Salk Institute for Biological Studies
Martin W. Hetzer, Asako McCloskey, Brandon H. Toyama, Travis Berggren
La Jolla, CA
Most healthy individuals experience declines in cognitive performance as they age, suggesting that neuronal functionality decays over time. This is not surprising as neurons are particularly long-lived compared to most other cell types. It is therefore critical to untangle the molecular and cellular factors that maintain a youthful neuronal proteome and counteract age-associated or neurodegenerative phenotypes in long-lived neurons. However, mechanisms governing the maintenance and aging of the proteome are largely unknown because technological challenges have limited the analysis of protein turnover in adult tissues. To overcome current limitations, a team of Salk Institute investigators developed pulse-chase labeling of rats in combination with quantitative mass spectrometry and multi-isotope image mass spectrometry. Using this strategy, they identified long-lived proteins (LLPs) that persist in neurons for years. Importantly, these LLPs perform key regulatory functions, and both neuronal functional decline and specific loss of LLPs in neurons occur during normal aging. The team plans to decipher the mechanisms underlying the functional integrity of LLPs over long periods of time in rat brain neurons, and determine whether their eventual decline contributes to pathologies in the brain. The proposed work could provide new avenues for studying the normal aging process and for developing therapies against age-related disease.
Seattle Children's Research Institute
Mark Majesky, Kathleen Millen, Daryl Okamura, Adrian Piliponsky, Joshua Akey, Jay Shenure
The long-term goal of this project is to enable regenerative wound repair in humans. A multidisciplinary team of investigators from Seattle Children’s Research Institute and the University of Washington will focus on learning how the African spiny mouse (genus Acomys), a terrestrial mammal with remarkable natural regenerative abilities, reacts to tissue injury and restores organ function without fibrosis or scar formation. The team plans to acquire, assemble and annotate the whole genome sequence of Acomys, and to develop CRISPR-Cas9 genome editing methods for Acomys embryonic stem and somatic cells. Furthermore, they propose to generate transgenic animals to allow investigators to test hypotheses about gene function in regenerative wound repair. Once completed, the Acomys genome sequence and the transgenic animals will be made publically available. The team will also investigate Acomys’ ability to regenerate internal organs after injury. The investigators believe that the lack of scar formation in Acomys could shed light on how tissue fibrosis, which causes many types of organ failure, develops over time, and in this context propose methods to alter the course of fibrotic disease to promote regenerative wound healing.
University of California, Berkeley
Jennifer Doudna, Alex Marson, Peidong Yang
CRISPR-Cas9 is a powerful technology for engineering genomic sequences. Efforts are underway to use CRISPR-Cas9 directly in human tissues, but delivery efficiency has been limited, especially in human T cells where genome engineering would be a crucial tool to dissect genetic mechanisms controlling immune homeostasis. A team of University of California, Berkeley and University of California, San Francisco investigators proposes a comprehensive approach to delivering Cas9 protein-RNA complexes into primary human T cells that will be of broad biomedical utility. The team plans to develop a delivery system that is: 1) efficient and simple to use; 2) independent of T cell stimulation or other ex vivo manipulations of cells; and, 3) tolerated by T cells with minimal toxicity. Nanowires will serve as ‘nano-syringes,’ transiently puncturing the cell membrane to deliver the genome editing machinery without inflicting permanent cell damage. This interdisciplinary effort aims to adapt nanowire technology for high throughput genetic engineering of human T cells for experimental and therapeutic purposes. Genome engineering T cells will offer insights into the genetic basis of immune-mediated diseases, providing a tool to test the cellular consequences of coding and non coding DNA variation. This project also lays the foundation for a new generation of T cell based immunotherapies for infections, autoimmunity and cancer.
University of California, Merced
Victor Muñoz, Mourad Sadqi, Zifan Wang
A major challenge in biosensor research is to achieve specific sensing at nanoscale (molecular) resolutions in living cells and in real time. Proteins would be ideal scaffolds because of their high specificity and tunable affinity for binding, and their built-in mechanism for transducing signals through conformational changes. However, reaching nanoscale resolution in real time requires single-molecule devices that produce analog outputs. This is a serious limitation because typical proteins behave as molecular switches with inherently binary outputs: in the presence of a specific molecule to which a protein binds tightly, the unfolded protein folds to its correct conformation and produces a signal such as fluorescence; whereas in the absence of the ligand molecule the protein unfolds and the signal stops altogether. A team of investigators at University of California, Merced proposes to develop single-molecule biosensors based on their previous discovery of “downhill folding” proteins, i.e., proteins that fold and unfold gradually through a continuum of partially-folded intermediates not separated by free-energy barriers. These proteins are theoretically and computationally predicted to detect other molecules in an analog mode, much like a rheostat. The team plans to apply the “folding coupled to binding” biosensor-design principle to proteins purposely engineered to fold downhill and implement them with gradual fluorescent signals. These novel sensors are expected to display wide dynamic range, ultrafast response and analog readouts at the single molecule level. To implement this idea, the team will employ a multidisciplinary approach that combines protein engineering and design, single-molecule fluorescence microscopy, nuclear magnetic resonance, theoretical modeling, and high-performance computational methods.
University of Oregon
Richard Taylor, Darren Johnson, Benjamin Alemán, Miriam Deutsch, Christopher Niell
A team of researchers at the University of Oregon proposes to simulate, fabricate and test a novel electronic-nerve interface (interconnect). Their objective is to engineer bioinspired interconnects that will have the same geometry as the nerves they interface with. The investigators posit that designing electronics that mimic the repetitive fine branching, or fractal, pattern seen in nature, such as at the dendritic ends of neurons, could radically improve electrical stimulation of nerves in the human retina, the brain, the limbs and other parts of the body. As a test application, these new electronics would be used to restore sight in mice with retinal degeneration. If successful, the bioinspired interconnects would allow victims of retinal diseases to see in greater detail and under more realistic lighting conditions compared to retinal implants using conventional interconnects. By manipulating the optical properties so that fractal branches absorb light at different wavelengths, it might be possible to simulate color vision. The ultimate goal is to restore vision to the point that recipients can read text and facial expressions, which are capabilities critical for functioning in society. The developed interconnects could also address other neurological disorders, such as Parkinson’s disease and depression, and improve nerve connections to prosthetic limbs.
Plamen Ch. Ivanov, Ednan Bajwa, Julian Goldman, Susan Redline
Investigators at Boston University, in collaboration with scientists from Massachusetts General Hospital and Brigham and Women’s Hospital at Harvard Medical School, will develop the first atlas representing the network of dynamical interactions among organ systems in the human body. The investigators plan to develop a theoretical framework and the first tools to explore quantitatively the way in which organ systems coordinate their functions and collectively interact as a network to produce distinct physiological states such as stages of sleep, coma, or multiple organ failure. This system-integrative approach will lay the foundations of a new field, Network Physiology. The researchers will also develop a novel platform capable of simultaneously recording organ output signals and directly relating them to physiological states and disease conditions. The work could have a large impact as it may determine, for the first time, fundamental mechanisms that govern organ network interactions and their evolution across physiological states, and may lead to next-generation ICU monitoring devices and more comprehensive assessment of drug effects based on novel information derived from networks of organ interactions. In addition, the investigators will build a database of network maps as a reference for normal and dysfunctional physiological conditions.
Christopher Moore, Ute Hochgeschwender, Diane Lipscombe, Barry Connors, Julie Kauer
Real-time detection of subcellular events coupled to feedback control could prove revolutionary. Current solutions detect only extracellular signals, and require implanted hardware and computer-based detection algorithms. Brown University investigators are solving this problem with an all-molecular approach that allows cells to sense and correct their own activity patterns. They would engineer cells to express bioluminescent proteins that produce light only when local calcium levels increase. Light-sensitive proteins such as optogenetic effectors detect this light and, in turn, generate context-dependent outcomes, including suppression or excitation of the host cell. The team will focus this development on calcium, as brief calcium increases are essential for driving contraction in muscles, information processing in neurons and insulin release in pancreatic cells. These new tools will have broad research and clinical applications, including sensing and discontinuing aberrant activity before it can cause harm. This approach can potentially be adapted to re-wire communication between cells, as light generated in one cell can be detected by sensors in a partner. The investigators will test these methods in neurons, smooth muscle and pancreatic beta cells. This molecular feedback system can be applied to any signal that bioluminescent enzymes can detect (e.g., ATP, cAMP, pH), further expanding the impact of this new self-regulation mechanism.
University of California, Santa Cruz
David Haussler, Benedict Paten
Santa Cruz, CA
Since the sequencing of the human genome was announced in 2000, much has been learned about the remarkable level of genomic variations in diverse human populations. However there is no framework to reliably compare these variations to understand their role in health and disease. This project, led by University of California, Santa Cruz researchers, aims to construct a new representation of the human reference genome, the Human Genome Variation Map (HGVM), which will accommodate all common human genomic variations using a new mathematical platform. This graph-based structure will augment the existing human reference genome, providing a means to name, identify, and analyze all common variations precisely and reproducibly. The HGVM data model will integrate into the standard application programming interface (API) for representing genomic variation being developed by the Global Alliance for Genomics and Health, placing it at the center of a burgeoning set of standards for sharing genomic data. The HGVM will be progressively scaled from prototypes for pilot regions to a complete resource that incorporates data from all major public sources of genetic variation, including common, complex structural variations and satellite DNA. It will include tools necessary for working with this rich structure to gain a deeper understanding of human genetic variation and its association with phenotypes relevant to health and disease. The HGVM will be public and freely available to all.
University of North Carolina, Chapel Hill
Jeremy Purvis, Jeanette Cook
Chapel Hill, NC
The cell division cycle is a fundamental biological process that impacts development, disease and biotechnology. Although the cell cycle involves a complex sequence of molecular events, our current knowledge of this process comes largely from “snapshot” measurements that provide little information about the underlying dynamics. In this project, the investigators will employ an interdisciplinary approach to assemble the first real-time map of the human cell cycle. The team will construct and validate a suite of novel fluorescent biosensors to visualize key molecular, metabolic, and developmental events throughout the cell cycle in normal epithelial cells, stem cells, and differentiated stem cell descendants. With these new reagents, they will use time-lapse microscopy and automated image analysis to track cell cycle transitions at the single cell level. A novel computer algorithm will be used to assemble the image sequences into a continuous timeline. Combined, these strategies will reveal the precise order of molecular events, their rates of change, cellular locations, and molecular interdependencies. Ultimately, this effort will produce the first comprehensive, interactive cell cycle model that can be accessed via a publicly available interface and used to explore new relationships among cell cycle events.