Questa produzione e il lavoro sperimentale presentato nel presente documento riflettono l&#39;aiuto dedicato ed entusiasta di Reynaldo Pareja gentilmente supportata da Lina Aguirre, Gilberto Córdoba, William Mera, Ximena Bonilla, e Darío Viveros. Ringraziamo anche John Miles e tre revisori anonimi per i commenti che hanno contribuito a migliorare il video e manoscritto.

1. Piante <ol><li> Talee singola a partire da piante mature sono utilizzati come unità sperimentali. Le talee sono tagliati a 10 cm per garantire uniformità di piantine. </li><li> Per prevenire la contaminazione da agenti patogeni delle piante, le talee vengono lavati in una soluzione di ipoclorito di sodio al 3% per 2 minuti e poi sciacquate con acqua di rubinetto thoughroughly. </li><li> Ogni taglio è piantato in ca. 36 g di terreno sterile in un tubo cilindrico in PVC (5,3 centimetri di diametro esterno di 6,5 cm di altezza) chiuso all&#39;estremità inferiore con un bicchiere di polistirolo e ricoperto con una boccola in PVC. Il taglio è tenuta in posizione da un anello di gommapiuma posto nel foro centrale della boccola. </li><li> Le piante sono fecondati e innaffiato secondo necessità per mantenere l&#39;umidità del terreno adeguato. </li><li> Le piante cresceranno per un mese prima di essere sfidato da insetti, permettendo lo sviluppo delle radici superficiali, che servono come siti di alimentazione per la root-xilema al seno ninfe spittlebug. </li><li> Tubi in PVC con barbatelle sono invertite per 8 giorni prima di infestazione per stimolare ulteriormente la crescita delle radici superficiali, in parte aggiustamento per le differenze inerenti l&#39;architettura radice. Questa fase richiede una fonte artificiale di luce, che mettiamo a disposizione per 24 ore al giorno. Per evitare fughe di confondimento da erbivori con resistenza erbivoro, singolo impianto si replica con radici superficiali poche o nessuna, dopo questo trattamento inversione sono eliminati dalla esperimento. Questa eliminazione deve essere rara, mai comportare l&#39;eliminazione del genotipo che rappresentano. </li></ol> 2. Insetti <ol><li> Spittlebugs adulti sono raccolti dal campo, identificato per specie, e la deposizione delle uova introdotto a gabbie in cui vengono forniti fogliame di brachiariagrasses suscettibili di nutrirsi. Le femmine depongono le uova nel suolo contenute in un vassoio superficiale rimovibile posto sul fondo delle gabbie. </li><li> Dopo alcuni giorni, il terreno è sospeso in acqua e passato attraverso una serie di setacci (42, 60 e 150 mesh) per raccogliere le uova. Sospendere il materiale setacciato in una soluzione salina al 30% provoca le uova mature di galleggiare, lasciando dietro di sé le uova immature e qualsiasi materia organica residua. </li><li> Per prevenire la contaminazione da agenti patogeni degli insetti, le uova vengono disinfettati in una soluzione di ipoclorito di sodio allo 0,5% per 5 minuti e poi sciacquate con acqua distillata. </li><li> Ciascuna pianta (unità sperimentale) è infestata sulla superficie del suolo con sei uova mature. Solo uova secolari, vicino al cova vengono selezionati per infestazioni. Essi possono essere identificati dalla presenza di due punti rossi nella loro parte anteriore, corrispondente agli occhi dell&#39;insetto; segna rosa nella loro parte posteriore, corrispondente al addome dell&#39;insetto, e completamente ampliato opercolo. </li><li> Cova successo è confermato 4 d più tardi, a questo punto le uova non schiuse è sostituito da un spittlebug neonato dalla nostra colonia. </li><li> Impianti di prova sono disposti in un disegno randomizzato blocco completo con sei repliche per ogni genotipo. </li></ol> 3. Valutazioni <ol><li> L&#39;esperimento è valutato circa. 30 d dopo l&#39;infestazione (tempi esatti variano a seconda della specie di insetti studiato). </li><li> I danni sono valutati su una scala 1-5 visivo, dove 1 corrisponde a nessun danno visibile e 5 corrisponde ad un impianto di morti (Figura 1). Punteggio danni impianto fornisce una stima della tolleranza. </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="Figura 1" /> Figura 1. Danni scala usata per valutare la tolleranza al spittlebug erbivori in Brachiaria spp. 1 = nessun danno rilevabile; 2 = fogliame 0-25% necrotico, 3 = 25-50% foglie necrotiche; 4 = 50-75% foglie necrotiche; 5 = fogliame 75-100% necrotico. <ol start="3"><li> Sopravvivenza degli insetti viene valutata contando gli insetti di raggiungere sia il loro instar finale o stadio adulto (Figura 2). Punteggio per la sopravvivenza di insetti fornisce una stima della antibiosi. </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="Figura 2" /> Figura 2. Spittlebug sviluppo di prima instar (a sinistra) per adulti (a destra). Solo gli insetti raggiungono almeno i loro instar finale (frecce) alla data di valutazione sono contati come &quot;sopravvissuti&quot;. 4. Rappresentante risultati I risultati di un rappresentante ibridi Brachiaria test di screening per la resistenza alla Aeneolamia spittlebug reducta sono presentati nella Figura 3. <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="Figura 3" /> Figura 3. I risultati di uno screening ibridi Brachiaria per la resistenza alle spittlebug Aeneolamia reducta. Consideriamo piante resistenti se la loro risposta alla erbivori cade nella metà inferiore di questa trama. Aumenta antibiosi da destra a sinistra e la tolleranza aumenta dall&#39;alto verso il basso. Le frecce indicano la nostra controlli resistenti.

<ol>
	<li>Painter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect resistance in crop plants. , (1951).</li><li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antixenosis-a+new+term+proposed+to+define+Painter's+'Nonpreference'+modality+of+resistance.">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li><li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+agricultural,+genetic,+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm.">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li><li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+damage+of+grassland+spittlebugs+(Homoptera+Cercopidae)+in+Colombia:+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens.">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li><li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species.">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li><li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana.">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li><li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+to+spittlebugs+(Homoptera:+Cercopidae)+in+accessions+of+Brachiaria+spp.">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li><li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+(Homoptera:+Cercopidae).">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li><li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+and+tolerance+to+five+species+of+spittlebug+(Homoptera:+Cercopidae)+in+Brachiaria+spp.:+implications+for+breeding+for+resistance.">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li><li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for evaluating insect resistance in crop plants. , (1994).</li><li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+for+rearing+spittlebugs+(Homoptera:+Cercopidae).">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li><li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+adult+spittlebugs+(Hemiptera:+Cercopidae)+in+Brachiaria+spp.:+methods+and+categories+of+resistance.">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Discriminare tra i meccanismi di resistenza erbivoro può illuminare le attività di selezione delle colture 3. Resistenza sulla base di antibiosi forte potrebbe, in alcune circostanze, creare pressioni selettive per biotipi parassiti più aggressivi. D&#39;altra parte, la resistenza basata sulla tolleranza potrebbe consentire popolazioni di parassiti di aumentare la tolleranza fino alla fine è sopraffatto. Selezione per la resistenza durevole, quindi, richiede un&#39;attenta considerazione di quello che meccanismi di resistenza a conferire maggior stabilità per una determinata coltura / infestanti sistema [vedi Kennedy et al. (1987) per un dibattito approfondito sul tema] 3. Questo video dimostrativo si basa su diversi studi precedenti progettato per stimare il contributo relativo di antibiosi e tolleranza alla resistenza spittlebug in brachiariagrasses 6-9. In contrasto con test a scelta o esperimenti sul campo, nessun test a scelta garantire che tutte le piante ricevono la stessa pressione erbivoro, fornendo così controllare le differenze derivanti da comportamenti erbivoro (ad esempio, la preferenza di accoglienza, aggregazione). Per questo motivo, però, non test a scelta non sono adatti per valutare la resistenza da antixenosis. Artificialmente allevamento colonie di insetti è il metodo preferito per ottenere gli insetti di prova per test di screening resistenza 10. Colonie di fornire una fonte affidabile e uniforme di spittlebugs di un&#39;età noto per esperimenti da condurre in qualsiasi momento dell&#39;anno 11. Può tuttavia essere necessario infondere periodicamente individui selvatici nella colonia per garantire che la colonia non si discosta da popolazioni geneticamente settore pertinente 10. Di valutare l&#39;idoneità di questi erbivori allevati artificialmente, e monitorare eventuali cambiamenti nelle loro aggressività nel tempo, è importante che tutte le proiezioni sono idonei controlli dei livelli di resistenza nota. Più parametri devono essere attentamente definito per progettare un apposito test di screening resistenza. A pochi parametri chiave abbiamo preso in considerazione per progettare il nostro test includono l&#39;età delle piante ospiti, la specie e lo stadio di sviluppo del spittlebugs, il livello di infestazione, e la durata della pianta-insetto contact 8,9,12. Diverse fasi di sperimentazione e affinamento protocollo può essere necessario per giungere ad una rapida, conveniente, affidabile, test che predice in maniera adeguata resistenza erbivoro in campo.

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

Le piante possono resistere al danno erbivoro attraverso tre meccanismi principali: antixenosis, antibiosi e tolleranza 1. Antixenosis è il grado in cui si evita la pianta quando l&#39;erbivoro è in grado di selezionare altre piante 2. Antibiosi è il grado in cui la pianta influisce sulla idoneità della erbivori si nutrono di esso 1. Tolleranza è il grado in cui la pianta può sopportare o riparare i danni causati dal erbivoro, senza compromettere la crescita del erbivoro e riproduzione 1. La durata di resistenza erbivoro, in un ambiente agricolo dipende in larga misura dal meccanismo di resistenza favorito durante le attività di selezione delle colture 3. Abbiamo dimostrato un non-scelta esperimento progettato per stimare il contributo relativo di antibiosi e tolleranza alla resistenza spittlebug in Brachiaria spp. Diverse specie di erbe africane del Brachiaria genere sono preziose foraggi e piante pascolo nel neotropicale, ma possono essere messi a dura prova da numerose specie autoctone di spittlebugs (Hemiptera: Cercopidae). 4 Per valutare la loro resistenza alla spittlebugs, le piante sono propagate vegetativamente- di talee e lasciato crescere per circa un mese, che consente la crescita delle radici superficiali su cui spittlebugs può alimentare. A quel punto, ciascuna pianta è individualmente sfidati con sei uova spittlebug vicino cova. Infestazioni possano progredire per un mese prima di valutare i danni delle piante e la sopravvivenza degli insetti. Punteggio danni impianto fornisce una stima della tolleranza, mentre il punteggio sopravvivenza degli insetti fornisce una stima della antibiosi. Questo protocollo ha facilitato il nostro obiettivo coltivazione delle piante per migliorare la resistenza spittlebug in brachiariagrases commerciali 5.

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Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Questo video spiega i meccanismi di resistenza pianta ospite di erbivori e dimostra una non-scelta di test che stima il contributo relativo di antibiosi e tolleranza per spittlebug resistenza in<em&gt; Brachiaria</em&gt; Spp.

Caratterizzano Erbivoro meccanismi di resistenza: Spittlebugs su Brachiaria Spp. come esempio

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

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Biology

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

13.7K Views.  CIAT. Questo video spiega i meccanismi di resistenza pianta ospite di erbivori e dimostra una non-scelta di test che stima il contributo relativo di antibiosi e tolleranza per spittlebug resistenza in Brachiaria Spp.

Video: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Studi genetici di proteine ​​umane di riparazione del DNA Utilizzando lievito come sistema modello

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Ricerca

Biologia

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Volatili Blueberry erbivori indotta e intra-impianto di segnalazione

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Misurare il osmotica dell&#39;acqua Permeabilità Coefficiente (P<sub&gt; F</sub&gt;) Di celle sferiche: protoplasti impianto isolato come un esempio

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La tecnica infiltrazione-centrifugazione per l&#39;estrazione di apoplastico fluido da foglie delle piante Utilizzo<em&gt; Phaseolus vulgaris</em&gt; Come un esempio

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

FITC-Destrano di imaging come Reporter per esocitosi regolata

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

Calibrazione e utilizzo delle prestazioni del microcoil MRM dimostrato sulle radici della trogola Medicago a 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

Esplorare gli epitrascrittomi m6A e m5C sull'infezione virale: un esempio di HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Caratterizzazione delle proprietà meccaniche della parete cellulare primaria in organi vegetali viventi mediante microscopia a forza atomica

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

Un metodo di elettroporazione per trasformare Rickettsia spp. con un vettore shuttle fluorescente che esprime proteine in linee cellulari di zecche

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Studio del meccanismo di trasporto dello zinco nelle cellule di mammifero con il saggio FluoZin-3

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Autore in primo piano: Esplorare la regolazione cellulare dello zinco attraverso la funzionalità ZnT1 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Caratterizzazione dei trasportatori di zinco nei mammiferi mediante un saggio di trasporto dello zinco in vitro

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...