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https://www.readbyqxmd.com/read/28217801/nucleic-acid-detection-using-bret-beacons-based-on-bioluminescent-protein-dna-hybrids
#1
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.
February 20, 2017: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/28195704/highly-potent-cell-permeable-and-impermeable-nanoluc-luciferase-inhibitors
#2
Joel R Walker, Mary P Hall, Chad A Zimprich, Matt 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 42k PubChem compound library with low μM IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nM, cell permeable inhibitors...
February 14, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28183248/new-horizons-on-molecular-pharmacology-applied-to-drug-discovery-when-resonance-overcomes-radioligand-binding
#3
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
#4
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...
February 3, 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
#5
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
#6
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...
January 30, 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
#7
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...
January 20, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28087810/akt1-lkb1-and-yap1-revealed-as-myc-interactors-with-nanoluc-based-protein-fragment-complementation-assay
#8
Xiu-Lei 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. Here 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...
January 13, 2017: Molecular Pharmacology
https://www.readbyqxmd.com/read/28050273/flavoprotein-minisog-cytotoxisity-can-be-induced-by-bioluminescence-resonance-energy-transfer
#9
E I Shramova, G M Proshkina, S P Chumakov, Yu M Khodarovich, S M Deyev
In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line...
October 2016: Acta Naturae
https://www.readbyqxmd.com/read/27966527/five-colour-variants-of-bright-luminescent-protein-for-real-time-multicolour-bioimaging
#10
Kazushi Suzuki, Taichi Kimura, Hajime Shinoda, Guirong Bai, Matthew J Daniels, Yoshiyuki Arai, Masahiro Nakano, Takeharu Nagai
Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins...
December 14, 2016: Nature Communications
https://www.readbyqxmd.com/read/27919754/neutralization-takes-precedence-over-igg-or-iga-isotype-related-functions-in-mucosal-hiv-1-antibody-mediated-protection
#11
Rena D Astronomo, Sampa Santra, Lamar Ballweber-Fleming, Katharine G Westerberg, Linh Mach, Tiffany Hensley-McBain, Laura Sutherland, Benjamin Mildenberg, Georgeanna Morton, Nicole L Yates, Gregory J Mize, Justin Pollara, Florian Hladik, Christina Ochsenbauer, Thomas N Denny, Ranjit Warrier, Supachai Rerks-Ngarm, Punnee Pitisuttithum, Sorachai Nitayapan, Jaranit Kaewkungwal, Guido Ferrari, George M Shaw, Shi-Mao Xia, Hua-Xin Liao, David C Montefiori, Georgia D Tomaras, Barton F Haynes, Juliana M McElrath
HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching...
2016: EBioMedicine
https://www.readbyqxmd.com/read/27853830/exploiting-nanoluc-luciferase-for-smartphone-based-bioluminescence-cell-biosensor-for-anti-inflammatory-activity-and-toxicity
#12
Luca Cevenini, Maria Maddalena Calabretta, Antonia Lopreside, Giuseppe Tarantino, Annalisa Tassoni, Maura Ferri, Aldo Roda, Elisa Michelini
The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed...
December 2016: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/27786307/coupling-optogenetic-stimulation-with-nanoluc-based-luminescence-bret-ca-sensing
#13
Jie Yang, Derrick Cumberbatch, Samuel Centanni, Shu-Qun Shi, Danny Winder, Donna Webb, Carl Hirschie Johnson
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...
October 27, 2016: Nature Communications
https://www.readbyqxmd.com/read/27779402/detection-and-activity-profiling-of-synthetic-cannabinoids-and-their-metabolites-with-a-newly-developed-bioassay
#14
Annelies Cannaert, Jolien Storme, Florian Franz, Volker Auwärter, Christophe P Stove
Synthetic cannabinoids (SCs) are the largest group of compounds currently monitored in Europe by the EU Early Warning System on new psychoactive substances. Emerging recreational use of these products has led to multiple cases of adverse health effects and even death. In contrast to marijuana, where Δ(9)-tetrahydrocannabinol (Δ(9)THC) is metabolized to only one major active metabolite, it has been reported that several major phase I metabolites of SCs remain biologically active, exerting cannabinoid (CB) receptor affinity, potency, and efficacy greater than those of Δ(9)THC...
December 6, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/27687581/sod1-dimerization-monitoring-using-a-novel-split-nanoluc-nanobit
#15
Kentaro Oh-Hashi, Yoko Hirata, Kazutoshi Kiuchi
In the present study, we applied a highly sensitive NanoLuc-based technology to understand the status of superoxide dismutase 1 (SOD1) within mammalian cells. Two fragments of NanoLuc (NanoBit), large N-terminal and small C-terminal regions, were fused with wild-type (wt) and mutant human SOD1 (hSOD1) genes and transfected into cells. Luciferase activity through NanoBit assembly was only detected in NanoBit-tagged wtSOD1-expressing cells. Furthermore, the developed NanoLuc system was used to investigate the role of protein-protein interactions in the pathogenesis of amyotrophic lateral sclerosis (ALS)...
October 2016: Cell Biochemistry and Function
https://www.readbyqxmd.com/read/27624517/the-use-of-a-novel-nanoluc-based-reporter-phage-for-the-detection-of-escherichia-coli-o157-h7
#16
Dandan Zhang, Claudia P Coronel-Aguilera, Patricia L Romero, Lynda Perry, Udit Minocha, Carla Rosenfield, Andrew G Gehring, George C Paoli, Arun K Bhunia, Bruce Applegate
Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27588207/use-of-a-new-proximity-assay-nanobret-to-investigate-the-ligand-binding-characteristics-of-three-fluorescent-ligands-to-the-human-%C3%AE-1-adrenoceptor-expressed-in-hek-293-cells
#17
Mark Soave, Leigh A Stoddart, Alastair Brown, Jeanette Woolard, Stephen J Hill
Previous research has indicated that allosteric interactions across the dimer interface of β 1-adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pKi values at the human β 1-adenoceptor, which may result from such allosterism interactions. Three fluorescent β 1-adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor-bound fluorescent ligand and the N-terminal NanoLuc tag of a human β 1-adrenoceptor expressed in HEK 293 cells (NanoBRET)...
October 2016: Pharmacology Research & Perspectives
https://www.readbyqxmd.com/read/27547982/dual-readout-bret-fret-sensors-for-measuring-intracellular-zinc
#18
Stijn J A Aper, Pieterjan Dierickx, Maarten Merkx
Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn(2+). However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative...
October 21, 2016: ACS Chemical Biology
https://www.readbyqxmd.com/read/27518620/using-%C3%AE-lactamase-and-nanoluc-luciferase-reporter-gene-assays-to-identify-inhibitors-of-the-hif-1-signaling-pathway
#19
Thai Khuc, Chia-Wen Amy Hsu, Srilatha Sakamuru, Menghang Xia
The hypoxia-inducible factor 1 (HIF-1) is a transcriptional factor involved in the regulation of oxygen within cellular environments. In hypoxic tissues or those with inadequate oxygen concentrations, activation of the HIF-1 transcription factor allows for subsequent activation of target gene expression implicated in cell survival. As a result, cells proliferate through formation of new blood vessels and expansion of vascular systems, providing necessary nourishment needed of cells. HIF-1 is also involved in the complex pathophysiology associated with cancer cells...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27515429/application-of-nanoluc-to-monitor-the-intrinsic-promoter-activity-of-grp78-using-the-crispr-cas9-system
#20
Kentaro Oh-Hashi, Eri Furuta, Junpei Norisada, Fumimasa Amaya, Yoko Hirata, Kazutoshi Kiuchi
In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock-in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose-regulated protein 78 (GRP78) gene. The N-terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin-resistant gene through the 2A peptide sequence and used as a knock-in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc-based CRISPR/Cas9 system is a very useful tool to isolate gene-edited cells and to characterize the endogenous promoter activity for genes of interest...
October 2016: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
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