Questo studio è stato sostenuto dal Public Health Service concedere AI-34431 (a DAH) dell&#39;Istituto Nazionale di Allergologia e Malattie Infettive e da Fondi VA Medical Research (a DAH).

1. Fissazione di Leptospira di vetrini <ol><li> Leptospira interrogans è una classe BSL2 patogeno. Lavorando con cellule vive che richiede una gestione appropriata, ad esempio i guanti, camice e fasi di pipettamento in una sterile-cappuccio. </li><li> Crescere Leptospira in EMJH medium11, integrato con siero di coniglio 1% a 30 ° C fino a raggiungere metà e la fine-log fase (densità di 5 x 10 7 a 5 x 10 8 cellule / ml) per circa 6 giorni. </li><li> Raccolta della cultura per centrifugazione a ~ 2000 xg per 7 minuti a temperatura ambiente. </li><li> Eliminare il surnatante tramite aspirazione e risospendere delicatamente il pellet in tampone fosfato (PBS) -5 mM MgCl 2, pH7.2 ad una concentrazione finale di 5 x 10 8 cellule / ml. </li><li> Aggiungere 1 ml di sospensione cellulare in ciascun pozzetto di diapositive da camera a due ben vetro e incubare a 30 ° C per 80 min per consentire alle cellule di aderire. </li><li> Rimuovere con attenzione il liquido contenente cellule non legato per aspirazione. </li><li> Fissare i batteri rimasti intatti alle diapositive di vetro con l&#39;aggiunta di 1 ml / pozzetto del 2% paraformaldeide in PBS-5 mM MgCl 2. Incubare per 40 min a 30 ° C. Questi diapositive saranno per la valutazione della superficie esposta proteine. </li><li> Vetrini di controllo per valutare sotto la superficie delle proteine, la membrana esterna permeabilize fissando con 1 ml / pozzetto del 100% ghiacciata metanolo. Incubare a -20 ° C per 20 min. Metanolo agisce in diversi modi: si permeabilizes la membrana esterna, denaturates le proteine, e le cellule correzioni alle diapositive di vetro. </li><li> Rimuovere gli agenti di fissaggio mediante aspirazione. </li></ol> 2. Etichettatura con anticorpi specifici <ol><li> Blocca legame non specifico con l&#39;aggiunta di 1 ml / pozzetto di tampone di bloccaggio (Difco Leptospira arricchimento EMJH). Incubare a 30 ° C per 90 min. </li><li> Diluire l&#39;anticorpo specifico (sieri immuni di coniglio o di anticorpi monoclonali di topo, coniglio qui sieri policlonali specifici per OmpL54 10, FlaA1 12 e OmpL1 13) e pre-immune coniglio ascetico sieri o il mouse fluido che non contengono anticorpi (quando utilizzati come controlli negativi) in tampone di bloccaggio. Diluizioni per ogni anticorpo devono essere determinati empiricamente a seconda del titolo anticorpale, antigene-anticorpo reattività e l&#39;abbondanza di proteine ​​all&#39;interno della cellula. La gamma usuale è 1:50 a 1:600. </li><li> Rimuovere il tampone di bloccaggio tramite aspirazione e aggiungere 1 ml / pozzetto di diluire gli anticorpi primari. </li><li> Incubare a 30 ° C per 1 ora. </li><li> Asportare il liquido mediante aspirazione e lavare i pozzetti tre volte con PBS (1 ml / pozzetto). </li></ol> 3. Visualizzazione delle leptospire <ol><li> Aggiungere 1 ml / pozzetto di Alexa Fluor 488-etichettati anticorpi secondari (o anti-rabbit IgG di capra o di capra anti-topo IgG) diluito 1:2000 e fluorescenti acido nucleico macchia, 4&#39;6-diamidino-2-fenil-indolo dicloridrato ( DAPI) diluito ad una concentrazione finale di 0,25 mg / ml in tampone di bloccaggio. Questo passaggio garantisce il rilevamento di legame degli anticorpi e la presenza di tutte le spirochete indipendente di legame degli anticorpi, rispettivamente. </li><li> Incubare i vetrini a 30 ° C per 45 min. </li><li> Asportare il liquido mediante aspirazione e lavare i pozzi due volte con PBS e una volta con acqua distillata (1 ml / pozzetto). </li><li> Rimuovere le camere e la striscia adesiva da vetrini e asciugare all&#39;aria per circa 10 min. </li><li> Aggiungi prolungare oro anti-sbiadimento mezzo di montaggio (2 x 20 l per vetrino) e una x 24 50 vetrino mm. </li><li> Incubare per una notte a temperatura ambiente al buio. Questo passaggio è necessario per permettere al mezzo di montaggio per la cura (indurimento). </li><li> Sigillare il coprioggetto con smalto. </li><li> Visualizza la colorazione al microscopio a fluorescenza utilizzando un grigio-azzurro / blu rilevamento filtro DAPI e un filtro di rilevamento verde per Alexa Fluor 488. Per garantire che una rappresentazione accurata dei campioni è fatto, assicuratevi di valutare l&#39;intera area della camera per ogni campione. </li><li> Registrare i dati da immagini di un campo rappresentante per ogni campione. Quando l&#39;imaging Alexa Fluor 488 fluorescenza, utilizzare lo stesso tempo di esposizione per tutti i campioni. Usando un tempo coerente esposizione da consentire il confronto più accurato dei risultati con gli antigeni di test rispetto ai controlli. </li></ol> 4. Rappresentante dei risultati: Esempi di risultati superficie IFA con le cellule Leptospira interrogans utilizzando anticorpi contro la superficie e sub-superficiali proteine ​​sono mostrati nella Figura 2. Un test è considerato positivo quando fluorescenza sostanziale viene rilevato in campioni con leptospire intatto per una proteina di interesse che è superficie esposta (fig. 2A). Di solito, in un test positivo (qui, superficie esposta OmpL54), circa lo stesso intensità di fluorescenza si osserva nelle cellule sia integro e permeabilizzate (Fig. 2A) È essenziale includere un controllo negativo per l&#39;integrità della membrana esterna, utilizzando anticorpi contro un sottosuolo , preferibilmente periplasmatico proteine ​​(ad esempio leptospiral FlaA1, fig. 2B). Solo quando i dati mostrano che la membrana esterna è rimasta intatta during dell&#39;esperimento, cioè di anticorpi contro un sub-superficiale di proteine ​​non reagiscono alle cellule intatte (non fluorescenza o fluorescenza molto più debole rispetto al campione permeabilizzate come in fig. 2B), si può concludere che la proteina (s) di interesse superficie esposta. L&#39;inclusione degli esperimenti con pre-sieri immuni (nel caso degli anticorpi policlonali) come un ulteriore controllo negativo è essenziale per dati univoci e non deve cedere a fluorescenza sostanziale cellule intatte o permeabilizzate (Fig. 2A, 2C). Macchia DAPI è necessario per visualizzare leptospire quando i risultati negativi (nessun Alexa Fluor 488 fluorescenza) siano rispettate. Controcolorazione con ioduro di propidio è un&#39;alternativa accettabile per DAPI. Questo metodo è qualitativa piuttosto che quantitativa come brillante fluorescenza può essere dovuto alla superficie più esposta epitopi antigenici essere riconosciuto in un singolo antigene rispetto a altri antigeni di abbondanza uguali ma con un minor numero di epitopi di superficie esposta. Pertanto le intensità di fluorescenza può essere utilizzato solo per confrontare la reattività degli anticorpi stessi, come le cellule intatte contro permeabilizzate e intensità di fluorescenza non tra proteine ​​diverse. Questa distinzione è importante quando un risultato ambiguo, dove si ottiene molto meno fluorescenza si osserva nelle cellule intatte che in cellule permeabilizzate (Fig. 2C). Tale risultato indica che la proteina è in una posizione sotto la superficie (con alcune non specifica colorazione di fondo delle cellule intatte), gli anticorpi sono vincolanti preferenzialmente a non nativi epitopi che sono esposti dopo permeabilizzazione metanolo o di una proteina potrebbe avere più siti di localizzazione, come descritto per le proteine ​​Erp e Elp di un altro spirocheta, Borrelia burgdorferi 14. In entrambi i casi, un risultato ambiguo IFA come questo richiede approcci alternativi come biotinilazione superficie o proteolisi superficie per determinare se la proteina di interesse superficie esposta o meno 10. <img src="/files/ftp_upload/2805/2805fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso per la superficie di immuno-fluorescenza saggio di Leptospira interrogans spirochete. In primo luogo, 1 ml di sospensione cellulare viene aggiunto in ciascun pozzetto della due-e diapositive da camera (almeno due vetrini per ogni esperimento) e incubate per leptospire di aderire. Successivamente, leptospire sono fissati alle diapositive da camera con l&#39;aggiunta di 1 ml / pozzetto del 2% paraformaldeide (PFA) per i campioni con intatto membrana esterna (OM) e 1 ml / pozzetto del 100% metanolo freddo per i campioni con permeabilizzate OM. Successivamente, leptospire vengono incubati con gli anticorpi riconoscimento superficie esposta proteine ​​con anticorpi di controllo per le proteine ​​del sottosuolo (Primario Ab) a 30 ° C per 90 min. Poi, gli anticorpi primari sono rilevati mediante incubazione con 1 ml / pozzetto di Alexa Fluor 488 anticorpi secondari coniugati. Infine, le cellule vengono visualizzati al microscopio a fluorescenza. <img src="/files/ftp_upload/2805/2805fig2.jpg" alt="Figura 2"/> Figura 2. Un&#39;analisi superficiale delle proteine ​​leptospiral immunofluorescenza. Leptospira interrogans spirochete sono stati sondati con sieri immuni e pre-immune, ed i risultati negativi sono stati accoppiati con un contrasto DAPI per dimostrare la presenza di spirochete. Una fluorescenza verde filtro è stato usato per rilevare Alexa Fluor 488 anticorpo marcato secondaria legame con le proteine ​​di superficie esposta e blu / grigio-azzurro fluorescenza filtro per rilevare legame al DNA delle cellule DAPI. A) leptospire sondato con anticorpi riconoscere una proteina di superficie esposta, OmpL54 10. B) Le cellule sondato con anticorpi contro una proteina periplasmatico sottosuolo, FlaA1 (controllo negativo). C) Le cellule sondato con anticorpi contro leptospiral porina, OmpL1. Vincolante di sieri di coniglio leptospire sono stati rilevati con Alexa Fluor 488 coniugato capra anti-coniglio frammenti di IgG. Tutte le immagini sono prese dopo 4 secondi di esposizione a lungo.

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Nessun conflitto di interessi dichiarati.

La nostra tecnica IFA superficie è simile a quello utilizzato per dimostrare l&#39;esposizione superficie di vari borrelial 15, 16 e leptospiral 12, 17 proteine. I dettagli del metodo superficie IFA qui descritti sono stati progettati per ridurre al minimo la manipolazione di cellule, nel tentativo di mantenere l&#39;integrità della membrana esterna sfruttando la tendenza delle cellule Leptospira di aderire alle diapositive di vetro. Il metodo prevede una concentrazione più bassa (2% versus 4%) ...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Due pozzetti camera di vetrini </td><td> Lab-Tek </td><td> 177380 </td><td></td></tr><tr><td> Siero di coniglio </td><td> Rockland Immunochemicals </td><td> D209-00-0100 </td><td></td></tr><tr><td> Leptospira arricchimento EMJH </td><td> BD Difco </td><td> 279510 </td><td></td></tr><tr><td> Alexa Fluor 488 di capra anti-IgG di coniglio (H + L) </td><td> Invitrogen / Molecular Probes </td><td> A-11034 </td><td></td></tr><tr><td> Alexa Fluor 488 capra anti-topo IgG (H + L) </td><td> Invitrogen / Molecular Probes </td><td> A-11029 </td><td></td></tr><tr><td> 4&#39;6-diamidino-2-fenil-indolo dicloridrato (DAPI) </td><td> Invitrogen / Molecular Probes </td><td> D1306 </td><td></td></tr><tr><td> Prolungare oro anti-sbiadimento mezzo di montaggio </td><td> Invitrogen / Molecular Probes </td><td> P36930 </td><td> A temperatura ambiente prima dell&#39;uso </td></tr><tr><td> Fisherfinest Premium di vetro di copertura </td><td> Pescatore </td><td> 12-544-14 </td><td></td></tr><tr><td> Microscopio a fluorescenza </td><td> Zeiss </td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

Proteine ​​della superficie batterica sono coinvolti in contatto diretto con le cellule ospiti e l&#39;assorbimento delle sostanze nutritive dall&#39;ambiente 1. Per questo motivo, localizzazione cellulare in grado di fornire intuizioni sul ruolo funzionale delle proteine ​​batteriche. Localizzazione superficiale delle proteine ​​batteriche è un passo fondamentale verso l&#39;identificazione dei fattori di virulenza coinvolti nei meccanismi di patogenicità. Metodi per frazionamento leptospiral 2-5 membrane può essere selettivo per una certa classe di proteine ​​di membrana esterna (OMP), come lipoproteine ​​vs OMP transmembrana, e quindi portare a errata classificazione. Questo probabilmente è dovuto a differenze strutturali e come essi sono associati alla membrana esterna. Le lipoproteine ​​sono associati alle membrane attraverso una interazione idrofobica tra N-terminale porzione lipidica (tre acidi grassi) e dei fosfolipidi doppio strato lipidico 6, 7. Al contrario, OMP transmembrana sono tipicamente integrate nel doppio strato lipidico da anfipatiche β-fogli disposti in una botte struttura simile 8, 9. Inoltre, la presenza di una proteina nella membrana esterna non garantisce necessariamente che la proteina oi suoi domini sono esposte sulla superficie. Spirochete membrane esterne sono noti per essere i metodi fragile e occorre, pertanto coinvolgono la manipolazione dolce delle cellule e l&#39;inclusione di sub-superficie proteina controlli per valutare l&#39;integrità della membrana esterna. Qui, presentiamo una immunofluorescenza saggio (IFA) per valutare direttamente l&#39;esposizione di proteine ​​sulla superficie leptospire intatto. Questo metodo si basa sul riconoscimento delle proteine ​​di superficie leptospiral da antigene-anticorpi specifici. Qui, gli anticorpi specifici per OmpL54 10 sono detetcted aftero vincolanti per natale, epitopi superficie esposta. Confronto di reattività anticorpi contro le cellule intatte permeabilizzate consente la valutazione della distribuzione cellulare e se non è selettivo una proteina presente sulla superficie leptospiral. L&#39;integrità della membrana esterna deve essere valutato utilizzando anticorpi ad una o più proteine ​​del sottosuolo, preferibilmente situato nel periplasma. Il metodo IFA superficie può essere utilizzato per analizzare l&#39;esposizione superficie di qualsiasi proteina leptospiral a cui gli anticorpi specifici sono disponibili. Sia l&#39;utilità e la limitazione del metodo dipende dal fatto che gli anticorpi utilizzati sono in grado di legarsi agli epitopi nativi. Dal momento che gli anticorpi sono spesso sollevate contro proteine ​​ricombinanti, di epitopi nativi, superficie esposta proteine ​​potrebbe non essere riconosciuto. Tuttavia, il metodo superficie IFA è uno strumento prezioso per lo studio delle componenti intatte superfici batterica. Questo metodo può essere applicato non solo per leptospire, ma anche spirochete e altri batteri gram-negativi. Per i più forti conclusioni sulla superficie-esposizione di OMP, un approccio globale che coinvolga diversi metodi di localizzazione delle cellule si consiglia 10.

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Un metodo efficace per valutare l&#39;esposizione superficiale di proteine ​​leptospiral è descritto. Il metodo è specificamente progettato per evitare l&#39;interruzione della membrana esterna delle cellule fragili leptospiral. Questa tecnica richiede l&#39;impiego di diversi controlli negativi per valutare l&#39;integrità della membrana esterna e la specificità della reazione anticorpale.

Immuno-fluorescenza saggio di Leptospiral superficie esposti Proteine

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

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Immunology and Infection

15.7K Views.  University of California, Los Angeles. Un metodo efficace per valutare l'esposizione superficiale di proteine ​​leptospiral è descritto. Il metodo è specificamente progettato per evitare l'interruzione della membrana esterna delle cellule fragili leptospiral. Questa tecnica richiede l'impiego di diversi controlli negativi per valutare l'integrità della membrana esterna e la specificità della reazione anticorpale.

Video: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Una tecnica di microscopia TIRF per Real-time, Imaging simultanea del TCR e delle sue proteine ​​associate segnalazione

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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Immunologia e infezioni

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Saggio di placca per Norovirus Murine

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Metodologia per la generazione efficiente di proteine del virus vaccinia taggate fluorescentmente

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Fenomica fagi: Approcci fisiologici per caratterizzare Novel virali Proteine

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Saggio altamente sensibile per la misura dell&#39;attaccamento Arenavirus-cell

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

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

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

Ottimizzazione di un Quantitative Micro-neutralizzazione Assay

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

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificazione di sequenze di localizzazione Plasmodesmal in proteine In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Targeting dei tioli cisteina per in Vitro site-specific glicosilazione delle proteine ricombinanti

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.