这项工作是由阿兰。和伊迪丝L.沃尔夫慈善基金，圣路易斯和巴恩斯犹太医院基金会赞助。属Shmuylovich和E戈什由美国心脏协会的中心地带加盟博士前奖学金的发放进行了部分支持。朱南接受部分支持来自华盛顿大学的康普顿奖学金项目和艺术学院与科学"暑期本科生研究奖。南Mossahebi获得了部分支持物理系。

获取超声图像，并对其进行分析，以获得PDF格式参数的过程详述如下。虽然心导管被提及在下面的受试者选择部分，所描述的方法只适用于超声心动图的部分。在导管部分的描述被列入为基础的模型预测的独立验证，是无关的电子波通过 PDF形式主义的分析。此前数据采集，所有科目提供签署知情同意参与这项研究符合机构审查委员会（人类研究保护办公室）在华盛顿大学医学院。 注:所有的软件程序（以及如何使用它们教程）本节描述可以从下载<a href="http://cbl1.wustl.edu/SoftwareAgreement.htm" target="_blank">http://cbl1.wustl.edu/Softw</a>areAgreement.htm 1，选题注:在心血管生物物理实验室数据库中的所有对象必须同时进行超声心动图及进行心导管检查和他们的医生进行诊断心导管转介。该数据库的入选标准是:1）没有任何显著瓣膜异常，2）无室壁运动异常或束支传导阻滞的心电图，3）存在一个令人满意的超声心动图的窗口有清晰可辨的E-和A波。 2，超声心动图数据采集<ol><li>记录为依据的超声心动图标准16与美国社会的所有科目的完整的2D /超声多普勒研究。注:超声心动图筛查，记录在标准的临床成像仪由超声医师。如果需要，额外的经胸超声心动图记录可核查的目的进行一个合适的，高逼真度导管后我国推进到LV的同时测量左室血流动力学。 </li><li>在仰卧位影像科。在非研究设置，可以使用标准的左横向定位，而不方法的损失一般性。获得使用2.5 MHz传感器心尖四腔观，与门控在1.5-5毫米的二尖瓣瓣叶的提示和垂直于平面的MV（最小化取向的影响之间的定向样品体积上看到彩色M型多普勒），壁滤波器设置为1（125赫兹）或2（250赫兹），调整以显示画面的全高度，并调整为利用输出的动态范围，而不会走样的速度刻度的优点基线。 </li><li>同门为2.5毫米，位于二尖瓣环的横向和间隔药水样本量进行多普勒组织成像。 </li><li>保存为DICOM格式多普勒检查回声机记录的DVD与SIMULTaneously记录心电图（ECG）。 </li></ol> 3，多普勒图像处理与常规分析注意:本节介绍了两个自定义的MATLAB程序。所述第一程序在步骤3.1中所述的第二程序中的步骤3.2-3.5中描述。所有的软件程序（以及如何使用它们教程）可以从下载<a href="http://cbl1.wustl.edu/SoftwareAgreement.htm" style="font-size: 14px; line-height: 28px;" target="_blank">http://cbl1.wustl.edu/SoftwareAgreement.htm</a> <ol><li>从DICOM格式和视频图像转换为位图（BMP）文件（使用自定义的MATLAB程序）。注意:下面描述的，以适应多普勒E-波和组织多普勒E&#39;-波的过程示于图1。 </li><li>上加载其他自定义的MATLAB程序的位图图像文件来衡量传统二尖瓣血流参数，如E 峰 ，A 峰 ，E 杜尔 </suB&gt; E&#39; 峰值 ，A" 峰 等 。并裁剪图像的PDF分析。通过心电图分析表明选择了明显的静脉流的轮廓和完整的心动周期的图像。 </li><li>图像中的标志的时间采样速率（在横轴上以像素/秒测量）和速度的采样率（像素/（米/秒测量）沿垂直轴）。通过记录和标记连续的R峰（或心电图的任何显着的特征）在图像上找出完整的心动周期。 </li><li>马克的二尖瓣多普勒E和所选择的心动周期的A-波或组织多普勒E&#39;-和A&#39;-波。 <ol><li>选择多普勒E波峰值点即 。 E 峰 ，（或E&#39; 峰 ）和标记使用的高峰连接到开始为指导，以配合电子波（或E&#39;波）的加速斜坡行波的开始。波开始被用来计算从开始的间隔时间，以PEAK流表示为在E-波（或E&#39;-波）加速时间（AT）。 </li><li>标记E波（或E&#39;-波）利用峰连接到端部为导向，以匹配减速斜率的线的末端。这被用来计算从峰值到表示为减速时间（DT）的基线的时间间隔。从开始到波的结束的时间间隔是E-波（E 杜尔 = AT + DT）的持续时间。该计划将指导通过与适当的指示，整个过程用户。 </li></ol></li><li>马克使用相同的程序，在E波的A波。同时与E-和A波标志着程序计算E 峰 / A 峰比值。 注:该程序保存为包含E和只有A波裁剪图像的显着海浪。该方案还创建具有裁剪和测量参数为每个节拍的数据文件。 </li></ol>静脉流的4自动拟合使用PDF形式主义<ol&gt; <li>做多普勒E-的自动装配和A波和组织多普勒E&#39;-和A&#39;-波轮廓是使用自定义的LabVIEW程序18,19。 <ol><li>加载裁剪的图像，并且该程序自动地计算出最大速度包络线（MVE）。通过设置这样的MVE近似于静脉流阈值， 如图1中选择的MVE的发病和终止定义的MVE的点可沿时间轴由操作者进行选择，使得只MVE提供良好的对应点向波的实际选择的部分被用作输入用于随后的装配。 </li></ol></li><li>注意:用户选择的MVE点是输入到该自动适合于速度作为时间使用Levenberg-马夸特（迭代）算法的函数的PDF模型溶液的计算机程序。嵌合完成的要求，即临床（输入）之间的均方误差数据（MVE）和PDF模型预测的轮廓最小化。由于模型是线性时，获得了用作输入每个派生多普勒E波MVE唯一的一组参数。因此数值上产生独特的K，C和X O值，每个E波和K&#39;，C&#39;，而x O&#39;每个E&#39;波。 </li><li>在该事件的拟合是显然不理想时的配合被叠加在电子波（或E&#39;-波）图像（ 即该算法试图适应包括在MVE例如噪声）通过使用多个修改MVE /少点，从而改变该模型预测的轮廓与PDF参数因之变形，以达到更好的配合。 </li></ol>保存数据时，适当的PDF适合已经生成。注:该程序被写入自动保存图像数据，并包含PDF文本参数文件和轮廓信息。 从上面描述的方法获得的PDF格式参数可以用来阐明新生理和正常和病理生理学区分，如下面的代表结果部分详述。

<ol>
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Physiol. 101, 92-101 (2006).</li><li>Kreyszig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced Engineering Mathematics. , (2011).</li><li>Press, W. H., Teukolsky, S. A., Vetterling, W. T., Flannery, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Numerical recipes 3rd Edition: The Art of Scientific Computing. , (2007).</li><li>Claessens, 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=The+Parametrized+Diastolic+Filling+Formalism:+Application+in+the+Asklepios+Population.">The Parametrized Diastolic Filling Formalism: Application in the Asklepios Population.</a> Am. Soc. Mech. Eng. Summer Bioengineering Conference Proceedings. , (2011).</li><li>Chung, C. S., Kov&#225;cs, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Increasing+Heart+Rate+on+Deceleration+Time,+Velocity+Time+Integral,+and+E/A.">Consequences of Increasing Heart Rate on Deceleration Time, Velocity Time Integral, and E/A.</a> Am. J. Cardiol. 97, 130-136 (2006).</li></ol>

The authors have no competing financial interests.

按照我们的方法学重点，这有利于获得准确和有意义的结果的方法的关键环节突出显示。 超声心动图美国超声心动图学会（ASE）有胸研究16的性能准则。期间的回声检查，有众多的影响图像质量的因素。这超出了超声医师的控制因素包括:热像仪的技术能力被利用，心脏率，患者的体型，在位置的个体差异，解剖结构的方向和"回声窗​​"的质量，...

由卡茨1于1930年开创性的研究表明，哺乳动物左心室由是机械抽气泵，从那时起一直致力于解开舒张的运作多的努力开始灌装。多年以来，侵入性方法，可供舒张功能（DF）2-16临床或科研评价的唯一选择。在20世纪70年代，但是，技术进步和发展，超声心动图终于给了心脏病学家和生理学家实用的工具，东风无创性的表征。 如果没有一个统一的因果理论或范式关于如何当它填补了心脏舒张的作品，研究人员提出了一种基于临床特征的关系很多phenomenologic指标。的曲线，迅速上升，并在早，迅速填充落入二尖瓣血流速度轮廓的形状，例如，被近似为三角形和舒张复nction索引是从几何特征限定了三角形的（高度，宽度，面积等 ）。在超声心动图技术的进步已经允许组织运动，应变和应变率填充到被测量，例如在，并带来了现象学指数的新作物各技术进步与临床特征相关。不过，指数仍然相关，而不是因果关系，很多指标都是相同的底层生理不同的措施。这并不奇怪，因此，东风目前使用的临床指标有限的特异性和灵敏度。 为了克服这些限制的参数化舒张功能（PDF格式）形式主义，因果运动，左室充盈是由动机，并采用舒张的抽吸泵生理学集总参数模型的开发和验证17。该模型舒张功能（如表现在曲线形状的静脉流轮廓）按照阻尼谐波振荡运动的规则。该方程阻尼谐波振荡运动是基于牛顿第二定律和可写，每单位质量，如: <img alt="式（1）" fo:content-width="1in" src="/files/ftp_upload/51471/51471eq1.jpg" /> 式（1） 这种线性2阶差分方程有三个参数:K -腔僵硬，C -粘弹性/松弛，和×○ -振荡器的初始位移/预紧力。该模型预测，不同临床观察舒张图案变化在这三个模型参数的数值的结果。基于PDF形式主义和经典力学，电子波可以归类为欠阻尼或过阻尼运动的制度来确定。许多研究<suP&gt; 17-21已经证实，临床上记录的E波的轮廓和PDF模型预测的轮廓展现高超的协议，并已阐明三个PDF格式的血流动力学/生理类似物参数21。这个过程从临床记录的E波的数据中提取模型参数是在下面的方法的详细。 与东风在目前临床使用的典型指标，PDF格式模型的三个参数是基于因果关系。如下面的方法所讨论的，舒张生理学的额外的索引可以从这些基本参数和从应用程序中的PDF形式主义的舒张其他方面比静脉流。在这项工作中，静脉流和生理关系的基于PDF的分析，可以从PDF方法绘制的方法，其参数与导出指标进行说明。另外，它表明，从它们衍生的PDF参数或索引可以逗从负载的外部效应内在相距室性可以提供关联到传统的侵入性定义的参数和正常和病理组之间可以分化。

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Equipment/ Software</td>
			<td style="width:159px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Philips iE33</td>
			<td style="width:159px;">Philips (Andover, MA.)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabVIEW 6.0</td>
			<td style="width:159px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td>Version 6.0.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MATLAB</td>
			<td style="width:159px;">MathWorks</td>
			<td style="width:119px;"></td>
			<td>Version R2010b</td>
		</tr>
	</tbody>
</table>

quantification of global diastolic function by kinematic modeling-based analysis of transmitral flow via the parametrized diastolic filling formalism

定量心脏功能的评估仍然是生理学家和临床医生的一个挑战。尽管历史上有创方法都包含唯一的手段可用，无创成像方式的发展（超声心动图，MRI，CT），具有高时空分辨率为定量舒张功能评估的新窗口。超声心动图是同意的标准舒张功能的评估，但目前临床使用的索引仅仅是利用所选室尺寸（M模式）或血液/组织运动（多普勒）波形的功能，而结合了运动本身的生理因果决定因素。所有的左心室（LV）的启动按作为机械吸泵灌装承认允许根据适用于所有室内运动定律全球舒张功能进行评估。什么从另一个区分各个心脏的运动是GOV方程的参数ERNS灌装。因此，参数化舒张期充盈的发展（PDF格式）形式主义表明，临床上观察到的早期二尖瓣流量的整个范围（多普勒E波）模式是非常好的契合了阻尼振荡运动的规律。这允许按照因果机制（反冲启动吸）能产生3（数字）独特的集总参数，其生理类似物室刚度（K），粘弹性/休闲（c）和负载（X个人电子波的分析O）。静脉流（多普勒电子波）的记录是在临床心脏病学的标准做法，因此，超声心动图的记录方法只简要回顾。我们的重点是确定从日常记录的电子波数据的PDF参数。作为突出的结果表明，一旦PDF参数已经从负载的适当数量而获得不同的E-波的不变拟tigator是免费使用的参数或者参数的构造指标（如储存能量半KX O 2，最大的AV压力梯度KX O，舒张功能的负载独立指数等 ），并选择生理或病理生理的角度进行量化。

多普勒波形代表了四种不同类型的填充图案的使用上文详述的方法（正常，假性，延迟松弛，缩窄限制性的）示于图2的图2A中示出的正常模式，其中，其本身是从假性难以区别图案。 图2B显示延迟的放松和图2C显示了严重的舒张功能障碍相关的紧缩限制性格局。为清楚起见，将PDF模型预测拟合叠加在图像上。传统的回声参数（E 峰 ，A ...

Watch this Scientific Journal Video about Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism at JoVE.

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

准确，因果关系为基础的全球舒张功能的定量已经被通过参数化舒张肺静脉流灌装（PDF格式）形式主义的运动造型为基础的分析来实现的。 PDF生成唯一的僵硬，放松和负载参数和阐发了"新"的生理，同时提供功能障碍的敏感和特异的指标。

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

quantification-global-diastolic-function-kinematic-modeling-based

Research

JoVE Journal

Bioengineering

11.1K 次观看.  Washington University in St. Louis. 准确，因果关系为基础的全球舒张功能的定量已经被通过参数化舒张肺静脉流灌装（PDF格式）形式主义的运动造型为基础的分析来实现的。 PDF生成唯一的僵硬，放松和负载参数和阐发了"新"的生理，同时提供功能障碍的敏感和特异的指标。 

视频: Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

科研

生物工程

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

用 Imagej 插件对小梁骨的外边界辅助分割和量化

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

微血管中场流的空间时间分析

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

机械测试的注入性量化

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

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