The Experts below are selected from a list of 11247 Experts worldwide ranked by ideXlab platform
Jennifer A. Prescher - One of the best experts on this subject based on the ideXlab platform.
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Rapid multicomponent Bioluminescence Imaging via substrate unmixing
2019Co-Authors: Colin M Rathbun, Krysten A. Jones, William B Porterfield, Anastasia A. Ionkina, Zi Yao, Jennifer A. PrescherAbstract:Engineered luciferases and luciferins have dramatically expanded the scope of Bioluminescence Imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Here we report a strategy for rapid, multiplexed Imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent Imaging in mice was also achieved on the minutes-to-hours time scale, a vast improvement over conventional protocols.
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Advances in Bioluminescence Imaging: New probes from old recipes
Current opinion in chemical biology, 2018Co-Authors: Zi Yao, Brendan S. Zhang, Jennifer A. PrescherAbstract:Bioluminescent probes are powerful tools for visualizing biology in live tissues and whole animals. Recent years have seen a surge in the number of new luciferases, luciferins, and related tools available for Bioluminescence Imaging. Many were crafted using classic methods of optical probe design and engineering. Here we highlight recent advances in bioluminescent tool discovery and development, along with applications of the probes in cells, tissues, and organisms. Collectively, these tools are improving in vivo Imaging capabilities and bolstering new research directions.
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Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging
ChemBioChem, 2018Co-Authors: Brendan S. Zhang, Krysten A. Jones, David C. Mccutcheon, Jennifer A. PrescherAbstract:: New applications for Bioluminescence Imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for Bioluminescence Imaging.
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orthogonal luciferase luciferin pairs for Bioluminescence Imaging
Journal of the American Chemical Society, 2017Co-Authors: Krysten A. Jones, David C. Mccutcheon, William B Porterfield, Colin M Rathbun, Miranda A Paley, Jennifer A. PrescherAbstract:Bioluminescence Imaging with luciferase–luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for Imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme–substrate “hits” were identified. These tools produced light when complementary enzyme–substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent Imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and ...
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Orthogonal Luciferase–Luciferin Pairs for Bioluminescence Imaging
Journal of the American Chemical Society, 2017Co-Authors: Krysten A. Jones, David C. Mccutcheon, William B Porterfield, Colin M Rathbun, Miranda A Paley, Jennifer A. PrescherAbstract:Bioluminescence Imaging with luciferase–luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for Imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme–substrate “hits” were identified. These tools produced light when complementary enzyme–substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent Imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and ...
Stephen C. Miller - One of the best experts on this subject based on the ideXlab platform.
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Bioluminescence Imaging in mice with synthetic luciferin analogues.
Methods in enzymology, 2020Co-Authors: Spencer T. Adams, Stephen C. MillerAbstract:Luciferase enzymes from bioluminescent organisms can be expressed in mice, enabling these rodents to glow when treated with a corresponding luciferin substrate. Light emission occurs where the expression of the genetically-encoded luciferase overlaps with the biodistribution of the administered small molecule luciferin. Here we discuss differences between firefly luciferin analogues for Bioluminescence Imaging, focusing on transgenic and adeno-associated virus (AAV)-transduced mice.
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beyond d luciferin expanding the scope of Bioluminescence Imaging in vivo
Current Opinion in Chemical Biology, 2014Co-Authors: Spencer T. Adams, Stephen C. MillerAbstract:The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive Imaging in live mice. However, photon emission from the luciferase is crucially dependent on the chemical properties of its substrate, D-luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of Bioluminescence Imaging.
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a synthetic luciferin improves Bioluminescence Imaging in live mice
Nature Methods, 2014Co-Authors: Melanie S Evans, Jennifer A. Prescher, Miranda A Paley, Spencer T. Adams, Joanna P Chaurette, Gadarla Randheer Reddy, Neil Aronin, Stephen C. MillerAbstract:Firefly luciferase is the most widely used optical reporter for noninvasive Bioluminescence Imaging (BLI) in rodents. BLI relies on the ability of the injected luciferase substrate D-luciferin to access luciferase-expressing cells and tissues within the animal. Here we show that injection of mice with a synthetic luciferin, CycLuc1, improves BLI from existing luciferase reporters and enables Imaging in the brain that could not be achieved with D-luciferin.
Gary D Luker - One of the best experts on this subject based on the ideXlab platform.
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dual reporter Bioluminescence Imaging with nanoluc and firefly luciferase
Methods of Molecular Biology, 2018Co-Authors: Anne E Gibbons, Kathryn E Luker, Gary D LukerAbstract:Bioluminescence Imaging is a powerful, broadly utilized method for noninvasive Imaging studies in cell-based assays and small animal models of normal physiology and multiple diseases. In combination with molecular engineering of cells and entire organisms using luciferase enzymes, Bioluminescence Imaging has enabled novel applications including studies of protein-protein interactions, ligand-receptor interactions, cell trafficking, and drug targeting in mouse models. We describe use of a novel luciferase enzyme derived from Oplophorus gracilirostris, NanoLuc, in cell-based assays Bioluminescence Imaging of tumor-bearing mice. We also combine NanoLuc with another luciferase enzyme, firefly luciferase, to image multiple signal transduction events in one Imaging session.
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Bioluminescence Imaging of reporter mice for studies of infection and inflammation.
Antiviral research, 2010Co-Authors: Kathryn E Luker, Gary D LukerAbstract:In vivo Bioluminescence Imaging offers the opportunity to study biological processes in living animals, and the study of viral infections and host immune responses can be enhanced substantially through this Imaging modality. For most studies of viral pathogenesis and effects of anti-viral therapies, investigators have used recombinant viruses engineered to express a luciferase enzyme. This strategy requires stable insertion of an Imaging reporter gene into the viral genome, which is not feasible for many RNA viruses, and provides data on the viral component of pathogenesis but not on the host. Genetically engineered mice with luciferase reporters for specific viral or host genes provide opportunities to overcome these limitations and expand applications of Bioluminescence Imaging in viral infection and therapy. We review several different types of reporter mice for Bioluminescence Imaging, including animals that permit in vivo detection of viral replication, trafficking of immune cells, activation of key genes in host immunity to viral infection, and response to tissue damage. By utilizing luciferase enzymes with different emission spectra and/or substrates, it is possible to monitor two different biologic processes in the same animal, such as pathogen replication and sites of tissue injury. Combining Imaging reporter viruses with genetically engineered reporter mice is expected to substantially enhance the power of Bioluminescence Imaging for quantitative studies of viral and host factors that control disease outcome and effects of established and new therapeutic agents.
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Real-time Bioluminescence Imaging of viral pathogenesis.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Kathryn E Luker, Gary D LukerAbstract:Mouse models are used commonly to study viral infection and define viral and host determinants of infection and disease morbidity. Conventional studies of viral infection in mice rely upon euthanizing cohorts of animals at multiple time points to identify sites of infection, quantify viral titers, and determine host immune responses. This experimental paradigm precludes longitudinal studies of infection and response to treatment in the same animal and assumes that progression of infection and pharmacodynamics of therapeutic agents are identical in all mice. To enable repetitive, quantitative studies of viral infection in mouse models, we and others are using noninvasive Bioluminescence Imaging to track viral infection, dissemination, and effects of host immune mediators on disease. In this chapter, we detail experimental protocols for Bioluminescence Imaging of viral infections in living mice.
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Transgenic reporter mouse for Bioluminescence Imaging of herpes simplex virus 1 infection in living mice
Virology, 2006Co-Authors: Kathryn E Luker, Tracey L. Schultz, Joseph D. Romine, David A. Leib, Gary D LukerAbstract:Abstract Bioluminescence Imaging allows spatial and temporal progression of viral infection to be detected and quantified in living mice, thereby providing a new approach for studies of viral–host pathogenesis. It has been necessary to construct and validate recombinant reporter viruses that express firefly luciferase to investigate viral replication and spread with this Imaging technology. This strategy greatly limits the ability to analyze multiple strains of virus and/or existing viral mutants, and reporter viruses also may be attenuated relative to the respective parental viruses. To facilitate Bioluminescence Imaging of herpes simplex virus type 1 (HSV-1), we developed a transgenic reporter mouse that uses the promoter from HSV-1 thymidine kinase to control expression of firefly luciferase. Infection with HSV-1 activated expression of firefly luciferase in corneal and flank models of infection, and amounts of Bioluminescence increased in proportion to increasing input titers of virus. Imaging could detect infection with three different strains of HSV-1 with the following relative rank order of Bioluminescence produced at the site of infection: McKrae > 17 > KOS. Corneal infection with as few as 1 × 10 3 pfu strain McKrae was detectable above background levels. By comparison, infection with vaccinia virus did not affect Bioluminescence in the reporter mouse. Collectively, these data establish a new transgenic reporter mouse for infection with HSV-1, thereby enabling in vivo Bioluminescence Imaging studies of HSV-1 pathogenesis without constructing new reporter viruses.
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noninvasive Bioluminescence Imaging of herpes simplex virus type 1 infection and therapy in living mice
Journal of Virology, 2002Co-Authors: Gary D Luker, David Piwnicaworms, Patrick J Bardill, Julie L Prior, Christina M Pica, David A. LeibAbstract:Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen that infects ca. 80% of all persons by adulthood (33). During acute infection, HSV-1 enters the body at epithelial and mucosal surfaces, where viral replication and subsequent lysis of epithelial cells and fibroblasts lead to infection of sensory neurons and intraaxonal transport of virus to neuronal cell bodies in sensory ganglia. Further viral replication occurs within ganglia, and HSV-1 may reemerge from the nervous system during acute infection and cause disease by zosteriform spread within the same dermatome as the initial site of infection (3, 9, 15). Ultimately, viral gene expression in sensory ganglia is repressed and nonproductive latent infection is established (23). In response to poorly defined stimuli, HSV-1 may reactivate from latency and produce new infectious virus. Both primary infection with HSV-1 and reactivation from latency can cause morbidity and some mortality from diseases such as blinding keratitis and encephalitis. Studies of HSV-1 in mouse models typically rely upon observations of cutaneous manifestations of disease, cultures from accessible epithelial surfaces, and sacrifice of infected mice to determine distribution and titer of virus. Although these experimental methods have allowed investigators to define viral and host genes that regulate replication and virulence of HSV-1 in vivo (10, 18, 19, 24, 30), these assay techniques preclude real-time monitoring of the kinetics and extent of disease progression in the same animal. Therefore, potentially significant insights from animal-to-animal variations in host-pathogen relationships may be missed with conventional protocols for determining viral infection and therapeutic response. Spread of HSV-1 to unexpected anatomic sites also may not be identified because the infected tissue is not assayed for virus. Thus, noninvasive, whole-body Imaging of HSV-1 infection in living mice could provide new insights into disease pathogenesis and treatment. Recent advances in biotechnology have enabled in vivo Imaging of luciferase reporter proteins in living mice by using a cooled charge-coupled device (CCD) camera (6, 34). Because Bioluminescence Imaging has minimal background activity, this technology is very sensitive for detecting light emitted from luciferases. As little as 38 pg of firefly (Photinus pyralis) luciferase (FL) per g of liver tissue has been detected with Bioluminescence Imaging in vivo (20). d-Luciferin, the substrate for FL, crosses cell membranes and penetrates the intact blood-brain barrier after intraperitoneal (i.p.) injection in mice, allowing this reporter protein to be imaged in any anatomic site. At the concentrations used for Bioluminescence Imaging, d-luciferin is nontoxic and nonimmunogenic, so serial Imaging examinations can be performed with the same mouse. Using region-of-interest (ROI) analysis of images to quantify light emission, Bioluminescence measured in vivo correlates highly with FL activity in lysates of extracted tissues (26, 34). Although light is attenuated and scattered by hair and other overlying tissues (5), Bioluminescence Imaging has been used to detect and localize FL in tumor xenografts and parenchymal organs, including lung, liver, and brain tissues (1, 26, 34). Bacterial infections also have been detected in vivo with this Imaging modality by using bacteria expressing the lux operon (11, 12). In addition to FL and bacterial luciferase, a recently published study has demonstrated the feasibility of Imaging luciferase from Renilla reniformis (RL) at various anatomic sites in living mice (2). FL and RL each have unique substrates and show distinct kinetics of light production in vivo. RL activity peaks within 1 min and is undetectable by ca. 10 min after intravenous injection of the cognate RL substrate coelenterazine, whereas light from FL is maximal at 3 to 4 min and declines slowly over 30 min. Therefore, FL and RL potentially could be used as reporters for Imaging two different molecular events in vivo. In the present study, we determined the feasibility of using Bioluminescence Imaging to monitor infection with HSV-1 in living mice. We used a recombinant HSV-1 strain KOS virus (KOS/Dlux/oriL) that expresses FL and RL from early gene promoters (27). Replication and spread of KOS/Dlux/oriL in the mouse ocular model of infection does not differ significantly from wild-type strain KOS, making this reporter virus an effective model for studying pathogenesis of HSV-1. FL activity from KOS/Dlux/oriL was detected by Bioluminescence Imaging at all tested sites of infection. Although Bioluminescence from RL also could be imaged in the ocular model of infection, our results indicate that pharmacokinetics and bioavailability of substrate may limit applications of this reporter protein for in vivo Imaging of HSV-1. Using ROI analysis of photon emission, we demonstrate that differences in viral titer can be quantified by Bioluminescence Imaging of FL, allowing real-time Imaging of disease progression and response to therapy.
Spencer T. Adams - One of the best experts on this subject based on the ideXlab platform.
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Bioluminescence Imaging in mice with synthetic luciferin analogues.
Methods in enzymology, 2020Co-Authors: Spencer T. Adams, Stephen C. MillerAbstract:Luciferase enzymes from bioluminescent organisms can be expressed in mice, enabling these rodents to glow when treated with a corresponding luciferin substrate. Light emission occurs where the expression of the genetically-encoded luciferase overlaps with the biodistribution of the administered small molecule luciferin. Here we discuss differences between firefly luciferin analogues for Bioluminescence Imaging, focusing on transgenic and adeno-associated virus (AAV)-transduced mice.
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beyond d luciferin expanding the scope of Bioluminescence Imaging in vivo
Current Opinion in Chemical Biology, 2014Co-Authors: Spencer T. Adams, Stephen C. MillerAbstract:The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive Imaging in live mice. However, photon emission from the luciferase is crucially dependent on the chemical properties of its substrate, D-luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of Bioluminescence Imaging.
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a synthetic luciferin improves Bioluminescence Imaging in live mice
Nature Methods, 2014Co-Authors: Melanie S Evans, Jennifer A. Prescher, Miranda A Paley, Spencer T. Adams, Joanna P Chaurette, Gadarla Randheer Reddy, Neil Aronin, Stephen C. MillerAbstract:Firefly luciferase is the most widely used optical reporter for noninvasive Bioluminescence Imaging (BLI) in rodents. BLI relies on the ability of the injected luciferase substrate D-luciferin to access luciferase-expressing cells and tissues within the animal. Here we show that injection of mice with a synthetic luciferin, CycLuc1, improves BLI from existing luciferase reporters and enables Imaging in the brain that could not be achieved with D-luciferin.
Miranda A Paley - One of the best experts on this subject based on the ideXlab platform.
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orthogonal luciferase luciferin pairs for Bioluminescence Imaging
Journal of the American Chemical Society, 2017Co-Authors: Krysten A. Jones, David C. Mccutcheon, William B Porterfield, Colin M Rathbun, Miranda A Paley, Jennifer A. PrescherAbstract:Bioluminescence Imaging with luciferase–luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for Imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme–substrate “hits” were identified. These tools produced light when complementary enzyme–substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent Imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and ...
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Orthogonal Luciferase–Luciferin Pairs for Bioluminescence Imaging
Journal of the American Chemical Society, 2017Co-Authors: Krysten A. Jones, David C. Mccutcheon, William B Porterfield, Colin M Rathbun, Miranda A Paley, Jennifer A. PrescherAbstract:Bioluminescence Imaging with luciferase–luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for Imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme–substrate “hits” were identified. These tools produced light when complementary enzyme–substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent Imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and ...
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a synthetic luciferin improves Bioluminescence Imaging in live mice
Nature Methods, 2014Co-Authors: Melanie S Evans, Jennifer A. Prescher, Miranda A Paley, Spencer T. Adams, Joanna P Chaurette, Gadarla Randheer Reddy, Neil Aronin, Stephen C. MillerAbstract:Firefly luciferase is the most widely used optical reporter for noninvasive Bioluminescence Imaging (BLI) in rodents. BLI relies on the ability of the injected luciferase substrate D-luciferin to access luciferase-expressing cells and tissues within the animal. Here we show that injection of mice with a synthetic luciferin, CycLuc1, improves BLI from existing luciferase reporters and enables Imaging in the brain that could not be achieved with D-luciferin.
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expedient synthesis of electronically modified luciferins for Bioluminescence Imaging
Journal of the American Chemical Society, 2012Co-Authors: David C. Mccutcheon, Miranda A Paley, Rachel C Steinhardt, Jennifer A. PrescherAbstract:Bioluminescence Imaging with luciferase enzymes requires access to light-emitting, small-molecule luciferins. Here, we describe a rapid method to synthesize d-luciferin, the substrate for firefly luciferase (Fluc), along with a novel set of electronically modified analogues. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogues produced with this approach emit light with Fluc in vitro and in live cells. Collectively, our work increases the number of substrates that can be used for Bioluminescence Imaging and provides a general strategy for synthesizing new collections of luciferins.