Los autores agradecen al técnico dental Nicola Lanfranconi para producir el modelo de referencia principal y la Compañía Alicona por su continuo apoyo a la mejora del software de escaneo.

1. Preparación de las muestras / Base <ol><li> Aplicar una base plana alrededor de la muestra. Coloque la muestra en la mesa de exploración. Orientar la superficie oclusal al plano horizontal (figura 1). </li></ol> 2. Análisis de Software <ol><li> Inicie el programa y luego el módulo de medición de laboratorio (Figura 2) </li><li> Coloque la muestra en el centro de la mesa de exploración. </li><li> Elija el lente de aumento correcto. Para los objetos grandes, como arco barridos completos, utilice el objetivo de 5X. </li><li> Mueva la óptica de exploración utilizando el ratón 3D hasta que se muestre la superficie de la muestra en la ventana de visualización en vivo (Figura 3). </li><li> Utilice el control de sensor para ajustar la exposición y el contraste para conseguir los parámetros óptimos de digitalización. Para las superficies de metal, utilice una exposición entre 400 y 800 microsegundos y un contraste entre 0,3 y 0,8 (Figura 4). </li><li> Compruebe la calidad de imagen mediante la comprobación en el botón&quot;Demostración de la calidad de imagen en la vista previa.&quot; </li><li> Establezca los parámetros correctos en la sección &quot;Control de Medición&quot;. El tipo de medición es &quot;3D Conjunto de datos&quot;; ImageField Type es &quot;ImageField General&quot; (Figura 5). </li><li> Haga clic en &quot;Nuevo ImageField&quot;. </li><li> Definir el volumen de exploración. El software tiene las coordenadas de delimitación del volumen de medida. <ol><li> Mueva la muestra al nivel más alto y más bajo de exploración y haga clic en &quot;Añadir Position&quot; en ambos puntos. El valor &quot;Z Rango&quot; muestra la altura real del volumen de análisis (Figura 6). </li><li> Mueva el espécimen a los boundings XY del volumen de exploración. Haga clic en &quot;Add Position&quot; para definir el eje X y longitudes del eje Y. La dimensión del volumen de exploración se muestra en la pestaña &quot;Información&quot; y debe ser superior a las dimensiones de la muestra de 1 cm en el eje X y el eje Y. </li></ol></li><li> Compruebe el número de &quot;puntos&quot;. El software es capaz de escanear 100 millones de puntos de la superficie en una exploración &quot;ImageField&quot;. El número realde puntos supera este límite. &quot;Diezmar&quot; el número del punto de medida por una &quot;disminución de resolución lateral&quot; del tamaño de punto. <ol><li> Haga clic en &quot;Configuración avanzada&quot; (Figura 7). </li><li> Mueva el control deslizante &quot;Lateral disminución de resolución&quot; a la derecha hasta que el número de &quot;puntos&quot; se reduce por debajo de 100 M (Figura 7). La disminución de resolución reduce la resolución lateral de los puntos de la superficie, lo que resulta en un tamaño más grande de píxeles del objeto escaneado. Para asegurar los mejores resultados de escaneo, se aumenta la resolución vertical. </li></ol></li><li> Haga clic en &quot;Medición Inicio&quot;. Esto iniciará el &quot;Modo Previsualizar&quot;. El software realiza un pre-escaneo con las dimensiones X e Y de los ejes seleccionados. </li><li> Una vez completado el pre-escaneo, seleccione la región de interés. Esto ayuda a reducir el tamaño del archivo y el tiempo de barrido (Figura 8). </li><li> Seleccione todas las partes del pre-escaneo, excepto la muestra y dos campos de medición alrededor de la muestra, que contiene la base plana (Figura 9). </li><li> Haga clic en &quot;Inicio&quot; para iniciar la exploración. </li><li> Controle el análisis en la &quot;ImageViewer&quot; con el ratón y pulse el botón izquierdo del ratón (Figura 10). </li><li> Cierre la ventana y haga clic en &quot;Ver Pseudo Solo Color&quot;. </li><li> Haga clic en &quot;Configuración&quot; y &quot;Pseudo colorear&quot;, y seleccione &quot;repetibilidad&quot;. </li><li> Ajuste el &quot;Max.&quot; valorar a 0.2 y haga clic en &quot;Aplicar intervalo&quot;. </li><li> Control de la repetibilidad, que debe ser igual para las zonas con la misma inclinación y el material. En particular, la base alrededor de la muestra debe mostrar una repetibilidad homogénea (Figura 11). </li><li> Haga clic en &quot;Base de datos&quot;, y guardar el escaneo en la carpeta correspondiente. </li><li> Exportar la exploración a diferentes formatos de archivo, si es necesario. Haga clic en &quot;File/Export/3D Data As / ....&quot; El protocolo puede ser una pausa en ese punto y continuó más tarde. </li></ol> 3. Análisis Diferencia <ol><li> Utilice la opción &quot;3D-Editor&quot; para cortar la base. Esta área no será utilizado para la diferenciaanálisis. </li><li> Comparar y analizar dos exploraciones, inicie el software &quot;Medición de la variación&quot; (Figura 12). </li><li> Elija el segundo modelo para comparar. </li><li> Haga clic en &quot;Automático Rough Alineación&quot; para llevar a cabo un primer partido de los modelos. </li><li> Haga clic en &quot;Ajuste Manual&quot; y alinee los modelos con la rotación y mover hasta que están en la misma orientación (Figura 13). </li><li> Haga clic en &quot;Alineación automática&quot; y &quot;Aplicar&quot; para comenzar el mejor algoritmo de ajuste del modelo a juego óptima. </li><li> Haga clic en &quot;Diferencias&quot; para ver el mapa de diferencias de los dos modelos emparejados. </li><li> Seleccione un rango de color adecuado para mostrar las desviaciones (Figura 14). Guarde el patrón de desviación visual como una captura de pantalla para su análisis. </li></ol> Haga clic en &quot;Estadísticas&quot; para mostrar los valores estadísticos de las diferencias. Elija el tamaño de clase 1 micras y guardar los datos del histograma a un archivo de texto para la comparación estadística (Figura 15).

<ol>
	<li>Wettstein, F., Sailer, I., Roos, M., H&#228;mmerle, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+study+of+the+internal+gaps+of+zirconia+and+metal+frameworks+for+fixed+partial+dentures.">Clinical study of the internal gaps of zirconia and metal frameworks for fixed partial dentures.</a> Eur. J. Oral Sci. 116 (3), 272-279 (2008).</li><li>Persson, A. S., Oden, A., Andersson, M., Sandborgh-Englund, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitization+of+simulated+clinical+dental+impressions:+virtual+three-dimensional+analysis+of+exactness.">Digitization of simulated clinical dental impressions: virtual three-dimensional analysis of exactness.</a> Dent. Mater. 25 (7), 929-936 (2009).</li><li>Del'Acqua, M. A., Arioli-Filho, J. N., Compagnoni, M. A., Mollo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=de+A+Accuracy+of+impression+and+pouring+techniques+for+an+implant-supported+prosthesis.">de A Accuracy of impression and pouring techniques for an implant-supported prosthesis.</a> Int. J. Oral Maxillofac. Implants. 23 (2), 226-236 (2008).</li><li>Schneider, D., Grunder, U., Ender, A., H&#228;mmerle, C. H. F., Jung, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+gain+and+stability+of+peri-implant+tissue+following+bone+and+soft+tissue+augmentation:+1-year+results+from+a+prospective+cohort+study.">Volume gain and stability of peri-implant tissue following bone and soft tissue augmentation: 1-year results from a prospective cohort study.</a> Clinical Oral Implants Research. 22 (1), 28-37 (2011).</li><li>Windisch, S. I., Jung, R. E., Sailer, I., Studer, S. P., Ender, A., H&#228;mmerle, C. H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+optical+method+to+evaluate+three-dimensional+volume+changes+of+alveolar+contours:+a+methodological+in+vitro+study.">A new optical method to evaluate three-dimensional volume changes of alveolar contours: a methodological in vitro study.</a> Clinical Oral Implants Research. 18 (5), 545-551 (2007).</li><li>Caputi, S., Varvara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensional+accuracy+of+resultant+casts+made+by+a+monophase,+one-step+and+two-step,+and+a+novel+two-step+putty/light-body+impression+technique:+an+in+vitro.">Dimensional accuracy of resultant casts made by a monophase, one-step and two-step, and a novel two-step putty/light-body impression technique: an in vitro.</a> 99 (4), 274-281 (2008).</li><li>Hoyos, A., Soderholm, K. J. <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+tray+rigidity+and+impression+technique+on+accuracy+of+polyvinyl+siloxane+impressions.">Influence of tray rigidity and impression technique on accuracy of polyvinyl siloxane impressions.</a> Int. J. Prosthodont. 24 (1), 49-54 (2011).</li><li>Luthardt, R. G., Kuhmstedt, P., Walter, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+computer-aided+evaluation+of+three-dimensional+changes+in+gypsum+materials.">A new method for the computer-aided evaluation of three-dimensional changes in gypsum materials.</a> Dent. Mater. 19 (1), 19-24 (2003).</li><li>Mehl, A., Ender, A., M&#246;rmann, W., Attin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+testing+of+a+new+intraoral+3D+camera.">Accuracy testing of a new intraoral 3D camera.</a> International Journal of Computerized Dentistry. 12 (1), 11-28 (2009).</li><li>Fickl, 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=Dimensional+changes+of+the+ridge+contour+after+socket+preservation+and+buccal+overbuilding:+an+animal+study.">Dimensional changes of the ridge contour after socket preservation and buccal overbuilding: an animal study.</a> J. Clin. Periodontol. 36 (5), 442-448 (2009).</li><li>Vlaar, S. T., vander Zel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+dental+digitizers.">Accuracy of dental digitizers.</a> Int. Dent. J. 56 (5), 301-309 (2006).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+complete-arch+dental+impressions:+a+new+method+of+measuring+trueness+and+precision.">Accuracy of complete-arch dental impressions: a new method of measuring trueness and precision.</a> 109 (2), 121-128 (2013).</li><li>Ender, A., Mehl, A. <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+scanning+strategies+on+the+accuracy+of+digital+intraoral+scanning+systems.">Influence of scanning strategies on the accuracy of digital intraoral scanning systems.</a> Int. J. Comput. Dent. 16 (1), 11-21 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+arch+scans:+conventional+versus+digital+impressions--an+in-vitro+study.">Full arch scans: conventional versus digital impressions--an in-vitro study.</a> Int. J. Comput. Dent. 14 (1), 11-21 (2011).</li><li>Meer, W. J., Andriessen, F. S., Wismeijer, D., Ren, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+intra-oral+dental+scanners+in+the+digital+workflow+of+implantology.">Application of intra-oral dental scanners in the digital workflow of implantology.</a> PLoS One. 7 (8), (2012).</li></ol>

Los autores declaran que no tienen intereses en competencia financieros u otros conflictos de intereses.

La precisión es una exigencia básica en la medicina dental. El escáner de referencia es capaz de escanear objetos pequeños y grandes con una alta veracidad y precisión. Con el método óptimo de exploración, incluso superficies dentales detallados morfológicas individuales pueden ser escaneados con una alta resolución y repetibilidad. Con los diferentes niveles de aumento del escáner, estructuras morfológicas macro y micro se pueden adquirir. Es posible explorar una variedad de materiales de modelo. <p cla...

La precisión es de un gran interés en muchos campos de la medicina dental. Sustitución de tejido duro dental necesita una prótesis de ajuste exacto para garantizar el funcionamiento adecuado y prevenir la destrucción aún más la estructura dentaria remanente 1,2. Dentaduras parciales fijas y prótesis totales son especialmente críticos para guarnición exacta en estructuras de apoyo como los dientes o implantes 3 ​​preparadas. Esto es por qué se necesita una reproducción de alta precisión, especialmente en el campo de impresiones dentales y flujo de trabajo de laboratorio dental. Sin embargo, otros ámbitos de tratamiento dental también se benefician de un resultado métrica precisa y veraz, para verificar el éxito del tratamiento y evaluar nuevas estrategias de tratamiento, por ejemplo, el aumento de tejidos blandos y duros, la vigilancia de la erosión y la abrasión, tratamientos periodontales, y los tratamientos de ortodoncia 4,5. En muchos de estos campos, procedimientos de validación actuales son medidas lineales distancia con pinzas o microscopios 6,7. Estos methods se limitan a sólo unos puntos de medición e información limitado de tres dimensiones (3D) los cambios de la zona de pruebas. Métodos de medición más recientes incluyen la captura óptica o radiográfica de toda la superficie del objeto de prueba 8,9. Aquí, toda la superficie o el volumen se mide y se muestran como un objeto 3D en la pantalla de ordenador. Mediciones lineales son posibles, así como las superposiciones de modelos de diferentes tiempos de exploración. Con esta superposición, es posible una evaluación de los cambios en la superficie en cada punto de exploración. Esto permite la posibilidad de seguimiento de un área específica o mostrar deformaciones en los tres ejes de coordenadas. Además, los cambios volumétricos se pueden medir 10. El punto límite con estos nuevos métodos es la exactitud del escáner, que se utiliza para capturar el objeto de prueba. Ninguno de los cambios dentro de la exactitud de la referencia de escáner se puede dividir en los cambios de el objeto de prueba o errores de escaneo. Precisión de escaneo es a menudo un valor dado por el fabricantecante deriva de escaneo de objetos pequeños y calibradas 11. Este error de exploración mínima es diferente al escanear objetos grandes como un arco dental. Precisión consta de veracidad y precisión. Veracidad es la desviación del objeto escaneado desde su geometría real. La precisión es la desviación entre las exploraciones repetidas (ISO 5725-1). En este estudio, un nuevo escáner óptico de referencia, sobre la base de la técnica de exploración variación de foco, se introdujo para escanear especímenes de solo diente hasta modelos de arco completo con más alta precisión. Este escáner de referencia se utiliza como una base para varios estudios, comparando la precisión de impresión dental a partir de técnicas convencionales y digitales 12-14 y para proyectos reales relativas a la oclusión dental y la abrasión de los materiales dentales. El objetivo de este estudio es proporcionar información básica de la precisión del escáner de referencia y algunas posibilidades de utilizar este dispositivo en el campo de la investigación dental.

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr>
			<td>Reference model</td>
			<td></td>
			<td></td>
			<td>individual non-precious metal model, derived from a patient impression</td>
		</tr>
		<tr>
			<td>Araldit repair</td>
			<td>Huntsmen Advanced Material, Basel, Switzerland</td>
			<td></td>
			<td>used for making the base of the reference model</td>
		</tr>
		<tr>
			<td>CamBase</td>
			<td>Dentona, Dortmund, Germany</td>
			<td></td>
			<td>Type IV dental ston for pouring conventional impressions</td>
		</tr>
		<tr>
			<td>Identium</td>
			<td>Kettenbach, Eschenburg, Germany</td>
			<td></td>
			<td>Vinylsiloxanether impression material for conventional impression</td>
		</tr>
		<tr>
			<td>inEOS model holder</td>
			<td>Sirona Dental Systems, Bensheim, Germany</td>
			<td></td>
			<td>used for fixing stone models at the reference scanner</td>
		</tr>
		<tr>
			<td>Accutrans</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>used for making the base of thestone models</td>
		</tr>
		<tr>
			<td>President putty</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>mix with accutrans for betterstability of the base</td>
		</tr>
		<tr>
			<td>Alicona Infinite Focus</td>
			<td>Alicona Imaging, Graz, Austria</td>
			<td></td>
			<td>Reference scanner&#160;</td>
		</tr>
	</tbody>
</table>

accuracy in dental medicine, a new way to measure trueness and precision

Escáneres de referencia se utilizan en la medicina dental para verificar una gran cantidad de procedimientos. El interés principal es verificar los métodos de impresión, ya que sirven como base para las restauraciones dentales. La limitación actual de muchos escáneres de referencia es la falta de precisión de escaneo de grandes objetos como arcos dentales completos, o la posibilidad limitada para evaluar las superficies detalladas de los dientes. Un nuevo escáner de referencia, basado en la técnica de escaneo de variación de enfoque, se evaluó con respecto a la más alta precisión local y general. Un protocolo de exploración específica se ensayó para escanear la superficie del diente original a partir de impresiones dentales. Además, se verificaron diferentes materiales modelo. Los resultados mostraron una alta precisión de exploración del escáner de referencia con una desviación media de 5,3 ± 1,1 m para veracidad y 1,6 ± 0,6 micras para la precisión en caso de arco barridos completos. Los métodos actuales de impresión dental mostraron mucho más alto desviaciones (veracidad: 20,4 ± 2,2 micras, de precisión: 12,5 ± 2,5 micras) THan la precisión de escaneo interno del escáner de referencia. Los objetos más pequeños, como la superficie de un solo diente pueden ser escaneados con una precisión aún mayor, lo que permite el sistema para evaluar la erosión y la pérdida de superficie dental abrasivo. El escáner de referencia se puede utilizar para medir las diferencias de una gran cantidad de campos de investigación dental. Los diferentes niveles de ampliación combinada con una alta precisión local y general se pueden utilizar para evaluar los cambios de los dientes individuales o restauraciones hasta arco cambios completos.

La figura 16A muestra el análisis de una muestra de yeso. Para verificar los parámetros óptimos de exploración, el control de la capacidad de repetición para el material. Una sección de la muestra se analiza con diferentes ajustes de contraste y brillo y la repetibilidad se comprueba después de cada exploración para encontrar los ajustes óptimos de exploración. Las superficies metálicas muestran los mejores resultados con un menor contraste 1,0 y materiales de yeso o de resina con un mayor co...

Watch this Scientific Journal Video about Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision at JoVE.com

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

La precisión es una demanda importante en la medicina dental. Para verificar la precisión, se necesitan escáneres de referencia. En este artículo se presenta un nuevo escáner de referencia con un método de exploración ajustado para adquirir una amplia variedad de morfologías dentales con alta veracidad y precisión.

Precisión en Medicina Dental, Una nueva forma de medir veracidad y precisión

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

accuracy-dental-medicine-new-way-to-measure-trueness

Research

JoVE Journal

Medicine

13.3K vistas.  University of Zürich.  La precisión es una demanda importante en la medicina dental. Para verificar la precisión, se necesitan escáneres de referencia. En este artículo se presenta un nuevo escáner de referencia con un método de exploración ajustado para adquirir una amplia variedad de morfologías dentales con alta veracidad y precisión. 

Video: Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

Técnica para recopilar fungiformes (gusto) Las papilas de la lengua humana

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

Investigación

Medicina

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Rapid Point-of-Care Ensayo de la eficacia anticoagulante enoxaparina en sangre entera

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

La detección de Modificadores melanoma utilizando un modelo de pez cebra Tumor autóctona

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetría intrauterina para medir la presión del ratón contráctil<em&gt; En Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Un dispositivo sencillo de preparar rápidamente enteros Montes del Intestino Ratón

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

Audacia, la agresión y ensayos para formar bancos de pez cebra de comportamiento Síndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

La traqueostomía abierta ácido gástrico aspiración modelo murino de lesión pulmonar aguda Resultados en máxima no letal aguda lesión pulmonar

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Biofilm oral toma de muestras para análisis de microbioma en niños sanos

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Realización de ejercicio máximo y submáximo resistencia pruebas para medir las respuestas fisiológicas y biológicas al ejercicio agudo en seres humanos

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...