Jennifer A. Prescher - US grants

This Integrative Graduate Education and Research Traineeship (IGERT) award initiates a novel model for interdisciplinary graduate training in biophotonics across the biomedical sciences, physical sciences, and engineering. Biophotonics technologies provide powerful capabilities to probe and manipulate biological components and processes. Their utilization in the life sciences and medicine represents an estimated annual economic impact of $50 billion. This program aims to produce the next generation of biophotonics leaders to make transformative advances in the development and application of new tools for biological and medical discovery and maintain global U.S. leadership in biotechnology, pharmaceutical and medical device industries.
Intellectual Merit: This IGERT award creates a hands-on training program that integrates physics, chemistry, engineering, and life-science principles across spatial and temporal scales. The interaction of, and collaboration between, biomedical scientists, physical scientists and engineers throughout the graduate traineeship will drive advances in biophotonics technologies, computational methods, and molecular probes to solve important problems in bio-molecular, cellular, tissue, and whole organismal systems.
Broader Impacts: The BEST IGERT project will promote dissemination of an innovative education framework aimed towards a diverse cadre of scientists and engineers. Moreover, IGERT faculty and trainees will engage vigorously in a spectrum of outreach, dissemination, recruitment, retention, and career development activities that leverages the commitment of multiple units within UC-Irvine, industry in Southern California, and a nationwide network of faculty contacts, including those at minority serving institutions, to inform the public and broaden participation by students from underrepresented groups.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to establish new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries, and to engage students in understanding the processes by which research is translated to innovations for societal benefit.

DESCRIPTION (provided by applicant): A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales-including those involved in cancer progression and migration-requires new imaging tools. The long-term goal of our work is to develop general strategies for macroscopic, multi-cell tracking in living organisms. The objective of this application is to engineer novel bioluminescent tools for multi-cellular imaging in vivo. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging utilize the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. Our central hypothesis is that the substrate-binding interface of firefly luciferase can be re-engineered to generate a panel of mutant enzymes that accept chemically distinct luciferins. When the mutants and analogs are mixed together, robust light emission will be produced when complementary enzyme-substrate partners interact. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Synthesize and identify light-emitting luciferins; 2) Generate complementary luciferases and screen for orthogonal pairs; and 3) Image tumor heterogeneity with orthogonal probes. Under the first aim, we will utilize divergent chemistries developed in our lab to access light-emitting small molecules. In the second aim, we will employ a combination of mutagenesis and screening assays to identify luciferase enzymes that catalyze light emission with the synthesized molecules. In the third aim, the enzyme-substrate pairs will be utilized to address the roles of distinct cellular subsets in heterogeneous tumor models. Our approach is highly innovative, as it combines a unique blend of chemical and biological techniques to fill a long-standing void in imaging capabilities. The proposed research is significant, as the bioluminescent tools will enable the direct interrogation of cell networks not currently possible with existing toolsets. Such studies will provide some of the first macroscopic images of tumor heterogeneity and may fundamentally change existing views on cancer progression and therapeutic approaches. Additionally, similar to other imaging technologies, the bioluminescent probes will likely inspire new discoveries in a broad spectrum of fields.

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Prescher, Jennifer

Imaging technologies have revolutionized our understanding of cancer progression by enabling researchers to 'look' into cells and tissues and visualize biological features in real time. While powerful, current imaging techniques are restricted in their ability to analyze cell movements and interactions over large time and length scales?processes crucial to metastatic disease. This NSF-CAREER application proposes the development of new imaging probes for visualizing tumor spread in vivo. The PI and her trainees will generate bioluminescent tools that produce light only when two distinct cell types come into close proximity (e.g., when tumor cells infiltrate distant tissues). Collectively, these tools will enable global surveys of cancer spread and will provide some of the first noninvasive, macroscopic views of metastases in preclinical models. The imaging probes will also be implemented in two outreach activities, including a luminescence screening laboratory (for undergraduates) and imaging demonstrations (for middle school students). The PI's outreach program also includes an innovative seminar series to introduce graduate students to a diverse set of scientific careers and highlight the impacts of scientific professions in society at large.


This CAREER award will result in a number of broader impacts. First, the proposed imaging tools will enable the direct interrogation of metastatic cancer cells not currently possible with existing toolsets. Such studies will provide macroscopic images of tumor spread and may fundamentally change existing views on malignancy. Second, the proposed tools will likely inspire new discoveries in diverse areas of science, as imaging technologies are widely used in materials research, neurobiology, and numerous other fields. Third, the imaging probes will be incorporated into a variety of educational outreach programs that will impact large numbers of students. These students will learn about the challenges and excitement associated with imaging and research in general. Last, the proposed outreach work will expose students from diverse backgrounds to the impacts of science in society by highlighting unique career opportunities.

Equal access to scientific inquiry and STEM careers is an important goal for the National Science Foundation and for the nation as a whole. However, minority students are not participating in the scientific enterprise in proportion to their representation within the general population. One barrier is the lack of connections between scientists at minority-serving institutions (MSIs) and at primarily white institutions (PWIs). This disconnect leads to a self-perpetuating cycle of primarily PWI graduates entering the highest ranks of academia and industry, relying on networks of trust that favor elite institutions in areas such as research collaborations. One possible solution is to build new faculty-based networks that thrive on mutual trust and exchange between MSIs and PWIs. This workshop will convene a group of faculty from MSIs and PWIs to begin a conversation about how to develop such a network.

The two-day workshop, to be held June 25-26, 2018 at the University of California, Irvine, will engage approximately 40 faculty members (20 from MSIs and 20 from PWIs) in a series of exercises to facilitate the development of partnerships across institutional types, and to begin the planning stages for writing proposals to support the partnerships. Faculty will meet each other, engage in discussion about model partnerships, and formulate ideas that could be used for grant proposals to be submitted to private foundations and national funding agencies. Workshop assessment will be conducted using surveys to measure how social networks have changed because of the meeting, and to follow up with partnerships established at the meeting to determine if they led to the submission or awarding of grants. Workshop discussions will be aggregated to develop a policy paper on best practices in developing MSI-PWI networks, which will be circulated publicly via posting on the University of California, Irvine website for the Division of Teaching and Learning, and by public presentation at relevant conferences and regional meetings.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Chemical tools can provide unique insights into biomolecule structure and function. Included in this group are bioorthogonal chemical reporters?biocompatible functional groups that can target diverse classes of biomolecules and be selectively ligated with various probes. These reporters and reactions enable biomolecules to be analyzed in their native environments. Despite their past successes and continued potential, most chemical reporters have been slow to transition to biological studies and the scientific community at large. Many reporters are too large or insufficiently stable for routine cellular use, or lack chemical versatility. The long-term goal of our work is to develop robust, easy-to-access chemical probes for tracking and controlling biomolecules in cells and tissues. The objective of this application is to develop one reagent?cyclopropenone?as a general and versatile chemical reporter. Cyclopropenones harbor unique features for biological application. They are small, reactive with bioorthogonal soft nucleophiles (e.g., phosphines), and highly tunable. In addition to tagging biomolecules, cyclopropenones can be used to control biomolecule function via phosphine-mediated crosslinking and decaging chemistries. Thus, from a single cyclopropenone unit, one?in theory?can assemble, analyze, and activate biomolecules of interest. The versatility and accessibility of such reagents can provide a ?one-stop-shop? for tool users, bringing chemical probes more rapidly into the hands of non-specialists. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Tune cyclopropenone reactivity for broad-spectrum biomolecule analyses; (2) Exploit ?latent? cyclopropenone reactivities for biomolecule assembly; and (3) Establish chemical triggers of biomolecule function. Under the first aim, we will examine the scope of the cyclopropenone-phosphine ligation, and identify scaffolds with suitable stability and reactivity profiles. In the second aim, we will capitalize on ?latent? cyclopropenone reactivities to assemble biomolecule conjugates. In the third aim, we will develop cyclopropenone triggers that can be used to crosslink and activate biomolecules of interest. Our approach is highly innovative, as it capitalizes on a unique reaction mechanism to access multi-functional bioorthogonal properties. The proposed research is significant, as it will provide versatile, easy-to-use chemical tools that are applicable to a broad spectrum of biomedical research. The probes will also enable experiments not possible with existing toolsets. Additionally, like other chemical technologies, the proposed reagents will likely inspire new discoveries in diverse fields.

Project Summary A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales?including those involved in cancer progression and migration?requires new imaging tools. The long-term goal of our work is to develop general strategies for macroscopic, multi-cell tracking in living organisms. The objective of this application is to engineer novel bioluminescent tools for sensitive, multi-cellular imaging in vivo. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging use the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. Over the previous granting period, we demonstrated that the substrate-binding interface of firefly luciferase can be re- engineered to generate panels of mutant enzymes that accept chemically distinct luciferins. When mutants and analogs are mixed together, light emission is produced only when complementary enzyme-substrate partners interact. Several pairs of orthogonal enzymes and substrates were identified, but they remain weak emitters and not suitable for sensitive imaging in vivo. Our central hypothesis is that improved orthogonal imaging tools can be generated using a combination of rational design and screening. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Identify the molecular determinants of orthogonality for lead pair optimization; 2) Generate orthogonal probes with improved tissue penetrance; and 3) Image tumor heterogeneity with expanded orthogonal toolsets. Under the first aim, we will use crystallography and deep-sequencing analyses to examine enzyme-substrate interactions responsible for orthogonality. These insights will be used to optimize existing orthogonal luciferase-luciferin pairs. In the second aim, we will prepare bioluminescent tools with red-shifted emission spectra. These tools will provide more sensitive imaging in vivo. In the third aim, the enzyme-substrate pairs will be used to address the roles of distinct cellular subsets in heterogeneous tumor models. Methods to rapidly differentiate the orthogonal probes in vivo will also be developed. Our approach is highly innovative, as it combines a unique blend of chemical, biological, and computational techniques to fill a long-standing void in imaging capabilities. The proposed research is significant, as the bioluminescent tools will enable the direct interrogation of cellular networks not currently possible with existing toolsets. Such studies will provide some of the first macroscopic images of tumor heterogeneity and may fundamentally change existing views on cancer progression. Additionally, similar to other imaging technologies, the bioluminescent probes will likely inspire new discoveries in a broad spectrum of fields.

Project Summary A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales?including those involved in cancer progression and migration?requires new imaging tools. Grant GM107630 aims to develop general strategies for macroscopic, multi-cell tracking with bioluminescent tools in living organisms. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging use the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. The PI and her team have demonstrated that the substrate-binding interface of firefly luciferase can be re-engineered to generate panels of mutant enzymes that accept chemically distinct luciferins. When mutants and analogs are mixed together, light emission is produced only when complementary enzyme-substrate partners interact. Ongoing work seeks to generate improved orthogonal imaging tools using a combination of rational design and screening. Our goals include (1) uncovering the molecular determinants of orthogonality (via crystallography and deep-sequencing analyses) for lead pair optimization; 2) generating orthogonal probes with improved tissue penetrance; and 3) imaging tumor heterogeneity with expanded orthogonal toolsets. Our research would be dramatically accelerated by the purchase of an all-in-one fluorescence microscope. Successful translation of the orthogonal luciferases in vivo requires benchmarking the bioluminescent enzymes against established markers (e.g., fluorescent proteins). Luciferase-fluorescent protein constructs also provide a ?one-stop-shop? for tool users, enabling macro-scale imaging and analyses (via bioluminescence) and micro-scale imaging/ex vivo analyses (via fluorescence). The fluorescence microscope will also enable the development of probes that can demarcate cellular location and function within heterogeneous systems. Such tools will further augment our studies of tumor-immune cell interactions. The proposed research is significant, as the imaging tools will enable the direct interrogation of cellular networks not currently possible with existing toolsets. Such studies may fundamentally change existing views on cancer progression. Additionally, similar to other imaging technologies, the probes will likely inspire new discoveries in a broad spectrum of fields.

This project develops a portfolio of molecular tags that emit strong signals when stimulated by mid-infrared (MIR) light. These molecular MIR tags are designed to light up selected parts in the cell when illuminated with an MIR microscope. The MIR tags developed by the investigators enable the visualization of molecular diffusion and chemical conversion, including numerous metabolic processes in cells and tissues, processes that have hitherto remained out of reach for MIR microscopy. This development transforms the MIR microscope from an instrument for inspecting static images of cells and tissue to a technology for studying dynamic processes such as cellular cholesterol uptake, protein synthesis and lipogenesis. This capability opens up new opportunities for investigating cellular processes that are difficult to study with standard optical microscopy methods. The investigators are committed to broaden the participation of a diverse pool of students by providing summer research training to students from HBCUs through the Access to Careers in Engineering and Sciences (ACES) program.

MIR imaging, typically in the form of Fourier transform infrared (FTIR) microscopy, is a label-free imaging tool based on molecular vibrational contrast. MIR labels or tags can significantly improve the specificity of MIR imaging, yet MIR labels have so far not been used for imaging purposes. The use of MIR labels would expand the complementary vibrational palette in IR microscopy, opening up a new catalogue of biorthogonal molecular probes based on IR transitions, and offering strategies for super-multiplex imaging. In this project, the PI and co-PI develop a portfolio of chemical motifs that exhibit an exceptionally strong IR-response. They will use these motifs as vibrational tags of small molecules, including cholesterol, glucose, nucleic acids, amino acids and other metabolites. In addition, the team will design probes suitable for fluorescence encoded infrared (FEIR) excitation and detection, enabling multiplex labeling studies in the MIR microscope with sensitivities that reach the single molecule limit.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.