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Orders Norzivirales and Timlovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Selby and Peterlin (1990) expressed the first chimeric MS2 coat derivative, namely the coat fused to the HIV-1 Tat, in eukaryotic cells. The product did not form VLPs (but this study paved the long way for the tethering techniques, another breakthrough) but formed non-VLP application of the RNA phages. The two others historically important non-VLP MS2 coat fusions were performed with the (i) LexA protein that started the so-called three-hybrid approach and (ii) green fluorescence protein (GFP) that gave onset to the pioneering visualization (or imaging) studies in living cells. It is remarkable here that, by the tethering studies in eukaryotic cells, the MS2 coat was provided with the HA tag, an epitope 98–106 from human influenza hemagglutinin (Kim YK et al. 2005). Then, the SNAP domain of O-6-alkylguaninalkyltranferase was fused to the MS2 coat with an idea to link the MS2-SNAP fusion protein to DNA when the latter would be labeled with the SNAP substrate benzylguanine (Paul et al. 2013).
Advances in Genome Editing
Published in Yashwant Pathak, Gene Delivery, 2022
Different approaches in nanostructures are investigated by various researchers for genome editing for varied purpose. Recently, Bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica nanoparticles were investigated for intracellular delivery of the Cas9-sgRNA ribonucleoprotein complex. Gene-editing was observed with an efficiency of about 40 percent as measured by GFP gene knockdown of HT1080-GFP cells, with no notable change in the morphology of the cells (Salekdeh et al., 2021). In another study, biomimetic cancer cell coated zeolitic imidazolate frameworks were reported for genome editing carrying CRISPR-Cas9. Incubation of C3-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells. A three-fold repression in the enhanced green fluorescent protein expression was observed (Alyami et al., 2020). CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence using DNA-functionalized Au nanoparticle to detect breast cancer gene-1 with very high sensitivity in 30 minutes (Choi et al., 2021).
Antimicrobial Preservative Efficacy and Microbial Content Testing*
Published in Philip A. Geis, Cosmetic Microbiology, 2020
Scott V.W. Sutton, Philip A. Geis
A second approach is to utilize an alternative technology (avoiding the use of plate counts). Relatively little has been published on this topic, perhaps as the need for a 28-day sampling period lessens the impact of a rapid enumeration of survivors. However, studies of several technologies have been reported in the literature. Impedance techniques are the most commonly reported for this application (93,94). Connolly et al. compared impedance, direct epifluorescence (DEFT-MEM), and ATP bioluminescence for applicability to preservative testing and found that only impedance yielded results similar to the traditional method (95). Another approach that shows promise, at least for E. coli, is the use of green fluorescent protein to monitor viability. Casey and Nguyen (96) utilized a genetically engineered strain of E. coli that contained the green fluorescent protein, which provided an easily distinguished marker for viable cells. When this strain is used, less than 2 minutes per sample is required for the determination of viable cells.
Application of mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles in suppression of laryngeal cancer by targeting cancer stem cells
Published in Drug Delivery, 2023
Xiaoyan Xu, Tianhao Zhou, Xudong Wei, Xuelian Jiang, Jiyan Cao
Hep-2 cells were transfected with plasmid pEGFP-C3, encoding enhanced green fluorescent protein, and the transfection was observed in vitro by inverted fluorescence microscopy. Hep-2 was inoculated and cultured in 24-well cell culture plates such that cell fusion reached 70-80% in each well at the time of administration. The mPEG-CS-RGD/pEGFP-C3 nanosuspension was added to the wells after filtration and de-sterilization. The naked gene group, liposome group, mPEG-CS-cRGD group and mPEG-CS-cRGD/PTX group were established. After 6h of administration, the culture medium was replaced to complete medium and continued for 24 h. The green fluorescent protein (GFP) in the cells was observed by inverted fluorescence microscopy. The expression of green fluorescent protein (GFP) was observed by inverted fluorescence microscopy.
Virtual screening and zebrafish models in tandem, for drug discovery and development
Published in Expert Opinion on Drug Discovery, 2023
David Hernández-Silva, Francisca Alcaraz-Pérez, Horacio Pérez-Sánchez, Maria Luisa Cayuela
Green fluorescent protein (GFP) transgenic reporter. Zebrafish is a viable whole animal model for monitoring cell or biological processes [91]. This is supported by the optical clarity of the embryo in combination with advancements in imaging technologies. Seventeen flavonoids as inhibitors or of angiogenesis were identified in almost 36,043 compounds from the Traditional Chinese Medicine (TCM) database [92]. They were identified after computational screening against the crystal structure of human COX-2, human mPGES-1, and the 3D structure of CYP4A11 (Arachidonic acid metabolic enzymes), which all play an important role in glioma angiogenesis. Using a transgenic zebrafish line that expresses GFP in endothelial cells (Tg(fli1a:EGFP), isoliquiritigenin exhibited the most potent antiangiogenic activities. Therefore, in silico and zebrafish screening working in tandem is an efficient strategy for screening multiple-target inhibitors to block a biological process.
In vitro cell-based models of drug-induced hepatotoxicity screening: progress and limitation
Published in Drug Metabolism Reviews, 2022
Maryam Mirahmad, Reyhaneh Sabourian, Mohammad Mahdavi, Bagher Larijani, Maliheh Safavi
Multiple fluorescent probes are reported to be employed to quantify various oxidative responses like ROS production in cytosol or nucleus, lipid peroxidation, mitochondrial superoxide, and DNA damage signaling in hepatocytes as a screening strategy (Donato and Tolosa 2021). Fluorescent probes have been tagged to specified proteins to detect their cellular dynamics. Gene tagging can be performed by either inserting the tag into the native locus of the genome or by placing a tagged copy of the gene into another location of the genome. Green fluorescent protein (GFP) is an example of genetically encoded protein that is used to image live cells and localize proteins. By measuring alterations in the expression of transcription factors and stress response markers (like GFP-tagged sulfiredoxin 1), it is possible to determine stress responses like Nrf2, and to monitor oxidative stress through HCI (Wink et al. 2017, 2018).