Role of Preschool and Primary School Children in Epidemics of Influenza A in a Local Community in Japan during Two Consecutive Seasons with A(H3N2) as a Predominant Subtype

Enhanced influenza surveillance was implemented to analyze transmission dynamics particularly driving force of influenza transmission in a community during 2011/12 and 2012/13 seasons in Odate City, Japan. In these two consecutive seasons, influenza A(H3N2) was the predominant influenza A subtype. Suspected influenza cases were tested by commercial rapid test kits. Demographic and epidemiological information of influenza positive cases were recorded using a standardized questionnaire, which included age or age group, date of visit, date of fever onset, and the result of rapid test kit. Epidemiological parameters including epidemic midpoint (EM) and growth rate (GR) were analyzed. In 2012/13 season, numbers of influenza A positive cases were significantly lower among preschool (212 cases) and primary school (224 cases) children than in 2011/12 season (461 and 538 cases, respectively). Simultaneously, total influenza A cases were also reduced from 2,092 in 2011/12 season to 1,846 in 2012/13 season. The EMs in preschool and primary school children were earlier than EMs for adult and all age group in both 2011/12 and 2012/13 seasons. The GR in 2012/13 season was significantly lower than that in 2011/12 season (0.11 and 0.18, respectively, p = 0.003). Multiple linear regression analysis by school districts revealed that GRs in both seasons were significantly correlated with the incidence of school age children. Our findings suggest that preschool and primary school children played an important role as a driving force of epidemics in the community in both 2011/12 and 2012/13 seasons. The reduction of total influenza A cases in 2012/13 season can be explained by decreased susceptible population in these age groups due to immunity acquired by infections in 2011/12 season. Further investigations are needed to investigate the effect of pre-existing immunity on influenza transmission in the community.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

A lycoperdaceous fungus, Arachnion album (Agaricales, Arachniaceae), newly found in Japan

Arachnion album was collected in Osaka and Hyogo Prefectures, western Honshu, Japan. This is a new record of this genus for Japan. Macro- and microscopic features of the species are described and illustrated based on the Japanese specimens, and comparisons with some related taxa of A. album are given.     

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

Activation of the T‐cell receptor (TCR) and that of the B‐cell receptor (BCR) elicits tyrosine‐phosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large‐scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine‐phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine‐phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine‐phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level.     

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.     

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

Human γδ T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.¹ The majority of γδ T cells in peripheral blood have the Vγ9Vδ2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.¹Therefore, γδ T cells are attractive candidate effector cells for cancer immunotherapy. Vγ9Vδ2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;² and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.³ In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of γδ T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and γδ T cells activation.³ Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to γδ T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating γδ T cells with aminobisphosphonates. ⁴ Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. ⁵ Monocytes that accumulate IPP become antigen-presenting cells and stimulate Vγ9Vδ2 T cells in the peripheral blood.⁶ Based on these mechanisms, we developed a technique for large-scale expansion of γδ T cell cultures using zoledronate and interleukin-2 (IL-2).⁷ Other methods for expansion of γδ T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)⁸ or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).⁹ All of these methods allow ex vivo expansion, resulting in large numbers of γδ T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded γδ T cells display CD27^-CD45RA^- effector memory phenotype and thier function can be evaluated by IFN-γ production assay. ⁷Download video file.^{(76M, mov)}     

Modification of secondary head-forming activity of microinjected ∆β-catenin mRNA by co-injected spermine and spermidine in Xenopus early embryos

Polyamines are essential for cell growth and differentiation. In Xenopus early embryos, per embryo level of spermine is extremely low as compared with that of spermidine. To disclose the possible function of polyamines in Xenopus early embryos, we tested the effect of co-injection of spermine and spermidine on the functioning of exogenously microinjected in vitro-synthesized, ?β-catenin mRNA which is known to induce a secondary head after being microinjected into a ventral vegetal blastomere in 8-celled Xenopus embryos. Microinjection of ?β-catenin mRNA in fact induced a secondary axis with a secondary head, and co-injection of spermine or spermidine suppresses induction of the secondary head and secondary axis without drastic effects like induction of immediate cell death or execution of apoptosis at blastula stage. The inhibitory effects were dosage dependent, and at lower doses the inhibition was mainly on secondary head formation rather than on secondary axis formation. We performed similar experiments using GFP mRNA and confirmed that expression of GFP mRNA was also suppressed by co-injection of spermine. We mixed ?β-catenin mRNA with different amounts of spermine and performed electrophoresis on agarose gels, with a finding that the prior mixing greatly suppressed mRNA migration. These results suggest that an excess amount of spermine as well as spermidine exerts inhibitory effects on mRNA translation, and that the inhibition may be due to direct binding of polyamines to mRNA and a reduction of negative charges on mRNA molecules that might also induce the formation of cross link-like networks among mRNAs.     

Effects of temperature,salinity and their interaction on growth of the benthic dinoflagellate Ostreopsis cf. ovata (Dinophyceae) from Japanese coastal waters

Benthic dinoflagellates of the genus Ostreopsis produce palytoxin (PTX)‐like compounds. The worldwide distributed Ostreopsis ovata/O. cf. ovata is potentially responsible for outbreaks of human health problems around the coasts of tropical, subtropical, and temperate regions. The present study examined growth responses of an O. cf. ovata strain s0662 collected from coastal waters of Japan with 35 different combinations of temperature (15–35°C) and salinity (20–40) and discusses the bloom dynamics of the organism in Japanese coastal environments. The O. cf. ovata strain s0662 tolerated a wide range of temperature (17.5–35°C) and salinity (25–40). Results of a two‐way ANOVA showed significant effects of temperature‐salinity interaction on growth rates and biomass yields of the O. cf. ovata strain (F(24,70) > 127, P < 0.001). The strain showed a maximal growth rate (1.03 divisions day^?1) and biomass yield (240 relative fluorescence) at temperature 25°C and salinity 30. The high growth rates of over 1.0 division day^?1 were obtained in conditions of temperature 25–30°C and salinity 30–35, which indicates that strain s0662 prefers high temperature and salinity conditions. The growth rates of O. cf. ovata under the optimal conditions were higher than those of other benthic toxic‐dinoflagellates, Coolia monotis, Gambierdiscus toxicus, and Prorocentrum lima (Dinophyceae) previously reported. Taken together, we suggest that O. cf. ovata is able to grow faster than the other benthic dinoflagellates in waters of high temperature and salinity. The physiological feature probably confers an ecological advantage on O. cf. ovata in the bloom development during warmer seasons in Japan and may be responsible for outbreaks of PTX‐like poisoning in the region especially during the warmer seasons.