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Order Tymovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Shukla et al. (2014a) engineered the PVX virions displaying green fluorescent protein (GFP) or mCherry as N-terminal coat protein fusions, using the FMDV 2A approach and producing a 1:3 fusion protein to coat ratio. These particles allowed the infection of N. benthamiana plants to be visualized clearly. The infection of plants with the recombinant GFP-PVX and mCherry-PVX particles was documented by fluorescence imaging, structural analysis, and genetic characterization to determine the stability of the chimeras and optimize the molecular farming protocols. Then, the fluorescent mCherry-PVX filaments were used as probes for optical imaging in human cancer cells and a preclinical mouse model. The cell viability assays and histological analysis following the administration of mCherry-PVX indicated the biocompatibility and rapid tissue clearance of the particles. The authors concluded that such particles could be functionalized with additional cancer-specific detection ligands to provide tools for molecular imaging, allowing the investigation of molecular signatures, disease progression/recurrence, and the efficacy of novel therapies (Shukla et al. 2014a).
Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
DNA fragments incorporating the coding sequences of the genes of interest can be cloned in frame with N- or C-terminally tagged GFP and fRFP using established (e.g. pEGFP-N1, Clontech; pmKate2-N, Evrogen) or bespoke vectors. It is recommended that for each target protein, a GFP and an fRFP tagged construct be created because, in our experience, the nature of the protein-tag can have an effect on protein expression, even if the reasons for this are not necessarily clear. It is further important to keep in mind that GFP has severely diminished fluorescence intensity when in acidic compartments (Haupts et al., 1998), such as the lysosome, and that FRET will work poorly or not at all when fluorescent proteins are on opposite sides of the membrane. Therefore, it is recommended to use known protein structures or topologies to place the fluorescent tag on the protein terminus that is cytosolic for each target protein. In cases where protein topology is unknown, both the N- and C-termini should be separately tagged for each fluorescent protein. fRFP is recommended over shorter wavelength RFPs (e.g. DsRed or mCherry, Clontech) because the longer wavelength excitation spectrum of fRFP has essentially no overlap with the 488-nm laser, meaning very little background signal will arise from direct laser stimulation.
Scanning Angle Interference Microscopy (SAIM)
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Cristina Bertocchi, Timothy J. Rudge, Andrea Ravasio
SAIM is feasible for fixed samples as well as live-cell imaging. To avoid loss of accuracy we do recommend special attention to the choice of fluorophores. Although SAIM does not require special fluorophores, the fluorophores should have high photostability to minimize photobleaching during the imaging scanning sequence and should be bright with high quantum yield to provide a good signal-to-noise ratio. Among the fluorescent proteins and synthetic dyes compatible with SAIM, genetically encoded fluorescent proteins have the principal advantage of being small and suitable for live-cell imaging and are capable of achieving maximal labeling specificity, removing any possible problems associated with nonspecific labeling. Furthermore, they do not require fixation or permeabilization procedures that could affect cellular nanostructure. Some fluorescent proteins successfully used in SAIM include green fluorescent proteins such as EGFP and mEmerald, red fluorescent protein mCherry,33 and photoconverted tdEOS25 that has excellent brightness and photostability. In addition, chromobodies (generated by the fusion of a fluorescent protein to a nanobody34), a novel species of extremely small antibodies that are endogenously synthesized within cultured cells, could possibly be used to prepare samples for SAIM.
Folic acid: a potential inhibitor against SARS-CoV-2 nucleocapsid protein
Published in Pharmaceutical Biology, 2022
Yu-meng Chen, Jin-lai Wei, Rui-si Qin, Jin-ping Hou, Guang-chao Zang, Guang-yuan Zhang, Ting-ting Chen
A cell experiment was designed to assess the role of folic acid in treating COVID-19. A red fluorescent protein derived from mushroom coral, mCherry is used to label and trace certain molecular and cellular components. RNAi is an RNA-dependent gene-silencing phenomenon that can be triggered by shRNA, as well as a strong and versatile silencer of various genes (Xin et al. 2012). SARS-CoV-2 N is a structural protein that binds directly to viral RNA and provides stability; it has two RNA-binding domains, and its forms or regulates biomolecular condensates in vivo through interactions with RNA and key host cell proteins (Cascarina and Ross 2020). SARS-CoV-2 N encoded by SARS-CoV-2 plays a vital role in inhibiting RNA interference in cells (Carlson et al. 2020; Mu et al. 2020; Yoshimoto 2020). The cell experiment was designed based on these theories. The shRNA expression inhibits expression of the red fluorescent protein mCherry, and SARS-CoV-2 N expression inhibits the effect of shRNA-mediated RNAi. The change in mCherry expression reflected the effect of folic acid on SARS-CoV-2 N. As a result, after the addition of folic acid, the inhibitory effect of SARS-CoV-2 N on RNAi was weakened, and mCherry expression decreased. The result showed that folic acid inhibited the biological activity of SARS-CoV-2 N, suggesting that folic acid might disrupt RNA, and thus have an anti-viral effect. This needs to be further studied in subsequent experiments. It was preliminarily shown that SARS-CoV-2 N could be a target for folic acid in COVID-19 treatment.
High-sugar, high-fat, and high-protein diets promote antibiotic resistance gene spreading in the mouse intestinal microbiota
Published in Gut Microbes, 2022
Rong Tan, Min Jin, Yifan Shao, Jing Yin, Haibei Li, Tianjiao Chen, Danyang Shi, Shuqing Zhou, Junwen Li, Dong Yang
The donor bacterium (MEC-5) isolated from mouse feces was identified as Escherichia coli. Donor strains were chromosomally tagged with mCherry encoding constitutive red fluorescence and the tac promoter expressing mCherry was encoded upstream.55 In addition, RP4 plasmids harboring tetracycline (tet) and kanamycin (km) resistance genes were appended to a genetically encoded expressible green fluorescent protein (GFP) gene. Next, MEC-5-mCherry was electroporated with the plasmid RP4-GFP-TetRKmR. As a result, both red and green fluorescence occurs in donor cells, but upon plasmid transfer to a fecal bacterium, the transconjugants display green fluorescence due to GFP expression, which can be detected and sorted by fluorescence microscopy or fluorescent-activated cell sorting (Supplementary File S1).
Discovery of CFTR modulators for the treatment of cystic fibrosis
Published in Expert Opinion on Drug Discovery, 2021
Miquéias Lopes-Pacheco, Nicoletta Pedemonte, Guido Veit
Very recently, a novel assay exploiting high-content imaging methodology has been developed [50]. This assay provides simultaneous measurements of both CFTR function and CFTR membrane proximity based on the expression of two fluorescent proteins: a cytosolic mCherry protein and a HS-YFP fused to the intracellular N-terminal of CFTR [50]. The mCherry expression allows image segmentation and accurate localization of the cell membrane by marking the border of cells, but it is also useful as an internal standard for the normalization of YFP-CFTR expression [50]. The time course of YFP quenching in response to extracellular iodide addition informs on anion conductance [50]. At the same time, evaluation of fluorescent signals corresponding to total cellular YFP-CFTR and YFP-CFTR within the membrane-proximal zone provides a readout of efficiency of CFTR maturation and trafficking, as well as of the overall rates of biosynthesis and degradation of the protein [50].