Este trabajo fue financiado por los Institutos Nacionales de Salud subvenciones F32 NS078958 (DTB) y R01 AG033620 (BG).

1. Ensamble Vuelo Tester <ol><li> Secure cilindro vuelo a Anillo de soporte 1 con abrazaderas de cadena. (Deje aproximadamente 3 cm por debajo del cilindro para pesar la cápsula.) (Nota: El cilindro de vuelo que utilizamos es de 90 cm de largo con un diámetro de 13,5 cm.) </li><li> Inserte pesan plato con una fina capa de aceite mineral por debajo del cilindro de vuelo. </li><li> Embudo seguro a Anillo soporte 2 con una abrazadera de anillo y abrazadera de la garra. Ajustar la altura del embudo de modo que el fondo del embudo esté a ras con la parte superior del cilindro de vuelo. (Nota: el diámetro de la punta del embudo debe ser menor que el diámetro exterior de los viales colocados en los tubos de caída de manera que los viales no se caigan a través de.) </li><li> Inserte el tubo de llenado en la parte superior del embudo y fijar con una abrazadera de la garra. (Nota: Nosotros usamos un tubo de caída que es de 25 cm de largo de Goteo volar-que contiene los viales desde esta altura permite la expulsión constante de todas las moscas con fuerza uniforme El diámetro interior del tubo de caída debe ser sligh..TLY más grande que el diámetro exterior del vial para permitir que el vial a gota libremente.) </li><li> Cortar hoja (s) de poliacrilamida al tamaño apropiado. (Nota: Para ayudar en la inserción y extracción de la hoja, la anchura debe ser ligeramente menor que la circunferencia interior del cilindro de vuelo). </li><li> Aplique una capa delgada de Maraña-Trap a la hoja. Deje reposar durante 1 hora antes de su uso. (Nota: Deje suficiente espacio en la parte superior e inferior de la hoja (aproximadamente 3 cm) sin recubrir para agarrar la hoja de inserción / extracción.) </li><li> Inserte la hoja de poliacrilamida en el cilindro de vuelo. </li><li> Montar la pista de cámara utilizando soportes de pino. (Nota: asegúrese de que la parte inferior de la pista se puede apoyar la cámara sin bloquear la lente Consulte la Figura 1B.). </li><li> Añadir tapones y el tornillo en su lugar. (Nota: colocar los tapones en lugares que permitan la cámara para ver la totalidad de la lámina de plástico en el modo panorámico.) </li></ol> 2. Ejecutar Experimento <ol> &lt;li&gt; Recoger viales de moscas para ensayar. Para obtener los mejores resultados, no use más de 20 moscas / vial. </li><li> Golpee suavemente moscas a fondo del vial, desconecte y luego insertar en el tubo de llenado y suelte vial. (Nota: El vial se cae el tubo de caída hasta que llega a la apertura estrecho embudo Cuando el vial golpea el embudo, las moscas son expulsados ​​en el cilindro de vuelo.). </li><li> Levante el tubo de caída para eliminar el vial vacío. (Nota: Varios frascos de moscas del mismo grupo de prueba se pueden ensayar en una hoja de poliacrilamida sola Encontramos que hasta 200 moscas (10 viales de 20 moscas cada uno) se pueden probar y tomar imágenes claras en una sola hoja.. </li><li> Retire la hoja de plástico y colóquelo en una superficie plana y blanca. (Nota: cartulina blanca se puede utilizar si las mesas de trabajo son de color oscuro.) </li><li> Montar la pista de cámara sobre la lámina de plástico. La cámara debe ser lo suficientemente alta por encima de la hoja de tener la parte superior e inferior de la hoja en el campo de visión. </li><li> Deslice la cámara a lo largo de the pista mientras mantiene pulsado el botón de &quot;captura&quot; para adquirir una imagen panorámica. </li><li> El número de moscas que aterrizan en el aceite puede contarse manualmente para cada ensayo. </li><li> Repetir los pasos 2.2 a 2.7 para todas las condiciones en un experimento dado. Las moscas pueden ser retirados de la hoja entre cada ensayo. Por otra parte, varias hojas se pueden usar, con una hoja nueva para cada ensayo. </li></ol> 3. Recolección de Datos <ol><li> Abrir archivos de imagen utilizando el software ImageJ. </li><li> Recorte de imágenes si es necesario para incluir sólo el área de la superficie de aterrizaje. (Esta es la zona recubierta de Maraña-Trap.) </li><li> Convertir imágenes a 8 bits en escala de grises. </li><li> Crear un &quot;umbral&quot; para filtrar el fondo blanco. (Imagen → Ajuste → Umbral). </li><li> Establezca los parámetros para identificar cada vuelo usando el menú de &quot;analizar partículas&quot;. (Analizar → Analizar Partículas) Definir los parámetros utilizados para identificar una partícula. Con nuestra creó, nos encontramos con que el uso de unárea de 5 a 90 pixeles 2 y una circularidad de 0.4-1.0 identificará con precisión todas las muestras. </li><li> Medir la ubicación de cada marcha utilizando la lista generada de coordenadas para cada partícula. Coordenada x en píxeles se puede convertir en centímetros para calcular la altura de aterrizaje. </li><li> Importar tabla en una hoja de cálculo (como Microsoft Excel). </li></ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

Los autores no tienen conflictos de intereses a revelar.

Utilizando los métodos descritos aquí, hemos sido capaces de evaluar rápidamente el rendimiento de vuelo de un gran número de mutantes de Drosophila, que proporciona una mayor eficiencia que antes. Para nuestros experimentos, separamos rutinariamente a hombres y mujeres y criarlos a baja densidad (menos de 20 moscas / vial) para limitar la agresión que pudiera dañar las alas. Otra consideración importante es controlar adecuadamente las diferencias en el rendimiento de vuelo debido a las diferencias en el...

Drosophila ha sido utilizado para estudiar la base genética de comportamiento 1, y los investigadores han ideado un número de maneras de analizar varios tipos de conducta 2-6. Las moscas han sido particularmente útil para proporcionar modelos útiles de trastornos neuromusculares 7. Un ensayo común utilizado para estudiar el comportamiento del aparato locomotor es el rendimiento de vuelo. El probador de vuelo original es útil para identificar los vuelos mutantes defectuosos y para la evaluación cuantitativa de la habilidad de vuelo 1, pero tiene varios inconvenientes que limitan su aplicación para las pantallas de alto rendimiento: el uso de cilindros de petróleo recubierto es complicado y engorroso, ciertas características, tales como la longitud del cilindro y la introducción de las moscas en el tubo con fuerza variable a reducir la precisión cuantitativa, y es difícil de recuperar moscas en vivo desde el probador. Para superar estas limitaciones, se ha modificado el probador de vuelo para incluir una serie de mejoras. Hemos añadido una &quot;gota maser &quot;para introducir las moscas para eliminar la variabilidad entre los experimentos y los usuarios. Utilizamos láminas acrílicas removibles recubiertas con un adhesivo que permite la fácil limpieza y recuperación de las moscas individuales. Hemos aumentado la longitud del tubo de vuelo para mejorar la precisión y la fiabilidad cuantitativa. Finalmente , usamos una cámara digital y software de imágenes para calcular las alturas de aterrizaje de moscas. Creemos que estas mejoras sean de utilidad para todos los laboratorios interesados ​​en la realización de cribados genéticos a gran escala para los defectos de funcionamiento de vuelo.

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an improved method for accurate and rapid measurement of flight performance in drosophila

Drosophila ha demostrado ser un sistema modelo útil para el análisis del comportamiento, incluyendo el vuelo. El probador de vuelo involucrado caída inicial vuela en un aceite recubierto probeta; altura aterrizaje proporciona una medida de la capacidad de vuelo mediante la evaluación de cómo las moscas lejos caerán antes de producir empuje suficiente para hacer contacto con la pared del cilindro. Aquí se describe una versión actualizada del probador de vuelo con cuatro mejoras importantes. En primer lugar, hemos añadido un &quot;tubo de caída&quot; para asegurarse de que todas las moscas entran en el cilindro de vuelo a una velocidad similar entre los ensayos, lo que elimina la variabilidad entre los usuarios. En segundo lugar, hemos sustituido el recubrimiento de aceite con láminas de plástico desmontables revestidos en Maraña-Trap, un adhesivo diseñado para capturar insectos vivos. En tercer lugar, se utiliza un cilindro más largo para permitir la discriminación más precisa de la capacidad de vuelo. En cuarto lugar se utiliza una cámara digital y software de imágenes para automatizar la puntuación de rendimiento de vuelo. Estas mejoras permiten el rapIdentificación, evaluación cuantitativa del comportamiento de vuelo, útil para grandes conjuntos de datos y pantallas de genética a gran escala.

<html> Figura 1A muestra un esquema del montaje probador de vuelo actualizado. Figura 1B ilustra el diseño de la pista permite la cámara para tomar una imagen panorámica sin bloquear el campo de visión. Los resultados representativos se muestran en la Figura 2, en el que el rendimiento de vuelo de moscas mutantes Slowpoke, que tienen un vuelo conocido defecto de 8-10, se comparan con los de tipo salvaje Canton-S vuela. Control de moscas a...

Watch this Scientific Journal Video about An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila at JoVE.com

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Aquí se describe un método para la medición rápida y exacta de funcionamiento de vuelo en Drosophila, lo que permite detección de alto rendimiento.

Un método mejorado para la rápida y precisa medición de rendimiento en vuelo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

an-improved-method-for-accurate-rapid-measurement-flight-performance

Research

JoVE Journal

Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

11.6K vistas.  University of Wisconsin-Madison.  Aquí se describe un método para la medición rápida y exacta de funcionamiento de vuelo en Drosophila, lo que permite detección de alto rendimiento. 

Video: An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

De alta resolución de medición del comportamiento de olor-Driven en larvas de Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Un Sistema de Anclaje magnética para investigar control mediado de vuelo visual y olfativa en Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Seguimiento visual mediada olor durante el vuelo en Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Un método mejorado para el aislamiento de ARN a partir de pino taeda ( P. taeda L.) y otras especies de coníferas

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Protocolo de entrenamiento de resistencia y ensayos de funcionamiento de los longitudinales de Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medición de la esperanza de vida en<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Un método innovador para exosome cuantificación y medición de tamaño

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

La medición de la tasa metabólica en<em&gt; Drosophila</em&gt; Mediante respirometría

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...