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

Biomechanical Characteristics of Long Stair Climbing in Healthy Young Individuals in a Real-World Study Using a Wearable Motion Analysis System

Background: Stair climbing is a part of the basic activities of daily living. Previous biomechanical analyses of stairs have been conducted in the laboratory, resulting in only a few steps. Therefore, the biomechanical characteristics of long stair climbing in the real world remain unclear. The purpose of this study was to identify differences in kinematic and kinetic in the lower limb between the beginning and end phases of long stair climbing in an outdoor environment using a wearable motion analysis system. Eight subjects (four males and four females) were included in the data analysis (age: 23.6 &plusmn; 0.5 years). The long stair was 66 consecutive steps out of 202 stone steps. A wearable motion analysis system comprised six inertial measurement units and foot pressure sensors. The maximum ankle joint flexion angle in the end phase was significantly increased more than in the beginning phase (p &lt; 0.001). On the other hand, the other kinematic, kinetic, and stair climbing speeds showed no significant difference between the phases. The findings indicated that fatigue during long stair climbing might increase ankle dorsiflexion to compensate for forwarding propulsion

Biomechanical Characteristics of Long Stair Climbing in Healthy Young Individuals in a Real-World Study Using a Wearable Motion Analysis System

Background: Stair climbing is a part of the basic activities of daily living. Previous biomechanical analyses of stairs have been conducted in the laboratory, resulting in only a few steps. Therefore, the biomechanical characteristics of long stair climbing in the real world remain unclear. The purpose of this study was to identify differences in kinematic and kinetic in the lower limb between the beginning and end phases of long stair climbing in an outdoor environment using a wearable motion analysis system. Eight subjects (four males and four females) were included in the data analysis (age: 23.6 ± 0.5 years). The long stair was 66 consecutive steps out of 202 stone steps. A wearable motion analysis system comprised six inertial measurement units and foot pressure sensors. The maximum ankle joint flexion angle in the end phase was significantly increased more than in the beginning phase (p < 0.001). On the other hand, the other kinematic, kinetic, and stair climbing speeds showed no significant difference between the phases. The findings indicated that fatigue during long stair climbing might increase ankle dorsiflexion to compensate for forwarding propulsion

Effectiveness of Combined Treatment using X-rays and a Phosphoinositide 3-kinase Inhibitor, ZSTK474, on Proliferation of HeLa cells in vitro and in vivo

ZSTK474 is a novel orally applicable PI3K-specific inhibitor and strongly inhibits cancer cell proliferation. To explore further the antitumor effect of ZSTK474 for future clinical usage, combined effects with radiation were studied. The proliferation of HeLa cells was inhibited by the treatment with X-rays alone or ZSTK474 alone. The combination treatment with X-rays and ZSTK474 applied after 24 h post-irradiation for 8 days remarkably enhanced the cell growth inhibition. The combined effect was also observed for the clonogenic survival with continuous ZSTK474 treatment. Western blotting showed enhanced phosphorylation of Akt and GSK-3beta by X-irradiation, whereas the phosphorylation was inhibited by the ZSTK474 treatment alone. The treatment with ZSTK474 after X-irradiation also inhibited the phosphorylation. Combination of ZSTK474 and X-rays also remarkably inhibited xenograft tumor growth. The combined treatment with X-rays and ZSTK474 had a great therapeutic potential than the radiation or the drug therapy alone both in vitro and in vivo