Journal articles: 'Reporter luciferase assay'

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Kenda, Maša, Jan Vegelj, Barbara Herlah, Andrej Perdih, Přemysl Mladěnka, and Marija Sollner Dolenc. "Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids." International Journal of Molecular Sciences 22, no. 13 (June 28, 2021): 6927. http://dx.doi.org/10.3390/ijms22136927.

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Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

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Siebring-van Olst, Ellen, Christie Vermeulen, Renee X. de Menezes, Michael Howell, Egbert F. Smit, and Victor W. van Beusechem. "Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening." Journal of Biomolecular Screening 18, no. 4 (October 30, 2012): 453–61. http://dx.doi.org/10.1177/1087057112465184.

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The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

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Hancock, Michael K., Myleen N. Medina, Brendan M. Smith, and Anthony P. Orth. "Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts." Journal of Biomolecular Screening 12, no. 1 (November 12, 2006): 140–44. http://dx.doi.org/10.1177/1087057106296046.

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Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

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Wu, Chun, Chie Suzuki-Ogoh, and Yoshihiro Ohmiya. "Dual-reporter assay using two secreted luciferase genes." BioTechniques 42, no. 3 (March 2007): 290–92. http://dx.doi.org/10.2144/000112428.

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Cheng, Zhijie, Denise Garvin, Aileen Paguio, Pete Stecha, Keith Wood, and Frank Fan. "Luciferase Reporter Assay System for Deciphering GPCR Pathways." Current Chemical Genomics 4 (December 21, 2010): 84–91. http://dx.doi.org/10.2174/1875397301004010084.

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Barriscale, Kathy A., Sharon A. O’Sullivan, and Tommie V. McCarthy. "A single secreted luciferase-based gene reporter assay." Analytical Biochemistry 453 (May 2014): 44–49. http://dx.doi.org/10.1016/j.ab.2014.02.019.

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Kolb, Alfred J., and Kenneth Neumann. "Luciferase Measurements in High Throughput Screening." Journal of Biomolecular Screening 1, no. 2 (March 1996): 85–88. http://dx.doi.org/10.1177/108705719600100207.

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Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

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Hannah, Rita R., Martha L. Jennens-Clough, and Keith V. Wood. "MipTec Proceedings: Transcriptional Assay Screens Using Renilla Luciferase as an Internal Control." JALA: Journal of the Association for Laboratory Automation 3, no. 4 (September 1998): 41–44. http://dx.doi.org/10.1177/221106829800300406.

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In cell biology research and pharmaceutical discovery, physiological responses of mammalian cells are commonly screened using transcriptional assays. Although firefly luciferase is widely used because of its rapid and simple assay, greater precision can be achieved using a second reporter as an internal control. Renilla luciferase serves as an efficient internal control because it can be measured as easily and rapidly using the same instrument. The Dual-Luciferase™ Reporter (DLR™) Assay developed at Promega measures both reporters sequentially within each sample, which eliminates the need to separate the test sample into aliquots for each assay. The expression of each reporter is independently quantitated using selective assay conditions based on their distinctive chemical characteristics. The firefly luciferase is initiated first by the addition of LAR II to the sample or cell lysate. Following measurement of the luminescent output, the firefly reaction is rapidly quenched and the Renilla reaction is simultaneously activated by addition of Stop & Glo™ Reagent, and the luminescence is measured a second time. The DLR™ Assay allows quantitation of both reporters within 4 seconds per well using a 96-well luminometer equipped with two reagent injectors. Multi-well plates may be processed even more rapidly using a CCD-based imaging system.

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Didiot, Marie-Cecile, Sergio Serafini, Martin J. Pfeifer, Frederick J. King, and Christian N. Parker. "Multiplexed Reporter Gene Assays: Monitoring the Cell Viability and the Compound Kinetics on Luciferase Activity." Journal of Biomolecular Screening 16, no. 7 (June 21, 2011): 786–93. http://dx.doi.org/10.1177/1087057111407768.

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High-throughput screening assays with multiple readouts enable one to monitor multiple assay parameters. By capturing as much information about the underlying biology as possible, the detection of true actives can be improved. This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth.

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Nieuwenhuijsen, Bart W., Youping Huang, Yuren Wang, Fernando Ramirez, Gary Kalgaonkar, and Kathleen H. Young. "A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions." Journal of Biomolecular Screening 8, no. 6 (December 2003): 676–84. http://dx.doi.org/10.1177/1087057103258287.

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To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GαZand RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40-to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

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Unterreiner, Vincent, Yvonne Ibig-Rehm, Marjo Simonen, Hanspeter Gubler, and Daniela Gabriel. "Comparison of Variability and Sensitivity between Nuclear Translocation and Luciferase Reporter Gene Assays." Journal of Biomolecular Screening 14, no. 1 (November 21, 2008): 59–65. http://dx.doi.org/10.1177/1087057108328016.

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High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non—image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC50 values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC50 values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities. ( Journal of Biomolecular Screening 2009:59-65)

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Rawson, Jonathan M. O., Alice Duchon, Olga A. Nikolaitchik, Vinay K. Pathak, and Wei-Shau Hu. "Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors." Viruses 13, no. 2 (January 24, 2021): 173. http://dx.doi.org/10.3390/v13020173.

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The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

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Liu, Xuemei, Jeffrey A. Kramer, Yi Hu, James M. Schmidt, Jianghong Jiang, and Alan G. E. Wilson. "Development of a High-Throughput Human HepG2 Dual Luciferase Assay for Detection of Metabolically Activated Hepatotoxicants and Genotoxicants." International Journal of Toxicology 28, no. 3 (May 2009): 162–76. http://dx.doi.org/10.1177/1091581809337166.

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Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45β reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG2 cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45β promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants.

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Ura, Seiji, Hiroshi Ueda, Jun Kazami, Genji Kawano, and Teruyuki Nagamune. "Single cell reporter assay using cell surface displayed Vargula luciferase." Journal of Bioscience and Bioengineering 92, no. 6 (January 2001): 575–79. http://dx.doi.org/10.1016/s1389-1723(01)80319-4.

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Yun, Chi, and Ramanuj DasGupta. "Luciferase Reporter Assay in Drosophila and Mammalian Tissue Culture Cells." Current Protocols in Chemical Biology 6, no. 1 (March 2014): 7–23. http://dx.doi.org/10.1002/9780470559277.ch130149.

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Martin, Chris S., Patricia A. Wight, Anna Dobretsova, and Irena Bronstein. "Dual Luminescence-Based Reporter Gene Assay for Luciferase andβ-Galactosidase." BioTechniques 21, no. 3 (September 1996): 520–24. http://dx.doi.org/10.2144/96213pf01.

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Tombácz, Kata, Duncan Mwangi, Dirk Werling, and Amanda J. Gibson. "Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle." Innate Immunity 23, no. 4 (February 27, 2017): 329–35. http://dx.doi.org/10.1177/1753425917695445.

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PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

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George, Samantha E., Peter J. Bungay, and Louise H. Naylor. "Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation." Journal of Biomolecular Screening 2, no. 4 (June 1997): 235–40. http://dx.doi.org/10.1177/108705719700200408.

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A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

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Bronstein, I., C. S. Martin, J. J. Fortin, C. E. Olesen, and J. C. Voyta. "Chemiluminescence: sensitive detection technology for reporter gene assays." Clinical Chemistry 42, no. 9 (September 1, 1996): 1542–46. http://dx.doi.org/10.1093/clinchem/42.9.1542.

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Abstract A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.

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Pulkina, A. A., E. A. Romanovskaya-Romanko, A. S. Mustafaeva, A. Yu Egorov, and M. A. Stukova. "Rapid Neutralizing Antibody Assessment Using Influenza Viruses with Luciferase Reporter." Biotekhnologiya 37, no. 2 (2021): 81–91. http://dx.doi.org/10.21519/0234-2758-2021-37-2-81-91.

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Influenza viruses cause an acute respiratory infection, especially in the autumn-winter period. They are characterized by a high mutation frequency and cause annual seasonal epidemics. The detection of virus-neutralizing antibodies is an important criterion in the assessment of population immunity and the influenza vaccine effectiveness. In this study, a method for determining the titer of virus-neutralizing antibodies in blood serum has been developed. A new test, called the luciferase neutralization assay, uses measurement of a bioluminescent signal as a detection method. The influenza A reporter viruses of various subtypes were constructed that encode the nanoluciferase protein in the non-structural NS1 protein reading frame. The developed method was used to compare paired sera of volunteers before and after their immunization with a seasonal influenza vaccine. The proposed method was also compared with certified antibody assay methods: neutralization reaction and hemagglutination inhibition reaction. The tests showed a high correlation, while the luciferase neutralization assay reduced the time and simplified the detection procedure. microneutralization, reporter virus, influenza virus, bioluminescence, nanoluciferase The study was supported by the grant of the President of the Russian Federation for young PhDs (no. 075-15-2019-226); and also by the grant of the Government of Saint-Petersburg for undergraduate and graduate students (September 25, 2018, no 124).

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Sun, Yinglu, Jinfeng Zhao, Suyash Bhimgonda Patil, Jingjing Fang, Jun Liu, and Xueyong Li. "Improved dual luciferase reporter (DLR) assay to determine the protein stability." Analytical Biochemistry 612 (January 2021): 114021. http://dx.doi.org/10.1016/j.ab.2020.114021.

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Dyer, Benjamin W., Fernando A. Ferrer, Donna K. Klinedinst, and Ronald Rodriguez. "A Noncommercial Dual Luciferase Enzyme Assay System for Reporter Gene Analysis." Analytical Biochemistry 282, no. 1 (June 2000): 158–61. http://dx.doi.org/10.1006/abio.2000.4605.

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Zhang, Huateng, Haixiu Bai, Tianyu Jiang, Zhao Ma, Yanna Cheng, Yubin Zhou, Lupei Du, and Minyong Li. "Quenching the firefly bioluminescence by various ions." Photochemical & Photobiological Sciences 15, no. 2 (2016): 244–49. http://dx.doi.org/10.1039/c5pp00432b.

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McNabb, David S., Robin Reed, and Robert A. Marciniak. "Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces cerevisiae." Eukaryotic Cell 4, no. 9 (September 2005): 1539–49. http://dx.doi.org/10.1128/ec.4.9.1539-1549.2005.

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ABSTRACT A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.

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Ji, Changhua, Jun Zhang, Nick Cammack, and Surya Sankuratri. "Development of a Novel Dual CCR5-Dependent and CXCR4-Dependent Cell-Cell Fusion Assay System with Inducible gp160 Expression." Journal of Biomolecular Screening 11, no. 1 (October 18, 2005): 65–74. http://dx.doi.org/10.1177/1087057105282959.

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In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC 50data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSOon this assay systemwere assessed, and a 2-to 3-fold increase in luciferase activitywas observed in the presence of 0.05% to2% DMSO. Although cell-cell fusion efficiencywas enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.

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Mudge, SR, WR Lewis-Henderson, and RG Birch. "Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants." Functional Plant Biology 23, no. 1 (1996): 75. http://dx.doi.org/10.1071/pp9960075.

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Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

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Anbarasu, Sivaraj. "Luciferase Reporter Phage Assay for Anti Tuberculosis Screening: Current Status and Challenges." Bioscience Biotechnology Research Communications 13, no. 3 (August 25, 2020): 1236–44. http://dx.doi.org/10.21786/bbrc/13.3/39.

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Chen, Feng, Zhen Xu, Jing Lu, Xiang Lü, Wen-li Mu, Ya-jun Wang, De-pei Liu, and Chih-chuan Liang. "Gaussia Luciferase Reporter Assay for Assessment of Gene Delivery Systems in Vivo." Chinese Medical Sciences Journal 25, no. 2 (June 2010): 95–99. http://dx.doi.org/10.1016/s1001-9294(10)60029-6.

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Connelly, Colleen M., Meryl Thomas, and Alexander Deiters. "High-Throughput Luciferase Reporter Assay for Small-Molecule Inhibitors of MicroRNA Function." Journal of Biomolecular Screening 17, no. 6 (March 12, 2012): 822–28. http://dx.doi.org/10.1177/1087057112439606.

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MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3′ untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.

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Ciulla, Daniel, Rebecca Mancusi, Alexandra Psaras, Sahib Ghotra, and Brian Callahan. "Improved Sonic Hedgehog Protein Autoprocessing Assay in Cells Using Luciferase Reporter System." FASEB Journal 34, S1 (April 2020): 1. http://dx.doi.org/10.1096/fasebj.2020.34.s1.01785.

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Comley, John C. W., Tony Reeves, and Phil Robinson. "A 1536 Colorimetric SPAP Reporter Assay: Comparison with 96- and 384-Well Formats." Journal of Biomolecular Screening 3, no. 3 (April 1998): 217–25. http://dx.doi.org/10.1177/108705719800300308.

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We report the successful miniaturization of a functional cell-based reporter gene assay. Utilizing interleukin-1beta (IL-1/β)-induced secreted placental alkaline phosphatase (SPAP)-catalyzed colorimetric readout, we reduced the assay volume to 10 μl using a Greiner 1536-well microplate. Our experiences of assay development, liquid handling (using a Hydra® 96; Robbins Scientific, Sunnyvale, CA), and detection (using the SpectraImage and SpectraFluor-Plus plate readers, Tecan Austria GmbH, Grodig, Austria) in 1536 wells are discussed. The effect of a set of 1,280 compounds in this SPAP reporter assay were compared between 96-, 384-, and 1536-well formats and were shown to be very similar. We conclude that cell-based reporter gene assays using SPAP-catalyzed color readouts are sensitive and highly reproducible in 1536-well plates and should be considered as a cost-effective alternative to luciferase reporters for miniaturized assay formats. Finally, we review the prospects for the implementation of routine HTS in 1536-well plates based around the instrumentation investigated.

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Yu, Lei, Chuncui Jia, Wenrong Yao, Dening Pei, Xi Qin, Chunming Rao, and Junzhi Wang. "Development and Validation of a Reporter-Cell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc." Molecules 24, no. 21 (October 25, 2019): 3845. http://dx.doi.org/10.3390/molecules24213845.

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Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc). Sgp130-Fc could inhibit the expression of luciferase induced by IL-6/sIL-6Rα complex, and the dose–response curve fitted the four-parameter logistic model, with 50% inhibitive concentration (IC50) being about 500 ng/mL and detection range between 40 and 5000 ng/mL. Both the intra-assay and inter-assay coefficient of variation (CV) were below 10.0%, and the accuracy estimates ranged from 94.1% to 106.2%. The assay indicated a good linearity (R² = 0.99) in the range of 50% to 150% of optimized initial concentration. No significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay (unpaired t test, p = 0.4960, n = 6). The dose-response effect and copy number of the luciferase gene was basically unchanged after long-term culture (up to passage 60), demonstrating the stability of CHO/SIE-Luc cells. These results suggested that the new reporter assay was suited to routine potency determination of therapeutic sgp130-Fc.

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Skarpidi, Evangelia, George Vassilopoulos, Qiliang Li, and George Stamatoyannopoulos. "Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin." Blood 96, no. 1 (July 1, 2000): 321–26. http://dx.doi.org/10.1182/blood.v96.1.321.

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Abstract Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

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Skarpidi, Evangelia, George Vassilopoulos, Qiliang Li, and George Stamatoyannopoulos. "Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin." Blood 96, no. 1 (July 1, 2000): 321–26. http://dx.doi.org/10.1182/blood.v96.1.321.013k48_321_326.

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Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.

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Jung, Soo Bin, Chae young Lee, Kwang-Ho Lee, Kyu Heo, and Si Ho Choi. "A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency." Nucleic Acids Research 49, no. 15 (June 4, 2021): e85-e85. http://dx.doi.org/10.1093/nar/gkab467.

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Abstract CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.

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Omokoko, Tana A., Uli Luxemburger, Shaheer Bardissi, Petra Simon, Magdalena Utsch, Andrea Breitkreuz, Özlem Türeci, and Ugur Sahin. "Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity." Journal of Immunology Research 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/9540975.

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Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughputin vitrocytotoxicity assays that efficiently analyze immune effector functions. The gold standard51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferasein vitrotranscribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the51Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

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Xin, Lili, Jianshu Wang, Guoqiang Fan, Bizhong Che, Kaiming Cheng, and Guangzhu Dong. "Comparative oxidative stress elicited by nanosilver in stable HSPA1A promoter-driven luciferase reporter HepG2 and A549 cells." Toxicology Research 5, no. 5 (2016): 1298–305. http://dx.doi.org/10.1039/c6tx00195e.

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Kolb, Alfred J., and Kenneth Neumann. "Beyond the 96-Well Microplate: Instruments and Assay Methods for the 384-Well Format." Journal of Biomolecular Screening 2, no. 2 (March 1997): 103–9. http://dx.doi.org/10.1177/108705719700200209.

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A new microplate scintillation and luminescence counter (TopCount™ HTS, Packard) has been developed for counting both 96- and 384-well microplates. The first assay methods tested in the 384-well plate were the scintillation proximity assay and the luciferase reporter gene assay. The binding curve and luciferase induction were identical in either plate format. The signal-to-noise ratio (maximum binding divided by nonspecific binding) in the 384-well plate is equal to or greater than those measured in the 96-well format resulting in a comparable assay window. Reagent pipetting for SPA, including the scintillating beads, was performed with the MultiPROBETm 104 Automated Liquid Handling System (Packard, Meridan, CT). This instrument has both the positioning and pipetting accuracy to support the 384-well microplate; therefore, making complete automation practical. With little or no optimization, both the SPA and the luciferase reporter gene assay could be miniaturized to 50 ,Il volumes or less.

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Terstappen, G. C., A. Giacometti, E. Ballini, and L. Aldegheri. "Development of a Functional Reporter Gene HTS Assay for the Identification of mGluR7 Modulators." Journal of Biomolecular Screening 5, no. 4 (June 2000): 255–61. http://dx.doi.org/10.1177/108705710000500408.

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For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC50 of 0-9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS.

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Hsieh, Ya-Ju, Luen Hwu, Chien-Chih Ke, Skye Hsin-Hsien Yeh, Chien-Feng Lin, Fu-Du Chen, Hsin-Ell Wang, Kang-Ping Lin, Ran-Chou Chen, and Ren-Shyan Liu. "Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging." BioMed Research International 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/605358.

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Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase(fl), monomeric red fluorescence protein(mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant(ttksr39)were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro andin vivowere determined by luciferase reporter assay,H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels ofH-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak forin vivoimaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter constructDsRedm-fl-ttksr39for more effective and sensitivein vivoanimal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.

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Murray, Robert W., Earline P. Melchior, Jeanne C. Hagadorn, and Keith R. Marotti. "Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors." Antimicrobial Agents and Chemotherapy 45, no. 6 (June 1, 2001): 1900–1904. http://dx.doi.org/10.1128/aac.45.6.1900-1904.2001.

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ABSTRACT The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extractS. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system.

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Larocque, Louise, Alex Bliu, Ranran Xu, Abebaw Diress, Junzhi Wang, Rongtuan Lin, Runtao He, Michel Girard, and Xuguang Li. "Bioactivity Determination of Native and Variant Forms of Therapeutic Interferons." Journal of Biomedicine and Biotechnology 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/174615.

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The traditional antiviral assays for the determination of interferon potency are reported to have considerable variability between and within assays. Although several reporter gene assays based on interferon-inducible promoter activities have been reported, data from comprehensive validation studies are lacking and few studies have been conducted to analyze the variant forms of interferons, which could have undesirable clinical implications. Here, a reporter gene assay employing a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter was developed and validated. The assay was found to be more sensitive, with a larger detection range than the antiviral assay. Several cytokines tested did not interfere with the test, suggesting the assay possesses a certain degree of selectivity. Moreover, the robustness of the assay was demonstrated by minimal variations in the results generated by different analysts and cell passage number (up to 52 passages). Finally, the method was employed to analyze several interferon variants (interferon-α2a) and we found that the aggregated form has completely lost its potency; while a modest loss of bioactivity in oxidized interferon was observed (approx. 23%), the deamidated form essentially retained its activity.

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Conway, Shaun, Sarah J. Canning, H. Edward Howell, Elaine S. Mowat, Perry Barrett, Janice E. Drew, Phillipe Delagrange, Daniel Lesieur, and Peter J. Morgan. "Characterisation of human melatonin mt1 and MT2 receptors by CRE-luciferase reporter assay." European Journal of Pharmacology 390, no. 1-2 (February 2000): 15–24. http://dx.doi.org/10.1016/s0014-2999(99)00914-0.

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SHIRASUGI, Ichiro, Yoichi SAKAKIBARA, Masao YAMASAKI, Kazuo NISHIYAMA, Takashi MATSUI, Ming-Cheh LIU, and Masahito SUIKO. "Novel Screening Method for Potential Skin-Whitening Compounds by a Luciferase Reporter Assay." Bioscience, Biotechnology, and Biochemistry 74, no. 11 (November 23, 2010): 2253–58. http://dx.doi.org/10.1271/bbb.100466.

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Wider, Diana, and Didier Picard. "Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry." PLOS ONE 12, no. 12 (December 8, 2017): e0189403. http://dx.doi.org/10.1371/journal.pone.0189403.

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Tannous, Bakhos A. "Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo." Nature Protocols 4, no. 4 (April 2009): 582–91. http://dx.doi.org/10.1038/nprot.2009.28.

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Shi, Yihui, Jaehyeon Park, Chandraiah Lagisetti, Wei Zhou, Lidia C. Sambucetti, and Thomas R. Webb. "A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore." Bioorganic & Medicinal Chemistry Letters 27, no. 3 (February 2017): 406–12. http://dx.doi.org/10.1016/j.bmcl.2016.12.056.

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Smirnova, N. A., A. I. Osipyants, A. Yu Khristichenko, D. M. Hushpulian, S. V. Nikulin, T. A. Chubar, A. A. Zakhariants, V. I. Tishkov, I. G. Gazaryan, and A. A. Poloznikov. "HIF2 ODD-luciferase reporter: the most sensitive assay for HIF prolyl hydroxylase inhibitors." Russian Chemical Bulletin 67, no. 1 (January 2018): 150–56. http://dx.doi.org/10.1007/s11172-018-2051-5.

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Yasunaga, Mayu, Kazutoshi Murotomi, Hiroko Abe, Tomomi Yamazaki, Shigeaki Nishii, Tetsuya Ohbayashi, Mitsuo Oshimura, et al. "Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3." Journal of Biotechnology 194 (January 2015): 115–23. http://dx.doi.org/10.1016/j.jbiotec.2014.12.004.

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Pathe-Neuschäfer-Rube, Andrea, Frank Neuschäfer-Rube, and Gerhard P. Püschel. "Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals." Toxins 13, no. 4 (March 30, 2021): 247. http://dx.doi.org/10.3390/toxins13040247.

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The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.

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Journal articles on the topic 'Reporter luciferase assay'

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