Name: computational reconstruction of pancreatic islets as a tool for structural and functional analysis
Uploaded: 2022-03-09T13:30:00
Description: Watch this Scientific Journal Video about Computational Reconstruction of Pancreatic Islets as a Tool for Structural and Functional Analysis at JoVE.com

G.J. Félix-Martínez 感谢 CONACYT（墨西哥国家科学与技术委员会）和墨西哥城自治大学电气工程系对该项目的支持。我们感谢Danh-Tai Hoang博士，Manami Hara博士和Junghyo Jo博士在与研究界分享小岛建筑方面的出色工作和慷慨。

注：该协议的示意图如图 1所示。分步说明如下（有关协议每个步骤中使用的控制面板的详细信息，请参阅 补充文件 1 ）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63351/63351fig01.jpg"> 图 1：流程图。 描述在 IsletLab 中实现的协议的顺序的流程图。 <a href="https://www.jove.com/files/ftp_...

<ol>
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L., 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=Beta-cell+hubs+maintain+Ca2++oscillations+in+human+and+mouse+islet+simulations.">Beta-cell hubs maintain Ca2+ oscillations in human and mouse islet simulations.</a> Islets. 10 (4), 151-167 (2018).</li><li>Watts, M., Ha, J., Kimchi, O., Sherman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+glucagon+secretion:+the+&#946;/&#945;/&#948;+model.">Paracrine regulation of glucagon secretion: the &#946;/&#945;/&#948; model.</a> American Journal of Physiology. Endocrinology and Metabolism. 310 (8), 597-611 (2016).</li><li>F&#233;lix-Mart&#237;nez, G. J., Mata, A., God&#237;nez-Fern&#225;ndez, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructing+human+pancreatic+islet+architectures+using+computational+optimization.">Reconstructing human pancreatic islet architectures using computational optimization.</a> Islets. 12 (6), 121-133 (2020).</li><li>Hellman, B., Salehi, A., Gylfe, E., Dansk, H., Grapengiesser, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+generates+coincident+insulin+and+somatostatin+pulses+and+antisynchronous+glucagon+pulses+from+human+pancreatic+islets.">Glucose generates coincident insulin and somatostatin pulses and antisynchronous glucagon pulses from human pancreatic islets.</a> Endocrinology. 150 (12), 5334-5340 (2009).</li><li>Hellman, B., Salehi, A., Grapengiesser, E., Gylfe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+mouse+islets+respond+to+glucose+with+an+initial+peak+of+glucagon+release+followed+by+pulses+of+insulin+and+somatostatin+in+antisynchrony+with+glucagon.">Isolated mouse islets respond to glucose with an initial peak of glucagon release followed by pulses of insulin and somatostatin in antisynchrony with glucagon.</a> Biochemical and Biophysical Research Communications. 417 (4), 1219-1223 (2012).</li><li>F&#233;lix-Mart&#237;nez, G. 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上述协议概述了一种使用新型计算算法重建和分析胰岛结构的实用方法。这项工作的主要目标是使胰岛研究界能够推导出定量指标，以表征胰岛结构的形态学和连接特性， 并通过 计算模拟评估这些特性的可能功能影响。
虽然该协议中采用的算法先前已在12，19中进行了详细描述，但由于其相对复杂性，缺乏直接和用户友好的...

胰腺分为称为头部，颈部，身体和尾巴的区域，每个区域具有不同的结构，功能和解剖位置1，2。从功能的角度来看，胰腺可以分为内分泌和外分泌系统，前者负责分泌严重参与葡萄糖稳态调节的激素，而后者 通过 酶分泌到十二指肠1中来促进食物消化。胰岛构成胰腺的内分泌组织，负责胰高血糖素、胰岛素和生长抑素的分泌，分别由 ɑ、β 和 δ 细胞分泌3。除了其内在的调节机制外，这些细胞 还通过 直接电通信（β细胞和可能的β和δ细胞之间）以及旁分泌和自分泌信号传导4，5，6进行调节。这两种机制都高度依赖于胰岛结构（即胰岛内不同类型细胞的组成和组织）7，8。重要的是，胰岛结构在糖尿病存在的情况下被改变，结果最有可能干扰胰岛内的通信9，10。
胰岛的研究涉及广泛的实验方法。其中，使用荧光技术来确定胰岛中不同细胞的数量，位置和类型，使得能够研究胰岛11，12，13的结构和形态学特性，并更好地了解健康和疾病的功能影响。作为补充，胰腺细胞14，15，16和最近的胰岛12，17，18，19的计算模型在过去几十年中已被用于评估难以甚至不可能通过实验解决的方面。
在该协议中，我们的目标是通过概述重建胰岛结构的方法，通过定量指标分析其形态和连通性属性，并执行基本模拟以评估胰岛属性的功能含义，从而弥合实验和计算工作之间的差距。
下面描述的方案基于专门为胰岛研究而设计的计算算法。总之，在方案的第一步中，使用Félix-Martínez等人最近提出的算法从实验数据中重建胰岛结构.19 ，其中通过4'，6-二脒-2-苯基吲哚（DAPI）染色获得的核位置和通过免疫荧光鉴定的细胞类型（如Hoang等人详细描述的那样11，12）。） 在迭代优化过程中进行处理。这导致确定每个细胞的最佳大小和位置，并获得由非重叠细胞组成的胰岛。其次，基于重构架构，识别小区间接触点，以确定连通性，生成相应的胰岛网络，使用户能够获得定量指标，进一步描述小岛架构（关于重构算法的细节可以在关于该主题19的原始工作中查阅）。最后，使用Hoang等人提出的 建模方法进行基本功能模拟，其中，基于实验观察到的激素分泌的脉动性质20，21，每个细胞被视为振荡器，因此该胰岛被表示为遵循重建胰岛的连通性特性的耦合振荡器网络。
鉴于该协议中使用的算法的计算复杂性，所涉及的所有步骤都在独立的应用程序22 中实现，其主要目标是将这些计算工具接近所有感兴趣的读者，无论他们在使用专用软件或编程语言方面的经验水平如何。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CUDA-capable NVIDIA graphics card</td>
			<td></td>
			<td></td>
			<td>Required for the functional simulations</td>
		</tr>
		<tr>
			<td>IsletLab</td>
			<td></td>
			<td></td>
			<td>https://github.com/gjfelix/IsletLab (Follow the instructions to download and install the application.)</td>
		</tr>
	</tbody>
</table>

computational reconstruction of pancreatic islets as a tool for structural and functional analysis

胰岛的结构特性是胰岛素、胰高血糖素和生长抑素分泌细胞功能反应的关键，因为它们 通过 电、旁分泌和自分泌信号传导影响胰岛内通讯。在该协议中，首先使用一种新的计算算法从实验数据中重建胰岛的三维结构。接下来，获得重建胰岛的形态学和连接特性，例如不同类型细胞的数量和百分比，细胞体积和细胞间接触。然后，使用网络理论通过网络派生的指标（如平均度、聚类系数、密度、直径和效率）来描述胰岛的连通性属性。最后，所有这些属性都通过使用耦合振荡器模型的计算仿真进行功能评估。总体而言，在这里我们描述了一个分步工作流程，在IsletLab中实现，IsletLab是一个专门为胰岛研究和模拟开发的多平台应用程序，以应用一种新的计算方法来表征和分析胰岛，作为实验工作的补充。

使用Félix-Martínez等人提出的方法重建胰岛，高度依赖于优化算法（在重建设置中定义）的参数。图3直观地显示了一个示例，其中显示了使用不同参数集获得的重建小岛。首先，在图3A中，显示了包含初始数据中包含的86.6%的细胞的重建（588个细胞中的509个，初始温度= 1，迭代因子= 1，接受因子= 1）。当初始温度以及迭代和接受因子增?...

Watch this Scientific Journal Video about Computational Reconstruction of Pancreatic Islets as a Tool for Structural and Functional Analysis at JoVE.com

Computational Reconstruction of Pancreatic Islets as a Tool for Structural and Functional Analysis

在该协议中，胰岛使用专用多平台应用程序中实现的计算算法进行重建和分析。

胰岛的计算重建作为结构和功能分析的工具

Structural properties of pancreatic islets are key for the functional response of insulin, glucagon, and somatostatin-secreting cells, due to their ...

该协议意义重大，因为它描述了重建胰岛架构的计算工作负载，分析其形态和连通性属性，并通过计算模拟评估其功能含义。主要优点是它提供了一种实现高性能计算算法的方法，以补充胰岛研究领域的理论和实验工作。这种方法对于比较健康和改变的小岛的形态和连接特性或比较来自不同动物物种的胰岛结构特别有用。首先，验证是否安装了 GCC 和 NVCC 编译器。通过执行命令打开终端，如果系统无法识别这些命令中的任何一个，请按照文本中提供的说明进行操作。打开终端并转到 IsletLab 文件夹。通过在终端中执行命令来创建新环境。通过执行命令激活新环境。通过在终端中执行命令来启动 IsletLab 应用程序。要准备输入数据，请将输入胰岛数据组织在一个四列文件中，其中第一列是像元类型，第二列、第三列和第四列分别是输入像元的 X、Y 和 Z 坐标，从而确保输入文件不包含列标题。单击加载初始小岛按钮，然后选择包含输入数据的文件以生成初始小岛、三维表示和相应的统计数据。要配置重建过程，请单击重建设置按钮并修改优化参数。单击“确定”按钮以保存参数值。单击重建小岛按钮以打开重建日志窗口。单击“运行”按钮以启动重建过程。通过分析统计面板的“最终胰岛”选项卡中显示的优化统计数据来评估重建过程的结果，重点是最大化重建小岛中包含的实验细胞的百分比，或者等效地最小化重叠次数。如果根据用户目标，实验统计信息的百分比被视为较低，请单击“文件”菜单，然后选择“重新启动”。单击加载初始小岛按钮，然后选择包含输入数据的文件以生成初始小岛。然后在重建设置中增加初始温度、迭代系数和接受系数，并重复前面描述的胰岛重建过程，直到获得满意的结果。单击重建设置，然后选择接触公差以定义像元间接触公差。单击“确定”保存参数值。单击“单元格到单元格联系人”按钮以识别密切接触中的单元格。单击构建网络按钮以生成胰岛网络并计算关联的网络矩阵。切换到界面配置面板的“仿真”选项卡。选择所需的固有频率模式，恒定或随机。单击配置固有频率按钮以定义振荡器的频率（以赫兹为单位）。选择初始阶段的所需模式，常量或随机。单击配置交互组件按钮以在“交互强度”窗口中定义单元到单元的交互参数。通过定义总仿真时间、时间步长和保存系数来配置仿真。定义可用于执行仿真的块、线程和计算平台功能的数量。单击运行模拟按钮以打开模拟日志窗口。单击“运行”按钮开始模拟并监视该过程，直到显示图例“请关闭窗口以继续”。然后关闭“模拟日志”窗口以观察模拟结果。单击“文件”，然后在菜单栏中选择“导出项目”。选择要在其中保存项目文件的目录，然后单击“确定”按钮。在重建设置中使用次优参数集获得胰岛重建，包括86.6%的初始细胞。增加初始温度，迭代和接受因子导致重建胰岛中初始细胞的百分比更高，分别为93.37%和99.15%。获得了重建过程的收敛图，证明了重叠细胞的进化作为温度的函数。从重建的胰岛结构中识别单元间接触取决于接触容差参数的值。仅确定了290个细胞间接触，值为1微米，而对于两个微米，确定的总背景分别增加到636和731。获得了网络图，该图提供了连接在一起的细胞间接触的可视化表示。仿真结果表明，α细胞和β细胞完全异相振荡，而δ细胞与α细胞和β细胞异相振荡。胰岛的振荡行为主要由α细胞的振荡主导，其他细胞群的贡献很小。获得了胰岛同步指数图，该图提供了胰岛细胞之间振荡的相相一致性以及胰岛细胞之间同步性随时间变化的测量。所有衍生的指标和功能模拟都依赖于重建过程，因此在重建的胰岛中实现最高百分比的初始细胞是关键。通过该程序获得的重建胰岛可以进一步用于通过包括胰岛细胞的详细生物物理描述来开发更现实的胰岛计算模型。

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

病人的具体建模之心：的心室纤维方向估计

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

液体中脉冲激光烧蚀制备金属纳米粒子：研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

化重构法 (的) 对肠道干细胞和小生境细胞功能分析的研究

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

胰岛血气 StarPEG Nanocoating 的表面工程

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...