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https://www.readbyqxmd.com/read/29601823/cholesterol-modulates-the-binding-properties-of-human-relaxin-family-peptide-receptor-3-with-its-ligands
#1
Jia-Hui Wang, Meng-Jun Hu, Xiao-Xia Shao, Dian Wei, Ya-Li Liu, Zeng-Guang Xu, Zhan-Yun Guo
Relaxin family peptide receptor 3 (RXFP3) is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. As an A-class G protein-coupled receptor, RXFP3 is an integral plasma membrane protein with seven transmembrane domains, yet influence of the membrane lipids on its function remains unknown. In the present study, we disclosed that cholesterol, an essential membrane lipid for mammalian cells, modulated the binding properties of human RXFP3 with its ligands...
March 27, 2018: Archives of Biochemistry and Biophysics
https://www.readbyqxmd.com/read/29526710/a-split-luciferase-based-trimer-formation-assay-as-a-high-throughput-screening-platform-for-therapeutics-in-alport-syndrome
#2
Kohei Omachi, Misato Kamura, Keisuke Teramoto, Haruka Kojima, Tsubasa Yokota, Shota Kaseda, Jun Kuwazuru, Ryosuke Fukuda, Kosuke Koyama, Shingo Matsuyama, Keishi Motomura, Tsuyoshi Shuto, Mary Ann Suico, Hirofumi Kai
Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3-α5 chains (α3-α5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of α3/α4/α5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular α(IV) chain and the absence of high-throughput screening (HTS) platforms to assess α345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion α345(IV) proteins to monitor α345(IV) trimerization of wild-type and clinically associated mutant α5(IV)...
February 21, 2018: Cell Chemical Biology
https://www.readbyqxmd.com/read/29523687/mini-g-protein-probes-for-active-g-protein-coupled-receptors-gpcrs-in-live-cells
#3
Qingwen Wan, Najeah Okashah, Asuka Inoue, Rony Nehmé, Byron Carpenter, Christopher G Tate, Nevin A Lambert
G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefitted greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active state GPCRs...
March 9, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29497379/molecular-evidence-of-adenosine-deaminase-linking-adenosine-a-2a-receptor-and-cd26-proteins
#4
Estefanía Moreno, Júlia Canet, Eduard Gracia, Carme Lluís, Josefa Mallol, Enric I Canela, Antoni Cortés, Vicent Casadó
Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans...
2018: Frontiers in Pharmacology
https://www.readbyqxmd.com/read/29476720/a-luciferase-immunoprecipitation-assay-for-the-detection-of-proinsulin-insulin-autoantibodies
#5
Yun Ling, Pengjun Jiang, Nan Li, Qianhua Yan, Xin Wang
AIM: Luciferase immunoprecipitation (LIPS) assays show good sensitivity and specificity in testing islet specific autoantibodies for diagnosis of type 1 diabetes (T1D). However, there are currently no LIPS assays available for detecting proinsulin/insulin autoantibody (IAA) previously. We here developed a LIPS assay to measure IAA using nano luciferase (NanoLuc)-proinsulin fusion protein. METHODS: The NanoLuc-proinsulin fusion protein expression plasmid was constructed and transfected to COS1 cells...
February 21, 2018: Clinical Biochemistry
https://www.readbyqxmd.com/read/29463029/nanoluciferase-reporter-gene-system-directed-by-tandemly-repeated-pseudo-palindromic-nfat-response-elements-facilitates-analysis-of-biological-endpoint-effects-of-cellular-ca-2-mobilization
#6
Wei Zhang, Terunao Takahara, Takuya Achiha, Hideki Shibata, Masatoshi Maki
NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+ /calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+ -response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+ -induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization...
February 18, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29446920/homogeneous-noncompetitive-luminescent-immunodetection-of-small-molecules-by-ternary-protein-fragment-complementation
#7
Yuki Ohmuro-Matsuyama, Hiroshi Ueda
The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) in vitro. Since the detection of antigen-induced approximation of the two antibody variable region fragments V H and V L by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two C-terminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which V H and V L were each fused and expressed in Escherichia coli cells...
February 15, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29408532/mitochondrial-transgene-expression-via-an-artificial-mitochondrial-dna-vector-in-cells-from-a-patient-with-a-mitochondrial-disease
#8
Takuya Ishikawa, Kana Somiya, Reina Munechika, Hideyoshi Harashima, Yuma Yamada
To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV-mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO-Porter, a liposome-based carrier for delivering a cargo to mitochondria via membrane fusion...
March 28, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/29379384/construction-and-cloning-of-reporter-tagged-replicon-cdna-for-an-in-vitro-replication-study-of-murine-norovirus-1-mnv-1
#9
Muhammad Khairi Ahmad, Yasser M Tabana, Mowaffaq Adam Ahmed, Doblin Anak Sandai, Rafeezul Mohamed, Ida Shazrina Ismail, Nurulisa Zulkiflie, Muhammad Amir Yunus
Background: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression...
December 2017: Malaysian Journal of Medical Sciences: MJMS
https://www.readbyqxmd.com/read/29371669/development-of-novel-fluorescent-histamine-h1-receptor-antagonists-to-study-ligand-binding-kinetics-in-living-cells
#10
Leigh A Stoddart, Andrea J Vernall, Monica Bouzo-Lorenzo, Reggie Bosma, Albert J Kooistra, Chris de Graaf, Henry F Vischer, Rob Leurs, Stephen J Briddon, Barrie Kellam, Stephen J Hill
The histamine H1-receptor (H1R) is an important mediator of allergy and inflammation. H1R antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor H1R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor...
January 25, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29352444/activation-of-the-anti-aging-and-cognition-enhancing-gene-klotho-by-crispr-dcas9-transcriptional-effector-complex
#11
Ci-Di Chen, Ella Zeldich, Yuexuan Li, Andrea Yuste, Carmela R Abraham
Multiple lines of evidence show that the anti-aging and cognition-enhancing protein Klotho fosters neuronal survival, increases the anti-oxidative stress defense, and promotes remyelination of demyelinated axons. Thus, upregulation of the Klotho gene can potentially alleviate the symptoms and/or prevent the progression of age-associated neurodegenerative diseases such as Alzheimer's disease and demyelinating diseases such as multiple sclerosis. Here we used a CRISPR-dCas9 complex to investigate single-guide RNA (sgRNA) targeting the Klotho promoter region for efficient transcriptional activation of the Klotho gene...
February 2018: Journal of Molecular Neuroscience: MN
https://www.readbyqxmd.com/read/29351998/detection-of-experimental-and-clinical-immune-complexes-by-measuring-ship-1-recruitment-to-the-inhibitory-fc%C3%AE-riib
#12
Richard J Stopforth, Robert J Oldham, Alison L Tutt, Patrick Duriez, H T Claude Chan, Brock F Binkowski, Chad Zimprich, Dun Li, Philip G Hargreaves, Mei Cong, Venkat Reddy, Maria J Leandro, Geraldine Cambridge, Anja Lux, Falk Nimmerjahn, Mark S Cragg
Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor...
March 1, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/29305766/a-novel-lips-assay-for-insulin-autoantibodies
#13
Daniela Liberati, Rebecca C Wyatt, Cristina Brigatti, Ilaria Marzinotto, Maurizio Ferrari, Elena Bazzigaluppi, Emanuele Bosi, Ben T Gillard, Kathleen M Gillespie, Frans Gorus, Ilse Weets, Eric Balti, Lorenzo Piemonti, Peter Achenbach, Alistair J K Williams, Vito Lampasona
AIMS: Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. METHODS: We developed (pro)insulin antigens with alternative placements of a NanoLuc™ luciferase reporter (NLuc)...
March 2018: Acta Diabetologica
https://www.readbyqxmd.com/read/29280616/recombinant-peptidomimetic-nano-luciferase-tracers-for-sensitive-single-step-immunodetection-of-small-molecules
#14
Yuan Ding, Xiude Hua, He Chen, Fengquan Liu, Gualberto González-Sapien, Minghua Wang
Phage borne peptides isolated from phage libraries have proven to be valuable reagents for the development of small-molecule immunoassays. However, the large size, low diffusion rate, and biological nature of the phage particles create some limitations to their use and require secondary reagents for its detection. In this work, we explore the use of the Nano luciferase (NanoLuc) as a fusion partner to generate recombinant tracers for immunoassay development. The imidaclothiz peptidomimetic C2-15 that specifically binds to the anti-imidaclothiz monoclonal antibody (mAb) 1E7 was fused to NanoLuc, both at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15)...
February 6, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29208980/long-term-drug-modification-to-the-surface-of-mesenchymal-stem-cells-by-the-avidin-biotin-complex-method
#15
Yukiya Takayama, Kosuke Kusamori, Mika Hayashi, Noriko Tanabe, Satoru Matsuura, Mari Tsujimura, Hidemasa Katsumi, Toshiyasu Sakane, Makiya Nishikawa, Akira Yamamoto
Mesenchymal stem cells (MSCs) have various functions, making a significant contribution to tissue repair. On the other hand, the viability and function of MSCs are not lasting after an in vivo transplant, and the therapeutic effects of MSCs are limited. Although various chemical modification methods have been applied to MSCs to improve their viability and function, most of conventional drug modification methods are short-term and unstable and cause cytotoxicity. In this study, we developed a method for long-term drug modification to C3H10T1/2 cells, murine mesenchymal stem cells, without any damage, using the avidin-biotin complex method (ABC method)...
December 5, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29178343/prdm14-directly-interacts-with-heat-shock-proteins-hsp90%C3%AE-and-glucose-regulated-protein-78
#16
Chiharu Moriya, Hiroaki Taniguchi, Satoru Nagatoishi, Hisayoshi Igarashi, Kouhei Tsumoto, Kohzoh Imai
PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2)...
February 2018: Cancer Science
https://www.readbyqxmd.com/read/29170442/inhibition-of-the-inflammatory-response-to-stress-by-targeting-interaction-between-pkr-and-its-cellular-activator-pact
#17
Stephanie Dabo, Patrick Maillard, Milagros Collados Rodriguez, Marianne Doré Hansen, Sabrina Mazouz, Donna-Joe Bigot, Marion Tible, Geneviève Janvier, Olivier Helynck, Patricia Cassonnet, Yves Jacob, Jacques Bellalou, Anne Gatignol, Rekha C Patel, Jacques Hugon, Hélène Munier-Lehmann, Eliane F Meurs
PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy...
November 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29132917/nanobret-approaches-to-study-ligand-binding-to-gpcrs-and-rtks
#18
REVIEW
Leigh A Stoddart, Laura E Kilpatrick, Stephen J Hill
Recent advances in the development of fluorescent ligands for G-protein-coupled receptors (GPCRs) and receptor tyrosine kinase receptors (RTKs) have facilitated the study of these receptors in living cells. A limitation of these ligands is potential uptake into cells and increased nonspecific binding. However, this can largely be overcome by using proximity approaches, such as bioluminescence resonance energy transfer (BRET), which localise the signal (within 10nm) to the specific receptor target. The recent engineering of NanoLuc has resulted in a luciferase variant that is smaller and significantly brighter (up to tenfold) than existing variants...
November 10, 2017: Trends in Pharmacological Sciences
https://www.readbyqxmd.com/read/29121422/investigating-the-hepatitis-b-virus-life-cycle-using-engineered-reporter-hepatitis-b-viruses
#19
Hironori Nishitsuji, Keisuke Harada, Saneyuki Ujino, Jing Zhang, Michinori Kohara, Masaya Sugiyama, Masashi Mizokami, Kunitada Shimotohno
Chronic infection with hepatitis B virus (HBV) increases the risk of developing fibrosis, cirrhosis or hepatocellular carcinoma. Current therapies are limited to type-I interferons and/or nucleos(t)ide analogues; however, these are only partially effective. The development of novel anti-HBV agents for new treatment strategies has been hampered by the lack of a suitable system that allows the in vitro replication of HBV. Studies of virus infection/replication at the molecular level using wild-type HBV are labor-intensive and time-consuming...
January 2018: Cancer Science
https://www.readbyqxmd.com/read/29114997/in-vitro-modeling-of-hiv-proviral-activity-in-microglia
#20
Lee A Campbell, Christopher T Richie, Yajun Zhang, Emily J Heathward, Lamarque M Coke, Emily Y Park, Brandon K Harvey
Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection...
December 2017: FEBS Journal
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