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  • br Synthesis and mechanism of fluorescent DNA


    Synthesis and mechanism of fluorescent DNA-CuNMs
    Application of fluorescent DNA-CuNMs
    Summary and conclusions In summary, we introduce recent research progress in the synthesis and various applications of DNA-CuNMs. DNA-CuNMs with novel catalytic, electrical and optical properties can be obtained on various DNA scaffolds with special sequence design through chemical and electrochemical methods simply, rapidly and cheaply. On account of the advantages of fluorescent DNA-CuNMs, such as the large Stokes shift, high fluorescence efficiency, good photostability and non-toxic, DNA-CuNMs fluorophores gradually become a multipurpose tools promising for applications in logic gate construction, staining and biosensing of DNAs and RNAs, ions, proteins and enzymes, small molecules and so on (Fig. 7).
    Future perspectives
    Introduction Transplant recipients are maintained on immunosuppressive therapy to prevent rejection and loss of the allograft. The major immunosuppressive agents that are available in various combination regimens are glucocorticoids, azathioprine, mycophenolate mofetil (MMF), mizoribine, cyclosporine, tacrolimus, everolimus, rapamycin, and belatacept (Halloran, 2004; Ventura-Aguiar et al., 2016). Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) that inhibits mTOR complex 1 (mTORC1) and regulates factors involved in several crucial cellular functions, such as protein synthesis, regulation of angiogenesis, lipid biosynthesis, mitochondrial biogenesis and function, cell cycle, and autophagy (Granata et al., 2016). Many clinical studies have shown that everolimus or rapamycin decreases the risk of CMV infection in transplant recipients (Andreassen et al., 2014; Brennan et al., 2011; Hocker et al., 2016; Lehmkuhl et al., 2009; Nashan et al., 2012; Radtke et al., 2016; Strueber et al., 2016; Tedesco-Silva et al., 2015; Vigano et al., 2010; Vitko et al., 2005; Webster et al., 2006). mTORC1 regulates translation by phosphorylation of eIF4E-binding protein and p70 S6 kinase, and cell growth via phosphorylation of p70 S6 kinase and eukaryotic initiation factor binding protein. CMV replication is dependent on the activation of mTOR and is Granzyme B Inhibitor Z-AAD-CH2Cl inhibited by rapamycin and everolimus (Clippinger et al., 2011a, b; Moorman and Shenk, 2010; Roy and Arav-Boger, 2014). Although extensive studies on the effects of everolimus on CMV without pretreatment have been performed, the underlying mechanisms for decreased episodes of CMV replication are not well understood (Clippinger et al., 2011a, b; Kudchodkar et al., 2007; Kudchodkar et al., 2004; Moorman and Shenk, 2010; Roy and Arav-Boger, 2014). The anti-CMV activity of mTOR inhibitors has been shown immunologically by their mechanism of action, an improvement in CMV-specific CD8/CD4 T cell responses (Havenith et al., 2013; Roy and Arav-Boger, 2014). We have characterised the effects of cyclosporine, tacrolimus, prednisolone, MMF, mizoribine, and azathioprine on CMV plaque formation and replication in treated Granzyme B Inhibitor Z-AAD-CH2Cl (Kuramoto et al., 2010; Shiraki et al., 1990). Prednisolone and cyclosporine enhance CMV growth, and tacrolimus slightly suppresses CMV growth (Shiraki et al., 1991a). These effects on CMV growth by immunosuppressants may be due to a modification of cellular metabolism influencing CMV growth. Mizoribine (Halloran, 2004), which is used mainly in Japan (Ushigome et al., 2016; Yoshimura et al., 2013) and China (Shi et al., 2017), and MMF are inosine monophosphate dehydrogenase inhibitors and used for transplantation. Mizoribine dose-dependently suppresses CMV growth, whereas MMF does not. Mizoribine treatment of CMV-infected cultures resulted in the isolation of two mizoribine-resistant mutants, indicating the direct inhibition of CMV function for replication by mizoribine (Kuramoto et al., 2010).
    Materials and methods
    Discussion Because everolimus alleviates CMV infection in transplant recipients, we first tried to characterise the anti-CMV action without pretreatment, as other researchers have done (Kudchodkar et al., 2004, 2007; Moorman and Shenk, 2010). Although CMV replication was not significantly affected by various concentrations of everolimus without preinfection treatment, we found that preinfection treatment and its length were important to demonstrate the suppressing activity of everolimus on spreading CMV infection. Everolimus reduced virus adsorption and the spread of CMV infection, and it reduced and delayed viral DNA synthesis in the cells and the release of viral DNA from cells to the culture supernatant. Everolimus treatment of the cells caused formation of smaller plaques and a reduction to approximately 40% in the total number of infected cells in the CMV-infected culture on day 9, corresponding to two to three replication cycles. The average plaque size was reduced from 0.11 mm2 to 0.04 mm2 (36.4%), and this was similar to the total number of infected cells assessed by the infectious centre assay. This reduction in the total number of infected cells to 40% in the plate (two dimensions) on day 9 corresponded to 0.4 × 0.4 = 0.16 in the solid (three dimensions), indicating that the volume of CMV lesions was reduced to one-sixth by everolimus. Low and delayed CMV replication was supported by the delay and reduction in viral DNA synthesis and viral release in everolimus-treated cultures. The mechanisms of this everolimus anti-CMV action may be complex processes, and many cellular factors might contribute to anti-CMV activity. CMV replication is highly dependent on cellular factors, and everolimus modifies many cellular transcriptional and translational processes related to CMV replication. The target of everolimus, mTORC1, regulates translation and cell growth, and the importance of pretreatment of the cells indicated that many factors might contribute to the observed anti-CMV activity. The mechanism of the suppression of CMV is not clearly understood, although extensive studies on the action of everolimus on CMV have been performed (Clippinger et al., 2011a, b; Kudchodkar et al., 2007; Kudchodkar et al., 2004; Moorman and Shenk, 2010; Roy and Arav-Boger, 2014).