La investigación descrita en este artículo se realizó en las instalaciones del BMIT en la Fuente de Luz canadiense, que cuenta con el apoyo de la Fundación canadiense para la Innovación, las Ciencias Naturales y el Consejo de Investigación de Ingeniería de Canadá, la Universidad de Saskatchewan, el Gobierno de Saskatchewan, Western Economic Diversification Canada, el Consejo Nacional de Investigación de Canadá y los Institutos Canadienses de Investigación de la Salud. Los autores quisieran agradecer a los científicos de la fuente de luz canadiense, en particular a Adam Webb, Denise Miller, Sergey Gasilov y Ning Zu por la asistencia en la configuración y solución de problemas de los sistemas de microscopios SkyScan SRμCT y de haz blanco. También queremos agradecer a Beth Dalzell de la Facultad de Medicina y Ciencias de la Vida de la Universidad de Toledo y al Dr. Jeffrey Wenstrup de la Universidad Médica del Noreste de Ohio por el acceso a muestras cadavéricas para este estudio. JM Andronowski cuenta con el apoyo de fondos de investigación de puesta en marcha proporcionados por la Universidad de Akron y una beca del Instituto Nacional de Investigación y Desarrollo de la Justicia en Ciencias Forenses con fines de justicia penal (2018-DU-BX-0188). RA Davis cuenta con el apoyo de una asistente de posgrado proporcionada por la Universidad de Akron. Los equipos y suministros utilizados para el corte y la aserración fueron comprados por fondos de puesta en marcha proporcionados por la Universidad de Akron y la subvención de la NSF EAR-1624242 a CW Holyoke.

<ol>
	<li>Andronowski, J. M., Crowder, C., Soto Martinez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+the+analysis+of+bone+microstructure:+New+dimensions+in+forensic+anthropology.">Recent advancements in the analysis of bone microstructure: New dimensions in forensic anthropology.</a> Forensic Sciences Research. 3 (4), 278-293 (2018).</li><li>Wysokinski, T. W., 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=Beamlines+of+the+biomedical+imaging+and+therapy+facility+at+the+Canadian+light+source+-+part+3.">Beamlines of the biomedical imaging and therapy facility at the Canadian light source - part 3.</a> Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 775, 1-4 (2015).</li><li>Carter, Y., Suchorab, J. L., Thomas, C. D. L., Clement, J. G., Cooper, D. 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=Normal+variation+in+cortical+osteocyte+lacunar+parameters+in+healthy+young+males.">Normal variation in cortical osteocyte lacunar parameters in healthy young males.</a> Journal of Anatomy. 225 (3), 328-336 (2014).</li><li>Carter, Y., Thomas, C. D. L., Clement, J. G., Cooper, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+osteocyte+lacunar+density,+volume+and+morphology+in+women+across+the+lifespan.">Femoral osteocyte lacunar density, volume and morphology in women across the lifespan.</a> Journal of Structural Biology. 183 (3), 519-526 (2013).</li><li>Langer, 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=X-Ray+Phase+Nanotomography+Resolves+the+3D+Human+Bone+Ultrastructure.">X-Ray Phase Nanotomography Resolves the 3D Human Bone Ultrastructure.</a> PLoS ONE. 7 (8), 35691 (2012).</li><li>Peyrin, F., Dong, P., Pacureanu, A., Langer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-+and+Nano-CT+for+the+Study+of+Bone+Ultrastructure.">Micro- and Nano-CT for the Study of Bone Ultrastructure.</a> Current Osteoporosis Reports. 12 (4), 465-474 (2014).</li><li>Dong, P., 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=3D+osteocyte+lacunar+morphometric+properties+and+distributions+in+human+femoral+cortical+bone+using+synchrotron+radiation+micro-CT+images.">3D osteocyte lacunar morphometric properties and distributions in human femoral cortical bone using synchrotron radiation micro-CT images.</a> Bone. 60, 172-185 (2014).</li><li>Gauthier, R., 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=3D+micro+structural+analysis+of+human+cortical+bone+in+paired+femoral+diaphysis,+femoral+neck+and+radial+diaphysis.">3D micro structural analysis of human cortical bone in paired femoral diaphysis, femoral neck and radial diaphysis.</a> Journal of Structural Biology. 204 (2), 182-190 (2018).</li><li>Giuliani, A., 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=Bisphosphonate-related+osteonecrosis+of+the+human+jaw:+A+combined+3D+assessment+of+bone+descriptors+by+histology+and+synchrotron+radiation-based+microtomography.">Bisphosphonate-related osteonecrosis of the human jaw: A combined 3D assessment of bone descriptors by histology and synchrotron radiation-based microtomography.</a> Oral Oncology. 82, 200-202 (2018).</li><li>Andronowski, J. 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Los autores no tienen nada que revelar.

No ha habido un protocolo integral y estandarizado para adquirir muestras uniformes y cilíndricas de núcleo óseo cortical para imágenes SRμCT de alta resolución con configuraciones limitadas de FOV. El protocolo detallado aquí llena ese vacío proporcionando un tutorial completo sobre cómo adquirir muestras de núcleo óseo cortical de tamaño consistente para imágenes SRμCT y la posterior visualización y extracción precisas de datos microarquiitecturales. Hemos demostrado que nuestro protocolo proporciona un...

Con los recientes avances en la tecnología de imágenes, ahora es factible adquirir datos de imágenes de rayos X con muy alta resolución. Los sistemas micro-CT de escritorio (μCT) son el estándar actual para el hueso canceloso por imágenes debido a su naturaleza no destructiva1. Sin embargo, cuando las características microestructurales por imágenes del hueso cortical, el uso de μCT ha sido más limitado. Debido a las restricciones de resolución, los sistemas de escritorio no pueden alcanzar la resolución necesaria para crear imágenes de características microestructurales más pequeñas que los poros corticales, como las lagunas de osteocitos. Para esta aplicación, SRμCT es ideal debido a la mayor resolución de estos sistemas1. Por ejemplo, los experimentos en la Fuente de Luz Canadiense (CLS) en las líneas de haz de imágenes y terapia biomédicas (BMIT)2 han producido imágenes con voxels de hasta 0,9 μm. Estudios previos1,3,4,5 han utilizado esta resolución para adquirir proyecciones y posteriores renderizaciones tridimensionales (3D) a partir de especímenes óseos corticales de huesos largos humanos(Figura 1)para cuantificar la densidad de lacunar de osteocitos4,6,7,8,9 y variación en la forma de lacunar y tamaño3 a lo largo de la vida humana y entre los sexos. Otros estudios han demostrado la presencia de bandas de osteones en humanos10,un fenómeno previamente reconocido como asociado sólo con mamíferos no humanos en la literatura antropológica forense.
Para lograr una resolución excepcional, el haz de rayos X debe centrarse finamente dentro del campo de visión (FOV), que a menudo limita el tamaño máximo de la muestra a unos pocos milímetros de diámetro. Actualmente, no ha habido procedimientos completos y estandarizados descritos en la literatura que describa la adquisición de muestras óseas que cumplan estas restricciones. El centrado de muestras dentro del FOV es fundamental para garantizar que 1) la muestra permanezca centrada a medida que gira 180° durante la toma de imágenes, y 2) artefactos de escaneo son limitados ya que no hay truncamiento de imagen. En otras palabras, ninguna porción de la muestra fuera del FOV interfiere con el haz que entra en su punto focal dentro del FOV. Si esto ocurre, el algoritmo de reconstrucción se ve privado de algunos de los datos de atenuación necesarios para una reconstrucción totalmente correcta. Vale la pena señalar además que los escaneos de 360° (rotación completa) minimizan los efectos del endurecimiento del haz, pero aumentan los artefactos causados por la desalineación y el movimiento de la muestra durante la toma de imágenes. Por lo tanto, mientras que una exploración de 360° normalmente generará datos más limpios, el tiempo de imagen se duplica y por lo tanto se debe abordar un compromiso entre el costo experimental y la calidad de los datos.
Un aspecto importante y a menudo pasado por alto de los experimentos de imágenes óseas es la técnica de preparación de muestras precisa y replicable realizada antes de la exploración. Los estudios que incorporan métodos SRμCT en sus experimentos mencionan brevemente su protocolo de muestreo, pero los autores proporcionan poco o ningún detalle con respecto a la metodología particular utilizada para reunir sus especímenes. Muchos de estos estudios mencionan el corte de bloques óseos rectilíneos de dimensiones arbitrarias, pero generalmente no proporcionan más información sobre las herramientas o materiales de incrustación utilizados3,4,10,11,12,13,14. Algunos investigadores utilizan comúnmente herramientas rotativas portátiles (por ejemplo, Dremel) para eliminar bloques rectilíneas de hueso de una región de interés (ROI)3,4,10,11,12,13,14. Este método da como resultado muestras de tamaño no uniforme que pueden ser mayores que la FOV, lo que aumenta la probabilidad de artefactos de exploración y truncamiento de imagen. Estos especímenes a menudo requieren más refinación utilizando una sierra de diamantes y obleas de precisión (por ejemplo, Buehler Isomet). La adquisición de muestras con dimensiones coherentes (hasta las dos centésimas/mm) es fundamental para garantizar que los datasets adquiridos sean de la más alta calidad y que los resultados posteriores sean replicables.
La limitada notificación de la metodología de adquisición de muestras añade una capa adicional de dificultad al intentar emplear y/o validar métodos realizados en un estudio anterior. Actualmente, los investigadores deben ponerse en contacto directamente con los autores para obtener más detalles sobre sus procedimientos de muestreo. El protocolo detallado aquí proporciona a los investigadores biomédicos una técnica de muestreo completamente documentada, replicable y rentable. El objetivo principal de este artículo es proporcionar un tutorial completo sobre cómo adquirir muestras de núcleo óseo cortical de tamaño consistente utilizando una prensa de perforación de molino y broca de diamante para la visualización y extracción precisas de datos microarquiitecturales. Este método se modifica a partir de procedimientos utilizados para recoger rutinariamente cilindros uniformes de diámetro pequeño (1-5 mm) de bloques de materiales duros en mecánica de roca de alta presión15,16,17,18,19.

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			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:133px;">22-327379</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fixturing clamps for XY machine table for mill/drill</td>
			<td style="width:120px;">MSC Industrial Supply</td>
			<td style="width:133px;">#04804571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass microscope slides</td>
			<td style="width:120px;">Ted Pella</td>
			<td style="width:133px;">26005</td>
			<td>75x50mm slides, 1mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass slide chuck</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">#112488</td>
			<td>Large enough to hold 75x50mm glass slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Hot plate capable of reaching 140 &#176;C</td>
			<td style="width:120px;">ThermoScientific</td>
			<td style="width:133px;">HP88850105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:120px;">NAPCO</td>
			<td>Model 4200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isocut Fluid</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111193032</td>
			<td>Lubricant; 30mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Jeweler&#39;s diamond coring drill bit</td>
			<td style="width:120px;">Otto Frei</td>
			<td style="width:133px;">#119.050</td>
			<td>2mm inner diameter hollow stem coring bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NRecon</td>
			<td style="width:120px;">Bruker microCT</td>
			<td style="width:133px;">v.1.6.10.2</td>
			<td>Download and install at https://www.bruker.com/products/microtomography.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oscillating saw</td>
			<td style="width:120px;">Harbor Freight</td>
			<td style="width:133px;">62866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oven-safe glass dishes</td>
			<td style="width:120px;">Pyrex</td>
			<td style="width:133px;">1117715</td>
			<td>Glass food storage container</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Precision slow-speed saw (Isomet 1000)</td>
			<td style="width:120px;">Buehler</td>
			<td style="width:133px;">111280160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Razor blades</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">25181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Shallow aluminum tins</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">B01MRWLD0R</td>
			<td>~8cm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Specimen cups</td>
			<td style="width:120px;">Amazon</td>
			<td style="width:133px;">616784425436 885334344729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tergazyme detergent</td>
			<td style="width:120px;">Alconox</td>
			<td style="width:133px;">1304-1</td>
			<td>1.8kg box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ultrasonic cleaner</td>
			<td style="width:120px;">MTI Corporation</td>
			<td style="width:133px;">KJ201508006</td>
			<td></td>
		</tr>
	</tbody>
</table>

a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct

El hueso es un tejido dinámico y mecánicamente activo que cambia de estructura sobre la vida útil humana. Los productos del proceso de remodelación ósea han sido estudiados sustancialmente utilizando técnicas bidimensionales tradicionales. Los recientes avances en la tecnología de imágenes de rayos X a través de tomografía micro-computarizada de escritorio (μCT) y tomografía micro-computarizada de radiación sincrotrón (SRμCT) han permitido la adquisición de escaneos tridimensionales de alta resolución (3D) de un campo de visión más grande (FOV) que otras técnicas de imágenes 3D (por ejemplo, SEM) proporcionando una imagen más completa de las estructuras microscópicas dentro del hueso cortical humano. Sin embargo, la muestra debe centrarse con precisión dentro del FOV para limitar la aparición de artefactos de rayas conocidos por afectar el análisis de datos. Estudios anteriores han reportado la adquisición de bloques óseos rectilíneas de forma irregular que resultan en artefactos de imágenes debido a bordes irregulares o truncamiento de imagen. Hemos aplicado un protocolo de muestreo geológico (coring) para adquirir especímenes de núcleo óseo cortical de tamaño consistente para experimentos SRμCT desde el aspecto anterior de la femora humana. Este método de coring es eficiente y mínimamente destructivo para el tejido. Crea muestras cilíndricas uniformes que disminuyen los artefactos de imagen por naturaleza de ser isométricos durante la rotación y proporcionan una longitud de trayecto uniforme para los haces de rayos X a lo largo del escaneo. El procesamiento de imágenes de datos tomográficos de rayos X de muestras con forma irregular y de cored confirma el potencial de la técnica para mejorar la visualización y el análisis de la microarquitectura ósea cortical. Un objetivo de este protocolo es ofrecer un método confiable y repetible para la extracción de núcleos óseos corticales que es adaptable para varios tipos de experimentos de imágenes óseas de alta resolución. Un objetivo general de la obra es crear una adquisición de hueso cortical estandarizado para SRμCT que sea asequible, consistente y directa. Este procedimiento puede ser adaptado por investigadores en campos relacionados que comúnmente evalúan materiales compuestos duros como antropología biológica, geociencias o ciencias materiales.

El método descrito de muestreo básico demostró ser altamente eficaz y eficiente. La perforación de muestras utilizando este protocolo permitió la adquisición de muestras de tamaño consistente de >300 para experimentos en la línea de haz CLS BMIT-BM2,con un FOV de ~2 mm a 1,49 μm de tamaño voxel. Para validar la consistencia del diámetro del núcleo, se tomaron tres mediciones a lo largo de la longitud (superior, media, inferior) de un subconjunto de núcleos femorales anteriores humanos...

Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Empleamos un protocolo geológico (coring) de muestreo para adquirir muestras óseas corticales de tamaño uniforme para experimentos SRμCT desde el aspecto anterior de la femora humana. Este método es mínimamente destructivo, eficiente, resulta en muestras cilíndricas que minimizan los artefactos de imagen de formas de muestra irregulares y mejora la visualización y análisis microarquiitectural.

Una guía de sección, corte y procesamiento de imágenes para adquisición y análisis de muestras óseas corticales de alto rendimiento para Micro-TC sincrotrón

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

a-sectioning-coring-image-processing-guide-for-high-throughput

Research

JoVE Journal

Biology

5.0K vistas.  The University of Akron. Empleamos un protocolo geológico (coring) de muestreo para adquirir muestras óseas corticales de tamaño uniforme para experimentos SRμCT desde el aspecto anterior de la femora humana. Este método es mínimamente destructivo, eficiente, resulta en muestras cilíndricas que minimizan los artefactos de imagen de formas de muestra irregulares y mejora la visualización y análisis microarquiitectural.

Video: A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guía práctica para Phylogenetics para no expertos

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Investigación

Biología

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Un ensayo de unión de alto rendimiento MHC II para el análisis cuantitativo del péptido epítopos

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

De alto rendimiento de imagen Análisis de esferoides tumorales: una aplicación de software fácil de usar para medir el tamaño de esferoides forma automática y precisa

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

De alto rendimiento, de múltiples imágenes Cryohistology de tejidos mineralizados

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Aislamiento, purificación y diferenciación de los precursores de osteoclastos de la médula ósea de rata

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Evaluación de la estructura ocular global después del vuelo espacial mediante un método de imagen de tomografía microcalculable (Micro-CT)

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

Una plataforma de alto rendimiento para el cultivo y la obtención de imágenes 3D de organoides

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Elucidación de la estructura y ocurrencia de glicanos mediante perfiles de polímeros de microarrays

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

Autor destacado: Protocolo MAPP - Avance del análisis de glicanos 

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

Perfil de polímeros de microarrays (MAPP) para análisis de glicanos de alto rendimiento

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...