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

Forfatterne takker støtten mottatt fra Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp, for genereringen av AdipoPH-mus, Inmmunometabolism and Cell Signaling Laboratory, og National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) for all eksperimentell støtte. Vi takker den økonomiske støtten fra Sao Paulo Research Foundation (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Dyrestudier ble utført i samsvar med University of Campinas Guide for the Care and Use of Laboratory Animals (protokoll CEUA #5810-1/2021).
1. Molekylær kloning
<ol><li>Design av single guide RNA (sgRNAer)<ol><li>Design sgRNA for CRISPR-aktivering ved hjelp av CHOPCHOP, tilgjengelig på https://chopchop.cbu.uib.no/, eller et annet egnet verktøy. Bruk følgende parametere for å designe sgRNA rettet mot Prdm16-genet: Mål: Prdm16; I: Muskulus; Bruk: Crispr/Cas9; For: Aktiv...

<ol>
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En av de mest nyttige ikke-redigeringsapplikasjonene til CRISPR-teknologi er undersøkelsen av genfunksjon gjennom aktivering av endogene gener ved bruk av CRISPRa-systemer6. SPH er en kraftig CRISPRa som opprinnelig ble beskrevet for å indusere omdannelsen av astrocytter til aktive nevroner ved å målrette flere nevrogene gener14. I denne studien ble AdipoSPH vist å være et egnet verktøy for å undersøke beige fettbiologi ved å aktivere uttrykket av endogen Prdm16 i...

Beige (eller brite) adipocytter er frakoblingsprotein 1 (UCP1) -uttrykkende og mitokondrierike adipocytter som ligger i hvite fettvev (WAT) depoter. Beige fett kommer fra en delmengde av adipocytprogenitorer eller modne hvite adipocytter som respons på kald eksponering og andre stimuli 1,2. Beige adipocytter kan konvertere energi til varme på en UCP1-avhengig eller uavhengig måte3. Uavhengig av sin termogene funksjon, kan beige fett også forbedre metabolsk helse på andre måter, for eksempel sekresjon av adipokiner og antiinflammatoriske og antifibrotiske aktiviteter. Studier på mus og mennesker har vist at induksjon av beige fett forbedrer hele kroppen glukose og lipid homeostase3. Men selv om vår kunnskap om beige fettbiologi har utviklet seg raskt de siste årene, er de fleste av dens metabolske fordeler og relaterte mekanismer fortsatt ikke fullt ut forstått.
Klyngede regelmessig interspacede korte palindromiske repetisjoner (CRISPR) ble først beskrevet i eukaryote celler som et verktøy som er i stand til å generere et dobbeltstrengbrudd (DSB) på et bestemt sted i genomet gjennom nukleaseaktiviteten til Cas9-proteinet 4,5. Cas9 styres av et syntetisk enkeltveis RNA (sgRNA) for å målrette mot en bestemt genomisk region, noe som fører til en DNA DSB. I tillegg til å bruke nukleasen Cas9 til redigeringsformål, har CRISPR-Cas9-teknologien utviklet seg til å bli brukt som et sekvensspesifikt genreguleringsverktøy6. Utviklingen av et katalytisk inaktivt Cas9-protein (dCas9) og assosiasjonen av transkripsjonsmoduler som er i stand til å forbedre genuttrykk, har gitt opphav til CRISPR-aktiveringsverktøy (CRISPRa). Flere CRISPRa-systemer har dukket opp, for eksempel VP647,8, synergistisk aktiveringsmekler (SAM) 9, SunTag10,11, VPR12,13 og SunTag-p65-HSF1 (SPH) 14, som kombinerer komponentene til SAM- og SunTag-aktivatorer. Det har nylig blitt vist at det induserte uttrykket av nevrogene gener i N2a-neuroblaster og primære astrocytter er høyere ved bruk av SPH sammenlignet med andre CRISPRa-systemer14, noe som demonstrerer SPH som et lovende CRISPRa-verktøy.
Her benyttet vi oss av en tidligere utviklet SPH mus14 for å generere en betinget musemodell som uttrykker SPH spesifikt i adipocytter ved bruk av adiponektin Cre-avstamningen (AdipoSPH). Ved å bruke et adenoassosiert virus (AAV) som bærer gRNA rettet mot det endogene Prdm16-genet, ble (hvit til beige konvertering) av inguinal WAT (iWAT) indusert for å øke ekspresjonen av det termogene genprogrammet. Videre økte in vitro Prdm16-overekspresjon oksygenforbruket. Derfor gir denne protokollen en allsidig PH-musemodell for å utforske mekanismene for beige fettutvikling i fettvev.

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			<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>
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		<tr>
			<td>Amicon Ultra Centrifugal Filter</td>
			<td>Merckmillipore</td>
			<td>UFC510008</td>
			<td>100 KDa</td>
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		<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>
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		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
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		<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>
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		<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>
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</table>

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

Clustered regularly interspaced short palindromic repeats (CRISPR) teknologi har ført til en revolusjon i biologi, og nyere verktøy har blitt brukt langt utover den opprinnelig beskrevne genredigering. CRISPR-aktiveringssystemet (CRISPRa) kombinerer det katalytisk inaktive Cas9 (dCas9) proteinet med distinkte transkripsjonsmoduler for å indusere endogent genuttrykk. SunTag-p65-HSF1 (SPH) er en nylig utviklet CRISPRa-teknologi som kombinerer komponenter av synergistiske aktiveringsformidlere (SAMs) med SunTag-aktivatorene. Dette systemet tillater overekspresjon av enkle eller flere gener ved å designe et tilpasset enkeltveis RNA (sgRNA). I denne studien ble en tidligere utviklet SPH mus brukt til å generere en betinget mus som uttrykker SPH i adipocytter (adiponektin Cre avstamning), kalt AdipoSPH. For å indusere en hvit-til-beige fett () fenotype, ble et adenoassosiert virus (AAV) som bærer sgRNA rettet mot det endogene Prdm16-genet (en veletablert transkripsjonsfaktor relatert til brun og beige fettutvikling) injisert i det inguinale hvite fettvevet (iWAT). Denne musemodellen induserte uttrykket av endogen Prdm16 og aktiverte det termogene genprogrammet. Videre forbedret in vitro SPH-indusert Prdm16-overekspresjon oksygenforbruket av beige adipocytter, og fenokopierte resultatene fra en tidligere Prdm16 transgen musemodell. Dermed beskriver denne protokollen en allsidig, kostnadseffektiv og tidseffektiv musemodell for å undersøke fettvevsbiologi.

AdipoSPH mus ble utviklet av avl SPH og Adipoq-Cre musestammer. Begge musestammene var i en hybrid C57BL6J-DBA/2J bakgrunn (ifølge den kommersielle leverandøren; se materialtabell). PH-muselinjen ble opprinnelig beskrevet av Zhou et al.14.
In vivo beige adipocyttutvikling gjennom AdipoSPH-mediert Prdm16-overekspresjon For å evaluere kapasiteten til modellen beskrevet i denne studien for å utvikle beige a...

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

Denne protokollen presenterer bruk av CRISPR SunTag-p65-HSF1 (SPH) i adipocytter (AdipoSPH) som en alternativ strategi til adenoassosiert virus (AAV) for å undersøke beige fettbiologi. In vivo injeksjon av AAV-bærende sgRNA rettet mot det endogene Prdm16-genet er tilstrekkelig til å indusere beige fettutvikling og forbedre det termogene genprogrammet.

Undersøkelse av beige fettbiologi og metabolisme ved hjelp av CRISPR SunTag-p65-HSF1 aktiveringssystem

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

Denne protokollen presenterer SPH CRISPR Activation System som en alternativ strategi til konvensjonelle virale vektorer for å utføre forsterkningsfunksjonsanalyser i adipocytter. Det tillater overekspresjon av enkle eller flere adipocytter endogene gener i det komplekse cellulære miljøet av fettvev ved å levere et tilpasset enkelt guide-RNA ved hjelp av et adenoassosiert virus. De utfordrende trinnene i denne protokollen er relatert til kloning av RNA med én guide og produksjon og injeksjon av adenoassosiert virus i fettvevet.For å begynne, design enkeltguide RNA eller sgRNA for CRISPR-aktivering ved hjelp av chopchop eller et annet egnet verktøy. Design sgRNA rettet mot PRDM16-genet ved å bruke følgende parametere: Mål som PRDM16 i en Mus-muskulus ved hjelp av en CRISPR / Cas9 og for som aktivering. Legg overheng til sgRNA for å matche Sac1-begrensningsstedet i vektorryggraden PAAVU6GRNACBHM-kirsebær.Inkluder 5'AGCT 3'hvor n tilsvarer nukleotider. Oppnå 3'sgRNA omvendt komplementsekvens ved hjelp av det angitte verktøyet. Deretter, for glødning av enkeltstrengede komplementære oligonukleotider, tilsett en mikroliter av hvert 5'og 3'enkeltstrenget oligonukleotid, en mikroliter T4-ligasebuffer, 0,5 mikroliter T4 polynukleotidkinase og 6,5 mikroliter vann for et endelig reaksjonsvolum på 10 mikroliter.Anneal de komplementære enkeltstrengede oligonukleotidene ved hjelp av en termosyklist ved 37 grader Celsius i 30 minutter og 95 grader Celsius i fem minutter, etterfulgt av en nedtrappingshastighet på fem grader Celsius per minutt. Deretter tilsettes for ligering av glødet sgRNA-oligonukleotider 25 nanogram plasmid PAAVU6GRNACBHM-kirsebær til to mikroliter glødet sgRNA-oligonukleotider, en mikroliter Sac1-enzym, to mikroliter 10X T4 DNA-ligasebuffer, en mikroliter T4 DNA-ligase og vann til et endelig reaksjonsvolum på 10 mikroliter. Bruk en termosyklist, utfør ligering ved å inkubere reaksjonsblandingen i 15 sykluser ved 37 grader Celsius i fem minutter og 25 grader Celsius i fem minutter etterfulgt av å holde ved fire grader Celsius.Deretter transformerer du de kompetente Escherichia coli DH10B-cellene med fire mikroliter av ligeringsproduktet ved varmesjokk ved 42 grader Celsius i 45 sekunder og spres på en agarplate inneholdende 10 mikrogram per milliliter Ampicillin. Bekreft transformerte kolonier ved Colony Polymerase Chain Reaction eller PCR ved hjelp av PCR Master Mix, velg kolonien og bland den med fem mikroliter Master Mix, 0,1 mikroliter universell primer, 0,1 mikroliter sgRNA omvendt primer og fem mikroliter vann. Kjør PCR ved hjelp av innledende denaturering ved 94 grader Celsius i to minutter, etterfulgt av 35 sykluser med denaturering ved 94 grader Celsius i 20 sekunder, glødning i 30 sekunder ved 60 grader Celsius, forlengelse ved 72 grader Celsius i 30 sekunder, og et siste forlengelsestrinn ved 72 grader Celsius i fem minutter.Etter PCR, oppløs DNA ved hjelp av agarosegelelektroforese i 0,5X TAE-buffer ved 90 volt i 30 minutter. Send inn positive prøver for Sanger-sekvensering ved hjelp av universell primer. Deretter renser du plasmidet fra en positiv klon ved hjelp av et plasmidrensesett i henhold til produsentens instruksjoner.Plasser den bedøvede musen i liggende stilling og barber et lite område på flankene proksimalt til hofteleddene for inguinal hvitt fettvev eller IWAT-injeksjoner med barbermaskin. Tilsett hårfjerningskrem i fem minutter. Fjern gjenværende krem med vann for å unngå hudforbrenninger.Desinfiser huden ved hjelp av tre alternerende runder med påføring av povidonjodoppløsningen på huden med rent gasbind og 70% alkohol. Deretter gjør du et snitt på en til to centimeter med sterilisert saks i det proksimale området av leddene og holder huden åpen ved hjelp av tang for å avsløre fettdepotet. WAT kan festes til huden på begge sider og strekker den fra begynnelsen fra baksiden og mot testiklene.Bruk tang, trekk fettdepotet forsiktig oppover gjennom snittet for å sikre at injeksjonen er på riktig sted og dybde. Fyll mikrolitersprøyten med 2,5 mikroliter av adenoassosiert virus eller en AAV som inneholder sgRNA rettet mot det endogene PRDM16-genet. Sett kanylen forsiktig i en 30-45 graders vinkel inn i IWAT.Gjenta injeksjonen fem ganger på forskjellige steder i vevet for å homogent infisere hele IWAT fettputen. Plasser musen på varmeputen til bevisstheten er gjenvunnet. Overvåk dyret hvert 10. til 15. minutt til det gjenoppretter helt.Etter at dyret gjenvinner bevisstheten, observer lokomotivprofileringen, som skal være lineær og ikke ha tegn på nød eller smerte. Se de stromale vaskulære cellene eller SVF-ene avledet fra adipo PH-mus IWAT til en 6-brønnplate som inneholder komplett DMEM-medium i en til to timer, aspirer mediet, vask brønnen to ganger ved hjelp av PBS, og erstatt den med friskt, komplett medium. Inkuber cellene ved 37 grader Celsius, 5% karbondioksid og 95% fuktighet til cellene når 70-80% konfluens.På dag 0 induserer du differensiering ved å behandle cellene med induksjonsmediet og etter to dager erstattes induksjonsmidlet med vedlikeholdsmediet. Igjen etter to dager, på dag fire, erstatt vedlikeholdsmediet med et nytt vedlikeholdsmedium i 2-3 dager. Bytt vedlikeholdsmedium hver 48. time til preadipocytter er fullstendig differensiert til adipocytter.Observer de modne adipocytter ved hjelp av lysmikroskopi, da de differensierte cellene ser ut til å være lastet med lipiddråper. Etter å ha dyrket cellene på en 6-brønns kulturplate med hele mediet til cellene når 70-80% konfluens, bland 5,6 * 10 ^ 10 viralt genom per mikroliter AAV-bærende sgRNA PRDM16 med to milliliter komplett medium og heksadimetrinbromid. Transdusere cellene ved å erstatte hele mediet og tilsette hele mediet som inneholder AAV.Inkuber de transduserte cellene i 12 timer ved 37 grader Celsius, 95% fuktighet og 5% karbondioksid. Splitt og se cellene som beskrevet for celleproliferasjon og differensiering til beige adipocytter. Immunfluorescensbildene fra IWAT av adipo PH-mus viste uttrykket av dCas9 i både kontroll- og PRDM16-gruppene.SPH-indusert PRDM16-uttrykk induserte tydelig en utbredt akkumulering av multiokulære beige adipocytter i IWAT. Videre viste kvantitativ PCR økt ekspresjon av PRDM16 og det termogene genprogrammet i PRDM16-gruppen sammenlignet med kontrollgruppen. Tilsvarende bekreftet in vitro genekspresjonsanalyse det økte uttrykket av det endogene PRDM16-genet og termogene gener i PRDM16-overekspresjonsgruppen sammenlignet med kontrollen.Det SPH-induserte PRDM16-uttrykket resulterte i høyere basalt og maksimalt oksygenforbruk enn i kontrollceller. Det er viktig at dataene indikerte forbedret ukoblet respirasjon i PRDM16-gruppen sammenlignet med kontrollgruppen. Adipo PH-modellen er egnet for å undersøke flere gener og regulatoriske elementer i adipocytter.Denne metoden åpner muligheten for en dypere forståelse av beige fettbiologi.

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.

Bakteriell Detection &amp; Identification hjelp elektrokjemiske sensorer

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

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.

Anatomiske rekonstruksjoner av Human Cardiac venøse systemet ved hjelp av kontrast-computertomografi av Perfusjons-fast Prøver

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.

Eksperimentell Undersøkelse av sekundære strøm Structures Nedstrøms av en modell Type IV Stent Svikt i en 180 ° Buet Artery Test Seksjon

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.

Stillasstøttet transplantasjon av øyer i epididymisk fettpute av diabetiske mus

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.

Bruke Syntetisk biologi Ingeniør levende celler som grensesnitt med Programmerbare Materials

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.

Effektiv generasjon og redigering av mater-gratis IPSCs fra menneskelige bukspyttkjertelen celler ved hjelp av 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 ...

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.

Intra-CARDIAC side-avfyring lys kateter for overvåking Cellular metabolisme bruker transmuralt absorbansen spektroskopi av Perfusert pattedyr hjerter

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 Genova redigering av Saccharomyces cerevisiae og opprettelse av gjær 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.

Bygging av CRISPR Plasmids og påvisning av Knockout effektivitet i pattedyrceller gjennom en 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. ...

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.

Måling av 3D In-vivo skulderkinematikk ved hjelp av biplanet videoradiografi

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