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https://www.readbyqxmd.com/read/29208980/long-term-drug-modification-to-the-surface-of-mesenchymal-stem-cells-by-the-avidin-biotin-complex-method
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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