Mark W. Albers
Massachusetts General Hospital
The Olfactory Neural Circuit as a Systems Level Model of Neurodegenerative Diseases
Neurodegenerative disease will increasingly plague our society as modern medicine augments the number of people reaching their seventh, eighth, and ninth decades. These pathological processes erode the functional integrity of susceptible neural circuits. Much progress has been gained in identifying mutations and gene products that underlie these disease processes, particularly early onset Alzheimer's disease (AD) and Parkinson's disease (PD). While these findings have led to the development of mouse models of these diseases, little is known at the systems level about how these disease genes impact the function of a specific neural circuit. We have developed an experimental approach to elucidate the actions of genes that cause neurodegenerative disease in a single mammalian neural circuit. Using mouse genetics, we expressed an AD gene exclusively in primary olfactory sensory neurons in a conditional manner. Characterization of this model uncovered a population of mouse olfactory neurons that undergo enhanced cell death and axon mistargeting in a non-cell autonomous fashion in the presence of this AD gene. In this proposal, we outline a series of studies to extend our analysis of this mouse model by employing: 1) longitudinal in vivo functional imaging using multiphoton microscopy, and 2) primary olfactory neuron culture that can be adapted to high-throughput screens for molecules that reverse the cell death phenotype. Hits from this screen can be rapidly tested in the mouse model by intranasal delivery to the olfactory epithelium. In addition, we propose to extend this experimental approach to genes associated with other neurodegenerative diseases such as PD. Elucidation of the actions of these disease genes at a systems level with novel functional and cellular outcomes from our neural circuit mouse model and characterization of additional neurodegenerative disease genes in this model system hopefully will contribute to the development of effective therapies.
University of California, San Diego
Chemically Amplified Response Strategies for Medical Sciences
A new, amplified response strategy for inducing multi-photon-driven processes noninvasively in living systems will be investigated. This strategy should enable optical and thus remote control or activation of substances inside living systems noninvasively, with a previously unattainable control of depth. The impact of such remote control is large and broad, allowing previously invasive procedures to be performed noninvasively, and previously inaccessible target sites to be reached for both treatment and diagnosis. Multi-photon phenomena allow unparalleled spatiotemporal control, and where longer wavelengths are employed, deeper penetration into turbid bulk media such as tissue. Despite the revolutionary impact these phenomena have had on neuroscience, microscopy, and lithography, it has been generally very difficult to apply this technique in vivo to stimulate biomaterials, diagnostics, and drug delivery systems. Currently there are no reported systems for in vivo multi-photon-responsive materials. The dogma is that not enough photons can reach the materials to initiate a response. The amplified response strategy we aim to explore is inspired by one that has revolutionized the electronics industry with the advent of chemically amplified photoresistors for the fabrication of computer chips. When a single responsive molecular unit, repetitively embedded in a material, simultaneously absorbs two photons, the changes in that molecular unit will cause the system to unravel entirely.
Euan A. Ashley
Nanoscale Approaches to Allelic Silencing in Myocardial Disease States
Hypertrophic cardiomyopathy is the most common inherited cardiovascular disease. It affects one in 500 of the population. It is the most frequent cause of sudden death in young people. Despite groundbreaking work in defining the genetic basis for the disease, leading to the description of more than 400 predominantly missense mutations in more than 8 largely sarcomeric genes, therapies remain palliative. In this application, I outline an innovative approach to the treatment of the underlying genetic defect in hypertrophic cardiomyopathy. Dominant negative “poison peptide” transmission, together with a proof-of-principle phenotype reversal in a transgenic mouse model, provide the rationale. The combination of allele-specific RNA silencing and creative biological and physical approaches to cellular specificity provide the mechanism and route of delivery. Specificity provides the single greatest challenge, and I outline several innovative approaches to overcoming this. Potential translation to patients is a theme woven throughout the strategic plan. We begin with mutations identified in families from the Stanford Hypertrophic Cardiomyopathy Center; carry out basic RNA biology in an attempt to selectively silence these mutations in cell lines; and move progressively through organ models, then small animals, to a preclinical large animal model. The approach we outline is not limited to hypertrophic cardiomyopathy. The ability to selectively modify a single mutated allele and deliver the therapy in vivo could revolutionize the treatment of diverse genetic diseases and finally fulfill the therapeutic promise of the Human Genome Project. Finally, I argue that the most innovative ideas would never have been noticed without timing: With the genetic basis of the disease laid out, and many critical techniques on the verge of significant advancement, the time is right for a new approach.
Duke University School of Medicine
Discovering New Regulators of CFTR and Fluid Secretion in Zebrafish
Most internal organs are built around fluid-filled tubes, and control of fluid secretion is essential for their development and function. Defects in fluid secretion have been linked to some of the most prevalent genetic and acquired pathological conditions, including cystic fibrosis, polycystic kidney disease, and secretory diarrheas. At the cellular level, fluid secretion is driven by directional salt ion transport, which is then followed by water. Several key channels and pores responsible for ion and water transport have been identified. However, we still need to understand how fluid secretion functions as a developmental force and how different processes that depend on fluid secretion are coordinated at the whole-organism level. To address these fundamental problems, I have embarked on a fully integrated approach based on zebrafish genetics and physiology. My focus is on the cystic fibrosis transmembrane conductance regulator, a chloride channel that is the major regulator of fluid secretion in vertebrates. The proposed research plan will lead to new insights into: how fluid pressure shapes development and the responses elicited at the cellular level by this force, CFTR function, and how CFTR activity is regulated in vivo and in real time during development. We will also carry out a forward genetic screen to identify mutations controlling CFTR-dependent and -independent fluid secretion. These approaches will establish a new genetic and physiologic model system for studying the functional regulation and developmental potential of fluid secretion and CFTR activity.
University of Texas M.D. Anderson Cancer Center
Connecting the Selection of Noisy Gene Expression Deviants to Genetic Evolution
Drug resistance of pathogenic cell populations causes the failure of therapy and represents a major challenge for today’s medicine. Early drug resistance has been shown to rely on intricate gene expression patterns across the cell population. However, current techniques of gene expression control (gene deletion, overexpression, knockdown, etc.) are aimed to control only the average gene expression across the cell population and are therefore insufficient to study how gene expression properties other than the mean affect drug resistance. This is creating a widening gap in knowledge, as we and others have recently demonstrated that gene expression characteristics other than the mean (such as the variance or cellular memory of deviant expression states) are just as important as the mean for cell population survival during drug treatment. Here we propose to develop novel, versatile, and modular gene constructs to control various expression characteristics of any gene in any organism. Negative feedback-based constructs will permit precise, linear inducer-dependent control of gene expression in every cell of the population. Positive feedback-based constructs will allow us to adjust the cellular memory (rate of stochastic expression fluctuations). We will use these constructs to control diverse expression characteristics of a drug-resistance gene and study how these expression properties affect cell survival during drug treatment and initiate the evolution of genetic drug resistance in a yeast cell population. We will develop multiscale, stochastic models to explain the mechanisms underlying the experimental observations. We can now directly visualize the expression of a drug resistance gene at the single-cell level. The innovation consists of bridging molecular- and cell-population dynamics and connecting stochastic gene expression fluctuations to genetic evolution in experiment and simulation. The results might transform our current understanding of drug resistance and might substantially improve future therapeutic strategies.
University of Pittsburgh
Defining Mechanisms Controlling Stem Cell Fate During Differentiation
Regenerative medicine largely relies on cell-based therapy for treatment of degenerative diseases such as diabetes, heart failure, and Parkinson’s disease. Embryonic stem cells and recently established induced pluripotent stem cells will be able to contribute significantly in generating a renewable source of transplantable, fully functional cells. In spite of diverse efforts in deriving mature cellular phenotypes from these pluripotent cell types, what is lacking is a thorough understanding of the mechanism governing differentiation and lineage commitment of these pluripotent cells, hence resulting in incremental advancement of the field. My objective is to address the complex differentiation process from a completely different approach, which will have the potential to shift paradigms in regenerative medicine and stem cell bioengineering as a whole. The objective of the proposed research is to develop an insightful mechanistic understanding of the process of differentiation through an integrated experimental and theoretical approach organized around three basic questions: 1) How do the transcription factors interact in controlling and deciding on a cell lineage? 2) How do environmental perturbations influence these networks toward desired lineage? 3) How do gene and protein networks operating at the cellular level govern the tissue functionality at the systems level? These questions will be addressed in a system of embryonic stem cells differentiating to pancreatic islets, using a bottom-up approach where molecular-level information will be integrated to predict tissue-level functionality. Successful completion of this project will directly impact cellular therapy-based regenerative medicine and will pave the way for mechanistic understanding of disease progression and potential therapeutic intervention.
Edward B. Brown, III
University of Rochester Medical Center
Exploiting Collagen Organization to Predict and Prevent Tumor Metastasis
The extent and nature of the ordering of collagen fibers within a tumor has significant influence on metastasis: In murine breast tumor models, tumor cells move toward blood vessels along fibers that are visible via second harmonic generation (SHG), and SHG is exquisitely sensitive to molecular ordering. Tumor cells that move along SHG fibers are significantly faster than those moving independently, and SHG-associated motility is correlated with metastatic ability. Furthermore, the tumor-host interface contains radially oriented SHG fibers associated with tumor cells invading the surrounding tissue. Lastly, we have shown that treatment of tumors with relaxin, known to alter metastatic ability, alters collagen ordering as detectable by SHG. Consequently, we believe that the process of establishing ordered fibers offers an exciting, and currently unexploited, therapeutic target. To take advantage of this, we must first learn the cellular players and molecular signals by which collagen ordering is induced. Therefore, in this application we propose to determine the key cells and signals which influence the ordering of collagen in breast tumors. The tumor-draining lymph node is the first bridgehead for many metastasizing tumor cells, and we have exciting preliminary data suggesting that changes in collagen ordering within the node are evident (via SHG) before clinical detection of metastatic cancer, therefore we will also determine the key cells and signals which influence the ordering of collagen in the draining lymph node. Additionally, we will determine if SHG measures of collagen ordering in breast tumors and draining nodes are clinically useful predictors of metastatic outcome in breast cancer patient biopsies. This project has a high impact because it has two independent pathways to clinical relevance, by developing promising antimetastatic drug targets, and by developing an optical method to predict metastatic ability.
Children’s Hospital Boston
Analysis of Stem Cell Dynamics and Differentiation by Cellular Barcoding
The changing demographics of developed nations underscore the need for regenerative medicine approaches to combat the clinical and financial burden of degenerative diseases. The basic understanding of how tissues are normally maintained by their resident stem cells is, therefore, key for pursuing regenerative approaches. Though a great deal of knowledge has been gained through the use of traditional experimental approaches over the past two decades, limitations and drawbacks of these techniques have precluded us from gaining a complete understanding of stem cell function, particularly in the in vivo setting. The goal of my New Innovator proposal is to develop a novel experimental paradigm for the study of stem cell biology. In this model, individual stem cells in a population are uniquely and genetically tagged in situ and these unique genetic tags, or barcodes, can then be used to dynamically monitor individual stem cell activity, lifespan, and differentiation in highly complex populations over time. We believe that this model can give us an unprecedented, high-resolution picture of the inner workings of a complex and dynamic stem cell system, and allow us to answer long-standing biological questions. Our findings ultimately may uncover conserved mechanisms of stem cell maintenance that are perturbed in old age or other disease contexts. Though this proposal will address the biology of the normal and malignant hematopoietic (blood-forming) stem cell compartments, the modular nature of our model makes easily adaptable to any tissue. Our model is also suitable, among other things, for the study of aging and immunological problems.
University of Michigan
A Biochip for Point-of-Care HIV/AIDS Diagnosis in the Developing World
HIV/AIDS is one of the most destructive pandemics in human history, responsible for more than 25 million deaths. More than 30 million people live with limited or no access to therapeutic treatments, mainly due to the high cost of highly active antiretroviral therapies (HAART) and current diagnostic tests as well as due to the lack of basic infrastructure (e.g., lack of electricity, no trained personnel) that can support these tests. The need for innovative, inexpensive diagnostic instrumentation technology that can be used in resource-limited settings is immediate. While programs that offer free HAART are being implemented in resource-limited settings, no diagnostic tests are available for evaluating the efficacy of HAART provided for the reasons mentioned above. Efficient management of HAART requires monitoring the course of HIV infection over time. The World Health Organization recommends the CD4+ T-cell count test for monitoring the clinical status of HIV individuals in resource-limited settings. We propose to develop a portable, inexpensive, MEMS (MicroElectroMechanical Systems)-based, imaging system for counting the absolute number of CD4 cells from 1 µl of whole blood. We use the term “imaging system” to denote the different approach we follow for counting CD4 cells: Rather than reading single cells one by one (as is done with flow cytometry), our system can image simultaneously thousands of individual cells, pre-assembled on the surface of a biochip. Although the proposed imaging system can replace current expensive cell-counting instrumentation, our goal is to develop a system that can reach the end-user wherever limited infrastructure is present and no access to a hospital or clinic is possible. Such technology will not only make it possible to monitor the efficacy of an individual’s HAART in the developing world, but it will make more medicines available by identifying patients who need a treatment from patients who do not need it.
Brigham and Women's Hospital
Prevalence, Risk Factors and Consequences of Complex M. tuberculosis Infections
Tuberculosis (TB) is an infectious disease of global importance; in 2006, there were more than 9 million incident cases and 1.7 million deaths attributable to TB. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB and the convergence of the HIV and TB epidemics are threats to effective TB control. Furthermore, evidence exists that previous M. tuberculosis infection confers limited immunity to re-infection, that an individual can simultaneously harbor more than one distinct strain of M. tuberculosis, that distinct lineages of M. tuberculosis differ in their virulence characteristics, and that M. tuberculosis diversifies within a host. Each of these factors contributes to the within-host complexity of M. tuberculosis infection and presents complications for the treatment of individuals and the control of disease in populations. I propose an observational study among individuals starting treatment for TB in Lima, Peru, and Pietermaritzburg, South Africa, to evaluate the prevalence, risk factors, and consequences of complex M. tuberculosis infection. I will: 1) estimate the site-specific prevalence of multiple-strain M. tuberculosis infection and clonal heterogeneity among individuals at the time of initial diagnosis, 2) determine the host- and strain-related risk factors for multiple-strain infection and clonal heterogeneity, 3) evaluate the effect of multiple-strain infection and clonal heterogeneity on early response to standard first-line treatment regimens, and 4) develop mathematical models to examine the individual- and population-level effects of multiple-strain infection and clonal heterogeneity.
University of California, Davis, School of Medicine
Transmission and Virulence of Mycobacterium tuberculosis
Why do some individuals who are exposed to Mycobacterium tuberculosis become infected, while others do not? Of those who are exposed and infected, why do some individuals rapidly progress to active disease, while others remain asymptomatic, latently infected, or progress to active disease decades later or not at all? The observed heterogeneity in individuals' responses to exposure and infection might be explained by differences in the transmission characteristics of M. tuberculosis and its virulence in the human host. Our goals are to: 1) identify the gene expression profiles of M. tuberculosis in sputa from patients with latent TB infection and active disease; 2) identify sets of genes that characterize the early infection, pro-inflammatory immune response post-infection, and active disease stages of M. tuberculosis in the human host; and 3) identify differential gene expression patterns attributable to different strains, including drug-resistant and pan-susceptible strains of M. tuberculosis. We propose a case-control study of tuberculosis patients, their infected and uninfected contacts, and neighborhood healthy controls. From enrolled study participants, we will obtain information, a cough sample with sputa, and a blood sample. The sets of gene expression patterns—diagnostic signatures—could be translated into new, accurate, and rapid diagnostic tests. We will recruit and enroll patients in Shanghai, China, a city with over 6,000 new TB cases annually and the point of departure for many migrants to the United States. Our discoveries will have a significant impact on the tuberculosis epidemic worldwide, a global health emergency that caused 9.2 million new cases and killed 1.7 million persons in 2006. We seek strategies to target scarce public health resources to prevent new cases of active tuberculosis in the United States and globally.
Elva D. Diaz
University of California, Davis
Generation of Tumor Stem Cell Lines for Directed Therapeutics of Brain Cancer
Tumors of the central nervous system (CNS) represent nearly one quarter of all childhood cancers. Although progress has been made in the treatment of some types of childhood cancer, the outcome for children with primary CNS tumors has remained bleak and little advancement has been made in the last decade. In addition, due to the adverse effects of the tumor on brain development or the treatment required to control its growth, survivors of childhood brain tumors often have severe neurodevelopmental defects that negatively impact their quality of life. Thus, there is a need for better treatments specific for childhood brain tumors. Current models suggest that only a few atypical cells within the cancerous mass are responsible for the initiation, growth, and recurrence of brain tumors. These transformed cells have both the defining properties of neural stem cells and the ability to initiate cancer, thus these cells are referred to as “brain tumor stem (BTS) cells.” While the isolation of neural stem cells is fairly well established, the isolation of BTS cells remains a difficult and complex issue, suggesting the need for innovative approaches to isolate and characterize these cells. The development of induced pluripotent stem cells (somatic cells that have been reprogrammed to an embryonic-like pluripotent state by retroviral-mediated introduction of specific transcription factors) represents a powerful new approach that might alleviate such confounding issues. Thus, the goals of the proposed project are: 1) to reprogram brain tumor cells toward a more stem-like phenotype, 2) to characterize the tumorigenic potential of such reprogrammed tumor stem-like cell lines, and 3) to identify chemical compounds that specifically target the reprogrammed tumor stem-like cells. Completion of these studies will provide a directed strategy for novel therapeutics to specifically target the cellular population responsible for the initiation, growth, and recurrence of pediatric brain tumors.
Adam J. Engler
University of California, San Diego
“Smart” Materials to Engineer a More Complete Stem Cell Niche
One of the recent paradigm shifts in stem cell biology and regenerative medicine has been the discovery that stem cells can begin to differentiate into adult tissue cells when exposed to intrinsic properties of the extracellular matrix (ECM), such as matrix structure, elasticity, and composition. ECM regulation of stem cells has also been shown to be as sensitive as well-studied soluble growth factors, and together in the body, they comprise the stem cell niche, or “microenvironment.” However, these cues have typically been studied as isolated stimuli where no single cue, whether a growth factor or an ECM property, has been sufficient to generate the appropriate type of differentiated cells for a given regenerative cell therapy. Moreover, as stem cells mature in the body during development, their microenvironment is highly spatially and temporally controlled, yet our ability to dynamically regulate the niche as the body does has not been developed and is probably a critical requirement for developing differentiated cells from stem cells. Therefore, I propose to substantially advance the field of stem cell biology by developing a new hybrid hydrogel system using a unique combination of conventional polymer chemistries. These gels, comprised of hyaluronic acid-co-acrylamide polymer, should present spatially and temporally controlled matrix properties that mimic their presentation during development. When combined with spatially patterned growth factors, these cues could more accurately recapitulate the development of a specific tissue ex vivo, which may improve the differentiated cell sources used for cell-based therapeutic applications.
Columbia University College of Physicians and Surgeons
Investigating the Potential of Endogenous RNAi in Mediating Adaptation to Environment
RNA interference (RNAi) provides defense against exogenous nucleic acids, such as viruses and transposons, in diverse organisms. The production of short interfering RNAs (siRNAs) antisense to the viral or transposon sequences is a hallmark of the RNAi response. The discovery of the endogenous antisense siRNAs (endo-siRNAs) matching thousands of protein-coding sequences in C. elegans and identification of similar molecules in Drosophila and mammals poses a question about their function. Our recent microarray analysis of genes misregulated in the RNAi pathway mutants in C. elegans revealed preferential targeting by the RNAi components and endogenous short RNAs of genes whose inactivation is beneficial for stress resistance and lifespan extension, such as genes encoding translation factors. We propose that pools of endogenous short RNAs in C. elegans are subject to natural selection. Therefore, the composition of siRNAs in populations is adjusted in response to the environmental changes to achieve maximum fitness. The goal of this project is to test the above model. We will select populations of C. elegans resistant to specific environmental conditions and test these populations for the accumulation of endo-siRNAs antisense to very specific genes whose inactivation allows survival under the tested condition. We already established a correlation between thermotolerance and accumulation of endo-siRNAs specific to translation initiation factors. In addition, natural selection for survival on the nematocidal drugs ivermectin and levamisole will be used to generate C. elegans strains resistant to drugs due to epigenetic endo-siRNA-based inactivation of specific genes. Proving the existence of a siRNA-based epigenetic natural selection would represent a fundamental breakthrough in basic science. Epigenetic RNAi-based mechanisms are not likely to be limited to lower organisms and may be involved in the immune escape and drug resistance of malignant tumors and in other cases when cells evolve to escape the action of therapeutic agents.
Ira M. Hall
University of Virginia
Extent, Origin, and Control of Structural Variation in Mammalian Genomes
Mammalian genomes have a complex physical structure shaped by myriad duplications, deletions, and rearrangements, and this structure varies considerably among the populations and individuals of a species. These "structural variations" are of special importance to our understanding of evolution and disease because single mutational events can affect large phenotypic changes and because mutation rates vary dramatically among different genomic loci. We are only in the very early stages of understanding how structurally plastic genomes truly are and why they are this way. Massively parallel paired-end DNA sequencing now offers the opportunity, in theory, to reconstruct the architecture of entire genomes on a routine basis. However, the practical utility of these methods remains limited by the significant computational challenges posed by proper data interpretation and by cost. Over the past year, we have developed novel experimental and computational tools, and we are now close to our initial goal of being able to comprehensively map structural variation in mammalian genomes, at reasonable cost, and with modest computing power. We propose to apply these tools to examine structural variation in three especially revealing contexts: among diverse mouse strains with shared genealogical origins, among related mouse colonies separated by ~2,000 generations of breeding, and among single cells from diverse somatic lineages of the body and brain. In each case we will systematically identify and characterize "hotspot" loci that mutate at elevated rates. These studies will yield an unbiased evaluation of the extent and origin of structural variation in mammalian genomes and will enable us pursue our final goal: to develop a high-throughput platform for identifying factors that affect structural mutation rates. This work has immediate relevance to medicine, considering that structural genomic variation has emerged as a major cause of both inherited and spontaneous human disease.
Engineering 3D In Vitro Niches to Reveal Fundamentals of Cellular Biomechanics
The development of tissue culture techniques by Ross Granville Harrison in 1907 has been cited as one of the ten greatest discoveries in medicine and enabled monumental advances in biological understanding. Despite the enduring importance of in vitro culture in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940s: Cells are cultured on hard, flat substrates and surrounded by homogeneous solutions of medium that do little to recreate the exquisite microenvironments found in vivo. Cells are well known to respond to multiple cues found within their in vivo niches, e.g., concentration gradients of soluble and tethered biochemicals, matrix rigidity, patterns of matrix ligands, and interactions with other cell types; however, few methods exist to recapitulate these cues in in vitro cell culture studies. To address these limitations, I propose creating versatile, three-dimensional in vitro niches with precise spatial and temporal resolution of cellular cues. These three-dimensional microenvironments will be fabricated using innovative and transdisciplinary approaches that combine advances in protein engineering, biomaterials, and microfluidics with traditional cell biology protocols. As a model system, these in vitro niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration. NPC chemotaxis within gradients of soluble factors is hypothesized to be contextual and reliant on additional biomechanical cues from the 3D matrix. The presence of NPCs within specific niches of the brain opens up the tantalizing possibility that the adult central nervous system may be able to regenerate following injury or disease if NPCs were induced to migrate to sites of need. The development of quantitative, in vitro mimics of in vivo niches will have a profound impact on biomedical research by enabling scientists to test entirely new hypotheses about the interactions between different cells and their three-dimensional microenvironments.
Engineering of Cell Shape and Intracellular Organization
A bacterial cell is much more than the sum of its parts. Most cellular functions are critically impacted not only by regulation of the genome and proteome, but also by the shape of the cell and how the shape dictates the localization of intracellular components. The ability to systematically manipulate cell shape will ultimately provide a powerful suite of applications in antibiotic drug development, synthetic biology, and biosensing. My laboratory will leverage insight from evolutionary, synthetic, and cell biological approaches to inform our ongoing development of quantitative, biophysical models of bacterial cell shape determination and growth. We have already successfully used modeling to predict the cell shape response to antibiotic treatment. We will focus our efforts on exploiting other predictions generated from quantitative models to re-engineer cell shape and redesign the intracellular localization landscape. For the period of this award, three design targets will be pursued that leverage our expertise in biophysical modeling of cell shape to probe key features of cell growth: 1) We will explore the evolutionary origins of cell shape determination by transplanting foreign cytoskeletal elements between closely related bacteria. 2) We will program specific intracellular organizational phenotypes to dynamically reengineer cell shape. 3) We will determine the tension sensitivity of the growth machinery to elucidate potential feedback mechanisms for cell shape maintenance. These targets will strategically expand the experimental focus of my laboratory. Success will address many longstanding questions of how cells determine their shape and how they utilize shape to regulate complex intracellular processes such as cell division. The physical principles of organization are likely to appear in diverse biological contexts, in both bacterial cells and in higher organisms. Ultimately, we will challenge our understanding of cell shape determination by transforming shape into an experimentally tunable parameter.
Sanjay K. Jain
Johns Hopkins University School of Medicine
Novel Imaging Biomarkers to Address Fundamental Controversies in TB Pathogenesis
Recognizing that tuberculosis (TB) is still one of the leading causes of human death, the international health community has set ambitious targets to control TB by 2050. Unfortunately, this target cannot be achieved with current tools and requires the development and use of better anti-TB drugs/vaccines. Since Mycobacterium tuberculosis adapts to a quiescent physiological state—“dormancy”—and successfully evades anti-TB drugs and host immune responses for decades, understanding the kinetics of adaptive bacterial responses and the host-microenvironment is essential for developing better anti-TB drugs/vaccines. However, current tools for assessing bacterial-host kinetics in animal models are limited to analyzing postmortem tissues. Artifacts introduced during sacrifice/processing make them less reliable. Moreover, lesion-specific characteristics are generally not assessed separate from the whole organ. Since a different animal is sacrificed at every time point, bacterial-lesion kinetics in an individual animal can also never be assessed. We have pioneered the development of imaging biomarkers to assess M. tuberculosis bacterial burden in animal models. In this proposal, we will develop novel imaging biomarkers that will not only permit assessment of M. tuberculosis burden but also allow monitoring and localization of both adaptive bacterial responses and the host microenvironment (inflammation, hypoxia, and early immunity), in the same, live animal, over several time points. These tools will be utilized to address fundamental controversies in TB pathogenesis that cannot be tackled using current tools: 1) Are host tissue inflammation and hypoxia a sanctuary for “dormant” M. tuberculosis? 2) Where does “dormant” M. tuberculosis reside? 3) Is innate immunity required for controlling initial M. tuberculosis infection? Knowledge gained from this proposal will provide unique insights for developing better anti-TB drugs/vaccines. By permitting cost-effective, cross-species preclinical assessment, these tools will also dramatically reduce the time required for “bench-to-bedside” translation. Finally, since these tools are easily translatable, preclinical validation will lay the groundwork for their future use in humans.
Kevin A. Janes
University of Virginia
Stochastic Control of Abnormal Morphogenesis Induced by the ErbB2 Oncoprotein
Cancer is a stochastic disease whose biology has been studied almost exclusively with deterministic approaches. Why? In this application, I propose to exploit the apparent randomness of cellular transformation to uncover new mechanisms involved in tumorigenesis. My focus is the ligandless receptor tyrosine kinase, ErbB2, which is overexpressed in 20–30% of breast cancers and is the target of anticancer drugs such as Herceptin® and Tykerb®. In a 3D in vitro culture model of mammary-acinar morphogenesis, inducible activation of ErbB2 causes hyperproliferative multiacinar structures that in many ways are reminiscent of early-stage breast tumors. Importantly, the penetrance of the phenotype is incomplete—only a random fraction of the cultured acini exhibit the morphogenetic defect when ErbB2 is activated. How this fraction is specified and the mechanism by which a multiacinus initiates are unknown. My hypothesis is that acute differences (dichotomies) in gene expression develop among acini and give rise to the distinct 3D phenotypes induced by ErbB2. The transcriptional dichotomies that exist before the appearance of the multiacinar phenotype will be the ones most likely to control it. However, without seeing the phenotype, it is impossible to know which ErbB2 structures will go on to develop abnormally. To overcome this challenge, we will use a new technique, called “stochastic profiling,” that I developed for discovering transcriptional dichotomies in a seemingly uniform cell population. We will apply stochastic profiling to a series of conditional ErbB2 homo- and heterodimer pairs that have different penetrances for the multiacinar phenotype. By mapping the transcriptional dichotomies to the differences in penetrance among dimer pairs, we will link upstream acinus-specific expression programs to downstream morphogenetic heterogeneities. The results from this project could explain mechanistically why only a fraction of ErbB2-overexpressing breast cancers respond positively to ErbB2-targeted therapeutics.
Melissa Lambeth Kemp
Georgia Institute of Technology
Redox Regulation of Cellular Information Processing
Elevated concentrations of extracellular reactive oxygen species (ROS) are hallmarks of inflammation, and decades of medical research have focused on suppression of these molecules to treat pathologies as diverse as rheumatoid arthritis, cancer, and atherosclerosis with mixed results. More recently, researchers have discovered that these same molecules are produced during the course of normal signal transduction. In order to effectively treat inflammation, we must understand these distinct roles for reactive oxygen species. I propose an innovative research program that will elucidate the role of hydrogen peroxide, a key ROS, in normal cell signaling through computational models and laboratory experiments. This research will lead to a new, quantitative understanding of ROS and facilitate the development of effective antioxidant treatments for inflammation. This project will use three complementary approaches to evaluate the complex regulatory role of hydrogen peroxide on receptor-induced signaling. First, we will develop computational network models describing redox regulation of proteins in a time-dependent manner. Secondly, we are designing new methods to detect oxidative changes on multiple proteins simultaneously. These assays will allow investigation of the relationships between phosphorylation of signal transduction molecules and reversible thiol modifications. Finally, we have created a series of cell lines in which key components of the redox network have been perturbed that demonstrate augmentation and attenuation of receptor signaling. These lines will be used to systematically investigate the efficiency of three receptor networks–a pro-inflammatory cue (TNF-α), anti-inflammatory cue (TGF-β), and antigenic response (TCR)–under different oxidative environments. The results of these studies will provide the first computational modeling platform capable of interpreting incongruous literature reports of oxidative effects on cellular information processing. This project leverages my unique experience at the interface of immunology, systems biology, and metabolism to address a fundamental mechanism of cellular regulation critical for a large class of therapeutic drugs.
Children’s Hospital Boston
Towards the Neuronal Correlates of Visual Awareness
The brain, a physical system composed of neurons and synapses, can give rise to what seems to be the least physical property of all: consciousness. How this transformation takes place has preoccupied generations of clinicians and scientists. Advances in neuroscience over the last several decades make it possible to enquire into the neural circuits responsible for consciousness. This proposal focuses on one particular aspect of conscious experience: the neuronal mechanisms and circuits that underlie visual awareness. We propose to study visual awareness using a combination of neurophysiology, psychophysics, and electrical stimulation. We take advantage of a rare opportunity to examine activity in the human occipital and temporal lobes using neurophysiology at high spatial and temporal resolution (neurons and milliseconds) while subjects report their perceptions. We propose two experiments where visual perception is dissociated from the visual input: binocular rivalry and motion-induced blindness. In both cases, perception changes in spite of a constant visual input. We investigate where, when, and how neuronal responses along the ventral visual cortex (from primary visual cortex to inferior temporal cortex) change their activity patterns with the perceptual alterations. Furthermore, we ask whether those neurophysiological responses are sufficient to elicit perception by electrically stimulating local circuits. Impairments in conscious processing can be devastating and are at the core of such seemingly diverse conditions as epilepsy, vegetative coma, schizophrenia, and autism. Furthering our understanding of the link between brains and minds in the context of vision will pave the way for addressing other aspects of consciousness and may have profound implications in changing how we think about and address these challenging disorders.
Medical College of Wisconsin
Genetic Approaches to Protein-Protein Interactions Mediating Antibiotic Resistance
Antibiotic-resistant bacteria, such as vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), are major causes of hospital-acquired infections and are driving forces of an escalating health crisis. We will help address the burgeoning antibiotic resistance problem by leveraging the power of bacterial genetics via unbiased genetic selections to: 1) comprehensively identify protein-protein interactions in cellular pathways that result in antibiotic resistance, and 2) discover small molecules that disable these protein-protein interactions. The proposed research will jointly exploit our expertise in the development of genetic strategies and our ongoing interest in elucidating the fundamental mechanisms of bacterial antibiotic resistance. By focusing on protein-protein interactions, this research promises to: 1) reveal new insights into the underlying biology of antibiotic resistance mechanisms and their integration into the physiological processes of the bacterial host, 2) define new targets (in the form of protein-protein interactions) for innovative therapeutics to treat infections caused by drug-resistant pathogens, and 3) identify novel small-molecule drug candidates with unique modes of action. Our experimental design possesses critical strategic advantages. For example, our analyses will be done within the native context of the drug-resistant bacterial host (e.g., not by in vitro screens on isolated proteins), which will enable us to capture any potential, but as yet unknown, effects of dynamic cellular processes or post-translational modifications on key protein-protein interactions. Furthermore, we will employ powerful genetic selections capable of rapidly sifting through immense libraries to reveal even rare hits that, by definition, are functional in a physiological context. Collectively, these strategies will enable the discovery of unknown, unpredictable, and novel biological insights, not accessible by conventional means, that will be exploited to discover new candidate therapeutics with efficacy against drug-resistant bacterial infections.
Siavash K. Kurdistani
University of California, Los Angeles, David Geffen School of Medicine
A Blueprint for Oncogenic Epigenetic Reprogramming
While cancer is a genetic disease, the cancerous cellular state is associated with multiple epigenetic alterations, including aberrant DNA methylation and histone modification patterns. A significant challenge in cancer biology is to elucidate the precise order of epigenetic alterations during tumor initiation and progression and their contributions to the transformed phenotype. To meet this challenge, one requires a model of cellular transformation that is temporally traceable from a normal to a malignant state. Cancer cell lines are not necessarily good models, as they have already accumulated hundreds to thousands of genetic and epigenetic alterations. Here, I propose to study the oncogenic transformation of normal human cells by viral oncoproteins as a model to determine the precise epigenetic reprogramming events occurring along the path of neoplastic transformation. Viral oncoproteins such as the adenovirus small e1a or papillomavirus E7 have been extraordinarily useful in delineating the central molecular players that regulate cell proliferation such as the retinoblastoma (RB) and p53 tumor suppressors. Our work has recently elucidated a defined global epigenetic reprogramming by one viral oncoprotein, e1a, that forces normal cells to escape quiescence—a hallmark of cancer. Importantly, e1a directly implements a precise and coordinated mechanism of regulation of thousands of host cell genes leading to cellular transformation by interacting and rearranging specific epigenetic modifiers across the whole genome in a time-dependent manner. This provides a powerful model that is amenable to time series measurements with phenotypically defined endpoints, enabling one to delineate the successive order of epigenetic alterations that contribute to oncogenic transformation. By understanding how e1a orchestrates a specific sequence of epigenetic alterations for cellular transformation, we should learn greatly about the functions and mechanisms of fundamental epigenetic processes in normal biology and human disease, especially cancer.
Naa Oyo A. Kwate
Columbia University Mailman School of Public Health
Immunologic Effects and a Structural “Countermarketing” Intervention: Racism, the HPA Axis, and African American Health
The overall aims of this project are to explore the effects of multiple levels of racism on the immune function and overall health of urban African Americans and to test a novel structural-level intervention to reduce the negative impact of racism. Significant disparities in major chronic illnesses, including cardiovascular disease, cancer, and overweight/obesity, continue to describe the current picture of health for African Americans. Researchers have long understood stress to play a critical role in negative health outcomes, and particular attention has been paid to the hypothalamic-pituitary-adrenocortical (HPA) axis as a key pathway. Because experiences with racism constitute a significant stressor in the lives of African Americans, understanding the ways in which racism activates the axis will have profound meaning in addressing the determinants of African American health disparities. More broadly, given the role of the HPA axis in many systems, the proposed research has the potential to uncover critical information on the impact of social processes on overall physical and mental health and to delineate unstudied paths in brain-behavior relationships. In two sub-studies, the proposed research will enroll urban African Americans from New York City in longitudinal investigations. Study 1 is an investigation of how neighborhood-level institutional racism and perceived individual and collective racism affect immune and metabolic function and overall physical health, psychological wellbeing, and health behaviors. Study 2 is a neighborhood-level intervention to minimize the likelihood of internalized racism via a racism "countermarketing" campaign. Outdoor advertising will be employed to deliver stark facts about American inequality in predominantly African American neighborhoods, thereby raising consciousness and minimizing negative health outcomes. Taken together, the proposed research attempts to answer two critical unanswered questions in biomedical and behavioral research: 1) How does racism get into the body? and 2) What do we do about it?
Rutgers, The State University of New Jersey, New Brunswick
Combinatorial Approaches for Studying Multiple Cues Regulating Human Pluripotent Stem Cell (hPSC) Fate
Human pluripotent stem cells (hPSCs) are promising resources as cell-based therapies for the debilitating injuries caused by many neurodegenerative diseases. However, controlling hPSC differentiation into lineage-specific neural cells is one of the most important problems needed to be addressed before their potential for neuroregenerative medicine can be fully realized. A detailed insight into the functions of extracellular microenvironments and intrinsic cellular regulators which dynamically regulate the hPSC neurogenesis into neural/neuronal cells is a prerequisite for addressing the aforementioned challenges. However, functions of hPSC microenvironments are much more complicated to investigate because of our lack of knowledge about the multiple signals inducing differentiation and limited methods available for investigation. Therefore, the primary focus of our study is to develop innovative methods to identify optimal cues for hPSC differentiation into subtype-specific neurons and genetic manipulation of hPSCs using nonviral, siRNA-based transfection tools. Our innovative approaches will allow for the establishment of novel cell-based assay tools and siRNA-based genetic manipulation tools for selective and efficient neurodifferentiation of hPSCs. Moreover, efforts will be made to integrate these studies into one multianalytic microfluidics platform for synchronized control of microenvironmental cues and intrinsic cellular regulators. The PI’s research experiences in nanoscale biomaterials, functional genomics, and stem cell biology and current interdisciplinary research programs aiming at investigating cellular interactions within microenvironments would be critical to develop the aforementioned innovative tools.
Daniel A. Lim
University of California, San Francisco
Chromatin-Based Cellular Memory in Neural Stem Cells
This proposal addresses two fundamental questions at the crossroads of epigenetics, stem cell biology, and regenerative medicine that relate to the chromatin-based cellular memory system. 1) Do chromatin modifications at specific genetic loci predict the progressive “restriction” of differentiation potential that occurs in neural stem cells during brain development and into adulthood? 2) Can cellular memory systems be partially “erased” or “reset” at the chromatin level in precursor cell populations to broaden their developmental potential? These studies should greatly advance our understanding of how precursor cells “remember” both their temporal and positional identities as well as determine whether this cellular memory system can be manipulated for novel therapeutic strategies. Three areas of impact are: developmental neurobiology, where results shed light on epigenetic mechanisms of neuronal and glial differentiation; regenerative medicine, where insight gained may suggest novel methods of cell fate specification; and cancer biology, where results may reveal how certain chromatin derangements can promote brain tumors. First, we propose investigating the changes in chromatin modifications that occur along a neural stem cell continuum from the embryo and into adulthood. Our proposed methods utilizing cells acutely isolated from the brain represent a significant advancement upon current cell culture-based studies. To accomplish this, we must innovate new, integrative approaches for chromatin study. We will also employ novel and as of yet unproven approaches to “reset” chromatin memory of cell identity with the purpose of altering cell fate. Given that these ideas concerning the chromatin basis of cellular memory and strategic epigenetic manipulation are new and relatively untested, the level of risk in our proposal is substantially higher than in traditional investigator grants. We wish to embark on this tangent from our current studies to broaden the impact of our research and explore fundamental principles of cellular memory in stem cell biology.
University of California, San Francisco
Characterization of the Role of CpA Methylation in Neuronal Plasticity
Our brain displays an astonishing degree of plasticity. Experiences from a constantly changing environment generate, modify, and eliminate synapses and alter the function of our neurons. Extensive research over the last three decades has demonstrated that long-term potentiation is a process that requires enduring changes in gene expression. Although transcription factors mediate most of these changes, it is the covalent modifications on DNA and chromatin that render this changes long-lasting. Among these “so called” epigenetic changes, DNA methylation is the only one that cannot be enzymatically reversed. DNA methylation on CpG islands is a well established mechanism of gene silencing. Here, we show that we discovered a novel epigenetic modification, the methylation of CpA dinucleotides. Using a novel, genome-wide method to detect CpA methylation in primary neurons, we made the remarkable observation that CpA methylation appears only on actively transcribed genes. Moreover, our preliminary data suggest that this modification can be modulated by neuronal activity, exactly like the transcription status of the genes that it marks. An irreversible modification that can enhance or modulate gene expression could have profound consequences in neuronal plasticity. Therefore, we propose experiments that will dissect the role of CpA methylation in gene expression and neuronal function.
Andre G. Machado
Cleveland Clinic Lerner College of Medicine-CWRU
Deep Brain Stimulation of the Ventral Anterior Limb of the Internal Capsule for Modulation of the Affective Sphere of Chronic Neuropathic Pain
Chronic neuropathic pain is a common cause of disability in the population. Most treatment options for patients with medically refractory neuropathic pain, such as spinal cord stimulation, thalamic deep brain stimulation, or intrathecal infusion of narcotics, are aimed at producing analgesia and are known to have limited efficacy. We propose an innovative, neuromodulation-based approach to treat patients with central thalamic pain syndrome, a particularly severe form of neuropathic pain characterized by relentless anesthesia dolorosa resulting from injury to the thalamic sensory pathways. We depart from the traditional goal of intervening in the sensory discriminative neural pathways of pain transmission in order to produce analgesia. Instead, we plan to target with deep brain stimulation (DBS) the ventral area of the anterior limb of the internal capsule (ALIC), a region densely populated by fibers related to neural networks related to the control of behavior and emotion. By electrically stimulating these networks, we expect to modulate the affective sphere of patients with otherwise intractable pain and, consequently, reduce pain-related disability. We hypothesize that the improvement in pain-related disability associated with modulation of the affective pain sphere will not be dependent on analgesic effects. For this reason, the visual analog scale will be used as a secondary outcome measure to control for pain levels and analgesia, but the primary outcome measure of the study will be the pain disability index. Patients enrolled in this research will undergo baseline and post-DBS double-blinded evaluations for a period of six months, followed by chronic open-label stimulation. The neural circuits of emotion control and the effects of DBS upon these networks will be studied at regular intervals with functional magnetic resonance imaging and magnetoencephalography.
University of Minnesota Medical School
Maximizing CD8 T Cells for Protection
HIV has claimed >25 million lives. Two decades of research, but no vaccine. Theoretically, generation of CD8 T cell immunity may succeed where traditional, neutralizing antibody-dependent vaccines have failed. But nascent efforts have consistently failed to prevent chronic SIV infection in monkeys following a stringent challenge, and the first human HIV CD8 T cell vaccine trial was a complete failure. Why? Is a preventative CD8 T cell vaccine impossible, as many now suggest? We hypothesize that past efforts have been crippled by a safety-first approach and relative ignorance of the importance of memory CD8 T cell quantity, quality, and location to protection. Current approaches are largely refinements of past failures. A bolder approach is long overdue, at least in animal models where safety requirements are less stringent. What could SIV-specific memory CD8 T cells accomplish if they were 500-fold more plentiful than what is established by current vaccination strategies? What if these cells were preferentially located at common portals of viral entry and destroyed infected host tissue quickly upon contact? If a vaccine converted most CD8 T cells into HIV- or SIV-specific memory cells, could they fully protect the host and eliminate the infection completely? Or are CD8 T cells incapable of fulfilling this goal, even under ideal scenarios? This proposal will answer these essential questions. Moreover, it will test the limits of memory CD8 T cell generation and define CD8 T cell correlates of protection. If successful, this study may demonstrate that a preventative HIV vaccine is theoretically possible.
J. Rodrigo Mora
Massachusetts General Hospital / Harvard Medical School
Reassessing the Physiological Role of Gut-Specific Lymphocyte Homing: Implications for Autoimmunity and Tolerance
Oral immunological tolerance is an essential although poorly understood phenomenon by which the immune system becomes “nonresponsive” against antigens administered via the intestinal mucosa. Although this process is vital for the co-existence with nonpathogenic intestinal antigens, such as food and normal microbial flora, the mechanisms responsible for oral tolerance remain poorly understood. Lymphocyte migration (homing) is essential for protective and pathological immune responses, and we and others have demonstrated that gut-associated, antigen-presenting dendritic cells instruct lymphocytes to express gut-specific homing receptors, integrin α4β7, and chemokine receptor CCR9, by a mechanism dependent on the vitamin A metabolite retinoic acid (RA). Importantly, RA also contributes to the generation of regulatory T lymphocytes (TREG), which have been shown to be important for the establishment of oral tolerance. Given that RA induces gut-homing lymphocytes and also promotes TREG differentiation, I hypothesize that RA is essential for the establishment of oral tolerance 1) by inducing gut-tropism and “sequestering” potentially pathogenic T lymphocytes in the intestinal mucosa, and 2) by promoting TREG differentiation in the gut. If successful, my work will highlight a new physiological role of gut homing in the establishment of oral tolerance, providing also a straightforward approach to induce immune tolerance using RA, which could be used in the treatment of autoimmune diseases.
Alysson R. Muotri
University of California, San Diego School of Medicine
Modeling Autism with Human Pluripotent Cells
Autism and autism-spectrum disorders (ASD) are highly heritable, complex neurodevelopmental diseases where different gene combinations may play a role in different individuals. Nevertheless, the study of mutations in specific genes is helping to characterize the molecular mechanism responsible for subtle alterations in the nervous system, perhaps pointing to a general mechanism for the disorder. Here, we propose a novel approach to study ASD. Using Rett syndrome (RTT) as a pilot disease, we developed an in vitro system deriving induced pluripotent stem cells (iPSC) from RTT patients’ fibroblasts. RTT patients have several autistic features and are part of ASD. RTT patients have defined mutations in the X-linked gene encoding the methyl-CpG binding protein 2 (MeCP2). RTT patients’ reprogrammed cells can generate human neurons carrying different types of MeCP2 mutations. Deep sequencing will be used to analyze gene expression during the transition steps of differentiation, simulating early stages of human neural development. The system will allow us to study the relationship of gene expression of coding and non-coding RNAs to the cellular and network phenotypes, such as neuronal arborization, synapse formation, and network electrophysiology. Moreover, we will use a chimeric brain system to study the effects of environment in human RTT neurons. In a future step, we will repeat the strategy using different single-gene mutations that also lead to the autistic diagnosis. The data generated will help to reveal and understand possible common mechanisms present in ASD.
Massachusetts General Hospital / Harvard Medical School
Engineering Sensitive Microfluidic Multiplex Technology for Isolating Circulating Endothelial Progenitor and Tumor Cells to Study Angiogenesis and Metastasis in Cancer Development and Progression
Despite major strides in understanding of the molecular basis of cancer and cancer therapeutics, the complexities of the metastatic process remain poorly understood. Especially in colorectal cancer, understanding has been severely hampered by limited knowledge about the cells that cause the disease to metastasize through the bloodstream. Circulating cells of several lineages are thought to participate in angiogenesis, tumor growth, and metastasis. Among these, circulating tumor cells (CTCs) shed from the primary and metastatic carcinomas presumably give rise to bloodborne metastases, whereas circulating endothelial progenitor cells (CEPCs) from adult bone marrow initiate the premetastatic niche. This hypothesis gives rise to the “seed (CTCs) and soil (CEPCs)” concept. Although current models explain distinct and important aspects of metastasis, no single model can explain the sum of the cellular changes apparent in human cancer progression and metastasis. I will investigate the inextricable relationship between CTCs and CEPCs and their roles in carcinogenesis and metastasis. I propose to take a radical but integrated technology- and biology-based translational approach using microfluidic engineering tools to identify and study the biological relevance of these rare cells in peripheral blood. This approach will seek to answer the following questions: 1) Do the levels of CEPCs and CTCs in early and late stages of colon cancer correlate with each other along with tumor volume and clinical course? 2) Can dynamic changes in their load during the course of treatment plan predict the clinical outcome of the therapy? 3) Are there any changes to phenotypic and biological characteristics of these cells that distinguish prognostic subtypes? 4) What is the effect of CEPCs on CTCs when cocultured, and what is the fundamental biology of interaction? 5) Can we expand these cells in vitro to identify the true “metastatic precursors” or “cancer stem cells” and to determine biomarkers of angiogenesis and metastasis as potential therapeutic targets?
Robert Wood Johnson Medical School/University of Medicine and Dentistry of New Jersey
Computational Design of a Synthetic Extracellular Matrix
The extracellular matrix (ECM) is a complex network of collagens, laminins, fibronectins, and proteoglycans that provides a surface upon which cells can adhere, differentiate, and proliferate. Defects in the ECM are the underlying cause of a wide spectrum of diseases. The ECM mediates endothelial cell polarity and under normal conditions can suppress pre-oncogenic transitions to a neoplastic state. We are constructing artificial, de novo, collagen-based matrices using a hierarchic computational approach. These matrices will be physically characterized in the laboratory and used to probe the role of chemical and spatial organization in the ECM on the tumor-forming potential of adhered cells. We are using a two-stage, computational strategy to construct an artificial ECM. A key technology is protCAD (protein Computer Automated Design), a software platform developed in our laboratory specifically for computational protein design. In the first stage, the sequences of short collagen-like modules are designed to independently assemble into trimers of programmed stability and specificity. These modules are then covalently connected using flexible peptide linkers to facilitate the self-assembly of controlled, higher-order structures such as networks and fibrils. Encouraging experimental characterization of the first-generation collagen designs suggests that our computational strategy is likely to succeed. Synthetic ECMs will be useful in biomedical research and translational applications. Mammalian cells will be grown on anisotropic, self-assembling nanostructured matrices to assay effects on cell polarity, cytoskeletal orientation, and morphology. We will explore the ability of artificial matrices to suppress cell proliferation in the presence of various oncogenic signals. This will provide a powerful system for studying molecular aspects of the matrix biology of cancer. Successfully designed matrices will be applied to engineering safer artificial tissues.
Diane Joyce Ordway
Colorado State University
Immune Modulation by Highly Virulent Clinical Isolates of M. tuberculosis
The global epidemic of tuberculosis continues unabated. A particularly dangerous family of clinical strains is the "W-Beijing" family, many of which are highly drug-resistant (MDR). The substantial and relatively rapid transmission of these strains across the world has led people to believe that these particular isolates have some capacity or property to resist, and even subvert, the host immune response. Innovative work in the applicant's research group has uncovered the possibility that at least some of these isolates can induce Foxp3+ regulatory T cells that interfere with the proper expression of protective immunity in the mouse. Moreover, in the highly relevant guinea pig model, the applicant has published new flow cytometric technology to allow (for the very first time) the definition of the host pulmonary immune response. In this second model, many of these W-Beijing strains cause extremely severe lung pathology, far worse than that seen using "laboratory strains" of Mycobacterium tuberculosis. This is a very important point, because all new vaccine candidates to date have only been tested against these laboratory strains. In this proposal, we will use innovative new transgene mouse models to address the role of newly identified T cell subsets in modulating or subverting host immunity to this very serious group of pathogens, using our state-of-the-art Level III biosafety facilities. In addition, we will continue to develop innovative new methods to apply these studies to the (currently reagent-limited) guinea pig model. These studies will include three W-Beijing strains that cause a range (moderate to extremely severe) of lung pathology and several characterized examples of drug- sensitive/drug-resistant genetically matched pairs to test the concept that acquisition of drug resistance reduces bacterial fitness, and hence virulence.
University of California, Los Angeles
Towards Mega-Throughput, Label-Free Genomics and Proteomics: Revolutionizing Microarray Technologies Using Lensless On-Chip Holographic Imaging and Nano-Plasmonics
Microarrays provide a high-throughput platform for various key studies in functional genomics, proteomics, epigenetics, medical diagnostics, and even tissue engineering. Together with advanced biochemical detection, imaging, and bioinformatics technologies, it is now possible to cost-effectively monitor the expression behavior of genes, proteins, or other biomarkers, as well as screening the genome and proteome content of various cell lines, on-chip drug profiling, or even detection of single-nucleotide-polymorphism. Therefore, microarray technologies provide a vital platform for performing high-throughput screening experiments that shed light on our understanding of cellular, genomic, and proteomic processes occurring at the nanoscale. In this proposal, we aim to create the next generation of microarray technologies to achieve an unprecedented mega-throughput, i.e., label-free imaging of millions of DNA/protein microspots would be feasible per second. We term the broad umbrella of these revolutionary technologies as Nano-plasmonic LUCAS. Specifically, we aim achieve a throughput of >120 cm2/second or >4.5 million spots/second for highly sensitive and label-free imaging of DNA/protein microarrays, which constitutes a speed improvement of >3 orders of magnitude when compared to the state of the art. Label-free imaging is especially important in not perturbing the natural biochemical, physical, and structural properties of the original molecule of interest. It also makes the measurements much more quantitative, significantly improving the data quality; eliminates inconvenient labeling steps which further reduces the cost; and avoids cross-reactivity issues among secondary probes, which can significantly improve the detection of weak or transitional molecular interactions. This mega-throughput capability will revolutionize the speed of progress that is taken in proteomics/genetics research by orders of magnitude that could eventually lead to the development of improved strategies/therapies for combating previously intractable biomedical problems and various diseases, including cancer. Furthermore, the Nano-plasmonic LUCAS platform does not require any lenses, microscope objectives, or other bulk optical components, and therefore offers an extremely compact on-chip platform that can easily be merged with microfluidic systems to permit point-of-care operation.
Christine K. Payne
Georgia Institute of Technology
Intracellular Delivery and Targeting of Nanoparticles
Nanoparticles have important biomedical applications ranging from the treatment of human disease with gene therapy to understanding basic cellular functions with fluorescent probes. It is now possible to synthesize nanoparticles with nearly any property or size, direct them to specific cells, and functionalize them to target intracellular locations, but delivering nanoparticles across the plasma membrane to reach their targets remains a challenge. We have recently demonstrated the first method for noninvasive delivery of semiconductor nanoparticles, known as quantum dots (QDs), to the cytosol of multiple cells simultaneously. Delivery requires pyrenebutyrate in combination with a cationic peptide for direct interaction of the QD with the plasma membrane. The ability of QDs to cross the plasma membrane offers exciting possibilities for the delivery of other nanoparticles to living cells. The first goal of this research program is to determine the molecular and cellular mechanism of pyrenebutyrate-mediated delivery and extend it to other nanoparticles with the ultimate goal of targeting specific intracellular sites with nanoparticles of choice. Novel imaging methods, including single-particle tracking fluorescence microscopy, will be used to probe the motion of the nanoparticle and its interaction with pyrenebutyrate as it moves across the plasma membrane and through the cytosol. As pyrenebutyrate-mediated delivery may not be suitable for all nanoparticles and all applications, we will also work to develop a suite of cytosolic delivery and targeting methods that are based on the well-characterized endosomal uptake pathways. Both delivery methods will be carried out in conjunction with studies of cellular response to nanoparticles that aim to optimize delivery and minimize disruption. In the course of this research, QDs, which are sufficiently bright for imaging at the single-particle level, will be used to probe fundamental questions of cellular transport, including diffusion through the crowded cellular environment, vesicle-mediated transport, and nuclear targeting.
Anna A. Penn
Stanford University School of Medicine
Fetal Brain Damage: A Placental Disorder
The placenta has long been underappreciated and understudied by the scientific community. Improper function of this critical organ causes fetal abnormalities, premature labor, and the most common disease of pregnancy, preeclampsia. Despite the importance of the placenta, our understanding of its role in fetal development, especially at a molecular level, is crude. Sadly, our understanding of placental function may be compared to the knowledge of kidney function 50 years ago—we can describe the anatomy, but not the biology. My overarching goal is to use new molecular techniques to understand placental function and its relationship to fetal outcomes. Here my specific goal is to investigate how placental hormones shape fetal brain development. As an endocrine organ, the placenta produces a wide array of neuroactive hormones. This endocrine function can be disrupted in many ways—by abnormal gene expression, infection, prematurity—resulting in long-term damage. Preterm birth, affecting one-tenth of all deliveries, provides the most extreme case of hormone loss, but I hypothesize that it is just one of many cases in which placental dysfunction leads to brain damage. I will develop a series of animals in which individual hormones are specifically removed from the placenta at precise times during development. This system will allow the first direct, definitive tests of the placenta as a key regulator of fetal brain development. Both established hormones, such as progestins and oxytocin, and hormones that we have recently identified as neuromodulators made by the placenta, such as secretin, will be assessed. These experiments are likely to provide fundamental new insights in placental physiology and neurodevelopment, help redefine disorders such as cerebral palsy, autism and schizophrenia as disorders of the placenta, and open new avenues to therapeutic treatments to improve neurological outcome in fetuses and infants at high risk of developmental brain damage.
Patrick L. Purdon
Massachusetts General Hospital / Harvard Medical School
A Neural Systems Approach to Monitoring and Drug Delivery for General Anesthesia
General anesthesia is a drug-induced, reversible condition comprised of five behavioral states: loss of consciousness, amnesia, analgesia, lack of movement, and hemodynamic stability. Over 100,000 patients annually receive general anesthesia in the United States for surgical and medical procedures, yet the mechanisms for general anesthesia remain a mystery of modern medicine. While considerable safety improvements in anesthetic drugs and monitoring have been made over the past several decades, anesthesia-related morbidity remains a significant medical problem. Approximately 1 in 500 patients experience postoperative recall of events during surgery, which can result in post-traumatic stress disorder. Up to 41% of elderly patients suffer from postoperative cognitive dysfunction, with long-term deficits found in 13% of such patients. The human and economic costs of postoperative cognitive dysfunction will continue to grow significantly as the population of the United States ages and requires more frequent surgical and medical intervention. Other frequent side effects of general anesthesia include cardiovascular and respiratory depression, nausea, and vomiting. These instances of anesthesia-related morbidity occur because anesthetic drugs affect the entire central nervous system, not just the specific brain areas required to produce the state of general anesthesia. Because we are unable to monitor brain activity intraoperatively within the desired target brain systems, it is a challenge at present to balance the desired anesthetic state against unintended side effects. Furthermore, we lack the means to deliver anesthetic drugs specifically to anesthesia-related brain systems in order to avoid side effects mediated in other brain systems. We present here an innovative research program that will develop neural systems-based anesthetic monitoring and drug delivery to eliminate anesthesia-related morbidity. The proposed studies will result not only in dramatically improved anesthesia care in the long term, but will also result in fundamental discoveries and developments in systems neuroscience, neuroimaging, and drug delivery.
Engineering Ubiquitin Ligases to Investigate Protein Aggregation and Neurodegeneration
Protein degradation lies at the heart of biological processes from signal transduction to cell cycle regulation. Compromised clearance of misfolded proteins from cells is the leading cause of human diseases such as neurodegenerative disorders. A common theme manifested in neurodegeneration is the accumulation of insoluble protein aggregates in the brain. However, the role of protein aggregation in the pathophysiology of neurodegeneration remains controversial. It remains a formidable task to remove protein aggregates from the affected neurons. In this proposal, we are taking a bold and innovative approach to attacking this exceedingly difficult problem: harnessing the ubiquitin/proteasome system to investigate protein aggregation and neurodegeneration. This proposal builds upon our previous development of methods to engineer single-chain ubiquitin ligase CHIP. We successfully established a novel strategy that enables us to alter the substrate binding specificity of CHIP without affecting its ligase activity. Using Huntington’s disease (HD) as a model, we propose herein to create recombinant ubiquitin ligases targeting the disease protein huntingtin (Htt) for ubiquitination. Our long-term goal is to define the structural features that determine the ligase activity of engineered ubiquitin ligases, elucidate the molecular mechanisms underlying protein aggregation and neurodegeneration, and evaluate the therapeutic potential of Htt-specific ubiquitin ligases. If successful, the engineered ubiquitin ligases will represent an unprecedented level of control over protein function in somatic cells, which would have direct impact on proteomic research by introducing novel “protein knockout” tools. In addition, the results of this project will provide unique insights into the fundamental cellular and molecular mechanisms underlying protein quality control and the pathophysiology of neurodegeneration. Application of these findings may help to delay or reverse the detrimental effects of neurodegeneration. Ultimately, it will serve as a prototype for the treatment of other neurodegenerative disorders, as well as non-neuronal human diseases.
Pacific Northwest National Laboratory
A Universal Multiplex Assay System for High-Throughput Clinical Applications
Recent advances in genomics, proteomics, and metabolomics make the “omics” technologies powerful discovery-based tools for identifying candidate biomarkers for human diseases; however, it has not been successful so far to establish new biomarkers for clinical practice by utilizing these technologies. The main bottleneck lies in the lack of effective tools for high-throughput validation. To overcome this bottleneck I propose to develop a novel, “reagent-free,” mass spectrometry-based universal multiplex assay system that will provide high-throughput quantitative measurements for hundreds of low-abundance protein and metabolite analytes, independent of antibody-based reagents. The goal for this technology platform is to achieve a profound advance over current MS-platforms by providing >1000-fold enhancement in analyte signal intensities, sufficient for detecting low-abundance species, and >5000-fold improvement in resolving power for extremely high-specificity detection. These advances will be achieved by developing and integrating 1) a novel subambient pressure ionization source with nanoelectrospray array, 2) advanced ion-funnel interfaces, 3) novel multistage gas-phase ion mobility technology (differential mobility analyzer coupled to field asymmetric ion mobility spectrometry) for separating and selecting analytes of interest, and (4) a new triple-stage pentaquadrupole (QqQqQ) mass spectrometer for further isolating as well as detecting ions. The optimized platform will have a potential analytical throughput of >100 samples per day, sensitivity for broadly analyzing low-abundance candidate biomarkers without enrichment, and the multiplexing power to monitor up to 1000 analytes simultaneously. At least 3-4 orders of magnitude enhancement in sensitivity or detection dynamic range (i.e., to a level comparable or superior to current ELISA) is anticipated, a significant advance over current assay platforms. Such a novel assay system is a disruptive technology that will revolutionize many areas of biomedical research, current medical practice, and the future of health care, as well as the biomedical research field in general through instrument commercialization.
Tufts University School of Medicine
Molecular Analysis of Functional Neural Circuits
A functional neural circuit consists of a group of connected neurons that collaborate to execute a specific function of the brain. The understanding of the molecular mechanisms that underlie the development, maintenance, and experience-dependent modification of functional neural circuits is incomplete due to limitations of existing methods. I propose to generate a transgenic mouse that addresses these limitations by exploiting two recent methodological advances. The first advance is the TetTag mouse, which is a transgenic mouse that can be used to genetically tag a single functional neural circuit. This tag enables the selective molecular analysis of neurons that have a shared function. This method is more sensitive to changes within a single functional neural circuit than other available methods, which have to rely on spatial criteria and thereby include neurons that do not participate in the circuit of interest. The second advance is the Translating Ribosome Affinity Purification (TRAP) method, which enables purification of actively translated messenger RNA from a genetically defined group of neurons. TRAP analysis reflects changes at both the transcriptional and translational level, while other available methods only detect transcriptional changes. I will combine TetTag and TRAP within a single transgenic mouse to generate the first tool that enables the comprehensive analysis of all translational events within a single functional neural circuit. Neurons tagged with the TetTag mouse during fear conditioning provide a stable neural correlate of the fear memory. I will use the TetTag/TRAP mouse to purify actively translated messenger RNA from these tagged neurons in order to detect the protein synthesis events that underlie the storage of a memory. The TetTag/TRAP mouse can be used for the molecular analysis of various functional neural circuits, including those involved in memory, addiction, epilepsy, circadian rhythms, spinal cord regeneration, pain, brain development, and neuronal cell death.
Theresa M. Reineke
Illuminating the Mechanistic Pathways of Polymer-Mediated Nucleic Acid Delivery
The wealth of information being obtained from genomic, proteomic, and glycomic research is allowing researchers to unravel the intricate genetic and epigenetic mechanisms associated with human health and disease. The intracellular delivery of nucleic acids to study these processes offers unprecedented promise for revolutionizing biomedical research and drug development. However, the nucleic acid delivery vehicle plays a central yet elusive role in dictating the efficacy, safety, mechanisms, and kinetics of gene regulation in a spatial and temporal manner, thus having a far-reaching impact in health-related research. To this end, we have developed several novel carbohydrate-containing polymers that have shown outstanding affinity to encapsulate polynucleotides into nanoparticles (polyplexes) and facilitate highly efficient intracellular delivery without toxicity. The goals of this project directly commence from our previous work where we aim to examine our wide-range of delivery vehicles for their mechanistic pathways and kinetics of nucleic acid encapsulation and intracellular transport from the cell surface to their final intracellular destination. We plan to examine 10 different carbohydrate-based polymers synthesized in our laboratory for their delivery mechanisms and kinetics with three polynucleotide forms: plasmid DNA, oligodeoxynucleotide decoys, and small interfering RNA, in two cell types, H9C2(2-1) and HeLa cells. The research program highlighted herein is driven by three specific goals: 1) to unravel the molecular-level interactions between structurally diverse yet analogous polymeric delivery vehicles and differing nucleic acid types and to correlate these interactions with the biological stability and mechanisms of the subsequent polyplexes, 2) to understand the interactions of these various polyplex types with cell surface glycosaminoglycans and compare polyplex structure to receptor selectivity and mechanisms of cellular uptake in two cell types, and 3) to decipher the intracellular trafficking pathways in a spatial and temporal manner from uptake to the final destination for each polyplex form with the two model cell types.
John L. Rinn
Beth Israel Deaconess Medical Center / Broad Institute of MIT and Harvard
RNA and Chromatin Formation: From Discovery to Mechanism
A major outstanding challenge in biology is to understand how the exact same genomic sequence present in every cell takes on alternate epigenetic landscapes to confer a myriad of cellular functions, all while using ubiquitous cellular machinery. In addition to histone modifications and DNA methylation, RNA has been long thought to be involved in the establishment and inheritance of these epigenetic states, but is far less understood. Indeed, recently three examples of large noncoding RNAs (HOTAIR, XIST, and AIR) have been discovered that share a common theme: They physically associate with chromatin-remodeling complexes and are required to guide chromatin formation at specific genomic loci. Although these examples suggest a general mechanism, it is still unclear to what extent RNA plays a role in chromatin formation and the mechanisms by which this guidance occurs. Here we propose to comprehensively and systematically address the roles of large noncoding RNAs in the formation of chromatin structure. We will accomplish this by: 1) identifying and characterizing large noncoding RNAs that physically associate with chromatin remodeling complexes genome-wide across multiple yet related cell contexts, 2) defining the sites of regulation and the guidance mechanism to these genomic loci, and 3) identifying how these molecules and their mechanisms are misregulated in human disease. Together, our multifaceted experimental and computational approaches aim to “crack the code” of epigenetic establishment and maintenance. This will transform our understanding of genome regulation and establish a new paradigm for RNA in the guidance of chromatin formation.
Pardis Christine Sabeti
Host and Pathogen Evolution in Lassa Fever
Disease-causing pathogens are among the most intriguing forces shaping human evolution, as they have a tremendous impact on our genome and themselves evolve over time. A genome-wide survey of human variation identified two genes biologically linked to Lassa fever as among the strongest signals of natural selection in West Africans. Lassa fever is a severe hemorrhagic disease endemic in West Africa, and our findings suggest it is an ancient selective force driving the emergence of genetic resistance. While poorly understood, Lassa fever has arguably the greatest potential impact of all infectious diseases of humans because of its unique status as both an immediate public health crisis and a category A potential bioterrorist agent. With the aim to pursue the intriguing signal of natural selection linked to Lassa fever, we first set out to address critical gaps in knowledge, capacity, and diagnostics. We established a basic diagnostic and research lab in Irrua, Nigeria, where yearly outbreaks of Lassa fever occur with population exposure of ~30%. Preliminary data suggests our initial measures have significantly reduced fatality from an estimated 65% to 20% among Lassa fever cases. We now aim to design a robust, field-deployable diagnostic, based on genome sequencing of diverse strains, to rapidly detect and distinguish Lassa virus strains. This work addresses immediate public health needs and sets the foundations for research into the genetic factors in both virus and human that underlie resistance to Lassa fever found among many West Africans. The ultimate goal of our work is to identify natural mechanisms of defense and illuminate the evolutionary adaptations that have allowed humans to withstand some of our most complex and challenging selective agents. Moreover, these efforts will create new opportunities in Lassa virus research, including investigations of viral pathogenicity and evolution and development of novel vaccines.
Schepens Eye Research Institute / Harvard Medical School
Bioengineering of Bruch's Membrane for the Treatment of Age-Related Macular Degeneration
Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries for those over 55. AMD is a complex and multifactorial disease described as two distinct types, dry and wet, leading to central vision loss. Dry AMD is characterized by the subretinal accumulation of deposits (drusen) and is associated with the progressive atrophy of the retinal pigment epithelium (RPE), choriocapillaris (vasculature supplying the photoreceptors), and retinal neurons. Dry AMD can progress to the proliferative form, wet AMD, where pathological and highly permeable vessels grow into the subretinal space. There is no treatment for dry AMD, and the current anti-angiogenic therapies for wet AMD, though effective at reducing vessel growth and permeability, do not address the underlying pathogenesis. Thus, the need for new therapeutic approaches is clear. Abnormalities in the RPE and the Bruch’s membrane (BrM), on which the RPE sit, are central to the development of AMD. Therefore, we are proposing a novel transplantation strategy to replace the degenerative RPE/BrM with the goal of preserving the choriocapillaris integrity and photoreceptor function. Previous attempts to transplant the RPE have failed largely because BrM alterations were not addressed. The aim of this proposal is to develop a co-culture system of RPE and endothelial cells on a biodegradable, biocompatible poly(ε-caprolactone) (PCL) polymer to bioengineer a RPE/Bruch’s membrane complex. The differentiation of the RPE and the formation of the BrM-like matrix on the PCL scaffold will be evaluated by immunohistochemistry, gene expression analysis, and electron microscopy techniques. Finally, the therapeutic potential of the RPE-PCL transplantation for patients with AMD will be determined in animal models of RPE damage. Results of these studies may permit the development of strategies aimed at replacing the diseased subretinal tissue with a bioengineered RPE/BrM prosthesis that could represent a therapeutic solution for all forms of AMD.
Fred Hutchinson Cancer Research Center
Cellular Cooperation and Cheating: An Experimental and Mathematical Analysis
Cooperation is widespread and has been postulated to drive major transitions in evolution. A cooperator pays a cost to benefit others, and when reciprocated, it gains a net benefit. However, Darwinian selection favors "cheaters" that consume benefits without paying a fair cost. Many cooperative systems have evolved sophisticated cheater recognition/exclusion mechanisms. How did cheater-resisting mechanisms evolve from simple cooperative systems? To address this question, I created a genetically tractable cooperative system that can be observed as it evolves, step-by-step, from its inception toward increased stability. It consists of two engineered, nonmating yeast strains–a red-fluorescent R strain that requires adenine and releases lysine and a yellow-fluorescent Y strain that requires lysine and releases adenine. I observed that: 1) the system is viable, able to grow from low density to saturation in the absence of adenine and lysine supplements, over a wide range of conditions; 2) system viability requirements could be calculated from growth, death, and metabolic properties of the two cooperating strains; and 3) the system evolved increased system viability, with the minimum cell density required for system viability reduced 100-fold. My group will: 1) Discover the diversity of changes that increase system viability. Pro-cooperation changes must act through benefiting self and/or partner. Properties of evolved strains will be measured and their relative contributions to enhanced cooperation will be quantified. 2) Determine mechanisms of cheater tolerance. After introducing a cheater that consumes but does not release metabolites, we will select for cooperator/cheater cocultures with increased cheater tolerance and delineate mechanisms. 3) Investigate the possibility of spatial structure stabilizing cooperation. We will compare viability requirements and cheater tolerance of the cooperative system in a well-mixed liquid culture (no spatial structure) with those on an agar pad (with spatial structure). We hope to quantitatively understand the evolution of cooperation and cheater tolerance.
Justin L. Sonnenburg
Stanford University School of Medicine
Discovery of Gut Microbiota-Targeted Small Molecules: New Tools and Therapeutics
The composition and function of the human intestinal microbiota is tightly linked to diverse aspects of host biology. Several diseases, including obesity and inflammatory bowel diseases, have been associated with altered microbiota composition. While much research is currently aimed at a genomic definition of the microbiota in both healthy and diseased states, there is a paucity of studies aimed at understanding this community at a mechanistic and ecological level and how changes in its function and composition directly impact host biology. The question of whether disease-associated alterations in microbiota composition are a cause or symptom of disease is difficult to address due to the current lack of tools that allow us to test the effect of perturbations in microbiota structure and function on the host in a controlled experimental setting. And once we are able to identify a pathologic microbiota definitively, how will we return it to a healthy state? The goal of this research proposal is to identify small molecules that can alter the microbiota at the level of function and composition, providing: 1) tools to aid investigation of altered microbiotas in model organisms and 2) a model pipeline for identifying a new class of therapeutics that targets the intestinal microbiota. The ability to monitor host responses in gnotobiotic mice colonized with a normal human gut microbiota provides an unprecedented capacity to search for compounds that will be useful in human medicine. While others have speculated on the promise of targeting the microbiota to manipulate human health, specific plans of how this would be achieved are lacking. This proposal lays out a plan to screen for compounds that target specific taxa of the microbiota, characterize the targeted microbiota in vivo, and determine the impact on host biology.
The Discovery of MicroRNAs That Predict Chemotherapeutic Responsiveness of Cancer
The vast majority of cancer deaths result from the metastatic spread of cancer cells to distal organs. Systemic chemotherapy can prevent metastasis in some patients by killing microscopic tumor cells throughout the body. Chemotherapy can also dramatically reduce the size of metastases in some advanced-stage patients. Interestingly, these standard chemotherapeutic regimens are administered to hundreds of thousands of patients without prior knowledge of the sensitivity of individual patients’ cancer cells to such treatments. If we could identify the chemotherapeutic responsive and resistant subsets of patients at diagnosis, innumerable patients would be spared from the risks, side effects, and expense of ineffective chemotherapy and instead offered alternative and experimental therapies in the upfront setting. Furthermore, the identification of such biomarkers could provide mechanistic insights into the molecular underpinnings of chemotherapeutic resistance. Working in breast cancer, we recently discovered a set of human microRNAs that strongly suppress metastasis in a robust mouse model of breast cancer. These microRNAs act as biomarkers since their expression levels in primary tumors predict future metastatic relapse, thus guiding clinical decisionmaking. Colorectal cancer is a highly prevalent and aggressive disease entity with significantly fewer treatment options than breast cancer. We propose to apply a conceptually and technically innovative, systematic, and multidisciplinary approach to discover chemotherapeutic-response predictive microRNAs through an experimental approach that integrates molecular, in vitro, in vivo, and human clinical insights. We will validate the power of these microRNA biomarkers through prospective in vivo human studies. If successful, we envision this powerful approach applied to other common cancers. The identification of such microRNAs will not only be of tremendous clinical value now, it will also lay the foundation for future mechanistic and synthetic efforts aimed at generation of novel, microRNA-based therapeutic agents for the prevention and treatment of cancer metastasis.
Jerilyn A. Timlin
Sandia National Laboratories
Multiplexed Measurements of Protein Dynamics and Interactions at Extreme Resolutions
My goal is to develop state-of-art imaging technology that can measure protein complex formation and protein networks in a multiplexed fashion with spatial resolution beyond that of optical microscopy. At present, a major limitation to clarifying the dynamics of a particular signaling cascade is the inability to visualize multiple (>4) proteins and their interactions simultaneously in real time in the living cell. This is due in part to the interference of spectrally similar species (including cellular autofluorescence) and the mismatch between the spatial resolution of the confocal microscope and the scale of protein interactions. Computational and experimental approaches can help to elucidate many of these interactions, but not all. Specialized microscopy methods have been developed to address some aspects of the problem, but to date, no technology has demonstrated true multiplexed (simultaneous, not sequential) detection of >4 proteins and their complex formation in living cells at spatial resolutions >100 nm. This type of detection is critical for unraveling protein interaction network details, and my proposed work will address that. Specifically, I will: 1) implement novel emission-scanning hyperspectral confocal microscopy hardware to collect information from large numbers of fluorescent species simultaneously at spatial resolution beyond that of the optical microscope, and 2) develop corresponding algorithms to spectrally unmix the 6D (X, Y, Z, excitation l, emission l, and time) ../images and provide accurate measurements of fluorophore concentrations even in the presence of energy transfer. This creative approach alleviates limitations of existing multicolor technology by extending my expertise in live-cell, hyperspectral imaging technology into the "super-resolution" realm. Its success will be enabled by robust, multivariate image analysis algorithms. This advance will have far-reaching impact in exploring signaling pathways and networks in biology and biomedicine.
University of California, Los Angeles
Cellular Quiescence and Brain Tumor Stem Cells
The discovery of tumor stem cells in human brain tumors has greatly changed the biological and clinical views of treatment-refractory brain cancer. Targeted therapies causing tumor stem cells to differentiate, undergo apoptosis, or die therefore represent a novel therapeutic strategy to treat recurrent tumors. The goals of this proposal are to identify genes that confer the tumor stem cell quiescence and to develop new brain tumor therapies based on the blockade of cellular quiescence in order to potentiate the treatment efficacy of radiation and chemotherapy. We have established several tumorigenic CD133+ glioblastoma (GBM) stem cell lines, which are directly derived from patients’ primary tumors that are recurrent and had previous treatment. Functional and molecular studies revealed that the slow-growing CD133+ GBM stem cells expressed a series of tumor suppressor/quiescence-associated genes but are capable of clonal self-renewal and spontaneous re-entry to the cell cycle to generate highly proliferative CD133- progeny that can populate tumor spheres in cultures and reconstitute a malignant tumor in mouse brain. We therefore hypothesize that GBM stem cells use reversibility of cellular quiescence to escape treatment followed by regenerating a new tumor upon treatment removal. We will perform a loss-of-function RNA interference screen for molecular targets of GBM tumor stem cell quiescence and test whether knockdown of quiescence factors will improve the treatment efficacy of radiotherapy and chemotherapy. Thus, the innovative treatment strategy proposed here aims to redirect reversible, viable arrested tumor stem cells toward non-reversible senescence, apoptosis, or terminal differentiation upon radiotherapy or chemotherapy. Preventing tumor stem cells from re-entering the cell division cycle after treatment shall greatly diminish the recurrence rate of GBM tumor.
Erik M. Ullian
University of California, San Francisco, School of Medicine
The Role of Astrocytes in Plasticity and Disease
Astrocytes are the most abundant cell type in the human brain, yet we still do not fully understand the impact of astrocytes on human disease. In this proposal we will begin to uncover the role of astrocytes in regulating cortical plasticity using several new technologies, including quantitative mass spectroscopy and microfluidic chambers developed to rapidly identify astrocyte factors that are released in response to the paracrine signals acetylcholine (ACh) or norepinephrine (NE) and that are likely to impact plasticity in both the developing and adult brain. Additionally, we will take advantage of the outstanding stem cell and proteomic centers here at UCSF to ask whether astrocytes derived from somatic cells from individuals on the autism spectrum secrete altered levels of synaptogenic factors. Using a combined microfluidic chamber and imaging system we will screen for effects on synapse formation and function using astrocytes derived from autism patients and familial controls. These studies have the potential to uncover the role of glial cells both in regulating normal plasticity and in disease states.
University of Minnesota Medical School
Understanding the Persistence of Immune-Mediated Chronic Diseases
Autoimmunity and asthma are immune-mediated diseases which can last for the lifetime of an individual, causing financial, physical, psychological, and societal burdens. Many of these diseases are characterized by symptomatic periods or flares, followed by a time of remission. Multiple factors, such as stress and infections, are believed to be responsible for this pattern. During these diseases, T cells are persistently stimulated by self or environmental antigens. Like autoimmune diseases, exposure to pathogens, which cause a chronic infection, also results in constant T cell stimulation by persistent antigens. I have recently demonstrated that new, pathogen-specific T cells are constantly produced during the chronic phase of infection, well after the initial infectious burst has resolved. This continued thymic output was required for maintaining the immune response. As common features can pertain to different immunological situations, the hypothesis to be tested in this proposal is whether the generation of T cells specific for tissue and environmental antigens in autoimmunity and asthma during established disease is a factor responsible for perpetuation of tissue damage. This hypothesis will be tested in different models of diabetes, multiple sclerosis, and a novel model of asthma. In addition, manipulation of thymic output during these chronic diseases, as well as during chronic infections, will determine whether newly developed T cells can be programmed to dampen atopic or autoimmune diseases or augment antimicrobial immunity. If successful, these studies may offer a novel explanation for the chronicity of certain immunological diseases and provide a new paradigm on which to base therapeutic intervention.
Leor S. Weinberger
University of California, San Diego
Developing Transmissible Antivirals by Exploiting Gene-Expression Circuitry
Emerging and established viral diseases take an enormous toll on human health. Current treatment approaches are unlikely to halt epidemic spread of many viruses, notably HIV-1, due to prohibitive costs of treatment (i.e., access), compliance issues, rapid viral mutation, and the influence of hard-to-reach high-risk viral “superspreaders.” We propose to shift the treatment paradigm toward developing Therapeutic Infectious Pseudoviruses (TIPs) that require the pathogen to replicate. TIPs would transmit along a pathogen's normal transmission route, reaching precisely those high-risk populations that most require therapy. TIPs co-opt wild-type virus packaging elements, decreasing disease-progression in vivo and reducing disease transmission on a population scale. We have demonstrated that an anti-HIV TIP could mutate with equal speed and under evolutionary selection to maintain its parasitic relationship with wild-type virus, thereby overcoming viral mutational escape. Since TIPs replicate conditionally (i.e., piggyback), treatment compliance and cost issues are eliminated. A precedent for the safety of TIPs exists in the oral polio vaccine (a live-attenuated vaccine), which exhibits limited spread and is being used in the polio eradication campaign. To develop candidate TIPs, we will capitalize upon our expertise in HIV-1 transcriptional circuitry. We discovered that HIV-1 exploits stochastic gene expression to control entry into a dormant state (proviral latency). By targeting a cellular gene (SirT1) essential for viral feedback, we have biased HIV-1 toward dormancy and diminished reactivation. We will exploit this innovative strategy of forcing viruses into dormancy by utilizing our single-cell imaging methods to conduct high-throughput imaging screens for therapeutic candidates that promote viral latency. Next, these candidate TIPs will be analyzed in novel microfluidic chemostats that maintain homeostatic infection and allow viral evolution in an in vivo-like setting. By integrating these approaches with predictive models, we will develop a revolutionary therapy to halt the spread of HIV/AIDS and other infectious diseases.
University of Texas Southwestern Medical Center
Neurogenesis De Novo in the Adult Central Nervous System
Trauma, stroke, and neurodegenerative disease result in neuronal loss, which leads to morbidity and mortality. A major advancement to mitigate these conditions would be to harness the ability to regenerate lost neurons. Although neural stem cells (NSCs) and neurogenesis normally exist in adult brain, the scarcity and restricted localization render them inadequate for regeneration. Cell transplantation is currently the strategy of choice to deliver new neuronal cells, but this approach is inefficient and cumbersome due to limited cell survival and poor integration into the functional neural networks following transplantation. In a highly novel approach to adult neurogenesis studies based on recent findings, we hypothesize that endogenous glial cells can be directly converted into neurogenic NSCs so that de novo-generated neurons will repopulate damaged brain regions. This hypothesis is based on our extensive studies using an essential nuclear receptor for NSCs and on the recent advancement of induced pluripotent stem (iPS) cells. We previously revealed that nuclear receptor TLX is not only essential for adult neurogenesis but is also sufficient to convert differentiated astrocytes into NSCs in culture. Although somatic cells from various tissues can be reprogrammed to iPS cells, it is not clear whether somatic cells can also be directly induced to form NSCs and neurons. Through transcriptional reprogramming, we propose to convert astrocytes and microglia, the two most proliferative glia cells during CNS damage, into NSCs and neurons. Using regulated expression of TLX in cultured cells and in transgenic mice, we will induce astrocytes to become proliferative, multipotent NSCs and then differentiate them into neurons. In addition, by using combinations of transcription factors, we will directly reprogram astrocytes or microglia to NSCs in cell culture and in adult mouse brains. Our long-term goal is to repopulate the damaged CNS regions using the patient’s endogenous non-neuronal cells.
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