The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

1. Progettazione di esperimenti <ol><li> Determinare il volume di nanocompositi rame necessari per la sintesi. Su tale base, scegliere un numero di flaconi di piccolo volume (25 centimetri 2), o flaconi grandi come indicato di seguito nella preparazione dei materiali. </li><li> Per questa sintesi, utilizzare un incubatore a 37 ° con il 5% di CO 2 e almeno il 40% di umidità. Assicurarsi che tale incubatrice è disponibile e che non verrà ripetutamente disturbato durante il periodo di sintesi (circa 24 ore). ATTENZIONE: l&#39;apertura e la chiusura ripetuta dell&#39;incubatrice sarà certamente causare sbalzi di temperatura che possono provocare la sintesi alterata delle strutture nanocompositi. </li></ol> 2. Preparazione dei Materiali <ol><li> Preparare tutti materiali freschi prima dell&#39;inizio di un esperimento, aggiungendo materiali solidi a solventi destra prima sintesi deve iniziare. Mantenere soluzioni stock di cistina e di partenza di rame materiale a liquido per tempi lunghi brima l&#39;esperimento non è raccomandato e può portare a risultati variabili. Una volta aperto dal fornitore, tenere materiali secchi avvolgendo la parte superiore del contenitore con parafilm partenza. Nota: il seguente protocollo viene utilizzato come esempio per la reazione in 25 cm 2 cella pallone di coltura con 7 ml di cistina, 6643 ml di acqua sterile, e 350 ml di CNPS. </li><li> Preparare una soluzione di 2 mg / ml di nanoparticelle rame pesando almeno 2 mg di CNPS. Indossare guanti monouso durante questa fase per evitare possibili contatti di CNPS con la pelle. Mettere le nanoparticelle in un vuoto sterile 16 ml di vetro fiala. <ol><li> Per la fiala contenente CNPS, aggiungere acqua deionizzata sterile il volume appropriato per ottenere una soluzione 2 mg / ml e agitare la soluzione per 20 sec per fornire dispersione delle nanoparticelle prima dell&#39;inizio di sintesi (almeno 1 ml di volume totale è raccomandato). Non riempire il più di metà strada fiala con l&#39;acqua come questo inibisce la miscelazione con il vortex. CNPS will risolvere rapidamente al fondo del flacone e apparirà di colore scuro (grigio e il nero). </li><li> Sonicare la soluzione CNP per 17 minuti a temperatura ambiente per fornire la dispersione massima di CNPS prima dell&#39;inizio della sintesi. Controllare periodicamente per assicurarsi che CNPS stia mescolando causa di ultrasuoni. Dopo una sonicazione successo, CNPS rimangono sospese in soluzione per almeno 30 minuti e la soluzione sarà di colore scuro. </li></ol></li><li> Pesare massa sufficiente di cistina per ottenere una soluzione / ml 72,9 mg per la sintesi. Poiché cistina non è direttamente solubile in acqua, posizionare la cistina pesati in un recipiente di pesata antistatico. <ol><li> Per la pesatura recipiente contenente cistina, aggiungere sufficiente volume di sterili, 1 M NaOH, in modo che la cistina scioglie completamente. Ad esempio, sciogliere 7,29 mg di cistina completamente in 100 ml di 1 M NaOH, per fare un / ml soluzione 72,9 mg. </li><li> Per mantenere condizioni di sterilità, eseguire questo passaggio in una cappa coltura tissutale flusso sterile. ATTENZIONE: NaOHa 1 M concentrazione caustica, così indossare guanti monouso durante questa fase per evitare il contatto di NaOH concentrato con la pelle </li></ol></li><li> Lavorando in una cappa coltura tissutale sterili, aggiungere 7 ml di cistina con 6643 ml di acqua sterile alla beuta di sintesi sterili prima, e lasciate incubare per 30 min in incubatore a 37 ° C con il tappo pallone ventilato (sciolto) per fornire efficace miscelazione. Risospendere il / soluzione CNP ml 2 mg vortexando per 30 secondi, in quanto CNPS sarà risolto dopo la fase di ultrasuoni. <ol><li> Aggiungere la soluzione CNP sufficiente al pallone di sintesi (con tecnica sterile) per mantenere i seguenti rapporti tra i componenti: combinare 1 parti cistina, 50 parti CNPS, e 949 parti di acqua sterile in 25 cm 2 pallone di coltura cellulare per iniziare la sintesi. Ad esempio, per un volume di sintesi 7 ml, unire 7 ml di soluzione madre cistina, 350 ml di CNPS, e 6643 ml di acqua sterile. Rimettere il tappo sul pallone e stringere in modo che sia secure. </li><li> Dopo che unisce tutti i componenti per la sintesi, mescolare delicatamente nel pallone agitando 4-5 volte. Posizionare beuta nel CO 2 incubatore e sfiatare la beuta allentando il tappo in modo che ci sia lo scambio di gas dentro e fuori del pallone durante la sintesi. </li></ol></li><li> Lasciare che la sintesi di eseguire in incubatrice per circa 24 ore. Durante la sintesi, si può osservare, con microscopia e occhio, formazione di compositi altamente lineari. Nota: Il processo di formazione delle strutture può accadere improvvisamente nel senso che le strutture sono inizialmente difficili da individuare, quindi aspetto procede rapidamente a una densità crescente. La formazione può quindi avvenire prima 24 ore. Il processo può anche essere osservato ad occhio volta strutture diventano più grandi e loro densità aumenta. Mentre la generazione delle strutture può essere osservato nel tempo al microscopio e occhio a successivi punti di tempo, interrompendo continuamente le condizioni di sintesi e temperatura porterà a scarsarisultati di sintesi. </li><li> Terminare sintesi di biocompositi dal strettamente tappatura matraccio sintesi e memorizzare il vaso in frigorifero (4 ° C). Strutture, una volta generati, rimangono stabili in questa forma per almeno un anno. Etichettare il pallone con condizioni di sintesi, compresi i componenti utilizzati, la data della sintesi, e il tempo di incubazione della sintesi prima della terminazione. </li></ol> 3. Sintesi Utilizzando solfato di rame <ol><li> Eseguire la sintesi di auto-assemblaggio, sostituendo CNPS con sale solfato di rame. Usando una tecnica sterile, sciogliere almeno 2 mg di solfato di rame in quantità sufficiente di acqua deionizzata sterile per ottenere uno / ml soluzione 2 mg. I cristalli di solfato di rame facilmente andare in soluzione a questa concentrazione, ma vortice il flaconcino se necessario, e ispezionare ad occhio per garantire che tutti i cristalli sono dissolti. </li><li> Dopo la preparazione del solfato di rame, effettuare sintesi come descritto in precedenza, ma sostituendo CNPS con solfato di rame. Nota: nanocompositi auto-assemblati con solfato di rame come materiale di partenza sono stati trovati ad essere molto più consistente in forma finale che per le strutture sintetizzate da CNPS. </li><li> Terminare la sintesi di biocompositi solfato di rame come per i compositi CNP (punto 2.6) e archiviarli a lungo termine a 4 ° C. </li></ol> 4. Caratterizzazione e manipolazione dei biocompositi Post-sintesi <ol><li> Caratterizzare biocompositi derivati ​​da CNPS e da solfato di rame dal bianco al microscopio ottico 9 e microscopia elettronica 9. <ol><li> Per la caratterizzazione e l&#39;ispezione di biocompositi post-sintesi mediante microscopia a luce bianca, utilizzare un microscopio invertito come compositi si depositerà alla superficie inferiore del pallone entro pochi minuti dalla posa del matraccio piatta, e può quindi essere messo a fuoco. Utilizzare l&#39;impostazione in campo chiaro sul microscopio per massimizzare il contrasto tra biocompositi e il mezzo liquido. Compositi derivati ​​da CNPS e poliziottoper solfato saranno entrambi apparire chiaro opaco colore, ma che non hanno reagito aggregati CNP appare molto scuro. <ol><li> Utilizzare una fotocamera digitale collegata al microscopio per catturare le immagini dei compositi. Viene osservata una gamma di lunghezze per le singole strutture. </li></ol></li><li> Per la caratterizzazione e l&#39;ispezione di biocompositi post-sintesi e dopo conservazione a 4 ° C, permettono fiaschi di venire a temperatura ambiente per almeno 15 minuti come fiaschi formeranno condensazione inizialmente dopo la rimozione dal frigorifero, che oscurare efficace focalizzazione nello svolgimento immagini di microscopia. Dopo aver lasciato il raggiungimento dell&#39;equilibrio di RT, pulire le superfici superiore e inferiore del pallone con un tovagliolo di carta pulito per ottimizzare la qualità delle immagini di microscopia. </li><li> Quando si lavora con o immagini compositi che sono stati conservati a lungo termine, il vortice fiasco per 30 sec a dissociarsi ciuffi di composti che si formano mentre in frigorifero. Dopo vortex, ispezionare le strutture con un microfono invertitoROSCOPE per garantire che gli aggregati sono dissociate, e ripetere vortex se necessario. </li><li> Utilizzare invertita microscopia luce bianca per valutare l&#39;efficacia della sintesi per un dato esperimento utilizzando CNPS. Ad esempio, documentare la presenza o l&#39;assenza di non reagito CNPS in fiaschi di sintesi utilizzati per biocompositi CNP-derivati ​​da palloni con diversi parametri come il tempo di sintesi. Nota: individuale CNPS sono troppo piccoli per osservare con un microscopio ottico, ma non reagito CNP aggregati appariranno come tondeggiante e oggetti scuri, in contrasto con il CNP-compositi sintetizzati con successo che avrà un rapporto di alta aspetto, forma lineare, e avrà una gamma di differenti lunghezze. Evitare di realizzare sintesi per troppo tempo di un periodo di tempo prima della terminazione, come questo si tradurrà in strutture di tipo altamente ramificate &quot;riccio&quot;, che sono difficili da disperdere in singole strutture volta formate. </li><li> Utilizzare invertita microscopia ottica bianca per valutare l&#39;efficaciadella sintesi per un dato esperimento utilizzando solfato di rame. Poiché solfato di rame va completamente in soluzione con questo protocollo, la soluzione apparirà meno scuro rispetto alla soluzione dalla sintesi utilizzando CNPS. Documentare la dimensione e la portata dei compositi solfato di rame confrontando palloni con differenti condizioni di sintesi, quali il tempo di sintesi prima della terminazione. Nota: composti sintetizzati con successo mostreranno una gamma di diverse lunghezze. Evitare di realizzare sintesi per troppo tempo di un periodo di tempo prima della terminazione, come questo si tradurrà in aggregati altamente ramificati di compositi, alcuni dei quali saranno &quot;urchin-like&quot; nella struttura, e che sono difficili da disperdere in singole strutture volta formate . </li></ol></li><li> Per concentrare biocompositi post-sintesi, soluzioni di centrifugazione di compositi in una provetta da centrifuga. Aggiungere 6 ml di una delle strutture CNP-derivati ​​o strutture solfato derivato rame in una provetta da centrifuga da 15 ml. Centrifugare per 10 minutia 500 xg a RT per formare un pellet. Per i volumi più piccoli, aggiungere 500 ml di strutture in soluzione di 0,6 ml tubi di dimensioni. Centrifugare a 2.000 xg a temperatura ambiente per almeno 10 minuti per formare un pellet. <ol><li> Dopo centrifugazione per un tempo sufficiente (almeno 10 min per microfuges), salvare il pellet osservabile nella parte inferiore del tubo dove le strutture sono concentrati rimuovendo accuratamente il liquido supernatante sopra il pellet. Strutture Biocomposite derivate da solfato di rame appaiono di colore blu e strutture derivate da CNPS sono più scuro (grigio al nero). </li><li> Aggiungere più compositi di questo tubo e ripetere il processo nello stesso tubo di concentrare strutture se desiderato. Per disperdere i pellets concentrati, aggiungere il volume desiderato di soluzione al tubo, e vortex per 10-30 sec. </li></ol></li><li> Strutture Sonicare volta formate, per spostare la dimensione media della popolazione (lunghezza) delle strutture a valori più bassi. Strutture Luogo in acqua deionizzata sterile e sonicare a Least 10 min. Utilizzando questo processo, nel corso del tempo, strutture vengono frammentati e più piccola della lunghezza media (vedi figura 6 del testo). Le modifiche ai documenti di dimensioni compositi con diversi tempi di sonicazione usando un microscopio a luce bianca invertito e fotocamera digitale. </li></ol>

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II. Copper (II) with Cystine and Related Ligands.</a> Inorganic Chemistry. 2, 843-849 (1963).</li><li>Hallman, P., Perrin, D., Watt, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+computed+distribution+of+copper+(II)+and+zinc+(II)+ions+among+seventeen+amino+acids+present+in+human+blood+plasma.">The computed distribution of copper (II) and zinc (II) ions among seventeen amino acids present in human blood plasma.</a> Biochem. J. 121, 549-555 (1971).</li><li>Jensen, L. S., Maurice, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sulfur+amino+acids+on+copper+toxicity+in+chicks.">Influence of sulfur amino acids on copper toxicity in chicks.</a> The Journal of nutrition. 109, 91-97 (1979).</li><li>Lee, Y., Choi, J. R., Lee, K. J., Stott, N. 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Authors have nothing to disclose.

Nel valutare potenziali effetti tossici di nanomateriali compreso CNPS, è stato osservato che nel lungo termine, CNPS furono trasformati da una distribuzione di particelle inizialmente più dispersa ad una forma aggregata maggiore (Figura 2). In alcuni casi, queste formazioni altamente aggregati che sono stati prodotti nel piatto di coltura cellulare, in condizioni biologiche, formate proiezioni altamente lineari da dell&#39;aggregato centrale, che ricordano rame precedentemente descritto contengono &q...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

L&#39;obiettivo di questo protocollo è quello di descrivere la sintesi di due nuovi biocompositi con strutture ad alto rapporto-aspetto. I biocompositi consistono di rame e cistina, sia con nanoparticelle di rame (CNPS) o solfato di rame contribuendo componente metallico. Sintesi è condotta in un liquido in condizioni biologiche (37 ° C) e la forma compositi autoassemblati dopo 24 ore. Una volta formati, questi composti sono altamente stabili in entrambi i liquidi e in una forma secca. I compositi ridimensionare dal nano alle micro gamma di lunghezza, e da pochi micron a 25 nm di diametro. Emissione di campo microscopia elettronica a scansione con spettroscopia a dispersione di energia dei raggi X (EDX) ha dimostrato che lo zolfo è presente nelle strutture lineari NP-derivati, mentre era assente dal materiale di partenza CNP, confermando cistina come fonte di zolfo nei nanocompositi finali . Durante la sintesi di questi nano e micro-compositi lineari, una vasta gamma di lunghezze di structures è formata nel serbatoio di sintesi. Sonicazione della miscela liquida dopo la sintesi è stata dimostrata per aiutare a controllare dimensione media delle strutture diminuendo la lunghezza media con un tempo di sonicazione. Poiché le strutture formate sono altamente stabili, non agglomerato, e si formano in fase liquida, centrifugazione può essere utilizzato anche per aiutare a concentrare e segreganti compositi formati.

La figura 1 mostra una schematica diagramma di flusso delle fasi di sintesi per formare i biocompositi lineari descritti in questo lavoro. CNPS o rame solfato come materiali di partenza sono combinati con acqua sterile per formare un / ml soluzione 2 mg, questa soluzione viene miscelata e sonicato per fornire un impasto omogeneo, e questa soluzione di rame viene quindi miscelata nel seguente rapporto per sintesi: 949 parti sterile acqua: 50 parti miscela di rame: cistina soluzione stock 1 parte. I volum...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Qui vi presentiamo un protocollo per sintetizzare nuovi, ad alto rapporto di aspetto biocompositi in condizioni biologiche e in mezzi liquidi. I biocompositi scala da nanometri a micrometri di diametro e lunghezza, rispettivamente. Nanoparticelle di rame (CNPS) e solfato di rame in combinazione con cistina sono le componenti chiave per la sintesi.

Generazione di scalabili, Metallic Tacchi-Aspect nanocompositi Rapporto in un Biologica Liquid Media

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K Views.  Centenary College of Louisiana.  Qui vi presentiamo un protocollo per sintetizzare nuovi, ad alto rapporto di aspetto biocompositi in condizioni biologiche e in mezzi liquidi. I biocompositi scala da nanometri a micrometri di diametro e lunghezza, rispettivamente. Nanoparticelle di rame (CNPS) e solfato di rame in combinazione con cistina sono le componenti chiave per la sintesi. 

Video: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Estrazione dei lipidi cellulari per analisi mirate Stabile diluizione isotopica cromatografia liquida-spettrometria di massa

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Ricerca

Bioingegneria

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Test microtrazione Environmentally-controllato di meccanica-adattivi nanocompositi polimerici per<em&gt; Ex vivo</em&gt; Caratterizzazione

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

In situ TEM di assemblee biologiche in liquido

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Sintesi di nanofibre cheratina-based per Ingegneria Biomedica

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Alternando microgel ibrida Gelatina di campo magnetico-Responsive per Controlled Drug uscita

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

Preparazione di Monodomain Liquid Crystal Elastomeri e cristallo elastomero Nanocompositi Liquid

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatizzato robot Liquid Handling Assembly di dispositivi modulari del DNA

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Sintesi di biocompatibili a cristalli liquidi in elastomero Schiume come impalcature cellulari per 3D spaziali colture cellulari

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantificazione simultanea di kynurenine selezionate analizzate dalla cromatografia liquida-spettrometria di massa in mezzo raccolto da colture cellulari tumorali

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Generazione scalabile di organoidi cerebellari maturi da cellule staminali pluripotenti umane e caratterizzazione da immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...