Name: fruit volatile analysis using an electronic nose
Uploaded: 2012-03-30T13:00:00
Duration: 11 min 2 s
Description: Watch this Scientific Journal Video about Fruit Volatile Analysis Using an Electronic Nose at JoVE.com

Forfatterne takker Bill Copes (Harris Moran Seed Company, Davis) for at give melon frugt til denne analyse. Dette projekt er støttet af specialafgrøder Research Initiative konkurrencedygtige tilskud Program ikke give. 2009-51181-05783 fra USDA National Institute for Fødevarer og Landbrug.

1. Prøvefremstilling <ol><li> Harvest frugter på det ønskede modningen. Skylles med ledningsvand for at fjerne snavs og støv. </li><li> Vælg frugt til analyse baseret på fraværet af eksterne og interne fejl, og størrelse homogenitet. </li><li> Skær frugten på langs i kiler, der skal anvendes for flygtige prøvetagning. Hvis det er relevant, fjerne hud, frø, frø hulrum væv, eller pit. Frugt væv valg skal være konsekvent gennem hele forsøget og tage hensyn til variationer inden for en enkelt frugt (dvs. få prøve lige fra den ækvatoriale, blomstre og stilkenden dele). </li><li> Kombiner den valgte frugt væv, bland det med henblik på at randomisere det, og derefter afvejes 200 g ind i en blender. </li><li> Tilsæt 200 ml mættet CaCl2-opløsning (372,5 g ved 20 ° C, i 500 ml deioniseret vand) og 50 pi af en 100 mM opløsning af 2-methylbutyl isovalerat i methanol. CaCI 2 er beregnet til at virke som en inhibitor af enzymatisk acvitet, som kan opstå efter opskæring og homogenisering af frugtkødet. 2-methylbutyl isovalerat er tilføjet som en intern standard for at overvåge for eventuelle tab af flygtige forbindelser under homogeniseringen processen. </li><li> Homogenisere blandingen i en laboratorieblender (Waring, USA) i 30 sekunder ved 18.000 rpm og derefter straks hældes i glasflaske og forsegling med Teflon låg. Holde homogenat i flasken ved stuetemperatur, indtil alle prøver fremstilles. </li><li> Efter udhældning af homogenatet i flasken, vent 10 minutter for at tillade separation af skummet fra væsken, derefter afpipetteres 5 ml portioner af væske uden skum, i 20 ml glas ravfarvede hætteglas og forsegle hætteglas med stål skruelåg forsynet med Teflon / silicone septa. Denne fremgangsmåde er egnet til melon og pære homogenat præparat. Hvis andre frugter anvendes til analysen, kan et centrifugeringstrin være påkrævet. Derfor fjerne skummet og centrifugeres væsken for at pelletere partikler, kunnehindre pipetten. Forbered mindst tre hætteglas pr prøve at tjene som teknisk gentagelser. </li><li> På dette tidspunkt kan prøver analyseres øjeblikkeligt eller flash-frosset i flydende nitrogen og opbevaret ved ultra-lav temperatur (-80 ° C) til senere analyse. </li><li> For frosne prøver fjernes på analysen dage prøverne fra fryseren og tillade dem at tø i en time ved stuetemperatur. Efter optøning, og før analyse hætteglashætte med en ny en, der har en ren, tør septum udskifte. Hvis skillevæggen ikke udskiftes, kan vand kondenseres på skillevæggen under optøning trækkes ind i instrumentet og beskadige den. </li></ol> 2. Gaskromatografi-overflade Acoustic Wave (GC-SAW) Set-up og Data Acquisition <ol><li> Læg den passende analysemetode på zNose. Til analyse af esteren-rige flygtige profil melon, er vores parametre i MicroSense udgave 5.44.22 software (Newbury Park, CA, USA) som følger: headspace sugning iindløb i 20 sekunder ved 30 ml/min.-1 via pumpe; indgangstemperatur på 200 ° C; Tenax fælde temperatur på 225 ° C; bæregas (helium renhed 99,999%) strømningshastighed på 2,9 ml min-1, søjle (DB-5 kolonne, en m x 0,25 mm ID x 0,25 um filmtykkelse) temperaturprogram fra 45 ° C til 180 ° C med en hastighed på 10 ° C sek-1; sensor temperatur på 40 ° C; ventil ved 165 ° C. Den totale analysetid er 1 minut per prøve. </li><li> Tilslut en kanyle af rustfrit stål med ikke-udkerningen tip til zNose indløb og rense systemet flere gange med den omgivende luft, indtil basislinjen er stabil og ingen toppe større end 200 Counts (Ct) er fundet. </li><li> Tune instrumentet under anvendelse af en opløsning af ligekædede alkaner (C6-C14). Melodien resultat anvendes af instrumentet software til at konvertere retentionstiden for de eluerede toppe fra tidsenheder til Kovats Index (KI) enheder. Følgelig, når systemet er indstillet, er retentionstiderne rapporteret i KI enheder. </li&gt; <li> Før analysen tillader prøven at ækvilibrere i 30 minutter. For at analysere en af ​​de prøvehætteglas, indsætte en nål ind i hætteglasset septum at aflaste. Sæt derefter nålen tilsluttet instrumentet indløb i hætteglasset septum og indlede headspace prøvetagning. Analyser mindst tre teknisk replikater per prøve. </li><li> Manuelt starte instrumentet ved at klikke på &quot;Play&quot; knappen er pumpen aktiveres og trækker dampene præsenterer over prøven. Ved afslutningen af ​​analysen vises et kromatogram på skærmen, og sensoren bliver automatisk opvarmes til 150 ° C i 10 sekunder for at rense den. Når systemet status kassen knappen bliver grønt igen, instrumentet er klar til at analysere en anden prøve. </li><li> At sikre en stabil basislinie og passende system rengøring køre mindst én luft råemnet mellem hver prøve. For at overvåge for eventuelle flygtige forurening fra hætteglasset og hætte, analysere to hætteglas-emner (tomme hætteglas med hætte), i begyndelsen og slutningen af ​​dagen. &lt;/ Li&gt; </ol> 3. Data Export og analyse <ol><li> Eksportere data i en Microsoft Excel-fil efter erhvervelsen ved hjælp af &quot;Peak logging&quot; funktionen i MicroSense software. Når data eksporteres, skal du tilføje kolonner, der indeholder etiketter til variabler og gentagelser. </li><li> Omdan data format for lettere manipulation ved hjælp af Python (version 2,6, frit tilgængelig on-line) script vi udviklede, kaldet &quot;reform_data.py&quot; (se figur 1 for et eksempel på dataformat før og efter brug af scriptet &quot;reform_data. py &quot;). Navnet på kildefilen (xls-format) og arknavn for input-data, samt det ønskede filnavn for output (xls-format), der redigeres direkte i scriptet. </li><li> Start &quot;kim_interface.py&quot; (også skrevet i Python 2,6, se figur 2), og importere data fra filen genereres i det foregående trin. Konkret er analysen baseret på visning og analysere det antal gange hver KI værdi blev påvist(&quot;Ki hits&quot;). Således programmet viser et søjlediagram af KI hits for hver KI værdi. </li><li> Vurdere KI hits specifikke delmængder af prøver, analysere hver gruppe af teknisk gentagelser sammen. For at gøre dette, analysere enhver behandling eller variabel separat ved at markere / afmarkere de tilsvarende felter. Se figur 2 billedtekst detaljeret beskrivelse af den grafiske brugergrænseflade (GUI) funktioner. </li><li> Efter identifikation af bredden af ​​hver KI vindue ved hjælp af GUI, vælges tilfældigt nogle af de tilsvarende kromatogrammer i Microsense software og evaluere overlappende toppe mellem det tekniske replikater. Se figur 3 for et eksempel på overlejrede kromatogrammer af to tekniske gentagelser. </li><li> Når KI vinduet individueres, skal du bruge &quot;Flet&quot;-funktion til rådighed i GUI at fusionere de KIS, der falder i vinduet, i den mest befolkede KI. Ved at bruge denne funktion, er toppe mærket med en række Ki-værdier samles under en enkelt KI etiket, allowing behandling af sådanne toppe som en enkelt variabel. For at gøre dette, skal du først klikke på &#39;Flet&#39; knappen for at aktivere funktionen og vælge den mest befolkede KI som centrum af vinduet ved at venstre-klikke på den tilsvarende bar. Når stangen er blevet valgt, den ændrer farve og bliver grøn. Hvis du vil flette de Kis der falder ind under vinduet i den valgte KI, skal du højreklikke på de tilsvarende barer, hvilket forårsager barer til røde, mens en blå bjælke af den tilsvarende længde lægges oven i det centrale KI (se figur 4 ). Når alle de valgte KIS er blevet fusioneret ind i den relevante centrale KI, skal du klikke på &#39;Merge&#39; knappen igen for at acceptere de ændringer, dette medfører &quot;Flet&quot;-knappen for at slå gul. I tilfælde af fejl, er den »unmerge &#39;knappen også tilgængelig. For at splitte, skal du klikke på &quot;unmerge&quot; knappen i GUI, så højre-klik på den røde bjælke, du ønsker at splitte. Fra rød, vender bar blå. Klik på &#39;unmerge&#39; knappen igen for at acceptere ændringerne. </li><li> Hvis man forsøger at incorrectly fusionere to toppe i en enkelt prøve i en enkelt Ki-værdi, er en fejlmeddelelse trykt. Under sådanne omstændigheder, se nøje kromatogrammet og omdefinere KI vinduet i den pågældende region. </li><li> Når alle de fusionerende operationer er blevet udført, skal du gemme filen. </li><li> Før der fortsættes med statistisk analyse, er chromatogrammer af luft og hætteglasset råemner analyseret for at kontrollere for eventuelle forureninger. Når KI af toppene i emnerne er blevet identificeret, trækkes den del af top detekteres i luft-og / eller hætteglas-emnet fra arealet af toppen til stede i prøven. Fortsæt derefter med statistiske analyser. </li></ol> 4. Repræsentative resultater Den elektroniske næse var i stand til at påvise forskelle i flygtige profiler mellem melon frugt høstet på forskellige udløb trin (figur 5). Tyve KI vinduer blev identificeret på tværs af alle prøver. En analyse af varians viste, at 14 toppe forværringcted den elektroniske næse varierede betydeligt mellem modenhed trin. I figur 6, er log af de gennemsnitlige peak områder i disse 14 komponenter planlagt at vise forskelle i peak mængderne mellem to løbetid faser, tidlig modne og fuldmodne frugt. <img alt="Figur 1" src="/files/ftp_upload/3821/3821fig1.jpg" /> Figur 1. Eksempler på dataformat eksporteres fra instrumentet software (A) og efter transformation under anvendelse af &quot;reform_data.py&quot; script (B) udføres. For at lette data manipulation og analyse, er alle de unikke Kis identificeret på tværs af alle de prøver, så de data, der er omorganiseret med oplysningerne om prøverne i rækker og toparealet i kolonner, svarende til unikke KIS. Hvis en top ikke detekteres en Ki-værdi i en prøve, den tilsvarende celle forbliver tomt. <img alt="Figur 2" src="/files/ftp_upload/3821/3821fig2.jpg" /> Figur 2. Skærmbillede fra skrifternet fil &quot;kim_interface.py&quot;. Plottet i centrum viser antallet af hits pr KI versus KI. &#39;Hit pr KI&#39; er antallet af prøver, hvor et højdepunkt med den pågældende KI blev påvist. På venstre side er der tre gule bokse styrer de markerede data. De udviser parametre til at opdele datasæt (behandlinger, replikater, kvalitative variabler osv.). I denne figur er de (fra top til bund): Sort, plantning dato og modningen ved høst. På den nederste: ved at klikke på de 3 barer og flytte den blå bjælke til venstre eller til højre, kan man vælge den mindste og den maksimale værdi af KI rækkevidde, og den minimale arealet (&quot;Threshold&quot;). Til højre: den &quot;Flet&quot;-knappen giver mulighed for sammenlægning udvalgte KIS ved manuelt at klikke på barer i plottet. Den &quot;unmerge &#39;knappen gør det muligt at vende processen for udvalgte tilfælde. <img alt="Figur 3" src="/files/ftp_upload/3821/3821fig3.jpg" /> Figur 3. Overlejrede kromatogrammer (i<html> sort og rød) af to tekniske replikater fra melon flygtige headspace at illustrere et skift i retentionstid. <img alt="Figur 4" src="/files/ftp_upload/3821/3821fig4.jpg" /> Figur 4. Eksempel KI fusionerende procedure. I den centrale plot, repræsenterer den grønne bjælke (Central KI) den mest befolkede KI, som er blevet valgt som centrum for KI vinduet. KI X og KI Y er Kis falder i vinduet af interesse og de skal flettes ind i den centrale KI. Ved at højre-klikke på KI X bar, bliver det rødt, og på samme tid, vises en blå linje af samme længde af KI X bar, på toppen af ​​den grønne. Ved at gentage den samme fremgangsmåde for KI Y er længden af ​​den blå bjælke (fusioneret KIS) vil øge den tilsvarende længde. Når alle Kis er blevet tilføjet, ved at klikke på det grønne &#39;Flet&#39; knappen, de fusionerende proces ender, gemmes ændringerne, og knappen farven bliver gul. / Files/ftp_upload/3821/3821fig5.jpg &quot;/&gt; Figur 5. To kromatogrammer af melon prøver høstet på forskellige udløb faser tidlig modne (øverst) og fuldt modne (bund), for at illustrere evnen af den elektroniske næsen at påvise forskelle i flygtige mængder. <img alt="Figur 6" src="/files/ftp_upload/3821/3821fig6.jpg" /> Figur 6. Radar afbildning, der viser topareal af 14 komponenter i to melon prøver ved to forskellige udløb trin, tidlig modne og fuldt modne. Toparealerne er rapporteret i logaritmisk skala til at visualisere sammenligningen. Tallene i slutningen af ​​hver ray repræsenterer de tilsvarende Kovats Indices.

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Vi har intet at afsløre.

Elektroniske næser repræsenterer en lovende metode til hurtig, objektiv vurdering af aromaprofiler fra frugter eller flygtige-rige prøver. Men skift i retentionstid udgør en udfordring for peak identifikation og kan føre til misfortolkning af de data, når to kromatogrammer ikke er perfekt afstemt. Visuel inspektion af chromatogrammer indikerede, at variation af retentionstiderne blandt prøver ofte forårsaget samme top, der skal mærkes med lidt forskellige Ki-værdier (ca. ± 10). Dette blev omsat til en overdrevet antallet af unikke opdaget KIS. For at drage fordel af de faktiske omstændigheder, der (a) forskellige forbindelser er til stede på forskellige løbetid stadier og (b) teknisk gentagelser er ca ens, to edb-baserede scripts (&quot;kim_merge.py&quot;, som indeholder rutiner til håndtering af data sæt, og &quot;kim_interface.py&quot;, som tilvejebringer en grafisk brugergrænseflade (GUI)) blev udviklet til systematisksammenligne prøver i en semi-automatisk mode, hvilket reducerer den nødvendige tid til kromatogram analyse af store datasæt. Disse programmer tillader konsolidering, hvor det er relevant, af toppe mærket med en række Ki-værdier under en enkelt KI etiket. Dette tjener to vigtige formål: (a) det muliggør en statistisk analyse til at behandle sådanne toppe som en enkelt variabel, og (b) den letter maksimal identifikation og sammenligning med andre systemer og offentliggjorte værdier. Resultaterne præsenteret her viser, at melon prøver kunne blive diskrimineret baseres på modenhed og aroma profilering ved hjælp af zNose system i kombination med tilstrækkelig KI identifikation. Dette udgør en lovende ny teknologi til analyse af flygtige stoffer, der kan anvendes til kvalitetskontrol programmer.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Calciumchlorid </td><td> MP Biomedicinsk </td><td> 195088 </td><td></td></tr><tr><td> 2-methylbutyl isovalerat </td><td> SAFC Global </td><td> W350613 </td><td> ≥ 98%, naturlig, FCC </td></tr><tr><td> Methanol </td><td> Fisher Scientific </td><td> A411-4 </td><td></td></tr><tr><td> Hætteglas </td><td> Sigma / Supelco </td><td> SU860098 </td><td></td></tr><tr><td> Kasket </td><td> Sigma / Supelco </td><td> SU860101 </td><td></td></tr><tr><td> Laboratorieblender </td><td> Waring Laboratory Science </td><td> 7009G </td><td> 2-trins blandeapparat, 1 - liters glasbeholder </td></tr><tr><td> Flaske </td><td> Fisher Scientific </td><td> 06-414-1C </td><td> Pyrex, 500 ml, polypropylen plug-forsegling </td></tr><tr><td> Needle </td><td> Electronic Sensor Technology </td><td> TLC101046 </td><td> Sidehul luer </td></tr><tr><td> Alkaner løsning </td><td> Electronic Sensor Technology </td><td></td><td> C6-C14-alkaner opløsning i methanol </td></tr><tr><td> zNose </td><td> Electronic Sensor Technology </td><td> Model 4500 </td><td></td></tr><tr><td> DB-5 GC søjle </td><td> Electronic Sensor Technology </td><td> SYS4500C5 </td><td></td></tr><tr><td> MicroSense </td><td> Electronic Sensor Technology </td><td> Version 5.44.22 </td><td></td></tr><tr><td> Python 2,6 </td><td></td><td></td><td> Frit tilgængelige on-line </td></tr><tr><td> &quot;Reform_data.py&quot; og &quot;kim_interface.py&quot; scripts</td><td></td><td></td><td> Scripts til rådighed som supplerende materiale på JOVE </td></tr></tbody></table>

fruit volatile analysis using an electronic nose

Talrige og forskelligartede fysiologiske ændringer sker under frugtmodning, herunder udvikling af en specifik flygtig blanding, der karakteriserer frugt aroma. Modenhed ved høst er en af de vigtigste faktorer, der påvirker smagen kvaliteten af frugt og grøntsager 1. Valideringen af ​​robuste metoder, der hurtigt at vurdere frugt modenhed og aroma kvalitet ville muliggøre forbedret styring af avancerede avlsprogrammer, produktion praksis og postharvest håndtering. I løbet af de sidste tre årtier har megen forskning blevet udført på at udvikle såkaldte elektroniske næser, som er apparater i stand til hurtigt opdage lugt og smag 2-4. I øjeblikket er der adskillige kommercielt tilgængelige elektroniske næser i stand til at udføre flygtig analyse, baseret på forskellige teknologier. Den elektroniske næse anvendes i vores arbejde (zNose, EST, Newbury Park, CA, USA), består af ultra-hurtige gaskromatografi kombineret med en overflade akustisk sensor (UFGC-SAW). Denne teknologi er allerede blevet testet for sin evne til at overvåge kvaliteten af de forskellige råvarer, herunder påvisning af forringelse af æble 5, modenhed og råd evaluering mango 6, aroma profilering af thymus arter 7 c 6 flygtige forbindelser i druer, 8; karakterisering af vegetabilsk olie 9 og påvisning af adulterants i jomfru kokosolie 10. Dette system kan udføre de tre store trin aromaanalyse: headspace prøveudtagning, adskillelse af flygtige forbindelser, og afsløring. I et minut, bliver output, et kromatogram, der produceres og efter en skylning cyklus, er instrumentet er klar til yderligere analyse. De opnåede resultater med zNose kan sammenlignes med de øvrige gaskromatografiske systemer ved beregning af Kovats Indices (KI). Når instrumentet er blevet indstillet med en alkan-standardopløsning, er retentionstiderne konverteres automatisk tilKIS. Imidlertid små ændringer i temperatur og strømningshastighed forventes at forekomme over tid, hvilket retentionstider at drive. Også afhængigt af polariteten af søjlen stationære fase, kan reproducerbarheden af KI beregninger varierer fra flere indeks enheder 11. En række programmer og grafiske grænseflader blev derfor udviklet til at sammenligne beregnede KIS blandt prøver i en semi-automatiseret måde. Disse programmer reducere den tid, der kræves for kromatogram analyse af store datasæt og minimere risikoen for misfortolkning af data, når kromatogrammerne ikke perfekt afstemt. Vi præsenterer en fremgangsmåde til hurtig flygtig forbindelse analyse frugt. Prøveforberedelse, datafangst og håndtering procedurer er også diskuteret.

Watch this Scientific Journal Video about Fruit Volatile Analysis Using an Electronic Nose at JoVE.com

Fruit Volatile Analysis Using an Electronic Nose

En hurtig metode til flygtig forbindelse analyse frugt er beskrevet. De flygtige forbindelser til stede i frirummet i en homogenat af prøven er hurtigt separeret og detekteret med ultra-hurtige gaskromatografi (GC) kobles med en overflade akustisk bølge (SAW) sensor. En procedure for håndtering af data og analyse er også drøftet.

Frugt flygtige Analyse Brug af en elektronisk næse

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

fruit-volatile-analysis-using-an-electronic-nose

Research

JoVE Journal

Biology

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Isolering og Biofysisk Undersøgelse af Frugt neglebånd

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

Analyse af SNARE-medieret membranfusion anvendelse af en enzymatisk Cellefusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Laboratoriet protokol for genetiske Gut indhold analyser af akvatiske Macroinvertebrates brug af gruppe-specifikke rDNA primere

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Simple Homemade Tools to Handle Fruit Flies&amp;#8212;&lt;em&gt;Drosophila melanogaster&lt;/em&gt;

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

Enkle hjemmelavede værktøjer til at håndtere frugt fluer —Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

Aktuel anvendelse Bioassay til kvantificering af insekticidtoksicitet for myg og bananfluer

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

Protokoller for CRISPR/Cas9 Mutagenese af den orientalske bananflue Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...