Systems pharmacology approach to investigate the mechanism of Artemisia argyi in treating rheumatic diseases.

Artemisia argyi (AA) has been proven to be effective in the adjuvant treatment of rheumatism (RA), but the mechanism of its action in RA is not clear. This study aims to clarify the molecular mechanism of AA as a potential therapy for RA by using network pharmacology. The TCM systems pharmacology (TCMSP) was used to screen the active components of AA, and identification of the potential target genes of active compounds and rheumatism was performed with PharmMapper and GeneCards, respectively. Construction of complex target networks and protein-protein interaction networks was based on the Cytoscape software. The biological functions and pathway analysis of targets and effective targets were analyzed using DAVID. Our study demonstrated that 105 target genes were associated with these active compounds and RA. ALB, AKT1, and MAPK1 were the first three hub genes, and the metabolic and signaling pathways related to these hub genes were remarkably abundant. Results showed that AA might play a role in RA by affecting multiple targets and multiple ways, reflecting that TCM was characterized by multicomponents and multitargets. AA has the potential to be a promising new candidate for the treatment of RA and has value for further research and development.

Preparation of a polyclonal antibody against the non-structural protein, NSs of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is newly discovered virus which is the member of the order Bunyavirales, family phenuiviridae, phlebovirus genus. Its genome is composed of 3 segments of negative-sense RNA L, M and S. NSs is a non structure protein encoded by S segment which is important for viral replication and virulence. NSs protein of SFTSV is only involved in the regulation of host innate immune responses and suppression of IFN-promoter activities. So, the exact functions of this protein need to be studied deeply. To understand the exact role of NSs from SFTSV in viral replication and host immune response, a qualified antibody against this protein is required. In this study, NSs gene of SFTSV, was cloned into a bacterial expression vector (pGEX-6P-1) and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. The SFTSV NSs fusion protein was purified using Glutathione Sepharose 4B and utilized as an antigen to immunize rabbits and obtain an anti-SFTSV NSs polyclonal antibody. Proper expression of the fusion protein and polyclonal antibody specificity were confirmed by western blotting and immunofluorescence analyses. The polyclonal antibody recognized NSs from SFTSV specifically. This is the first report that NSs can form viroplasm-like structures not only in infected cells but also in transfected cells with NSs plasmids. This polyclonal antibody will be useful for future studies of NSs functions.

The effects of autophagy on the replication of Nelson Bay orthoreovirus.

BACKGROUND: Nelson Bay orthoreovirus (NBV) was first isolated over 40 years ago from a fruit bat in Australia. Normally, NBV does not cause human diseases, but recently several NBV strains have been associated with human respiratory tract infections, thus attracting clinical attention. Autophagy, an evolutionarily conserved process in eukaryotic cells, degrades intracellular substrates, participates in multiple physiological processes, and maintains cellular homeostasis. In addition, autophagy is intimately involved in viral infection. METHODS: A new strain of NBV, isolated from a patient with a respiratory tract infection who returned to Japan from Bali, Indonesia, in 2007, was used in this study. NBV was rescued using a reverse genetics system involving cotransfection of BHK cells with 11 plasmids (pT7-L1 MB, pT7-L2 MB, pT7-L3 MB, pT7-M1 MB, pT7-M2 MB, pT7-M3 MB, pT7-S1 MB, pT7-S2 MB, pT7-S3 MB, pT7-S4 MB, and pcDNA3.1-T7), yielding NBV-MB. Recovered viruses were confirmed by immunofluorescence. The effect of NBV-MB on autophagy was evaluated by measuring the LC3-I/II proteins by immunoblot analysis after infection of BHK cells. Furthermore, after treatment with rapamycin (RAPA), 3-methyladenine (3-MA), chloroquine (CQ), or plasmid (GFP-LC3) transfection, the changes in expression of the LC3 gene and the amount of LC3-I/II protein were examined. In addition, variations in viral titer were assayed after treatment of BHK cells with drugs or after transfection with plasmids pCAGM3 and pCAGS3, which encode virus nonstructural proteins µNS and σNS, respectively. RESULTS: NBV-MB infection induced autophagy in host cells; however, the level of induction was dependent on viral replication. Induction of autophagy increased viral replication. By contrast, inhibiting autophagy suppressed NBV replication, albeit not significantly. The NBV-MB nonstructural protein µNS was involved in the induction of autophagy with viral infection. CONCLUSIONS: NBV-MB infection triggered autophagy. Also, the NBV nonstructural protein µNS may contribute to augmentation of autophagy upon viral infection.

Association of XPD and XRCC1 genetic polymorphisms with hepatocellular carcinoma risk.

AIM: XRCC1 and XPD are two major repair genes involved in nucleotide excision repair (NER), which is reported to be associated with risk of several cancers. We explored the association of XRCC1 and XPD polymorphisms with the risk of HCC. METHODS: A total of 410 cases with HCC and 410 health controls were collected. XRCC1 Arg194Trp, XRCC1 Arg399Gln, XPD Lys751Gln and XPD Asp312Asn genotyping was performed by duplex polymerase-chain-reaction with the confronting-two-pair primer (PCR-CTPP) method. RESULTS: XRCC1 194Trp/Trp was strongly significantly associated with an increased risk of HCC cancer when compared with the wide-type genotype (OR=2.26, 95% CI=(1.23-5.38). Individuals carrying the XRCC1 399Gln/ Gln showed increased risk of HCC (OR=1.74, 95%CI=1.06-2.74). The XPD 751Gln/Gln and Gln allele genotype were associated with strong elevated susceptibility to HCC (OR=3.51 and 1.42, respectively). CONCLUSION: These results suggest that polymorphisms in XRCC1 and XPD may have functional significance in risk of HCC.