This work was supported in part by the Alan A. and Edith L. Wolff Charitable Trust, St. Louis, and the Barnes-Jewish Hospital Foundation. L. Shmuylovich and E. Ghosh were partially supported by predoctoral fellowship awards from the Heartland Affiliate of the American Heart Association. S. Zhu received partial support from the Washington University Compton Scholars Program and the College of Arts and Sciences&#8217; Summer&#160;Undergraduate&#160;Research&#160;Award. S. Mossahebi received partial support from the Department of Physics.&#160;

The procedure for acquiring echocardiographic images and analyzing them to obtain the PDF parameters is detailed below. Although cardiac catheterization is mentioned in the subject selection portion below, the methodology described applies only to the echocardiographic portion. The description of the catheterization portion was included for independent validation of model based predictions and is unrelated to the analysis of E-waves via the PDF formalism. Prior to data acquisition, all subjects provide signed, informed consent for participation in the study in accordance with the Institutional Review Board (Human Research Protection Office) at Washington University School of Medicine.

NOTE: All software programs (along with tutorials on how to use them) described in this section can be downloaded from <a href="http://cbl1.wustl.edu/SoftwareAgreement.htm" target="_blank">http://cbl1.wustl.edu/SoftwareAgreement.htm</a>
1. Subject Selection
NOTE: All subjects in the Cardiovascular Biophysics Laboratory Database had simultaneous echocardiography and cardiac catheterization performed and were referred by their physicians for diagnostic cardiac catheterization. The database inclusion criteria are: 1) absence of any significant valvular abnormalities, 2)&#160;absence of wall motion abnormalities or bundle branch block on ECG, 3) presence of a satisfactory echocardiographic window with clearly identifiable E- and A-waves.
2. Echocardiographic Data Acquisition
<ol>
	<li>Record a complete 2D/echo-Doppler study for all subjects in accordance with American Society of Echocardiography criteria16. NOTE: The screening echocardiograms were recorded on a standard clinical imager by a sonographer. If desired, additional transthoracic echocardiographic recording can be performed for verification purposes after a suitable, high fidelity catheter is advanced into the LV to measure LV hemodynamics simultaneously.</li>
	<li>Image subjects in the supine position. In a nonresearch setting,&#160;standard left lateral positioning can be used without loss of generality of the method. Obtain apical four-chamber views using a 2.5 MHz transducer, with the sample volume gated at 1.5-5 mm directed between the tips of the mitral valve leaflets and orthogonal to the MV plane (to minimize alignment effects as seen on color M-mode Doppler), the wall filter set at 1 (125 Hz) or 2 (250 Hz), the baseline adjusted to take advantage of the full height of the display and the velocity scale adjusted to exploit the dynamic range of the output without aliasing.</li>
	<li>Perform Doppler tissue imaging with the sample volume gated at 2.5 mm and positioned at the lateral and septal potions of the mitral annulus.</li>
	<li>Save Doppler examinations in DICOM format in the echo machine and record on DVD with simultaneously recorded electrocardiogram (ECG).</li>
</ol>
3. Doppler Image Processing and Conventional Analysis
NOTE: This section describes two&#160;custom MATLAB programs. The first program is described in step 3.1 and the second program is described in steps 3.2-3.5. All software programs (along with tutorials on how to use them) can be downloaded from&#160;<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>Convert images from the DICOM format and video to bitmap (.bmp) files (using a custom MATLAB program). NOTE: The procedure described below to fit Doppler E-waves and tissue Doppler E&#8217;-waves is shown in Figure 1.</li>
	<li>Load the bitmap image files on another custom MATLAB program to measure conventional transmitral flow parameters such as Epeak, Apeak, Edur, E&#8217;peak, A&#8217;peak, etc. and crop the images for PDF analysis. Select images with discernible transmitral flow contour and complete cardiac cycle as indicated by ECG for analysis.</li>
	<li>Mark the time sampling rate (measured in pixels/s on the horizontal axis) and velocity sampling rate (measured in pixels/(m/sec) along the vertical axis) in the images. Identify the complete cardiac cycle by noting and marking consecutive R peaks (or any distinct feature of the ECG) on the image.</li>
	<li>Mark the transmitral Doppler E- and A-wave or tissue Doppler E&#8217;- and A&#8217;- wave in the selected cardiac cycle.
	<ol>
		<li>Select the Doppler E-wave peak point i.e. Epeak, (or E&#8217;peak) and mark the start of the wave using the line connecting the peak to the start as a guide to match the acceleration slope of the E-wave (or E&#8217;-wave). The start of the wave is used to calculate the interval from start to peak flow denoted as the E-wave (or E&#8217;-wave) acceleration time (AT).</li>
		<li>Mark the end of the E-wave (or E&#8217;-wave) using the line connecting the peak to the end as a guide to match the deceleration slope. This is used to calculate the interval from the peak to the baseline denoted as the deceleration time (DT). The interval from start to end of the wave is the duration of the E-wave (Edur = AT+DT). The program guides the user through the entire process with appropriate instructions.</li>
	</ol>
	</li>
	<li>Mark the A-wave using a similar procedure as the E-wave. With both the E- and A-waves marked the program calculates the Epeak/Apeak ratio. 
	NOTE: The program saves the marked waves as cropped images containing the E- and A-waves only. The program also creates a data file with the cropping and measured parameters for each beat.</li>
</ol>
4. Automated Fitting of Transmitral Flow Using the PDF Formalism
<ol>
	<li>The automated fitting of Doppler E- and A-wave and tissue Doppler E&#8217;- and A&#8217;- wave contours is done using a custom LabView program18,19.
	<ol>
		<li>Load the cropped image, and the program automatically calculates the maximum velocity envelope (MVE). Select the MVE by setting the threshold such that MVE approximates transmitral flow as shown in Figure 1. The onset and termination of the points that define the MVE can be selected along the time-axis by the operator such that only MVE points that provide good correspondence to the actual selected portion of the wave are used as input for the subsequent fitting.</li>
	</ol>
	</li>
	<li>NOTE: The user-selected MVE points are the input to the computer program that automatically fits the PDF model solution for velocity as a function of time using a Levenberg- Marquardt (iterative) algorithm. The fitting is accomplished with the requirement that the mean square error between the clinical (input) data (MVE) and the PDF model predicted contour be minimized. Since the model is linear, a unique set of parameters is obtained for each Doppler E-wave derived MVE used as input. Thus numerically unique k, c, and xo values are generated for each E-wave and k&#8217;, c&#8217;, and xo&#8217; for each E&#8217;-wave.</li>
	<li>In the event the fit is obviously suboptimal when the fit is superimposed on the E-wave (or E&#8217;-wave) image (i.e. the algorithm attempted to fit noise included in the MVE for example) modify the MVE by using more/less points, thereby modifying the model predicted contour with consequent modification of PDF parameters to achieve a better fit.</li>
</ol>
Save the data when the appropriate PDF fit has been generated. NOTE: The program is written to automatically save the data in image and text files containing the PDF parameters and the contour information. 
The PDF parameters obtained from the procedure described above can be used to elucidate new physiology and distinguish between normal and pathological physiology as detailed in the Representative Results section below.

<ol>
	<li>Katz, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+played+by+the+ventricular+relaxation+process+in+filling+the+ventricle.">The role played by the ventricular relaxation process in filling the ventricle.</a> Am. J. Physiol. 95, 542-553 (1930).</li><li>Frais, M. A., Bergman, D. W., Kingma, I., Smiseth, O. A., Smith, E. R., Tyberg, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dependence+of+the+time+constant+of+left+ventricular+isovolumic+relaxation+on+pericardial+pressure.">The dependence of the time constant of left ventricular isovolumic relaxation on pericardial pressure.</a> Circulation. 81, 1071-1080 (1990).</li><li>Weiss, J. L., Frederiksen, J. W., Weisfeldt, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+determinants+of+the+time-course+of+fall+in+canine+left+ventricular+pressure.">Hemodynamic determinants of the time-course of fall in canine left ventricular pressure.</a> J. Clin Invest. 58, 751-760 (1976).</li><li>Weisfeldt, M. L., Weiss, J. L., Frederiksen, J. W., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+incomplete+left+ventricular+relaxation:+Relationship+to+the+time+constant+for+isovolumic+pressure+fall.">Quantification of incomplete left ventricular relaxation: Relationship to the time constant for isovolumic pressure fall.</a> Eur. Heart J. 1, 119-129 (1980).</li><li>Thompson, D. S., 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=Analysis+of+left+ventricular+pressure+during+isovolumic+relaxation+in+coronary+artery+disease.">Analysis of left ventricular pressure during isovolumic relaxation in coronary artery disease.</a> Circulation. 65, 690-697 (1982).</li><li>Ludbrook, P. A., Bryne, J. D., Kurnik, P. B., McKnight, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+reduction+of+preload+and+afterload+by+nitroglycerin+on+left+ventricular+diastolic+pressure-volume+relations+and+relaxation+in+man.">Influence of reduction of preload and afterload by nitroglycerin on left ventricular diastolic pressure-volume relations and relaxation in man.</a> Circulation. 56, 937-943 (1977).</li><li>Tyberg, J. V., Misbach, G. A., Glantz, S. A., Moores, W. Y., Parmley, W. 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Res. 48, 813-824 (1981).</li><li>Suga, 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=Systolic+pressure-volume+area+(PVA)+as+the+energy+of+contraction+in+Starling&#8217;s+law+of+the+heart.">Systolic pressure-volume area (PVA) as the energy of contraction in Starling&#8217;s law of the heart.</a> Heart Vessels. 6, 65-70 (1991).</li><li>Murakami, T., Hess, O., Gage, J., Grimm, J., Krayenbuehl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diastolic+filling+dynamics+in+patients+with+aortic+stenosis.">Diastolic filling dynamics in patients with aortic stenosis.</a> Circulation. 73, 1162-1174 (1986).</li><li>Baan, J., 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=Continuous+measurement+of+left+ventricular+volume+in+animals+and+humans+by+conductance+catheter.">Continuous measurement of left ventricular volume in animals and humans by conductance catheter.</a> Circulation. 70, 812-823 (1984).</li><li>Falsetti, H. 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The authors have no competing financial interests.

In keeping with our methodologic focus, the key aspects of the methods that facilitate obtaining accurate and meaningful results are highlighted.
ECHOCARDIOGRAPHY
The American Society of Echocardiography (ASE) has guidelines for the performance of transthoracic studies16. During an echo exam, there are a multitude of factors that affect image quality. Factors that are beyond the control of the sonographer include: technical capabilities of the imager bein...

Pioneering studies by Katz1 in 1930 revealed that the mammalian left ventricle initiates filling by being a mechanical suction pump, and much effort since then has been devoted to unraveling the workings of diastole. For many years, invasive methods were the only options available for clinical or research assessment of diastolic function (DF)2-16. In the 1970s, however, technical advancements and developments in echocardiography&#160;finally gave cardiologists and physiologists practical tools for noninvasive characterization of DF.
Without a unifying causal theory or paradigm for diastole regarding how the heart works when it fills, researchers proposed numerous phenomenologic indexes based on correlation with clinical features. The curvilinear, rapidly rising and falling shape of the transmitral blood flow velocity contour during early, rapid filling, for example, was approximated as a triangle and diastolic function indexes were defined from geometric features (height, width, area, etc.) of that triangle. Technical advancements in echocardiography have allowed tissue motion, strain, and strain rate during filling to be measured, for example, and each technical advancement brought with it a new crop of phenomenological indexes to be correlated with clinical features. However, the indexes remain correlative and not causal and many indexes are different measures of the same underlying physiology. It&#8217;s not surprising, therefore, that currently employed clinical indexes of DF have limited specificity and sensitivity.
To overcome these limitations the Parametrized Diastolic Filling (PDF) formalism, a causal kinematic, lumped parameter model of left ventricular filling that is motivated by and incorporates the suction-pump physiology of diastole was developed and validated17. It models diastolic function (as manifested by the curvilinear shapes of transmitral flow contours) in accordance with the rules of damped harmonic oscillatory motion. The equation for damped harmonic oscillatory motion is based on Newton&#8217;s Second Law and can be written, per unit mass, as:
<img alt="Equation 1" fo:content-width="1in" src="/files/ftp_upload/51471/51471eq1.jpg" />&#160; &#160; Equation&#160;1
This linear 2nd order differential equation has three parameters: k- chamber stiffness, c- viscoelasticity/relaxation, and xo- the oscillator&#8217;s initial displacement/preload. The model predicts that the different clinically observed diastolic filling patterns are the result of variation in the numerical value of these three&#160;model parameters. Based on the PDF formalism and classical mechanics, E-waves can be classified as being determined by under-damped or over-damped regimes of motion. Numerous studies17-21 have validated that clinically recorded E-wave contours and PDF model predicted contours show superb agreement&#160;and have elucidated the hemodynamic/physiologic analogues of the three&#160;PDF parameters21. The process for extracting model parameters from clinically recorded E-wave data is detailed in the methods below.
Unlike typical indexes of DF in current clinical use, the PDF model&#8217;s three&#160;parameters are causality based. As discussed in the methods below, additional indexes of diastolic physiology can be derived from these fundamental parameters&#160;and from application of the PDF formalism to aspects of diastole other than transmitral flow. In this work, methods of PDF-based analysis of transmitral flow and the physiologic relations that can be drawn from the PDF approach, its parameters and the derived indexes are described. Additionally, it is shown that the PDF parameters or indexes derived from them can tease apart intrinsic chamber properties from the external effects of load&#160;can provide correlates to traditional invasively defined parameters&#160;and can differentiate between normal and pathologic groups.

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

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Doppler waveforms representative of the four different types of filling patterns (normal, pseudonormal, delayed relaxation, constrictive-restrictive) using the method detailed above are shown in Figure 2. Figure&#160;2A shows the normal pattern, which, by itself is indistinguishable from the pseudonormal pattern. Figure 2B shows a delayed relaxation and Figure 2C shows a constrictive-restrictive pattern associated with severe diastolic dysfunction. For c...

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

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

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

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11.1K Views.  Washington University in St. Louis. Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation, and load parameters and elucidates 'new' physiology while providing sensitive and specific indexes of dysfunction.

Video: 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.

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

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

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.

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.

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.

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.

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.

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

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.

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.

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.