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https://www.readbyqxmd.com/read/28771321/activity-based-detection-of-consumption-of-synthetic-cannabinoids-in-authentic-urine-samples-using-a-stable-cannabinoid-reporter-system
#1
Annelies Cannaert, Florian Franz, Volker Auwärter, Christophe P Stove
Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for 'unknown' compounds...
August 3, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28768857/molecular-function-analysis-of-rabies-virus-rna-polymerase-l-protein-by-using-an-l-gene-deficient-virus
#2
Kento Nakagawa, Yuki Kobayashi, Naoto Ito, Yoshiyuki Suzuki, Kazuma Okada, Machiko Makino, Hideo Goto, Tatsuki Takahashi, Makoto Sugiyama
While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells [20013240291] transfected to express the L protein...
August 2, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28733215/development-of-nanoluc-bridging-immunoassay-for-detection-of-anti-drug-antibodies
#3
Nidhi Nath, Rod Flemming, Becky Godat, Marjeta Urh
Anti-drug antibodies (ADAs) are generated in-vivo as an immune response to therapeutic antibody drugs and can significantly affect the efficacy and safety of the drugs. Hence, detection of ADAs is recommended by regulatory agencies during drug development process. A widely accepted method for measuring ADAs is "bridging" immunoassay and is frequently performed using enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence (ECL) platform developed by Meso Scale Discovery (MSD). ELISA is preferable due to widely available reagents and instruments and broad familiarity with the technology; however, MSD platform has gained wide acceptability due to a simpler workflow, higher sensitivity, and a broad dynamic range but requires proprietary reagents and instruments...
July 18, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28726090/flavoprotein-minisog-bret-induced-cytotoxicity-depends-on-its-intracellular-localization
#4
E I Shramova, G M Proshkina, S M Deyev, R V Petrov
It is proposed to use the bioluminescent resonance energy transfer to solve the problem of creating the internal light sources in photodynamic therapy of cancer. Energy donor in the developed system is the oxidized form of the luciferase NanoLuc substrate furimamide, and acceptor is the phototoxic fluorescent protein miniSOG. It is shown that, in the proposed system, the photoinduced cytotoxicity of flavoprotein miniSOG in vitro depends on the intracellular localization, and the cytotoxic effect is 48% for the cytoplasmic localization of the fusion protein, 65% for the mitochondrial localization, and 69% for the membrane localization...
May 2017: Doklady. Biochemistry and Biophysics
https://www.readbyqxmd.com/read/28670623/bright-bioluminescent-bret-sensor-proteins-for-measuring-intracellular-caspase-activity
#5
Anniek den Hamer, Pieterjan Dierickx, Remco Arts, Joost S P M de Vries, Luc Brunsveld, Maarten Merkx
FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination...
June 23, 2017: ACS Sensors
https://www.readbyqxmd.com/read/28631012/rapid-preparation-of-bioluminescent-tracers-for-relaxin-family-peptides-using-sortase-catalysed-ligation
#6
Jia-Hui Wang, Xiao-Xia Shao, Meng-Jun Hu, Dian Wei, Wei-Han Nie, Ya-Li Liu, Zeng-Guang Xu, Zhan-Yun Guo
Relaxin family is a group of peptide hormones with a variety of biological functions by activating G protein-coupled receptors RXFP1-4. We recently developed bioluminescent tracers for their receptor-binding assays by chemical conjugation with the ultrasensitive NanoLuc reporter. To simplify preparation of the bioluminescent tracers, in the present study, we established a sortase-catalysed ligation approach using the chimeric R3/I5 as a model. Following catalysis by recombinant sortase A, a NanoLuc reporter carrying the LPETG sortase recognition motif at the C-terminus was efficiently ligated to an R3/I5 peptide carrying four successive Gly residues at the A-chain N-terminus, via the formation of a peptide bond between the C-terminal LPET sequence of NanoLuc and the A-chain N-terminal Gly residue of R3/I5...
June 19, 2017: Amino Acids
https://www.readbyqxmd.com/read/28624460/identification-of-kx2-391-as-an-inhibitor-of-hbv-transcription-by-a-recombinant-hbv-based-screening-assay
#7
Keisuke Harada, Hironori Nishitsuji, Saneyuki Ujino, Kunitada Shimotohno
Antiviral therapies for chronic hepatitis B virus (HBV) infection that are currently applicable for clinical use are limited to nucleos(t)ide analogs targeting HBV polymerase activity and pegylated interferon alpha (PEG-IFN). Towards establishing an effective therapy for HBV related diseases, it is important to develop a new anti-HBV agent that suppresses and eradicates HBV. This study used recombinant HBV encoding NanoLuc to screen anti-HBV compounds from 1827 US Food and Drug Administration approved compounds and identified several compounds that suppressed HBV infection...
August 2017: Antiviral Research
https://www.readbyqxmd.com/read/28521201/validation-of-the-use-of-an-artificial-mitochondrial-reporter-dna-vector-containing-a-cytomegalovirus-promoter-for-mitochondrial-transgene-expression
#8
Yuma Yamada, Takuya Ishikawa, Hideyoshi Harashima
Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system...
May 10, 2017: Biomaterials
https://www.readbyqxmd.com/read/28510347/bioluminescent-antibodies-for-point-of-care-diagnostics
#9
Lin Xue, Qiuliyang Yu, Rudolf Griss, Alberto Schena, Kai Johnsson
We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore...
June 12, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28392095/real-time-analysis-of-the-binding-of-fluorescent-vegf165a-to-vegfr2-in-living-cells-effect-of-receptor-tyrosine-kinase-inhibitors-and-fate-of-internalized-agonist-receptor-complexes
#10
Laura E Kilpatrick, Rachel Friedman-Ohana, Diana C Alcobia, Kristin Riching, Chloe J Peach, Amanda J Wheal, Stephen J Briddon, Matthew B Robers, Kris Zimmerman, Thomas Machleidt, Keith V Wood, Jeanette Woolard, Stephen J Hill
Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF165a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF165a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET)...
July 15, 2017: Biochemical Pharmacology
https://www.readbyqxmd.com/read/28374838/a-novel-tool-for-monitoring-endogenous-alpha-synuclein-transcription-by-nanoluciferase-tag-insertion-at-the-3-end-using-crispr-cas9-genome-editing-technique
#11
Sambuddha Basu, Levi Adams, Subhrangshu Guhathakurta, Yoon-Seong Kim
α-synuclein (α-SYN) is a major pathologic contributor to Parkinson's disease (PD). Multiplication of α-SYN encoding gene (SNCA) is correlated with early onset of the disease underlining the significance of its transcriptional regulation. Thus, monitoring endogenous transcription of SNCA is of utmost importance to understand PD pathology. We developed a stable cell line expressing α-SYN endogenously tagged with NanoLuc luciferase reporter using CRISPR/Cas9-mediated genome editing. This allows efficient measurement of transcriptional activity of α-SYN in its native epigenetic landscape which is not achievable using exogenous transfection-based luciferase reporter assays...
April 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28336075/engineering-bret-sensor-proteins
#12
Remco Arts, Stijn J A Aper, Maarten Merkx
FRET-sensors have become important tools for intracellular imaging, but their dependence on external illumination presents some limitations, such as photobleaching and phototoxicity, which limit measurements over extended periods of time. Fluorescence measurements also suffer from autofluorescence and light scattering, which hampers in vivo imaging and measurements in strongly absorbing and scattering media such as blood. In principle, these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28217801/nucleic-acid-detection-using-bret-beacons-based-on-bioluminescent-protein-dna-hybrids
#13
Wouter Engelen, Kayleigh M van de Wiel, Lenny H H Meijer, Bedabrata Saha, Maarten Merkx
Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.
March 2, 2017: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/28195704/highly-potent-cell-permeable-and-impermeable-nanoluc-luciferase-inhibitors
#14
Joel R Walker, Mary P Hall, Chad A Zimprich, Matthew B Robers, Sarah J Duellman, Thomas Machleidt, Jacquelynn Rodriguez, Wenhui Zhou
Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors...
April 21, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28183248/new-horizons-on-molecular-pharmacology-applied-to-drug-discovery-when-resonance-overcomes-radioligand-binding
#15
Larissa Pernomian, Mayara Santos Gomes, Josimar Moreira Dornelas, Carlos Henrique Tomich de Paula da Silva, Jaoaquín María Campos Rosa, Cristina Ribeiro De Barros Cardoso
One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G protein-coupled receptors (GPCR)...
February 8, 2017: Current Radiopharmaceuticals
https://www.readbyqxmd.com/read/28167276/detection-and-monitoring-pla2r-autoantibodies-by-lips-in-membranous-nephropathy
#16
COMPARATIVE STUDY
Peter D Burbelo, Laurence H Beck, Meryl Waldman
Autoantibodies against the M-type phospholipase A2 receptor (PLA2R) are specific markers for primary membranous nephropathy (MN). Quantification of PLA2R autoantibodies is an important, noninvasive tool that facilitates the diagnosis and monitoring of primary MN. In this report we describe a highly quantitative luciferase immunoprecipitation systems (LIPS) assay for detecting PLA2R autoantibodies. For these studies, a cDNA fragment encoding the first 858 amino acids of PLA2R protein was cloned to generate N-terminal antigen fusion constructs with Gaussia luciferase (Gluc) and Nano luciferase (NanoLuc) reporters...
May 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28161795/a-novel-bret-based-binding-assay-for-interaction-studies-of-relaxin-family-peptide-receptor-3-with-its-ligands
#17
Jia-Hui Wang, Xiao-Xia Shao, Meng-Jun Hu, Dian Wei, Ya-Li Liu, Zeng-Guang Xu, Zhan-Yun Guo
Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage...
February 4, 2017: Amino Acids
https://www.readbyqxmd.com/read/28137864/entirely-plasmid-based-reverse-genetics-system-for-rotaviruses
#18
Yuta Kanai, Satoshi Komoto, Takahiro Kawagishi, Ryotaro Nouda, Naoko Nagasawa, Misa Onishi, Yoshiharu Matsuura, Koki Taniguchi, Takeshi Kobayashi
Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme...
February 28, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28115169/development-of-a-bimolecular-luminescence-complementation-assay-for-rgs-g-protein-interactions-in-cells
#19
Christopher R Bodle, Michael P Hayes, Joseph B O'Brien, David L Roman
Cell based assessment tools and screening platforms are the preferred paradigm for small molecule identification and validation due to selectively identifying molecules with cellular activity and validation of compound activity against target proteins in their native environment. With respect to Regulator of G Protein Signaling (RGS) proteins, current cell based methodologies are either low throughput or monitor downstream signaling consequences. The increasing number of reports indicating RGS function in various disease pathogeneses highlights the need for a robust RGS inhibitor discovery and characterization paradigm...
April 1, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28087810/akt1-lkb1-and-yap1-revealed-as-myc-interactors-with-nanoluc-based-protein-fragment-complementation-assay
#20
Xiulei Mo, Qi Qi, Andrei A Ivanov, Qiankun Niu, Yin Luo, Jonathan Havel, Russell Goetze, Sydney Bell, Carlos S Moreno, Lee A D Cooper, Margaret A Johns, Fadlo R Khuri, Yuhong Du, Haian Fu
The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2...
April 2017: Molecular Pharmacology
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