Este trabajo fue apoyado por becas de los Grandes Lagos DOE Centro de Investigación en Bioenergía (DOE BER Oficina de Ciencia-DE-FC02 07ER64494). Estamos muy agradecidos con el Dr. Zhang Xudong y sus miembros del grupo para las discusiones técnicas útiles y valiosos comentarios sobre la finalización del protocolo.

1. Preparación de la muestra y la extracción de ácido <ol><li> Liofilizar las muestras de suelo después de la recolección de campo. </li><li> Triturar y homogeneizar muestras de suelo utilizando un molino de bolas, molino de suelo, o un mortero y almirez. </li><li> Pesar muestras de suelo (que contiene&gt; 0,3 mg de N) en un matraz de 25 ml hidrólisis. </li><li> Añadir 10 ml de HCl 6 M en cada frasco de la hidrólisis, llene N 2 gas en los frascos, y la tapa. </li><li> Hidrolizar a 105 ° C en una incubadora durante 8 horas utilizando un interruptor de tiempo automático. </li></ol> 2. Purificación de la muestra <ol><li> Retirar los frascos de la incubadora y dejar enfriar a temperatura ambiente. </li><li> Añadir 100 ml de patrón interno de myo-inositol (1 mg mL -1 de agua) a cada matraz, mezclar por agitación. </li><li> Establecer embudos de plástico que desembocan en 200 ml en forma de pera frascos con ST / NS 24/40 articulaciones, establecidos en las tazas de plástico para la estabilidad. </li><li> Doble Whatman # 2 Círculos cualitativos (11 cm de diámetro) en cuartos y poner en embudos. </li&gt; <li> Agitar cada matraz hidrólisis, y verter purines en el embudo para filtrar (que además puede Enjuagar con ~ 3 ml de agua). </li><li> Secar el filtrado utilizando un evaporador rotatorio a ~ 45 ° C, aplicando vacío. </li><li> Volver a suspender el residuo seco de cada matraz pera con 3 ~ 5 ml de agua (uso de baño de ultrasonidos, si lo desea), y se vierte en un tubo de 40 ml de teflón, enjuagar el matraz con una segunda parte alícuota de agua. </li><li> Ajustar el pH a 6,6 a 6,8 con solución 1M de KOH para precipitar los iones metálicos y otras moléculas orgánicas. </li><li> Retirar los precipitados por centrifugación a 2000 rcf durante 10 minutos. </li><li> Verter el sobrenadante en un tubo de vidrio 40 ml, cubrir la abertura del tubo con parafilm, congelación a -20 ° C, a continuación, hacer agujeros en la parafina. </li><li> Liofilizar el sobrenadante congelado para eliminar todo el líquido. </li><li> Disolver el residuo con 3 ml de metanol seco, agitación a fondo (uso de baño de ultrasonidos, si lo desea), tapar el tubo, y centrifugar a 2000 fcr durante 10 minutos para resolverlas sales. </li><li> Transferir el sobrenadante a un vial de reacción 3 ml cónico; evaporar a sequedad por la máquina RapidVap a 45 ° C (o bajo una suave corriente de gas nitrógeno seco, si se desea). </li><li> Para cada vial, añadir 100 ml de recuperación estándar de N-metilglucamina (1 mg mL -1 en agua) y 1 ml de H 2 O, cubrir la boca del vial con Parafilm, congelar a -20 ° C, perforar la parafina, y luego congelar en seco . </li><li> Hacer normas: añadir 100 l de ácido murámico (0,5 mg mL -1 en metanol), 100 l de glucosamina (1 mg mL -1 de agua), 100 l de myo-inositol (1 mg mL -1 de agua), la N-100 l metilglucamina (1 mg mL -1 de agua) y 1 ml de H 2 O, parafina cada vial, congelar a -20 ° C, perforar la parafina, y luego congelar en seco. </li></ol> 3. Derivatización <ol><li> Preparar reactivo de derivatización que contiene 32 mg ml -1 hidrocloruro de hidroxilamina y 40 mg ml -1 4-dimetilamino-PYridine en piridina-metanol (4:1 v / v). </li><li> Añadir 300 l del reactivo de derivatización para cada uno de los reactivials, tapa herméticamente, y mezclar bien. </li><li> Ponga los frascos en un baño de agua 75-80 ° C durante 35 minutos (así se sellan los viales de evitar que el agua para entrar en los viales). </li><li> Saque los frascos del baño de agua, y deje enfriar a temperatura ambiente. </li><li> Añadir 1 ml de anhídrido acético a cada uno de los 3 ml reactivials, tapa herméticamente, y mezclar bien, a continuación, recalentar en 75-80 ° C baño de agua durante 25 minutos. </li><li> Saque los frascos del baño de agua, y deje enfriar a temperatura ambiente. </li></ol> 4. Separación y Medición <ol><li> Añadir 1,5 ml de diclorometano a cada frasco, tapa bien, y mezclar bien. </li><li> Añadir 1 ml de HCl 1 M a cada vial, tapa y mezclar bien para permitir que las soluciones para sentarse inalterado hasta que las dos frases separadas, aspirar y desechar la parte superior (acuosa) en fase inversa usando pipeta 1000 l. </li><li> En la misma fAshion, se extrae la fase orgánica 3 veces pero con 1 ml de H 2 O (con la última etapa de lavado, tener especial cuidado para asegurar que toda la fase acuosa superior se ha eliminado). </li><li> Se seca la solución final utilizando RapidVap a 45 ° C (o en seco con gas nitrógeno, si se desea). </li><li> Disolver en 300 l de acetato de etilo-hexano (1:1 v / v) y transferencia a 2 ml de color ámbar con tapón de rosca viales con inserto de un pequeño volumen y tapa. </li><li> Para la cuantificación, el análisis por GC-FID con una columna de sílice fundida capilar no polar: 30 m de largo, 0,25 mm de diámetro, espesor de la película 0,25 m; fase estacionaria 5% fenil-, el 95% de metil-polisiloxano (DB-5 o equivalente) con hidrógeno o helio como gas portador, en 0,5 ml min -1 flujo constante. La cromatografía es mejor en las primas &quot;inerte&quot; y las fases de gas portador de hidrógeno, pero es aceptable en las variedades más comunes y con helio. Los ajustes del detector son 300 ° C; 400 ml min -1 aire y 30 ml min -1 tanto para el nitrógeno y elgases de hidrógeno maquillaje. La inyección y los parámetros del horno son como sigue: 1 l de inyección de división (30:1) con la entrada de GC fijado a 250 ° C; temperatura del horno inicial, 120 ° C; mantener 1 min; aumentar la temperatura del horno a 10 ° C min. -1 a 250 ° C, tienen 2,5 minutos;. la rampa a 270 ° C a 20 ° C min -1; mantener 2 minutos; la rampa a 290 ° C a 20 ° C min -1, mantenga 5 min. Ajustar el caudal del gas portador de manera que el eluir inositol, glucosamina y derivados de ácido murámico a 250 ° C, y los N-metilglucamina eluye a 270 ° C. </li></ol> 5. Derivado de validación <ol><li> El uso de ionización suave LC-ESI-TOF-MS para identificar el ion molecular y determinar el peso molecular del derivado. </li><li> El uso de GC-EI-MS-SIM o GC-IE-MSMS para la mejora de la sensibilidad para investigar los iones específicos de la derivada. </li><li> El uso de múltiples reactivos marcados con isótopos para la preparación de los derivados, y luego usar el cambio de masade aquellos derivados marcadas isotópicamente en la EM para investigar ión molecular y los fragmentos de iones. </li></ol> 6. Los resultados representativos El protocolo del método comprende principalmente cuatro pasos: digestión ácida, la purificación de la muestra, y la determinación derivatización GC (Figura 1). Un ejemplo del análisis de glucosamina y ácido murámico de las existencias de estándar y de una muestra de suelo se muestra en la Figura 2. Además de la glucosamina y ácido murámico, manosamina y galactosamina (dos isómeros de glucosamina) también se puede determinar simultáneamente utilizando el método. Con base en los factores de respuesta de las normas con respecto a la norma interna de myo-inositol, podemos cuantificar estos biomarcadores en muestras de suelo. El estándar de recuperación ha sido utilizado para controlar cualitativamente el proceso de derivatización. Los esquemas para la formación del ácido acetato aldononitrile derivatizado glucosamina y murámico se muestra en la Figura 3 </strong&gt;. Las estructuras propuestas de los derivados se determinó por GC-EIMS-SIM para la mejora de la sensibilidad, o ionización suave LC-ESI-TOF-MS 8; las estructuras propuestas de los fragmentos de iones formados en la ionización de electrones se estudiaron mediante la comparación de los espectros de iones del Las muestras preparadas con incorporaciones de isótopos diferentes 11. La Figura 4 muestra el cambio de masa del ion dominante m / z 187 de acetato de aldononitrile derivatizado glucosamina en tres escenarios, preparados con agentes no-etiquetados, anhidrita D-acético, y U -13 C-glucosamina. Otra información detallada y explicaciones pueden ser remitidos a nuestro publicaciones recientes de 8, 11. Esta estrategia podría servir como modelo para estudiar la química derivada. <img alt="Figura 1" src="/files/ftp_upload/3767/3767fig1.jpg" /> Figura 1. Medición diagrama de flujo del protocolo para el análisis de la glucosamina y ácido murámico en el suelomuestras. El protocolo contiene principalmente 4 pasos: digestión ácida, Purificación, la derivación y la determinación de GC. <img alt="Figura 2" src="/files/ftp_upload/3767/3767fig2.jpg" /> Figura 2. GC-FID cromatogramas de acetatos aldononitrile de inositol, la glucosamina, ácido murámico, manosamina, galactosamina y metilglucamina de las normas y el suelo. <img alt="Figura 3" src="/files/ftp_upload/3767/3767fig3.jpg" /> Figura 3. Esquemas para la formación del ácido acetato aldononitrile derivatizado glucosamina y murámico. El número 1 representa la reacción de nitrilo. El número 2 representa acetilación. <a href="http://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg"><img alt="Figura 4" src="/files/ftp_upload/3767/3767fig4.jpg" /></a> Figura 4. Los espectros de masas de acetato aldononitrile derivatizado glucosamina asociada con la estruc ión dominantees bajo modo EI preparado con (A) los agentes no-etiquetados, (B) de D-acético anhidrita, (C) U -13 C-glucosamina. Estrella representa pesado átomo o grupo isótopo isótopo.

<ol>
	<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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No tenemos nada que revelar.

El GC presentó basado en el método para el análisis de ácido aldononitrile acetato derivado de glucosamina y murámico proporciona un método relativamente rápido para cuantificar los aminoácidos, los azúcares extraídos del suelo. Los derivados son químicamente estables, y se puede determinar en un análisis. El método no se limita a las muestras de suelo, y puede ser simplificado para muestras de matriz no-suelo. La bomba de vacío utilizado en este método está construido para s...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Ácido murámico </td><td> Sigma-Aldrich </td><td> M2503-25MG </td></tr><tr><td> D-(+)-glucosamina clorhidrato </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> Myo-inositol </td><td> Fisher Scientific </td><td> A307003G025 </td></tr><tr><td> El metanol (seco) </td><td> Acros orgánicos </td><td> AC326950010 </td></tr><tr><td> 4-dimetilamino-piridina </td><td> Acros orgánicos </td><td> AC148270050 </td></tr><tr><td> Acetato de etilo </td><td> VWR International </td><td> BJGC100-4 </td></tr><tr><td> Hydroxlamine clorhidrato de </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> El anhídrido acético </td> &lt;td&gt; Fisher Scientific </td><td> A10-100 </td></tr><tr><td> Diclorometano (cloruro 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> El ácido clorhídrico (6 M) </td><td> Fisher Scientific </td><td> S456-4 </td></tr><tr><td> Hidroxilamina clorhidrato de N -15 </td><td> Icono de los servicios </td><td> IN5280 </td></tr><tr><td> Anhídrido acético -2 H (D 6 C 4 O 3) </td><td> Acros orgánicos </td><td> AC174670050 </td></tr><tr><td> D-glucosa-U -13 C </td><td> Cambridge laboratorio de isótopos </td><td> CLM-1396-1 </td></tr><tr><td> Amonio sufate -15 N </td><td> Cambridge laboratorio de isótopos </td><td> NLM-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Nombre del equipo </td><td> Empresa </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> La hidrólisis frasco </td><td> Fisher Scientific </td><td> 06 423A </td></tr><tr><td> Derivatización microvial </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> Un sistema de la serie 1200 de Agilent HPLC acoplado a un 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

Los enfoques cuantitativos que caracterizan a los microorganismos son cruciales para una comprensión más amplia de la situación y función microbiana dentro de los ecosistemas. Las estrategias actuales para el análisis microbiológico de laboratorio incluyen tanto los tradicionales que dependen de la cultura y las técnicas basadas en la extracción directa y la determinación de ciertos biomarcadores 1 y 2. Pocos entre la diversidad de especies microbianas que habitan en el suelo pueden ser cultivadas, por lo que dependen de la cultura-los métodos de introducir sesgos significativos, una limitación ausente en el análisis de biomarcadores. El ácido de glucosamina, manosamina, galactosamina y murámico han sido bien servido como medidas tanto de los vivos y los muertos masa microbiana, de ellos el ácido glucosamina (más abundante) y murámico (únicamente a partir de células bacterianas) son los componentes más importantes en los sistemas de suelo 3 , 4. Sin embargo, la falta de conocimiento en el análisis restringe la divulgación amplia de los pares científicos. Entre todas las exipicadura de los métodos analíticos, la derivación de aldononitrile acetatos seguido por GC-basado en el análisis se ha convertido en una buena opción con respecto al óptimo equilibrio entre precisión, sensibilidad, sencillez, buena separación cromatográfica, y la estabilidad en el almacenamiento de la muestra 5. A continuación, presentamos un protocolo detallado para un análisis fiable y relativamente simple de la glucosamina y ácido murámico del suelo después de su conversión al acetatos aldononitrile. El protocolo comprende principalmente cuatro pasos: digestión ácida, la purificación de la muestra, la derivatización y la determinación de GC. El procedimiento paso a paso se modifica de acuerdo con el ex publicaciones 6, 7. Además, se presenta una estrategia para validar la estructura del ion molecular del derivado y sus fragmentos de iones formados en la ionización de electrones. Se aplicaron GC-EI-MS-SIM, LC-ESI-TOF-MS y reactivos marcados con isótopos para determinar el peso molecular de acetato derivado aldononitrile la glucosamina y el murácido amídico, se utilizó el cambio de masa de isótopos marcados derivados en el espectro de iones para investigar los fragmentos de iones derivados de cada 8. Además de la elucidación teórica, la validación de ión molecular del derivado y sus fragmentos de iones será útil para los investigadores que utilizan δ 13 C o fragmentos de iones de estos biomarcadores en estudios bioquímicos 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

Se describe un protocolo método para el análisis de GC-basada de los derivados aldonitrile acetato de glucosamina y ácido murámico extraída del suelo. Para esclarecer el mecanismo químico, también presentan una estrategia para confirmar la estructura del derivado y los fragmentos de iones formados en la ionización de electrones.

GC-basado en la detección de ácido Aldononitrile acetato derivado de glucosamina y murámico de la determinación de residuos microbiana en el suelo

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 vistas.  University of Wisconsin, Madison. Se describe un protocolo método para el análisis de GC-basada de los derivados aldonitrile acetato de glucosamina y ácido murámico extraída del suelo. Para esclarecer el mecanismo químico, también presentan una estrategia para confirmar la estructura del derivado y los fragmentos de iones formados en la ionización de electrones.

Video: 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 en la naturaleza y de laboratorio - Entrevista

Investigación

Biología

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.

La detección directa de la actividad de acetato de formación de la enzima cinasa de acetato

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.

El uso de quimiostatos en Microbiana Biología 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.

El aislamiento de los microorganismos del suelo nativo con potencial para romper biodegradables Mantillo películas de plástico utilizados en la 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.

Pyrosequencing para la identificación microbiana y caracterización

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.

Determinación de la oxidación de ácidos grasos y la lipogénesis en el ratón hepatocitos primarios

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.

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por 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.

Los métodos para la determinación de las tasas de glucosa y la oxidación de ácidos grasos en el corazón de rata aislado de Trabajo

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.

Un análisis de la competencia la placa como una evaluación preliminar rápida de supresión de la enfermedad

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.