Name: customization of aspergillus niger morphology through addition of talc micro particles
Uploaded: 2012-03-15T13:00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles at JoVE.com

Forfatterne ønsker å takke for økonomisk støtte gitt av den tyske Research Foundation (DFG) gjennom forskningssamarbeid senteret SFB 578 &quot;Fra Gene til Produkt&quot; ved Technische Universität Braunschweig, Tyskland.

1. Reactor Oppsett og Start av Dyrking 4 bioreaktor jordbearbeiding totalt blir gjennomført. <ol><li> Bruk en 3 L rørt tank bioreaktor med en fungerende volum på 2,2 L for dyrking av A. Niger SKAn1015. </li><li> Hell 72,6 g glukose monohydrat inn i reaktoren, og fylle den opp med 1,9 L avionisert vann. </li><li> Installer reaktoren utstyr som 3 Bafflene, en to seks-blader disk turbin impeller, en pH elektrode, en gass innløp med luft filter, en avkjølende finger, en avtrekksluft kjøler med luft filter, en nedsenking tube for sterilt prøvetaking med luft filter og inngang slanger for medium, podestoff, syre og base. </li><li> Autoklaver reaktoren ved 121 ° C i 20 min. </li><li> Koble reaktoren med syre-base reservoar (2 M HCl, 2 M NaOH) og de tilsvarende pumper og sette opp en pH-verdi på 5,0 ± 0,05 med pH kontrollenheten. </li><li> Lenke reaktoren med kjøling-vann system og sette varmekapperundt reaktortanken. </li><li> Installer temperaturføleren med tilhørende kontrollenhet og sette opp en temperatur på 37 ± 0,1 ° C ved temperaturstyringsenhet. </li><li> Monter agitasjon motor på toppen av reaktoren og bringe det inn i tjeneste med en uro hastighet på 200 min -1. </li><li> Legg til sterile minimal vekst medium 11 gjennom en av de sterile innløpet slangene (250 ml). For middels forberedelse alle komponentene ble autoklaveres ved 121 ° C i 20 min og blandet sammen. For tre jordbearbeiding komponentene inkluderer talkum pulver (3MgO • 4SiO 2 • H 2 O) i konsentrasjonene av 1 g / L (reaktor 2), 3 g / L (reaktor 3) og 10 g / L (reaktor 4). Før bruk, re-suspendere mikropartikler i 50 mM natriumacetat buffer (pH 6,5) og legge den til sterilt medium. I kontroll kulturer (uten partikler) erstatte mikro partikkel fjæringen med 50 mM natriumacetat buffer (pH 6,5). </li><li> Legg sporeoppløsning (podestoff) ens utarbeidet i Wucherpfennig et. al (2011) 11 gjennom en av de sterile innløpet slangene (50 ml) slik at konsentrasjonen av sporer utgjør 1x10 6 ml -1. Inokuleringen markerer starten på dyrking (h = 0 h). </li><li> Start lufting med en rate på 1,0 L min -1. </li></ol> 2. Steril Prøvetaking etter 24, 48 og 72 timer av Dyrking <ol><li> Ta 50 ml sterilt kultur buljong i en flacon tube. </li><li> Bruk utvalget for bestemmelse av biomasse tørrvekt, β-fructofuranosidase aktivitet og mikroskopisk analyse. </li></ol> 3. Fastsettelse av biomasse Tørrvekt etter 24, 48 og 72 timer av Dyrking <ol><li> Biomasse prøvene skal tas minst i to eksemplarer. </li><li> Vekt en cellulose filter med en mikro skalaer etter tørking i eksikkator og legg filteret i en Büchner trakt med tilkoblet vann-jet vakuumpumpe. </li><li> Filtrere et definert prøvevolum (f.eks 10 ml) ennd skylle filteret med 10 ml avionisert vann for å fjerne middels forbindelser fra biomasse. </li><li> Rynke filteret en gang i midten, legg den i et glass petriskål og sette det inn i en kupé tørketrommel til vekt constancy (minst 24 h). </li><li> Avkjøl filteret i en eksikkator og måle vekten. </li><li> Beregn biomasse tørrvekt som differansen mellom vekten av filter med og uten tørket biomasse fordelt på brukte prøvevolum. </li></ol> 4. Fastsettelse av ekstracellulær enzymatisk aktivitet av β-fructofuranosidase av Gud / POD-essay etter 24, 48 og 72 timer av Dyrking Oppbevar prøvene på is hele tiden mens du arbeider med dem. <ol><li> Filter 1,5 mL kultur suspensjon gjennom en celluloseacetat filter ved å trykke på kulturen suspensjonen med en sprøyte gjennom filteret i en reaksjon tube. Oppbevar reaksjonsrøret ved -20 ° C til bruk. </li><li> For reaksjonsblandingen bruke 20mL prøve og legge 200 mL av 1,65 M sukrose løst i 0,05 M fosfat buffer (pH 5,4) for å starte reaksjonen fra sukrose til glukose. Cary ut reaksjonen i alle fall i to eksemplarer. </li><li> Inkuber reaksjonsblandingen ved 40 ° C i 20 min i en oppvarming blokk. Stopp reaksjonen ved å inkubere ved 95 ° C i 10 min i en oppvarming blokk. Kjøl reaksjonsblandingen ved å lagre den på is og spinne ned det kondenserte vannet ved sentrifuger ved 13.000 g for 10 min ved 4 ° C. </li><li> Til konto pH og temperatur avhengig spalting av sukrose til glukose, gjennomføre en tom verdi ved å bruke 20 mL avionisert vann i stedet for 20 mL prøve. </li><li> Til konto for gjenværende glukose i kulturen kjøttkraft, utføre en negativ kontroll for hver prøve. For at sluttbruk 20 mL av prøven og inaktiverer β-fructofuranosidase ved oppvarming ved 95 ° C i 10 min før for å legge til sukrose og inkuberes som beskrevet i trinn 4.3. </li><li> Fortynn prøvene slik at følgende målermåles adsorpsjon er i den kalibrerte verdiområdet. </li><li> Gjennomføre alle enzymatiske essay i tre eksemplarer. Påfør 2 mL fortynnet prøve i en mikro titer plate godt. Påfør en standard serie for kalibrering på mikro titer plate ved hver med 2 mL av ti glukoseoppløsninger med ti forskjellige kjenner konsentrasjoner (fra 1 mm til 15 mm) i stedet for 2 mL prøve. For nullpunktet kalibrering bruke 2 mL avionisert vann i stedet for 2 mL prøve. </li><li> Tilsett 200 mL av reagensen løsning til hver brønn ved hjelp av en multi pipette. </li><li> Inkuber blandingen i 10 minutter ved romtemperatur. </li><li> Oppbevar mikro titer platen ved 6 ° C til mål (noen få timer på de fleste). </li><li> Mål absorpsjon ved 450 nm ved hjelp av en 96-brønn Sunrise mikro plateavleseren og Magellan innhenting av data programvare. Still blandetiden opp til 5 sek. og resten perioden fram til 1 sek. </li><li> Åpne resultatet diagrammet med et regneark og konstruere en kalibrering linje ved hjelp av standard område: glucose konsentrasjon = a X absorpsjon + b </li><li> Beregn aktivitet: aktivitet = (absorpsjon X fortynningsfaktoren-blank verdi) X a + b </li><li> Regne ut den β-fructofuranosidase aktivitet ved å beregne forskjellen mellom virksomheten i utvalget og den egnet negativ kontroll. </li><li> Beregn spesifikk produktivitet for følgende bruk ved å ta hensyn til biomasse tørrvekt og β-fructofuranosidase aktivitet 11. </li></ol> 5. Mikroskopi og automatisert bildeanalyse etter 72 timers Dyrking <ol><li> Sted rundt 3 ml kultur suspensjon i en plast petriskål ogfortynne dem med fysiologisk natriumkloridoppløsning før morfologiske strukturer blir separert. </li><li> Plasser petriskål under et mikroskop, som har en integrert eller tilkoblet kamera. Erverve og lagre rundt 100 bilder (figur 2) av morfologiske strukturer per prøve. Vær oppmerksom på at på hvert bilde på minst ett objekt er helt avbildet. </li><li> Åpne alle bilder av den samme prøven med bildebehandling programmet ImageJ 12. Konverter bildene til svart og hvitt ved hjelp av prosessen verktøyet &quot;Lag Binary&quot; (figur 2). For å bruke kommandoen til hele serien av bilder bruker du en makro kode som følger. løpe (,, Gjør Binary &quot;) </li><li> Åpne bearbeidede binære bilder med ImageJ igjen. Beregn formen faktorer Feret diameter, anslått areal, omkrets, sirkularitet, sideforhold, rundhet und soliditet for hvert bilde med analysere verktøyet &quot;Set Measurement&quot;. For å bruke kommandoen til en Series av bilder bruker du en makro kode som følger. kjøre (&quot;8-bit&quot;); kjøre (&quot;Gjør Binary&quot;); kjøre (&quot;Set Scale ...&quot;, &quot;avstand = X kjent = 1000 pixel = 1 enhet = mikrometer global&quot;); kjøre (&quot;Sett Mål ...&quot;, &quot;areal omkrets form feret grense skjerm viderekobler = Ingen desimal = 3&quot;); kjøre (&quot;Analyze Partikler ...&quot;, &quot;size = 10000-Infinity sirkularitet = 0,00 til 1,00 showet = Outlines display&quot;); Bestem X som antall piksler som korrelerer til 1000 mikrometer ved å konstruere en rett linje over en skala bar. Correlate antall piksler i rett linje med lengden av skalaen bar i mikrometer. </li><li> Åpne resultatet diagrammet inneholder form factor verdiene for hvert bilde med et regneark. Beregn en Morfologi nummer som følger for hvert bilde. <img alt="Formel 1" src="/files/ftp_upload/4023/4023eq1.jpg" /></li><li> Beregn middelverdi og standardavvik for morfologi tallet tar hensyn til alle bilder av en prøve. </li&gt; <li> Bruk en grafisk og data analyse program for grafisk korrelasjon av morfologi tallet med den spesifikke produktivitet og bestemme den matematiske forhold ved matematisk regresjon. </li></ol> 6. Representative Resultater Gjennom tilførsel av talkum mikro partikler A. Niger Skan 1015 morfologi er forandret fra en sann pellet morfologi til en spredt eller mycelial morfologi. Mens pellet morfologi er utstilt ved normaltilstand en mycelial morfologi er skapt av tilskudd av medium med 10 g / L av talkum mikropartikler (figur 4.). Samtidig aktiviteten av β-fructofuranosidase øker rundt tre ganger 3-5. Et tilskudd på 1 eller 3 g / L av talkum pulver fører til en spredt morfologi, med en doblet fructofuranosidase aktivitet (figur 4.). Mikro partikkel avhengige morfologi kan være omfattende beskrevet av Morphology nummer som kan beregnes ved hjelp av parametere bestemt ved automatisk bildeanalyse. Perfekt runde og glatte pellets vil i mikroskopiske bilder vises som perfekte sirkler. For slike partikler morfologi nummeret har en verdi på 1. Den minste fragment av mycelial morfologi kan forenkles som en endimensjonal linje gir en Morfologi antall 0. Alle mellomliggende morfologiske former som avlange uregelmessige pellets eller klumper vil derfor ha verdier mellom 0 og 1. Ganske store partikler vil resultere i en høy, sopp partikler med en stor overflate eller avlange partikler, i en ganske lav Morfologi nummer 11. Ved normaltilstand morfologi i reaktoren en utstillinger en Morfologi rekke rundt 0,8. Morfologi i reaktoren 4 med 10 g / l talkum pulver har en Mn rundt 0,1. Morfologi tall for reaktorer 2 og 3, med talkum pulver konsentrasjoner av 1 og 3 g / L, ligger mellom disse ytterpunktene, viser en spredt Morphologi. Siden micro partikkel avhengig morfologi er nært beslektet med β-fructofuranosidase produktivitet, er en matematisk sammenheng av morfologi nummer og produktivitet som ligner på Figur 5 innhentet. <img alt="Figur 1" src="/files/ftp_upload/4023/4023fig1.jpg" /> Figur 1. Generelle ordningen for eksperimentell design og den analytiske prosedyren. A. Niger er dyrket (med eller uten mikropartikler) i en 3 L rørt tank bioreaktor for 72 timer. Etter 24, blir 48 og 72 ha prøve som er tatt for bestemmelse av biomasse tørr vekt og β-fructofuranosidase aktivitet, som igjen brukes til beregning av den spesifikke produktivitet. Etter 48 timers soppens morfologi blir undersøkt av mikroskop og preget av digital bildeanalyse. Relevante parametere for bildeanalyse kombineres til en Morfologi tall, noe som er matematisk korrelert med spesifikke produktivitet. <imgalt = &quot;Figur 2&quot; src = &quot;/ files/ftp_upload/4023/4023fig2.jpg&quot; /&gt; Figur 2. Steps av bildebehandlingen for mikroskopisk-genererte bilder av morfologiske strukturer fra A. Niger. Trinn 1: image oppkjøpet av mikroskop. Trinn 2: image forbedring om nødvendig. Trinn 3: image binarization, svart-hvitt (binær) image generert på ImageJ. Trinn 4: det binære bildet er behandlet av og uønsket objekt slettes. Trinn 5: morfologisk analyse er utført med &quot;Analyze partikler&quot; funksjonen til åpen kildekode-program ImageJ. <img alt="Figur 3" src="/files/ftp_upload/4023/4023fig3.jpg" /> Figur 3. Ulike morfologiske former for A. Niger avhengig av konsentrasjonen av tilsatte mikropartikler. Med økende konsentrasjon av mikropartikler lagt til pelletstørrelse kan være nettopp redusert ned til små kjerne-skall pellets, små flokker og selv fritt spredt mycel. Morfologi prosjektering av Aspergillus Niger SKAn1015 av micro partikkel tilskudd i neddykket kultur. Uten mikropartikler (A), 10 mg / L (B), 0,1 g / L (C), 0,2 g / L (D), 0,3 g / L (E), 0,6 g / L (F), 1,0 g / L (G), 1,5 g / L (H), 2,0 g / L (I), 2,5 g / L (J), 3,0 g / L (K), 3,5 g / L (L), 4,0 g / L (M ), 4,5 g / L (N), 5,0 g / L (O), 10 g / L (P), 15 g / L (Q), 20 g / L (R), 30 g / L (S) og 40-50 g / L (T). Bilder ble tatt av lysmikroskopi etter 72 timer av kultivering. <img alt="Figur 4" src="/files/ftp_upload/4023/4023fig4.jpg" /> Figur 4. Fructofuranosidase aktivitet i avhengighet av talkum mikro partikkelkonsentrasjonen 1 g / L (reaktor 2), 3 g / L (reaktor 3) og 10 g / L (reaktor 4). Reactor 1 er ikke supplert med mikropartikler, her ved dyrking er utført under vanlige forhold. <img alt="Figur 5" src="/files/ftp_upload/4023/4023fig5.jpg" /> Figur 5. Representant god korrelasjon (R 2 = 0,91) av Morfologinummer og den spesifikke produktivitet. Morfologi nummer plottes (abscissen) mot den spesifikke produktivitet (samordner). Ulineær regresjon gir den eksponentielle korrelasjon.

<ol>
	<li>Wucherpfennig, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Applied Microbiology. 72, 89-136 (2010).</li><li>Driouch, H., H&#228;nsch, R., Wucherpfennig, T., Krull, R., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzyme+production+by+bio-pellets+of+Aspergillus+niger:+Targeted+morphology+engineering+using+titanate+microparticles.">Improved enzyme production by bio-pellets of Aspergillus niger: Targeted morphology engineering using titanate microparticles.</a> Biotechnology and Bioengineering. 109, 462-471 (2012).</li><li>Driouch, H., Roth, A., Dersch, P., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+bioprocess+for+production+of+fructofuranosidase+by+recombinant+Aspergillus+niger.">Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger.</a> Applied Microbiology and Biotechnology. 87, 2011-2024 (2010).</li><li>Driouch, H., Roth, A., Dersch, P., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fungi+in+good+shape:+Microparticles+for+tailor-made+fungal+morphology+and+enhanced+enzyme+production.">Filamentous fungi in good shape: Microparticles for tailor-made fungal morphology and enhanced enzyme production.</a> Bioengineered Bugs. 2, 100-104 (2011).</li><li>Driouch, H., Sommer, B., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+engineering+of+Aspergillus+niger+for+improved+enzyme+production.">Morphology engineering of Aspergillus niger for improved enzyme production.</a> Biotechnology and Bioengineering. 105, 1058-1068 (2010).</li><li>Kaup, B. -. 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Modifikasjonen av sopp morfologi har vært av interesse for bioteknologi ettersom mange tiår. Ulike studier har forsøkt å variere valgte prosessen parametere som pH-verdi, strøminntak, temperatur, medium næringsstoffer eller podestoff konsentrasjon en, men lider heller upresis og ufullstendig kontroll av morfologi, høye energikostnader, hemming virkninger eller produkt ustabilitet, derimot, tilskudd av mikropartikler gir en presis prosjektering av sopp morfologi gjennom finjustert variasjon av partikkelstørrelse og konsentrasjon. Dette åpner nye muligheter for å bruke mikropartikler for optimalisering og for skreddersydd design av høy produserende morfologi i bioteknologisk produksjon med A. Niger og andre trådformede mikroorganismer. Den digitale bildeanalyse er en lett reproduserbar metode for å karakterisere sopp makro morfologi. Men rekke parametere for størrelse, form og overflate pregetter av morfologiske strukturer som er beskrevet i litteraturen gjør rask vurdering av sopp morfologi komplisert. Det presenteres Morfologi tallet som en kombinasjon av relevante parametere, unngår denne mangelen, og kan brukes ikke bare for en omfattende karakterisering av morfologiske strukturer, men også for direkte matematisk sammenheng med produktivitet. Dette gjør igjen en estimering av produktiviteten ved gitte morfologi og derfor en tilpasning av morfologi for prosessen trenger mulig. Bruke Morfologi tallet, er det mulig å skille mellom ulike pellet og klumper seg morfologi 4,5. For videre utvikling av Morfologi antall hensynet til fraktalen dimensjonen synes å være lovende. En fraktal dimensjon gir en måling av kompleksitet og masse fylle egenskaper for et objekt 13, og er derfor forutbestemt for helhetlig karakterisering av mycelial morfologi. CReation av et høyt produserende mycelial morfologi, derimot, kan føre til problemer med en prosess, særlig i stor skala dyrking, fordi mycelial veksten skjemaet er tidligere vist å utstille mye større kultur kjøttkraft viskositeter to. Dette fører til problemer med varme-og massetransport og dannelse av stagnerende ikke blandede soner, som krever en høyere makt innspill og gjøre dyrking dyrere å drive en. Derfor forholdet mellom sopp morfologi og kultur buljong viskositeten bør vurderes ved endring av morfologi og bli innlemmet i ytterligere modeller.

Table of Equipment:
<table border="1">
	<tbody>
		<tr>
			<td>
			Equipment</td>
			<td>
			Company</td>
			<td>
			Catalogue Number/model</td>
		</tr>
		<tr>
			<td>
			autoclave</td>
			<td>
			Systec</td>
			<td>
			V150</td>
		</tr>
		<tr>
			<td>
			B&uuml;chner funnel (plastic)</td>
			<td>
			VWR</td>
			<td>
			-</td>
		</tr>
		<tr>
			<td>
			cellulose filter (for biomass dry weight)</td>
			<td>
			Sartorius Stedim Biotech</td>
			<td>
			Filter Discs Grade 389</td>
		</tr>
		<tr>
			<td>
			cellulose acetate filter (for air filtration at reactor)</td>
			<td>
			Sartorius stedim biotech</td>
			<td>
			Midisart 200 PTFE</td>
		</tr>
		<tr>
			<td>
			cellulose acetate filter (for enzyme activity)</td>
			<td>
			Sartorius Stedim Biotech</td>
			<td>
			Midisart NML</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Eppendorf</td>
			<td>
			Centrifuge 5415R</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Heraeus</td>
			<td>
			Biofuge fresco</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Heraeus sepatech</td>
			<td>
			Varifuge 3.0R</td>
		</tr>
		<tr>
			<td>
			compartment dryer (105 &deg;C)</td>
			<td>
			Heraeus</td>
			<td>
			Kelvitran t</td>
		</tr>
		<tr>
			<td>
			control unit (temperature)</td>
			<td>
			Jumo</td>
			<td>
			Jumo iTron 08</td>
		</tr>
		<tr>
			<td>
			control unit (pH-value)</td>
			<td>
			meredos</td>
			<td>
			pH Control 2</td>
		</tr>
		<tr>
			<td>
			desiccator</td>
			<td>
			Duran</td>
			<td>
			Vacuum stable</td>
		</tr>
		<tr>
			<td>
			Falcon tubes</td>
			<td>
			Omnilab</td>
			<td>
			FALC352070</td>
		</tr>
		<tr>
			<td>
			heating block 40 &deg;C</td>
			<td>
			Biometra</td>
			<td>
			TB1 Thermoblock</td>
		</tr>
		<tr>
			<td>
			heating block 95 &deg;C</td>
			<td>
			HLC</td>
			<td>
			HBT 130</td>
		</tr>
		<tr>
			<td>
			micro plate reader</td>
			<td>
			Tecan</td>
			<td>
			Sunrise-Microplate-Reader</td>
		</tr>
		<tr>
			<td>
			micro scales</td>
			<td>
			Sartorius</td>
			<td>
			CP 225</td>
		</tr>
		<tr>
			<td>
			microscope (digital inverted)</td>
			<td>
			AMG</td>
			<td>
			EVOS xl</td>
		</tr>
		<tr>
			<td>
			micro pipettes and tips (different sizes)</td>
			<td>
			Omnilab</td>
			<td>
			5283303
			5283298 
			5283299 
			5283300</td>
		</tr>
		<tr>
			<td>
			micro titer plate</td>
			<td>
			Nunc</td>
			<td>
			MaxiSorp</td>
		</tr>
		<tr>
			<td>
			multi pipette and tips</td>
			<td>
			Eppendorf/ Omnilab</td>
			<td>
			5283611/ 5283611</td>
		</tr>
		<tr>
			<td>
			pH-electrode</td>
			<td>
			Schott</td>
			<td>
			pH-Meter CG840</td>
		</tr>
		<tr>
			<td>
			reaction tubes</td>
			<td>
			Roth</td>
			<td>
			E518.1</td>
		</tr>
		<tr>
			<td>
			scale</td>
			<td>
			Sartorius</td>
			<td>
			CP 3202 S</td>
		</tr>
		<tr>
			<td>
			stirred tank bioreactor with equipment</td>
			<td>
			Applikon Biotechnology</td>
			<td>
			2L Bioreactor set</td>
		</tr>
		<tr>
			<td>
			syringe</td>
			<td>
			Eppendorf</td>
			<td>
			Combitips Plus 5 mL</td>
		</tr>
	</tbody>
</table>
Table of Reagents:
<table border="1">
	<tbody>
		<tr>
			<td>
			Name of the reagent</td>
			<td>
			Company </td>
		</tr>
		<tr>
			<td>
			Acetic acid</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Disodium hydrogen phosphate</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			Ethanol (95%)</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Glucose monohydrate, (&alpha;-D-)</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Glucose oxidase (Typ II from Aspergillus niger)</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Hydrochloride acid (37 % w/v)</td>
			<td>
			Fiedel-de Ha&euml;n</td>
		</tr>
		<tr>
			<td>
			Hydrous magnesium silicate</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Monopotassium phosphate</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			o-dianoisidine dihydrochloride</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Peroxidase (Typ II from horseradish)</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Sodium acetate</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Sodium hydroxide</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			Sucrose, D-(+)</td>
			<td>
			Fluka</td>
		</tr>
		<tr>
			<td>
			Water (deionized)</td>
			<td>
			-</td>
		</tr>
	</tbody>
</table>
Table of Solutions and Medium Composition:
<table border="1">
	<tbody>
		<tr>
			<td>
			Solution</td>
			<td>
			Components</td>
			<td>
			Amount</td>
		</tr>
		<tr>
			<td>
			50 mM sodium acetate buffer (pH 6.5)</td>
			<td>
			Sodium acetate
			Bring to volume with deionized water
			Adjust at pH 6.5 with acetic acid</td>
			<td>
			4.1 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M monopotassium phosphate solution</td>
			<td>
			Monopotassium phosphate
			Bring to volume with deionized water</td>
			<td>
			6.805 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M disodium hydrogen phosphate solution</td>
			<td>
			Disodium hydrogen phosphate
			Bring to volume with deionized water</td>
			<td>
			7.1 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M phosphate buffer (pH 7.0)</td>
			<td>
			0.05 M disodium hydrogen phosphate solution
			Bring to volume with 0.05 M monopotassium phosphate solution</td>
			<td>
			61.2 mL</td>
		</tr>
		<tr>
			<td>
			0.05 M phosphate buffer (pH 5.4)</td>
			<td>
			0.05M disodium hydrogen phosphate solution
			Bring to volume with 0.05 M monopotassium phosphate solution</td>
			<td>
			3 mL</td>
		</tr>
		<tr>
			<td>
			1.65 M sucrose solution</td>
			<td>
			D-(+)-sucrose
			Bring to volume with phosphate buffer (pH 5.4)</td>
			<td>
			564.8 g L-1</td>
		</tr>
		<tr>
			<td>
			reagent solution</td>
			<td>
			o-Dianisidin-Dihydrochlorid
			Ethanol (95%)</td>
			<td>
			25 mg
			10 mL</td>
		</tr>
		<tr>
			<td>
			Glucose reagent solution</td>
			<td>
			Glucose oxidase
			Peroxidase
			Phosphate buffer (pH 7.0)
			reagent solution</td>
			<td>
			10.5 mg
			3 mg
			90 mL
			10 mL</td>
		</tr>
	</tbody>
</table>

customization of aspergillus niger morphology through addition of talc micro particles

Den trådformede sopp A. Niger er en mye brukt belastning i et bredt spekter av industrielle prosesser fra mat til farmasøytisk industri. En av de mest spennende og ofte ukontrollerbart kjennetegner denne trådformede organismen er dets komplekse morfologi. Det spenner fra tette kuleformede pellets til viskøs mycelia (figur 1). Ulike prosessparametre og ingredienser er kjent for å påvirke sopp morfologi en. Siden optimal produktivitet korrelerer sterkt med en bestemt morfologisk form, representerer sopp morfologi ofte flaskehalsen av produktiviteten i industriell produksjon. En rett frem og elegant tilnærming til nøyaktig kontroll morfologisk form er tillegg av uorganiske uløselige mikropartikler (som hydrous magnesium silikat, aluminiumsoksid eller titan silikat oksid) til kultur medium bidrar til økt enzymproduksjon 2-6. Siden det er en obvIOUs korrelasjon mellom mikro partikkel avhengige morfologi og enzym produksjon er det ønskelig å matematisk koble produktivitet og morfologiske utseende. Derfor en kvantitativ presis og helhetlig morfologiske beskrivelse er målrettet. Derfor presenterer vi en metode for å generere og karakterisere mikro partikkel avhengige morfologiske strukturer og å korrelere sopp morfologi med produktiviteten (figur 1) som muligens bidrar til en bedre forståelse av morphogenesis av trådformede mikroorganismer. Den rekombinant belastningen A. Niger SKAn1015 dyrkes i 72 timer i en 3 L rørt tank bioreaktor. Ved tilsetting av talkum mikropartikler i konsentrasjoner på 1 g / L, 3 g / l og 10 g / L før inokulering en rekke morfologiske strukturer er reproduserbart generert. Sterile prøver tatt etter 24, 48 og 72 timer for bestemmelse av vekst fremgang og aktivitet av produsert enzymet. Detdannet produktet er av høy verdi enzymet β-fructofuranosidase, en viktig biocatalyst for neo-sukker-formasjonen i mat eller farmasøytisk industri, som katalyserer blant annet reaksjonen av sukrose til glukose 7-9. Derfor innebærer kvantifisering av glukose etter at sukrose mengden av produsert β-fructofuranosidase. Glukose kvantifisering er laget av en Gud / POD-Assay 10, som er modifisert for high-throughput analyser i 96-brønnen mikro titer plater. Fungal morfologi etter 72 timer er undersøkt av mikroskop og preget av digital bildeanalyse. Dermed blir partikkel form faktorer for sopp makro morfologi som Feret diameter, anslått areal, omkrets, sirkularitet, sideforhold, rundhet und soliditet beregnes med åpen kildekode bildebehandlingsprogram ImageJ. Relevante parametere er kombinert til en dimensjonsløs morfologi nummer (Mn) 11, som muliggjør en omfattende karakterisering avsopp morfologi. Den nære sammenhengen av morfologi antall og produktivitet fremheves av matematisk regresjon.

Watch this Scientific Journal Video about Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles at JoVE.com

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

En metode for å nettopp skape og til omfattende karakterisere morfologi trådformede sopp<em&gt; Aspergillus niger</em&gt; Er beskrevet, noe som gjør den matematiske sammenhengen av morfologisk utseende og produktivitet.

Tilpasning av Aspergillus niger Morfologi Gjennom Tilsetting av talkum mikropartikler

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most ...

customization-aspergillus-niger-morphology-through-addition-talc

Research

JoVE Journal

Immunology and Infection

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

Emballasje HIV-eller FIV-baserte Lentivector Expression konstruerer og transduksjon av VSV-G Pseudotyped viruspartikler

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Påvisning av Fluorescent Nanoparticle Interaksjoner med Primær immun celle subpopulasjoner av flowcytometri

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modellering livssyklusen til Ebola virus Under biosikkerhetsnivå 2 forhold med viruslignende partikler som inneholder Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Optimalisering av en kvantitativ Micro-nøytralisering analyse

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

Produksjon av Pseudotyped partikler for å studere svært patogene Coronaviruses i en Biosafety nivå 2

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Måle fagocytose av Aspergillus fumigatus Conidia av menneskelige leukocytter bruker Flow flowcytometri

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

Produksjon av høy titer smittsomme influensa pseudotyped partikler med konvolutt glykoproteiner fra svært patogene H5N1 og avian H7N9 virus

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Kvantifisere humant norovirus viruslignende partikler binding til commensal bakterier ved hjelp av flow cytometri

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Infeksjon av sebrafisk larver med Aspergillus sporer for analyse av vert-patogen interaksjoner

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

Veiledet maskinlæring for semikvantifisering av ekstracellulært DNA i Glomerulonefrit

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...