Baylor College of Medicine
Susan Rosenberg, Kyle Miller, Christophe Herman
Baylor College of Medicine investigators, in collaboration with a researcher from the University of Texas at Austin, propose to create ‘freeze-frame’ proteins that can trap and fluorescently label the DNA intermediates in genomic instability reactions in living cells from bacteria to human. These tools allow a new kind of in-vivo biochemistry in which transient reaction intermediates are “frozen” in living cells, quantified as fluorescent foci, mapped in the genome, and proteins that act on them identified. The freeze-frame proteins will allow identification of functions of hundreds of genes in large networks previously discovered to promote genome instability, most by unknown means. At present, research in genome instability focuses on enzymes rather than DNA structures that they affect. Genetic and other approaches can miss pathways that process damaged DNA due to the presence of redundant and parallel pathways. Likewise, therapies that target the enzymes often fail because of rapid evolution that can lead to resistance. The team would first create optimal proteins engineered to target DNA lesions called double-stranded breaks. Subsequently, the team would create a panel of freeze-frame proteins targeted to various structures present during DNA damage and repair. If successful, the proposed freeze-frame proteins could provide a direct and sensitive method to identify the amounts, types, and causes of DNA damage that occurs due to aging, stress, and diseases such as cancer and neurodegeneration and possibly point to new therapeutic directions.
Eric Ortlund, Graeme Conn
An important unanswered question in biology is how proteins evolved to recognize other proteins, small molecules, DNA and RNA. These interactions, which drive intricate cell signaling networks, rely on biomolecules that often perform multiple functions. Emory University researchers propose to use a methodology known as ancestral gene resurrection to understand the mechanisms by which these biomolecules evolved their current functions. Ancestral gene resurrection is the process of using information from the evolutionary tree to reconstruct ancient genes which can then be used to generate the RNAs and proteins they encode. The investigators will derive ancient transcription factors and noncoding RNAs and determine the evolutionary steps that enabled them to acquire their unique functions. They will use the ancient molecules as tools to discover the forces that shaped the specific molecular interactions driving gene expression. A number of state-of-the-art cellular and biophysical methods will be applied to understand how DNA sequence changes drove changes in cell signaling. Their findings may help researchers generate predictive knowledge regarding how DNA sequence drives gene regulation, enabling the generation of algorithms designed to predict the functional outcome of transcription factor binding. Moreover, the work will likely have significant impact on understanding the biophysical principles underlying the structure-function relationships in protein-DNA and protein-RNA complexes.
Laura-Ann Petitto, Melissa Malzkuhn, Arcangelo Merla, David Traum, Brian Scassellati
A team based at Gallaudet University proposes a comprehensive project to advance the science of language learning in infants. The work builds on a sequence of major discoveries led by principal investigator Laura-Ann Petitto who found that deaf babies are sensitive to the rhythmic patterning of human language’s phonological structure, even though these patterns are conveyed silently on the hands in signed languages. She assembled a multidisciplinary team from the University of Southern California, Yale University, and the University of Chieti, Italy to apply state-of-the-art technologies to study and to develop methods for enhancing early language acquisition. The goal is to create a learning tool that provides the core components of language’s rhythmic patterning to babies during critical periods of brain development. The “RAVE,” or “Robot AVatar thermal-Enhanced” prototype, will be placed near a baby’s crib to facilitate sensitivity to language patterns. The Robot will be interfaced with the thermal infrared imaging that signals when the infant is in a peaked emotional and attentional state and most “ready to learn.” A baby’s interactive eye gaze with the robot will trigger a virtual human to provide rhythmically patterned nursery rhymes in a visual language, with speech options. If successful, the work will advance new understanding of the human learning potential and could have a significant impact in the field of human cognitive neuroscience, especially pertaining to language acquisition.
Nationwide Children's Hospital
Veronica Vieland, Jayajit Das, Susan Hodge, Sang-Cheol Seok
Biomedical research relies on the statistical assessment of the strength of the evidence for or against hypotheses based on scientific data. A variety of measures are used as evidence measures such as p-values. But our standard measures of evidence lack calibration: A given change in any particular measure does not always correspond to the same amount of change in the strength of evidence; different measures are on fundamentally different scales; and any one of them may indicate decreasing strength of evidence while the evidence strength is actually increasing. Just as failure to properly calibrate experimental equipment can lead to scientific errors, uncalibrated evidence measures can lead to erroneous interpretations of biological data. The goal of this proposal is develop an absolute (context-independent) measure of the strength of evidence, through a novel information-dynamic paradigm (IDP). Combining theory development with high-performance computing we will extend the current prototype IDP to general statistical models and apply it to two distinct biomedical fields, human statistical genetics and biophysical modeling of the immune system.
University of Georgia
Michael Tiemeyer, Stephen Dalton, Marcus Fechheimer, Charles Schwartz, Richard Steet, Kevin Strauss, Lance Wells
The cell surfaces of all multicellular organisms are enveloped within a coating of carbohydrates called glycans which are linked to proteins and lipids. These glycans comprise the interface at which cells interact with each other and with their environment. Individual cell types express characteristic glycans in order to broadcast their identity, ensure productive interactions with other cells and protect themselves from pathogens. A growing number of human diseases disrupt glycosylation and neural cells are particularly sensitive to this perturbation. University of Georgia investigators, in collaboration with researchers from Greenwood Genetic Center (Greenwood, SC) and the Clinic for Special Children (Strasburg, PA), propose to identify and characterize the glycosylation changes that affect cognitive disorders, for example, familial Alzheimer’s disease (FAD), autism spectrum disorder (ASD) and X-linked intellectual disability (XLID). They have developed several state-of-the-art mass spectrometry-based analytical tools and would apply these tools for high throughput mass spectrometry to obtain the glycomic profiles of diseased and normal human neural cells and assess how an altered glycome impacts cell-specific functions. The investigators would also screen for modifiers of glycosylation as potential therapeutic leads for ameliorating glycomic deficiencies. If successful the project would advance current technologies for glycomic analyses and directly link altered glycosylation with cognitive diseases.
University of Illinois at Urbana-Champaign
Susan Martinis, Steven Blanke, Jie Chen, Lin-Feng Chen, Raven Huang, Zaida Luthey-Schulten
Aminoacyl-tRNA synthetases (AARSs) constitute a group of enzymes whose primary function is to translate the nucleotide codes contained in genes into their corresponding amino acids, the building-blocks of proteins. Because AARSs are essential for the translation of genetic information into proteins, they are found in all forms of life. University of Illinois researchers propose to determine the structures and characterize the functions of newly discovered genetic variants of these enzymes in humans that function in pathways other than translation. Using twenty mammalian leucyl-tRNA synthetases (LeuRS) as a model, the PIs will create a unique platform that can be used to systematically interrogate the function of over 250 AARS variants identified to date. This platform will consist of cutting-edge biochemical and cell based assays to determine variant localization and function(s). The platform will also include computational methodologies for structure-function analyses, as well as sophisticated intracellular investigations. Recent studies indicate that AARS genes can produce proteins whose functions vary from an acute inflammatory response to cholesterol transport. One already identified alternate function for a LeuRS variant is in cancer cell proliferation and inhibitors of this variant are being considered as cancer therapeutics. The project’s impact is expected to be broad as the team will likely uncover new mechanisms for how proteins acquire alternate functions.
Hereditary Disease Foundation
Nancy Wexler, Robert Darnell, Jean Paul Vonsattel
New York, NY
The team’s goal is to discover and understand the mode of action of genetic modifiers of age of onset and other symptoms of Huntington’s disease (HD). For over two decades Hereditary Disease Foundation researchers characterized its extensive Venezuelan HD kindred cohorts. A genome-wide linkage scan of this unique population demonstrated strong statistical support for three genetic modifier loci for age of HD onset. The investigators propose to identify the specific genetic variants responsible for modification of age of onset for HD. Neurological, psychiatric and cognitive examinations, well preserved brain tissue and germ line DNA represent an unprecedented resource for carrying out this study. They will conduct next-generation whole genome sequencing of key family groups, in-depth transcriptome analysis of brain regions, and genome-wide epigenetic analysis on germ line DNA at the New York Genome Center. The unique combination of resources in this project provides an extraordinary opportunity to unravel the pathway for age of onset. Results from this project could be applied to other inherited and other neurological diseases.
Northern Arizona University
Kiisa Nishikawa, Brent Nelson, Christopher Mann, Matthew Gage
The research team will develop new techniques for characterizing protein interactions that will test a radical new theory on muscle contraction. The prevailing “sliding filament” theory fails to adequately predict muscle behavior during natural movements. PI Nishikawa’s winding filament hypothesis represents a paradigm shift for the theory of muscle contraction. She proposes that the titin protein binds to actin upon calcium influx and winds on thin filaments as they are rotated by the cross-bridges during force development. She has demonstrated that the hypothesis fills existing gaps between experiment and theory. In order to directly characterize the proposed interactions, the team will develop and validate methods at or beyond the current state-of-the-art. First, single molecule atomic force spectroscopy will be used to quantify interactions between synthetically produced titin fragments and actin. Second, the investigators will use fluorescence resonance energy transfer reporters in a cell culture system and subsequently in transgenic mice to visualize and quantify these interactions in real time in living muscle fibers. Third, to observe the winding of titin on actin filaments, electron holographic tomography will be used to generate 3D images. The latter two approaches will require improvement of these technologies to yield sufficient data to either validate or disprove the winding filament hypothesis. Even if the hypothesis is disproved, a set of improved technologies would add value to the field of biological mechanics. If correct, her hypothesis could inspire new approaches for ameliorating neuromuscular disease and injury.
Salk Institute for Biological Studies
Clodagh O'Shea, Mark Ellisman
La Jolla, CA
To fit within the nucleus, DNA assembles into chromatin and coils into spatially defined territories that determine if genes are active or silent through poorly understood mechanisms. The higher order coding structures of the human genome have not been visualized within an intact cell and remain one of the most intractable challenges in biology. To overcome this, PI Clodagh O’Shea has identified a cell permeable fluorescent small molecule that binds specifically to DNA and upon excitation can be used to paint its surface with an electron dense polymer that enables the 3D ultrastructure of chromatin to be visualized at nucleosome resolutions. O’Shea and UCSD co-PI Mark Ellisman will combine this technique, called ChromEM, with metal nanoparticles targeted to specific genes. They will apply the combined technology to visualize how viruses cause rearrangement of the 3D chromatin structure in the infected cell’s nucleus to modulate gene activity. If successful, the methodology would reveal important clues to the relationship between chromatin structure and gene expression. These technological innovations and conceptual advances will have exciting applications that impact many aspects of biomedical science.
Manu Prakash, Zev Bryant
Two early career investigators at Stanford University propose to determine how individual molecular components assemble into complex cellular-scale systems. Their test case will be to design dynamic functional assemblies of engineered molecular motors derived from naturally occurring proteins that transport molecules inside the cell. The engineered proteins will actively move cargo such as nanoparticles along tracks made of cytoskeletal proteins, and will collectively drive bulk fluid flows. Zev Bryant will systematically engineer specifically tailored and dynamically controllable molecular-scale components, and Manu Prakash will engineer cellular-scale environments and model hydrodynamic interactions in order to program biomimetic far-from-equilibrium phenotypes. The team will reconstitute large systems of motors and filaments in confined microfluidic geometries, and then apply state-of-the-art microscopy methods to quantify the dynamics of individual motor molecules, the motion of nanoscale cargos, and the bulk fluid flow driven by these motors. If successful, a connection would be made between the atomic-resolution design of molecular motor properties and mesoscale behavior of cellular fluid flow dynamics. This would be a significant milestone in creating the capacity for engineering minimal systems that organize matter and respond to environmental signals with capabilities that have so far been confined to living cells.
University of California, San Francisco
Bo Huang, Noelle L'Etoile, Geeta Narlika, Lei Qi, Jonathan Weissman, Chao-Ting Wu
San Francisco, CA
A multidisciplinary team of investigators, led by early career investigator Bo Huang at UCSF, will develop advanced labeling tools that would allow tracking of such elements as genes and DNA binding proteins in living cells and animals. They will develop an image-based approach to monitor these elements to study biological processes such as gene regulation in cancer cells and stem cell differentiation. This technology platform consists of fluorescence bar-coding to track live cell genome organization and chromosome dynamics. The team will also engineer fluorescent reporters to identify specific histone modifications which will allow them to monitor the epigenetic status of target genes. The team will further create a C. elegans platform so that these studies can be performed in an intact organism. If successful, the studies would reveal where genetic information is accessed within the cell nucleus in real time. This advanced imaging platform, when fully established, would find applications in other biological science research.
University of California, Santa Barbara
Tod Kippin, Kevin Plaxco, Tom Soh
Santa Barbara, CA
The complexity of the interplay of dynamic neuronal signals in the brain has implications for human behavior. A UCSB team of engineers and a neuroscientist proposes to develop analytical tools that could continuously track multiple small molecules including psychoactive drugs and neurotransmitters in the brain of awake and ambulatory rodents. The technology is based on the team’s recently developed electrochemical sensors, composed of small DNA molecules called aptamers. These can be engineered to bind tightly to specific target molecules thereby triggering an electrical signal. These sensors, supported on micron-scale gold electrodes inserted into a rodent brain, will, for the first time, establish the time-synchronized relationships between drug neuropharmacokinetics as well as the neurotransmitter dynamics and behaviors that they induce. The success of this project could help to answer some of the most fundamental questions in addiction biology. It will also validate a powerful new approach for studying the dynamic molecular changes that form the biological bases of behavior.