Multinucleation of Incubated Cells and Their Morphological Differences Compared to Mononuclear Cells

Some cells cultured in vitro have multiple nuclei. Since cultured cells are used in various fields of science, including tissue engineering, the nature of the multinucleated cells must be determined. However, multinucleated cells are not frequently observed. In this study, a method to efficiently obtain multinucleated cells was established and their morphological properties were investigated. Initially, we established conditions to quickly and easily generate multinucleated cells by seeding a Xenopus tadpole epithelium tissue-derived cell line (XTC-YF) on less and more hydrophilic dishes, and incubating the cultures with medium supplemented with or without Y-27632—a ROCK inhibitor—to reduce cell contractility. Notably, 88% of the cells cultured on a less hydrophilic dish in medium supplemented with Y-27632 became multinucleate 48 h after seeding, whereas less than 5% of cells cultured under other conditions exhibited this morphology. Some cells showed an odd number (three and five) of cell nuclei 72 h after seeding. Multinucleated cells displayed a significantly smaller nuclear area, larger cell area, and smaller nuclear circularity. As changes in the morphology of the cells correlated with their functions, the proposed method would help researchers understand the functions of multinucleated cells

Decoding the Effect of Hydrostatic Pressure on TRPV1 Lower-Gate Conformation by Molecular-Dynamics Simulation.

In response to hydrostatic pressure, the cation channel transient receptor potential vanilloid 1 (TRPV1) is essential in signaling pathways linked to glaucoma. When activated, TRPV1 undergoes a gating transition from a closed to an open state that allows the influx of Ca2+ ions. However, the gating mechanism of TRPV1 in response to hydrostatic pressure at the molecular level is still lacking. To understand the effect of hydrostatic pressure on the activation of TRPV1, we conducted molecular-dynamics (MD) simulations on TRPV1 under different hydrostatic pressure configurations, with and without a cell membrane. The TRPV1 membrane-embedded model is more stable than the TPRV1-only model, indicating the importance of including the cell membrane in MD simulation. Under elevated pressure at 27.6 mmHg, we observed a more dynamic and outward motion of the TRPV1 domains in the lower-gate area than in the simulation under normal pressure at 12.6 mmHg. While a complete closed-to-open-gate transition was not evident in the limited course of our MD simulations, an increase in the channel radius at the lower gate was observed at 27.6 mmHg versus that at 12.6 mmHg. These findings provide novel information regarding the effect of hydrostatic pressure on TRPV1 channels

Novel biaxial tensile test for studying aortic failure phenomena at a microscopic level

<p>Abstract</p> <p>Background</p> <p>An aortic aneurysm is a local dilation of the aorta, which tends to expand and often results in a fatal rupture. Although larger aneurysms have a greater risk of rupture, some small aneurysms also rupture. Since the mechanism of aortic rupture is not well understood, clarification of the microstructure influencing the failure to rupture is important. Since aortic tissues are stretched biaxially <it>in vivo</it>, we developed a technique to microscopically observe the failure of an aortic rupture during biaxial stretch.</p> <p>Methods</p> <p>A thinly sliced porcine thoracic aortic specimen was adhered to a circular frame and pushed onto a cylinder with a smaller diameter to stretch the specimen biaxially. To induce failure to rupture at the center, the specimen was thinned at the center of the hole as follows: the specimen was frozen while being compressed with metal plates having holes, which were 3 mm in diameter at their centers; the specimen was then sliced at 50-μm intervals and thawed.</p> <p>Results</p> <p>The ratio of the thickness at the center to the peripheral area was 99.5% for uncompressed specimens. The ratio decreased with an increase in the compression ratio <it>ε</it>_c and was 47.3% for specimens with <it>ε</it>_c = 40%. All specimens could be stretched until failure to rupture. The probability for crack initiation within the cylinder was <30% and 100% for specimens with <it>ε</it>_c <10% and <it>ε</it>_c >30%, respectively. Among specimens ruptured within the cylinder, 93% of those obtained from the mid-media showed crack initiation at the thin center area.</p> <p>Conclusions</p> <p>Aortic tissues were successfully stretched biaxially until failure, and their crack initiation points were successfully observed under a microscope. This could be a very useful and powerful method for clarifying the mechanism of aortic rupture. We are planning to use this technique for a detailed investigation of events occurring at the point of failure when the crack initiates in the aortic aneurysm wall.</p

A Novel Method for Measuring Tension Generated in Stress Fibers by Applying External Forces

The distribution of contractile forces generated in cytoskeletal stress fibers (SFs) contributes to cellular dynamic functions such as migration and mechanotransduction. Here we describe a novel (to our knowledge) method for measuring local tensions in SFs based on the following procedure: 1), known forces of different magnitudes are applied to an SF in the direction perpendicular to its longitudinal axis; 2), force balance equations are used to calculate the resulting tensions in the SF from changes in the SF angle; and 3), the relationship between tension and applied force thus established is extrapolated to an applied force of zero to determine the preexisting tension in the SF. In this study, we measured tensions in SFs by attaching magnetic particles to them and applying known forces with an electromagnetic needle. Fluorescence microscopy was used to capture images of SFs fluorescently labeled with myosin II antibodies, and analysis of these images allowed the tension in the SFs to be measured. The average tension measured in this study was comparable to previous reports, which indicates that this method may become a powerful tool for elucidating the mechanisms by which cytoskeletal tensions affect cellular functions