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provides coniferyl ferulate(CAS#:850649-61-5) MSDS, density, melting point, boiling point, structure, formula, molecular weight etc. Articles of coniferyl ferulate are included as well.>> amp version: coniferyl ferulate
The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo. Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against Flaviviridae viruses.
IMPORTANCE The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinant Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals against Flaviviridae viruses.
Flaviviridae, reporter virus, antiviral screening, in vivo dynamics
Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag
Tomokazu Tamura,a Takasuke Fukuhara,corresponding authora Takuro Uchida,b Chikako Ono,a Hiroyuki Mori,a Asuka Sato,a Yuzy Fauzyah,a Toru Okamoto,a Takeshi Kurosu,c Yin Xiang Setoh,d Michio Imamura,b Norbert Tautz,e Yoshihiro Sakoda,f Alexander A. Khromykh,d Kazuaki Chayama,b and Yoshiharu Matsuuracorresponding authora
2018 Jan 15
In plants, cytokinin (CK) perception and signaling pathway is composed by a histidine kinase receptor (HK) and a response regulator (RR), the signal being mediated by a histidine phosphotransfer (HPt), as described in Arabidopsis, maize and rice. From database searches we identified in grapevine three HKs, three HPs, four A-type RRs and six B-type RRs, suggesting a common mechanism for grapevine. The phylogenetic analysis of these Vitis genes showed a variable but high degree of homology with Arabidopsis sequences. When sulfate was withdrawn from the culture medium (−S) of in vitro Vitis shoots, we assessed a significant reduction in shoot branching. To ascertain the crosstalk of S status with CK signaling in grapevine, control and −S grown shoots and control, −S and −CK cell suspensions were used as experimental systems. Real-time PCR was elected to quantify the expression of key genes. The expression of CK receptor genes was downregulated in −S cells while not affected in −CK cells. In differentiated shoots no response to −S was observed on those genes. A-type VvRRa4 was downregulated in −S or −CK cells while Vitis B-type RRs did not respond either to CK or S starvation. The results suggest that Vitis CK signaling pathway is affected by −S, although differently according to the model system. Transcription of Vitis apical meristem-identity genes VvWUS, VvCLV and VvSTM and axillary meristem genes VvBRC1, VvBRC2, VvLAS, VvRAX and VvREV was estimated and VvSTM and VvLAS showed to be downregulated in −S. Then, the expression levels of VvSTM and VvLAS make them strong candidates to be associated with the branching pattern of Vitis shoots in −S.
apical meristem genes, axillary meristem genes, cytokinin signaling genes, sulfur, vitis
Identification and expression of cytokinin signaling and meristem identity genes in sulfur deficient grapevine (Vitis vinifera L.)
João Fernandes, Silvia Tavares, and Sara Amanciocorresponding author