Gostaria de agradecer a Daniel Van Dyk do Centro para o Estudo do Comportamento Integrativa Animal (CISAB) para obter ajuda muito na concepção do modelo e acesso ao vídeo de arquivo. Christopher Evans (CISAB) prestou apoio técnico, Daniel Warner (Universidade de Sydney), desde o modelo empalhados, e Richard Peters (Australian National University), desde características iniciais do projeto.

Design de Animação Objeto de digitalização Adquirir lagarto empalhados. Digitalizar o modelo com um Konica Minolta VI 9i. Ela produz um objeto 3D (*. lwo e arquivos *. obj) em uma única malha poligonal de 50.000 polígonos. Lightwave ® v8.3 tem dois programas utilizados para animação 3D: Modeller e Layout. Projetos Lightwave ® Modeller e manipula o objeto. Lightwave programa de layout ® cria cenas de animação. Lightwave ® Modeller e Layout tem três dimensões de posicionamento, orientação e rotação: título (Y), pitch (X) eo banco (Z). Aquisição textura Fotografia um lagarto vivo usando um 12,8 megapixels Canon EOS 5D câmera digital de dois ângulos (frontal e ortogonais), três posições (frontal, ortogonais, ventral e dorsal), e três regiões do corpo partes (cabeça, corpo, cauda e membros) sobre uma folha branca de papel a ser branca equilibrado pela cor padronizada. Fusível fotografias juntos em Adobe ® Photoshop ® Elements 3.0. Criar um mapa de UV Atlas em Lightwave ® Modeller. O mapa UV quebra o objeto em cadeias de conexão polígonos. Captura de imagem de JPEG Atlas mapa UV com Grab V1.2 para criar uma imagem JPEG separado. Imbed como a camada de fundo para o Adobe ® Photoshop ® Elements 3.0. No Lightwave ® Modeller, polígonos destaque no mapa UV Atlas para identificar áreas específicas sobre o lagarto. Área de cultivo e sobrepô sobre o Atlas fundo UV mapa JPEG. Quando todos os fragmentos fotográficos estão em camadas sobre o mapa UV Atlas JPEG, retire o fundo e criar um único arquivo TIFF. Importar o arquivo TIFF em Lightwave ® Modeller e atribuir coordenadas UV. Skelegons implantação e Ossos Em Lightwave ® Modeller, skelegons foram projetados e embutida. Skelegons atuou como espaços reservados para os ossos. Criar 61 ossos. Criar skelegons para imitar o número de vértebras em um dragão Jacky, com uma skelegon de grandes dimensões utilizados para simular a cabeça. Criar uma coluna virtual espinhal do pescoço para baixo até o final da cauda. Fundir o forelimbs, que consistem em quatro skelegons cada um, a coluna vertebral. Quando todos skelegons são criados, enviar o objeto inteiro para Lightwave ® Layout, onde o skelegons devem ser transformados em ossos. Adicionando sombreamento Peso Mapas de peso independentes têm uma faixa de valor de -100% a +100% para distribuir uniformemente o movimento. Designados os membros, cauda e cabeça com positivo (+) valores do mapa de peso. Dar ao corpo um negativo - valor do mapa de peso (). Captura de vídeo de estímulo Archival Imagens de arquivo lagarto foi mostrado usando Sony gravador de vídeo digital MiniDV PAL jogador em um Sony Trinitron monitor para um lagarto vivo realizada em seu gabinete (para aquisição de vídeo inicial, consulte Ord e Evans, 2002; Ord, Peters, Evans, &amp; Taylor, 2002; Van Dyk e Evans, 2007). Respostas de lagartos foram gravados usando uma Canon (MV650i) filmadora digital colocado aproximadamente no metro do gabinete. Tamanho lagarto foi dimensionado para o tamanho representante do poleiro no monitor. Exportação como indivíduo imagens seqüenciais JPEG em Apple Quicktime ™ v7.0. Rotoscopia para replicação de estímulo &quot;Rotoscoping &#39;é uma técnica onde a animação objeto é sobreposta quadro a quadro no primeiro plano da seqüência de fundo (Gatsey, Middleton, Jenkins, &amp; Shubin, 1999). Enviar a animação para Lightwave ® Layout, onde a luz, câmera, objeto e características de fundo pode ser controlado para a cena. Selecione padrões motores de vídeo digital de arquivo e de exportação como uma seqüência de imagens (JPEG) usando o Apple QuickTime ™ Player 7.0. O objeto deve estar na vista da câmera. Importar o JPEG em primeiro lugar na seqüência de imagens em segundo plano. Manipular o objeto usando os ossos e sobrepô na posição atual que reflete a imagem de fundo. Cada quadro é para ser &quot;keyframed &#39;, tal que a posição do objeto é salvo. Substituir a imagem de fundo com a imagem seguinte consecutivos e manipular o objeto na nova posição. Repita o processo até que a seqüência seja concluída. Tornando a seqüência de Conclusão Clips seqüência grande ou processamento em lote são processados ​​usando renderização Farm Commander v2.9. Tornar Farm Commander (RFC) ligações de computadores para produzir uma série de processadores para auxiliar no processamento mais rápido. Quatro da Apple Mac G5 dual-processadores (oito threads) foram usados ​​para distribuir o processamento, reduzindo assim o tempo de quatro horas. Todas as sequências foram mantidas em PAL-DV padrão (720 pixels de resolução ¥ 576; 05:01 de compressão, a resolução horizontal 575 linhas; 25 frames 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>

Financiamento foi suportado pelo Prêmio de Pós-Graduação CISAB e do Departamento de Psicologia da Universidade Macquarie, eo Conselho de Pesquisa australiano. A pesquisa foi realizada de acordo com as diretrizes éticas da Universidade Macquarie animal Comitê de Ética (2003/014) e New South Wales National Parks and Wildlife Services (S11024).

Animações geradas por computador são cada vez mais se tornando uma ferramenta popular em que a investigar questões relacionadas com a comunicação em comportamento animal. Usando técnicas de animação, os pesquisadores podem se concentrar em aspectos específicos do projeto do sinal, e manipular os recursos que anteriormente eram incapazes de ser investigado devido à tecnologia limitada. Apesar de animais convencionais encenado encontros ainda demonstrar princípios teóricos, técnicas de animação agora fornecer uma ferramenta so...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
</tr>
<tr>
<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create animation</td>
</tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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

</td>
</tr>
<tr>
<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>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

computer-generated animal model stimuli

Comunicação entre os animais é diverso e complexo. Os animais podem se comunicar usando auditiva, sísmicos, chemosensory, sinais elétricos, ou visual. Em particular, a compreensão das limitações no design sinal visual para a comunicação tem sido de grande interesse. Métodos tradicionais para a investigação de interações entre animais têm utilizado técnicas básicas de observação, encenado encontros, ou manipulação física da morfologia. Métodos menos invasivos têm tentado simular conspecíficos usando as ferramentas de reprodução bruto, tais como espelhos, imagens fixas ou modelos. Como a tecnologia tornou-se mais avançadas, reprodução de vídeo surgiu como mais uma ferramenta para examinar a comunicação visual (Rosenthal, 2000). No entanto, para mover um passo adiante, a aplicação de animação por computador permite que os pesquisadores agora especificamente para isolar os componentes críticos necessários para obter as respostas sociais de membros da mesma espécie, e manipular esses recursos para controlar as interações. Aqui, eu fornecer detalhes sobre como criar uma animação usando o dragão Jacky como modelo, mas este processo pode ser adaptado para outras espécies. Na construção da animação, eu escolhi usar Lightwave 3D para alterar a morfologia do objeto, adicionar a textura, instalar os ossos, e proporcionar sombreamento peso comparável que impede o movimento exagerado. A animação é então combinado para selecionar padrões motores para replicar características movimento crítico. Finalmente, a seqüência deve prestados em um clipe individual para a apresentação. Embora existam outras técnicas adaptáveis, este método em particular, tinha sido demonstrado ser eficaz em obter respostas tanto visível e social encenado interações.

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

Computer-Generated Animal Model Stimuli

Estímulos gerados por computador usando o dragão Jacky como modelo.

Gerados por computador Modelo Estímulos 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 visualizações.  Macquarie University. Estímulos gerados por computador usando o dragão Jacky como modelo.

Vídeo: Computer-Generated Animal Model Stimuli

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.

Criopreservação de embriões pré-implantatório de bovinos, ovinos e caprinos

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

Biologia

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.

Transferência de Embrião Utero-tubária e vasectomia no Rato Modelo

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.

Um protocolo para o uso de Gene definir análise de enriquecimento para identificar o modelo Animal adequado para investigação de translação

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.

Um modelo de cultura de tecidos de produção de estrogênio primário Granulosa bovina 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 biópsia da glândula mamária bovina

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

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Simple Homemade Tools to Handle Fruit Flies&amp;#8212;&lt;em&gt;Drosophila melanogaster&lt;/em&gt;

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

Ferramentas caseiras simples para lidar com moscas de frutas —Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

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.

Análise de espectrometria de massa para identificar a ubiquização do CENP-A marcado pelo 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.

Planarian Scrunching como uma leitura comportamental quantitativa para sensoriamento 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.

Cultura do tecido do córtex ovariano bovino

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.

Um modelo cirúrgico modificado de isquemia de membros traseiros em ApoE-/- Ratos usando uma incisão em 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 ...