization of critical biomolecular events (~20nm) while still retaining the ability to image across lateral distances (~cm) and at a significant depth (up to 3mm) into the tissue. For this project, they demonstrated the principles of feedback imaging in 3D and imaging in strongly scattering media. Their aim is to show that that these technologies can work together for live animal tissues in an upright microscopy configuration. The project’s biological focus is on cell migration in tissues, a fundamentally more complicated problem than cell migration in 2D. Cell migration is important in cancer metastasis, wound healing, tissue regeneration, stem cell proliferation and developmental biology. The resolution, speed, sensitivity and depth necessary to visualize and characterize the molecular dynamics at the basis of 3D migration have not yet been achieved. This proposed development will advance our knowledge of fundamental biological processes.   University of California, Los Angeles Mayank Mehta, Katsushi Arisaka, Bahram Jalali Los Angeles, CA $1,000,000 2010 While Einstein revealed the nature of physical space, the nature of mental representation of space is not well understood. The UCLA team lead by Mayank Mehta will address this question at the interface of physics and neurobiology using novel experimental tools and mathematical theories. They hypothesize that mental space is defined by a sequence of neuronal events, or neural ensemble activity patterns. To test this hypothesis, they will focus on a brain region called the hippocampus which is known to be involved in spatial perception and shows spatially selective activity. The team will measure the influence of behavior on hippocampal ensemble activity, including its rhythmicity and the precise spike timing of neurons using whole cell patch clamp, multi-electrode devices and two-photon microscopy. The sequence of neural events measured during natural navigation will be manipulated by a virtual reality system. The emergence of long-term spatial memories will be investigated by determining the influence of calcium influx on hippocampal synaptic plasticity that could permanently encode the sequence of events using distinct neural activity patterns and oscillations. The results could provide a new theory of how a mental representation of space is generated from environmental stimuli, behavior and brain’s internal dynamics.   University of California, Riverside Susan Wessler, Peter Atkinson, Jason Stajich Riverside, CA $1,000,000 2010 While draft genome sequences are available for the three mosquito species that are the subject of this proposal: Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus, active transposable elements (TEs) for efficient genetic analysis are not. Class 2 TEs isolated from other organisms are quickly inactivated when introduced into mosquitoes. The UCR team led by Susan Wessler, Peter Atkinson and Jason Stajich, will address this bottleneck through a novel strategy to exploit mosquito genome sequences to identify new mosquito TEs that may have superior mobility properties. They will focus their strategy in part on one type of TE called MITEs that were discovered in the laboratory of Dr. Wessler. As the most abundant component of the three mosquito genomes, MITEs have evolved mechanisms to evade genome surveillance and spread through populations. This project has five Aims. Aim 1 will use newly developed computer pipelines to annotate and characterize all class 2 TEs and their family relationships and identify candidate active TEs. Aim 2 will determine the small RNA profiles of the annotated TEs to find TEs that have evaded host surveillance. Aims 3 and 4 will test the mobility of candidate TEs by exploiting high throughput yeast and mosquito cell assays. In Aim 5, they will analyze a diversity panel of mosquito strains to detect TEs in the act of amplifying. The researchers seek to develop these new active mosquito TEs as incisive genetic tools which will directly increase the ability of researchers to identify mosquito genes responsible for the transmission of deadly human pathogens.   University of California, San Francisco Michael Fischbach, Pieter Dorrestein, David Relman, Justin Sonnenburg San Francisco, CA $1,000,000 2010 This multi-institutional team led by Michael Fischbach at UCSF has observed that antibiotic production is widespread among human gut bacteria. They propose to identify antibiotics produced by gut bacteria and characterize their role in modulating the composition of the gut microbiome. Antibiotic-mediated interactions could explain two intriguing observations from early data from the Human Microbiome Project: that the composition of the gut microbial community differs dramatically between people, and that even within individuals, the gut community consists of a stable ‘core community’ and a dynamic ‘auxiliary community’. This project will focus on 1) Functionally characterizing antibiotic production by gut microbes, 2) Using a new mass spec technology to create a molecular map of the gut community, and 3) Integrating these data into a quantitative network model of interspecies interactions in the gut community. These results may enable the team to begin modeling the dynamics of synthetic gut communities, and they will form a basis for studying how changes in host genetics (e.g., polymorphisms in innate immunity genes) and the environment (e.g., changes in diet) perturb the composition of the gut community.   University of Oregon William Cresko, Hui Zong Eugene, OR $1,000,000 2010 The team of William Cresko and Hui Zong at the University of Oregon propose to combine innovative cell labeling and genome sequencing technologies with analytical tools from evolutionary biology to identify causative genetic changes during cancer development. They will use a mouse glioma model developed by Dr. Zong’s lab to generate sporadic tumorigenic cells and unequivocally label them with Green Fluorescent Protein (GFP), thus revealing close interactions between cells that will become tumors and their normal neighboring neurons. These cells will be precisely collected at distinct tumorigenic phases using a Laser Capture Microdissection system. Next Generation Sequencing approaches developed in Dr. Cresko’s lab will be used for genomic and transcriptomic analysis of these cells, and the specific mutations causing tumor formation will be determined using multivariate evolutionary genetic analyses. This work may help identify causal genetic changes across the tumorigenic process, starting from the earliest stages of tumor formation, revealing the critical supporting roles of normal neurons to glioma cells and possibly leading to new methods for early detection and treatments of glioma and other tumors.   University of Washington Eberhard Fetz, Jeffrey Ojemann, Brian Otis, Babak Parviz Seattle, WA $1,000,000 2010 The brain is the world’s most powerful autonomous computer, whose operations remain to be understood and whose functions can be rescued when impaired. The computational capabilities of biological neural networks and silicon computers are complementary and could operate synergistically if directly connected. Recent advances in low-power electronic and biocompatible microfabrication technology will allow the team led by Eberhard Fetz to develop implantable computer systems that interact continuously with the brain. The project will develop and deploy autonomous recurrent brain-computer interfaces (R-BCI) that implement novel interactions between brain sites. By operating continuously, the R-BCI will allow the brain to learn to exploit these new resources to optimize its function. This novel paradigm opens many fundamentally new research directions, depending on the site of recording and stimulation, and the programmable transform between recorded activity and stimulation. The R-BCI also has numerous clinical applications for bridging damaged biological pathways and for strengthening weak neural connections. This project will also create a powerful new multi channel “Keck Active Electrode Array” with integrated electronics to implement minimally invasive recording and stimulation of large numbers of brain sites.   Grant Abstracts 2009   Princeton University William Bialek Princeton, NJ $1,000,000 2009 The three-year program entitled Physical Limits and Biological Function will support the principal investigators as they lead a group of postdoctoral fellows in collaboration with researchers at the Lewis-Sigler Institute and the Center for Theoretical Science, as part of a broader theoretical effort at the interface of physics and biology. The group will be investigating key theoretical questions about the way in which the building blocks of life are organized to solve the problems essential for an organism’s survival, development and reproduction. More specifically, they will explore the idea that life’s solutions to these problems are not merely consistent with the laws of physics, but in many cases have been driven to the limits of what the laws of physics allow. As theorists, their goal is to turn this hypothesis about operation near the physical limits into a predictive theory, in the physics tradition, from which many aspects of the underlying biological mechanisms can be predicted. They will search for common theoretical principles that cut across many levels of biological organization, from the early events of embryonic development in fruit flies to the dynamics of sensorimotor control in primates and from lifestyle choices in bacteria to human perception.   University of California, Berkeley Lydia Sohn Berkeley, CA $1,000,000 2009 Why do embryos form limbs while postnatal humans form scar tissue? This fundamental question remains elusive under existing medical research paradigms. Current knowledge of amphibian limb regeneration cannot explain the lack of functional tissue re-growth in adult mammals. Although limb regeneration via blastema has been observed in neonatal mice, little is known in cellular or molecular terms. Consequently, controlling and enhancing postnatal organogenesis for therapeutic ends appears to be an intractable challenge. Thus, the researchers seek to transform present understanding of the dramatic decline in regenerative capacity after birth by inventing technology capable of probing cellular and molecular determinants of regeneration in single mammalian cells. Their combined efforts will develop: 1) a novel cell-sorting tool to isolate from tiny tissue clusters the single cells that are pivotal to mammalian tissue regeneration; 2) new tools to study molecular mechanisms orchestrating the regenerative behavior at single cell levels; and 3) 3D ex-vivo systems to recreate the regenerative processes biosynthetically, thereby identifying molecular cues driving tissue regeneration. This single-cell approach may surmount prevailing limitations, thus advancing the frontiers of biomedicine and engineering.   University of California, San Diego Ralph J. Greenspan La Jolla, CA $1,000,000 2009 Demonstrating that there is a fundamental, unifying principle for the operation of biological networks, one that cuts across phylogeny and type of network, could revolutionize the natural sciences. This project will use a research strategy based on the concept of relational networks, in which the coordinated interactions among functional sectors of the network are more critical than the specific identities of any of the interacting components. The program combines experimental network perturbation and global monitoring in genetic and neuronal model systems, followed by theory development, going beyond the standard computational modeling to capture the essential, relational nature of the system at a higher level. Should there prove to be fundamental, universal principles underlying the relations and function of biological networks, they will have substantial implications not only for many areas of biology, including the prospects for synthetic cells and therapeutic developments, but also for the design and implementation of artificial networks in applications as diverse as computing, engineered devices and communications.   University of California, San Francisco Joseph DeRisi San Francisco, CA $1,000,000 2009 Among the greatest achievements of modern medicine is the development of vaccines, but for many important diseases, such as HIV, tuberculosis and malaria, the production of safe and effective vaccines has been elusive. This is especially true for malaria, a disease that disproportionally affects children. An important first step in developing a rationally designed synthetic vaccine is the identification of antigens and their epitopes that are the actual determinants of immunological protection. The investigators propose to develop a new platform for the production of programmable proteome-scale peptide arrays on beads using a novel approach, whereby millions of peptides can be made deterministically and inexpensively on spectrally encoded microbeads. They will then conduct immunoassays on these beads to map epitopes across the entire proteome, using rodent malaria as a model system. The overall goal is to correlate mapped epitopes with actual protection from disease to inform the vaccine design process. This proposal embodies several major technological and conceptual innovations, and beyond vaccine design, this approach has the potential for broad impact on many fields of biomedical research, including cancer diagnostics, autoimmunity and allergy.   Grant Abstracts 2008   Buck Institute Julie K. Andersen Novato, CA $1,500,000 2008 Scientists at the Buck Institute share a common goal: to understand aging. We have recently made key discoveries that point to a deep mechanistic relationship between disease and aging across diverse species and various age-related diseases. Our collective data suggest that, rather than viewing aging as a risk factor, it may be more accurately viewed as a principal causal factor of these disorders. We propose to test the hypothesis that age-related human disease is the result of segmentally-accelerated aging – in other words, that diseases of aging such as Alzheimer’s disease, prostate cancer and osteoporosis are part and parcel of the fundamental aging process itself, simply enhanced in the affected tissues. This is based in part on our recent discovery of an acceleration of the aging process in terms of oxidative modification to mitochondrial complex I (CI) in affected regions of the Parkinsonian versus the normal aging brain; selective inhibition of this enzyme complex has been long associated with Parkinson’s disease (PD). To test our hypothesis, we propose two experimental programs: (1) to undertake a functional analysis of mitochondrial post-translational modifications (PTMs) particularly in CI to determine which are critical for neuronal cell dysfunction, death and progression of PD; and (2) to test the generality of this mechanism by surveying Alzheimer’s disease models and post-mortem patient brain tissues for mitochondrial PTMs and their functional correlates particularly but not limited to mitochondrial complex IV whose inhibition has been suggested to be preferentially involved in this age-related disorder versus changes associated with normal aging.   J. David Gladstone Institutes Lennart Mucke San Francisco, CA $1,500,000 2008 The ability to control one’s movements is essential to life. Neural circuits involving the basal ganglia are critical for proper motor control, and disruption of these circuits leads to movement disorders such as Parkinson’s disease and Huntington’s disease. The striatum, which is the input nucleus of the basal ganglia, is a major site of activity-dependent plasticity in both health and disease. Because the striatum lies upstream of other basal ganglia nuclei, cellular and synaptic plasticity within this region alters the transfer of information throughout basal ganglia circuits. However, studies of the striatum have been hampered by difficulties identifying different types of cells during both in vitro and in vivo experiments. Here, we propose to utilize recently developed optical and genetic technologies to characterize the properties of striatal neurons and the neural circuits in which they are embedded. In vivo fiber optic technology will be used in conjunction with expression of channelrhodopsin and halorhodopsin to identify and directly drive neural activity in striatal neurons of awake behaving mice. These experiments will allow us to address how the rate and timing of activity in basal ganglia circuits are causally related to motor behavior. The ultimate goal is to develop a framework that will enable the rational design of novel therapies for devastating disorders affecting the striatum.   Northwestern University Teresa K. Woodruff Evanston, IL $1,600,000 2008 Little is known about the signaling networks that support the integration of the male and female germ cells into a new totipotent cell, the one-cell embryo. We propose that heretofore poorly understood inorganic signaling molecules initiate the massive changes in the physiology of a fertilized egg. Based on preliminary studies, the team hypothesizes that fluxes in zinc ions mediate the first definitive signal in embryonic development. This hypothesis will be tested by two approaches: one targets real time changes in the subcellular concentrations of free zinc and calcium in live cells and the other rigorously maps specific changes in the total zinc pools at the nanometer level. The mouse oocyte is an ideal model system to study this novel inorganic signaling pathway. It undergoes a clear developmental pattern of receptor-mediated events as it transitions from a dormant stage to a fully active state upon fertilization. Also, its large size facilitates spatial localization of key molecular players. New analytical tools will be developed to map the abundance of specific inorganic molecules and biological receptors.   Stanford University Karl Deisseroth Stanford, CA $1,500,000 2008 Excitable cells are the biological building blocks of brain, heart and muscle, and communicate and compute using tiny, transient electrical currents. Widespread throughout the body, these cells underlie remarkable behaviors, from orchestration of movement to high-level cognition. When they malfunction, these cells give rise to devastating diseases, ranging from heart failure to Parkinson’s disease to depression; however, the interventional tools currently available to deal with these conditions are exceedingly primitive, have severe side effects, and yield little or no understanding of healthy cells or of disease processes. New technology is required, powerful enough to address the high speed and structural complexity of these electrical tissues. This grant is for a four-year project to address this fundamental obstacle by developing and applying emerging optical technologies first developed at Stanford. This light-based bioengineering approach has the power to control cellular functioning in vivo with millisecond precision, and to control intracellular messengers in specific cell types, thereby opening new vistas of both investigation and healing. The project personnel will develop the science and technology of this approach and apply these tools for the first time to mammalian models of neurological, neuropsychiatric and cardiac disease, spanning the central, peripheral and autonomic nervous systems.   University of Colorado at Boulder Natalie G. Ahn Boulder, CO $1,200,000 2008 This proposal requests support to develop innovative research strategies in mass spectrometry in order to detect and characterize all proteins in a cellular “proteome” (i.e., all proteins present in a single cell type). Mass spectrometry is the most powerful tool for addressing this problem and worldwide efforts are underway to define the proteomes of organisms, tissues and fluids. However, such efforts are stymied by the inability to comprehensively observe all proteins in highly complex biological samples. The goal is to create innovative experimental and computational technologies that will enhance the accuracy and sensitivity of protein detection by mass spectrometry, and enable comprehensive protein identification. In order to expand the depth to which the proteome can be observed, funds will be used to purchase a high resolution mass spectrometry system with capabilities for electron transfer dissociation. With this instrument, new methods will be developed to overcome limitations in data collection and solve major hurdles in recovering information from large scale datasets. These will be applied to collaborative research efforts between eight laboratories, providing the possibility for unprecedented capabilities to answer questions that were previously impossible to address.