Apoyado por becas de los Institutos Canadienses de Investigación en Salud, la Red Canadiense de accidente cerebrovascular, y la Alberta Heritage Foundation para la Investigación Médica.

Rotorod aparato de caminar El rotorod era un cilindro giratorio, 4 cm de diámetro, fija 35 cm sobre el suelo y rodeado de plexiglás transparente. El cilindro rotorod estaba cubierta de revestimiento de goma de textura, lo que facilita la tracción. El suelo del aparato estaba cubierto con una capa de espuma para evitar lesiones cuando los animales se redujo. Un pequeño motor eléctrico siempre encendido para encender el rotorod a través de una correa de goma. El rotorod se estableció para girar una vez cada 5 s. Las grabaciones de vídeo y la cinemática Una cámara de video de alta velocidad digital se utilizó para filmar a los animales, con una velocidad de obturación de un 1000 de segundo. La parte frontal, lateral y trasera puntos de vista fueron filmadas mientras el animal caminaba en la rotorod. Una de dos brazos de Nikon, la fuente de luz fría MII y un juego de luces de fibra óptica se utiliza para proporcionar una iluminación adecuada durante la grabación. Las cintas fueron analizadas cuadro por cuadro con una Sony DSR 20 DV CAM cubierta. Los fotogramas de vídeo se capturaron con una tarjeta capturadora y un ordenador Macintosh. El procedimiento de prueba y análisis Con el fin de evaluar las diferencias entre los movimientos a pie de la 6-OHDA y el control de ratas, los registros de vídeo de post-quirúrgicas los días 15 - 30 (después de la recuperación de la lesión se podría esperar que en gran medida completa) fueron examinados por dos independientes observadores ciegos a las condiciones experimentales. El nigroestriatal 6-OHDA lesión inducida en el hemisferio derecho, lo que resulta en alteraciones duraderas en primer plano, y dejó las extremidades posteriores. Una escala de calificación se ha desarrollado para votar la postura de las ratas y los movimientos de la intensificación de los miembros anteriores (Whishaw et al., 2003). Los cinco componentes de la intensificación fueron calificados: <ol><li> la postura, la puntuación de la postura se hizo mediante el examen de la posición de la rata en el tambor; </li><li> levantamiento y la liberación, la parte del ciclo de intensificación en el que se levantó la pata del tambor; </li><li> realizar, la parte del ciclo de intensificación en el que se llevó a cabo la extremidad por encima del tambor como la rata se adelantó; </li><li> avance, la parte del alcance en el que se extendió la pierna para recuperar el contacto con el tambor, y </li><li> colocación, en el que se coloca la pata de nuevo en el tambor. </li></ol> Los siguientes criterios fueron utilizados para definir un movimiento normal: <ul><li> Postura: El cuerpo de la rata se orientó sobre el tambor con la cabeza bien arriba. </li><li> Levantar y soltar: El hombro, el codo y la muñeca en flexión son elevar la pata de la superficie del tambor. </li><li> Llevar: La pata se mantiene por encima de la superficie del tambor, las cifras están ligeramente flexionadas y las puntas de dígitos se centran en la línea media del cuerpo. Avance: La extremidad se extiende hacia adelante con los dedos en movimiento desde una posición semiflexionados a una posición extendida y abierta. </li><li> Colocación: La extremidad se extiende en el hombro, el codo y la muñeca y los dedos se abren para colocar la pata de adelante y en la superficie del tambor, con el dígito 2 (segundo dígito de la línea media), ubicado aproximadamente en el centro de la de la rata cuerpo. </li></ul> Cada movimiento se mide en una escala de 3 puntos: 1 = / el movimiento parecido al de una rata control, 0,5 = / el movimiento pareció un poco anormal, y 0 = / el movimiento era claramente anormal (Whishaw et al, 2003).. Esta escala de clasificación se utilizó para evaluar cinco movimientos caminar de cada rata en los días de postoperatorio 1, 3, 5, 7, 9 y 15. Las puntuaciones de las cinco medidas se promediaron para cada rata en cada día, y los resultados sometidos a un ANOVA con días como medida repetida. Resumen de rotorod caminar en 6-OHDA y el control de ratas La escala de calificación de movimiento se aplica a las sesiones de pruebas post-operatorio para el análisis de movimiento cualitativo. Durante las sesiones de prueba, el grupo de lesiones 6-OHDA recibió puntuaciones significativamente más bajas que el grupo de control, F (1,8) = 33,2, p &lt;0,001, pero no hubo un efecto significativo del día de la prueba, ni tampoco había un grupo importante por la interacción de prueba del día. Las puntuaciones de dar un paso eran entonces analizado por separado con las patas delanteras como una variable (por las ratas 6-OHDA la extremidad anterior contralateral a la lesión en comparación con la extremidad anterior ipsilateral a la lesión, para el control de ratas, la pata izquierda frente a la pata derecha). Hubo un efecto significativo de la extremidad anterior, F (1,40) = 15,8, p &lt;0,01, y hubo una interacción significativa del Grupo por la extremidad anterior, F (1,40) = 15,0, p &lt;0,01. Seguimiento de las pruebas t indicaron que la extremidad anterior contralateral a la lesión del grupo 6-OHDA recibió puntuaciones significativamente más bajas que la extremidad anterior ipsilateral a la lesión o las patas delanteras de las ratas de control, que no fue diferente. Con el fin de determinar qué componentes del movimiento contribuyó a los resultados pobres obtenidos por el grupo 6-OHDA, las medidas de la posturay los componentes individuales del ciclo de la intensificación de las extremidades anteriores ipsilateral y contralateral del grupo 6-OHDA se compararon con los valores obtenidos de las ratas control. El grupo 6-OHDA difieren en la postura y todos los componentes de refuerzo hecha por la extremidad contralateral, pero ninguno de los componentes de la extremidad ipsilateral (p &lt;0,05). Diferencias cualitativas y descriptivas de la postura en el control y las ratas 6-OHDA En un control de ratas, la cabeza se mantiene en posición horizontal, y la extremidad anterior de apoyo y las extremidades posteriores se sitúan en el tambor de la formación de un arco de soporte. Las ratas 6-OHDA suelen caminar con la cabeza hacia abajo con sus patas delanteras con discapacidad (de cara al espectador) coloca más cerca y más arriba en la superficie del tambor que se produjo a las extremidades ipsilateral o de los miembros de las ratas de control (ver video de una rata control puntuación de &quot;1&quot; por la postura, y un animal de 6-OHDA lesión con una puntuación de &quot;0&quot;). Por lo general, la anormalidad en la postura pueden ser vistos a simple vista desde la posición relativa de la cabeza de una rata. Extremidad anterior pisar En la secuencia de control, intensificando la extremidad anterior se levanta por la flexión del hombro, codo y muñeca. A medida que la extremidad se levanta, las cifras están ligeramente flexionadas, y el miembro se eleva muy por encima de la superficie del tambor. Cuando el miembro se avanza, se extiende a los hombros, codos y muñecas y que se coloca los dedos están extendidos y abiertos. En comparación con las ratas control, la amplitud de todos los componentes de las ratas DA-agotado en la extremidad contralateral lesión se atenúa. El miembro libera el tambor más tarde, se levanta menos, y se avanza una distancia más corta de lo que la extremidad anterior de control. Además, cuando el miembro está levantado, los dedos se flexionan más, y cuando la extremidad se coloca, los dígitos se extendió y abrió inferiores a los de una rata control. La extremidad anterior de control se levanta una vez que alcanza la parte superior del tambor, la muñeca se flexiona para levantar la pata hacia arriba y adelante, y se extiende la muñeca a medida que avanza la extremidad. Por el contrario, las ratas 6-OHDA es contralateral a la lesión de extremidad anterior se lleva más caudal por el movimiento del tambor, y por lo tanto versiones posteriores con la muñeca aún extendida. La flexión de la muñeca que parece ser pasivo y se produce como los dígitos se llevan hacia atrás por el movimiento del tambor, y cuando la extremidad anterior se adelanta, la garra y el antebrazo no se levantó claramente fuera de la superficie del tambor. La extremidad anterior de una rata control se extiende también hacia adelante y los dedos abiertos a medida que avanza la extremidad. A medida que la extremidad se coloca, se coloca con un movimiento de arpegio, con el dígito 5 en contacto con la superficie de la primera batería, seguido sucesivamente por los dígitos 4, 3, y 2. Por el contrario, el miembro de la rata 6-OHDA se levanta y se extendió menos, las cifras son menos extensas, y en contacto con la superficie del tambor casi al mismo tiempo una &quot;bofetada&quot;. El video muestra los movimientos de extremidad anterior en una intensificación del control de animales que fueron calificados como &quot;1&quot;, y la extremidad anterior movimientos de caminar en un animal de lesión 6-OHDA que se anotó principalmente como &quot;0&quot;. Las extremidades posteriores caminar También hubo deficiencias en las extremidades traseras contralateral a la lesión de las ratas 6-OHDA. A medida que el control de las extremidades traseras se mueve hacia atrás, parece empujar contra el giro del tambor y al hacerlo, los dedos del pie gira hacia afuera. A medida que la extremidad se mueve hacia delante, el pie se gira hacia el exterior. La extremidad posterior de la rata 6-OHDA se mueve directamente hacia atrás con la rotación del tambor, y en todo caso, gira ligeramente hacia el interior. A medida que la extremidad se mueve hacia delante, el pie se lleva directamente hacia adelante. El pie de los cambios de control de las ratas hacia el exterior y que se avanza, los dedos se mantienen más altos que el tobillo y los dedos del pie se extienden. Durante la colocación de cinco ratas control, dedo del pie en contacto con la superficie de la primera batería y el peso se distribuye a través de los dedos y la palma. Por el contrario, los dedos de la rata 6-OHDA se doblan durante transporte y la colocación se realiza sin un movimiento de arpegio. Los dedos de los pies de la rata de control son liberados en un patrón de arpegio invertido en los pies 5 se levanta primero y un dedo del pie se levanta pasado. Este movimiento había desaparecido en la rata lesión 6-OHDA, mientras que el tobillo ha mantenido su postura en flexión durante el movimiento de llevar. Una vez más los movimientos de avance de las ratas 6-OHDA se atenúan, con la extremidad inferior exorotated, levantó menos, y menos avanzado que el de la rata control. Como el control de ratas colocar sus dedos de los pies, se extienden y se abrió, de manera que los pies 5 puntos lateralmente con respecto al cuerpo de la rata. Toe 5 contacto con el tambor primero, seguido por los dedos restantes de tal manera que la rata apoya su peso sobre las puntas de los pies. Para la rata 6-OHDA, los dedos del pie son algo flexionadas cuando el pie se lleva adelante, los dedos del pie son más bajos en relación a la horizontal en el tobillo, hay menos apertura de los dedos del pie cuando el pie se coloca en el tambor. Longitud de la zancada es también algo más corto que el de las ratas control. Con el fin de cuantificar las diferencias entre control y la 6-OHDA movimientos de las extremidades traseras, los componentes de la elevación y la liberación, llevan, el progreso y la colocación fueron valorados en una escala de 3 puntos, al igual que las patas delanteras. Las medidas se realizaron de los registros de video hecho en el día postoperatorio 20, y tres ciclos de paso se clasificaron para cada rata. No hubo diferencias significativas entre los grupos, F (1,8) = 47,6, p &lt;0,001. Seguimiento de las pruebas t de los componentes indicó que todos los componentes de las ratas control recibieron una puntuación significativamente mayor que con los componentes de las ratas 6-OHDA. El video muestra los movimientos de las extremidades traseras pisar un control de animales que fueron calificados como &quot;1&quot;. Las extremidades posteriores pisando los movimientos en la 6-OHDA se muestra en el video se clasificaron principalmente con una puntuación de &quot;0&quot;.

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El propósito del presente estudio fue examinar si la prueba de habilidad rotorod roedores locomoción se puede utilizar para el examen cualitativo de la postura, dando un paso patas delanteras y la extremidad posterior intensificación de las ratas del sistema nervioso lesionado. Para el análisis, las ratas con disminuciones unilaterales DA y el control de ratas fueron de vídeo grabados desde puntos de vista frontal, lateral y posterior. Una escala de valoración de la postura y los movimientos de extremidades anteriores indicaron que lo...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

La prueba rotorod, en el que los animales caminen sobre un tambor giratorio, es ampliamente utilizado para evaluar el estado del motor en los roedores de laboratorio. El rendimiento se mide por el tiempo que un animal se mantiene hasta en el tambor en función de la velocidad del tambor. Aquí mostramos que la tarea es una fuente rica de información sobre los aspectos cualitativos de los movimientos de caminar. Debido a que los movimientos se realizan en una ubicación fija, que fácilmente se puede examinar utilizando métodos de alta velocidad de grabación de vídeo. El presente estudio se realizó para examinar el potencial de la rotorod para revelar los cambios cualitativos en los movimientos a pie de hemi-Parkinson ratas analógica, producida por la inyección de 6-hidroxidopamina (6-OHDA) en el paquete nigroestriatal derecho a agotar la dopamina nigroestriatal (DA ). A partir del día siguiente de la cirugía y luego periódicamente durante los próximos dos meses, las ratas fueron filmadas desde puntos de vista frontal, lateral y posterior, mientras caminaban en el rotorod. Comportamiento fue analizado por reproducir fotograma a fotograma de las grabaciones de vídeo. Las escalas de calificación de dar un paso comportamiento indica que las ratas hemi-Parkinson fueron dañados de forma crónica en su postura y en el uso de las extremidades contralateral a la DA-agotamiento. Las extremidades contralateral no sólo se muestra anomalías posturales y de movimiento, que participó menos en el inicio y el mantenimiento de propulsión que hizo las extremidades ipsilateral. Estos resultados no sólo revelan nuevos déficits unilateral secundaria a DA-agotamiento, pero también muestran que el rotorod puede proporcionar una sólida herramienta para el análisis cualitativo del movimiento.

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Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

La prueba rotorod se utiliza para evaluar el estado del motor en el movimiento de caminar de la hemi-Parkinson ratas analógica.

El uso de Rotorod como un método para el análisis cualitativo de la Ruta de la rata

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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12.9K vistas.  University of Lethbridge. La prueba rotorod se utiliza para evaluar el estado del motor en el movimiento de caminar de la hemi-Parkinson ratas analógica.

Video: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Investigación

Biología

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

El peldaño de la escalera a pie de la tarea: un sistema de puntuación y su aplicación práctica.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Análisis de la elaboración de un fenotipo morfológico en función de la concentración de proteínas en la levadura en ciernes

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microinyección de embriones de medaka para su uso como un organismo modelo genético

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Un método simple para imágenes Arabidopsis Uso de hojas perfluorodecalina como un medio de imágenes infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

El uso de quimiostatos en Microbiana Biología de Sistemas

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Un híbrido método de extracción de ADN para la Evaluación de las comunidades bacterianas cualitativa y cuantitativa de Aves de Corral en muestras de producción

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...