Esta investigación fue financiada en parte por subvenciones-en-Ayudas a la Investigación Científica para impugnar la investigación exploratoria (JK), e Investigación Científica (A) (JK y SM) del Ministerio de Educación, Cultura, Deportes, Ciencia y Tecnología, Japón . Un RIKEN FPR comunión (ICE) prestó apoyo adicional. Los autores expresan su agradecimiento a los Dres. Eisuke Chikayama, Yasuyo Sekiyama y Okamoto Mami de asistencia técnica con RMN y análisis estadísticos.

1. Preparación del filtro para eliminar extraíbles <ol><li> Utilizar 25-mm de diámetro de 0,22 micras de tamaño de poro Durapore filtros hidrófilos PVDF (Millipore). Poner el filtro en un vaso de precipitados limpio de 500 ml Pyrex usando unas pinzas. Pre-enjuague tres veces con agua destilada. Agitar bien como enjuague para evitar que los filtros se peguen entre sí. Añadir 300 ml de Milli-Q (Millipore) o agua equivalente de alta calidad. Autoclave para facilitar la eliminación completa de extraíbles de los filtros. </li><li> Vierta el Milli-Q y otra triple lavado de los filtros, esta vez con Milli-Q. El uso de filtros pinzas lugar individuales en una superficie limpia y seca (tal como papel de aluminio) y, o bien seco a una temperatura razonable (por ejemplo 37 ° C) o aire seco. Los filtros están ahora listos para usar. </li></ol> 2. La filtración del material de muestra <ol><li> Para demostrar este protocolo, un acero inoxidable de 3-Millipore lugar colector del filtro con las columnas de microanálisis de filtro de 25 mm con soportes de vidrioy una bomba mecánica se utilizan. Utilizando una técnica aséptica, colocar un único 25-mm de filtro en la base de la columna filtro, aplicar la columna y la abrazadera juntas. </li><li> Coloque 15 ml de muestra a la columna, abra la válvula de cierre en el colector del filtro, y encienda la bomba. Filtrar bajo una suave presión para minimizar la rotura de células (&lt;5 kPa). Otras bombas, tales como la mano o una bomba peristáltica puede ser adaptado a este protocolo. Para muestras de menor densidad, adiciones sucesivas de agua puede ser necesario, no deje pasar el filtro seco durante un período prolongado de tiempo entre adiciones de agua. </li><li> Para las muestras marinas, después de haber filtrado la muestra, usted puede realizar un enjuague de agua dulce opción para reducir los iones paramagnéticos residuales en la muestra y en el filtro. Esto puede ayudar con una afinación más precisa del imán del espectrómetro. Simplemente agregue suavemente una pequeña cantidad de agua y filtrar a través de su muestra recogida al final. </li><li> Una vez filtrado se termine, apague la bomba, y salir de la opa de la válvulan lo que todavía hay una presión negativa en el filtro. Retire la abrazadera y la columna del filtro. </li><li> Con una mano, utilice pinzas limpias para apoderarse de el filtro. Doble el filtro en sí mismo, pero no se pliegue. Con la otra mano utilizar el borde de un tubo estéril de microcentrífuga de 2 ml para sujetar el filtro. Suelte las pinzas luego usarlas para volver a agarrar ambos lados del filtro. Regrip en un ángulo de 45 ° al redil. </li><li> Coloque el filtro en el tubo de 2 ml y la liberación por lo que se abre con la cara mirando hacia dentro de la muestra. Usted puede colocar un máximo de dos filtros en el tubo de microcentrífuga estéril 2 ml de esta manera. Si se usan dos filtros, asegurar que se solapan tan poco como sea posible. Congelar de inmediato (al menos a -30 ° C). </li></ol> 3. Extracción de acuoso solubles Metabolitos <ol><li> Liofilizar las muestras durante la noche o por lo menos 10 horas. </li><li> Después de la liofilización, añadir una trituradora de acero inoxidable a cada tubo (Tokken). Añadir 750 l de potasio estandarizadatampón de fosfato de RMN en óxido de deuterio (2 H&gt; 90%) con 2,2-Dimetil-2-silapentane-5-sulfonato (DSS) estándar (KPI; 38,3 mM de KH 2 PO 4, 61,7 mM K 2 HPO 4, 0,1 DSS mM, pH 7,0, 90% de D 2 O) 2. </li><li> Soníquelos las muestras durante 5 minutos a 4 ° C en un sonicador de agua (Bioruptor, Diagenode) para eliminar el material celular del filtro. Retire los filtros con pinzas limpias. </li><li> Romper las células utilizando un molino triturador (1600 rpm) durante 5 minutos. </li><li> Incubar a 65 ° C con agitación (1400 rpm) en un agitador de banco superior (Eppendorf) durante 15 minutos. </li><li> Retirar el cerdo de metal con pinzas limpias, y centrifugar la muestra a 13.000 g durante 5 minutos. </li><li> Extraer el sobrenadante directamente a un tubo de RMN para espectroscopía de RMN. </li></ol> 4. Espectroscopia de RMN y análisis de datos <ol><li> Cargue la muestra en un espectrómetro de RMN (en este caso, un Bruker DRX-500 equipado con espectrómetro deTXI sonda con tres ejes de gradiente controlado por un equipo que ejecuta XWIN-RMN). </li><li> Obtener 1D 1 H espectros de RMN mediante métodos adecuados previamente publicados 2,15 desde la interfaz XWIN-RMN. En el presente estudio 1 H espectros de RMN se registraron en un espectrómetro de funcionamiento DRX-500 a 500,03 MHz a 298 señales de agua K. residuales fueron suprimidas por la secuencia de pulsos de Watergate, con un tiempo de repetición de 1,2 s. 128 transitorios se recogieron para obtener 32.000 puntos de datos por espectro. </li><li> Transferencia de los directorios de datos de RMN a un PC instalado el software NMRPipe 16. Procesar los datos en bruto y DSS conjunto como 0 ppm referencia a continuación, eliminar manualmente los espectros. Digitalizar los datos espectrales en un conjunto de valores discretos mediante la integración o &quot;hurgar en la basura&quot; con un software como rNMR, Automics, o utilizando el paquete FT2B a disposición del público en el sitio web eComics ( <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> ) 3,17. En ªes un ejemplo, los espectros se integraron entre 0,5 y 10,5 ppm en 0.032 ppm regiones integrales utilizando eComics y normalizadas a cualquiera de DSS o la intensidad de la señal total. Los datos de salida ahora se puede utilizar para el análisis estadístico de aguas abajo, tales como análisis de componentes principales (ACP) con paquetes de software libre como el R 18. </li></ol> 5. Los resultados representativos Un ejemplo de 1 H espectros de RMN obtenidos utilizando los métodos anteriores se muestran en la Figura 1. Estas muestras, a partir de dos puntos de tiempo de un experimento de microcosmos muestran claras diferencias debido a las actividades metabólicas de algas. El día 4 espectro muestra considerable abundancia de picos, particularmente en el intervalo de 3-4 ppm en comparación con el día 1 muestra. Estos picos se puede atribuir a los azúcares producidos por la floración de diatomeas en el microcosmos. En un experimento similar comparando el crecimiento de las comunidades de plancton natural en agua de mar artificial o natural, aproximaciones estadísticas sUCH como trama principal de análisis de componentes puntuación (PCA) derivado de espectros de RMN binned se puede utilizar para mostrar claras diferencias metabólicas entre los dos tratamientos (Fig. 2), mientras que las parcelas de carga puede identificar los picos en el espectro de que la forma de la distribución de los datos . Estos resultados pueden compararse con los datos de los niveles ómicas otros, tales como métodos de huellas genómicas (Fig. 3). Estos picos de RMN se pueden consultar de forma individual (por ejemplo, en el BMRB; <a href="http://www.bmrb.wisc.edu/">http://www.bmrb.wisc.edu/</a> ) 19, o los espectros de todo pueden ser analizados estadísticamente (por ejemplo, con SpinAssign en <a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken. jp /? action = nmr_search</a> ) 2. En este ejemplo, las diferencias entre los tratamientos se debieron a una gran cantidad de picos en la región de los azúcares (3,39 ppm a 4,04 ppm) de los espectros de metabolitos naturales de la comunidad de plancton, y varios picos característicos de las comunidades de agua de mar fueron tentativamente identified como lactato y formiato utilizando SpinAssign. <img src="/files/ftp_upload/3163/3163fig1.jpg" alt="Figura 1" /> Figura 1. Representante 1 H espectros de RMN obtenidos de las muestras procesadas usando este procedimiento. Las muestras fueron tomadas antes de microcosmos (día 1) y durante (día 4) un intenso florecimiento de diatomeas. Experimentos de RMN se realizaron en un espectrómetro Bruker DRX-500 con las señales normalizadas a la altura del pico de patrón interno (DSS; 0 ppm). <img src="/files/ftp_upload/3163/3163fig2.jpg" alt="Figura 2" /> Figura 2. Análisis de Componentes Principales (PCA) parcela puntuación de los espectros de RMN de metabolomes desechado de forma natural derivados de las comunidades microbianas planctónicas cultivadas en microcosmos con natural (rombos abiertos) o agua de mar artificiales (círculos negros). Hay claras diferencias metabólicas se puede observar en el diagrama de dispersión. Un diagrama de carga de este tipo de análisis a continuación, se puede utilizar para identificar picos distintos de importancia enel sistema; estos picos pueden ser analizadas según sea necesario. <img src="/files/ftp_upload/3163/3163fig3.jpg" alt="Figura 3" /> Figura 3. Un ejemplo de multi-ómicas análisis RMN combinando con los datos genómicos. Composición de la comunidad basada en electroforesis en gel con gradiente desnaturalizante del 18S (izquierda) y 16S (derecha), los genes rRNA de las mismas muestras que se analizan en la figura 2 también muestra distintos patrones de las comunidades microbianas naturales entre (rombos abiertos) y microcosmos de agua de mar artificiales (círculos negros). Esta correspondencia entre el metaboloma y el genoma de los sistemas naturales demuestra la utilidad de este enfoque.

<ol>
	<li>Bundy, J. G., Davey, M. P., Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+metabolomics:+a+critical+review+and+future+perspectives.">Environmental metabolomics: a critical review and future perspectives.</a> Metabolomics. 5, 3-21 (2008).</li><li>Chikayama, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+indices+for+simultaneous+large-scale+metabolite+detections+for+a+single+NMR+spectrum.">Statistical indices for simultaneous large-scale metabolite detections for a single NMR spectrum.</a> Anal. Chem. 82, 1653-1658 (2010).</li><li>Lewis, I. A., Schommer, S. C., Markley, 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=rNMR:+open+source+software+for+identifying+and+quantifying+metabolites+in+NMR+spectra.">rNMR: open source software for identifying and quantifying metabolites in NMR spectra.</a> Magn. Reson. Chem. 47, S123-S126 (2009).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiotic+gut+microbes+modulate+human+metabolic+phenotypes.">Symbiotic gut microbes modulate human metabolic phenotypes.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2117-2122 (2008).</li><li>Mochida, K., Furuta, T., Ebana, K., Shinozaki, K., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+exploration+of+metabolic+and+genomic+diversity+in+rice.">Correlation exploration of metabolic and genomic diversity in rice.</a> BMC Genomics. 10, 568 (2009).</li><li>Fukuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifidobacteria+can+protect+from+enteropathogenic+infection+through+production+of+acetate.">Bifidobacteria can protect from enteropathogenic infection through production of acetate.</a> Nature. 469, 543-547 (2011).</li><li>Kikuchi, J., Hirayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+stable+isotope+labeling+of+higher+plants+for+a+hetero-nuclear+multi-dimensional+NMR-based+metabolomics.">Practical aspects of stable isotope labeling of higher plants for a hetero-nuclear multi-dimensional NMR-based metabolomics.</a> Methods Mol. Biol. 358, 273-286 (2007).</li><li>Martin, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+top-down+systems+biology+view+of+microbiome-mammalian+metabolic+interactions+in+a+mouse+model.">A top-down systems biology view of microbiome-mammalian metabolic interactions in a mouse model.</a> Mol. Syst. Biol. 3, 112 (2007).</li><li>Mahrous, E. A., Lee, R. B., Lee, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+approach+to+lipid+profiling+of+mycobacteria+using+2D+HSQC+NMR+maps.">A rapid approach to lipid profiling of mycobacteria using 2D HSQC NMR maps.</a> J. Lipid Res. 49, 455-463 (2008).</li><li>Fukuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+characterization+of+bacterial+metabolic+dynamics+with+a+novel+profiling+technique,+real-time+metabolotyping.">Evaluation and characterization of bacterial metabolic dynamics with a novel profiling technique, real-time metabolotyping.</a> PloS ONE. 4, e4893 (2009).</li><li>Date, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+monitoring+approach+for+metabolic+dynamics+in+microbial+ecosystems+using+stable-isotope-labeling+technologies.">New monitoring approach for metabolic dynamics in microbial ecosystems using stable-isotope-labeling technologies.</a> J. Biosci. Bioeng. 110, 87-93 (2010).</li><li>Nakanishi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+omics+approach+identifies+nutrition-mediated+microbial+interactions.">Dynamic omics approach identifies nutrition-mediated microbial interactions.</a> J. Proteome Res. 10, 824-836 (2011).</li><li>Falkowski, P., Barber, R., Smetacek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogeochemical+controls+and+feedbacks+on+ocean+primary+production.">Biogeochemical controls and feedbacks on ocean primary production.</a> Science. 281, 200-207 (1998).</li><li>Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+of+aquatic+organisms:+the+new+'omics'+on+the+block.">Metabolomics of aquatic organisms: the new 'omics' on the block.</a> Mar. Ecol. Prog. Ser. 332, 301-306 (2007).</li><li>Sekiyama, Y., Chikayama, E., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+semipolar+solvent+system+as+a+step+toward+heteronuclear+multidimensional+NMR-based+metabolomics+for+13C-labeled+bacteria,+plants,+and+animals.">Evaluation of a semipolar solvent system as a step toward heteronuclear multidimensional NMR-based metabolomics for 13C-labeled bacteria, plants, and animals.</a> Anal. Chem. 83, 719-726 (2011).</li><li>Delaglio, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMRPipe:+A+multidimensional+spectral+processing+system+based+on+UNIX+pipes.">NMRPipe: A multidimensional spectral processing system based on UNIX pipes.</a> J. Biomol. NMR. 6, 277-293 (1995).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automics:+an+integrated+platform+for+NMR-based+metabonomics+spectral+processing+and+data+analysis.">Automics: an integrated platform for NMR-based metabonomics spectral processing and data analysis.</a> BMC Bioinformatics. 10, 83 (2009).</li><li>Eldon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioMagResBank.">BioMagResBank.</a> Nucleic Acids Res. 36, D402-D408 (2007).</li><li>Sekiyama, Y., Chikayama, E., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+polar+and+semipolar+plant+metabolites+throughout+extraction+processes+using+a+combined+solution-state+and+high-resolution+magic+angle+spinning+NMR+approach.">Profiling polar and semipolar plant metabolites throughout extraction processes using a combined solution-state and high-resolution magic angle spinning NMR approach.</a> Anal. Chem. 82, 1643-1652 (2011).</li></ol>

No hay conflictos de interés declarado.

El método de extracción de filtración y el metabolito ha demostrado aquí permite la biomasa planctónica microbiana que debe recogerse en cantidad suficiente para la metabolómica RMN. Mientras que la extracción acuosa de sólo solubles en metabolitos utilizando KPI y 1D 1 H RMN se demuestra, otros disolventes de extracción y enfoques espectroscópicos se puede utilizar. Un ejemplo útil es el uso de metanol deuterado como disolvente semipolar, que se ha demostrado para producir espectros de RMN superio...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 0,22 hidrófilas Durapore PVDF filtros de 25 mm </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Titular de Microanálisis de filtro de 25 mm, soporte de vidrio poroso </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3-el lugar del colector, de 47 mm, de acero inoxidable </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 deuterio, 2 H&gt; 90% </td><td> Campridge isótopos 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> Trituradoras de acero inoxidable incluidos </td></tr><tr><td> Thermomixer comodidad </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 al vacío </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> RMN </td><td> Bruker </td><td> DRX-500 con 5 mm de la sonda TXI </td><td></td></tr><tr><td> Herramienta de categorización espectral </td><td> Originalmente desarrollado </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> Herramienta de anotación de metabolitos y base de datos </td><td> Originalmente desarrollado </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

Ambiental metabolómica es un campo emergente que está promoviendo una nueva comprensión de cómo responden los organismos e interactuar con el medio ambiente y con los demás a nivel bioquímico 1. Resonancia magnética nuclear (RMN) es una de varias tecnologías, incluyendo la cromatografía de gases-espectrometría de masas (GC-MS), con una promesa considerable para este tipo de estudios. Ventajas de la RMN son que es adecuado para los análisis focalizados, proporciona información estructural y los espectros se pueden consultar en las costumbres cuantitativos y estadísticos en bases de datos recientemente disponibles de espectros individuales metabolito 2,3. Además, los datos espectrales de RMN se pueden combinar con los datos de los niveles de otras ómicas (genómica transcriptómica por ejemplo,) para proporcionar una comprensión más completa de las respuestas fisiológicas de los taxones entre sí y el medio ambiente 4,5,6. Sin embargo, RMN es menos sensible que otras técnicas de metabolómicos, lo que hace difícil APcapas de los sistemas microbianos naturales donde la población de la muestra pueden ser las concentraciones de baja densidad y el metabolito de bajo en comparación con los metabolitos de las bien definidas y fácilmente extraíbles, tales como fuentes de tejidos enteros, biofluidos o cultivos celulares. En consecuencia, los pocos estudios ambientales directos metabolómicos de los microbios realizados hasta la fecha se han limitado a base de la cultura-o puede ser definido fácilmente de alta densidad de los ecosistemas tales como los sistemas de acogida-simbionte, construidos co-culturas o manipulaciones del entorno intestinal en el etiquetado de isótopos estables puede ser utiliza también para mejorar las señales de RMN 7,8,9,10,11,12. Los métodos que facilitan la concentración y la recogida de los metabolitos del medio ambiente en concentraciones adecuadas para RMN se carece. Dado que la atención reciente se ha dado a la metabolómica medio ambiente de organismos en el medio acuático, donde la mayor parte del flujo de la energía y el material está mediada por la comunidad planctónica 13,14, hemos desarrollado un método para la concentraciónción y la extracción de toda la comunidad-metabolitos de microbios planctónicos sistemas de filtración. Comercialmente disponibles hidrófilos de poli-1 ,1-difluoroeteno (PVDF) filtros son especialmente tratados para eliminar completamente extraíbles, que de lo contrario pueden aparecer como contaminantes en los análisis posteriores. Estos filtros tratados se utilizan para filtrar las muestras ambientales o experimentales de interés. Filtros que contienen el material de la muestra húmeda se liofilizan y acuosa solubles metabolitos se extrae directamente por espectroscopía de RMN convencional utilizando un fosfato de potasio estandarizado tampón de extracción 2. Los datos derivados de estos métodos pueden ser analizados estadísticamente para identificar patrones significativos, o integrado con otros niveles de ómicas para la comprensión integral de la comunidad y la función del ecosistema.

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

Un método para la extracción de metabolitos microbianos de las comunidades planctónicas se presenta. Muestreo de toda la comunidad se logra por filtración en filtros especialmente preparados. Después de la liofilización, acuoso-solubles metabolitos se extraen. Este enfoque permite la aplicación de la metabolómica ambientales trans-ómicas las investigaciones de las comunidades microbianas naturales o experimentales.

La concentración de los metabolitos de las comunidades planctónicas de baja densidad para la metabolómica ambiental utilizando espectroscopia de Resonancia 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 vistas.  RIKEN Advanced Science Institute. Un método para la extracción de metabolitos microbianos de las comunidades planctónicas se presenta. Muestreo de toda la comunidad se logra por filtración en filtros especialmente preparados. Después de la liofilización, acuoso-solubles metabolitos se extraen. Este enfoque permite la aplicación de la metabolómica ambientales trans-ómicas las investigaciones de las comunidades microbianas naturales o experimentales.

Video: 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 en la naturaleza y de laboratorio - Entrevista

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Biología

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.

En el etiquetado in vitro de células estaminales embrionarias humanas de Resonancia 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.

El análisis de la expresión génica de las comunidades microbianas marinas con Transcriptómica 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.

La detección de óxido nítrico y el radical anión superóxido por espectroscopía de resonancia paramagnética de electrones de las células utilizando atrapadores

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.

Cuantificación de mezcla utilizando imágenes por resonancia 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.

Purificación de las transcripciones y los metabolitos de<em&gt; Drosophila</em&gt; Cabezales

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.

La eliminación de materiales exógenos de la parte exterior de congelados Núcleos para Investigar la antigua comunidades biológicas albergaba el interior

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 el uso de Drosophila como sistema modelo de invertebrado para ensayos de toxicidad en el laboratorio

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.

Síntesis de 68Ga dopado a base de óxido de hierro nanopartículas de doble emisión de positrones/proyección de imagen de resonancia magnética (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;