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Current in vivo Models for Brain Disorders
Published in Carla Vitorino, Andreia Jorge, Alberto Pais, Nanoparticles for Brain Drug Delivery, 2021
Marta Guerra-Rebollo, Cristina Garrido
In the specific case of brain tumours, one of the most utilised techniques to visualise drug delivery into the tumour is using BLI. The firefly luciferase (FLuc) can be used as a reporter gene and d-luciferin as a substrate. FLuc catalyses d-luciferin oxidation in the presence of adenosine triphosphate (ATP) and coenzyme A (CoA) producing O2 and photons which can be detected in vivo. Recording of the emitted light by photodetectors, capable of linear response allows for real-time measurements [57, 58]. This technique is based on the stable expression of luciferase by the brain tumour cell line selected for the experiment. Once the cells are implanted into the mouse brain, d-luciferin could be injected intraperitoneally into the host mice, where it is distributed throughout the mouse body and crosses the BBB. The intensity of the emitted light correlates to the size of the tumour and allows comparisons of tumours size across different animals but also within the same animal in different time points (Fig. 13.1) [59].
The Stress Response and Stress Proteins
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Martin E. Feder, Dawn A. Parsell, Susan L. Lindquist
Numerous studies have now demonstrated that some stress proteins can contribute to the refolding and reactivation of denatured proteins in vitro. A typical experiment involves denaturing a test protein (e.g., rhodanese, dihydrofolate reductase, luciferase, citrate synthase) with a chemical denaturant (e.g., urea, guanidine) or heat, removing the denaturant, and characterizing either the structure or the function of the test protein in the presence or absence of presumptive chaperones and their partner proteins. GroEL and GroES (the E. coli counterparts of hsp60 and hsp10, respectively) in combination have now repeatedly been shown to chaperone refolding and reactivation of a variety of test proteins (e.g., Figure 4); this activity requires ATP. To date, only two such studies have documented similar activity for DnaK (the E. coli counterpart of hsp70) and its partner proteins DnaJ and GrpE. In one study, reactivation of RNA polymerase required a 60-fold molar excess of DnaK.25 The other study26 examined firefly luciferase expressed in E. coli and showed that less excessive quantities of DnaJ, DnaK, and GrpE in combination could prevent the aggregation of heat-denatured luciferase and eventually release refolded luciferase; again, this activity requires ATP.
Optical-CT Imaging
Published in George C. Kagadis, Nancy L. Ford, Dimitrios N. Karnabatidis, George K. Loudos, Handbook of Small Animal Imaging, 2018
Xueli Chen, Dongmei Chen, Fenglin Liu, Wenxiang Cong, Ge Wang, Jimin Liang
Bioluminescent light is produced from the expression of luciferase enzymes, such as firefly luciferase, Renilla luciferase, and jellyfish luciferase. Cells encoded with the luciferase enzymes can serve as bioluminescent probes, which allow bioluminescent light emission. The BLT technique uses the luciferase enzyme gene as a reporter (Contag and Bachmann 2002). Oxidation of luciferin, which is injected into the animal, leads to the emission of bioluminescent light and occurs only in cells in which the luciferase enzyme is expressed by the reporter gene. The major attraction of this approach is that although absolute light levels are low, signal is produced only where luciferase is present, leading to extremely low background signals (Troy et al. 2004). BLT was first proposed by Wang’s group in 2003 (Wang et al. 2003). In mathematics, it is an inverse source problem. Based on an accurate light transport model, BLT aims to reconstruct the 3D spatial distribution and concentration of the bioluminescent probes inside a small living animal.
Advances in luminescence-based technologies for drug discovery
Published in Expert Opinion on Drug Discovery, 2023
Bolormaa Baljinnyam, Michael Ronzetti, Anton Simeonov
The dominant detection methodology for developing high-throughput assays in early-stage drug discovery has been fluorescence-based, owing in part to its robustness and ease of use. Fluorescence assays, however, are not necessarily the best performing and will typically present lower sensitivity than chemiluminescence methods. Chemiluminescent assays are not without weakness, themselves being very sensitive to experimental conditions, so their applicability in complex systems is very limited. Bioluminescent systems introduce a degree of environmental robustness emission with the signal emission occurring in an enzyme active site under biological conditions. Bioluminescence-based technologies are expected to play an essential role in future drug discovery paradigms, from hit identification to lead validation, owing to inherent advantages in the enzymatic light-producing reaction. Over the years, luciferases and their substrates have gone through multiple iterations for various applications in early-stage drug discovery. While the firefly luciferase protein has enjoyed immense popularity as a wildtype reporter, recent efforts have focused on improving specific functions or properties of nearly every luciferase described.
A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways
Published in Gut Microbes, 2022
Fereshteh Ghazisaeedi, Jochen Meens, Bianca Hansche, Sven Maurischat, Peter Schwerk, Ralph Goethe, Lothar H. Wieler, Marcus Fulde, Karsten Tedin
NF-κB-luciferase reporter derivatives of porcine (IPEC-J2/K6), human (Caco-2/C6), and mouse (MODE-K/H8) intestinal epithelial and IPEC-J2 MAPK/JNK(AP-1)-luciferase cell lines (IPEC-J2/D6) were constructed by infection of the cell lines with prepackaged, lentiviral vectors harboring either NF-κB- or JNK(AP-1)-luciferase reporter fusions with selection for puromycin resistance according to the manufactuerer´s instructions (Cignal Lenti Reporters, SA Biosciences, CLS-013 L and CLS-011 L, resp.). These luciferase fusion constructs encode a minimal promoter element (TATA box) preceded by a transcriptional response element specific for either NF-kB or AP-1. The luciferase is a mammalian codon-optimized, non-secreted form of the firefly luciferase gene, carrying a protein-destabilizing sequence to minimize long-term accumulation of the luciferase. After removal of dead, non-adherent cells, puromycin-resistant cells were allowed to form microcolonies, then pooled and diluted in fresh cell culture medium containing puromycin to a concentration of approximately ten cells/ml, and 100 µl of the suspension was used to seed each well of a 96-well plate. Clones derived from single cells under selection with puromycin were grown to monolayers, harvested and used to seed 25 cm2 flasks (Corning). All clones isolated in this manner were screened for their responses to TLR ligands and those showing the best background/induction ratios and dose responses were retained for further assays.
Altered expression levels of miR-144-3p and ATP1B2 are associated with schizophrenia
Published in The World Journal of Biological Psychiatry, 2022
Bo Pan, Yuting Wang, Yiwen Shi, Qianzhan Yang, Bing Han, Xiaoli Zhu, Yanqing Liu
The pmirGLO dual-luciferase miRNA target expression vector (Promega, USA) was transfected with the PCR amplified 3′ untranslated regions (3′-UTR) of ATP1B2 mRNA. In a 24-well plate, the luciferase construct containing wild-type (WT) or mutated binding site of ATP1B2 (constructed by General Biol (Anhui, China)) was transfected into target 293 T cells. This was followed by co-transfection with miR-144-3p mimics or vehicle using Lipofectamine 2000, to identify the binding site between ATP1B2 and miR-144-3p. Determination of luciferase activity after 48 h of transfection and normalisation with Renilla luciferase was done utilising a Dual-Luciferase Reporter System Kit (#E1910, Promega, Madison, WI, USA). The levels of firefly luciferase activities were obtained by normalising to Renilla luciferase activities and relative to a control.