Name: dual-dye optical mapping of hearts from ryr2r2474s knock-in mice of catecholaminergic polymorphic ventricular tachycardia
Uploaded: 2023-12-22T12:20:00
Description: Watch this Scientific Journal Video about Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia at JoVE.com

本研究得到了国家自然科学基金（81700308 至 XO，31871181 至 ML，82270334 至 XT）、四川省科技支撑计划（CN）（2021YJ0206 至 XO、23ZYZYTS0433、2022YFS0607 至 XT、2022NSFSC1602 至 TC）和广西师范大学化学与分子工程国家重点实验室（CMEMR2017-B08 至 XO）， MRC（G10031871181 至 ML02647、G1002082、ML）、BHF（PG/14/80/31106、PG/16/67/32340、PG/12/21/29473、PG/11/59/29004 ML）、BHF CRE 在牛津 （ML） 赠款。

使用体重为20-25g的雄性10至14周龄野生型小鼠或RyR2-R2474S / +小鼠（C57BL / 6背景）进行实验。所有程序均已获得中国四川西南医科大学动物护理和使用委员会（批准号：20160930）的批准，符合该机构运营所依据的国家指南。
1. 准备工作
<ol><li>库存解决方案<ol><li>布比司他汀储备液：在含有 2.924 mg （-） 布比司他汀粉末的原始烧瓶中加入 1 mL 100% 二甲基亚砜 （DM...

<ol>
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根据我们的经验，成功对小鼠心脏进行双染料光学成像的关键包括精心准备的溶液和心脏、染料上样、实现最佳信噪比以及减少运动伪影。
溶液的制备 克雷布斯解决方案对于成功的心脏实验至关重要。MgCl 2 和 CaCl2 储备溶液 （1 mol/L） 是预先制备的，考虑到它们的吸水性，并在所有其他组分都溶解在纯水中后加入克雷布斯溶液中，因为 Mg 2+ 和 ...

遗传性心脏病儿茶酚胺能性多形性室性心动过速 （CPVT） 表现为体力活动、应激或儿茶酚胺激发后多形性室性心动过速 （PVT） 发作，可恶化为可能致命的心室颤动 1,2,3,4 .在 1995 年首次报告为临床综合征后，最近的证据表明涉及七个基因的突变，这些基因都与肌浆网状 （SR） 存储在这种情况下的 Ca 2+ 释放有关：最常报道的编码 Ca 2+ 释放通道的 RyR2 编码 Ca2+ 释放通道 5,6、FKBP12.67、编码心脏钙螯合蛋白 8、TRDN 的 CASQ2编码连接 SR 蛋白 triadin 9，以及编码钙调蛋白 11,12 的 CALM1 9、CALM210 和 CALM3。这些基因型模式将心律失常事件归因于 SR 储存 Ca2+12 不受调节的病理释放。
SR 自发释放的 Ca 2+ 可以检测为 Ca 2+ 火花或 Ca 2+ 波，从而激活 Na+/Ca 2+ 交换剂 （NCX）。一个 Ca2+ 换三个 Na+ 的交换器产生向内电流，加速舒张去极化并将膜电压驱动到动作电位 （AP） 阈值。在 RyR2 敲入小鼠中，窦房结 （SAN） 中 RyR2R4496C 活性的增加导致 SAN 自动性意外降低，舒张期 I Ca，L 和 SR Ca 2+ 耗竭的 Ca2+ 依赖性降低，确定导致 CPVT 患者 SAN 功能障碍的亚细胞病理生理学改变13,14。在儿茶酚胺（包括异丙肾上腺素 （isoproterenol， ISO）激发后，背景胞质 [Ca 2+] 增加后，相关心肌细胞胞质 Ca2+ 波的发生更可能。
RyR2 介导的 Ca2+ 释放后 Ca2+ 信号转导响应动作电位 （AP） 激活的详细动力学变化，这可能是在完整心脏 CPVT 模型中观察到的室性心律失常的原因，仍有待确定所有报告的 RyR2 基因型12。本文介绍了小鼠野生型 （WT） 和杂合子 RyR2-R2474S/+ 心脏中 Ca2+ 信号和跨膜电位 （V m） 的高通量定位仪器设置和实验程序，并结合异丙肾上腺素攻击前后的程序化电起搏。该方案为分离小鼠心脏中CPVT疾病的机制研究提供了一种方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m syringe filter</td>
			<td>Medical equipment factory of Shanghai Medical Instruments Co., Ltd., Shanghai, China</td>
			<td>N/A</td>
			<td>To filter solution</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Guangzhou Jet Bio-Filtration Co., Ltd. China</td>
			<td>CFT011150</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Pasteur pipette</td>
			<td>Beijing Labgic Technology Co., Ltd. China</td>
			<td>00900026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>B. Braun Medical Inc.</td>
			<td>YZB/GER-5474-2014</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L PCR tube</td>
			<td>Sangon Biotech Co., Ltd. Shanghai. China</td>
			<td>F611541-0010</td>
			<td>Aliquote the stock solutions&#160; to avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Guangzhou Jet Bio-Filtration Co., Ltd. China</td>
			<td>CFT011500</td>
			<td>Store Tyrode&#39;s solution at 4 &#176;C for follow-up heart isolation</td>
		</tr>
		<tr>
			<td>585/40 nm filter</td>
			<td>Chroma Technology</td>
			<td>N/A</td>
			<td>Filter for calcium signal</td>
		</tr>
		<tr>
			<td>630 nm long-pass filter</td>
			<td>Chroma Technology</td>
			<td>G15604AJ</td>
			<td>Filter for voltage signal</td>
		</tr>
		<tr>
			<td>Avertin (2,2,2-tribromoethanol)</td>
			<td>Sigma-Aldrich Poole, Dorset, United Kingdom</td>
			<td>T48402-100G</td>
			<td>To minimize suffering and pain reflex</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Tocris Bioscience, Minneapolis, MN, United States</td>
			<td>SLBV5564</td>
			<td>Excitation-contraction uncoupler to&#160; eliminate motion artifact during mapping</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBK1794V</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>Custom-made thermostatic bath</td>
			<td>MappingLab, United Kingdom</td>
			<td>TBC-2.1</td>
			<td>To keep temperature of perfusion solution</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>(RNBT7442)</td>
			<td>Solvent for dyes</td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China</td>
			<td>YAF030</td>
			<td></td>
		</tr>
		<tr>
			<td>ElectroMap software</td>
			<td>University of Birmingham</td>
			<td>N/A</td>
			<td>Quantification of electrical parameters</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Evolve 512 Delta, Photometrics, Tucson, AZ, United States</td>
			<td>A18G150001</td>
			<td>Acquire images for optical signals</td>
		</tr>
		<tr>
			<td>ET525/36 sputter coated filter</td>
			<td>Chroma Technology</td>
			<td>319106</td>
			<td>Excitation filter</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBT4811V</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>Heparin Sodium</td>
			<td>Chengdu Haitong Pharmaceutical Co., Ltd., Chengdu, China</td>
			<td>(H51021209)</td>
			<td>To prevent blood clots in the coronary artery</td>
		</tr>
		<tr>
			<td>&#160;Iris forceps</td>
			<td>Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China</td>
			<td>YAA010</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>MedChemExpress, Carlsbad, CA, United States</td>
			<td>HY-B0468/CS-2582</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBS5003</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>MacroLED</td>
			<td>Cairn Research, Faversham, United Kingdom</td>
			<td>7355/7356</td>
			<td>The excitation light of fluorescence probes</td>
		</tr>
		<tr>
			<td>MacroLED light source</td>
			<td>Cairn Research, Faversham, United Kingdom</td>
			<td>7352</td>
			<td>Control the LEDs</td>
		</tr>
		<tr>
			<td>Mayo scissors</td>
			<td>Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China</td>
			<td>YBC010</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Optical signals sampling</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>BCBS6841V</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>MICRO3-1401</td>
			<td>Cambridge Electronic Design limited, United Kingdom</td>
			<td>M5337</td>
			<td>Connect the electrical stimulator and Spike2 software</td>
		</tr>
		<tr>
			<td>MyoPacer EP field stimulator</td>
			<td>Ion Optix Co, Milton, MA, United States</td>
			<td>S006152</td>
			<td>Electric stimulator</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBS2340V</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>NaH2PO4&#160;</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>BCBW9042</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBX3605</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>NeuroLog System</td>
			<td>Digitimer</td>
			<td>NL905-229</td>
			<td>For ECG amplifier</td>
		</tr>
		<tr>
			<td>OmapScope5</td>
			<td>MappingLab, United Kingdom</td>
			<td>N/A</td>
			<td>Calcium alternans and arrhythmia analysis</td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Huaian Teshen Medical Instruments Co., Ltd., Jiang Su, China</td>
			<td>T4-3904</td>
			<td></td>
		</tr>
		<tr>
			<td>OptoSplit</td>
			<td>Cairn Research, Faversham, United Kingdom</td>
			<td>6970</td>
			<td>Split the emission light for detecting Ca2+ and Vm&#160; simultaneously</td>
		</tr>
		<tr>
			<td>Peristalic pump</td>
			<td>Longer Precision Pump Co., Ltd., Baoding, China,</td>
			<td>BT100-2J</td>
			<td>To pump the solution</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>BIOFIL</td>
			<td>TCD010060</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Invitrogen, Carlsbad, CA, United States</td>
			<td>1899021</td>
			<td>To enhance the loading with Rhod2AM</td>
		</tr>
		<tr>
			<td>RH237</td>
			<td>Thermo Fisher Scientifific, Waltham, MA, United States</td>
			<td>1971387</td>
			<td>Voltage-sensitive dye</td>
		</tr>
		<tr>
			<td>Rhod-2 AM</td>
			<td>Invitrogen, Carlsbad, CA, United States</td>
			<td>1890519</td>
			<td>Calcium indicator</td>
		</tr>
		<tr>
			<td>Silica gel tube</td>
			<td>Longer Precision Pump Co., Ltd., Baoding, China,</td>
			<td>96402-16</td>
			<td>Connect with the peristaltic pump</td>
		</tr>
		<tr>
			<td>Silk suture</td>
			<td>Yuankang Medical Instrument Co., Ltd.,Yangzhou, China</td>
			<td>20172650032</td>
			<td>To fix the aorta</td>
		</tr>
		<tr>
			<td>Spike2</td>
			<td>Cambridge Electronic Design limited, United Kingdom</td>
			<td>N/A</td>
			<td>To record and analyze ECG data</td>
		</tr>
		<tr>
			<td>Stimulation electrode</td>
			<td>MappingLab, United Kingdom</td>
			<td>SE1600-35-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>T510lpxr</td>
			<td>Chroma Technology</td>
			<td>312461</td>
			<td>For light source</td>
		</tr>
		<tr>
			<td>T565lpxr</td>
			<td>Chroma Technology</td>
			<td>321343</td>
			<td>For light source</td>
		</tr>
	</tbody>
</table>

dual-dye optical mapping of hearts from ryr2r2474s knock-in mice of catecholaminergic polymorphic ventricular tachycardia

促心律失常性心脏病儿茶酚胺能性多形性室性心动过速 （CPVT） 表现为体力活动、应激或儿茶酚胺激发后多形性室性心动过速发作，可恶化为可能致命的心室颤动。小鼠心脏是用于模拟遗传性心律失常疾病（包括CPVT）的广泛物种。来自Langendorff灌注的小鼠心脏的跨膜电位（Vm）和钙瞬变（CaT）的同时光学映射有可能阐明心律失常发生的潜在机制。与细胞水平研究相比，光学映射技术可以测试一些电生理参数，如活化、传导速度、动作电位持续时间和CaT持续时间的测定。本文介绍了在异丙肾上腺素攻击前和异丙肾上腺素激发之前和期间，结合程序化电起搏，在小鼠野生型和杂合子 RyR2-R2474S/+ 心脏中高通量光学映射 CaT 和 V m 的仪器设置和实验程序。该方法为体外小鼠心脏制剂中CPVT疾病的机理研究提供了一种可行且可靠的方法。

在过去十年中，光学标测一直是研究复杂心律失常的流行方法。光学映射装置由EMCCD相机组成，每个像素的采样率高达1,000 Hz，空间分辨率为74 x 74 μm。它在信号采样期间实现了相当高的信噪比（图 1）。一旦朗根道夫灌注的心脏达到稳定状态并且染料上样完成，心脏在两个 530 nm LED 的照射下放置在等温室中，用于激励电压指示器 RH237 和 Ca2+ 指示器 Rhod-2 AM。发射光被?...

Watch this Scientific Journal Video about Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia at JoVE.com

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

该协议介绍了从受儿茶酚胺能多形性室性心动过速影响的野生型和敲入动物获得的小鼠心脏的双染料光学映射，包括具有高时间和空间分辨率的跨膜电压和细胞内Ca2 + 瞬变的电生理测量。

儿茶酚胺能多形性室性心动过速的 RyR2R2474S 敲入小鼠心脏的双染料光学映射

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes ...

该方法将有助于我们揭示与儿茶酚胺能多形性室性心动过速等疾病相关的心室停搏涂片的电生理特性和机制。利用该技术，我们可以在各种程序化的电起搏方案下同时获得膜电位和细胞间钙信号，特别适用于探索心律失常疾病（如CPVT）的潜在机制和动力学。为了成功进行此实验，请确保心脏灌注良好，适当的染料加载，激发-收缩解偶联和仔细的相机设置。首先，设置光学映射系统并打开相机，以在零下 50 摄氏度下稳定采样温度。将收获的小鼠心脏放入冷的CREB溶液中，以减缓新陈代谢并保护心脏。切除主动脉周围组织。使用定制的插管针进行插管，并用 4-0 丝线缝合固定。现在用Langendorff系统以每分钟3.5至4.0毫升的恒定速度灌注心脏。将一根小塑料管插入左心室，以释放腔室中溶液的充血，以避免过度预负荷，并将塑料管固定在插管针中。然后将两根导线放入浴槽的灌注液中，并打开心电图放大器盒和电刺激控制器的电源。接下来，启动参考的心电图或心电图软件并持续监测心电图。当心脏达到稳定状态时，在黑暗中执行后续步骤。将 blebbistatin CREBs 溶液混合物持续灌注到心脏中 10 分钟，以解除收缩与兴奋的耦合，并避免拍摄过程中出现收缩伪影。然后用红色手电筒检查心脏，确认宫缩完全停止。解偶联兴奋收缩后，在Langendorff灌注系统中用Rhod-2 AM工作溶液灌注心脏15分钟。在细胞内钙染料加载期间维持氧气供应。为防止多元 F-127 形成气泡，请在灌注系统中插入气泡捕集器。将 10 微升 RH 237 储备溶液稀释到 50 毫升灌注液中，并加载 10 分钟。完成双染料加载后，拍摄一系列照片。确认电压和钙信号足以进行分析。打开激发灯的两个 LED 并将其强度调整到适当的范围。将心脏放在检测设备下方，确保它被两个 LED 很好地照亮，将光斑直径调整到 2 厘米。将晶状体和心脏之间的工作距离设置为 10 厘米，以达到所需的采样率和空间分辨率。打开信号采样软件，同时控制相机并捕获电压和钙信号。启动 MyoPacer 场刺激器并将起搏模式设置为晶体管晶体管逻辑，每个脉冲的起搏持续时间为 2 毫秒。将初始强度设置为 0.3 伏。将一对铂电极连接到左心室顶点的心外膜上。使用心电图记录软件连续应用 30 次 10 赫兹 S1 刺激来测试心脏的舒张压阈值。逐渐增加电压幅度，直到实现一对一捕获。实施 S1-S1 方案以测量钙或动作电位替代物和恢复特性。从 100 毫秒的基本周期长度开始连续起搏心脏。在每个后续序列中将周期长度减少 10 毫秒，直到达到 50 毫秒。在刺激之前同时启动光学映射。要使用 S1-S2 刺激方案测量心室有效不应期，请从 100 毫秒的 S1-S1 起搏周期长度开始。在 60 毫秒处耦合 S2，并以 2 毫秒的步长递减递减，直到 S2 无法捕获异位 QRS 波群。对于心律失常诱发，给予永久 50 赫兹的突发起搏，并在等待两秒的休息间隔后进行相同的起搏发作。在连续高频起搏期间仔细监测心电图记录，以便在产生有趣的心律失常波时立即开始同步光学映射记录。继续使用电子倍增电荷耦合器件相机捕获图像。在图像采集软件中，按选择文件夹，加载图像，开始半自动海量视频数据分析过程。为分析输入正确的采样参数。手动设置图像阈值并选择感兴趣的区域。应用 3 x 3 像素高斯空间滤波器、Savitzky-Golay 滤波器和顶帽基线校正。然后按处理图像删除基线并计算电生理参数，如 APD-80 和 CATD-50。将起始时间、电位持续时间设置在峰值和终点处，在80%复极化处，用于计算APD-80。同样，将钙瞬态持续时间的开始时间定义为峰值，终点为80%弛豫。显示了 APD-80 和 CATD-80 热图中的典型迹线。异丙肾上腺素缩短了APD-80和野生型和儿茶酚胺能多形性室性心动过速或CPVT小鼠，但在异丙肾上腺素攻击后未发现差异。CATD-80 和 CPVT 小鼠在异丙肾上腺素攻击后比野生型更长，而治疗前没有显着性。根据电压信号，野生型和CPVT心脏在基线和异丙肾上腺素干预后对心外膜具有相同的传导能力。热图表明，CPVT小鼠在异丙肾上腺素攻击前后具有与野生型小鼠相同的传导能力。钙振幅交替分析显示，在14.29和16.67赫兹的连续S1-S1起搏期间，野生型心脏的钙信号在基线时保持稳定，而CPVT心脏则表现出频率依赖替。异丙肾上腺素激发后，CPVT心脏在S1-S1起搏期间表现出频率依赖替和钙信号，而野生型心脏不受影响。心动过速分析表明，在基线时 50 赫兹爆发起搏期间，野生型和 CPVT 心脏均表现出正常传导。在充满异丙肾上腺素后，CPVT心脏在50赫兹爆发起搏后表现出高频转子，而野生型心脏保持正常传导。按照该程序，使用雄激素型和野生型小鼠来说明这些模型或药物发明中的电生理和功能特性。光学标测是研究心律失常的有力工具，但由于荧光染料在激发收缩解偶联作用下的限制，它不能在临床上使用。随着高转计算技术的发展，针对不同目标分子的荧光技术的发展，心脏光学标测技术势必只能得到应用。

Research

JoVE Journal

Biology

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

Proper Care and Cleaning of the Microscope

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Major Components of the Light Microscope

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase Contrast and Differential Interference Contrast DIC Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Optical Mapping of Langendorff-perfused Rat Hearts

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Isolation of Embryonic Ventricular Endothelial Cells

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...