Disruption of cell polarity induced by TGF-β1 treatment prevented folding.

<p><i>(A)</i> F-actin (green) and gp135 (red) fluorescence. Cells were treated with TGF-β1 (1.5 ng/ml) for 2 days before fixation. Bar = 25 µm. <i>(B)</i> Time-lapse images of polarity-disrupted epithelial colonies after the gel overlay. Numbers in the images represent the observation time (h). Bar = 100 µm.</p

Inhibition of either integrin-β1 or Rac1, but not ROCK, delayed folding.

<p><i>(A)</i> The scatter plot shows the migration distance from the outer periphery to the leading edge for each treatment. Observation time corresponds to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099655#pone-0099655-g003" target="_blank">Fig. 3</a>. The equation used to calculate the average distance is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099655#s2" target="_blank">Materials and Methods</a>. The mean values of three independent experiments are shown. <i>(B)</i> Histogram indicating the mean ratio of the migration velocity in the presence of inhibitors. The ratio was calculated by dividing the migration velocity after inhibitor treatment by the velocity before treatment. The mean values are shown with SD (shown as error bars) from three independent experiments, *<i>p<0.05</i>, **<i>p<0.01</i>.</p

Temporal images of the computational simulation of epithelial sheet folding.

<p>The numbers above the images show relative time (s) from the start of modeling. <i>(A)</i> As a control, the parameters are modulated appropriately. <i>(B)</i> The rigidity of the upper substrate (red springs, the parameter in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099655#pone-0099655-g006" target="_blank">Fig. 6<i>D</i></a>) is increased. <i>(C)</i> The rigidity of the lower substrate (red springs) is increased.</p

Definitions and parameters of the simulation.

<p><i>(A)</i> 2D model of an MDCK cell: An MDCK cell is illustrated as a blue circle with a radius of <i>R</i>. <i>X-Z</i> section of an MDCK sheet is represented as a chain of the circles. The origin is the center of the chain. The circles are numbered in order from the negative edge of the chain, represented as <i>i</i> in subscripts; <i>j = i</i>+1. A white dot indicates the center of <i>i</i>^th blue circle, represented by <i>r_ci</i>. <i>(B)</i> Force causing random motion: Two black dots on the periphery of a circle move randomly on the <i>X-Z</i> plane. Their positions are represented as <i>r_ti</i> or <i>r_bi</i>; <i>t</i> or <i>b</i> in subscript indicate top or bottom, respectively. The two dots are connected by a spring with a natural length of <i>L</i>, (black line). <i>(C)</i> The repulsive force is represented by a spring between adjacent circles. <i>(D)</i> Shear tolerance: composed of pairs of parallel and crossed springs (black) between neighboring circles. <i>(E)</i> Elastic force generated by the surrounding substrate: represented by springs on the surface of the circles. <i>B_x</i> or <i>B_z</i> is initial <i>X</i> or <i>Z</i> coordinate of <i>r_ci</i>, respectively. The subscripted <i>p</i> or <i>n</i> indicates <i>B_x</i> of the positive or negative edge, respectively. <i>(F)</i> Migration force: represented as red dots that apply force to the coordinate origin. This force is applied to the <i>r_ti</i> at the edges.</p

Epithelial sheets formed luminal structures by folding after the collagen gel overlay.

<p><i>(A)</i> Time-lapse images of an epithelial colony after the collagen gel overlay. Images were acquired using a phase contrast microscope. Numbers in the images represent the relative time from the start of the observation. The orange dotted line corresponds to the horizontal axis of the kymograph in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099655#pone-0099655-g002" target="_blank">Fig. 2<i>B</i></a>. The orange arrowheads indicate the position of the leading edge of the MDCK sheets, and they correspond with the kymograph. Bar = 100 µm. <i>(B)</i> Kymograph of epithelial folding. <i>(C)</i> Fluorescent images of F-actin (green) and collagen (red). Bar = 20 µm. <i>(D)</i> Images were acquired using a confocal fluorescence microscope. Green represents AG-CAAX (plasma membrane domain). <i>X-Z</i> sectional views were merged from the area indicated by the dotted line in an <i>X-Y</i> sectional view. Bar = 20 µm.</p

Real-Time Measurement of Antiglaucoma Drugs in Porcine Eyes Using Boron-Doped Diamond Microelectrodes

The primary treatment for glaucoma, the most common cause of intermediate vision impairment, involves administering ocular hypotensive drugs in the form of topical eye drops. Observing real-time changes in the drugs that pass through the cornea and reach the anterior chamber of the eye is crucial for improving and developing safe, reliable, and effective medical treatments. Traditional methods for measuring temporal changes in drug concentrations in the aqueous humor employ separation analyzers such as LC–MS/MS. However, this technique requires multiple measurements on the eyes of various test subjects to track changes over time with a high temporal resolution. To address this issue, we have developed a measurement method that employs boron-doped diamond (BDD) microelectrodes to monitor real-time drug concentrations in the anterior chamber of the eye. First, we confirmed the electrochemical reactivity of 13 antiglaucoma drugs in a phosphate buffer solution with a pH of 7.4. Next, we optimized the method for continuous measurement of timolol maleate (TIM), a sympathetic beta-receptor antagonist, and generated calibration curves for each BDD microelectrode using aqueous humor collected from enucleated porcine eyes. We successfully demonstrated the continuous ex vivo monitoring of TIM concentrations in the anterior chambers of these enucleated porcine eyes. The results indicate that changes in intracameral TIM concentrations can be monitored through electrochemical measurements using BDD microelectrodes. This technique holds promise for future advancements in optimizing glaucoma treatment and drug administration strategies