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https://www.readbyqxmd.com/read/29027455/-construction-and-verification-of-nf-%C3%AE%C2%BAb-luciferase-reporter-gene-system
#1
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
#2
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
#3
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...
October 7, 2017: Gene
https://www.readbyqxmd.com/read/28972606/coelenterazine-analogues-emit-red-shifted-bioluminescence-with-nanoluc
#4
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
https://www.readbyqxmd.com/read/28965124/a-novel-bioluminescent-nanoluc-yeast-estrogen-screen-biosensor-nanoyes-with-a-compact-wireless-camera-for-effect-based-detection-of-endocrine-disrupting-chemicals
#5
Luca Cevenini, Antonia Lopreside, Maria Maddalena Calabretta, Marcello D'Elia, Patrizia Simoni, Elisa Michelini, Aldo Roda
The presence of chemicals with estrogenic activity in surface, groundwater, and drinking water poses serious concerns for potential threats to human health and aquatic life. At present, no sensitive portable devices are available for the rapid monitoring of such contamination. Here, we propose a cell-based mobile platform that exploits a newly developed bioluminescent yeast-estrogen screen (nanoYES) and a low-cost compact camera as light detector. Saccharomyces cerevisiae cells were genetically engineered with a yeast codon-optimized variant of NanoLuc luciferase (yNLucP) under the regulation of human estrogen receptor α activation...
September 30, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28960874/polymeric-spycatcher-scaffold-enables-bioconjugation-in-a-ratio-controllable-manner
#6
Lili Jia, Kosuke Minamihata, Hirofumi Ichinose, Kouhei Tsumoto, Noriho Kamiya
Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post-translational modification methods is particularly favored since the proteins can be site-specifically conjugated together with less activity loss...
September 27, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28955790/application-of-a-novel-hibit-peptide-tag-for-monitoring-atf4-protein-expression-in-neuro2a-cells
#7
Kentaro Oh-Hashi, Eri Furuta, Keito Fujimura, Yoko Hirata
A split NanoLuc assay system consisting of two fragments, large N-terminal and small C-terminal regions (NanoBiT), was developed to investigate protein-protein interactions within living cells. Interestingly, the replacement of five amino acids among 11 C-terminal amino acids dramatically increased affinity against the large N-terminal fragment, LgBiT, and the complex had NanoLuc luciferase activity. In this study, we first applied this small fragment, HiBiT, to elucidate the expression of ATF4 protein by transient overexpression of HiBiT-tagged ATF4...
December 2017: Biochemistry and Biophysics Reports
https://www.readbyqxmd.com/read/28941028/luciferases-with-tunable-emission-wavelengths
#8
Julien Hiblot, Qiuliyang Yu, Marina D B Sabbadini, Luc Reymond, Lin Xue, Alberto Schena, Olivier Sallin, Nicholas Hill, Rudolf Griss, Kai Johnsson
We introduce luciferases whose emission maxima can be tuned to different wavelengths by chemical labeling. The luciferases are chimeras of NanoLuc with either SNAP-tag or HaloTag7. Labeling of the self-labeling tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Luciferases with tunable colors have applications as reporter genes, for the construction of biosensors and in bioimaging.
September 22, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28929569/evaluation-of-chemical-chaperones-based-on-the-monitoring-of-bip-promoter-activity-and-visualization-of-extracellular-vesicles-by-real-time-bioluminescence-imaging
#9
Tomohisa Horibe, Nanako Okushima, Aya Torisawa, Ryutaro Akiyoshi, Yoko Hatta-Ohashi, Hirobumi Suzuki, Koji Kawakami
It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging...
September 20, 2017: Luminescence: the Journal of Biological and Chemical Luminescence
https://www.readbyqxmd.com/read/28892606/crispr-mediated-tagging-of-endogenous-proteins-with-a-luminescent-peptide
#10
Marie K Schwinn, Thomas Machleidt, Kris Zimmerman, Christopher T Eggers, Andrew S Dixon, Robin Hurst, Mary P Hall, Lance P Encell, Brock F Binkowski, Keith V Wood
Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating intracellular signaling and the effects induced by synthetic compounds is to overexpress recombinant reporter genes within a cell. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins...
September 11, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28869756/red-shifted-luciferase-luciferin-pairs-for-enhanced-bioluminescence-imaging
#11
Hsien-Wei Yeh, Omran Karmach, Ao Ji, David Carter, Manuela M Martins-Green, Hui-Wang Ai
Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues...
October 2017: Nature Methods
https://www.readbyqxmd.com/read/28771321/activity-based-detection-of-consumption-of-synthetic-cannabinoids-in-authentic-urine-samples-using-a-stable-cannabinoid-reporter-system
#12
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...
September 5, 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
#13
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 transfected to express the L protein...
October 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28733215/development-of-nanoluc-bridging-immunoassay-for-detection-of-anti-drug-antibodies
#14
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...
November 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28726090/flavoprotein-minisog-bret-induced-cytotoxicity-depends-on-its-intracellular-localization
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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