Me gustaría dar las gracias a Daniel Van Dyk en el Centro para el Estudio Integral del Comportamiento Animal (CISAB) para obtener ayuda mucho en el diseño del modelo y el acceso a los archivos de vídeo. Christopher Evans (CISAB) brindó apoyo técnico, Daniel Warner (Universidad de Sydney) constituyó el modelo disecados, y Richard Peters (Universidad Nacional de Australia), siempre cuenta con principios de diseño.

Diseño de Animación Objeto de análisis Adquirir lagarto taxidermia. Explorar el modelo con una Konica Minolta VI-9i. Que produce un objeto 3D (*. lwo y archivos *. obj) en una malla poligonal único de 50.000 polígonos. Lightwave ® v8.3 tiene dos programas que se utilizan para la animación 3D: Modeller y Diseño. Lightwave ® diseña Modeller y manipula el objeto. Lightwave programa Layout ® crea escenas de animación. Lightwave ® Modeller y diseño en tres dimensiones de la posición, orientación y rotación: título (Y), altura (X), y el banco (Z). Adquisición de textura Fotografía de un lagarto vivo con un 12,8 megapíxeles Canon EOS 5D cámara digital desde dos perspectivas (frontal y ortogonales), tres posiciones (frontal, ortogonales, ventral y dorsal), y tres regiones de las partes del cuerpo (cabeza, cuerpo, cola y extremidades) sobre una hoja de papel blanco que blanco equilibrada de colores estándar. Fusible de fotografías juntos en Adobe ® Photoshop ® Elements 3.0. Crear un mapa UV Atlas en Lightwave ® Modeller. El mapa UV rompe el objeto en las cadenas de conexión de los polígonos. Captura de imágenes JPEG de Atlas mapa UV con Grab V1.2 para crear una imagen JPEG por separado. Incrustan en la capa de fondo en Adobe ® Photoshop ® Elements 3.0. En el Modeller Lightwave ®, polígonos de relieve en el mapa UV Atlas para identificar áreas específicas de la lagartija. Superficie de cultivo y se superponen en el fondo Atlas UV mapa JPEG. Cuando todos los fragmentos fotográficos se superponen en el mapa de Atlas UV JPEG, eliminar el fondo y crear un único archivo TIFF. Importe el archivo TIFF en Lightwave ® Modeller y asignar coordenadas UV. Skelegons implantación y huesos En Lightwave ® Modeller, skelegons fueron diseñados y encajados. Skelegons actuó como marcadores de posición de los huesos. Crear 61 huesos. Crear skelegons para imitar el número de vértebras en un dragón de Jacky, con un skelegon de grandes dimensiones utilizados para simular la cabeza. Crear una columna virtual vertebral desde el cuello hasta la punta de la cola. Fusible de los miembros anteriores, que consisten en cuatro patas skelegons cada uno, a la columna vertebral. Cuando todos los skelegons se crean, envían a todo el objeto de Lightwave ® Diseño, donde el skelegons se transforman en huesos. Adición de sombreado Peso Mapas independientes de peso tienen un rango de valor de -100% a +100% para distribuir uniformemente el movimiento. Designó a los miembros, la cola y la cabeza con positivo (+) los valores de peso mapa. Dar al cuerpo una negativa - valor del peso del mapa (). Captura de vídeo de estímulo de archivo Imágenes de archivo lagarto se mostró de Sony MiniDV grabadora de vídeo digital PAL jugador en un monitor Sony Trinitron de un lagarto vivo a cabo en su recinto (para la adquisición inicial de video, ver Ord y Evans, 2002; Ord, Peters, Evans y Taylor, 2002; Van Dyk y Evans, 2007). Las respuestas de los lagartos se registraron con una cámara Canon (MV650i) cámara de vídeo digital colocada aproximadamente a un metro del recinto. Tamaño de lagarto se redujo al tamaño de representante de la perca en el monitor. Exportar como individual secuencial imágenes JPEG en Apple Quicktime ™ v7.0. Rotoscopia para la replicación de estímulo &quot;Rotoscopia&quot; es una técnica donde se superpone la animación de objetos fotograma a fotograma en el primer plano de la secuencia de fondo (Gatsey, Middleton, Jenkins, y Shubin, 1999). Enviar la animación Lightwave ® Diseño, donde se encuentra la luz, cámara, objeto y características del contexto controlado de la escena. Seleccione los patrones motores de vídeo digital y archivos de exportación como una secuencia de imágenes (JPEG) con Apple QuickTime ™ Player 7.0. El objeto debe ser en vista de la cámara. Importar la primera imagen JPEG en la secuencia de imágenes en el fondo. Manipular el objeto con los huesos y se superponen en la posición actual que refleja la imagen de fondo. Cada cuadro debe ser &quot;fotogramas clave&quot;, de tal manera que la posición del objeto se guarda. Vuelva a colocar la imagen de fondo con la imagen siguiente consecutivos y manipular el objeto en la nueva posición. Repita el proceso hasta que la secuencia ha finalizado. Representación de la secuencia de finalización Clips gran secuencia o el procesamiento por lotes se representan con Render Granja Comandante v2.9. Render Granja Commander (RFC) enlaza las computadoras para producir una serie de procesadores para ayudar en el procesamiento más rápido. Cuatro Apple Mac G5 de doble procesador (ocho hilos) se utiliza para distribuir la representación, lo que reduce el tiempo de cuatro horas. Todas las secuencias se mantuvieron en PAL-DV estándar (720 ¥ 576 píxeles de resolución, compresión 5:1, la resolución horizontal 575 líneas, 25 cuadros s-1).

<ol>
	<li>Gatesy, S. M., Middleton, K. M., Jenkins, F. A., Shubin, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+preservation+of+foot+movements+in+Triassic+theropod+dinosaurs.">Three-dimensional preservation of foot movements in Triassic theropod dinosaurs.</a> Nature. 399, 141-144 (1999).</li><li>Newtek, Inc, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lightwave+[8]+Reference+Manual.">Lightwave [8] Reference Manual.</a> , (2004).</li><li>Ord, T. J., Evans, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+video+playback+and+opponent+assessment+in+lizards.">Interactive video playback and opponent assessment in lizards.</a> Behav. Process. 59, 55-65 (2002).</li><li>Ord, T. J., Peters, R. A., Evans, C. S., Taylor, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+video+playback+and+visual+communication+in+lizards.">Digital video playback and visual communication in lizards.</a> Anim. Behav. 63, 879-890 (2002).</li><li>Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+considerations+and+techniques+for+constructing+video+stimuli.">Design considerations and techniques for constructing video stimuli.</a> Acta Ethol. 3, 49-54 (2000).</li><li>Van Dyk, D. A., Evans, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familiar-unfamiliar+discrimination+based+on+visual+cues+in+the+Jacky+dragon,+Amphibolurus+muricatus.">Familiar-unfamiliar discrimination based on visual cues in the Jacky dragon, Amphibolurus muricatus.</a> Anim. Behav. 74, 33-44 (2007).</li></ol>

El financiamiento fue apoyado por el Premio de Postgrado CISAB y el Departamento de Psicología de la Universidad Macquarie, y el Consejo Australiano de Investigación. La investigación fue realizada de acuerdo a las normas éticas de la Universidad de Macquarie Comité de Ética Animal (2003/014) y Nueva Gales del Sur Parques Nacionales y Vida Silvestre de Servicios (S11024).

Animaciones generadas por ordenador se están convirtiendo en una herramienta popular en el que investigar cuestiones relacionadas con la comunicación en el comportamiento animal. Mediante el uso de técnicas de animación, los investigadores pueden centrarse en aspectos específicos del diseño de la señal, y manipular las características que antes no podían ser investigadas debido a la limitada tecnología. Aunque los animales convencionales realizaron encuentros todavía demostrar los principios teóricos, técnicas de animación ofr...

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<td>New Item</td>
<td>Digital camera</td>
<td>Konica Minolta Holdings, Inc. </td>
<td>VI-9i</td>
<td>Used to acquire 3D mesh (done by New Dawn&#174; - Bexley North, NSW Australia)
 
</td>
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<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create animation</td>
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<tr>
<td>Canon</td>
<td>Digital camera</td>
<td>Canon, Inc.</td>
<td>EOS 5D</td>
<td>12.8 megapixels</td>
</tr>
<tr>
<td>Grab</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>v1.2</td>
<td>Extract texture from photographs and UV map</td>
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<td>Adobe&#174; Photoshop&#174; Elements</td>
<td>Software program</td>
<td>Adobe Systems Inc.</td>
<td>v3.0</td>
<td>Extract texture from photographs</td>
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<td>Render Farm Commander</td>
<td>Software program</td>
<td>Bruce Rayne&#169;</td>
<td>v2.9</td>
<td>Mass rendering across local area network

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<td>Mac Pro</td>
<td>Computer</td>
<td>Apple Computer, Inc.</td>
<td>&nbsp;</td>
<td>Four dual processors (eight threads); Create and render animation</td>
</tr>
<tr>
<td>Sony MiniDV</td>
<td>Digital video recorder</td>
<td>Sony Corporation</td>
<td>GV-D300E</td>
<td>Playback stimuli during stimulus acquisition</td>
</tr>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A</td>
<td>One monitor for stimulus acquisition</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior during stimulus acquisition</td>
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computer-generated animal model stimuli

La comunicación entre los animales es diversa y compleja. Los animales pueden comunicarse utilizando auditiva, sísmicos, señales quimiosensoriales, eléctrico o visual. En particular, la comprensión de las limitaciones en el diseño de señales visuales de la comunicación ha sido de gran interés. Los métodos tradicionales para la investigación de las interacciones con animales han utilizado las técnicas básicas de observación, que tuvo lugar los encuentros, o la manipulación física de la morfología. Métodos menos intrusivos han tratado de simular el uso de herramientas congéneres crudo de reproducción, tales como espejos, imágenes fijas, o modelos. Como la tecnología se ha vuelto más avanzados, la reproducción de vídeo se ha convertido en una herramienta más para examinar la comunicación visual (Rosenthal, 2000). Sin embargo, para dar un paso más, la aplicación de la animación por ordenador ahora permite a los investigadores aislar específicamente los componentes críticos necesarios para obtener las respuestas sociales de sus congéneres, y manipular estas características para el control de las interacciones. Aquí, brindo detalles sobre cómo crear una animación con el dragón Jacky como modelo, pero este proceso puede adaptarse para otras especies. En la construcción de la animación, que decidió utilizar Lightwave 3D para alterar la morfología de objetos, añadir textura, instalar los huesos, y proporcionar sombra peso similar que impide el movimiento exagerado. La animación se adecuan a seleccionar los patrones motores para replicar las características fundamentales del movimiento. Finalmente, la secuencia debe representado en un clip individual para su presentación. Aunque hay otras técnicas adaptables, este método en particular se había demostrado ser eficaz en la obtención de respuestas, tanto visible y social en una puesta de interacciones.

Watch this Scientific Journal Video about Computer-Generated Animal Model Stimuli at JoVE.com

Computer-Generated Animal Model Stimuli

Generada por ordenador utilizando los estímulos que el dragón Jacky como modelo.

Los estímulos generados por ordenador Modelo animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Research

JoVE Journal

Biology

10.8K vistas.  Macquarie University. Generada por ordenador utilizando los estímulos que el dragón Jacky como modelo.

Video: Computer-Generated Animal Model Stimuli

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

No invasiva En Vivo Pequeños Animales MRI y MRS: Procedimientos básicos Experimental

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Investigación

Biología

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

La criopreservación de embriones de preimplantación de ganado, ovejas y cabras

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

Transferencia de Embriones Utero-de trompas y vasectomía en el modelo de ratón

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Un protocolo para el uso de gen Set enriquecimiento análisis para identificar el modelo Animal adecuado para la investigación traslacional

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Un modelo de cultivo de tejidos de Granulosa bovino primario producción de estrógeno de las células

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

Técnicas de biopsia de glándula mamaria bovina

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Análisis de espectrometría de masas para identificar la ubiquitylación de CENP-A con etiqueta EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Exploración planarian como una lectura cuantitativa del comportamiento para la detección de estímulos nocivos

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Cultivo de tejido de corteza ovárica bovina

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

Un modelo quirúrgico modificado de isquemia de la extremidad posterior en ApoE-/- Ratones que usan una incisión en miniatura

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...