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In vivo bioluminescence imaging of labile iron accumulation in a murine model of infection.
Aron AT, Heffern MC, Lonergan ZR, Vander Wal MN, Blank BR, Spangler B, Zhang Y, Park HM, Stahl A, Renslo AR, Skaar EP, Chang CJ
(2017) Proc Natl Acad Sci U S A 114: 12669-12674
MeSH Terms: 2,2'-Dipyridyl, Acinetobacter Infections, Acinetobacter baumannii, Anemia, Iron-Deficiency, Animals, Cation Transport Proteins, Cations, Divalent, Disease Models, Animal, Ferric Compounds, Firefly Luciferin, Fluorescent Dyes, Gene Expression Regulation, Hepcidins, Homeostasis, Iron, Iron Overload, Iron Regulatory Protein 1, Iron Regulatory Protein 2, Luminescent Measurements, Mice, Mice, Transgenic, Quaternary Ammonium Compounds, Receptors, Transferrin, Signal Transduction, Transferrin
Show Abstract · Added March 15, 2018
Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.
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25 MeSH Terms
Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca sensing.
Yang J, Cumberbatch D, Centanni S, Shi SQ, Winder D, Webb D, Johnson CH
(2016) Nat Commun 7: 13268
MeSH Terms: Animals, Batrachoidiformes, Calcium, Fluorescence Resonance Energy Transfer, HEK293 Cells, HeLa Cells, Humans, Luciferases, Luminescence, Luminescent Measurements, Microscopy, Fluorescence, Optogenetics
Show Abstract · Added March 26, 2019
Optogenetic techniques allow intracellular manipulation of Ca by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.
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Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist-Induced Arrestin Recruitment to Modified and Unmodified G Protein-Coupled Receptors.
Donthamsetti P, Quejada JR, Javitch JA, Gurevich VV, Lambert NA
(2015) Curr Protoc Pharmacol 70: 2.14.1-2.14.14
MeSH Terms: Arrestin, Cell Membrane, GTP-Binding Proteins, HEK293 Cells, Humans, Ligands, Luminescent Measurements, Polyethyleneimine, Receptors, G-Protein-Coupled, Translocation, Genetic
Show Abstract · Added February 15, 2016
G protein-coupled receptors (GPCRs) represent ∼25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Because functionally selective or biased ligands activate one of these two pathways, they may be superior medications for certain diseases states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for two bioluminescence resonance energy transfer (BRET)-based assays used to monitor arrestin recruitment to GPCRs. One assay requires modification of GPCRs by fusion to a BRET donor or acceptor moiety, whereas the other can detect arrestin recruitment to unmodified GPCRs.
Copyright © 2015 John Wiley & Sons, Inc.
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10 MeSH Terms
Phosphorescence monitoring of hypoxic microenvironment in solid-tumors to evaluate chemotherapeutic effects using the hypoxia-sensitive iridium (III) coordination compound.
Zeng Y, Liu Y, Shang J, Ma J, Wang R, Deng L, Guo Y, Zhong F, Bai M, Zhang S, Wu D
(2015) PLoS One 10: e0121293
MeSH Terms: Adenocarcinoma, Animals, Antineoplastic Agents, Biological Transport, Cell Hypoxia, Cell Line, Tumor, Cell Survival, Cisplatin, Colonic Neoplasms, Coordination Complexes, Iridium, Luminescent Agents, Luminescent Measurements, Mice, Optical Imaging, Organometallic Compounds, Treatment Outcome, Tumor Microenvironment
Show Abstract · Added April 2, 2019
OBJECTIVES - To utilize phosphorescence to monitor hypoxic microenvironment in solid-tumors and investigate cancer chemotherapeutic effects in vivo.
METHODS - A hypoxia-sensitive probe named BTP was used to monitor hypoxic microenvironment in solid-tumors. The low-dose metronomic treatment with cisplatin was used in anti-angiogenetic chemotherapeutic programs. The phosphorescence properties of BTP were detected by a spectrofluorometer. BTP cytotoxicity utilized cell necrosis and apoptosis, which were evaluated by trypan blue dye exclusion and Hoechst33342 plus propidium iodide assays. Tumor-bearing mouse models of colon adenocarcinoma were used for tumor imaging in vivo. Monitoring of the hypoxic microenvironment in tumors was performed with a Maestro 2 fluorescence imaging system. Tumor tissues in each group were harvested regularly and treated with pathological hematoxylin and eosin and immunohistochemical staining to confirm imaging results.
RESULTS - BTP did not feature obvious cytotoxicity for cells, and tumor growth in low-dose metronomic cisplatin treated mice was significantly inhibited by chemotherapy. Hypoxic levels significantly increased due to cisplatin, as proven by the expression level of related proteins. Phosphorescence intensity in the tumors of mice in the cisplatin group was stronger and showed higher contrast than that in tumors of saline treated mice.
CONCLUSIONS - We develop a useful phosphorescence method to evaluate the chemotherapeutic effects of cisplatin. The proposed method shows potential as a phosphorescence imaging approach for evaluating chemotherapeutic effects in vivo, especially anti-angiogenesis.
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Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples.
Bastarache JA, Koyama T, Wickersham NE, Ware LB
(2014) J Immunol Methods 408: 13-23
MeSH Terms: Animals, Biomarkers, Bronchoalveolar Lavage Fluid, Electrochemical Techniques, Enzyme-Linked Immunosorbent Assay, Humans, Inflammation Mediators, Interleukin-6, Interleukin-8, Interleukins, Limit of Detection, Luminescent Measurements, Mice, Mice, Inbred C57BL, Predictive Value of Tests, Reagent Kits, Diagnostic, Reproducibility of Results, Species Specificity
Show Abstract · Added May 19, 2014
BACKGROUND - Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance.
METHODS - In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples.
RESULTS - We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations.
CONCLUSIONS - In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.
Published by Elsevier B.V.
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18 MeSH Terms
Bioluminescence imaging reveals dynamics of beta cell loss in the non-obese diabetic (NOD) mouse model.
Virostko J, Radhika A, Poffenberger G, Dula AN, Moore DJ, Powers AC
(2013) PLoS One 8: e57784
MeSH Terms: Aging, Animals, Diabetes Mellitus, Experimental, Disease Models, Animal, Female, Genetic Heterogeneity, Hyperglycemia, Imaging, Three-Dimensional, Insulin-Secreting Cells, Luminescent Measurements, Mice, Mice, Inbred NOD, Mice, Transgenic, Models, Biological, Time Factors
Show Abstract · Added October 24, 2013
We generated a mouse model (MIP-Luc-VU-NOD) that enables non-invasive bioluminescence imaging (BLI) of beta cell loss during the progression of autoimmune diabetes and determined the relationship between BLI and disease progression. MIP-Luc-VU-NOD mice displayed insulitis and a decline in bioluminescence with age which correlated with beta cell mass, plasma insulin, and pancreatic insulin content. Bioluminescence declined gradually in female MIP-Luc-VU-NOD mice, reaching less than 50% of the initial BLI at 10 weeks of age, whereas hyperglycemia did not ensue until mice were at least 16 weeks old. Mice that did not become diabetic maintained insulin secretion and had less of a decline in bioluminescence than mice that became diabetic. Bioluminescence measurements predicted a decline in beta cell mass prior to the onset of hyperglycemia and tracked beta cell loss. This model should be useful for investigating the fundamental processes underlying autoimmune diabetes and developing new therapies targeting beta cell protection and regeneration.
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15 MeSH Terms
High-fat diet acutely affects circadian organisation and eating behavior.
Pendergast JS, Branecky KL, Yang W, Ellacott KL, Niswender KD, Yamazaki S
(2013) Eur J Neurosci 37: 1350-6
MeSH Terms: Animals, Circadian Rhythm, Diet, High-Fat, Feeding Behavior, Luminescent Measurements, Male, Mice, Mice, Inbred C57BL, Motor Activity
Show Abstract · Added December 10, 2013
The organisation of timing in mammalian circadian clocks optimally coordinates behavior and physiology with daily environmental cycles. Chronic consumption of a high-fat diet alters circadian rhythms, but the acute effects on circadian organisation are unknown. To investigate the proximate effects of a high-fat diet on circadian physiology, we examined the phase relationship between central and peripheral clocks in mice fed a high-fat diet for 1 week. By 7 days, the phase of the liver rhythm was markedly advanced (by 5 h), whereas rhythms in other tissues were not affected. In addition, immediately upon consumption of a high-fat diet, the daily rhythm of eating behavior was altered. As the tissue rhythm of the suprachiasmatic nucleus was not affected by 1 week of high-fat diet consumption, the brain nuclei mediating the effect of a high-fat diet on eating behavior are likely to be downstream of the suprachiasmatic nucleus.
© 2013 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.
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9 MeSH Terms
Bioluminescence imaging of NADPH oxidase activity in different animal models.
Han W, Li H, Segal BH, Blackwell TS
(2012) J Vis Exp :
MeSH Terms: Animals, Female, Inflammation, Luminescent Measurements, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Animal, NADPH Oxidases, Reactive Oxygen Species
Show Abstract · Added March 5, 2014
NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response (1). Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47(phox), p67(phox), and p40(phox)) and Rac to a membrane-bound flavocytochrome (composed of a gp91(phox) and p22(phox) heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91(phox) -deficient mice and p47(phox)-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense (2, 13). In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase. Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion (14). Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species (15, 16). Kielland et al. (16) showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47(phox)-deficient mice. We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47(phox-/-) mice (2) in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47(phox-/-) mice enables us to delineate the specific contribution of ROS generated by p47(phox)-containing NADPH oxidase to the bioluminescent signal in these models. Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47(phox-/-) mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.
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11 MeSH Terms
Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue.
Innocenti F, Cooper GM, Stanaway IB, Gamazon ER, Smith JD, Mirkov S, Ramirez J, Liu W, Lin YS, Moloney C, Aldred SF, Trinklein ND, Schuetz E, Nickerson DA, Thummel KE, Rieder MJ, Rettie AE, Ratain MJ, Cox NJ, Brown CD
(2011) PLoS Genet 7: e1002078
MeSH Terms: 3' Untranslated Regions, African Continental Ancestry Group, Age Factors, Chromosome Mapping, Cloning, Molecular, European Continental Ancestry Group, Female, Gene Expression Profiling, Genetic Vectors, Genome, Human, Genome-Wide Association Study, Genotype, Hep G2 Cells, Hispanic Americans, Humans, Liver, Luminescent Measurements, Male, Polymorphism, Single Nucleotide, Principal Component Analysis, Promoter Regions, Genetic, Quantitative Trait Loci, Reproducibility of Results, Sex Factors, Transfection
Show Abstract · Added February 22, 2016
The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3'UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits.
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25 MeSH Terms
Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.
Pantel J, Williams SY, Mi D, Sebag J, Corbin JD, Weaver CD, Cone RD
(2011) Eur J Pharmacol 660: 139-47
MeSH Terms: Allosteric Regulation, Cyclic AMP, Drug Evaluation, Preclinical, Furocoumarins, HEK293 Cells, High-Throughput Screening Assays, Humans, Luminescent Measurements, Phosphodiesterase Inhibitors, Pilot Projects, Receptor, Melanocortin, Type 4, Reproducibility of Results, Signal Transduction, Structure-Activity Relationship, Sulfonamides, Time Factors
Show Abstract · Added December 10, 2013
The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β(2)AR agonists - the β(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.
Copyright © 2011 Elsevier B.V. All rights reserved.
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16 MeSH Terms