Name: investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system
Uploaded: 2023-01-06T14:20:00
Description: Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

Gli autori ringraziano il supporto ricevuto dal Centro Multidisciplinare para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp, per la generazione di topi AdipoSPH, il Laboratorio di Inmmunometabolismo e Segnalazione Cellulare e l'Istituto Nazionale di Scienza e Tecnologia sulla Fotonica Applicata alla Biologia Cellulare (INFABIC) per tutto il supporto sperimentale. Ringraziamo il sostegno finanziario della Fondazione di ricerca di San Paolo (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Gli studi sugli animali sono stati eseguiti in conformità con la Guida dell'Università di Campinas per la cura e l'uso degli animali da laboratorio (protocollo CEUA #5810-1/2021).
1. Clonazione molecolare
<ol><li>Progettazione di RNA guida singola (sgRNA)<ol><li>Progetta sgRNA per l'attivazione di CRISPR usando CHOPCHOP, disponibile su https://chopchop.cbu.uib.no/ o qualsiasi altro strumento adatto. Utilizzare i seguenti parametri per progettare sgRNA che ha come bersaglio...

<ol>
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Una delle applicazioni non-editing più utili della tecnologia CRISPR è l'interrogazione della funzione genica attraverso l'attivazione di geni endogeni utilizzando i sistemi CRISPRa6. SPH è un potente CRISPRa che è stato originariamente descritto per indurre la conversione degli astrociti in neuroni attivi prendendo di mira diversi geni neurogeni14. In questo studio, AdipoSPH ha dimostrato di essere uno strumento adatto per studiare la biologia del grasso beige attivand...

Gli adipociti beige (o brite) sono adipociti disaccoppianti che esprimono la proteina 1 (UCP1) e ricchi di mitocondri che risiedono all'interno di depositi di tessuto adiposo bianco (WAT). Il grasso beige emerge da un sottogruppo di progenitori adipocitari o adipociti bianchi maturi in risposta all'esposizione al freddo e ad altri stimoli 1,2. Gli adipociti beige possono convertire l'energia in calore in modo UCP1-dipendente o indipendente3. Indipendentemente dalla sua funzione termogenica, il grasso beige può anche migliorare la salute metabolica con altri mezzi, come la secrezione di adipochine e attività antinfiammatorie e antifibrotiche. Studi su topi e umani hanno dimostrato che l'induzione del grasso beige migliora il glucosio di tutto il corpo e l'omeostasi lipidica3. Tuttavia, sebbene la nostra conoscenza della biologia del grasso beige si sia evoluta rapidamente negli ultimi anni, la maggior parte dei suoi benefici metabolici e dei relativi meccanismi non sono ancora completamente compresi.
Le brevi ripetizioni palindromiche raggruppate regolarmente intervallate (CRISPR) sono state descritte per la prima volta nelle cellule eucariotiche come uno strumento in grado di generare una rottura del doppio filamento (DSB) in un sito specifico del genoma attraverso l'attività nucleasica della proteina Cas9 4,5. Cas9 è guidato da un RNA sintetico a guida singola (sgRNA) per colpire una specifica regione genomica, portando a un DSB del DNA. Oltre a utilizzare la nucleasi Cas9 per scopi di editing, la tecnologia CRISPR-Cas9 si è evoluta per essere utilizzata come strumento di regolazione genica specifico per sequenza6. Lo sviluppo di una proteina Cas9 cataliticamente inattiva (dCas9) e l'associazione di moduli trascrizionali in grado di migliorare l'espressione genica ha dato origine a strumenti di attivazione di CRISPR (CRISPRa). Sono emersi diversi sistemi CRISPRa, come VP647,8, mediatore di attivazione sinergica (SAM)9, SunTag10,11, VPR12,13 e SunTag-p65-HSF1 (SPH)14, che combina i componenti degli attivatori SAM e SunTag. Recentemente è stato dimostrato che l'espressione indotta di geni neurogeni nei neuroblasti N2a e negli astrociti primari è maggiore utilizzando SPH rispetto ad altri sistemi CRISPRa14, dimostrando SPH come un promettente strumento CRISPRa.
Qui, abbiamo sfruttato un topo SPH14 sviluppato in precedenza per generare un modello murino condizionale che esprime SPH specificamente negli adipociti utilizzando il lignaggio dell'adiponectina Cre (AdipoSPH). Utilizzando un virus adeno-associato (AAV) che trasporta il gRNA che ha come bersaglio il gene endogeno Prdm16, l'imbrunimento (conversione da bianco a beige) di WAT inguinale (iWAT) è stato indotto per aumentare l'espressione del programma genico termogenico. Inoltre, la sovraespressione in vitro di Prdm16 ha aumentato il consumo di ossigeno. Pertanto, questo protocollo fornisce un versatile modello murino SPH per esplorare i meccanismi di sviluppo del grasso beige all'interno del tessuto adiposo.

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		<tr>
			<td>3,3&#39;,5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>AAVpro 293T Cell Line</td>
			<td>Takarabio</td>
			<td>632273</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra Centrifugal Filter</td>
			<td>Merckmillipore</td>
			<td>UFC510008</td>
			<td>100 KDa</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) F-12, GlutaMAX&#8482; supplement</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td>high concentrations of glucose, amino acids, and vitamins</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelta Self-Opening Micro Scissors</td>
			<td>Fisher Scientific</td>
			<td>17-467-496</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cell Scrapers (100 pk)</td>
			<td>Fisher Scientific</td>
			<td>08-100-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>LigaFast Rapid DNA Ligation System</td>
			<td>Promega</td>
			<td>M8225</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep purification kit&#160;</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Model 701</td>
		</tr>
		<tr>
			<td>NEB 10-beta/Stable&#160;</td>
			<td>New England Biolabs</td>
			<td>C3019H</td>
			<td>E. coli competent cells</td>
		</tr>
		<tr>
			<td>pAAV2/8&#160;</td>
			<td>Addgene&#160;</td>
			<td>112864</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-U6-gRNA-CBh-mCherry</td>
			<td>Addgene&#160;</td>
			<td>91947</td>
			<td></td>
		</tr>
		<tr>
			<td>pAdDeltaF6&#160;</td>
			<td>Addgene&#160;</td>
			<td>112867</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Sigma-Aldrich</td>
			<td>23966</td>
			<td>Linear, MW 25000</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Rioqu&#237;mica</td>
			<td>510101303</td>
			<td>Antiseptic</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI enzyme</td>
			<td>New England Biolabs</td>
			<td>R0156</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design Premier Adson Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-079-741</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>475-40417</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M180B</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase buffer&#160;</td>
			<td>New England Biolabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK enzyme kit</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tramadol Hydrochloride</td>
			<td>SEM</td>
			<td>43930</td>
			<td></td>
		</tr>
		<tr>
			<td>Vidisic Gel&#160;</td>
			<td>Bausch + Lomb&#160;</td>
			<td>99620</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system

La tecnologia CRISPR (Clustered regularly Interspaced Short Palindromic Repeats) ha provocato una rivoluzione in biologia e strumenti recenti sono stati applicati ben oltre l'editing genetico originariamente descritto. Il sistema di attivazione CRISPR (CRISPRa) combina la proteina Cas9 cataliticamente inattiva (dCas9) con moduli di trascrizione distinti per indurre l'espressione genica endogena. SunTag-p65-HSF1 (SPH) è una tecnologia CRISPRa di recente sviluppo che combina componenti di mediatori di attivazione sinergica (SAM) con gli attivatori SunTag. Questo sistema consente la sovraespressione di geni singoli o multipli progettando un RNA a guida singola personalizzato (sgRNA). In questo studio, un topo SPH precedentemente sviluppato è stato utilizzato per generare un topo condizionale che esprime SPH negli adipociti (linea di adiponectina Cre), chiamato AdipoSPH. Per indurre un fenotipo di grasso bianco-beige (imbrunimento), un virus adeno-associato (AAV) che trasporta sgRNA mirato al gene endogeno Prdm16 (un fattore di trascrizione ben consolidato correlato allo sviluppo del grasso bruno e beige) è stato iniettato nel tessuto adiposo bianco inguinale (iWAT). Questo modello murino ha indotto l'espressione di Prdm16 endogeno e ha attivato il programma genico termogenico. Inoltre, la sovraespressione di Prdm16 indotta da SPH in vitro ha migliorato il consumo di ossigeno degli adipociti beige, fenocopiando i risultati di un precedente modello murino transgenico Prdm16. Pertanto, questo protocollo descrive un modello murino versatile, economico ed efficiente in termini di tempo per studiare la biologia del tessuto adiposo.

I topi AdipoSPH sono stati sviluppati allevando ceppi murini SPH e Adipoq-Cre. Entrambi i ceppi di topo erano in uno sfondo ibrido C57BL6J-DBA/2J (secondo il fornitore commerciale; vedi Tabella dei materiali). Il lignaggio dei topi SPH è stato originariamente descritto da Zhou et al.14.
Sviluppo di adipociti beige in vivo attraverso la sovraespressione di Prdm16 mediata da AdipoSPH Per valutare la capacità...

Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System

Questo protocollo presenta l'uso di CRISPR SunTag-p65-HSF1 (SPH) negli adipociti (AdipoSPH) come strategia alternativa al virus adeno-associato (AAV) per studiare la biologia del grasso beige. L'iniezione in vivo di sgRNA portatori di AAV mirati al gene endogeno Prdm16 è sufficiente per indurre lo sviluppo di grasso beige e migliorare il programma genico termogenico.

Studio della biologia e del metabolismo dei grassi beige utilizzando il sistema di attivazione CRISPR SunTag-p65-HSF1

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far ...

Questo protocollo presenta il sistema di attivazione SPH CRISPR come una strategia alternativa ai vettori virali convenzionali per eseguire saggi di funzione di guadagno negli adipociti. Consente la sovraespressione di geni endogeni adipociti singoli o multipli all'interno del complesso ambiente cellulare del tessuto adiposo fornendo un singolo RNA guida personalizzato utilizzando un virus adeno associato. Le fasi impegnative di questo protocollo sono legate alla clonazione del singolo RNA guida e alla produzione e iniezione di virus adeno-associato nel tessuto adiposo.Per iniziare, progetta un singolo RNA guida o sgRNA per l'attivazione di CRISPR usando chopchop o qualsiasi altro strumento adatto. Progettare l'sgRNA che prende di mira il gene PRDM16 utilizzando i seguenti parametri:Target come PRDM16 in un Mus musculus usando un CRISPR / Cas9 e per come attivazione. Aggiungi sporgenze allo sgRNA per abbinare il sito di restrizione Sac1 nella dorsale vettoriale PAAVU6GRNACBHM ciliegia.Includere 5'AGCT 3'dove n corrisponde ai nucleotidi. Ottenere la sequenza di complemento inverso di 3'sgRNA usando lo strumento indicato. Successivamente, per la ricottura di oligonucleotidi complementari a singolo filamento, aggiungere un microlitro di ogni oligonucleotide a singolo filamento 5' e 3', un microlitro di tampone T4 ligasi, 0,5 microlitri di polinucleotide chinasi T4 e 6,5 microlitri di acqua per un volume di reazione finale di 10 microlitri.Ricottura degli oligonucleotidi a singolo filamento complementari utilizzando un termociclatore a 37 gradi Celsius per 30 minuti e 95 gradi Celsius per cinque minuti, seguito da una velocità di discesa di cinque gradi Celsius al minuto. Quindi per la legatura degli oligonucleotidi sgRNA ricotti aggiungere 25 nanogrammi di ciliegia plasmide PAAVU6GRNACBHM a due microlitri di oligonucleotidi sgRNA ricotti, un microlitro di enzima Sac1, due microlitri di tampone 10X T4 DNA ligasi, un microlitro di T4 DNA ligasi e acqua per un volume di reazione finale di 10 microlitri. Utilizzando un termociclatore, eseguire la legatura incubando la miscela di reazione per 15 cicli a 37 gradi Celsius per cinque minuti e 25 gradi Celsius per cinque minuti seguita da tenere a quattro gradi Celsius.Successivamente, trasformare le cellule competenti di Escherichia coli DH10B con quattro microlitri di prodotto di legatura mediante shock termico a 42 gradi Celsius per 45 secondi e distribuire su una piastra di agar contenente 10 microgrammi per millilitro di ampicillina. Confermare le colonie trasformate mediante Colony Polymerase Chain Reaction o PCR utilizzando il Master Mix PCR, prelevare la colonia e mescolarla con cinque microlitri di Master Mix, 0,1 microlitri di primer universale, 0,1 microlitri di primer inverso sgRNA e cinque microlitri di acqua. Eseguire la PCR utilizzando la denaturazione iniziale a 94 gradi Celsius per due minuti, seguita da 35 cicli di denaturazione a 94 gradi Celsius per 20 secondi, ricottura per 30 secondi a 60 gradi Celsius, estensione a 72 gradi Celsius per 30 secondi e una fase di allungamento finale a 72 gradi Celsius per cinque minuti.Dopo la PCR, risolvere il DNA utilizzando l'elettroforesi su gel di agarosio in tampone TAE 0,5X a 90 volt per 30 minuti. Invia campioni positivi per il sequenziamento Sanger utilizzando primer universale. Quindi, purificare il plasmide da un clone positivo utilizzando un kit di purificazione del plasmide seguendo le istruzioni del produttore.Posizionare il topo anestetizzato in posizione supina e radere una piccola area sui fianchi prossimali alle articolazioni dell'anca per il tessuto adiposo bianco inguinale o iniezioni IWAT con un rasoio. Aggiungere la crema depilatoria per cinque minuti. Rimuovere la crema residua con acqua per evitare ustioni cutanee.Disinfettare la pelle usando tre cicli alternati di applicazione della soluzione di iodio povidone sulla pelle con una garza pulita e alcool al 70%. Quindi, praticare un'incisione da uno a due centimetri con forbici sterilizzate nella zona prossimale delle articolazioni e tenere la pelle aperta usando una pinza per esporre il deposito di grasso. Il WAT può essere attaccato alla pelle su entrambi i lati estendendolo dall'inizio dalla schiena e verso i testicoli.Utilizzando una pinza, tirare delicatamente il deposito di grasso verso l'alto attraverso l'incisione per assicurarsi che l'iniezione sia nella posizione e nella profondità corrette. Riempire la siringa da microlitri con 2,5 microlitri di virus adeno-associato o un AAV contenente l'sgRNA che ha come bersaglio il gene endogeno PRDM16. Inserire con cautela l'ago con un angolo di 30-45 gradi nell'IWAT.Ripetere l'iniezione cinque volte in diverse posizioni del tessuto per infettare in modo omogeneo l'intero cuscinetto adiposo IWAT. Posizionare il mouse sulla piastra riscaldante fino a quando la coscienza non viene riacquistata. Monitorare l'animale ogni 10-15 minuti fino a quando non si riprende completamente.Dopo che l'animale ha ripreso conoscenza osservare il profilo della locomotiva, che dovrebbe essere lineare e non avere segni di angoscia o dolore. Vedere le cellule vascolari stromali o SVF derivate da adipo SPH mouse IWAT in una piastra a 6 pozzetti contenente mezzo DMEM completo per una o due ore, aspirare il mezzo, lavare il pozzetto due volte usando PBS e sostituirlo con terreno fresco e completo. Incubare le cellule a 37 gradi Celsius, 5% anidride carbonica e 95% di umidità fino a quando le cellule raggiungono il 70-80% di confluenza.Al giorno 0, indurre la differenziazione trattando le cellule con il mezzo di induzione e dopo due giorni sostituire il mezzo di induzione con il mezzo di mantenimento. Di nuovo dopo due giorni, al quarto giorno, sostituire il mezzo di manutenzione con un nuovo mezzo di manutenzione per 2-3 giorni. Cambiare il mezzo di mantenimento ogni 48 ore fino a quando i preadipociti non sono completamente differenziati in adipociti.Osservare gli adipociti maturi usando la microscopia ottica poiché le cellule differenziate sembrano essere cariche di goccioline lipidiche. Dopo aver fatto crescere le cellule su una piastra di coltura a 6 pozzetti con il mezzo completo fino a quando le cellule raggiungono il 70-80% di confluenza, mescolare 5,6 * 10 ^ 10 genoma virale per microlitro di sgRNA trasportatore di AAV PRDM16 con due millilitri di terreno completo e bromuro di esadimetrina. Trasduci le celle sostituendo il mezzo completo e aggiungendo il mezzo completo contenente AAV.Incubare le cellule trasdotte per 12 ore a 37 gradi Celsius 95% di umidità e 5% di anidride carbonica. Dividi e vedi le cellule come descritto per la proliferazione cellulare e la differenziazione in adipociti beige. Le immagini di immunofluorescenza da IWAT di topi adipo SPH hanno dimostrato l'espressione di dCas9 sia nel gruppo di controllo che in quello PRDM16.L'espressione di PRDM16 indotta da SPH ha chiaramente indotto un accumulo diffuso di adipociti beige multioculari in IWAT. Inoltre, la PCR quantitativa ha rivelato una maggiore espressione di PRDM16 e del programma genico termogenico nel gruppo PRDM16 rispetto al controllo. Allo stesso modo, l'analisi dell'espressione genica in vitro ha confermato l'aumento dell'espressione del gene endogeno PRDM16 e dei geni termogenici nel gruppo di sovraespressione PRDM16 rispetto al controllo.L'espressione di PRDM16 indotta da SPH ha comportato un consumo di ossigeno basale e massimo più elevato rispetto a quello delle cellule di controllo. È importante sottolineare che i dati hanno indicato una maggiore respirazione disaccoppiata nel gruppo PRDM16 rispetto a quella nel gruppo di controllo. Il modello Adipo SPH è adatto per studiare più geni ed elementi regolatori all'interno degli adipociti.Questo metodo apre la possibilità di una comprensione più profonda della biologia del grasso beige.

investigation-beige-fat-biology-metabolism-using-crispr-suntag-p65

Research

JoVE Journal

Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Rilevamento e identificazione batterica Utilizzando Sensori elettrochimici

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Ricerca

Bioingegneria

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Ricostruzioni anatomiche del sistema umano venoso cardiaco utilizzando Contrasto-tomografia computerizzata di campioni di perfusione-fissi

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Indagine sperimentale di strutture flusso secondario a valle di una mancata Modello Tipo IV stent in una sezione di prova a 180 ° curvo Artery

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Il trapianto di isole nel supporto del grasso epididimico dei topi diabetici

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

Utilizzando biologia sintetica per Ingegnere cellule viventi che si interfacciano con materiali programmabili

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Efficiente generazione e la modifica di IPSCs privo di alimentatore da cellule pancreatiche umane utilizzando il sistema di CRISPR-Cas9

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Catetere leggero intra-cardiaco per il monitoraggio del metabolismo cellulare utilizzando la spettroscopia transmurale di riambente di cuori mammiferi perfusi

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a Multiplex Genome Editing di Saccharomyces cerevisiae e la creazione di Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Costruzione di plasmidi CRISPR e rilevamento dell'efficienza knockout nelle cellule di mammifero attraverso un sistema Dual Luciferase Reporter

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Misurazione della cinematica della spalla 3D in vivo utilizzando la videoradiografia biplanare

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...