Este trabajo fue apoyado por el Instituto Nacional de Salud, la concesión N º NIH / CNRR P41-RR 016 292 (para ACERT).

1. Materiales Todos los disolventes utilizados en este trabajo fueron de grado reactivo. La trampa de la vuelta, incluyendo N-terc-butil-α-phenylnitrone (PBN) y la etiqueta de giro estándar de 2,2,6,6-tetrametil-1-piperinyoxyl (TEMPO) se obtuvieron de Sigma y se utilizaron tal como se suministra. Los antioxidantes de plantas se obtiene comercialmente a partir de Swanson EE.UU., Inc.. 2. Preparación del humo del cigarrillo y el análisis de los radicales libres <ol><li> Para introducir antioxidantes en el filtro, el pycnogenol antioxidantes y extracto de semillas de uva fueron disueltos en etanol al 95%, mientras que el licopeno se disolvió en acetona. Los volúmenes de disolventes fueron diferentes dependiendo de la solubilidad antioxidante. </li><li> La cantidad de antioxidantes de 0,4 mg / filtro se utilizó. Que se revistieron con 10 mg de carbón activado. Para ello el carbón activado se agitó durante ~ 12 horas con la solución antioxidante en condiciones anaeróbicas, se filtra y se seca al vacío. </li&gt; <li> Los antioxidantes se introdujeron entonces en el filtro de acetato convencional (CA filtro). Para ello, el filtro se cortó en dos pedazos. La cubierta de plantas antioxidantes se inserta entre dos piezas del filtro y se envuelve con un trozo de cinta para formar un filtro - antioxidantes filtro sandwich (Fig. 1A). Este filtro combinado se unió posteriormente a las barras de cigarrillos que contengan tabaco. El control de filtro se realizó en la misma forma que el filtro antioxidante excepto que no se añadió antioxidante. </li><li> Antes de la simulación de fumar, los cigarrillos de investigación fueron desempaquetado y se mantienen en un ambiente de humedad constante (20 ° C, 60% de humedad relativa) utilizando una solución saturada de NaBr por un mínimo de 2 días. </li><li> Simulación de fumar para análisis de rutina se realizó a temperatura ambiente usando un dispositivo de fumar de un solo puerto que, como se muestra en la Fig. 1, consiste en un aspirador de agua o GAST DOA-P104-BN bomba de vacío / compresor de aire (Benton Harbor, Michigan) conectado con el giro que atrapanmontaje a través de un cruce con un extremo abierto. Bocanadas se llevaron a cabo conectando el extremo abierto que se mantuvo abierta entre calada y calada. El flujo de gas se comprobó mediante medidor de flujo y se fijó en ~ 2.2 SCFH = 17,5 ml / s mediante el ajuste de una válvula situada entre la bomba y el ano. </li><li> Los cigarrillos se fumaban la investigación bajo la condición de volumen bocanada de 35 ml con una duración de 2 segundos repite cada 60 segundos, similar a 1. Simulación de fumar para la estimación cuantitativa de los radicales libres en fase gaseosa se llevó a cabo en conjunción con el sistema de captura de giro como se muestra en la fig. 1. </li><li> Diez inhalaciones intensas (35 ml / puff) fueron tomadas por cada cigarrillo. Fase gaseosa de los radicales libres fueron recogidos por pasar la MCS a través de una plataforma de Cambridge filtro y se introduce en la solución de captura de giro (0,05 M PBN en benceno, 2,0 ml). </li><li> Después de la última dosis, la solución de captura de burbujas se reajustó a su volumen inicial (2,0 ml) con el benceno mismo. Una alícuota fue trasladado en un 25 ~0 mm de largo, 3 mm de diámetro del tubo de vidrio sellado en un extremo. </li><li> La solución de captura fue desoxigenada mediante un procedimiento de congelación y descongelación de la bomba. Que se congeló en nitrógeno líquido, y el vacío fue aplicado. Luego fue descongelado en atmósfera de argón, lo que permite burbujas de gas se escape, y se congela de nuevo. Después de este ciclo se repitió tres veces, el tubo se llama sellado al vacío y se utiliza en más medidas de ESR. Este paso es necesario porque el oxígeno, que es muy soluble en benceno, amplía las líneas de ESR de radicales orgánicos. Desoxigenación mejora considerablemente la relación señal-ruido. </li><li> X-Band ESR espectros se registraron en un espectrómetro Bruker EMX a una frecuencia de 9,34 GHz en condiciones normales. Los ajustes del espectrómetro utilizado en la mayoría de los experimentos fueron: el jardín central 3312.5G, escanear ancho 80G, la modulación de amplitud 0,5 G, la constante de tiempo 82 microsegundos, tiempo de exploración 40 seg. </li><li> Los aductos trampa de giro son relativamente estables en las condiciones del experimento, que en algunos casos reqle pedirá otra vez 25 acumulaciones, que normalmente se ~ 20 min. Sin embargo, después de 12 horas la intensidad de las señales de ESR en una solución de benceno disminuye en un factor de ~ 5. </li><li> Para cuantificar la concentración de la aducción de trampas, sus iniciales de la primera derivada del espectro de ESR se ha integrado. El espectro de absorción resultante muestra un fondo singlete amplio, muy probablemente debido a los productos de hollín / alquitrán contenidos en el humo. Después de la sustracción de estos antecedentes, el trío separado del aducto de captura se ha integrado una vez más (Fig. 2). </li></ol> 3. Resultados representante La mayoría de los radicales libres en la quema de cigarrillos de producción de humo (fase gas) son instantáneas e inestable. Con el fin de observar estos radicales una técnica trampa de spin se emplea. Captura fase gaseosa de los radicales libres por su transformación en un aducto de giro que es más estable y puede ser detectado por ESR (Fig. 1). En el presente estudio, la solución de spin-trampa de 0,05M PBN se utilizó para recoger el gas de humo fase de radicales libres, que son una mezcla de radicales de oxígeno, y se centra en carbono que son difíciles de separar 15. En nuestro caso, sin embargo, las constantes hiperfinos observa la división de un N = 13,7 g y 1,95 g H = son muy similares a los valores correspondientes para la captura de aductos de alcoxilo los radicales libres (Romanos) 2, lo que sugiere que son el producto principal. Hemos demostrado que las señales débiles ESR y reproducibilidad baja observada en nuestras mediciones iniciales (Fig. 2) se debieron a la humedad en el flujo de humo. Para resolver este problema, hemos añadido un nitrógeno líquido (LN2) trampa entre el filtro de Cambridge y la solución spin trampa. La LN 2 trampa elimina el agua del flujo de MCS de congelación rápida y la captura en la pared interior del tubo de vidrio. Esto mejoró las señales de ESR y ha permitido resultados altamente reproducibles (Fig. 3). La cantidad de radicales libres atrapados se determinó utilizando unmuestra de referencia. Para las muestras de control sin antioxidantes, la concentración de benceno en el aducto típicas estimadas por comparación de su espectro integral doble con un espectro integral doble de concentración conocida de TEMPO fue de 1,24 M (Fig. 2). Dado que la cantidad de aire que pasa a través de cada cigarrillo se fuma fue de ~ 350 ml, lo que da una estimación de la concentración de radicales libres en la fase gaseosa de MCS de 7.1X10 ~ -9 M, y un número total de radicales atrapados de la fase gaseosa de un cigarrillo entero de 1.5x10 ~ 15. Una estimación de la cantidad total de los radicales libres en el humo de un cigarrillo entero, incluyendo tanto el gas y la fase de partículas, fue ~ 10 16 radicales libres 9. Los diferentes niveles del efecto de barrido de la planta de antioxidantes sobre los radicales libres en fase gaseosa del humo del cigarrillo corriente principal se observaron. Sus tasas de recolección de residuos se presentan en la figura. 4. Licopeno y extracto de semilla de uva mostraron las tasas más altas mientras que los bajoser tasa se observó para el Pycnogenol (Fig. 4). <img alt="Figura 1" src="/files/ftp_upload/3406/3406fig1.jpg" /> Figura 1. Un diagrama de un mejor diseño de simulación de fumar para la recogida de fase gas-radicales libres en el humo del cigarrillo convencional (MCS) con trampa de spin. MCS fue dibujado por un aspirador de agua a través de CA filtro, filtro de Cambridge (filtro amarillo) y pasa a través de una trampa de nitrógeno líquido para eliminar el H 2 O. Los productos en fase gaseosa, finalmente fue a girar trampa y burbujea a través de la solución trampa giro. Antioxidantes planta fue colocada entre dos piezas de filtros de acetato convencional (ampliada en el círculo A) conectado a los cigarrillos para eliminar los radicales libres en el MCS. <img alt="Figura 2" src="/files/ftp_upload/3406/3406fig2.jpg" /> Figura 2. Una estimación cuantitativa de la concentración de aducto spin-captura en benceno requiere restar una señal de fondo amplio de la primera integraciónl espectro. Los parámetros de interacción hiperfina para el aducto spin-trampas son una H = 1,95 g, un N = 13,7 g. <img alt="Figura 3" src="/files/ftp_upload/3406/3406fig3.jpg" /> Figura 3. Pasando a través de LN2 trampa mejora sustancialmente la calidad de las señales de ESR obtenidos por spin-captura de MCS. <img alt="Figura 4" src="/files/ftp_upload/3406/3406fig4.jpg" /> Figura 4. Efecto de los antioxidantes naturales de la concentración de radicales libres en el MCS. La intensidad relativa de la señal son: control - 100%, el Pycnogenol - 55%, extracto de semilla de uva - 12%, el licopeno - un 10%.

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No hay conflictos de interés declarado.

Una estimación fiable del efecto de diferentes de radicales libres en el humo del tabaco requiere de una técnica reproducible para la detección cuantitativa de los radicales libres. Previamente 1, se ha demostrado que pequeñas cantidades de soluciones de mayor concentración de la trampa de espín en solventes no polares son más eficaces para atrapar los radicales libres del humo del tabaco. El humo del cigarrillo siempre contiene vapor de agua de la combustión de compuestos orgánicos y la humedad residual en tabaco, que pueden terminar en la captura de disolventes. Esta mezcla de agua en la solución trampa PBN giro disminuye sustancialmente el tiempo de vida de los aductos de giro de captura y la intensidad de sus señales de ESR. La eliminación de esta humedad por simple MCS pasa a través de un tubo en forma de U enfriado con nitrógeno líquido mejorado dramáticamente la calidad de los espectros de ESR en nuestros experimentos, a pesar de que alguna fracción de los radicales en fase gaseosa también podría ser atrapado en la superficie congelada. Con tsu técnica se comparó la eficacia relativa de los diferentes radicales de barrido compuestos naturales introducidos en los filtros de cigarrillos. Encontramos que el licopeno y extracto de semilla de uva inmediatamente después de la incorporación en los filtros de cigarrillos son capaces de recoger hasta el 90% de los radicales libres del MSC en fase gaseosa. Como de alta capacidad de barrido filas de estos compuestos naturales barata disponible entre las más eficientes informó protectores de los radicales libres como la hemoglobina y shikonine 8,14. Sin embargo, en nuestros filtros de cigarrillos cargados con experimentos estudiaron los antioxidantes naturales perdido una parte notable de su capacidad de recolección de residuos después de una semana de almacenamiento a temperatura ambiente. La solución de este problema se puede estimular la futura aplicación de licopeno y extracto de semilla de uva en los filtros de cigarrillos comerciales.

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

El consumo de cigarrillos está asociado con cánceres humanos. Se ha informado que la mayoría de las muertes por cáncer de pulmón son causados ​​por fumar cigarrillos 5,6,7,12. A pesar de los alquitranes del tabaco y productos relacionados en la fase de partículas del humo del cigarrillo son las principales causas de enfermedades relacionadas con carcinógenos y mutágenos, humo de cigarrillo contiene cantidades significativas de los radicales libres que también se consideran como un importante grupo de carcinógenos 9,10. Los radicales libres atacan los componentes celulares de la estructura de proteínas dañinas, los lípidos y las secuencias de ADN y aumentar el riesgo de desarrollar varios tipos de cánceres. Los radicales se inhala produce aductos que contribuyen a muchos de los efectos negativos para la salud del humo del tabaco en el pulmón 3. Se han realizado estudios para reducir los radicales libres del humo del cigarrillo para disminuir los riesgos de los daños inducidos por el tabaco. Se ha informado de que la hemoglobina y hemo-compuestos que contienen en parte podría limpiar el óxido nítrico, reactivaoxidantes y cancerígenas nitrosocompuestos volátil de 4 humo del cigarrillo. Un &quot;filtro biológico&quot; consistió en la hemoglobina y el carbono activado se utiliza para limpiar los radicales libres y de eliminar hasta el 90% de los radicales libres del humo del cigarrillo 14. Sin embargo, debido a la falta de efectividad de costos, no se ha comercializado con éxito. Otro estudio mostró un buen rendimiento de barrido de shikonin, un componente de la medicina china a base de hierbas 8. En el presente estudio, se presenta un protocolo común para la introducción de extractos antioxidantes naturales en el filtro del cigarrillo para eliminar los radicales libres en fase gaseosa del humo del cigarrillo y la medición del efecto de barrido de los radicales libres en fase gaseosa del humo del cigarrillo convencional (MCS) con spin-captura resonancia de spin electrónico (ESR) Espectroscopia 1,2,14. Hemos demostrado gran capacidad de barrido de licopeno y extracto de semilla de uva que podría apuntar a su futura aplicación en los filtros de cigarrillos. Una ventaja importante de estos profesionalespectivos carroñeros es que se puede obtener en grandes cantidades a partir de subproductos de la industria del tomate o el vino, respectivamente, 11,13

Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Spin-captura de la espectroscopía ESR fue utilizado para estudiar el efecto de la planta de antioxidantes licopeno, pycnogenol y el extracto de semilla de uva en la recolección de residuos en fase gaseosa radicales libres en el humo del cigarrillo.

Un protocolo para la detección y borrado en fase gaseosa radicales libres en el humo del cigarrillo Mainstream

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

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Bioengineering

19.2K vistas.  CDCF-AOX Lab. Spin-captura de la espectroscopía ESR fue utilizado para estudiar el efecto de la planta de antioxidantes licopeno, pycnogenol y el extracto de semilla de uva en la recolección de residuos en fase gaseosa radicales libres en el humo del cigarrillo.

Video: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Investigación

Bioingeniería

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocolo para Biofilm Streamer Formación en un dispositivo de microfluidos con Micro-pilares

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Hidrogel Nanoparticle recolección de plasma o de orina para la detección de proteínas de baja abundancia

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Evaluación del Impacto de la exposición repetida de Organotípicos 3D bronquiales y nasales Modelos Tissue Culture Whole humo del cigarrillo

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

encapsular citocromo<em&gt; c</em&gt; El aerogel de sílice en nanoarquitecturas sin metal nanopartículas Mientras Retienen bioactividad en fase gaseosa

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Un microcontrolador funciona dispositivo para la generación de extractos líquidos de Aerosol de humo y electrónicos cigarrillo cigarrillo convencional

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Rígida fijación de fijo y manchados, todo, escala milimétrica muestras para histología sin sección 3D de tomografía Micro computada

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Depuración catalítica de planta oxígeno reactivo especie In Vivo de nanopartículas de óxido de cerio aniónicos

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Un ensayo de liposomio único basado en microscopía de fluorescencia cuantitativa para detectar la inhomogeneidad compositiva entre liposomas individuales

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Protocolo de inyección lipídica para mediciones de cristalografía en serie en el sincrotrón australiano

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...