Children's Hospital of Wisconsin
Howard Jacob, David Dimmock, Stephen Duncan, Brian Link, James Verbsky, Fyodor Urnov, Elizabeth Worthey
A team of basic scientists and clinical investigators at the Children’s Hospital of Wisconsin (CHW), the Medical College of Wisconsin propose to develop a novel strategy for determining the molecular basis of rare undiagnosed diseases. CHW recently established a Genomic Medicine Clinic for rare diseases and will use whole genome sequencing (WGS) to advance a diagnostic odyssey for rare diseases. With WGS, CHW’s diagnosis rate for genetic diseases has increased to ~27% (compared with 5–10% in a standard genetics clinic), yet ~39% of patients remain undiagnosed because they have genetic variants of uncertain significance (VUS). The challenge that CHW’s team is taking on is to convert as many as possible of the undiagnosed cases into firm diagnoses. In collaboration with Sangamo BioSciences, they propose to build a high throughput strategy, involving gene editing technologies, to rapidly identify the functionality of VUSs in cellular assays or a vertebrate model system. The proposed approach for evaluating VUSs would allow the characterization of many genetic variants that have already been identified in clinical labs around the country but not yet linked to specific genetic diseases. If successful, this project could establish a generalizable strategy for screening genetic mutations for any disease whether or not there is existing knowledge about the disease.
Michael Levin, David Kaplan
Tufts University investigators propose to develop a new technology for vertebrate limb regeneration based on understanding and manipulating the bioelectrical properties of cells. While most of the field is focused on biochemical and gene networks, they are pioneering the molecular understanding and control of the regulation of limb development by endogenous cellular bioelectrical properties. If successful, the proposed experiments could establish bioelectricity as a central component of development and regeneration. The team will develop pharmacological and genetic techniques for manipulating natural bioelectrical gradients in vivo which will drive gene expression to initiate the regeneration of adult vertebrate limbs following amputation. Based on their findings, the PIs have engineered a unique prototype device called a “biodome” that can fit over a limb amputation site and could function as a bioreactor to promote regenerative repair in amphibians and eventually in rodents. This work could reveal novel pathways of biological regulation for complex tissue repair.
University of California, Davis
Angelique Louie, Jared Shaw, Yohei Yokobayashi
Non-invasive visualization of gene expression in deep and opaque tissues remains a challenge. Current molecular markers for gene expression work well in thin or transparent subjects, but it is difficult to optically probe the deeper regions of adult mammalian systems. This project would develop new imaging reagents for detecting gene expression using magnetic resonance imaging (MRI). The gene for a naturally florescent protein, luciferase, will be linked to the gene to be studied so that they are always expressed together. The energy of the light from luciferase will induce a structural change in nearby gadolinium probes, enabling them to be detected by MRI. Whereas traditional MRI reagents such as iron, cannot distinguish live from dead cells and are good for one time measurements only, the proposed strategy would allow for monitoring of gene expression over time as well as in deep tissues. If successful, this project could enable basic mechanistic studies such as tracking stem cells in vivo or observing cancer metastasis.
University of California, Irvine
Anthony James, Fred Gould
Modeling of the evolutionary consequences of movement of novel genotypes and selfish genetic elements into new populations often generates unexpected predictions that are rarely tested empirically. Rigorous experimental assessments of model predictions will be conducted to further a basic understanding of evolution and advance the new field of applied evolutionary biology. Research on invasive species that threaten biodiversity, agricultural productivity and human health provides opportunities for model evaluation. Specifically, releases of transgenic mosquitoes developed for population suppression or modification mimic natural intraspecific invasion by genes and elements. Outcomes generated by simple and complex models include unanticipated results due to non-random associations of physically unlinked genes interacting with genetic drift. Investigators from the University of California, Irvine and North Carolina State University will test these models in four scenarios in laboratory cage trials with the Asian malaria vector, Anopheles stephensi. This work will contribute to a basic understanding of multi-locus evolutionary dynamics, contribute to novel control strategies for a major human disease and provide guidance for research on other invasive species less amenable to laboratory testing.
University of California, Santa Barbara
Santa Barbara, CA
Apoptosis, programmed cell death, is an essential process in the development and homeostasis of multicellular organisms. Apoptosis can serve as a protective mechanism by eliminating abnormal or damaged cells. During apoptosis, a cascade of biochemical signals initiate cellular self-destruction. It is generally believed that once this signaling cascade is triggered, it cannot be reversed. Researchers, now at University of California, Santa Barbara, have discovered a cell biological phenomenon that overturns the dogma that progress towards cell death is irreversible. They found that many different cell types can survive and proliferate even after passing checkpoints previously believed to be points of no return. The investigators termed this process anastasis (Greek: rising to life), and posit that this mechanism evolved to salvage cells and limit permanent tissue damage caused by transient exposures to insults such as ischemia, radiation or toxins. The team will use state-of-the-art screening techniques to identify genes, mRNAs, microRNAs and proteins that could be responsible for anastasis. Finally, they will screen for small molecules that promote or inhibit anastasis. These studies could lead to interventions for either delaying healthy cell death (neurodegenerative disease) or hastening cell death (cancer).
University of Florida
Malcolm Maden, Brad Barbarzuk
It is generally accepted that mammals do not regenerate injured tissues but scar instead because of the induction of fibrosis at the site of injury which results in wound healing but prevents tissue regeneration. This project developed out of the discovery that the adult African spiny mouse, Acomys, can regenerate without scaring a repertoire of tissues (epidermis, hair, dermis, glands, smooth muscle and skeletal muscle) following skin removal or punches through the ear. No other mammal examined so far can do this. This project will identify the unique nature of the Acomys regenerative response by examining the cell type, the extracellular matrices, the transcriptome and the proteome following injury. The cells and tissues of the spiny mouse will be systematically compared with those of standard lab mice throughout the course of the healing process. Understanding the regenerative, non-scarring environment and the nature of the cells that initiate it could be a key to promoting mammalian tissue regeneration.
City of Hope
John Williams, Tijana Jovanovic-Talisman, David Horne, Jinha Park
Investigators at City of Hope, in collaboration with colleagues at the University of Hawaii, seek to advance a potentially transformative monoclonal antibody (mAb) based technology platform. The technology will provide new tools and methods for investigating and understanding molecular trafficking, especially at the cell surface, which will lay the foundation for improving cell-specific therapies for major diseases including cancer. The basis for this new platform is the team’s recent discovery of a meditope, a cyclic peptide that binds non covalently with high affinity and specificity to a buried site within the fragment antigen binding region (Fab) of cetuximab. Here, they propose to develop and optimize the meditope-antibody interaction to 1) gain fundamental insight into antibody mediated receptor trafficking, especially receptor internalization, 2) facilitate the delivery of imaging agents to monitor molecular and cellular dynamics, and 3) advance the cell-specific delivery of agents to detect and treat disease. The team will also develop a set of novel reagents that will be broadly applicable to basic and advanced methodologies in the life sciences such as imaging.
Sreekanth Chalasani, James Fitzpatrick
La Jolla, CA
A central challenge of modern neuroscience is to decode how an organism responds to external threats, particularly how neural circuits elicit appropriate behaviors. Neural circuits, including those in humans, are optimized to detect and trigger avoidance of threats in the environment, but most of the basic underlying molecular machinery, including synaptic signaling, is poorly understood. Complete understanding of these circuits requires classification of all participating neurons, their connections and their interactions with other systems (including sympathetic connections in the gut, circulation, musculature, etc.). However, this level of analysis is nearly impossible to achieve in a complex vertebrate system. One unconventional, yet rational approach to understand these behaviors is to dissect the relevant neural circuits in a simpler, more tractable model, Caenorhabditis elegans. The team has developed a novel model of threat behaviors using the interactions between C. elegans and a second nematode, an aggressor Pristionchus pacificus. This proposal will dissect the molecular signaling pathways that integrate external threats into behavioral and physiological responses using a combination of genetics, behavioral analysis, pharmacology, and advanced imaging methods. Findings from these analyses of threat-activated networks will provide significant insights into the nature of similar behaviors across species.
Our immune systems balance the vigilance required to kill cells compromised by infection or cancer with the tolerance needed to avoid harming healthy cells. The dynamic and complex repertoire of antigenic peptides nearly all cells present on major histocompatibility class I proteins (MHC 1) are the foundation of self-directed immune responses. Finding ways to sensitively and accurately measure these peptides (pMHC 1) is a long sought goal of immunologists. Past mass spectrometry-based efforts have been met with surprisingly low success rates, and were limited in their quantitative output. The team seeks to solve technical challenges that have hindered past efforts. By developing and applying a completely unbiased method for peptide analysis, they found that a surprisingly large proportion of pMHC 1 share essentially no homology with known proteins. While running counter to the established dogma, this finding opens new directions for the diagnosis and treatment of cancer. They will extend their methods in a quantitative fashion to exploit these non canonical peptides to better understand the antigenic contribution to B cell lymphomas. Specifically, the team will incorporate isotopic labeling into our peptide sequencing method, and use it to quantify how both canonical and non canonical pMHC 1 presentation changes with stress induction, and across increasingly neoplastic cell states.
University of California, San Diego
Nick Spitzer, Davide Dulcis
La Jolla, CA
Our perceptions, behaviors, emotions, memories and intelligence depend on the appropriate synthesis and release of specific neurotransmitters in the brain. Since the award of the Nobel Prize for the discovery of chemical synaptic transmission, it has been thought that transmitters are fixed and invariant throughout life. The team has recently discovered that sensory stimuli respecify transmitter expression during development of the nervous system. Their preliminary data from adult rats strongly suggest that different photoperiods stimulate transmitter switching between dopamine and somatostatin in neurons in the mature brain, overturning the dogma of transmitter stability, with resulting changes in behavior. Their goal is to confirm the ability of sensory stimuli to switch the neurotransmitters in the adult mammalian brain and to determine the effect of transmitter switching on behavior, in order to develop practical applications. The team’s approach will rely on a highly novel combination of immunocytochemistry, in situ hybridization, non-invasive imaging, behavioral tests and pharmacological cell ablation. They will quantify anatomical changes in the adult rat brain and correlate these findings with results of non-invasive imaging to accelerate throughput and analyze the effects of different stimuli on the same animal. They seek to identify the molecular mechanism of transmitter switching and the behavioral results.
University of California, San Francisco
San Francisco, CA
Detecting signals (“sensing”) and responding to them (“actuating”) are among the most fundamental abilities of living systems. This project aims to engineer new sensor/actuator systems that go beyond what exists in nature. Natural sensor/actuators can regulate very complex biological behaviors, such as production of chemicals in metabolism, formation of microbial communities in the human gut, or differentiation of cells in mammalian tissues. To be able to probe and precisely control such behavior, but not be limited to existing signals, would transform biological engineering and cell biology. The team proposes to develop a platform technology to computationally engineer proteins that convert sensing of small molecule signals into responses in living cells. Keck funding will allow them to fundamentally advance their ability to engineer such new biological functions by (i) inventing robotics-inspired approaches to reshape proteins for sensing new signals and (ii) optimizing the resulting designs to function in cells. The team’s results will enable them to begin to realize the enormous potential of engineered new sensor/actuators in testbed applications, including: (i) manufacturing valuable chemicals in microbes, (ii) building sensor/actuators that control communities of cells, and (iii) probing biology, where sensor/actuators would allow study of processes causing cancer and inflammation in new ways.