Anti-influenza Virus Activity of Methylthio-Formycin Distinct From That of T-705.

Seasonal influenza virus epidemics result in severe illness, and occasionally influenza pandemics cause significant morbidity and mortality, although vaccines and anti-influenza virus drugs are available. By screening an in-house library, we identified methylthio-formycin (SMeFM), an adenosine analog, as a potent inhibitor of influenza virus propagation. SMeFM inhibited the propagation of influenza A and B viruses (IC50: 34.1 and 37.9 nM, respectively) and viruses showing reduced susceptibility to baloxavir and neuraminidase inhibitors but not T-705 (Favipiravir). However, the combination of T-705 and SMeFM inhibited the propagation of the influenza virus not in an antagonistic but in a slightly synergistic manner, suggesting that SMeFM has targets distinct from that of T-705. SMeFM induced A-to-C transversion mutations in virus genome RNA, and SMeFM triphosphate did not inhibit in vitro viral RNA synthesis. Our results show that SMeFM inhibits the propagation of the influenza virus by a mechanism different from that of T-705 and is a potential drug candidate to develop for anti-influenza drug.

Translation of Hepatitis A Virus IRES Is Upregulated by a Hepatic Cell-Specific Factor.

Many viruses strongly prefer to infect certain cell types, a phenomenon known as "tropism." Understanding tropism's molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5' untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3' end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

Anti-influenza virus activity of a salcomine derivative mediated by inhibition of viral RNA synthesis.

Influenza virus infection is a major threat to global health. Although vaccines and anti-influenza virus drugs are available, annual influenza virus epidemics result in severe illness, and an influenza pandemic occurs every 20-30 years. To identify candidate anti-influenza virus compounds, we screened approximately 5,000 compounds in an in-house library. We identified MZ7465, a salcomine derivative, as a potent inhibitor of influenza virus propagation. We analyzed the antiviral propagation mechanism of the hit compound by determining the amounts of viral proteins and RNA in infected cells treated with or without the hit compound. Treatment of infected cells with MZ7465 decreased both viral protein and RNA synthesis. In addition, an in vitro assay showed that viral RNA synthesis was directly inhibited by MZ7465. These results suggest that salcomine and its derivatives are potential candidates for the treatment of influenza virus infections.

Encountered-Type Haptic Interface for Representation of Shape and Rigidity of 3D Virtual Objects.

This paper describes the development of an encountered-type haptic interface that can generate the physical characteristics, such as shape and rigidity, of three-dimensional (3D) virtual objects using an array of newly developed non-expandable balloons. To alter the rigidity of each non-expandable balloon, the volume of air in it is controlled through a linear actuator and a pressure sensor based on Hooke's law. Furthermore, to change the volume of each balloon, its exposed surface area is controlled by using another linear actuator with a trumpet-shaped tube. A position control mechanism is constructed to display virtual objects using the balloons. The 3D position of each balloon is controlled using a flexible tube and a string. The performance of the system is tested and the results confirm the effectiveness of the proposed principle and interface.

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases.

Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-ß and bone morphogenetic protein signaling.

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG­2, ­3, and ­5 from the bone marrow of green fluorescent protein (GFP)­transgenic mice. These cell lines clearly expressed mouse MSC markers Sca­1 and CD44, and SG­2 and ­5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)­ß superfamily. By contrast, SG­3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG­3 cells, which underwent osteogenesis in response to BMP, while TGF­ß receptor II was most highly expressed in SG­3 and ­5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF­ß1 in SG­2 cells but not in SG­3 or ­5 cells. Furthermore, TGF­ß1 clearly induced the expression of Smad­interacting transcription factor CCAAT/enhancer binding protein­ß in SG­2 but not in SG­3 or ­5 cells. These results demonstrated the establishment of TGF­ß­responsive SG­2 MSCs, BMP­responsive SG­3 MSCs and TGF­ß/BMP­unresponsive SG­5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF­ß superfamily affects the proliferation and differentiation status of MSCs in vivo.

Secreted caveolin-1 enhances periodontal inflammation by targeting gingival fibroblasts.

Caveolin-1 (Cav-1) is a membrane protein. Recently, it has been reported that secreted Cav-1 induces angiogenesis in inflammatory microenvironment. However, it is unclear that Cav-1 regulates gingival inflammation. Therefore, we investigated the Cav-1 function to periodontal cells. Expression of Cav-1 in human periodontitis tissues was examined pathologically. Secretion of Cav-1 from human gingival fibroblasts (HGFs) or human periodontal ligament cells (HPLFs) treated with IL-1ß and TNF-α was examined using Western blotting. Likewise, intracellular signals induced by Cav-1 were examined. Finally, we examined whether the secreted Cav-1 induces production of inflammatory mediators in HGFs using ELISA or qRT-PCR. Pathologically, high expression of Cav-1 was observed in human periodontitis tissues. Cav-1 secretion increased in both cultured HGFs and HPLFs treated with IL-1ß and TNF-α. Cav-1 induced phosphorylation of JNK and ERK, but not Stat3 in HGFs. Furthermore, Cav-1 increased proMMP-1 and VEGF secretion in HGFs, and the VEGF secretion was statistically suppressed by JNK inhibitor SP600125, but not ERK inhibitor PD98059. ProMMP-1 secretion was suppressed statistically by both SP600125 and PD98059. In addition, Cav-1 increased significantly MMP-1, -10 and -14 mRNA expressions, whereas no increase of TIMPs mRNA was observed in HGFs treated with Cav-1. These data suggest that secreted Cav-1 derived from periodontal fibroblastic cells enhances inflammation-related several proteases and VEGF secretion in HGFs via MAPKs pathway, resulting in progression of periodontitis through induction of tissue degradation or angiogenesis.

Efficient propagation of betanodavirus in a murine astrocytoma cell line.

Betanodavirus, a bipartite RNA virus of fishes and a member of Nodaviridae family, targets nervous tissues and is the causative agent of viral nervous necrosis in marine farmed fish. Betanodavirus is thought to be propagated only in fish cells because betanodavirus has only been isolated in fish and it is not well propagated in mammalian culture cells. However, the host specificity of betanodavirus has not yet been well analyzed. To analyze the host specificity of betanodavirus, various mammalian cells were screened for their permissiveness to betanodavirus. As a result, redspotted grouper nervous necrosis virus can be propagated efficiently in the murine astrocytoma cell line, DBT. The level of viral production in DBT was 10-fold-higher than in the fish cell line, E-11. This result is the first to demonstrate the efficient propagation of betanodavirus in mammalian cells and may help to elucidate the mechanism of the host specificity of betanodavirus.