Este trabalho foi suportado por concessões do DOE Grande Lakes Bioenergy Research Center (DOE BER escritório da Ciência DE-FC02-07ER64494). Somos gratos ao Dr. Xudong Zhang e seus membros de grupo para discussões técnicas úteis e valiosos comentários sobre a finalização do protocolo.

1. Preparação de amostras e extração de ácidos <ol><li> Liofilizar amostras de solo após a coleta de campo. </li><li> Moer e homogeneizar amostras de solo, utilizando um moinho de bolas, moinho de solo, ou um almofariz e um pilão. </li><li> Pesar amostras de solo (contendo&gt; 0,3 mg de N) para um balão de hidrólise de 25 mL. </li><li> Adicionar 10 mL de HCl 6M em cada frasco de hidrólise, preencher N gás 2 nos frascos, e tampa. </li><li> Hidrolisar a 105 ° C numa incubadora durante 8 horas, utilizando um interruptor temporizador automático. </li></ol> 2. Purificação da amostra <ol><li> Remover os frascos da incubadora; arrefecer à temperatura ambiente. </li><li> Adicionar 100 uL padrão interno de mio-inositol (1 mg mL -1 em água) a cada frasco e misturar por agitação. </li><li> Configure funis de plástico de drenagem em 200 ml de pêra com balões em forma de ST / NS 24/40 articulações, definidas em copos de plástico para a estabilidade. </li><li> Fold Whatman # 2 Círculos qualitativos (11 cm de diâmetro) em quartos e em conjunto funis. </li&gt; <li> Redemoinho de cada frasco de hidrólise, e despeje dejetos em funil para filtrar (você pode ainda Lavar com ~ 3 mL de água). </li><li> Seca-se o filtrado utilizando um evaporador rotativo a ~ 45 ° C sob vácuo, aplicando. </li><li> Ressuspender o resíduo seco de cada frasco de pêra com 3 ~ 5 mL de água (utilizar banho ultra-sónico, se desejado), e verter em um tubo de Teflon 40 mL; enxaguar o balão com uma segunda alíquota de água. </li><li> Ajustar o pH a 6,6-6,8 usando solução 1M de KOH a iões de metal precipitado e outras moléculas orgânicas. </li><li> Remover os precipitados por centrifugação a 2000 rcf durante 10 minutos. </li><li> Verter o sobrenadante para um tubo de vidro 40 mL, cobrir a abertura do tubo com parafilme, congelamento a -20 ° C, em seguida, criar buracos na parafilme. </li><li> Liofilizar o sobrenadante congelado para remover todo o líquido. </li><li> Dissolve-se o resíduo com 3 mL de metanol seco, vortex completamente (utilizar banho ultra-sónico, se desejado); tampa do tubo, e, em seguida, centrifugar a 2000 rcf durante 10 minutos para resolveros sais. </li><li> Transferir o sobrenadante para um frasco de reacção 3 mL cónico; evaporar até à secura por máquina RapidVap a 45 ° C (ou sob uma corrente suave de azoto gasoso seco, se desejado). </li><li> Para cada frasco, adicionar 100 uL de recuperação padrão N-metilglucamina (1 mg mL -1 em água) e 1 mL de H2O, cobrir as bocas dos frascos com parafilme, congeladas a -20 ° C, perfurar a parafilme e, em seguida seca congelar . </li><li> Fazer padrões: adicionar 100 uL de ácido murâmico (0,5 mg mL -1 em metanol), 100 uL de glucosamina (1 mg mL -1 em água), 100 uL de mio-inositol (1 mg mL -1 em água), 100 N-uL metilglucamina (1 mg mL -1 em água), e 1 mL de H2O, parafilme cada frasco, congelamento a -20 ° C, perfurar a parafilme, em seguida, liofilizar. </li></ol> 3. Derivatização <ol><li> Preparar o reagente de derivatização contendo 32 mg mL -1 cloridrato de hidroxilamina e 40 mg mL -1 4-dimetilamino-pyridine em piridina-metanol (4:1 v / v). </li><li> Adicionar 300 uL do reagente de derivatização para cada um dos reactivials, tampa firmemente, e vórtice completamente. </li><li> Coloque os frascos em banho-maria 75-80 ° C por 35 minutos (bem selados os frascos para evitar que a água entrar nos frascos). </li><li> Remover os frascos a partir do banho de água, e arrefecer à temperatura ambiente. </li><li> Adicionar 1 ml de anidrido acético a cada um dos reactivials de 3 mL tampão, firmemente, e vórtice completamente, em seguida aquecer em 75-80 ° C banho de água durante 25 minutos. </li><li> Remover os frascos a partir do banho de água, e arrefecer à temperatura ambiente. </li></ol> 4. Separação e Mensuração <ol><li> Adicione 1,5 mL de diclorometano a cada frasco tampa, com força, e vortex completamente. </li><li> Adicionar 1 mL de HCl 1M para cada frasco de tampa, com firmeza e vortex cuidadosamente para permitir que as soluções para sentar-se imperturbável até as duas frases em separado, aspirar e descartar a fase (aquosa) superior usando pipetador 1000 uL. </li><li> Na mesma fashion, extrair a fase orgânica 3 vezes, mas com 1 mL de H2O (com o passo de lavagem último, tomar cuidado especial para assegurar que toda a fase aquosa superior foi removida). </li><li> Seca-se a solução final utilizando RapidVap a 45 ° C (ou seca utilizando azoto gasoso, se desejado). </li><li> Dissolve-se em 300 uL de etilo acetato de etilo-hexano (1:1 v / v) e transferência para 2 mL de âmbar com tampa de rosca frascos com um volume pequeno-inserção e tampa. </li><li> Para a quantificação, analisar por GC-FID utilizando uma coluna capilar de sílica fundida não polar: 30 m de comprimento, 0,25 mm id, 0,25 espessura de filme UM; fase estacionária 5% fenil-, 95% metil-polissiloxano (DB-5 ou equivalente) com hidrogénio ou hélio como gás transportador, em 0,5 mL de fluxo -1 min constante. A cromatografia é melhor no prêmio &quot;inerte&quot; fases e gás transportador de hidrogênio, mas é aceitável para as variedades mais comuns e com hélio. As configurações são detectores 300 ° C, 400 mL min -1 de ar e 30 mL min -1, tanto para o nitrogênio emaquiagem gases hidrogênio. A injecção e os parâmetros do forno são como se segue: 1 injecção de divisão uL (30:1) com a entrada de GC fixado em 250 ° C; temperatura do forno inicial, de 120 ° C; segurar 1 min; aumentar a temperatura do forno a 10 ° C min. -1 a 250 ° C; segurar 2,5 min;. rampa até 270 ° C a 20 ° C min @ 1; segurar 2 minutos; rampa para 290 ° C a 20 ° C min -1, mantenha 5 min. Ajustar o transportador taxa de fluxo de gás de modo que a eluir inositol, glucosamina e derivados de ácido Murâmicos a 250 ° C, e os elui N-metilglucamina com 270 ° C. </li></ol> 5. Validação Derivada <ol><li> Use ionização mole LC-ESI-TOF-MS para identificar o ião molecular e determinar o peso molecular do derivado. </li><li> Use GC-EI-MS-SIM ou GC-EI-MSMS para aumento de sensibilidade para investigar os iões específicos do derivado. </li><li> Use vários reagentes isotopicamente marcados para a preparação dos derivados, e depois usar o deslocamento em massados derivados isotopicamente marcados em MS para investigar ião molecular e fragmentos de iões. </li></ol> 6. Os resultados representativos O protocolo do método compreende principalmente quatro etapas: digestão ácida, a purificação da amostra, a derivatização e GC determinação (Figura 1). Um exemplo da análise para a glucosamina e ácido murâmico das existências de padrão e de uma amostra de solo é mostrado na Figura 2. Além glucosamina e ácido murâmico, manosamina e galactosamina (dois isómeros de glucosamina) também pode ser determinada utilizando o método simultâneo. Com base em factores de resposta de padrões com relação ao padrão interno de mio-inositol, podemos quantificar estes biomarcadores em amostras de solo. O padrão de recuperação tem sido utilizada para monitorizar o processo qualitativamente derivatização. Os esquemas para a formação do ácido aldononitrile glucosamina e murâmico acetato derivatizado são mostrados na Figura 3 </strong&gt;. As estruturas propostas dos derivados foi determinada por GC-EIMS-SIM para aumento de sensibilidade, ou da ionização mole LC-ESI-TOF-MS 8; as estruturas propostas dos fragmentos de iões formados mediante ionização de electrões foram estudados por comparação dos espectros de iões do amostras preparadas com incorporações de isótopos diferentes 11. A Figura 4 mostra a mudança de massa do ião dominante m / z 187, de aldononitrile acetato de glucosamina derivatizado em três cenários, preparados com agentes não-etiquetados, D-acético anidrite, e -13 U C-glucosamina. Outras informações detalhadas e explicações podem ser encaminhados para nossas publicações mais recentes 8, 11. Esta estratégia poderia servir de modelo para estudar química derivada. <img alt="A Figura 1" src="/files/ftp_upload/3767/3767fig1.jpg" /> Figura 1. Fluxograma Medição do protocolo para a análise de glucosamina e ácido murâmico no soloAs amostras. O protocolo contém principalmente 4 etapas: digestão ácida, purificação, derivatização e determinação GC. <img alt="A Figura 2" src="/files/ftp_upload/3767/3767fig2.jpg" /> Figura 2. GC-FID cromatogramas de acetatos aldononitrile de inositol, glucosamina, ácido murâmico, manosamina, galactosamina e metilglucamina para normas e do solo. <img alt="A Figura 3" src="/files/ftp_upload/3767/3767fig3.jpg" /> Figura 3. Esquemas para a formação do ácido aldononitrile glucosamina e murâmico acetato derivatizado. O número 1 representa reacção de nitrilo. O número 2 representa acetilação. <a href="http://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg"><img alt="A Figura 4" src="/files/ftp_upload/3767/3767fig4.jpg" /></a> Figura 4. Os espectros de massa de acetato de glucosamina aldononitrile derivatizado associado com a estruturação ião dominantees sob modo EI preparado com (A) os agentes não-etiquetados, (B) D-acético anidrita, (C) U -13 C-glucosamina. Estrela representa pesado isótopo átomo ou grupo de isótopos.

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	<li>Zelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+patterns+of+phospholipids+and+lipopolysaccharides+in+the+characterisation+of+microbial+communities+in+soil:+a+review.">Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review.</a> Biology and Fertility of Soils. 29, 111-129 (1999).</li><li>Kirk, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+studying+soil+microbial+diversity.">Methods of studying soil microbial diversity.</a> Journal of Microbiological Methods. 58, 169-188 (2004).</li><li>Joergensen, R. 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O método GC apresentou baseado para a análise de aldononitrile ácido murâmico glucosamina e acetato de derivatizado proporciona um método relativamente rápido para quantificar estes açúcares aminados, extraídos do solo. Os derivados são quimicamente estáveis, e pode ser determinada em uma análise. O método não é restrito às amostras de solo, e pode ser simplificada para amostras de solo não-matriz. A bomba de vácuo usado neste método é construído para ser resistente a á...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Murâmico </td><td> Sigma-Aldrich </td><td> M2503-25mg </td></tr><tr><td> D-cloridrato de (+)-glucosamina </td><td> Sigma-Aldrich </td><td> G1514-100G </td></tr><tr><td> N-metil-D-glucamina </td><td> Sigma-Aldrich </td><td> M2004-500G </td></tr><tr><td> Mio-inositol </td><td> Fisher Scientific </td><td> A307003G025 </td></tr><tr><td> Metanol (seco) </td><td> Acros Organics </td><td> AC326950010 </td></tr><tr><td> 4-dimetilamino-piridina </td><td> Acros Organics </td><td> AC148270050 </td></tr><tr><td> Acetato de etilo </td><td> VWR International </td><td> BJGC100-4 </td></tr><tr><td> Cloridrato Hydroxlamine </td><td> Fisher Scientific </td><td> H330-100 </td></tr><tr><td> Piridina </td><td> Fisher Scientific </td><td> P368-500 </td></tr><tr><td> Anidrido acético </td> &lt;td&gt; Fisher Scientific </td><td> A10-100 </td></tr><tr><td> Diclorometano (cloreto de metileno) </td><td> Fisher Scientific </td><td> D37-500 </td></tr><tr><td> Hexano </td><td> Fisher Scientific </td><td> H303-4 </td></tr><tr><td> Ácido clorídrico (6M) </td><td> Fisher Scientific </td><td> S456-4 </td></tr><tr><td> Cloridrato de hidroxilamina -15 N </td><td> Ícone serviços </td><td> IN5280 </td></tr><tr><td> Anidrido acético -2 H (D 6 C 4 O 3) </td><td> Acros Organics </td><td> AC174670050 </td></tr><tr><td> D-glucose-U -13 C </td><td> Cambridge isótopo laboratório </td><td> CLM-1396-1 </td></tr><tr><td> Ammonium sufate -15 N </td><td> Cambridge isótopo laboratório </td><td> NLM-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Nome do equipamento </td><td> Companhia </td><td> Tipo </td></tr><tr><td> Rapid-Vap </td&gt; <td> Labconco </td><td> 790002 </td></tr><tr><td> Vacum bomba </td><td> KNF Neuberger </td><td> D-79112 </td></tr><tr><td> Frasco de hidrólise </td><td> Fisher Scientific </td><td> 06 423A </td></tr><tr><td> Microtubo derivatização </td><td> Fisher Scientific </td><td> 06-100E </td></tr><tr><td> GC </td><td> Hewlett-Packard </td><td> 6890 </td></tr><tr><td> MS </td><td> Hewlett-Packard </td><td> 5972 </td></tr><tr><td> LC-ESI-TOF-MS </td><td> Agilent </td><td> Um sistema Agilent 1200 series HPLC acoplado a um LC Agilent / MSD-TOF </td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

Abordagens quantitativas dos microrganismos que caracterizam são cruciais para uma compreensão mais ampla do estado microbiana e função dentro dos ecossistemas. As estratégias actuais para análise microbiana incluem ambos os tradicionais de laboratório de cultura dependentes de técnicas e aqueles com base em extracção directa e determinação de biomarcadores determinados 1, 2. Poucos entre a diversidade de espécies microbianas que habitam o solo podem ser cultivadas, de modo a cultura dependentes de métodos de introduzir enviesamentos significativos, uma limitação ausente na análise de biomarcadores. O ácido glucosamina, manosamina, galactosamina e murâmico têm sido bem como medidas de vivos e mortos massa microbiana, desses ácidos a glucosamina (mais abundante) e murâmico (exclusivamente a partir de células bacterianas) são componentes mais importantes nos sistemas de solo 3 , 4. No entanto, a falta de conhecimento sobre a análise restringe a ampla divulgação entre os pares científicos. Entre todas as existing métodos analíticos, derivatização para aldononitrile acetatos seguido por GC-análise baseada surgiu como uma boa opção em relação à melhor forma equilibrar precisão, sensibilidade, simplicidade, boa separação cromatográfica, e estabilidade durante o armazenamento da amostra 5. Aqui, apresentamos um protocolo detalhado para uma análise confiável e relativamente simples de glucosamina e ácido murâmico do solo após a sua conversão para acetatos aldononitrile. O protocolo é constituída principalmente por quatro etapas: digestão ácida, a purificação da amostra, a derivatização e determinação GC. O procedimento passo-a-passo é modificada de acordo com as publicações ex 6, 7. Além disso, apresentam-se uma estratégia para estruturalmente validar o ião molecular do derivado e fragmentos seus iões formada a partir de ionização de electrões. Foram aplicados GC-EI-MS-SIM, LC-ESI-TOF-MS e os reagentes isotopicamente rotulados para determinar o peso molecular de aldononitrile acetato de glucosamina derivatizado e murácido âmico; utilizou-se o deslocamento em massa de marcada com isótopos derivados no espectro de iões para investigar fragmentos de iões de cada derivados 8. Além disso para a elucidação teórica, a validação do ião molecular do derivado e fragmentos seus iões será útil para os investigadores que utilizam δ 13 C ou fragmentos de iões destes biomarcadores em estudos biogeoquímicos 9, 10.

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Nós descrevemos um método para o protocolo de análise de GC-Based dos derivados aldonitrile acetato de glucosamina e ácido murâmico extraído do solo. Para elucidação do mecanismo química, também apresentar uma estratégia para confirmar a estrutura do derivado e os fragmentos de iões formados mediante ionização de electrões.

GC baseado em detecção de ácido Aldononitrile Glucosamina e Murâmicos Acetato derivatizados à determinação do resíduo microbiana em solo

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

JoVE Journal

Biology

16.8K visualizações.  University of Wisconsin, Madison. Nós descrevemos um método para o protocolo de análise de GC-Based dos derivados aldonitrile acetato de glucosamina e ácido murâmico extraído do solo. Para elucidação do mecanismo química, também apresentar uma estratégia para confirmar a estrutura do derivado e os fragmentos de iões formados mediante ionização de electrões.

Vídeo: GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Microbial Communities in Nature and Laboratory - Interview

Comunidades microbianas na Natureza e Laboratório - Entrevista

Biologia

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Detecção direta da Atividade Acetato de formação do Acetato Quinase Enzima

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

The Use of Chemostats in Microbial Systems Biology

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

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

O Uso de quimiostatos em Microbial Biologia de Sistemas

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

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Isolamento de microrganismos do solo nativas com potencial para romper as biodegradáveis ​​Palha filmes plásticos usados ​​na agricultura

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pirosequenciamento de Identificação Microbiana e Caracterização

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Determinação da oxidação de ácidos gordos e Lipogênese em Rato Os hepatócitos primária

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Os métodos para a determinação das taxas de glicose e ácido gordo oxidação no coração isolado de rato de Trabalho

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

Um ensaio de competição de placa como uma rápida avaliação preliminar de supressão da doença

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.