Questo lavoro è stato sostenuto dal Fondo austriaco della scienza (FWF, P20619 e P22988 a BZ).

Microonde preparazione del campione assistita <ol><li> Prima dell&#39;inizio di preparazione del campione bisogna programmare il processore automatico per tessuti microonde microscopia elettronica (EM Leica AMW; Microsystems Leica, Vienna, Austria) con i seguenti protocolli (Tabella 1) per la preparazione del campione per microonde assistita TEM: <img src="/files/ftp_upload/2950/2950table1.jpg" alt="Tabella 1" /> Tabella 1. Protocollo per la preparazione del campione mediante irradiazione a microonde. Le colonne mostrano differenti (da sinistra a destra): <ol><li> Numero flaconcino (nr Vial.) Rappresenta l&#39;ordine in cui è caricato il flacone nel carosello del processore. </li><li> Fase del processo vero e proprio. </li><li> Reagenti nelle fiale. </li><li> Durata della fase attuale. </li><li> Temperatura massima che si raggiunge nella fiala prima irradiazione con microonde è spento. </li><li> Forno a microonde impostazione irradiazione: continua = incrementi di temperatura rapidaase, tenendo la temperatura impostata; Slope = aumento di temperatura moderata, temperatura finale raggiunta al termine; Pulsed = rapido aumento di temperatura, alimentazione spento finché la temperatura è scesa a 5 ° C, potenza ripreso a temperatura. </li><li> Potenza massima della radiazione a microonde. </li></ol></li><li> Fresco preparare le soluzioni per le diverse fasi descritte nel protocollo di preparazione del campione (per Agar 100 resina epossidica vedi 1.7), riempirle nei flaconi designati secondo il protocollo programmato (Tabella 1), caricare le fiale sulla giostra, quindi inserire il giostra nel processore tessuto microonde, e infine caricare il primo flaconcino in modo mono-camera. </li><li> Tagliare piccole sezioni di foglie (1mm 2) di Nicotiana tabacum infettati con virus del mosaico del tabacco (TMV), con una lama di rasoio in un piatto ceroplastica in un calo del 3% glutaraldeide (Agar Scientific Ltd., Stansted, Inghilterra) in 60mm Sørensen tampone fosfato (pH 7,2) a temperatura ambienteteratura. </li><li> Trasferire le sezioni con le pinzette sottili immediatamente nei cesti designati con una larghezza delle maglie di circa 200μm. Stack i cestelli uno sopra l&#39;altro e inserirle nella modalità mono-camera. Bisogna fare attenzione che i campioni sono costantemente ricoperti di soluzione fissativa durante il carico e accatastamento dei cestelli in modo che non si secchino. </li><li> Avviare il programmato in precedenza microonde assistita protocollo di preparazione del campione per la fissazione, la disidratazione e l&#39;infiltrazione. </li><li> Durante la preparazione del campione viene eseguita automaticamente dal processore tessuto microonde continuare con colorazione negativa con il materiale vegetale residuo come descritto nella sezione 3 (colorazione negativa). </li><li> Preparata fresca Agar 100 resina epossidica miscelando i seguenti componenti come descritto: riempimento 24g Agar 100, 16g dodecenyl anidride succinica, e 10 g di anidride metil nadic (per tutti i componenti vedere Agar Scientific Ltd., Stansted, Inghilterra) in una tazza di plastica, di calore a40 ° C e mescolare bene. Aggiungere 1,2 g di benzile dimetilammina e mescolare accuratamente. Riempire Agar 100 resina epossidica nella polimerizzazione designato forma appena prima del protocollo di preparazione del campione giunge al termine (ad esempio durante la fase 22 in tabella 1). </li><li> Dopo che il protocollo è terminata (dopo passo 22 nella tabella 1) rilasciare i cestelli sovrapposti contenenti i campioni infiltrate dalla modalità mono-camera nel flaconcino ultimo della giostra. Rimuovere la giostra dal dispositivo a microonde, Affianca i cestelli e caricarle utilizzando pinzette sottili nelle forme di polimerizzazione designati. Bisogna fare attenzione che i campioni sono sempre ricoperti di Agar 100 resina epossidica durante disimpilamento e il caricamento in modo che non si secchino. </li><li> Impilare le forme di polimerizzazione sopra l&#39;altro. Bisogna fare attenzione che i campioni sono sempre ricoperti di Agar 100 resina epossidica durante accatastamento e carico in modo che non si secchino. </li><li> Rimuovere le fiale precedentemente utilizzati dalla giostra del Tiss microondeue processore, caricarlo con i cesti impilati e inserire la giostra nel processore tessuto forno a microonde. </li><li> Avviare il protocollo di polimerizzazione programmato in precedenza (tabella 1). </li><li> Mentre polimerizzazione viene effettuata automaticamente dal processore tessuto microonde, esaminare griglie negativamente colorati con un microscopio elettronico a trasmissione [per esempio Philips CM10 TEM, FEI (ex Philips), Eindhoven, Paesi Bassi] ed eseguire analisi di immagine come descritto nel capitolo 3 e 4 ( colorazione negativa e analisi d&#39;immagine). </li><li> Dopo che il protocollo è terminata rimuovere le forme di polimerizzazione dal mono-modalità camera, Unstack le forme di polimerizzazione e polimerizzate rimuovere i blocchi contenenti i campioni. Essi sono ora pronti per essere sezionato con un microtomo. </li></ol> 2. Taglio e sezionamento <ol><li> Inserisci uno o più blocchi in porta-campioni separati per ultrasottile sezionamento con il campione di incollaggio superiore a circa 1 cm dal supporto. </li><li>Tagliare il blocco con un trimmer campione per TEM (ad esempio Leica Reichert Ultratrim, Leica Microsystems) in modo che una faccia blocco di max. 1 mm in lunghezza e in larghezza 200μm che contiene materiale fogliare quanto possibile si ottiene (Block Size faccia potrebbe dover essere adattato alle dimensioni del coltello diamante). </li><li> Sezione del blocco con un ultramicrotomo (es. Reichert Leica Ultracut S; Microsystems Leica) utilizzando un coltello di diamante con un angolo di 45 ° coltello (es. DIATOME Ultra 45, Gröpl, Tulln, Austria). Spessore della sezione deve essere regolato a circa 70 a 90 nm e la velocità di taglio dovrebbe essere di circa 1 mm / s. Raccogliere più sezioni con un (Agar Scientific Ltd.) di rame ricoperto di nichel o formvar 200 griglia a maglie quadrate. </li><li> Post-macchia le sezioni sulla griglia di partenza con citrato di piombo (Agar Scientific Ltd., citrato di piombo 1,1 g sciolti in 42 ml di acqua bidistillata e 8ml 1N NaOH) per 5 minuti in una capsula di Petri parzialmente pieni di NaOH per creare un senza CO 2 ambiente e per 15 minuti con acetato di uranile 1% (Agar Scientific Ltd.) disciolto in acqua distillata a temperatura ambiente. Lavare le griglie con acqua distillata per 1 minuto tra ogni fase post-colorazione. Far asciugare le griglie in una casella della griglia. </li><li> Esaminare le sezioni con un microscopio elettronico a trasmissione (es. Philips CM10 TEM, FEI, Eindhoven, Paesi Bassi). </li></ol> 3. Colorazione negativa <ol><li> Raccolto circa 100mg di TMV materiale infetto foglia e preparare linfa grezzo mediante omogeneizzazione del materiale per 2 minuti con una lama di rasoio in un vetrino in 100 microlitri di 60mm Søfrensen tampone fosfato (pH 7,2). </li><li> Trasferire 20μl di omogenato risultante sul primo pozzetto di un vetrino rivestito teflon con 4 o più pozzetti (in alternativa è anche possibile trasferire l&#39;omogeneizzato su un pezzo di parafilm). </li><li> Inserire una griglia formvar rivestito sopra l&#39;omogenato con l&#39;(Agar Scientific Ltd.) formvar lato patinato rivolto verso la caduta e incubate per 5 minuti. </li><li> Lavare la griglia 2 volte per 2 minuti ciascuno disponendo la griglia sopra due gocce di 200μl 60mm Sørensen tampone fosfato (pH 7,2). </li><li> Incubare la griglia per 1 minuto con una appena preparata di acido fosfotungstico al 2% (Agar Scientific Ltd.) in soluzione 60mm Sørensen tampone fosfato (pH 6,5). </li><li> Rimuovere la griglia e lasciare asciugare in una scatola griglia. </li><li> Esaminare la griglia con un microscopio elettronico a trasmissione (es. Philips CM10 TEM, FEI, Eindhoven, Paesi Bassi). Prendere almeno 10 immagini scelte a caso virioni colorazione negativa (almeno 10 o più virioni dovrebbero essere visibili su ogni immagine) con il microscopio elettronico a trasmissione ad un ingrandimento di primaria 21000X o superiore. Bisogna fare attenzione che tutte le immagini hanno lo stesso ingrandimento. </li></ol> 4. Analisi delle immagini <ol><li> Misurare la lunghezza e la larghezza di almeno 100 particelle scelte a caso singolo virus sulle micrografie prese dai campioni di colorazione negativa utilizzando qualsiasi software di analisi immagine del computer [per esempio cellule D (Olympus, Vita e Scienza dei Materiali Europe GmbH, Hamburg, Germania) con lo strumento di analisi di particelle o Optimas 6.5.1-(Media Cybernetics Inc., Bethesda, Maryland, USA)]. </li><li> Calcolare medie e deviazioni standard per ottenere una lunghezza e la larghezza media delle particelle virali individuate tramite metodi di colorazione negativa e TEM. </li><li> Confronta lunghezza e caratteristiche ultrastrutturali (ottenuta in 2.5) con dimensioni di virus e alterazioni ultrastrutturali indotte da malattie virali noti in letteratura per identificare chiaramente le malattie virali. </li></ol> 5. Rappresentante dei risultati: Dopo assistite campione microonde preparazione tipica alterazioni ultrastrutturali TMV indotte quali grandi aree contenenti virioni allineati in forma parallela potrebbe essere osservato con il microscopio elettronico a trasmissione nel citosol di infetto Nicotiana cellule tabacum (Fig. 1A). Inoltre, nella linfa grezza di TMV-infetta lascia particelle di TMV potrebbe essere osservata come flessuosi, astiformi strutture dopo colorazione negativa (Fig. 1B). L&#39;analisi delle immagini di 100 particelle virali ha rivelato una dimensione media di TMV di 280 nm di lunghezza e 17Nm di larghezza (Fig. 2). L&#39;ultrastruttura in TMV cellule infettate e le dimensioni dei virioni in questo studio sono risultati in accordo con proprietà ultrastrutturali TMV-indotte nel tabacco e l&#39;intervallo di dimensioni delle particelle di TMV precedentemente riportato in letteratura 9-15. <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="Figura 1" /> Figura 1. Micrografie elettroniche di trasmissione di TMV-cellule infettate da foglia e virioni. A) L&#39;immagine mostra l&#39;ultrastruttura delle cellule del mesofillo TMV-infettati foglie di Nicotiana tabacum dopo la preparazione del campione centrale assistita a microonde. Si noti la grande area di virioni allineate parallele che hanno accumulato nel citosol.C = cloroplasto con amido (St), M = mitocondrio, N = nucleo, V = vacuolo, Bar = 2μm. B) L&#39;immagine mostra virioni che sono stati individuati mediante colorazione negativa la linfa delle foglie infette. <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="Figura 2" /> Figura 2. Dimensione relativa di virioni. Distribuzione relativa della lunghezza e della larghezza del TMV-particelle dopo colorazione negativa la linfa delle foglie infette come apparivano al microscopio elettronico. I valori medi (media lunghezza / larghezza ± deviazione standard) sono stati calcolati da 100 virioni.

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Nessun conflitto di interessi dichiarati.

Forno a microonde assistita preparazione del campione impianto per TEM ha dimostrato di fornire i dati ultrastrutturali veloci e affidabili nel giro di poche ore 3,4,16. La conservazione multa strutturale di organelli e membrane ottenuti con il metodo utilizzato in questo studio era simile a campioni convenzionali e cryofixed 5,15 con il vantaggio di una drastica riduzione fissazione campione e l&#39;incorporamento di tempo da 3 giorni o più per circa 2 ore. Questo rappresenta il protocollo più veloce preparazione del campione per TEM attualmente disponibili in letteratura. Il metodo descritto in questo studio combinato microonde assistita impianto per la preparazione del campione TEM con metodi di colorazione negativa che ha consentito una identificazione chiara e rapida delle alterazioni ultrastrutturali TMV indotte e l&#39;agente virale stessa. TMV alterazioni indotte ultrastrutturali poteva essere analizzata dopo il taglio, sezionamento e post-colorazione entro circa 4 ore dopo l&#39;inizio del fissaggio nella trasmissione elettron microscopio. Utilizzando la modalità completamente automatica preparazione del provino liberato il ricercatore di condurre colorazione negativa nel frattempo, al fine di determinare le dimensioni e la larghezza del agente virale. Quindi, possiamo concludere che questo metodo permette una diagnosi chiara e rapida delle malattie delle piante virus in circa mezza giornata, che è di grande importanza per un uso futuro in esperimenti di agricoltura e scientifico in impianti fitopatologia. Poiché questo metodo potrebbe anche essere utilizzato per la diagnosi rapida di malattie umane e animali ha un grande potenziale per la futura applicazione in medicina veterinaria e patologia.

<table border="1"><tbody><tr><td> Nome del reagente / attrezzature </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Glutaraldeide </td><td> Agar Scientific Ltd. </td><td> R1312 </td><td></td></tr><tr><td> Osmio tetrossido </td><td> Agar Scientific Ltd. </td><td> R1022 </td><td></td></tr><tr><td> Agar resina 100 </td><td> Agar Scientific Ltd. </td><td> R1043 </td><td></td></tr><tr><td> Dodecenyl anidride succinica </td><td> Agar Scientific Ltd. </td><td> R1051 </td><td></td></tr><tr><td> Metile anidride nadic </td><td> Agar Scientific Ltd. </td><td> R1081 </td><td></td></tr><tr><td> Benzyl dimetilammina </td><td> Agar Scientific Ltd. </td><td> R1060 </td><td></td></tr><tr><td> Citrato di piombo </td><td> Agar Scientific Ltd. </td><td> R1210 </td><td></td></tr><tr><td> Acetato di uranile </td><td> Agar Scientific Ltd. </td><td> R1260A </td><td></td></tr><tr><td> Acido fosfotungstico </td><td> Agar Scientific Ltd. </td><td> R1213 </td><td></td></tr><tr><td> Formvar </td><td> Agar Scientific Ltd. </td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Leica EM AMW </td><td> Leica Microsystems </td><td></td><td></td></tr><tr><td> Leica (Reichert) Ultratrim </td><td> Leica Microsystems </td><td></td><td> Post più recente modello è disponibile </td></tr><tr><td> Leica (Reichert) Ultracut S </td><td> Leica Microsystems </td><td></td><td> Post più recente modello è disponibile </td></tr><tr><td> DIATOME Ultra 45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td> Philips CM10 TEM </td><td> FEI (ex Philips) </td><td> &lt;/ Td&gt; <td> Post più recente modello è disponibile </td></tr><tr><td> Cella D </td><td> Olympus Inc. </td><td></td><td></td></tr><tr><td> Optimas 6.5.1 </td><td> Media Cybernetics Inc. </td><td></td><td> Post più recente versione è disponibile </td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

Le indagini sulle alterazioni ultrastrutturali indotte dal virus sono spesso necessario individuare chiaramente malattie virali nelle piante. Con la preparazione del campione convenzionale per la microscopia elettronica a trasmissione (TEM) che tali indagini può richiedere diversi giorni 1,2 e non sono quindi adatti per una rapida diagnosi di malattie virali delle piante. Fissazione microonde può essere utilizzata per ridurre drasticamente il tempo di preparazione del campione per TEM con risultati simili ultrastrutturali come osservato dopo convenzionalmente preparazione del campione 3-5. Molti diversi dispositivi a microonde su misura disponibile, che può essere impiegata per il fissaggio successo e incorporamento di campioni biologici per TEM 5-8. In questo studio abbiamo dimostrato il virus del mosaico del tabacco (TMV) piante infettate Nicotiana tabacum che è possibile diagnosticare alterazioni ultrastrutturali in foglie in circa mezza giornata utilizzando preparazione del campione assistita microonde per TEM. Abbiamo scelto di compiere questo studio con un dispositivo a microonde disponibile in commercio come effettua la preparazione del campione quasi completamente automatico 5 in contrasto con gli altri dispositivi disponibili dove molti passi devono essere ancora eseguite manualmente 6-8 e sono quindi più tempo e consumare manodopera. Come preparazione del campione è eseguita in modo completamente automatico la colorazione negativa di particelle virali nel linfa dei restanti TMV-foglie infette e l&#39;esame del seguente ultrastruttura e le dimensioni possono essere eseguite durante la fissazione e l&#39;inclusione.

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Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Questo studio descrive un metodo che permette la diagnosi rapida e chiara delle malattie delle piante virus in circa mezza giornata, utilizzando una combinazione di preparazione dell&#39;impianto assistita campione microonde per microscopia elettronica a trasmissione e metodi di colorazione negativa.

Forno a microonde Assist diagnosi rapida di malattie virali piante da microscopia elettronica a trasmissione

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

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Immunology and Infection

13.4K Views.  University of Graz. Questo studio descrive un metodo che permette la diagnosi rapida e chiara delle malattie delle piante virus in circa mezza giornata, utilizzando una combinazione di preparazione dell'impianto assistita campione microonde per microscopia elettronica a trasmissione e metodi di colorazione negativa.

Video: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

La diagnosi rapida del virus dell&#39;influenza aviaria nei volatili selvatici: l&#39;uso di un Portable rRT-PCR e liofilizzato reagenti nel campo

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Ricerca

Immunologia e infezioni

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Struttura del virus HIV-1 Assemblee Capsidico da Cryo-microscopia elettronica e iterativo ricostruzione elicoidale reale spazio

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Analisi della accessibilità al solvente dei residui di cisteina in<em&gt; Mais Rayado Fino virus</em&gt; Particelle virus-simili Prodotto in<em&gt; Nicotiana benthamiana</em&gt; Piante e Cross-linking di peptidi a VLP

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Ad alta risoluzione microscopia elettronica del<em&gt; Helicobacter pylori</em&gt; Cag secrezione di tipo IV Sistema Pili Prodotto in diverse condizioni di Ferro Disponibilità

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

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.

Modellazione il ciclo di vita Ebola Virus Sotto livello di biosicurezza 2 condizioni con particelle virus-simili contenenti 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 ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologie per lo studio<em&gt; B. subtilis</em&gt; I biofilm come modello per Caratterizzare piccole molecole inibitori biofilm

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protocolli per Indagare la distribuzione Host-tessuti, trasmissione-mode, e Effetto sul Fitness ospite di un Densovirus in cotone bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

Antimicrobica sinergia test di stampante a getto d'inchiostro-assistita Checkerboard automatizzato matrice e il metodo di Time-kill manuale

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...