Name: a new ex vivo model for the evaluation of endoscopic submucosal injection material performance
Uploaded: 2018-10-19T13:30:00
Description: Watch this Scientific Journal Video about A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance at JoVE.com

这项工作得到了京都创新医学技术研究 & 发展支持系统的支持, 并由翻译研究计划提供;从日本医学研究与发展署 (艾湄湾) 实际应用创新医学技术 (TR SPRINT) 的战略推广。

下列议定书遵循京都府医学院的《动物保育指南》。
1. 使用猪胃制备标本
注意: 第一步是准备标本在前体模型中使用 (图 1)。猪胃壁的厚度在不同的胃区变化。使用猪胃的上部第三, 这是相对于人类的胃。排除不适当的标本, 因为纤维化, 没有发现黏膜下抬高。
<ol><li>将胃标本切成正方形, 尺寸约为 6 x 6 厘米。</li>
	<li>在-30 摄...

<ol>
	<li>Ono, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection+for+treatment+of+early+gastric+cancer.">Endoscopic mucosal resection for treatment of early gastric cancer.</a> Gut. 48 (2), 225-229 (2001).</li><li>Conio, M., Ponchon, T., Blanchi, S., Filiberti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection.">Endoscopic mucosal resection.</a> The American journal of gastroenterology. 101 (3), 653-663 (2006).</li><li>Soetikno, R. M., Gotoda, T., Nakanishi, Y., Soehendra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection.">Endoscopic mucosal resection.</a> Gastrointestinal endoscopy. 57 (4), 567-579 (2003).</li><li>Iishi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+resection+of+large+sessile+colorectal+polyps+using+a+submucosal+saline+injection+technique.">Endoscopic resection of large sessile colorectal polyps using a submucosal saline injection technique.</a> Hepato-gastroenterology. 44 (15), 698-702 (1997).</li><li>Katsinelos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+50%+dextrose+and+normal+saline+solution+on+their+ability+to+create+submucosal+fluid+cushions+for+endoscopic+resection+of+sessile+rectosigmoid+polyps.">A comparative study of 50% dextrose and normal saline solution on their ability to create submucosal fluid cushions for endoscopic resection of sessile rectosigmoid polyps.</a> Gastrointestinal endoscopy. 68 (4), 692-698 (2008).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+endoscopic+mucosal+resection+using+sodium+hyaluronate.">A novel method of endoscopic mucosal resection using sodium hyaluronate.</a> Gastrointestinal endoscopy. 50 (2), 251-256 (1999).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+successful+single-step+endoscopic+resection+of+a+40+millimeter+flat-elevated+tumor+in+the+rectum:+endoscopic+mucosal+resection+using+sodium+hyaluronate.">A successful single-step endoscopic resection of a 40 millimeter flat-elevated tumor in the rectum: endoscopic mucosal resection using sodium hyaluronate.</a> Gastrointestinal endoscopy. 50 (5), 701-704 (1999).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+and+safety+of+0.4%+sodium+hyaluronate+solution+as+a+submucosal+fluid+&amp;#34;cushion&amp;#34;+in+endoscopic+resection+for+gastric+neoplasms:+a+prospective+multicenter+trial.">Usefulness and safety of 0.4% sodium hyaluronate solution as a submucosal fluid &amp;#34;cushion&amp;#34; in endoscopic resection for gastric neoplasms: a prospective multicenter trial.</a> Gastrointestinal endoscopy. 67 (6), 830-839 (2008).</li><li>Yamamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+en-bloc+resection+of+large+superficial+tumors+in+the+stomach+and+colon+using+sodium+hyaluronate+and+small-caliber-tip+transparent+hood.">Successful en-bloc resection of large superficial tumors in the stomach and colon using sodium hyaluronate and small-caliber-tip transparent hood.</a> Endoscopy. 35 (8), 690-694 (2003).</li><li>Kishihara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+sodium+hyaluronate+solution+in+colorectal+endoscopic+mucosal+resection.">Usefulness of sodium hyaluronate solution in colorectal endoscopic mucosal resection.</a> Digestive endoscopy. 24 (5), 348-352 (2012).</li><li>Yoshida, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+mucosal+resection+with+0.13%+hyaluronic+acid+solution+for+colorectal+polyps+less+than+20+mm:+a+randomized+controlled+trial.">Endoscopic mucosal resection with 0.13% hyaluronic acid solution for colorectal polyps less than 20 mm: a randomized controlled trial.</a> Journal of gastroenterology and hepatology. 27 (8), 1377-1383 (2012).</li><li>Uraoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+glycerol+as+a+submucosal+injection+for+EMR.">Effectiveness of glycerol as a submucosal injection for EMR.</a> Gastrointestinal endoscopy. 61 (6), 736-740 (2005).</li><li>Conio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+performance+in+the+porcine+esophagus+of+different+solutions+used+for+submucosal+injection.">Comparative performance in the porcine esophagus of different solutions used for submucosal injection.</a> Gastrointestinal endoscopy. 56 (4), 513-516 (2002).</li><li>Moss, A., Bourke, M. J., Metz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind+trial+of+succinylated+gelatin+submucosal+injection+for+endoscopic+resection+of+large+sessile+polyps+of+the+colon.">A randomized, double-blind trial of succinylated gelatin submucosal injection for endoscopic resection of large sessile polyps of the colon.</a> The American journal of gastroenterology. 105 (11), 2375-2382 (2010).</li><li>Lee, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+EMR:+submucosal+injection+of+a+fibrinogen+mixture.">A new method of EMR: submucosal injection of a fibrinogen mixture.</a> Gastrointestinal endoscopy. 59 (2), 220-224 (2004).</li><li>Hurlstone, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMR+using+dextrose+solution+versus+sodium+hyaluronate+for+colorectal+Paris+type+I+and+0-II+lesions:+a+randomized+endoscopist-blinded+study.">EMR using dextrose solution versus sodium hyaluronate for colorectal Paris type I and 0-II lesions: a randomized endoscopist-blinded study.</a> Endoscopy. 40 (2), 110-114 (2008).</li><li>Huai, Z. Y., Feng Xian, W., Chang Jiang, L., Xi Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submucosal+injection+solution+for+endoscopic+resection+in+gastrointestinal+tract:+a+traditional+and+network+meta-analysis.">Submucosal injection solution for endoscopic resection in gastrointestinal tract: a traditional and network meta-analysis.</a> Gastroenterology research and practice. 2015, 702768 (2015).</li><li>Yandrapu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+saline+solution+versus+other+viscous+solutions+for+submucosal+injection+during+endoscopic+mucosal+resection:+a+systematic+review+and+meta-analysis.">Normal saline solution versus other viscous solutions for submucosal injection during endoscopic mucosal resection: a systematic review and meta-analysis.</a> Gastrointestinal endoscopy. , (2016).</li><li>Fernandez-Esparrach, G., Shaikh, S. N., Cohen, A., Ryan, M. B., Thompson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+reverse-phase+polymer+as+a+submucosal+injection+solution+for+EMR:+a+comparative+study+(with+video).">Efficacy of a reverse-phase polymer as a submucosal injection solution for EMR: a comparative study (with video).</a> Gastrointestinal endoscopy. 69 (6), 1135-1139 (2009).</li><li>Tran, R. T., Palmer, M., Tang, S. J., Abell, T. L., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+drug-eluting+elastomeric+polymer:+a+novel+submucosal+injection+material.">Injectable drug-eluting elastomeric polymer: a novel submucosal injection material.</a> Gastrointestinal endoscopy. 75 (5), 1092-1097 (2012).</li><li>Akagi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+alginate+as+an+ideal+submucosal+injection+material+for+endoscopic+submucosal+resection:+preliminary+experimental+and+clinical+study.">Sodium alginate as an ideal submucosal injection material for endoscopic submucosal resection: preliminary experimental and clinical study.</a> Gastrointestinal endoscopy. 74 (5), 1026-1032 (2011).</li><li>Eun, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+sodium+alginate+as+a+submucosal+injection+material+for+endoscopic+mucosal+resection+in+animal.">Effectiveness of sodium alginate as a submucosal injection material for endoscopic mucosal resection in animal.</a> Gut and Liver. 1 (1), 27-32 (2007).</li><li>Hirose, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+ex+vivo+model+for+evaluation+of+endoscopic+submucosal+injection+materials+performance.">Development of a new ex vivo model for evaluation of endoscopic submucosal injection materials performance.</a> Journal of the Mechanical Behavior of Biomedical Materials. 79, 219-225 (2018).</li></ol>

用于新模型的猪胃应在切除后立即储存在冰柜中, 并在冷冻后几个月内使用, 因为猪胃的新鲜度对于 SEH 测量是必不可少的。(我们使用冰冻和未解冻的胃标本测量了 seh, 并证实了 seh 测量结果没有差异。
胃标本的质量受猪胃个体差异的影响较大。因此, 建议在测量前排除具有许多褶皱的明显厚试样或试样。此外, 某些标本可能是由于纤维化而导致的 SEH 测量的不当标本。建议排?...

内窥镜黏膜下解剖 (ESD) 和内镜黏膜切除术 (EMR) 是目前常见的治疗早期胃肠道肿瘤1,2。将黏膜下注射材料 (SIM) 注入粘膜下层是 EMR 和 ESD 过程2,3的最重要步骤之一。高黏膜下抬高和黏膜下抬高的维护是安全地进行 EMR/ESD 的关键标准。
虽然正常生理盐水 (NS) 已被用作 SIM, 自内镜治疗4,5, 透明质酸钠 (HA) 被引入作为治疗在最近几年6,7。HA 由于其高性能8、9、10、11, 被广泛应用于内窥镜治疗作为高级 SIM 卡。目前, 在现有的模拟人之间进行了性能比较, 并开发了高性能的模拟 sim 卡, 以识别另一台高级5、12、13、14、 15,16,17,18。
使用猪胃标本的前体模型被用来评估 sim 的性能, 因为在人类胃肠道的 sim 性能估计是非常困难的19,20,21,22. 然而, 这种传统的体外模型非常简单, 并且有改进的余地。重现一个靠近人体胃肠粘膜的环境将能够准确评估 SIM 的性能。
在我们之前的研究中, 我们开发了一种新的体外模型, 可以通过对试样末端施加恒定的张力来详细评估各种模拟人生的表现。我们还证实了拟议的新的体外模型, 允许精确的她测量在统一条件下, 并详细比较各种类型的模拟人生23的表现。
在本研究中, 我们提出了新的体外模型的完整外观, 并通过视频和数字详细解释了新的体外模型的详细设置方法。新的前体模型由易于使用的部件组成, 可以快速设置。详细设置方法的说明将有助于传播新模型。

<table>
	<tbody>
		<tr>
			<td>weight (153.1 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fixed type pulley</td>
			<td>H.H.H. MANUFACTURING</td>
			<td>VS25</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel wire with a diameter of 0.45 mm</td>
			<td>Nissa Chain</td>
			<td>Cut wire Y-5</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel clip of width 147 mm</td>
			<td>KOKUYO</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel key wire with a length of 12 cm</td>
			<td>Nissa Chain</td>
			<td>P-702</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel S shaped hook</td>
			<td>TRUSCO NAKAYAMA</td>
			<td>TCS1.2</td>
			<td></td>
		</tr>
		<tr>
			<td>lockable stainless steel S-shaped hook</td>
			<td>Mizumoto Machine Mfg</td>
			<td>B2054</td>
			<td></td>
		</tr>
		<tr>
			<td>rectangular wooden base (45 x 60 cm)</td>
			<td>none</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber plate (5 x 5 cm)</td>
			<td>none</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>digital height gage</td>
			<td>Mitutoyo</td>
			<td>HDS-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5-mL syringe</td>
			<td>Terumo</td>
			<td>SS-02SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>23-gauge needle</td>
			<td>Terumo</td>
			<td>NN-2332R</td>
			<td></td>
		</tr>
		<tr>
			<td>MucoUp</td>
			<td>Boston Scientific</td>
			<td>none</td>
			<td>0.4% sodium hyaluronate (HA)</td>
		</tr>
		<tr>
			<td>saline (20 mL)</td>
			<td>Otsuka Pharmaceutical</td>
			<td>none</td>
			<td>normal saline (NS)</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7 software</td>
			<td>GraphPad Inc</td>
			<td>none</td>
			<td></td>
		</tr>
	</tbody>
</table>

a new ex vivo model for the evaluation of endoscopic submucosal injection material performance

提高黏膜下注射材料 (sim) 的性能对早期胃肠道肿瘤的内镜治疗具有重要意义。建立一个能准确评估 SIM 性能的前体模型是至关重要的, 因为开发高性能的模拟人生。在我们之前的研究中, 我们开发了一种新的体外模型, 可以通过对试样末端施加恒定的张力来详细评估各种模拟人生的表现。我们还证实, 拟议的新的体外模型允许精确的黏膜下高程高度 (SEH) 测量在统一条件下, 并详细比较各种类型的模拟人物的表现。在这里, 我们描述了新的体内模型, 并解释了这个模型的详细设置方法。由于新模型的所有部分都易于获得, 新模型的设置可以快速完成。使用新模型可以更准确地测量各种模拟器的 SEH。确定 SIM 性能的关键因素可以使用新模型进行标识。在确定因素后, SIM 开发速度将大幅增加。

在新的体内模型或常规的体外模型中, SEH 是随着时间的推移而测量的。使用常规模型测量的 SEH (ns) 的值 [ns 被注射到固定与引脚 (0.0 N) 的标本的黏膜下层) 是5.7 毫米 (0 分钟), 3.6 毫米 (5 分钟), 3.0 毫米 (10 分钟) 和2.2 毫米 (30 分钟)。这样, SEH 的值随注射后的时间增加而减小。使用 0.4% HA 而不是 NS 进行了类似的分析。SEH (0.4% 公顷) 的值为6.5 毫米 (0 分钟)、5.2 毫?...

Watch this Scientific Journal Video about A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance at JoVE.com

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

我们开发了一种新的体外模型, 对猪胃标本施加恒定的张力。这一发展使人们能够准确地评估各种模拟人生的表现 (黏膜下高程的高度和持续时间)。 介绍了此新模型的详细设置方法。

内镜下黏膜下注射材料性能评价的一种新的体内模型

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

该方法有助于回答生物医学领域有关医学关注问题的关键问题。该技术的主要优点是，与传统方法相比，可以更准确地蒸发各种亚粘膜注射事项的性能。首先从猪胃的上部三分之一切下六厘米六厘米的正方形，并立即将标本储存在零下30摄氏度。要设置牵引装置，请将不锈钢夹连接到钥匙线和 S 形钩，然后将钥匙线和挂钩连接到重量。将吊钩连接到导线的另一端，并在底座两端固定滑轮。然后将一个六到六厘米的橡胶板放在底座的中心，将解冻的胃样板放在橡胶板上。接下来，用牵引装置的夹子捏紧试样末端，通过两个滑轮悬挂重量，对试样施加恒定的张力，并使用数字仪表测量喷射前亚粘膜高程高度。然后将划片器固定到粘膜表面的高度，然后按预设，对高度计进行零点调整。要执行亚糖高程程序，请使用装有 23 量针的 2.5 毫升注射器，从试样边缘水平将两毫升 0.4%的 0.4% 氢化钠注射到亚糖体中。然后使用数字仪表立即测量亚粘度高程高度，并在注射后在指示的时间点。将抄写器固定到亚粘度高程的顶部时，记录高度仪表上显示的高度。在这些具有代表性的实验中，使用常规的、新的外体模型进行分析，在正常盐水或海洛酸注射后测量亚糖高程高度。虽然在注射任一溶液后，亚糖高程高度会随着时间而降低，但在这两种模型中，无论注射后的时间如何，在葫盐酸注射后产生的亚糖高高高度始终高于正常盐水注射后测量的高度。值得注意的是，与无张力常规测量模型相比，持续张力的应用降低了测量中的总体变异性。事实上，在3.0牛顿的张力下获得的亚木科高程高度明显低于1.5牛顿的亚木科高程高度。然而，与常张关系模型相比，使用传统模型观察到亚木面高程高度没有显著差异。开发后，该技术为生物医学领域的研究人员表达高性能亚粘膜注射材料用于内窥镜治疗铺平了道路。

Research

JoVE Journal

Bioengineering

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

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Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

建立了外科手术引起的小鼠模型，以调查的progranulin在骨性关节炎的保护作用

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...