Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells

<p>Abstract</p> <p>Background</p> <p>The specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including <it>m-Numb </it>and <it>p21 </it>mRNAs. <it>In vitro </it>experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating <it>Msi1 </it>expression are not yet clear.</p> <p>Results</p> <p>To identify the DNA region affecting <it>Msi1 </it>transcription, we inserted the fusion gene <it>ffLuc</it>, comprised of the fluorescent <it>Venus </it>protein and firefly <it>Luciferase</it>, at the translation initiation site of the mouse <it>Msi1 </it>gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the <it>Msi1 </it>transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for <it>Msi1 </it>transcription in NS/PCs.</p> <p>Conclusions</p> <p>A regulatory element for <it>Msi1 </it>transcription in NS/PCs is located in the sixth intron of the <it>Msi1 </it>gene. The 595-bp D5E2 intronic enhancer can transactivate <it>Msi1 </it>gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.</p

Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

<p>Abstract</p> <p>Background</p> <p><it>HOX </it>genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.</p> <p>Results</p> <p>In this study, we found high expression of the <it>HOXD9 </it>gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of <it>HOXD9 </it>in gliomas, we silenced its expression in the glioma cell line U87 using <it>HOXD9</it>-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that <it>HOXD9 </it>contributes to both cell proliferation and/or cell survival. The <it>HOXD9 </it>gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. <it>HOXD9 </it>siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of <it>HOXD9 </it>in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).</p> <p>Conclusions</p> <p>Our results suggest that <it>HOXD9 </it>may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.</p

RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways

Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P<0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI3 kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI3 kinase/Akt signaling

Quantifying the 60-Year Contribution of Japanese Zoos and Aquariums to Peer-Reviewed Scientific Research

With the shift in their social roles, modern zoos and aquariums are required to develop scientific research. Although zoos and aquariums worldwide have reported an increase in the number of papers they publish and the diversification of their fields in recent decades, the specific circumstances in Japan are slightly unclear. We listed peer-reviewed papers authored by Japanese zoos and aquariums using search engines and quantitatively evaluated the changes in the number of papers published over 62 years. Our results showed that papers published in Japan have increased remarkably since the 1990s, and research fields have diversified as in the rest of the world. In particular, joint research with research institutes has seen an upward trend, and the instances of English-language papers have increased. Meanwhile, the content of the research was biased. In zoos, research on animal welfare has been increasing, but the focus was heavily biased toward captive mammals. Aquariums contributed to the understanding of local ecosystems through the fundamental study of wildlife, but there were fewer papers on improving husbandry. Our results indicated that while research by Japanese zoos and aquariums is developing, research on welfare, conservation, and education regarding native endangered species must still be improved

Oncolytic Virus Therapy with HSV-1 for Hematological Malignancies

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Activity Status and issues of the Diversity Exchange Salon

［目的］ 　 多様性交流サロン「みんなのひろば☆きらり」（以下、「☆きらり」）は、2022年5 月から、地域活動を開始した。本調査は、「☆きらり」の活動の中心的な存在である学生運営委員（活動）の活動状況と課題を明らかにすることを目的とし行う。 ［調査方法］　 　 「☆きらり」の活動に参加している学生運営委員（活動）に研究依頼・説明書を配布し、補足説明した後、研究への参加の同意を得た。同意の得られた学生運営委員（活動） 8 名に、インタビュー調査の許可を得て、対面やリモート（zoom）で20分程度の半構造化面接を実施した。 ［結果］　 　 学生運営委員（活動）の活動状況は、【多様性のある方の参加】【人と人とのかかわり】【地域の居場所】【活動内容の検討】【活動の方法】【円滑な情報共有】【責任意識】の7 つのカテゴリーと29のコードに分類された。 　 「今後したいこと」については、【有効な広報活動】【地域交流の拡大】【学生運営委員（活動）の増員】【参加者の多様性】【多彩な活動内容】【地域の方との活動】【活動方法の工夫】【成長が伴う活動】の8 カテゴリー、29のコードに分類された。 ［考察・まとめ］　 　　調査結果から以下の４ つの内容が示された 　①活動を継続するためには、今後も円滑な情報共有が望まれる。 　② 今後したいことの実現のためには、学生自身が「地域の一員」であり、「多様性のある人」であることを自覚し、活動に参加することや「☆きらり」の目的を正しく認識することが必要である。 　③ 学生運営委員（広報）も地域活動の役割を担い、地域への貢献の一助となっていることが示唆された。学生運営委員（活動）との交流を図り、互いの思いや意見を確認し合ういながらすすめていくことが必要である。 　④ 学生運営委員（活動）はしっかりとした活動プロセスを踏まえ、有意義な活動につながるよう目標をもちながら実践していくことが望ましい。【査読なし】departmental bulletin pape