Neural stem cells (NSCs) have the ability to proliferate and differentiate into neurons and glia. Regulation of NSC fate by small molecules is important for the generation of a certain type of cell. The identification of small molecules that can induce new neurons from NSCs could facilitate regenerative medicine and drug development for neurodegenerative diseases. In this study, we screened natural compounds to identify molecules that are effective on NSC cell fate determination. We found that Kuwanon V (KWV), which was isolated from the mulberry tree (Morus bombycis) root, increased neurogenesis in rat NSCs. In addition, during NSC differentiation, KWV increased cell survival and inhibited cell proliferation as shown by 5-bromo-2-deoxyuridine pulse experiments, Ki67 immunostaining and neurosphere forming assays. Interestingly, KWV enhanced neuronal differentiation and decreased NSC proliferation even in the presence of mitogens such as epidermal growth factor and fibroblast growth factor 2. KWV treatment of NSCs reduced the phosphorylation of extracellular signal-regulated kinase 1/2, increased mRNA expression levels of the cyclin-dependent kinase inhibitor p21, down-regulated Notch/Hairy expression levels and up-regulated microRNA miR-9, miR-29a and miR-181a. Taken together, our data suggest that KWV modulates NSC fate to induce neurogenesis, and it may be considered as a new drug candidate that can regenerate or protect neurons in neurodegenerative diseases.

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IgG and IgM responses to human papillomavirus L1 virus-like particle as a function of dosing schedule and vaccine formulationMost commercialized virus-like particle (VLP) vaccines use aluminum salt as adjuvant, even though VLPs provoke adequate antibody responses without adjuvant. We do not have detailed knowledge of how adjuvant affects the profile of anti-VLP antibodies. Meanwhile, there is evidence that differences between vaccination protocols influence the glycosylation of antibodies, which may alter their effector functions. In the present study a murine model was used to investigate the effects of dosing schedule and adjuvant on the antibody profiles and glycosylation levels of antigen-specific antibody responses to human papillomavirus type 16 L1 (HPV16 L1) VLPs. Mice received subcutaneously 2,000 ng of antigen divided into 4 or 7 doses. The HPV16 L1 VLPs elicited > 4 log10 anti-HPV16 L1 IgG titers without adjuvant, and aluminum hydroxide as adjuvant increased IgG titers 1.3-to 4-fold and reduced the anti-HPV16 L1 IgG2a / anti-HPV16 L1 IgG1 ratio value (use of aluminum hydroxide reduced the ratio of the IgG2a). Immunization with HPV16 L1 VLPs in combination with Freund's adjuvant enhanced IgG titers 5-to 12fold. Seven-dose immunization markedly increased anti-HPV16 L1 IgM titers compared to four-dose immunization, as well as increasing the proportion of glycosylated antibodies. Our results suggest that antibody glycosylation can be controlled immunologically, and IgG and IgM profiles and glycosylation profiles of the vaccine-induced antibodies can be used as indicators reflecting the vaccine characteristics. These results indicate that the HPV16 L1 VLP dosing schedule can affect the quality of antigen-specific antibody responses. We suggest that dosing schedules should be noted in vaccination protocols for VLP-based vaccines.

Neural stem cells (NSCs) have the ability to proliferate and differentiate into various types of cells that compose the nervous system. To study functions of genes in stem cell biology, genes or siRNAs need to be transfected. However, it is difficult to transfect ectopic genes into NSCs. Thus to identify the suitable method to achieve high transfection efficiency, we compared lipid transfection, electroporation, nucleofection and retroviral transduction. Among the methods that we tested, we found that nucleofection and retroviral transduction showed significantly increased transfection efficiency. In addition, with retroviral transduction of Ngn2 that is known to induce neurogenesis in various types of cells, we observed facilitated final cell division in rat NSCs. These data suggest that nucleofection and retroviral transduction provide high efficiency of gene delivery system to study functions of genes in rat NSCs.