Este trabalho foi financiado pelo Instituto Nacional de Saúde, conceder No. NIH / NCRR P41-RR 016292 (para ACERT).

1. Materiais Todos os solventes utilizados neste trabalho foram de grau reagente. A armadilha de spin, incluindo N-terc-butil-α-phenylnitrone (PBN) eo rótulo de spin padrão 2,2,6,6-tetrametil-1-piperinyoxyl (TEMPO) foram obtidos da Sigma e foram usados ​​como fornecido. Os antioxidantes de plantas foram obtidas comercialmente a partir de Swanson, Inc. nos EUA. 2. Preparação de fumaça de cigarro e análise de radicais livres <ol><li> Introduzir antioxidantes no filtro, o Pycnogenol antioxidantes e extrato de semente de uva foram pela primeira vez dissolvido em etanol a 95%, enquanto o licopeno foi dissolvido em acetona. Os volumes de solvente foram diferentes, dependendo da solubilidade antioxidante. </li><li> A quantidade de antioxidantes de 0,4 mg / filtro foi usado. Eles foram, então, revestido com 10 mg de carvão ativado. Para este efeito, carvão ativado foi agitada por ~ 12 hrs com a solução antioxidante em condições anaeróbicas, filtrado e seco sob vácuo. </li&gt; <li> Antioxidantes foram então introduzidas no filtro de acetato de convencional (CA filtro). Para este propósito, o filtro foi cortado em dois pedaços. O revestimento vegetal antioxidantes foram inseridos entre dois pedaços do filtro e embrulhado com um pedaço de fita adesiva para formar um filtro - antioxidante-filtro sanduíche (Fig. 1A). Este filtro combinado foi posteriormente anexado ao varas de cigarros contendo tabaco. O controle de filtro foi feita da mesma forma como o filtro de antioxidantes, exceto nenhum antioxidante foi adicionado. </li><li> Antes da simulação de fumar, os cigarros pesquisa foram desembalados e mantidos em um ambiente de umidade constante (20 ° C, 60% de umidade relativa) usando uma solução de NaBr saturada para um mínimo de 2 dias. </li><li> Simulação de fumar para análise de rotina foi realizada à temperatura ambiente usando um dispositivo de fumo de um único porto que, como mostrado na Figura 1, consiste de um aspirador de água ou bomba de vácuo GAST DOA-BN-P104 / Air Compressor (Benton Harbor, Michigan) conectado para o spin-trappingmontagem através de um entroncamento com uma extremidade aberta. Puffs foram realizados, ligando a extremidade aberta que permaneceu aberta entre baforadas. O fluxo de gás foi verificada pelo medidor de vazão e fixada em 2,2 ~ SCFH = 17,5 mL / s através do ajuste de uma válvula colocada entre a bomba eo vent. </li><li> Os cigarros eram fumados de pesquisa sob a condição de volume de sopro de 35 ml para uma duração de 2 segundos repetida a cada 60 segundos, semelhante ao 1. Simulação de fumar para a estimativa quantitativa de fase gasosa de radicais livres foi realizada em conjunto com o sistema de armadilha de spin como mostrado na figura. 1. </li><li> Dez puffs intensa (35 mL / puff) foram tomadas para cada cigarro. Fase gasosa de radicais livres foram coletadas pela passagem do MCS através de um filtro Cambridge pad e depois introduzido na solução de spin trapping (0,05 M PBN em benzeno, 2,0 mL). </li><li> Após a última tragada, a solução armadilhas bolhas foi reajustado ao seu volume inicial (2,0 mL) com o benzeno mesmo. Uma alíquota foi transferida para uma 25 ~0 milímetros de comprimento, 3 milímetros tubo de vidro ID selado numa das extremidades. </li><li> A solução foi desoxigenada armadilhas usando um procedimento de congelamento-descongelamento-bomba. Foi congelados com azoto líquido, e um vácuo foi aplicado. Em seguida, ele foi descongelado sob atmosfera de argônio, permitindo que as bolhas de gás presas a escapar, e congelado novamente. Após este ciclo foi repetido três vezes, o tubo foi chama-selado a vácuo e usados ​​em outras medições ESR. Esta etapa é necessária porque o oxigênio, que é bem solúvel em benzeno, amplia linhas ESR de radicais orgânicos. Desoxigenação dramaticamente melhora a relação sinal-ruído. </li><li> X-Band ESR espectros foram registrados em um espectrômetro Bruker EMX em uma freqüência de 9,34 GHz em condições normais. As configurações espectrômetro utilizado na maioria dos experimentos foram: centro do campo 3312.5G, largura de digitalização 80G, modulação de amplitude 0.5G, constante de tempo 82 microsegundos, tempo de digitalização 40 segundos. </li><li> Os adutos armadilha de spin são relativamente estáveis ​​nas condições de experimento, que em alguns casos required 25 acumulações, que normalmente levava ~ 20 min. No entanto, após 12 horas a intensidade dos sinais de ESR em solução de benzeno diminuiu por um fator de ~ 5. </li><li> Para quantificar a concentração do aduto armadilhas, seu espectro ESR inicial de primeira derivada foi integrado. O espectro de absorção resultante mostra um fundo singlet amplo, provavelmente devido a fuligem / tar produtos contidos na fumaça. Após a subtração deste contexto, o trio separados do aduto armadilhas foi integrado mais uma vez (Fig. 2). </li></ol> 3. Resultados representante A maioria dos radicais livres na queima de cigarros produzidos fumaça (fase gasosa) são instantâneos e instável. A fim de observar esses radicais uma técnica armadilha de spin é empregado. Ele captura de fase gasosa de radicais livres, transformando-os em um aduto de spin que é mais estável e pode ser detectado por ESR (Fig.1). No presente estudo, a solução de spin-armadilha de 0,05M PBN foi usado para coletar o gás de fumaça fase de radicais livres, que são uma mistura de oxigênio e carbono-centrado radicais que são difíceis de separar 15. No nosso caso, no entanto, as constantes divisão observada hiperfinos um N = 13.7G e um H = 1.95G são muito semelhantes aos valores correspondentes para a captura de adutos alcoxil radicais livres (Rm) 2, sugerindo que eles são o produto principal. Nós mostramos que sinais fracos ESR e baixa reprodutibilidade observada em nossas medições iniciais (Fig. 2) foram devido à umidade do fluxo de fumaça. Para resolver esse problema, nós adicionamos uma armadilha de nitrogênio líquido (LN2) entre o filtro de Cambridge ea solução armadilha spin. A LN 2 armadilha de água removida do fluxo de MCS por congelamento rápido e capturá-lo na parede interna do tubo de vidro. Esta muito melhorada sinais ESR e permitiu resultados altamente reprodutível (Fig. 3). A quantidade de presos radicais livres foi determinada utilizando umamostra de referência. Para amostras de controle sem antioxidantes, a concentração de benzeno no aduto típico estimada por comparação de seu espectro dupla integral com um espectro de casal integrante de concentração conhecida de O TEMPO foi de 1,24 mM (Fig.2). Uma vez que a quantidade de ar passou por cada cigarro durante a fumar foi de ~ 350 ml, o que dá uma estimativa para a concentração de radicais livres na fase gasosa da MCS de ~ 7.1X10 -9 M, e um número total de radicais presos da fase gasosa de um cigarro inteiro de ~ 1.5x10 15. Uma estimativa para a quantidade total de radicais livres na fumaça de um cigarro inteiro, incluindo o gás ea fase particulada, foi de ~ 10 16 radicais livres 9. Diferentes níveis do efeito de limpeza de plantas antioxidantes em fase gasosa de radicais livres na fumaça do cigarro fluxo principais foram observados. Suas taxas de limpeza foram apresentados na fig. 4. Licopeno e extrato de semente de uva apresentaram as maiores taxas, enquanto baixaer taxa foi observada para picnogenol (Fig.4). <img alt="Figura 1" src="/files/ftp_upload/3406/3406fig1.jpg" /> Figura 1. Um diagrama de uma melhor concepção de fumar de simulação para a recolha de fase gasosa de radicais livres na fumaça do cigarro mainstream (MCS), utilizando armadilha de spin. MCS foi desenhada por um aspirador de água através CA filtro, então Cambridge filtro (filtro amarelo) e passou por uma armadilha de nitrogênio líquido para remover H 2 O. Os produtos fase gasosa finalmente fui para girar armadilha e borbulhar na solução armadilha spin. Antioxidante de plantas foi colocado entre dois pedaços de acetato de filtros convencionais (alargada em círculo A) ligado ao cigarro para varrer os radicais livres no MCS. <img alt="Figura 2" src="/files/ftp_upload/3406/3406fig2.jpg" /> Figura 2. A estimativa quantitativa para a concentração aduto-spin trapping em benzeno requer subtrair um sinal de fundo largo da integração primeiral espectro. Os parâmetros hiperfinos divisão para o aduto-spin trapping é um H = 1,95 G, um N = 13.7G. <img alt="Figura 3" src="/files/ftp_upload/3406/3406fig3.jpg" /> Figura 3. Passando por LN2 armadilha melhora substancialmente a qualidade dos sinais ESR obtidos por spin trapping de MCS. <img alt="Figura 4" src="/files/ftp_upload/3406/3406fig4.jpg" /> Figura 4. Efeito de antioxidantes naturais sobre a concentração de radicais livres no MCS. As intensidades de sinal são: controle - 100%, pycnogenol - 55%, extrato de semente de uva - 12%, o licopeno - 10%.

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A estimativa confiável do efeito de diferentes radicais livres catadores no fumo do tabaco requer uma técnica reprodutível para a detecção quantitativa de radicais livres. Anteriormente 1, demonstrou-se que pequenos volumes de soluções de maior concentração de spin armadilha em solventes não-polares são mais eficazes na captura de radicais livres do fumo do tabaco. A fumaça do cigarro contém sempre vapor de água provenientes da combustão de compostos orgânicos e da umidade residual do tabaco, que pode acabar no solvente trapping. Esta mistura de água na solução armadilha de spin PBN diminui substancialmente o tempo de vida do adutos armadilhas de spin ea intensidade de seus sinais ESR. Removendo a umidade por simples MCS passando através de um tubo em forma de U resfriado com nitrogênio líquido melhoraram consideravelmente a qualidade de espectros de ESR em nossos experimentos, apesar de alguma fração de fase gasosa radicais pode ser também presa na superfície congelada. Usando tsua técnica, comparamos a eficiência relativa de diferentes radical-limpeza compostos naturais introduzidos filtros de cigarro. Descobrimos que o licopeno e extrato de semente de uva imediatamente após a incorporação em filtros de cigarro são capazes de limpar até 90% de radicais livres a partir do MSC fase gasosa. Alta capacidade de limpeza, tais fileiras essas baratas disponíveis compostos naturais entre os mais eficientes reportados livre protetores radical como a hemoglobina e shikonine 8,14. No entanto, em nossos filtros de cigarro carregado com experimentos estudados antioxidantes naturais perdeu uma parte considerável de sua capacidade de limpeza depois de uma semana de armazenamento em temperatura ambiente. Resolver este problema pode estimular a futura aplicação de licopeno e extrato de semente de uva em filtros de cigarro comercial.

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

O tabagismo está associado a cânceres humanos. Tem sido relatado que a maioria das mortes por câncer de pulmão são causados ​​pelo tabagismo 5,6,7,12. Embora alcatrões tabaco e produtos afins na fase de partículas da fumaça do cigarro são as principais causas de doenças cancerígenas e mutagénicas relacionados, a fumaça do cigarro contém quantidades significativas de radicais livres que são também considerados como um importante grupo de substâncias cancerígenas 9,10. Os radicais livres atacam componentes da célula por danificar a estrutura da proteína, lipídios e sequências de DNA e aumentar os riscos de desenvolver vários tipos de cânceres. Radicais inalados produzem adutos que contribuem para muitos dos efeitos negativos sobre a saúde do fumo do tabaco no pulmão 3. Estudos têm sido realizados para reduzir os radicais livres da fumaça do cigarro para diminuir os riscos dos danos provocadas pelo tabagismo. Tem sido relatado que a hemoglobina e heme contendo compostos poderiam parcialmente scavenge óxido nítrico, reativaoxidantes e cancerígenos nitrosocompounds voláteis de 4 de fumaça de cigarro. A &#39;bio-filtro &quot;consistia de carbono hemoglobina e ativado foi utilizado para eliminar os radicais livres e para remover até 90% dos radicais livres da fumaça do cigarro 14. No entanto, devido ao custo ineficácia, não foi comercializada com sucesso. Outro estudo mostrou a eficiência de limpeza de boa shikonin, um componente da fitoterapia chinesa 8. No presente estudo, relatamos um protocolo comum para a introdução de extratos de antioxidantes naturais para o filtro de cigarro para limpeza de radicais livres em fase gasosa na fumaça do cigarro e medição do efeito scavenge em radicais livres em fase gasosa na fumaça do cigarro mainstream (MCS), utilizando-spin trapping ressonância paramagnética eletrônica (ESR) Espectroscopia 1,2,14. Nós mostramos capacidade de limpeza de alta de licopeno e extrato de semente de uva, que poderia apontar para sua futura aplicação em filtros de cigarro. Uma vantagem importante destes profissionaiscatadores pectiva é que eles podem ser adquiridos em grande quantidade a partir de subprodutos da indústria de tomate ou de vinho, 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-trapping espectroscopia ESR foi usado para estudar o efeito de antioxidantes vegetais licopeno, pycnogenol e extrato de semente de uva na limpeza de fase gasosa de radicais livres na fumaça do cigarro.

Um Protocolo para Detecção e eliminação em fase gasosa radicais livres do fumo do cigarro 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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19.2K visualizações.  CDCF-AOX Lab. Spin-trapping espectroscopia ESR foi usado para estudar o efeito de antioxidantes vegetais licopeno, pycnogenol e extrato de semente de uva na limpeza de fase gasosa de radicais livres na fumaça do cigarro.

Vídeo: 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.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

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

Bioengenharia

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 a formação de biofilme Streamer em um dispositivo micro com 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 nanopartículas colheita de plasma ou urina para detectar proteínas de baixa abundância

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.

Avaliação do Impacto da exposição repetida de Organotípicas 3D brônquica e cultura nasal Tissue Models para Whole fumo do cigarro

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.

encapsulando citocromo<em&gt; C</em&gt; Em Silica Aerogel nanoarquiteturas sem metal nanopartículas, mantendo bioatividade em fase gasosa

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.

Um microcontrolador operado dispositivo para a geração de extratos líquidos de aerossol de cigarro eletrônico e fumo cigarro 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 incorporação de fixo e manchado, inteiro, espécimes de milímetro-escala para histologia seção livre 3D por Microtomografia computadorizada

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.

Limpeza catalítica de planta reativas de oxigênio espécies na Vivo por nanopartículas de óxido de cério aniônicos

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.

Um ensaio de liposcopia única baseado em microscopia quantitativa para detectar a inhomogeneidade composicional entre lipossomos individuais

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 injeção de lipidico para medições de cristalografia serial no Síncrotron Australiano

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