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https://www.readbyqxmd.com/read/29446920/homogeneous-noncompetitive-luminescent-immunodetection-of-small-molecules-by-ternary-protein-fragment-complementation
#1
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
#2
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...
February 3, 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
#3
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
#4
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
#5
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...
January 19, 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
#6
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...
January 19, 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
#7
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)...
January 5, 2018: Acta Diabetologica
https://www.readbyqxmd.com/read/29280616/recombinant-peptidomimetic-nano-luciferase-tracers-for-sensitive-single-step-immunodetection-of-small-molecules
#8
Yuan Ding, Xiude Hua, He Chen, Fengquan Liu, Gualberto G Gonzalez-Sapienza, Ming-Hua 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 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)...
December 27, 2017: 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
#9
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-grp78
#10
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 needs to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer cells (TNBCs), which do not express the three most common types of receptors (estrogen receptors, progesterone receptors, and HER2)...
November 24, 2017: 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
#11
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
#12
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
#13
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...
November 9, 2017: Cancer Science
https://www.readbyqxmd.com/read/29114997/in-vitro-modeling-of-hiv-proviral-activity-in-microglia
#14
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...
November 8, 2017: FEBS Journal
https://www.readbyqxmd.com/read/29093094/characterization-of-recombinant-flaviviridae-viruses-possessing-a-small-reporter-tag
#15
Tomokazu Tamura, Takasuke Fukuhara, Takuro Uchida, Chikako Ono, Hiroyuki Mori, Asuka Sato, Yuzy Fauzyah, Toru Okamoto, Takeshi Kurosu, Yin Xiang Setoh, Michio Imamura, Norbert Tautz, Yoshihiro Sakoda, Alexander A Khromykh, Kazuaki Chayama, Yoshiharu Matsuura
The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes including fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses, and then examined the biological characteristics of the viruses...
November 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/29074234/development-of-a-rapid-and-quantitative-method-for-the-analysis-of-viral-entry-and-release-using-a-nanoluc-luciferase-complementation-assay
#16
Michihito Sasaki, Paulina D Anindita, Wallaya Phongphaew, Michael Carr, Shintaro Kobayashi, Yasuko Orba, Hirofumi Sawa
Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions...
October 23, 2017: Virus Research
https://www.readbyqxmd.com/read/29027455/-construction-and-verification-of-nf-%C3%AE%C2%BAb-luciferase-reporter-gene-system
#17
Zhilan Guo, Luyang Che, Jingzhe Li, Zhenxiao Sun, Changzhen Liu
To quantify the transcriptional activity of NF-κB and to screen drugs related to the regulation of NF-κB activation, we constructed a recombinant plasmid through deleting the original CMV promoter of retrovirus vector pQCXIP and inserting the NF-κB enhancer and NanoLuc luciferase sequence into the vector. Then, using the recombinant plasmid we constructed a cell line in which the expression of NanoLuc luciferase (NLuc) was regulated by NF-κB. The inserted sequences were verified by restriction endonuclease digestion and sequencing...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/29024447/the-influences-of-a-novel-anti-adhesion-device-thermally-cross-linked-gelatin-film-on-peritoneal-dissemination-of-tumor-cells-the-in-vitro-and-in-vivo-experiments-using-murine-carcinomatous-peritonitis-models
#18
Hiroe Miyamoto, Hiroyuki Tsujimoto, Tsunehito Horii, Yuki Ozamoto, Joe Ueda, Toshitaka Takagi, Naoto Saitoh, Akeo Hagiwara
To create anti-adhesive materials to be more effective and safer, we developed a thermally cross-linked gelatin film that showed superior anti-adhesive effects with excellent peritoneal regeneration. However, it may act as a convenient scaffold for tumor cell growth, thereby accelerating peritoneal dissemination when used in surgery for abdominal tumors. In this study, we tried to clarify this issue using mouse carcinomatous peritonitis models. First, we examined the in vitro tumor cell growth of mouse B16 melanoma or Colon26 cells on the gelatin film or the conventional hyarulonate/carboxymethylcellulose film...
October 10, 2017: Journal of Biomedical Materials Research. Part B, Applied Biomaterials
https://www.readbyqxmd.com/read/29017964/isolation-and-characterization-of-a-stress-responsive-gene-encoding-a-chrd-domain-containing-protein-from-a-halotolerant-green-alga
#19
Ryo Ishinishi, Hideyuki Matsuura, Satoshi Tanaka, Saaya Nozawa, Keisuke Tanada, Norihito Kawashita, Kazuhito Fujiyama, Hitoshi Miyasaka, Kazumasa Hirata
The genetic basis of stress resistance in extremophilic microalgae is not well studied. In this study, a gene of unknown function, the cluster58 or CL58 gene, was identified from the halotolerant green alga Chlamydomonas W80 and characterized. The CL58 gene encodes a protein containing a domain of unknown function, the CHRD domain, and a putative secretory signaling sequence at its N-terminus. The levels of CL58 mRNA increased in response to high copper levels and low temperatures. When the CL58 gene was heterologously expressed as a fusion gene with the NanoLuc luciferase gene in Chlamydomonas reinhardtii, a majority of the NanoLuc activity was detected in the culture medium compared with that in the intracellular fraction...
January 15, 2018: Gene
https://www.readbyqxmd.com/read/28972606/coelenterazine-analogues-emit-red-shifted-bioluminescence-with-nanoluc
#20
Anton Shakhmin, Mary P Hall, Thomas Machleidt, Joel R Walker, Keith V Wood, Thomas A Kirkland
We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc...
October 18, 2017: Organic & Biomolecular Chemistry
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