Questa ricerca è stata sostenuta in parte dal Grants-in-Aid per la ricerca scientifica per contestare la ricerca esplorativa (JK), e della Ricerca Scientifica (A) (JK e SM) dal Ministero della Pubblica Istruzione, Cultura, Sport, Scienza e Tecnologia, Giappone . Una borsa di studio RIKEN FPR (RCE) offerto un sostegno supplementare. Gli autori esprimono la loro gratitudine a Drs. Eisuke Chikayama, Yasuyo Sekiyama e Mami Okamoto per l&#39;assistenza tecnica con la NMR e analisi statistiche.

1. Preparazione filtro per rimuovere estraibili <ol><li> Utilizzare 25 mm di diametro 0,22 micron pori di dimensioni Durapore filtri PVDF idrofilo (Millipore). Mettere filtri in un bicchiere pulito da 500 ml Pyrex con una pinzetta. Pre-risciacquo tre volte con acqua distillata. Agitare così come si risciacquo per evitare che i filtri si attacchino tra loro. Aggiungere 300 ml Milli-Q (Millipore) o equivalente di alta qualità dell&#39;acqua. Autoclavare per facilitare la rimozione completa estraibili dai filtri. </li><li> Eliminare il Milli-Q e di nuovo triple lavare i filtri, questa volta con Milli-Q. Utilizzo dei filtri singoli pinzette posto su una superficie asciutta e pulita (come il foglio di alluminio) e sia asciutto ad una temperatura ragionevole (per esempio 37 ° C) o aria secca. I filtri sono ora pronti per l&#39;uso. </li></ol> 2. Filtrazione di materiale campione <ol><li> Per dimostrare questo protocollo, Millipore in acciaio inox a 3 posti collettore filtro con 25 mm di microanalisi colonne filtro con supporti in vetroe una pompa meccanica vengono utilizzati. Usando una tecnica asettica, inserire un singolo da 25 mm filtro sulla base della colonna filtro, applicare la colonna e fissarlo insieme. </li><li> Caricare 15 ml di campione alla colonna, aprire l&#39;otturatore sul collettore del filtro, e accendere la pompa. Filtrare sotto una leggera pressione per minimizzare la rottura delle cellule (&lt;5 kPa). Altre pompe, come la mano o una pompa peristaltica può essere adattato a questo protocollo. Per i campioni a bassa densità, successive aggiunte di acqua può essere necessaria, non lasciare andare il filtro a secco per un periodo prolungato di tempo tra le aggiunte di acqua. </li><li> Per i campioni marini, dopo aver filtrato il campione, è possibile eseguire un risciacquo acqua dolce opzionale per ridurre gli ioni paramagnetici residui nel campione e sul filtro. Questo può aiutare con più precisa messa a punto del magnete dello spettrometro. Basta aggiungere delicatamente una piccola quantità di acqua e filtrare attraverso il vostro campione prelevato alla fine. </li><li> Una volta che il filtraggio è finito, spegnere la pompa, e lasciare la valvola di Open quindi c&#39;è ancora pressione negativa sotto il filtro. Rimuovere il morsetto e la colonna filtro. </li><li> Con una mano, utilizzare pinzette pulite a prendere piede del filtro. Piegare il filtro tra sé, ma non piega. Con l&#39;altra mano usare il labbro di un sterile da 2 ml provetta a tenere premuto il filtro. Rilasciare le pinzette poi usarli per ri-afferrare entrambi i bordi del filtro. Regrip a 45 ° angolo di piega. </li><li> Posizionare il filtro nel tubo da 2 ml e il rilascio in modo che si apre con il lato rivolto verso l&#39;interno del campione. È possibile inserire fino a due filtri nel tubo da 2 ml sterile microcentrifuga in questo modo. Se si utilizzano due filtri, si sovrappongono garantire il meno possibile. Immediatamente congelare (almeno -30 ° C). </li></ol> 3. Estrazione di acquosa solubili metaboliti <ol><li> Liofilizzare i campioni durante la notte o per almeno 10 ore. </li><li> Dopo la liofilizzazione, aggiungere un frantoio in acciaio inox ad ogni provetta (Tokken). Aggiungere 750 microlitri di potassio standardizzatotampone fosfato NMR in ossido di deuterio (2 H&gt; 90%) con 2,2-dimetil-2-silapentane-5-solfonato (DSS) standard (KPI; 38,3 mM KH 2 PO 4, il 61,7 mM K 2 HPO 4, DSS 0,1 mM, pH 7,0, 90% D 2 O) 2. </li><li> Sonicare i campioni per 5 minuti a 4 ° C in un sonicatore acqua (Bioruptor, Diagenode) per rimuovere il materiale cella dal filtro. Rimuovere i filtri con pinzette pulite. </li><li> Frantumare le cellule utilizzando un frantoio a mulino (1600 rpm) per 5 minuti. </li><li> Incubare a 65 ° C con agitazione (1400 rpm) su un banco agitatore (Eppendorf) per 15 minuti. </li><li> Rimuovere il maiale metallo con pinzette pulite, e centrifugare il campione a 13.000 g per 5 minuti. </li><li> Disegnare il sopranatante direttamente ad un tubo NMR per spettroscopia NMR. </li></ol> 4. Spettroscopia NMR e Analisi dei Dati <ol><li> Caricare il campione in uno spettrometro NMR (qui, un Bruker DRX-500 equipaggiato con spettrometroSonda TXI con tre assi gradiente controllato da un computer che esegue XWIN-NMR). </li><li> Ottenere 1D 1 H spettri NMR con adeguati metodi precedentemente pubblicati 2,15 dal XWIN-NMR interfaccia. Nel presente studio 1 H NMR sono stati registrati su un sistema operativo DRX-500 spettrometro a 500,03 MHz a 298 K. acqua segnali residui sono state soppresse dalla sequenza di impulsi Watergate, con un tempo di ripetizione di 1,2 s. 128 transitori sono stati raccolti per ottenere 32.000 punti dati per spettro. </li><li> Trasferire le directory dei dati NMR ad un PC installato il software NMRPipe 16. Elaborare i dati grezzi e DSS set a 0 di riferimento ppm quindi manualmente eliminare gli spettri. Digitalizzazione dei dati spettrali in un insieme di valori discreti, integrando o &#39;categorizzazione&#39; con software come rNMR, Automics, o utilizzando il pacchetto FT2B pubblicamente disponibili sul sito web ECOMICS ( <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> ) 3,17. In thè, ad esempio, gli spettri sono stati integrati tra 0,5 e 10,5 ppm oltre 0.032 regioni ppm integrale con ECOMICS e normalizzati al DSS sia o intensità del segnale totale. I dati in uscita possono ora essere utilizzati per la valle analisi statistiche come analisi delle componenti principali (PCA) con pacchetti di software libero come R 18. </li></ol> 5. Risultati rappresentativi Un esempio di spettri 1 H NMR ottenuti utilizzando i metodi di cui sopra sono mostrati nella Figura 1. Questi campioni, da due punti di tempo di un esperimento microcosmo mostrano differenze evidenti a causa di alghe attività metaboliche. Il giorno 4 spettro mostra picchi di abbondanza considerevole, in particolare nell&#39;intervallo 3-4 ppm rispetto al giorno 1 campione. Questi picchi possono essere attribuiti agli zuccheri prodotti dal fiore diatomee nel microcosmo. In un esperimento simile a confronto la crescita delle comunità naturali plancton in acqua di mare artificiale o naturale, approcci statistici sale come principale trama analisi punteggio della componente (PCA) derivato da spettri NMR binned può essere usato per mostrare evidenti differenze metaboliche tra i due trattamenti (Fig. 2), mentre le trame possono identificare i picchi di carico all&#39;interno del spettri che forma la distribuzione dei dati . Tali risultati possono essere confrontati con i dati provenienti da altri livelli omiche, come da metodi di fingerprinting genomici (Fig. 3). Questi picchi NMR può essere interrogato singolarmente (ad esempio presso l&#39;BMRB; <a href="http://www.bmrb.wisc.edu/">http://www.bmrb.wisc.edu/</a> ) 19, o spettri intera possono essere analizzati statisticamente (per esempio con SpinAssign a <a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken. jp /? action = nmr_search</a> ) 2. In questo esempio, le differenze tra i trattamenti erano dovuti ad un abbondanza di picchi nella regione di zuccheri (3,39 ppm a 4,04 ppm) degli spettri di metaboliti naturali della comunità plancton, e numerosi picchi caratteristici delle comunità artificiali con acqua di mare sono stati provvisoriamente identified come lattato e formiato con SpinAssign. <img src="/files/ftp_upload/3163/3163fig1.jpg" alt="Figura 1" /> Figura 1. Rappresentativa spettri 1 H NMR ottenuti da campioni trattati con questa procedura. I campioni sono stati prelevati prima del Microcosmo (giorno 1) e durante (giorno 4) una fioritura di diatomee intenso. Esperimenti NMR sono state eseguite su uno strumento Bruker DRX-500 con segnali normalizzati al altezza picco dello standard interno (DSS; 0 ppm). <img src="/files/ftp_upload/3163/3163fig2.jpg" alt="Figura 2" /> Figura 2. Analisi delle componenti principale (PCA) plot punteggio spettri NMR cestinate da metabolomes delle comunità microbiche naturalmente derivati ​​planctonici coltivate in microcosmi con diamanti naturali (acqua di mare aperto) o artificiali (cerchi neri). Chiare differenze metaboliche può essere osservato nel grafico a dispersione. Una trama caricamento da una tale analisi può essere utilizzato per identificare picchi distinti di importanzail sistema, questi picchi può essere ulteriormente analizzato come necessario. <img src="/files/ftp_upload/3163/3163fig3.jpg" alt="Figura 3" /> Figura 3. Un esempio di multi-omiche analisi NMR combinazione con i dati genomici. Composizione della comunità sulla base di elettroforesi su gel gradiente denaturante 18S (a sinistra) e 16S (a destra) geni rRNA dai campioni stessi analizzati nella Figura 2 mostra anche diversi modelli di comunità microbiche tra i diamanti naturali (aperti) e artificiale (cerchi neri) microcosmi di acqua di mare. Tale corrispondenza tra metaboloma genoma e dai sistemi naturali dimostra l&#39;utilità di questo approccio.

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L&#39;estrazione filtrazione e metabolita metodo illustrato qui permette per la biomassa microbica planctoniche da raccogliere in quantità sufficiente per metabolomica NMR. Mentre solo di estrazione acquosa-solubili metaboliti utilizzando KPI e 1D 1 H NMR è dimostrata, altri solventi da estrazione e approcci spettroscopici possono essere utilizzati. Un esempio utile è l&#39;uso di metanolo deuterato come solvente semi-polare, che ha dimostrato di produrre spettri NMR superiore da campioni eterogenei ed è ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 0,22 micron idrofile Durapore filtri PVDF, 25 mm </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Holder microanalisi Filter, 25 mm, supporto di vetro poroso </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3-posto collettore, 47 mm, in acciaio inox </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> Ossido di deuterio, 2 H&gt; 90% </td><td> Campridge Isotope Laboratoties </td><td> DLM-4 </td><td></td<html> &gt; </tr><tr><td> DSS </td><td> Fluka </td><td> 92754 </td><td></td></tr><tr><td> Automill </td><td> Tokken </td><td> TK-AM4 </td><td> Frantoi in acciaio inox incluse </td></tr><tr><td> Thermomixer confort </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> Vacuum evaporatore </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> NMR </td><td> Bruker </td><td> DRX-500 con 5 mm TXI sonda </td><td></td></tr><tr><td> Spectral strumento di binning </td><td> Originariamente sviluppato </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> Annotazione strumento di Metabolita e database </td><td> Originariamente sviluppato </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

Metabolomica ambientale è un settore emergente che sta promuovendo una nuova comprensione in come gli organismi rispondere e interagire con l&#39;ambiente e l&#39;altro a livello biochimico 1. Risonanza magnetica nucleare (NMR) è una delle varie tecnologie, compreso il gas cromatografia-spettrometria di massa (GC-MS), con la promessa di un notevole per tali studi. I vantaggi di NMR sono che è adatto per le analisi non mirati, fornisce informazioni strutturali e spettri può essere interrogato in maniere quantitativi e statistici nei confronti di banche dati disponibili di recente dei singoli spettri metabolita 2,3. Inoltre, i dati NMR spettrali possono essere combinati con dati provenienti da altri livelli omiche trascrittomica (ad esempio, la genomica), per fornire una comprensione più completa delle risposte fisiologiche di taxa gli uni agli altri e l&#39;ambiente 4,5,6. Tuttavia, NMR è meno sensibile rispetto ad altre tecniche metabolomica, rendendo difficile apply ai sistemi microbici naturali dove campioni di popolazione possono essere le concentrazioni a bassa densità e basso rispetto al metabolita metaboliti ben definite e facilmente estraibili fonti, quali tessuti interi, biofluidi o cellulo-culture. Di conseguenza, i pochi studi ambientali diretti metabolomica di microbi eseguiti fino ad oggi sono stati limitati alla cultura-based o facilmente definito ad alta densità ecosistemi come host-simbionti sistemi, costruiti co-colture o manipolazioni dell&#39;ambiente intestinale in cui l&#39;etichettatura isotopi stabili possono essere inoltre utilizzati per migliorare segnali NMR 7,8,9,10,11,12. Metodi che facilitano la concentrazione e la raccolta dei metaboliti ambientali a concentrazioni adatte NMR mancano. Dal recente attenzione è stata data alle metabolomica ambientali degli organismi nell&#39;ambiente acquatico, in cui è mediata gran parte del flusso di energia e materiali da parte della comunità planctoniche 13,14, abbiamo sviluppato un metodo per la concentrazionezione e l&#39;estrazione di tutta la comunità-metaboliti microbici planctonici dai sistemi di filtrazione. Disponibili in commercio idrofile poli-1 ,1-difluoroethene (PVDF), i filtri sono appositamente trattati per rimuovere completamente estraibili, che possono altrimenti apparire come contaminanti nelle analisi successive. Questi filtri trattati vengono poi usati per filtrare i campioni ambientali o sperimentale di interesse. Filtri contenenti il materiale umido campione sono liofilizzato e acquosa metaboliti solubili vengono estratti direttamente per la spettroscopia NMR convenzionale utilizzando un tampone fosfato di potassio standardizzato di estrazione 2. I dati derivano da questi metodi possono essere analizzati statisticamente per identificare i modelli significativi, o integrato con gli altri livelli omiche per la comprensione globale della comunità e il funzionamento dell&#39;ecosistema.

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

Un metodo per l&#39;estrazione metabolita da parte delle comunità microbiche planctonici è presentato. Campionamento intera comunità si realizza mediante filtrazione su filtri appositamente preparati. Dopo liofilizzazione, acquoso-solubili metaboliti vengono estratti. Questo approccio consente per l&#39;applicazione della metabolomica ambientali trans-omiche indagini di comunità microbiche naturali o sperimentali.

Concentrazione dei metaboliti a bassa densità Comunità planctoniche per Metabolomica ambientali utilizzando la spettroscopia a risonanza magnetica nucleare

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 Views.  RIKEN Advanced Science Institute. Un metodo per l'estrazione metabolita da parte delle comunità microbiche planctonici è presentato. Campionamento intera comunità si realizza mediante filtrazione su filtri appositamente preparati. Dopo liofilizzazione, acquoso-solubili metaboliti vengono estratti. Questo approccio consente per l'applicazione della metabolomica ambientali trans-omiche indagini di comunità microbiche naturali o sperimentali.

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

Microbial Communities in Nature and Laboratory - Interview

Comunità microbica in natura e laboratorio - Intervista

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Biologia

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

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

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

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

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.

Analisi di espressione genica da comunità microbiche marine utilizzando Trascrittomica Ambientale

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.

Rilevamento di ossido nitrico e anione superossido radicale da spettroscopia di risonanza paramagnetica elettronica da cellule con 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 ...

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.

Quantificare miscelazione con risonanza magnetica

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.

Purificazione di Trascrizioni e metaboliti<em&gt; Drosophila</em&gt; Teste

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.

Rimozione di materiali esogene dalla parte esterna della congelati core per Indagare sulla antica comunità biologiche ospitava Dentro

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

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

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

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

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

Protocollo sperimentale per l'utilizzo di Drosophila come sistema modello invertebrati marini per test di tossicità in 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.

Sintesi di nanoparticelle Core-drogati di ossido di ferro di 68Ga per Dual tomografia a emissione di positroni/Imaging di risonanza magnetica (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;