Construction of an All-in-one Double-conditional shRNA Expression Vector

Gene silencing by RNA interference （RNAi） is widely used for assessing gene function. An important advance in the RNAi field was the discovery that plasmid-based RNAi can substitute for synthetic small interfering RNA in vitro and in vivo. However, constitutive and ubiquitous knockdown of gene expression by RNAi in mice can limit the scope of experiments because this process can lead to embryonic lethality, or result in compensatory overexpression of other genes such that no phenotypic abnormalities occur. Either way, analyses of the physiological roles of the gene of interest in adult mice are not possible. To overcome these limitations, we previously constructed a double-conditional short-hairpin RNA （shRNA） expression vector that can regulate shRNA expression in a spatio-temporal manner with a tetracycline-inducible floxed stuffer sequence selectively excised by application of Cre recombinase. In this study, we aimed to modify this vector to create an all-in-one vector that produces double-conditional transgenic mice through a single round of gene transfer to fertilized eggs. We added a coding region for nuclear localizing Cre （NCre） recombinase with a multi-cloning site for a cell-specific promoter into the double-conditional short-hairpin RNA （shRNA） expression vector that we previously constructed. Using Escherichia coli, we confirmed successful construction of the vector. First, we confirmed isopropyl-β-D-thiogalactopyranoside-induced expression of NCre recombinase through the lac operon as a specific promoter by western blotting. Second, we confirmed functional recombination of the floxed sequence of loxP-like TATA-lox by analysing restriction enzyme-digested fragments. This all-in-one double-conditional shRNA expression vector will be useful for reversible in vitro and in vivo knockdown of target gene expression, in target cells via promoter-specific expression of NCre, and at specific times by tetracycline application

Plasmodium falciparum: Differential Selection of Drug Resistance Alleles in Contiguous Urban and Peri-Urban Areas of Brazzaville, Republic of Congo

The African continent is currently experiencing rapid population growth, with rising urbanization increasing the percentage of the population living in large towns and cities. We studied the impact of the degree of urbanization on the population genetics of Plasmodium falciparum in urban and peri-urban areas in and around the city of Brazzaville, Republic of Congo. This field setting, which incorporates local health centers situated in areas of varying urbanization, is of interest as it allows the characterization of malaria parasites from areas where the human, parasite, and mosquito populations are shared, but where differences in the degree of urbanization (leading to dramatic differences in transmission intensity) cause the pattern of malaria transmission to differ greatly. We have investigated how these differences in transmission intensity affect parasite genetic diversity, including the amount of genetic polymorphism in each area, the degree of linkage disequilibrium within the populations, and the prevalence and frequency of drug resistance markers. To determine parasite population structure, heterozygosity and linkage disequilibrium, we typed eight microsatellite markers and performed haplotype analysis of the msp1 gene by PCR. Mutations known to be associated with resistance to the antimalarial drugs chloroquine and pyrimethamine were determined by sequencing the relevant portions of the crt and dhfr genes, respectively. We found that parasite genetic diversity was comparable between the two sites, with high levels of polymorphism being maintained in both areas despite dramatic differences in transmission intensity. Crucially, we found that the frequencies of genetic markers of drug resistance against pyrimethamine and chloroquine differed significantly between the sites, indicative of differing selection pressures in the two areas

Potential usefulness of 75-g oral glucose tolerance test using the flash glucose monitoring system in a comprehensive medical examination

目的：本研究では，人間ドック健診におけるフラッシュグルコースモニタリングシステム（FGM）を用いた75 g 経口ブドウ糖負荷試験（OGTT）の有用性を検討することを目的とした。方法：人間ドック健診を受診した健常ボランティア64 名を対象に，FGM（FreeStyle リブレPro®）センサーを装着して75 g OGTT を実施した。静脈血漿血糖値（PG）は60 分ごとに計3 回，FGM による間質液グルコース濃度（FGM-IG）は15 分ごとに計9 回測定し，PG とFGM-IG の関連を検討した。結果：PG とFGM-IG との間に有意な正の相関を認めた。FGM の精度を評価するコンセンサスエラーグリッド解析の結果，99.5 % の測定値が臨床的に利用可能とされる範囲内に該当し，平均絶対的相対的差異は13.7％であった。糖負荷前のFGM-IG はPG と比較して有意に低値であった。対象者64 名中60 名のOGTT において，PG とFGM-IG による判定が一致した。15 分ごとのFGM-IG の測定によって，60 分ごとのPG を用いた通常のOGTT では評価できないグルコース変動を捉えることが可能であった。結論：簡便かつ侵襲少なくグルコース濃度を測定できるFGM は，75 g OGTT において有用である可能性が示唆された

A Synergistic Antitumor Effect of Rituximab and Gamma Interferon Combined Therapy on Human CD20＋ B-Cell Lymphoma Cells

Rituximab （RTX） is an anti-CD20 human-mouse chimeric monoclonal antibody that exhibits antibody-dependent, cell-mediated cytotoxicity and complement-dependent cytotoxicity, resulting in an antitumor effect with immune cells or complement. RTX is approved for the treatment of many diseases including B cell lymphoma and rheumatoid arthritis. We examined whether combined RTX and gamma interferon （IFNγ） therapy provides a higher antitumor effect than RTX single therapy using B-cell lymphoma cells. In addition, we investigated the mechanisms underlying the antitumor effect. We treated tumor-derived cell lines with RTX alone, IFNγ alone, or a combination of RTX and IFNγ（RTX-IFNγ）. Untreated cells served as controls. We experimentally examined in vitro cell proliferation, conducted apoptosis and cell cycle assays, performed Western blotting to identify changes in the levels of proteins related to the cell cycle, and investigated tumor growth in a mouse xenograft experiment. Cell proliferation experiments indicated that RTX or IFNγ alone did not significantly suppress cell growth compared with the control, whereas treatment with RTX-IFNγ significantly suppressed cell proliferation. In vivo mouse experiments also showed that the administration of RTX-IFNγ significantly suppressed tumor growth compared to the single therapies. Some tumors in mice treated with RTX-IFNγ were completely resolved. The cell cycle assays revealed a significantly increased rate of cells in the G0/G1 phase following treatment with RTX-IFNγ compared with the other groups, and the levels of p27kip1 increased and the levels of cyclin E and Cdk 2 decreased in cells treated with RTX-IFNγ. Our findings suggested that RTX-IFNγ combined therapy directly affects cells by arresting the cell cycle at the G1/S checkpoint and had a synergistic antitumor effect compared to RTX single treatment of B-cell lymphomas. This combined therapy may change the mortality rate for B-cell lymphomas