Esta pesquisa foi financiada em parte pelo Grants-in-Aid para a Investigação Científica para impugnar pesquisa exploratória (JK), e da Investigação Científica (A) (JK e SM) do Ministério da Educação, Cultura, Desporto, Ciência e Tecnologia, do Japão . A Riken FPR comunhão (RCE) forneceu apoio adicional. Os autores expressam sua gratidão para com os drs. Eisuke Chikayama, Yasuyo Sekiyama e Mami Okamoto para assistência técnica com RMN e análises estatísticas.

1. Preparação filtro para remover Extraíveis <ol><li> Use 25-mm de diâmetro de poro de 0,22 um tamanho de filtros PVDF Durapore hidrofílico (Millipore). Coloque filtros em uma proveta de 500 ml limpa Pyrex usando uma pinça. Pré-lavagem três vezes com água destilada. Redemoinho bem como de enxaguar para impedir que os filtros se colem umas às outras. Adicionar 300 ml Milli-Q (Millipore) ou de água de alta qualidade equivalente. Autoclavar para facilitar a remoção completa de extraíveis a partir dos filtros. </li><li> Decantar a Milli-Q e novamente tripla lavagem dos filtros, desta vez com Milli-Q. O uso de filtros lugar pinças individuais sobre uma superfície limpa e seca (tal como folha de alumínio) e, ou seca a uma temperatura razoável (por exemplo 37 ° C) ou de ar seco. Os filtros agora está pronto para usar. </li></ol> 2. A filtração do material de amostra <ol><li> Para demonstrar este protocolo, a Millipore aço inoxidável 3 º lugar-tubo do filtro com colunas de 25 mm de microanálise de filtros de vidro com suportee uma bomba mecânica é utilizada. Utilizando uma técnica asséptica, colocar um filtro de 25 mm único na base da coluna de filtro, aplicar a coluna e pinça juntos. </li><li> Carregar 15 ml de amostra na coluna, abrir o obturador sobre o tubo do filtro, e ligar a bomba. Filtrar sob pressão suave para minimizar a quebra das células (&lt;kPa 5). Outras bombas, tais como mão ou uma bomba peristáltica podem ser adaptados a este protocolo. Para amostras de densidade mais baixa, adições sucessivas de água pode ser necessário, não deixe o filtro ficar seca por um período prolongado de tempo entre as adições de água. </li><li> Para amostras marinhas, depois de ter filtrado a sua amostra, você pode realizar uma de água doce opcional enxágüe para reduzir os íons paramagnéticos residuais em sua amostra e no filtro. Isso pode ajudar com mais ajuste preciso do magneto do espectrômetro. Simplesmente suavemente adicionar um pequeno volume de água e filtrar através de sua amostra recolhida no final. </li><li> Uma vez que a filtragem está terminado, desligue a bomba e deixar o ope válvulan por isso ainda há a pressão negativa sob o filtro. Remover o grampo e coluna de filtro. </li><li> Com uma mão, use uma pinça limpa para tomar posse do filtro. Dobre o filtro em si, mas não prega. Com a outra mão use o lábio de um tubo de microcentrífuga estéril 2-ml para segurar o filtro. Solte as pinças, em seguida, usá-los para voltar a agarrar ambas as bordas do filtro. Regrip em um ângulo de 45 ° ao redil. </li><li> Colocar o filtro para dentro do tubo 2-ml e libertação de modo que se abre com o lado da amostra virada para dentro. Você pode colocar até dois filtros para o tubo de microcentrífuga estéril 2 ml desta forma. Se usando dois filtros, assegurar que eles se sobrepõem tão pouco quanto possível. Imediatamente congelar (pelo menos -30 ° C). </li></ol> 3. Extracção do aquosa solúveis em metabolitos <ol><li> Liofilizar suas amostras durante a noite ou por pelo menos 10 horas. </li><li> Após liofilização, adicionar um triturador de aço inoxidável a cada tubo (Tokken). Adicionar 750 ul de potássio padronizadatampão fosfato de RMN em óxido de deutério (2 H&gt; 90%) com 2,2-Dimetil-2-silapentane-5-sulfonato de (DSS) padrão (KPI; 38,3 mM KH 2 PO 4, 61,7 mM de K 2 HPO 4, DSS 0,1 mM, pH 7,0, 90% de D 2 O) 2. </li><li> Sonicar as amostras durante 5 minutos a 4 ° C num sonicador água (Bioruptor, Diagenode) para remover o material celular a partir do filtro. Remover os filtros com pinças limpas. </li><li> Dissociação das células utilizando um triturador de moinho (1600 rpm) durante 5 minutos. </li><li> Incubar a 65 ° C com agitação (1400 rpm) num agitador de bancada (Eppendorf) durante 15 minutos. </li><li> Remover o porco de metal com uma pinça limpas, e centrifugar a amostra a 13.000 g durante 5 minutos. </li><li> Colhem-se o sobrenadante directamente para um tubo de RMN para espectroscopia de RMN. </li></ol> 4. Espectroscopia de RMN e Análise de Dados <ol><li> Carregue o seu exemplo em um espectrômetro de RMN (aqui, um Bruker DRX-500 equipado com espectrômetroTXI sonda com triplo-eixo gradiente controlado por um computador que executa Xwin-RMN). </li><li> 1D obter 1 H espectros de RMN utilizando métodos adequados previamente publicados 2,15 a partir da interface Xwin-RMN. No presente estudo 1 H espectros de RMN foram registados num espectrómetro de funcionamento DRX-500 em 500,03 MHz a 298 K. sinais de água residuais foram suprimida pela sequência de pulso Watergate, com um tempo de repetição de 1,2 s. 128 transientes foram coletadas para obter 32.000 pontos de dados por espectro. </li><li> Transfira os diretórios de dados de RMN para um PC instalado NMRPipe software 16. Processar os dados brutos e DSS definir como 0 ppm de referência, em seguida, manualmente fase do espectro. Digitalizar os dados espectrais em um conjunto de valores discretos, integrando ou &quot;binning-los&quot; com softwares como o rNMR, Automics, ou usando o pacote FT2B publicamente disponível no site da ECOMICS ( <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> ) 3,17. Em diaé exemplo, os espectros foram integrados entre 0,5 e 10,5 ppm ao longo de 0,032 ppm regiões integral usando ECOMICS e normalizado para tanto DSS ou a intensidade do sinal total. Os dados de saída agora pode ser usado para análise estatística jusante, tais como análise de componentes principais (PCA), utilizando softwares livres como o R 18. </li></ol> 5. Os resultados representativos Um exemplo de um espectros de RMN H obtidos utilizando os métodos acima são mostrados na Figura 1. Estas amostras, a partir de dois pontos de tempo de uma experiência de microcosmos mostram diferenças claras devido a algas actividades metabólicas. O espectro de 4 dias mostra abundância considerável de picos, particularmente na gama de 3-4 ppm em comparação com a amostra de 1 dia. Estes picos pode ser atribuída aos açúcares produzidos por floração diatomáceas dentro do microcosmo. Em um experimento semelhante comparando o crescimento de comunidades naturais de plâncton na água do mar artificial ou natural, abordagens estatísticas such como principal componente trama pontuação análise (PCA) derivado a partir de espectros de RMN binned pode ser usado para mostrar claras diferenças metabólicas entre os dois tratamentos (Fig. 2), enquanto que as parcelas de carregamento pode identificar picos dentro do espectro que forma a distribuição dos dados . Tais resultados podem ser comparados com dados de outros níveis ómicas, tais como a partir de métodos de impressão digital genómicas (Fig. 3). Estes picos de RMN podem ser consultados individualmente (por exemplo, no BMRB; <a href="http://www.bmrb.wisc.edu/">http://www.bmrb.wisc.edu/</a> ) 19, ou espectros inteiro pode ser analisados ​​estatisticamente (por exemplo, com SpinAssign em <a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken. jp / ação? = nmr_search</a> ) 2. Neste exemplo, as diferenças entre tratamentos foram devido a uma abundância de picos na região açúcares (3,39 ppm para 4,04 ppm) dos espectros de metabolitos naturais da comunidade de plâncton, e vários picos característicos para as comunidades água do mar artificial foram tentativamente identified como de lactato e formiato usando SpinAssign. <img src="/files/ftp_upload/3163/3163fig1.jpg" alt="A Figura 1" /> Figura 1. Representativas 1 H espectros de RMN obtido a partir de amostras processadas usando este procedimento. As amostras foram tomadas antes microcosmos (dia 1) e durante (dia 4) bloom de diatomáceas uma intensa. Experiências de RMN foram realizadas num Bruker DRX-500 com sinais normalizados para a altura do pico do padrão interno (DSS; 0 ppm). <img src="/files/ftp_upload/3163/3163fig2.jpg" alt="A Figura 2" /> Figura 2. Análise de Componentes Principais enredo pontuação (PCA) para espectros de RMN de binned metabolomes das comunidades microbianas de origem natural planctônicas cultivadas em microcosmos com naturais (diamantes abertos) ou água do mar artificial (círculos pretos). Limpar diferenças metabólicas pode ser observado no diagrama de dispersão. Um gráfico de carregamento de uma tal análise pode então ser usado para identificar picos distintos de importância nao sistema; estes picos pode ser ainda analisada conforme necessário. <img src="/files/ftp_upload/3163/3163fig3.jpg" alt="A Figura 3" /> Figura 3. Um exemplo de multi-omics análise combinando RMN com os dados genéticos. Composição da comunidade com base na electroforese em gel com gradiente desnaturante de 18S (esquerda) e 16S (direita) de genes de rRNA das mesmas amostras como analisados ​​na Figura 2 também mostra os padrões de microbianas distintas da comunidade entre naturais (diamantes abertos) e microcosmos artificiais (círculos pretos) de água do mar. Essa correspondência entre metaboloma e genoma dos sistemas naturais demonstra a utilidade dessa abordagem.

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O método de extração e filtração metabólito demonstrado aqui permite a biomassa microbiana planctônica a ser coletadas em quantidade suficiente para metabolômica RMN. Enquanto a extracção apenas de solução aquosa solúveis em metabolitos usando KPi e 1D 1 H RMN é demonstrado, outros solventes de extracção e abordagens espectroscópicas pode ser usado. Um exemplo útil é a utilização de metanol deuterado como solvente semi-polar, que tem sido mostrado para produzir espectros de RMN superiores...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> 0,22 hidrofílicos Durapore filtros PVDF, 25 mm </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Titular Microanálise do filtro, 25 mm, de frita de vidro suporte </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3 º lugar-colector, 47 mm, em aço inoxidável </td><td> Millipore </td><td> XX2504735 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Wako </td><td> 169-04245 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> Wako </td><td> 164-04295 </td><td></td></tr><tr><td> Óxido de deutério, 2 H&gt; 90% </td><td> Campridge Isotope Laboratoties </td><td> DLM-4 </td><td></td<html> &gt; </tr><tr><td> DSS </td><td> Fluka </td><td> 92754 </td><td></td></tr><tr><td> Automill </td><td> Tokken </td><td> TK-AM4 </td><td> Trituradores de aço inox incluído </td></tr><tr><td> Thermomixer conforto </td><td> Eppendorf </td><td> 5355 000.011 </td><td></td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 </td><td></td></tr><tr><td> Evaporador de vácuo </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> NMR </td><td> Bruker </td><td> DRX-500 com 5 mm de sonda TXI </td><td></td></tr><tr><td> Ferramenta binning Spectral </td><td> Originalmente desenvolvido </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> Ferramenta de anotação de metabólitos e banco de dados </td><td> Originalmente desenvolvido </td><td> SpinAssign </td><td>= Nmr_search &quot;&gt; http://prime.psc.riken.jp/?action=nmr_search </td></tr></tbody></table>

concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy

Metabolômica Ambiental é um campo emergente que está promovendo uma nova compreensão de como os organismos respondem e interagem com o ambiente e entre si, no nível bioquímico 1. Nuclear espectroscopia de ressonância magnética (RNM) é uma de várias tecnologias, incluindo cromatografia em fase gasosa-espectrometria de massa (GC-MS), com a promessa considerável para tais estudos. Vantagens de RMN são de que ele é adequado para análises não-alvo, fornece informações estruturais e espectros podem ser consultados de maneira quantitativa e estatística em bancos de dados recentemente disponíveis de espectros metabólito indivíduo 2,3. Além disso, dados espectrais de RMN podem ser combinados com dados de outros níveis genómica (transcritômica por exemplo, genómica) para fornecer uma compreensão mais abrangente das respostas fisiológicas de taxa para o outro e ao meio ambiente 4,5,6. No entanto, de RMN é menos sensível do que outras técnicas de metabolômica, tornando difícil a APply para sistemas microbianos naturais, onde as populações de amostras podem ser concentrações de baixa densidade e metabólito baixo em comparação com metabólitos de bem-definida e facilmente extraíveis fontes, tais como tecidos inteiros, fluidos biológicos ou de células-culturas. Por conseguinte, os poucos estudos directos ambientais metabolômica de micróbios realizadas até à data têm sido limitados a cultura à base de ou facilmente definido de alta densidade, tais como os ecossistemas hospedeiro simbióticas-sistemas, construídos co-culturas ou manipulações do ambiente intestino, onde rotulagem isótopo estável pode ser adicionalmente utilizado para aumentar os sinais de RMN 7,8,9,10,11,12. Métodos que facilitam a concentração e recolha de metabolitos ambientais em concentrações adequadas para RMN faltam. Uma vez que a atenção recente tem sido dada aos metabolômica ambientais de organismos dentro do meio aquático, onde a maior parte do fluxo de energia e material é mediada pela comunidade planctônicas 13,14, desenvolvemos um método para a concentraçãoção e extração de todo-comunidade metabólitos de sistemas microbianos planctônicos por filtração. Hidrofílicos disponíveis comercialmente poli-1 ,1-difluoroethene (PVDF) os filtros são especialmente tratado para remover completamente os extraíveis, que podem de outro modo aparecem como contaminantes em análises subsequentes. Estes filtros tratados são então usados ​​para filtrar amostras ambientais ou experimental de interesse. Filtros contendo o material da amostra molhada são liofilizadas e aquosa solúveis em metabolitos são extraídos directamente para a espectroscopia de RMN convencional, utilizando um tampão de extracção padronizados de potássio fosfato 2. Os dados derivados desses métodos podem ser analisados ​​estatisticamente para identificar padrões significativos, ou integrados com outros níveis genómica para compreensão abrangente da comunidade e função do ecossistema.

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Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Um método para a extração de metabólitos de comunidades planctônicas microbianas é apresentado. Amostragem comunidade inteira é obtida por filtração em filtros especialmente preparados. Após liofilização, aquosa solúveis em metabolitos são extraídos. Esta abordagem permite a aplicação da metabolômica ambientais para trans-genómica investigações de comunidades microbianas naturais ou experimentais.

Concentração de metabólitos de comunidades de baixa densidade fitoplanctônicas para Metabolomics ambiental utilizando Espectroscopia de Ressonância Magnética Nuclear

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

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12.7K visualizações.  RIKEN Advanced Science Institute. Um método para a extração de metabólitos de comunidades planctônicas microbianas é apresentado. Amostragem comunidade inteira é obtida por filtração em filtros especialmente preparados. Após liofilização, aquosa solúveis em metabolitos são extraídos. Esta abordagem permite a aplicação da metabolômica ambientais para trans-genómica investigações de comunidades microbianas naturais ou experimentais.

Vídeo: Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Microbial Communities in Nature and Laboratory - Interview

Comunidades microbianas na Natureza e Laboratório - Entrevista

Biologia

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Na rotulagem in vitro de células estaminais embrionárias humanas para Ressonância Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Analisando Expressão Gênica de comunidades microbianas Marinha usando transcriptômica Ambiental

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Detecção de Óxido Nítrico e ânion radical superóxido por espectroscopia de ressonância paramagnética eletrônica a partir de células usando armadilhas para spins

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Quantificar mistura usando Ressonância Magnética

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificação de Transcrições e metabolitos de<em&gt; Drosophila</em&gt; Cabeças

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Remoção de materiais exógenos da parte exterior dos núcleos congelados para investigar a antiga comunidades biológicas abrigava Dentro

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Protocolo experimental para o uso de drosófila como um sistema modelo invertebrados para testes de toxicidade em laboratório

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Síntese de nanopartículas de núcleo dopado com óxido de ferro Ga 68para tomografia por emissão de pósitrons Dual/imagem latente de ressonância magnética de (T1)

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;