Questo lavoro è stato sostenuto in parte da Alan A. e Edith L. Wolff Charitable Trust, St. Louis, e l&#39;Ospedale Fondazione Barnes-Jewish. L. Shmuylovich e E. Ghosh sono stati in parte supportati da borse di studio premi predoctoral dal Affiliato Heartland della American Heart Association. S. Zhu ha ricevuto il sostegno parziale della Washington University Compton Scholars Program e il College of Arts e Summer Undergraduate Research Award Scienze &#39;. S. Mossahebi ricevuto il sostegno parziale da parte del Dipartimento di Fisica.

La procedura per l&#39;acquisizione di immagini ecocardiografiche e analizzarli per ottenere i parametri PDF è descritto di seguito. Anche se cateterismo cardiaco è menzionato nella porzione selezione soggetto sotto, la metodologia descritta si applica solo alla parte ecocardiografica. La descrizione della porzione cateterizzazione è stata inclusa per la validazione indipendente del modello basato previsioni e non è correlato all&#39;analisi di E-onde tramite il formalismo PDF. Prima di acquisizione dei dati, tutti i soggetti forniscono firmato, il consenso informato per la partecipazione allo studio in conformità con la Institutional Review Board (Ufficio Protezione umano di ricerca) presso la Washington University School of Medicine. NOTA: Tutti i programmi software (insieme con tutorial su come usarli) descritte in questa sezione possono essere scaricati dal <a href="http://cbl1.wustl.edu/SoftwareAgreement.htm" target="_blank">http://cbl1.wustl.edu/Softw</a>areAgreement.htm 1 Oggetto Selezione NOTA: Tutti i soggetti presenti nel Database cardiovascolare Laboratorio di Biofisica avevano ecocardiografia simultanea e cateterismo cardiaco effettuato e sono stati indirizzati dai loro medici per la diagnostica cateterismo cardiaco. I criteri di inclusione del database sono: 1) assenza di significative anomalie valvolari, 2) assenza di anomalie di movimento della parete o blocco di branca di ECG, 3) la presenza di una finestra ecocardiografica soddisfacente con E-chiaramente identificabili e A-onde. 2 ecocardiografica Acquisizione Dati <ol><li> Registrare un completo studio / eco-Doppler 2D per tutti i soggetti, in conformità della Società Americana di Ecocardiografia criteri 16. NOTA: Gli ecocardiogrammi di screening sono stati registrati su un imager clinica standard da un ecografista. Se lo si desidera, ulteriori registrazione ecocardiografica transtoracica può essere eseguita per fini di verificas dopo un catetere adatto, l&#39;alta fedeltà è avanzata nella LV per misurare l&#39;emodinamica LV contemporaneamente. </li><li> Soggetti immagine nella posizione supina. In un ambiente nonresearch, posizionamento laterale sinistro standard può essere utilizzato senza perdita di generalità del metodo. Ottenere apicali vista quattro camere utilizzando un trasduttore 2,5 MHz, con il volume del campione gated a 1,5-5 mm diretti tra le punte dei lembi della valvola mitrale e ortogonale al piano MV (per minimizzare gli effetti di allineamento come visto sul colore di modo M Doppler ), il filtro parete impostato a 1 (125 Hz) o 2 (250 Hz), la linea di base adeguato per sfruttare tutta l&#39;altezza del display e la scala di velocità adeguato per sfruttare la gamma dinamica dell&#39;uscita, senza aliasing. </li><li> Eseguire l&#39;imaging Doppler tissutale con il volume del campione gated a 2,5 mm e posizionato le pozioni laterale e settale della mitrale. </li><li> Salva esami Doppler in formato DICOM nella macchina eco e registrare su DVD con simultelettrocardiogramma aneously registrati (ECG). </li></ol> Immagine 3 Doppler elaborazione e l&#39;analisi convenzionale NOTA: Questa sezione descrive due programmi personalizzati MATLAB. Il primo programma è descritta nel passo 3.1 e il secondo programma è descritto nei passaggi 3,2-3,5. Tutti i programmi software (insieme con tutorial su come usarli) possono essere scaricati da <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> Convertire le immagini dal formato DICOM e video in file bitmap (bmp) (utilizzando un programma MATLAB personalizzato). NOTA: La procedura descritta di seguito per adattarsi Doppler E-onde e Doppler tissutale E&#39;-onde è mostrato in Figura 1. </li><li> Caricare i file di immagine bitmap su un altro programma MATLAB personalizzato per misurare parametri di flusso transmitralico convenzionali come picco E, A picco, E dur </sub&gt;, E &#39;picco, A&#39; picco, ecc. e ritagliare le immagini per l&#39;analisi in formato PDF. Selezionare le immagini con contorno distinguibile flusso transmitralico e ciclo cardiaco completo come indicato dalla ECG per l&#39;analisi. </li><li> Seleziona il tasso di tempo di campionamento (misurata in pixel / s sull&#39;asse orizzontale) e frequenza di campionamento velocità (misurata in pixel / (m / sec) lungo l&#39;asse verticale) nelle immagini. Identificare il ciclo cardiaco completo osservando e marcatura picchi consecutivi R (o qualsiasi caratteristica distinta del ECG) sull&#39;immagine. </li><li> Segna il Doppler transmitralico E e A-onda o tessuto Doppler E&#39;- e A&#39;- onda nel ciclo cardiaco selezionato. <ol><li> Selezionare il Doppler E-onda punto di picco cioè. E picco, (o E &#39;di picco) e segnare l&#39;inizio dell&#39;onda utilizzando la linea che collega il picco all&#39;inizio come guida per abbinare la pendenza accelerazione della E-onda (o E&#39;-onde). L&#39;inizio dell&#39;onda viene utilizzato per calcolare l&#39;intervallo dall&#39;inizio alla peflusso ak denotato come l&#39;E-onda (o E&#39;-onde) tempo di accelerazione (AT). </li><li> Segna la fine della E-onda (o E&#39;-onde) utilizzando la linea che collega il picco alla fine come guida per abbinare la pendenza di decelerazione. Questo viene utilizzato per calcolare l&#39;intervallo dal picco alla linea di base indicato come il tempo di decelerazione (DT). L&#39;intervallo dall&#39;inizio alla fine dell&#39;onda è la durata della E-onda (E dur = AT + DT). Il programma guida l&#39;utente attraverso l&#39;intero processo con istruzioni appropriate. </li></ol></li><li> Mark l&#39;A-onda utilizzando una procedura simile a quella della E-onda. Con sia la E e A-onde ha segnato il programma calcola il picco rapporto E / A di picco. NOTA: Il programma salva le onde marcate come immagini ritagliate contenenti la E e solo A-onde. Il programma crea anche un file di dati con i parametri di ritaglio e misurati per ogni battito. </li></ol> 4 Montaggio automatico del flusso transmitralico utilizzando il formalismo PDF <ol&gt; <li> Il montaggio automatizzato di Doppler E e A-wave e del tessuto Doppler E&#39;- e contorni onda A&#39;- viene fatto utilizzando un programma LabView personalizzato 18,19. <ol><li> Caricare l&#39;immagine ritagliata, e il programma calcola automaticamente la busta velocità massima (MVE). Selezionare il MVE impostando la soglia così che MVE approssima flusso transmitralico come mostrato in Figura 1. L&#39;inizio e terminazione dei punti che definiscono la MVE può essere selezionato nel tempo asse dall&#39;operatore tale che solo MVE punti che forniscono buona corrispondenza alla porzione effettiva selezionata dell&#39;onda sono utilizzati come ingresso per il successivo montaggio. </li></ol></li><li> NOTA: I punti di MVE selezionati dall&#39;utente sono l&#39;ingresso al programma per computer che si adatta automaticamente la soluzione di modello PDF per la velocità in funzione del tempo utilizzando un algoritmo Levenberg- Marquardt (iterativo). L&#39;apparecchio è realizzato con la condizione che l&#39;errore quadratico medio tra la clinica (ingresso)dati (MVE) e il modello PDF contorno previsto essere ridotti al minimo. Dal momento che il modello è lineare, si ottiene un insieme unico di parametri per ciascun Doppler E-onda derivato MVE utilizzato come input. Così numericamente k, c, ed i valori x o unici sono generati per ogni E-wave e k &#39;, c&#39;, e x o &#39;per ogni E&#39;-onda. </li><li> Nel caso in cui la misura è ovviamente ottimale quando la misura si sovrappone su E-onda (o E&#39;-onde) immagine (cioè. L&#39;algoritmo ha tentato di adattarsi rumore incluso nel MVE ad esempio) modificare la MVE utilizzando più / meno punti, modificando in tal modo il modello previsto profilo con conseguente modifica dei parametri PDF per ottenere una migliore vestibilità. </li></ol> Salvare i dati quando è stato generato l&#39;appropriato adattamento PDF. NOTA: Il programma è scritto per salvare automaticamente i dati in immagini e file di testo contenenti i parametri di PDF ele informazioni di contorno. I parametri PDF ottenuti dalla procedura sopra descritta può essere utilizzata per chiarire nuova fisiologia e distinguere tra fisiologia normale e patologico, come descritto nella sezione Rappresentante dei risultati qui sotto.

<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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+shifts+in+the+diastolic,+left+ventricular,+pressure-volume+curve:+The+role+of+the+pericardium.">A mechanism for shifts in the diastolic, left ventricular, pressure-volume curve: The role of the pericardium.</a> Eur. J. Cardiol. 7, 163-175 (1978).</li><li>Suga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+a+left-ventricular+pumping+model+based+on+the+systolic+time-varying+pressure/volume+ratio.">Theoretical analysis of a left-ventricular pumping model based on the systolic time-varying pressure/volume ratio.</a> IEEE Trans. Biomed. Eng. 24, 29-38 (1977).</li><li>Raff, G. L., Glantz, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+loading+slows+left+ventricular+isovolumic+relaxation+rate.">Volume loading slows left ventricular isovolumic relaxation rate.</a> Circ. 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. L., Verani, M. S., Chen, C. J., Cramer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+pressure+differences+in+the+left+ventricle.">Regional pressure differences in the left ventricle.</a> Catheter Cardiovasc. Diag. 6, 123-134 (1980).</li><li>Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+diastolic+dysfunction.+Invasive+modalities.">Assessment of diastolic dysfunction. Invasive modalities.</a> Cardiol. Clin. 18 (3), 571-586 (2000).</li><li>Suga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+energetics:+from+EMAX+to+pressure-volume+area.">Cardiac energetics: from EMAX to pressure-volume area.</a> Clin. Exp. Pharmacol. Physiol. 30, 580-585 (2003).</li><li>Gottdiener, J. 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=American+Society+of+Echocardiography+recommendations+for+use+of+echocardiography+in+clinical+trials.">American Society of Echocardiography recommendations for use of echocardiography in clinical trials.</a> JASE. 17, 1086-1119 (2004).</li><li>Kov&#225;cs, S. J., Barzilai, B., P&#233;rez, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+diastolic+function+with+Doppler+echocardiography:+the+PDF+formalism.">Evaluation of diastolic function with Doppler echocardiography: the PDF formalism.</a> Am. J. Physiol. Heart Circ. Physiol. 252, H178-H187 (1987).</li><li>Hall, A. F., Aronovitz, J. A., Nudelman, S. P., 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=Automated+method+for+characterization+of+diastolic+transmitral+Doppler+velocity+contours:+Late+atrial+filling.">Automated method for characterization of diastolic transmitral Doppler velocity contours: Late atrial filling.</a> Ultrasound Med. Biol. 20, 859-869 (1994).</li><li>Hall, A. F., 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=Automated+method+for+characterization+of+diastolic+transmitral+Doppler+velocity+contours:+Early+rapid+filling.">Automated method for characterization of diastolic transmitral Doppler velocity contours: Early rapid filling.</a> Ultrasound Med. Biol. 20, 107-116 (1994).</li><li>Riordan, M. M., 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=Quantitation+of+Mitral+Annular+Oscillations+and+Longitudinal+'Ringing'+of+the+Left+Ventricle:+A+New+Window+into+Longitudinal+Diastolic+Function.">Quantitation of Mitral Annular Oscillations and Longitudinal 'Ringing' of the Left Ventricle: A New Window into Longitudinal Diastolic Function.</a> J. Appl. Physiol. 100, 112-119 (2006).</li><li>Kov&#225;cs, S. J., Meisner, J. S., Yellin, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+diastole.">Modeling of diastole.</a> Cardiol. Clin. 18, 459-487 (2000).</li><li>Riordan, M. M., 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=Diabetes+and+Diastolic+Function:+Stiffness+and+Relaxation+from+Transmitral+Flow.">Diabetes and Diastolic Function: Stiffness and Relaxation from Transmitral Flow.</a> Ultrasound Med. Biol. 31, 1589-1596 (2005).</li><li>Bauman, L., Chung, C. S., Karamanoglu, M., 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=The+peak+atrioventricular+pressure+gradient+to+transmitral+flow+relation:+kinematic+model+prediction+with+in+vivo+validation.">The peak atrioventricular pressure gradient to transmitral flow relation: kinematic model prediction with in vivo validation.</a> J. Am. Soc. Echocardiogr. 17 (8), 839-844 (2004).</li><li>Kov&#225;cs, S. J., Rosado, J., Manson-McGuire, A. L., Hall, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+Transmitral+Doppler+E-waves+Differentiate+Hypertensive+Hearts+From+Normal?.">Can Transmitral Doppler E-waves Differentiate Hypertensive Hearts From Normal?.</a> Hypertension. 30, 788-795 (1997).</li><li>Riordan, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Caloric+Restriction-+and+Exercise-Induced+Weight+Loss+on+Left+Ventricular+Diastolic+Function.">The Effects of Caloric Restriction- and Exercise-Induced Weight Loss on Left Ventricular Diastolic Function.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H1174-H1182 (2008).</li><li>Meyer, T. E., Kov&#225;cs, S. J., Ehsani, A. A., Klein, S., Holloszy, J. O., Fontana, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Caloric+Restriction+Slows+Cardiac+Aging+in+Humans.">Long-term Caloric Restriction Slows Cardiac Aging in Humans.</a> J. Am. Coll. Cardiol. 47, 398-402 (2006).</li><li>Riordan, M. M., 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=Absence+of+diastolic+mitral+annular+oscillations+is+a+marker+for+relaxation-+related+diastolic+dysfunction.">Absence of diastolic mitral annular oscillations is a marker for relaxation- related diastolic dysfunction.</a> Am. J. Physiol. Heart Circ. Physiol. 292, H2952-H2958 (2007).</li><li>Mossahebi, 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=Kinematic+Modeling-based+Left+Ventricular+Diastatic+(Passive)+Chamber+Stiffness+Determination+with+In-Vivo+Validation.">Kinematic Modeling-based Left Ventricular Diastatic (Passive) Chamber Stiffness Determination with In-Vivo Validation.</a> Annals BME. 40 (5), 987-995 (2012).</li><li>Zhang, W., Chung, C. S., Riordan, M. M., Wu, Y., Shmuylovich, L., 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=The+Kinematic+Filling+Efficiency+Index+of+the+Left+Ventricle:+Contrasting+Normal+vs.+Diabetic+Physiology.">The Kinematic Filling Efficiency Index of the Left Ventricle: Contrasting Normal vs. Diabetic Physiology.</a> Ultrasound Med. Biol. 33, 842-850 (2007).</li><li>Zhang, W., 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=The+Age+Dependence+of+Left+Ventricular+Filling+Efficiency.">The Age Dependence of Left Ventricular Filling Efficiency.</a> Ultrasound Med. Biol. 35, 1076-1085 (2009).</li><li>Courtois, M., Kov&#225;cs, S. J., Ludbrook, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitral+pressure-flow+velocity+relation.+Importance+of+regional+pressure+gradients+in+the+left+ventricle+during+diastole.">Transmitral pressure-flow velocity relation. Importance of regional pressure gradients in the left ventricle during diastole.</a> Circulation. 78, 661-671 (1988).</li><li>Zhang, W., Shmuylovich, L., 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=The+E-wave+delayed+relaxation+pattern+to+LV+pressure+contour+relation:+model-based+prediction+with+in+vivo+validation.">The E-wave delayed relaxation pattern to LV pressure contour relation: model-based prediction with in vivo validation.</a> Ultrasound Med. Biol. 36 (3), 497-511 (2010).</li><li>Shmuylovich, L., 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=A+load-independent+index+of+diastolic+filling:+model-based+derivation+with+in-vivo+validation+in+control+and+diastolic+dysfunction+subjects.">A load-independent index of diastolic filling: model-based derivation with in-vivo validation in control and diastolic dysfunction subjects.</a> J. Appl. 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.

In linea con il nostro obiettivo metodologico, sono evidenziati gli aspetti chiave dei metodi che facilitano ottenere risultati accurati e significativi. ECOCARDIOGRAFIA La Società Americana di Ecocardiografia (ASE) ha linee guida per la realizzazione di studi transtoracici 16. Durante un esame eco, ci sono una moltitudine di fattori che influenzano la qualità dell&#39;immagine. I fattori che sfuggono al controllo del ecografista includono: capacità...

Studi pionieristici di Katz 1 nel 1930 ha rivelato che il ventricolo sinistro mammiferi inizia il riempimento con l&#39;essere una pompa di aspirazione meccanica, e molto sforzo da allora è stata dedicata a svelare i meccanismi della diastole. Per molti anni, metodi invasivi erano le uniche opzioni disponibili per la valutazione clinica o di ricerca di funzione diastolica (DF) 2-16. Nel 1970, tuttavia, i progressi tecnici e gli sviluppi in ecocardiografia finalmente dato cardiologi e fisiologi strumenti pratici per la caratterizzazione non invasiva di DF. Senza una teoria causale di unificazione o di paradigma per quanto riguarda diastole come funziona il cuore quando si riempie, i ricercatori hanno proposto numerosi indici fenomenologici sulla base di correlazione con le caratteristiche cliniche. Il curvilineo, rapidamente salire e scendere la forma del transmitralico flusso di sangue velocità di contorno durante la prima, riempimento rapido, per esempio, è stata approssimata come un triangolo e diastolica fuindici nzioni sono state definite da caratteristiche geometriche (altezza, larghezza, zona, ecc.), di tale triangolo. Progressi tecnici in ecocardiografia hanno permesso il movimento dei tessuti, deformazione e velocità di deformazione durante il riempimento da misurare, per esempio, e ogni progresso tecnico ha portato con sé un nuovo raccolto di indici fenomenologici per essere correlata con caratteristiche cliniche. Tuttavia, gli indici rimangono correlativa e non causali e molti indici sono diverse misure della stessa fisiologia sottostante. Non sorprende, quindi, che gli indici clinici attualmente impiegati di DF hanno limitato la specificità e sensibilità. Per superare queste limitazioni il riempimento (PDF) formalismo parametrizzate diastolica, un cinematica causale, modello a parametri concentrati di riempimento ventricolare sinistro che è motivato dalla e incorpora la fisiologia di aspirazione-pompa di diastole è stato sviluppato e validato 17. Modella funzione diastolica (come manifestato dalle forme curvilineecontorni di flusso transmitralico) in conformità con le regole del moto oscillatorio armonico smorzato. L&#39;equazione per smorzato moto oscillatorio armonico si basa sulla seconda legge di Newton e può essere scritta, per unità di massa, come: <img alt="Equazione 1" fo:content-width="1in" src="/files/ftp_upload/51471/51471eq1.jpg" /> Equazione 1 Questo 2 ° equazione differenziale di ordine lineare ha tre parametri: k - rigidezza camera, c - viscoelasticità / relax, e x o - dell&#39;oscillatore iniziale spostamento / precarico. Il modello prevede che i diversi pattern di riempimento diastolico clinicamente osservati sono il risultato di variazione nel valore numerico di questi tre parametri del modello. Sulla base del formalismo PDF e meccanica classica, E-onde possono essere classificate come determinato da o over-smorzato regimi sotto-smorzate del moto. Numerosi studi <sup&gt; 17-21 hanno convalidato che clinicamente registrati contorni E-wave e il modello PDF previsti contorni mostrano accordo superbo e hanno chiarito le emodinamiche / analoghi fisiologici dei tre parametri PDF 21. Il processo per l&#39;estrazione di parametri del modello da clinicamente registrati dati E-onda è dettagliato nei metodi indicati. A differenza di indici tipici di DF in corrente uso clinico, tre parametri del modello PDF sono basati causalità. Come discusso nei metodi indicati, indici aggiuntivi di fisiologia diastolica possono essere derivate da questi parametri fondamentali e dall&#39;applicazione del formalismo PDF per aspetti di altri diastole di flusso transmitralico. In questo lavoro, metodi di analisi basati su PDF del flusso transmitralico e le relazioni fisiologiche che si possono trarre da un approccio PDF, i suoi parametri e gli indici derivati ​​sono descritti. Inoltre, è dimostrato che i parametri PDF o indici da essi derivati ​​possono prendere in giroa parte le proprietà intrinseche della camera dagli effetti esterni di carico in grado di fornire correlati ai parametri definiti invasivo tradizionali e possono distinguere tra i gruppi normali e patologici.

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			<td height="42" style="height:42px;width:216px;">Name of Equipment/ Software</td>
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			<td height="21" style="height:21px;width:216px;">Philips iE33</td>
			<td style="width:159px;">Philips (Andover, MA.)</td>
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			<td height="21" style="height:21px;width:216px;">LabVIEW 6.0</td>
			<td style="width:159px;">National Instruments</td>
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			<td>Version 6.0.2</td>
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			<td height="21" style="height:21px;width:216px;">MATLAB</td>
			<td style="width:159px;">MathWorks</td>
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			<td>Version R2010b</td>
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quantification of global diastolic function by kinematic modeling-based analysis of transmitral flow via the parametrized diastolic filling formalism

Valutazione della funzione cardiaca quantitativa rimane una sfida per i fisiologi e medici. Anche se i metodi invasivi storicamente hanno costituito l&#39;unico mezzo a disposizione, lo sviluppo di modalità di imaging non invasive (ecocardiografia, risonanza magnetica, TAC) con alta risoluzione temporale e spaziale fornisce una nuova finestra per la valutazione della funzione diastolica quantitativa. L&#39;ecocardiografia è il concordato standard per la valutazione della funzione diastolica, ma gli indici di corrente uso clinico semplicemente utilizzano caratteristiche di dimensione della camera (M-mode) o il movimento del sangue / tessuti (Doppler) forme d&#39;onda selezionata senza incorporare le determinanti causali fisiologiche del movimento stesso. Il riconoscimento che tutti ventricolo sinistro (LV) avviare la compilazione servendo come pompe di aspirazione meccanica permette la funzione diastolica globale per essere valutata sulla base di leggi del moto che si applicano a tutte le camere. Ciò che differenzia un cuore da un altro sono i parametri dell&#39;equazione di moto che govriempimento RER. Di conseguenza, lo sviluppo del riempimento diastolico parametrizzate (PDF) formalismo ha dimostrato che l&#39;intera gamma di clinicamente osservato precoce flusso transmitralico (E-Doppler a microonde) i modelli sono estremamente ben in forma con le leggi del moto oscillatorio smorzato. Ciò consente l&#39;analisi dei singoli E-onde in base a un meccanismo causale (aspirazione rinculo-iniziati) che produce tre (numericamente) parametri concentrati unici i cui analoghi fisiologica sono rigidità camera (k), viscoelasticità / rilassamento (c), e del carico (x o). La registrazione del flusso transmitralico (Doppler E-onde) è una pratica standard in cardiologia clinica e, quindi, il metodo di registrazione ecocardiografica è solo brevemente recensione. Il nostro obiettivo è la determinazione dei parametri di PDF da dati E-onda regolarmente registrati. Poiché i risultati evidenziati indicano, una volta che i parametri PDF sono stati ottenuti da un numero adeguato di carico variabile E-onde, gli invetigator è libero di utilizzare i parametri o costruire gli indici dei parametri (come l&#39;energia immagazzinata mezzo kx o 2, pressione massima pendenza AV kx o, Indice di carico indipendente della funzione diastolica, ecc.) e selezionare l&#39;aspetto della fisiologia o la fisiopatologia da quantificare.

Forme d&#39;onda Doppler rappresentante dei quattro diversi tipi di pattern di riempimento (normale, pseudonormale, rilassamento ritardato, costrittiva restrittivo) utilizzando il metodo sopra descritto sono illustrati nella Figura 2. Figura 2A mostra il modello normale, che, di per sé è indistinguibile dalla pseudonormale modello. Figura 2B mostra un ritardo relax e Figura 2C mostra un modello costrittiva-restrittiva associata a grave disfunzione diastolica. Per chia...

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

Preciso, a base di causalità-quantificazione della funzione diastolica globale è stato raggiunto cinematica analisi basato sulla modellazione del flusso transmitralico tramite il parametrizzate diastolica di riempimento (PDF) formalismo. PDF genera rigidità, relax, e parametri di carico unici e delucida &#39;nuovo&#39; fisiologia, fornendo indici sensibili e specifici di disfunzione.

Quantificazione di Global Funzione diastolica da Analisi basata su modellazione cinematica del flusso transmitralico tramite il formalismo di riempimento parametrizzate diastolica

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

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Bioengineering

11.1K Views.  Washington University in St. Louis.  Preciso, a base di causalità-quantificazione della funzione diastolica globale è stato raggiunto cinematica analisi basato sulla modellazione del flusso transmitralico tramite il parametrizzate diastolica di riempimento (PDF) formalismo. PDF genera rigidità, relax, e parametri di carico unici e delucida 'nuovo' fisiologia, fornendo indici sensibili e specifici di disfunzione. 

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.

Valutazione della consegna del gene polimerici Nanoparticelle per l&#39;analisi delle nanoparticelle Monitoraggio e High-throughput Citometria a Flusso

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

Ricerca

Bioingegneria

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.

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

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.

Elettrico Cell-substrato Impedance Sensing per la quantificazione dei Endothelial proliferazione, Barriera funzione e motilità

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.

La lignina down-regulation dei<em&gt; Zea mays</em&gt; Via dsRNAi e Klason lignina analisi

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.

Contorno esterno assistita segmentazione e quantificazione delle ossa Trabecular da un Plugin di Imagej

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.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

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.

Misurazione della proteinasi metallica della matrice cellulare globale e l'attività metabolica in idrogel 3D

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.

Quantificazione simultanea di kynurenine selezionate analizzate dalla cromatografia liquida-spettrometria di massa in mezzo raccolto da colture cellulari tumorali

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.

La quantificazione dell'iniettabilità mediante prove meccaniche

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.