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Toxicology Studies of Semiconductor Nanomaterials: Environmental Applications
Published in Suresh C. Pillai, Yvonne Lang, Toxicity of Nanomaterials, 2019
T. P. Nisha, Meera Sathyan, M. K. Kavitha, Honey John
Semiconductor QD-based biosensors target the specific biomolecules with the help of sensing ligands and the number of biomolecules is recognized from the change of luminescence. Besides the QD-based conventional biosensors, QD-based energy transfer sensors like FRET have also been developed. In QD-based FRET biosensors, QDs act as the donor and the fluorescence dye acts as acceptor and has been widely used for the detection of amino acids, insulin, intracellular pH, proteolytic activity assay, and monitoring DNA cleavage (Algar et al., 2014, Geissler et al., 2014). Similar to QD – FRET system, QD – BRET (bioluminescence resonance energy transfer) (Alam et al., 2014) and QD – CRET (chemiluminescence energy transfer) (Chen et al., 2014a) systems are also available (Figure 4.10).
Imaging of Intracellular Targets
Published in George C. Kagadis, Nancy L. Ford, Dimitrios N. Karnabatidis, George K. Loudos, Handbook of Small Animal Imaging, 2018
Two major approaches are pursued for optical imaging: bioluminescence reporter gene imaging using a construct encoding for Luciferase from the firefly Photinus pyralis (FLuc) and fluorescence imaging constructs using green fluorescent protein (GFP) (Misteli and Spector 1997; Gilad et al. 2007; Close et al. 2011). Bioluminescence imaging of FLuc requires the injection, usually intraperitoneally, of d-luciferin. The subsequent enzymatic turnover produces light, which allows the readout and semi-quantitation of the reporter gene activity. Luciferase is considered as an excellent marker for gene expression because of its lack of posttranslational modifications and a relatively short in vivo half-life of approximately 3 h. An example of such an approach is presented by Qayum et al. who used a luciferase reporter construct driven by three hypoxia-responsive element-binding sequences for hypoxia-inducible factor. This provided them with a quantifiable optical read-out of hypoxia. Following in vitro experiments, they used the same cell line in vivo to generate tumors subcutaneously in mice. Treatment of these tumors with signaling inhibitors of the RAS-PI3K-AKT pathway resulted in a decreased bioluminescence signal when compared to untreated animals, indicating a reduction in tumor hypoxia (Qayum et al. 2009). In contrast to bioluminescence imaging, animal fluorescence imaging is hampered by low signal-to-background ratios and autofluorescence (Troy et al. 2004). As a result, GFP fluorescence has generally been visualized in organs such as the lung or liver only during postmortem examinations. In the past years, a number of groups have developed several near-infrared fluorescent proteins that avoid overlapping emissions from tissue or organic compounds and enable real-time imaging to take place without interference from autofluorescence events. An interesting approach, taking advantage of both bioluminescence and fluorescence at the same time, was presented by Iglesias and Costoya (2009). They developed a genetically encoded biosensor that is induced by the presence of hypoxia. It comprised a regulatory module that contains a transcriptional enhancer able to bind the alpha subunit of the HIF-1 transcription factor, and a dual tracer formed by a fusion protein of a near-infrared fluorophore (mCherry) and FLuc. By fusing a fluorescent to a bioluminescent protein, they obtained a bioluminescence resonance energy transfer phenomenon, turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor.
Optical Imaging Probes
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Fluorescent proteins found in cnidarians (jellyfishes, anemones, and corals) function as acceptors of blue bioluminescence produced by aquaporins and luciferases. In other words, fluorescent proteins are essential components of bioluminescence resonance energy transfer (BRET) systems that are found in nature. In BRET, photons emitted by luciferases are absorbed by fluorescent proteins and excite the fluorophore, as a result a red-shifted (lower energy) photon can be emitted. Fluorophore of natural fluorescent proteins is spontaneously generated upon oxidation of folding-dependent proximal side groups of amino acids. Exogenous substrates and cofactors are not required for fluorescence of these proteins; however, the amino acid oxidation requires the presence of oxygen. Most of the fluorescent proteins are multimeric, for example, enhanced green fluorescent protein (EGFP) forms weak dimers while most of the red fluorescent proteins are tetramers. The most commonly used fluorescent protein in oncology is EGFP. This engineered fluorescent protein is a product of several stages of mutagenesis and codon usage design: Phe64→Leu mutation improved maturation at higher temperatures (37°C), 190 silent base changes for improving expression in mammals; Ser65→ Thr mutation to shift excitation peak to convenient 488 nm wavelength. The resultant protein is approximately twice as bright as the wild-type protein. Expression of fluorescent proteins can be used to monitor gene expression and protein localization in living organisms. The reason for this is the ability of EGFP to fold into a fluorescent product thereby forming an independent domain of many fusion proteins with little interference with the function of the fusion partner (18,19). Unfortunately, due to multimerization of red fluorescent proteins [e.g., sea anemone Discosoma striata red protein, DsRed (20)], the above fusion approach is not always successful because of the tendency of red fluorescence fusion proteins to aggregate and their slow maturation (i.e., appearance of a fluorescent product), which can take several hours and interfere with double fluorescent protein labeling experiments. These potential problems can be dealt by using mutagenesis (21): the removal of several positively charged N-terminal amino acids in DsRed2 mutant decreases the stability of multimers, and the introduction of several silent mutations yielded DsRed-Express, which matures within an hour after transfection of cells with cDNA. It has been reported that cervical carcinomas stably transfected with DsRed2 are easier to detect in vivo than EGFP-expressing tumors (22) (Fig. 2).
Unmasking allosteric-binding sites: novel targets for GPCR drug discovery
Published in Expert Opinion on Drug Discovery, 2022
Verònica Casadó-Anguera, Vicent Casadó
In order to study and discover unexpected allosteric interactions in GPCR oligomers and, then, for developing new allosteric drugs, experimental biochemical and biophysical methods are mandatory. Some of these methods are currently being applied in GPCR oligomerization research for detecting allosteric interactions between the respective ligands and/or protomers within oligomeric complexes. Saturation, competition, and dissociation radioligand-binding assays, homogeneous time resolved fluorescence (HTRF), complemented donor-acceptor resonance energy transfer (CODA-RET), receptor-G protein bioluminescence resonance energy transfer (BRET), and dynamic mass redistribution (DMR) assays, as well as the determination of intracellular signaling messengers such as cAMP, MAPK phosphorylation, β-arrestin recruitment, or the use of disturbing TM peptides for interfering oligomerization and, thus, the propagation of allosteric modulations between protomers are examples of these methods (see [27,50,135,136,167,168])
The structure of CLEC-2: mechanisms of dimerization and higher-order clustering
Published in Platelets, 2021
Eleyna M Martin, Malou Zuidscherwoude, Luis a Morán, Ying Di, Angel García, Steve P Watson
CLEC-2 dimers have been observed on the resting platelet membrane. Hughes et al [27]. cross-linked human platelet surface proteins, within 1.6 nm distance using Sulfo-EGS, and the subsequent complexes were immunoprecipitated and western blotted for CLEC-2. For non-stimulated platelets, a complex approximately twice the size of monomeric CLEC-2 was observed, likely consistent with the formation of a CLEC-2 dimer as no other platelet protein has yet been shown to associate with CLEC-2. In conjunction, the distribution of CLEC-2 on mouse platelets was visualized by immunogold labeling and electron microscopy. Approximately, one third of gold particles were found alone and half were found in pairs with the remainder forming higher-order structures. This indicates that CLEC-2 is present as both a monomer and dimer on resting platelets. Bioluminescence resonance energy transfer (BRET) was used to probe CLEC-2 proximity within a transfected HEK293T cell line. Energy transfer between luciferase-tagged and GFP-tagged CLEC-2 indicated that the two proteins are within 10 nm distance. The corresponding BRET efficiency and donor/acceptor ratio relationship observed for these experiments was consistent with the presence of CLEC-2 dimers[15]. Co-immunoprecipitation between overexpressed MYC and FLAG-tagged full length human CLEC-2 from HEK293T cells also provides supporting evidence that CLEC-2 forms dimers at the cell surface [15,32]. The fraction of CLEC-2 found as a monomer or a dimer in all of these studies was not determined, and knowledge of this would aid our understanding of CLEC-2 activation.
Structure-function relationship of the platelet glycoprotein VI (GPVI) receptor: does it matter if it is a dimer or monomer?
Published in Platelets, 2021
Joanne C. Clark, Foteini-Nafsika Damaskinaki, Yam Fung Hilaire Cheung, Alexandre Slater, Steve P. Watson
Our own studies using C-terminal tagged versions of GPVI provided further support for the presence of a dimer and/or higher order clusters of GPVI in transfected cell lines[57]. Using bioluminescence resonance energy transfer (BRET) of GPVI, we reported that GPVI produced a specific BRET signal in transfected HEK293T cells that was intermediate between that of a known monomer (CD2) and dimer (CTLA-4), with the degree of dimerization only marginally increased by expression of the FcRγ-chain[57]. This suggests that GPVI is expressed as a mixture of monomers and dimers and that this is not dependent on the FcRγ-chain. One surprising observation from this study however was that neither collagen nor convulxin were able to increase the BRET signal suggesting that binding may only have occurred to the dimeric form of the receptor or that clustering of the extracellular domain does not bring the intracellular tails close enough together to increase the signal. In addition, in the same study, we provided additional evidence of dimerization through the co-immunoprecipitation with myc- and flag-tagged versions of GPVI and CD2-GPVI chimeras, with dimerization being mediated by the extracellular domain of GPVI, and by the use of a chemical cross-linker which demonstrated the presence of dimers and higher order oligomers in platelets[57]. These observations strongly suggest that GPVI is expressed as a combination of monomers and dimers in a transfected cell line and raises the surprising possibility that ligands only bind to the dimeric form.