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

Les auteurs remercient le soutien reçu du Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp, pour la génération de souris AdipoSPH, du Laboratoire d’inmmunométabolisme et de signalisation cellulaire et de l’Institut national des sciences et technologies en photonique appliquée à la biologie cellulaire (INFABIC) pour tout le soutien expérimental. Nous remercions le soutien financier de la Fondation de recherche de Sao Paulo (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Les études animales ont été réalisées conformément au Guide de l’Université de Campinas pour le soin et l’utilisation des animaux de laboratoire (protocole CEUA #5810-1/2021).
1. Clonage moléculaire
<ol><li>Conception d’ARN guides uniques (ARNsg)<ol><li>Concevoir des sgRNA pour l’activation de CRISPR à l’aide de CHOPCHOP, disponible au https://chopchop.cbu.uib.no/, ou de tout autre outil approprié. Utilisez les paramètres suivants pour concevoir l’ARNg...

<ol>
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L’une des applications non éditrices les plus utiles de la technologie CRISPR est l’interrogation de la fonction des gènes par l’activation de gènes endogènes à l’aide de systèmes CRISPRa6. SPH est un puissant CRISPRa qui a été décrit à l’origine pour induire la conversion des astrocytes en neurones actifs en ciblant plusieurs gènes neurogènes14. Dans cette étude, AdipoSPH s’est avéré être un outil approprié pour étudier la biologie des graisse...

Les adipocytes beiges (ou brite) sont des adipocytes exprimant la protéine 1 (UCP1) et des adipocytes riches en mitochondries qui résident dans des dépôts de tissu adipeux blanc (WAT). La graisse beige émerge d’un sous-ensemble de progéniteurs adipocytaires ou d’adipocytes blancs matures en réponse à l’exposition au froid et à d’autres stimuli 1,2. Les adipocytes beiges peuvent convertir l’énergie en chaleur d’une manière dépendante ou indépendante de UCP13. Indépendamment de sa fonction thermogénique, la graisse beige peut également améliorer la santé métabolique par d’autres moyens, tels que la sécrétion d’adipokines et les activités anti-inflammatoires et anti-fibrotiques. Des études chez la souris et l’homme ont montré que l’induction de graisse beige améliore le glucose dans tout le corps et l’homéostasie lipidique3. Cependant, bien que nos connaissances sur la biologie de la graisse beige aient évolué rapidement ces dernières années, la plupart de ses avantages métaboliques et des mécanismes connexes ne sont pas encore entièrement compris.
Les courtes répétitions palindromiques groupées régulièrement espacées (CRISPR) ont d’abord été décrites dans des cellules eucaryotes comme un outil capable de générer une rupture double brin (DSB) à un site spécifique du génome par l’activité nucléase de la protéine Cas9 4,5. Cas9 est guidé par un ARN monoguide synthétique (sgRNA) pour cibler une région génomique spécifique, conduisant à un ADN DSB. En plus d’utiliser la nucléase Cas9 à des fins d’édition, la technologie CRISPR-Cas9 a évolué pour être utilisée comme outil de régulation génique spécifiqueà la séquence 6. Le développement d’une protéine Cas9 catalytiquement inactive (dCas9) et l’association de modules transcriptionnels capables d’améliorer l’expression génique ont donné naissance à des outils d’activation CRISPR (CRISPRa). Plusieurs systèmes CRISPRa ont vu le jour, tels que VP647,8, médiateur d’activation synergique (SAM)9, SunTag10,11, VPR12,13 et SunTag-p65-HSF1 (SPH)14, qui combine les composants des activateurs SAM et SunTag. Il a récemment été démontré que l’expression induite des gènes neurogènes dans les neuroblastes N2a et les astrocytes primaires est plus élevée en utilisant SPH par rapport aux autres systèmes CRISPRa14, démontrant SPH comme un outil CRISPRa prometteur.
Ici, nous avons profité d’une souris SPH14 précédemment développée pour générer un modèle murin conditionnel exprimant la SPH spécifiquement dans les adipocytes en utilisant la lignée Cre adiponectine (AdipoSPH). En utilisant un virus adéno-associé (AAV) portant l’ARNg ciblant le gène endogène Prdm16, le brunissement (conversion blanc en beige) de WAT inguinal (iWAT) a été induit pour augmenter l’expression du programme de gène thermogénique. De plus, la surexpression in vitro de Prdm16 a augmenté la consommation d’oxygène. Par conséquent, ce protocole fournit un modèle murin SPH polyvalent pour explorer les mécanismes de développement de la graisse beige dans le tissu adipeux.

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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 technologie CRISPR (Clustered Regular Interspaced Short Palindromic Repeats) a provoqué une révolution en biologie, et des outils récents ont été appliqués bien au-delà de l’édition de gènes décrite à l’origine. Le système d’activation CRISPR (CRISPRa) combine la protéine catalytiquement inactive Cas9 (dCas9) avec des modules de transcription distincts pour induire l’expression endogène des gènes. SunTag-p65-HSF1 (SPH) est une technologie CRISPRa récemment développée qui combine des composants de médiateurs d’activation synergiques (SAM) avec les activateurs SunTag. Ce système permet la surexpression d’un ou de plusieurs gènes en concevant un ARN monoguide personnalisé (sgRNA). Dans cette étude, une souris SPH précédemment développée a été utilisée pour générer une souris conditionnelle exprimant la SPH dans les adipocytes (lignée de l’adiponectine Cre), appelée AdipoSPH. Pour induire un phénotype de graisse blanche à beige (brunissant), un virus adéno-associé (AAV) porteur d’ARNg ciblant le gène endogène Prdm16 (un facteur de transcription bien établi lié au développement de la graisse brune et beige) a été injecté dans le tissu adipeux blanc inguinal (iWAT). Ce modèle murin a induit l’expression de Prdm16 endogène et activé le programme de gènes thermogéniques. De plus, la surexpression in vitro de Prdm16 induite par SPH a augmenté la consommation d’oxygène des adipocytes beiges, phénocopiant les résultats d’un précédent modèle murin transgénique Prdm16. Ainsi, ce protocole décrit un modèle murin polyvalent, rentable et rapide pour étudier la biologie du tissu adipeux.

Les souris AdipoSPH ont été développées en sélectionnant des souches de souris SPH et Adipoq-Cre. Les deux souches de souris étaient dans un fond hybride C57BL6J-DBA/2J (selon le fournisseur commercial; voir le tableau des matériaux). La lignée de souris SPH a été décrite à l’origine par Zhou et al.14.
Développement in vivo d’adipocytes beiges par surexpression de Prdm16 médiée par AdipoSPH<b...

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

Ce protocole présente l’utilisation de CRISPR SunTag-p65-HSF1 (SPH) dans les adipocytes (AdipoSPH) comme stratégie alternative au virus adéno-associé (AAV) pour étudier la biologie de la graisse beige. L’injection in vivo d’ARNg porteur d’AAV ciblant le gène endogène Prdm16 est suffisante pour induire le développement de graisse beige et améliorer le programme de gènes thermogéniques.

Étude de la biologie et du métabolisme des graisses beiges à l’aide du système d’activation 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 ...

Ce protocole présente le système d’activation SPH CRISPR comme une stratégie alternative aux vecteurs viraux conventionnels pour effectuer des tests de fonction de gain dans les adipocytes. Il permet la surexpression d’un ou de plusieurs gènes endogènes adipocytaires dans l’environnement cellulaire complexe du tissu adipeux en délivrant un ARN guide unique personnalisé à l’aide d’un virus associé à un adéno. Les étapes difficiles de ce protocole sont liées au clonage de l’ARN guide unique et à la production et à l’injection de virus adéno-associés dans le tissu adipeux.Pour commencer, concevez un ARN guide unique ou un ARNsg pour l’activation de CRISPR à l’aide de chopchop ou de tout autre outil approprié. Concevoir l’ARNg ciblant le gène PRDM16 en utilisant les paramètres suivants:Cibler en tant que PRDM16 dans un Mus musculus en utilisant un CRISPR / Cas9 et pour comme activation. Ajouter des surplombs à l’ARNg pour correspondre au site de restriction Sac1 dans la cerise du squelette vecteur PAAVU6GRNACBHM.Inclure 5'AGCT 3'où n correspond aux nucléotides. Obtenez la séquence inverse du complément 3'sgRNA à l’aide de l’outil indiqué. Ensuite, pour le recuit des oligonucléotides complémentaires simple brin, ajoutez un microlitre de chaque oligonucléotide simple brin 5' et 3', un microlitre de tampon ligase T4, 0,5 microlitre de polynucléotide kinase T4 et 6,5 microlitres d’eau pour un volume de réaction final de 10 microlitres.Recuit les oligonucléotides simple brin complémentaires à l’aide d’un thermocycleur à 37 degrés Celsius pendant 30 minutes et à 95 degrés Celsius pendant cinq minutes, suivi d’un taux de réduction progressive de cinq degrés Celsius par minute. Ensuite, pour la ligature des oligonucléotides d’ARNg recuits, ajoutez 25 nanogrammes de cerise plasmidique PAAVU6GRNACBHM à deux microlitres d’oligonucléotides d’ARNg recuits, un microlitre d’enzyme Sac1, deux microlitres de tampon ADN ligase 10X T4, un microlitre d’ADN ligase T4 et de l’eau à un volume de réaction final de 10 microlitres. À l’aide d’un thermocycleur, effectuer la ligature en incubant le mélange réactionnel pendant 15 cycles à 37 degrés Celsius pendant cinq minutes et 25 degrés Celsius pendant cinq minutes, puis en maintenant à quatre degrés Celsius.Ensuite, transformez les cellules DH10B compétentes d’Escherichia coli avec quatre microlitres du produit de ligature par choc thermique à 42 degrés Celsius pendant 45 secondes et étalez-les sur une plaque de gélose contenant 10 microgrammes par millilitre d’ampicilline. Confirmez les colonies transformées par réaction en chaîne de la polymérase des colonies ou PCR à l’aide du mélange maître PCR, choisissez la colonie et mélangez-la avec cinq microlitres de mélange maître, 0,1 microlitre d’apprêt universel, 0,1 microlitre d’amorce inverse d’ARNg et cinq microlitres d’eau. Exécutez la PCR en utilisant la dénaturation initiale à 94 degrés Celsius pendant deux minutes, suivie de 35 cycles de dénaturation à 94 degrés Celsius pendant 20 secondes, le recuit pendant 30 secondes à 60 degrés Celsius, l’extension à 72 degrés Celsius pendant 30 secondes et une étape finale d’allongement à 72 degrés Celsius pendant cinq minutes.Après PCR, résoudre l’ADN en utilisant l’électrophorèse sur gel d’agarose dans un tampon TAE 0,5X à 90 volts pendant 30 minutes. Soumettez des échantillons positifs pour le séquençage de Sanger à l’aide d’un apprêt universel. Ensuite, purifiez le plasmide d’un clone positif à l’aide d’un kit de purification plasmidique en suivant les instructions du fabricant.Placez la souris anesthésiée en décubitus dorsal et rasez une petite zone sur les flancs proximal aux articulations de la hanche pour les injections de tissu adipeux blanc inguinal ou les injections IWAT avec un rasoir. Ajouter la crème dépilatoire pendant cinq minutes. Enlevez la crème résiduelle avec de l’eau pour éviter les brûlures de la peau.Désinfectez la peau en utilisant trois cycles alternés d’application de la solution de povidone iodée sur la peau avec de la gaze propre et de l’alcool à 70%. Ensuite, faites une incision d’un à deux centimètres avec des ciseaux stérilisés dans la zone proximale des articulations et maintenez la peau ouverte à l’aide de forceps pour exposer le dépôt de graisse. Le WAT peut être attaché à la peau des deux côtés en l’étendant depuis le début du dos et vers les testicules.À l’aide d’une pince, tirez doucement le dépôt de graisse vers le haut à travers l’incision pour vous assurer que l’injection est au bon endroit et à la bonne profondeur. Remplissez la seringue de microlitre avec 2,5 microlitres du virus adéno-associé ou d’un AAV contenant l’ARNg ciblant le gène endogène PRDM16. Insérez délicatement l’aiguille à un angle de 30 à 45 degrés dans l’IWAT.Répétez l’injection cinq fois à différents endroits du tissu pour infecter de manière homogène l’ensemble du coussinet adipeux IWAT. Placez la souris sur le coussin chauffant jusqu’à ce que la conscience soit retrouvée. Surveillez l’animal toutes les 10 à 15 minutes jusqu’à ce qu’il se rétablisse complètement.Une fois que l’animal a repris conscience, observez le profilage de la locomotive, qui doit être linéaire et ne présenter aucun signe de détresse ou de douleur. Voir les cellules vasculaires stromales ou SVF dérivées de l’IWAT de souris adipo SPH dans une plaque à 6 puits contenant un milieu DMEM complet pendant une à deux heures, aspirer le milieu, laver le puits deux fois avec du PBS et le remplacer par un milieu frais et complet. Incuber les cellules à 37 degrés Celsius, 5% de dioxyde de carbone et 95% d’humidité jusqu’à ce que les cellules atteignent 70-80% de confluence.Au jour 0, induire la différenciation en traitant les cellules avec le milieu d’induction et après deux jours remplacer le milieu d’induction par le milieu d’entretien. Encore une fois après deux jours, au quatrième jour, remplacez le milieu d’entretien par un nouveau milieu d’entretien pendant 2-3 jours. Changez le milieu d’entretien toutes les 48 heures jusqu’à ce que les préadipocytes soient complètement différenciés en adipocytes.Observez les adipocytes matures à l’aide de la microscopie optique car les cellules différenciées semblent être chargées de gouttelettes lipidiques. Après avoir cultivé les cellules sur une plaque de culture à 6 puits avec le milieu complet jusqu’à ce que les cellules atteignent 70-80% de confluence, mélanger 5,6 * 10 ^ 10 % 10 génome viral par microlitre d’ARNg PRDM16 porteur d’AAV avec deux millilitres de milieu complet et du bromure d’hexadiméthrine. Transduisez les cellules en remplaçant le milieu complet et en ajoutant le milieu complet contenant AAV.Incuber les cellules transduites pendant 12 heures à 37 degrés Celsius 95% d’humidité et 5% de dioxyde de carbone. Divisez et voyez les cellules comme décrit pour la prolifération cellulaire et la différenciation en adipocytes beiges. Les images d’immunofluorescence de l’IWAT de souris adipo SPH ont démontré l’expression de dCas9 dans les groupes témoin et PRDM16.L’expression de PRDM16 induite par SPH a clairement induit une accumulation généralisée d’adipocytes beiges multioculaires dans l’IWAT. De plus, la PCR quantitative a révélé une expression accrue de PRDM16 et du programme de gènes thermogéniques dans le groupe PRDM16 par rapport au groupe témoin. De même, l’analyse in vitro de l’expression génique a confirmé l’expression accrue du gène endogène PRDM16 et des gènes thermogéniques dans le groupe de surexpression PRDM16 par rapport au groupe témoin.L’expression de PRDM16 induite par SPH a entraîné une consommation d’oxygène basale et maximale plus élevée que celle des cellules témoins. Il est important de noter que les données indiquaient une amélioration de la respiration non couplée dans le groupe PRDM16 par rapport à celle du groupe témoin. Le modèle Adipo SPH convient à l’étude de plusieurs gènes et éléments régulateurs au sein des adipocytes.Cette méthode ouvre la possibilité d’une compréhension plus profonde de la biologie de la graisse 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.

Bacterial Detection &amp; identification au moyen de capteurs électrochimiques

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

Recherche

Bio-ingénierie

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.

Reconstructions anatomiques du système veineux cardiaque humaine à l&#39;aide de contraste tomodensitométrie des échantillons Perfusion-fixes

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.

Etude expérimentale des structures de flux secondaire en aval d&#39;un échec Modèle Type IV Stent dans une section d&#39;essai de 180 ° Artère Curved

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.

Transplantation soutenue d&#39;échafaudage d&#39;îlots dans la pate épididimale de souris diabétiques

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.

Utilisation de la biologie synthétique à l&#39;ingénieur cellules vivantes Interface avec les matériaux programmables

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.

Génération efficace et l’édition de CISP exempte d’engraissement de cellules pancréatiques humaines en utilisant le système 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.

Cathéter de lumière à tir latéral intra-cardiaque pour la surveillance du métabolisme cellulaire à l'aide de la spectroscopie d'absorption transmurale des cœurs de mammifères perfusés

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

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.

Construction de plasmides CRISPR et détection de l’efficacité des knockouts dans les cellules mammifères grâce à un système de double 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.

Mesure de la cinématique 3D in vivo de l’épaule à l’aide de la vidéoradiographie biplanaire

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