Questo lavoro è stato supportato da sovvenzioni USDA 1999-35212-8680, 2005-01378, e 2010-04288-01, USDA Fondi Formula e dello Stato della Georgia di Medicina Veterinaria di Grant ricerca agricola. Il protocollo descritto in questa pubblicazione è stata sviluppata per l&#39;uso da studenti universitari come parte di corsi di ricerca diretto: MIBO 4900L, GENE 4960H, BCMB 4960L e 4960H BIOL presso l&#39;Università della Georgia e utilizzate dagli studenti su progetti diversi epidemiologia molecolare di ricerca nel Maurer laboratorio. Desideriamo ringraziare i numerosi studenti universitari che hanno svolto attività di ricerca in Maurer e laboratori Lee, e il nostro reparto di sedia, John Glisson per supportare i nostri sforzi per integrare l&#39;istruzione universitaria con la ricerca.

<ol>
	<li>Dauga, C., Zabrovskaia, A., Grimont, P. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+fragment+length+polymorphism+analysis+of+some+flagellin+genes+of+Salmonella+enterica.">Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.</a> J. Clin. Microbiol. 36, 2835-2843 .</li><li>Hong, Y., Liu, T., Lee, M. D., Hofacre, C. L., Maier, M., White, D. G., Ayers, S., Wang, L., Berghaus, R., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+screen+of+broth+enrichments+for+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg,+and+Typhimurium+using+an+allelotyping+multiplex+PCR+that+targets+O+and+H+antigen+alleles.">A rapid screen of broth enrichments for Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium using an allelotyping multiplex PCR that targets O and H antigen alleles.</a> J. Food Prot. 72, 2198-2201 (2009).</li><li>Hong, Y., Liu, T., Lee, H. o. f. a. c. r. e., Maier, C. L., White, M., Ayers, D. G., Wang, S., Berghaus, L., R, M. a. u. r. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.Rapid+screening+of+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg+and+Typhimurium+using+a+serologically-correlative+allelotyping+PCR+targeting+the+O+and+H+antigen+alleles.">J.Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.</a> BMC Microbiol. , (2008).</li><li>Liu, T., Liljebjelke, K., Bartlett, E., Hofacre, C. L., Sanchez, S., Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nested+PCR+to+detection+of+Salmonella+in+poultry+environments.">Application of nested PCR to detection of Salmonella in poultry environments.</a> J. Food Prot. 65, 1227-1232 (2002).</li><li>Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+limitation+of+molecular+techniques.">Rapid detection and limitation of molecular techniques.</a> Ann. Rev. Food Sci. Tech. 2, 259-279 (2011).</li><li>Moscoso, H., Alvarado, I., Hofacre, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+infectious+bursal+disease+virus+from+bursal+tissues+collected+on+FTA+filter+paper.">Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.</a> Avian Dis. 50, 391-396 (2006).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory. , (1989).</li><li>Wittwer, C. T., Fillmore, G. C., Hillyard, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+polymerase+chain+reaction+capillary+tubes+with+hot+air.">Automated polymerase chain reaction capillary tubes with hot air.</a> Nucleic Acids Res. 17, 4353-4357 (1989).</li></ol>

Nessun conflitto di interessi dichiarati.

La maggior parte dei test disponibili in commercio PCR per l&#39;individuazione dei geni Salmonella obiettivi unici al genere (es. invasioni). Tuttavia, non tutti Salmonella sono uguali nella loro capacità di causare malattie nell&#39;uomo o prevalenza nelle popolazioni animali. Il riconoscimento precoce delle differenze antigeniche in Salmonella LPS O-antigene e flagellinH1 e gli antigeni H2 ha portato alla differenziazione di Salmonella in oltre 2.500 sierotipi sulla base di queste differenze antigeniche. Ci sono 46 diversi antigeni O e 86 distinte flagellina (H) antigeni identificati nel genere Salmonella. Salmonella può possedere uno (H1) o due diversi (H1 e H2) flagellins. La diversa combinazione di O, H1 e H2 conto antigeni per i sierotipi di Salmonella 2.500 riconosciuto oggi. Un sierotipo viene assegnato sulla base della formula antigenica derivato dalla identificazione dei singoli antigeni O e H. La formula antigenica è scritto come O: H1: H2, e dove &quot;-&quot;, si riferisce all&#39;assenza di H1 o H2 flagellina e &quot;NT&quot; (non tipizzabile) per la salmonella negativo per l&#39;O-antigene. Ad esempio, il sierotipo Typhimurium è assegnato ad una S. enterica isolare con la formula antigenica B: i: 1,2 mentre Enteritidis è dato a un isolato con la formula D1: g, m: -. Questa variazione antigenica nella popolazione Salmonella è la manifestazione esteriore delle differenze a livello di nucleotidi che possono quindi essere facilmente sfruttato da PCR. Abbiamo identificato le differenze genetiche associate con specifici alleli O e H di sviluppare un allelotyping PCR per l&#39;individuazione S. enterica sierotipi: Enteritidis, Hadar, Heidelberg, e Typhimurium (3). La corretta assegnazione e la segnalazione di Salmonella serovar richiede test per tutti gli alleli associati a O: H1: formule antigeniche H2 per Enteritidis (D1: g, m: -), Hadar (C2: Z10: e, n, x), Heidelberg (B : r: 1,2), e Typhimurium (B: i: 1,2). Senza la conoscenza della allele O o antigene, il ritrovamento del g H1, allele m da solo non necessariamente identificare il isolare Salmonella Enteritidis come come gli altri sierotipi di Salmonella: Agona, California, e Montevideo, anche in possesso di questo stesso allele. Mentre la PCR allelotyping è stato originariamente progettato per rilevare il succitato sierotipi di Salmonella, questo test può identificare un ulteriore periodo di 19 sierotipi (Tabella 3). Il nostro allelotyping PCR è stato originariamente progettato per rilevare gli alleli O e H. In pratica, è diventato più pratico e conveniente per di identificare l&#39;O-antigene mediante test di agglutinazione su vetrino, con O-specifici antisieri, anziché eseguire l&#39;O allelotyping PCR quando si lavora con le culture Salmonella isolati. Tuttavia, si sconsiglia l&#39;uso del O allelotyping PCR se il laboratorio utilizza questo PCR come uno schermo (2) o per la tipizzazione isolati di Salmonella presentato in sede di FTA carte (6), e comprendono una Salmonella specifico PCR con la prima schermata dei campioni (4 ). Limitare la PCR allelotyping solo a quei campioni che sono identificati come Salmonella mediante PCR o test biochimici / antigenica. Il protocollo descritto in questo articolo è stato ottimizzato per l&#39;utilizzo con la tecnologia Idaho Rapidcycler, ad aria calda termociclatore che permette di far ridurre la quantità di reazione e le condizioni di reazione viene eseguito in meno tempo di quanto molti termociclatori blocco riscaldante. Per adattare questo protocollo per l&#39;utilizzo con un termociclatore blocco riscaldante può richiedere l&#39;ottimizzazione delle condizioni di reazione empiricamente abbassando / alzando la temperatura di ricottura; regolare le concentrazioni di fondo, oppure regolando MgCl2 concentrazioni. Per esempio, abbiamo dovuto adattare il protocollo per la Ricerca MJ termociclatore PTC-200 come segue. Lavorare con i volumi stessa reazione descritta nella tabella 1, lo stock di lavoro concentrationof è stato diluito il set fondo ho a 2,5 micron, la temperatura di annealing è stata abbassata da 55 ° C a 45 ° C, e tempi di incubazione alla denaturazione, appaiamento ed estensione passi è stato allungato a 20 s per i / g, m allelotyping PCR. Avendo gli opportuni controlli positivi e negativi sono essenziali nella start up iniziale e di ottimizzazione per qualsiasi PCR, inclusi i protocolli pubblicati. Si consiglia di acquisire i sierotipi di Salmonella noto, in particolare Anatum (E1: e, h: 1,6), Enteritidis (D1: g, m: -), Hadar (C2: Z10: e, n, x), Heidelberg (B: r : 1,2), Montevideo (C1: g, m, s: 1,2,7) e Typhimurium (B: i: 1,2), e un laboratorio di Escherichia coli K12 ceppo (DH5α, LE393, JM109, ecc ) come controlli positivi e negativi rispettivamente per il test PCR allelotyping descritto in questo articolo. Molti di questi sierotipi di Salmonella serviranno come controlli positivo o negativo, a seconda di quale allele è testato. Alcune precauzioni e le linee guida devono essere seguite durante l&#39;esecuzione di questo e qualsiasi altra diagnostico PCR. In primo luogo, la separazione fisicadi preparazione modello, PCR set-up, e l&#39;elettroforesi su gel è fondamentale nel prevenire possibili PCR riporto contaminazione e segnalazione erronea di falsi positivi. Inoltre, l&#39;inclusione di controlli adeguati è necessario in ogni routine PCR come questi controlli identificare i problemi che si presentano e facilitare l&#39;interpretazione dei risultati (5). Più importante, la coerenza è fondamentale, soprattutto per quanto riguarda le condizioni elettroforetica per amplicone (PCR prodotto) andestimation rilevamento di dimensioni amplicone. Per illustrare questo punto, abbiamo volutamente sospeso elettroforesi prima di quanto previsto nella Figura 1A. Gli standard di peso molecolare (corsie 1 e 13) e il controllo positivo (corsia 2) non sono sufficientemente separati da stimare con precisione le dimensioni o osservare la separazione di frammenti di DNA dove ci sono piccole differenze (~ 50 pb) in dimensioni. Un&#39;adeguata separazione di frammenti di DNA, in particolare gli standard di peso molecolare, è essenziale come report positivi dipende stima accurata della dimensione amplicone e distintivi &quot;vero positivo&quot; da non specifici ampliconi che vengono a volte osservata nella gestione quotidiana di una PCR (5 ). Ad esempio, vi è un non-specifico amplicone in Figura 1B, corsia 7 (~ 700 bp nel formato). Aveva elettroforesi stato interrotto troppo presto, quando il fronte del colorante è stato solo nel punto a metà strada nel gel di agarosio, ci sarebbero differenze sono minime tra i 500 bp e 700 bp frammenti di DNA. Pertanto, campione in corsia 7 sarebbe stato erroneamente riportato come positivo sia per i e g, m alleli. Infine, bisogna riconoscere i limiti associati a qualsiasi prova. Molti dei allelotyping H1/H2 PCR non hanno il potere discriminatorio di discernere sottili differenze genetiche in alleli appartenenti al H2 1,2; 1,5, 1,6, 1,7 complesso antigene, e, n, x / e , n, Z15 complesso antigene e l&#39;antigene H1 G complesso, che comprende l&#39;, allele g m. Uno o due cambiamenti di aminoacidi rappresentano gli epitopi diversi presenti in questi antigeni flagellari. Era quindi difficile per noi a progettare primers in grado di sfruttare queste differenze a volte singolo paia di basi della sequenza del gene flagellina (3). Per esempio, sierotipi di Salmonella Dublin (D1: g, p: -) e Berta (D1: f, g, t: -) sono positivi alla PCR con la nostra g, mallelotyping primer. I primer allelotyping H2 non può distinguere Typhimurium (B: i: 1,2) da Lagos (B: i: 1,5), Heidelberg (B: r: 1,2) da Bradford (B: r: 1,5), Winneba (B: r: 1,6), o Remo (B: r: 1,7), o Hadar (C2: Z10: e, n, x) da Glostrup (C2: Z10: e, n, Z15). Mentre la probabilità di incontrare alcuni di questi sierotipi: Lagos, Bradford, Winneba, Remo o Glustrup è remoto, è una possibilità e richiede uno provvedendo disclaimer sulla limitazione dei test &#39;o in un altro test di conferma. In conclusione, questo PCR-based sierotipizzazione identifica rapidamente S. enterica sierotipi Enteritidis, Hadar, Heidelberg e Typhimurium, e O, H1, H2 e gli alleli associati con 19 sierotipi aggiuntivi. Questo test è un rapido e conveniente alternativa al tradizionale approccio microbiologico sierotipizzazione di Salmonella.

<table border="1"><tbody><tr><th> Nome del materiale </th><th> Tipo </th><th> Azienda </th><th> Numero di catalogo </th><th> Commento </th></tr><tr><td> Luria Bertani </td><td></td><td> Pescatore </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Provette per microcentrifuga </td><td> 1,5 ml di capacità </td><td> Pescatore </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Etanolo </td><td> 200 prova </td><td> Pescatore </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> dH 2 O </td><td> Biologia Molecolare Grado </td><td> Sigma </td><td></td><td> (O qualsiasi fonte comparabile; PCR solo) </td></tr><tr><td> 10 x PCR buffer </td><td> 20 mM MgCl 2 </td><td> Idaho Tecnologia </td><td></td><td> (Buffer viene fornito con BSA, Ficoll e Tartrazina) </td></tr><tr><td></td><td> 30 mM MgCl 2 </td><td></td><td></td><td></td></tr><tr><td></td><td> 40 mM MgCl 2 </td><td></td><td></td><td></td></tr><tr><td> PCR primer </td><td></td><td></td><td></td><td></td></tr><tr><td> DMSO </td><td></td><td></td><td></td><td></td></tr><tr><td> dNTP </td><td></td><td> Roche </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Taq DNA polimerasi </td><td></td><td> Denville </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Pipette capillari </td><td> Un volume di 10 microlitri </td><td> Idaho Tecnologia </td><td></td><td></td></tr><tr><td> Rapido Cycler </td><td></td><td> Idaho Tecnologia </td><td></td><td></td></tr><tr><td> Agarosio </td><td> Bassa EEO; Grado Biologia Molecolare </td><td> BioRad </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> 50 X TAE tampone o buffer TBE 5X </td><td></td><td> Pescatore </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Bromuro di etidio </td><td></td><td> BioRad </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Orizzontale, sommersi gel elettroforesi da camera con muffa gel </td><td></td><td> BioRad </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Alimentazione </td><td></td><td> BioRad </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr><tr><td> Molecular Imager Gel Doc sistema </td><td></td><td> BioRad </td><td></td><td> (O qualsiasi fonte comparabili) </td></tr></tbody></table> Tabella 4. Materiale

an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium

Gli attuali test commerciale PCR per l&#39;identificazione dei geni bersaglio Salmonella unica di questo genere. Tuttavia, ci sono due specie, sei sottospecie, e oltre 2.500 diversi sierotipi di Salmonella, e non tutti sono uguali nel loro significato per la salute pubblica. Per esempio, trovare S. enterica sottospecie IIIa Arizona in una fattoria strato tavolo uovo è insignificante rispetto all&#39;isolamento di S. sottospecie enterica sierotipo Enteritidis I, la principale causa di salmonellosi legato al consumo di uova da tavola. Sierotipi sono individuati sulla base di differenze antigeniche in lipopolisaccaride (LPS) (antigene O) e flagellina (H1 e H2 antigeni). Queste differenze antigeniche sono l&#39;aspetto esteriore della diversità dei geni e alleli del gene associata a questo fenotipo. Abbiamo sviluppato un allelotyping, PCR multiplex che le chiavi sulle differenze genetiche tra i quattro maggiori S. sottospecie enterica sierotipi che ho trovato nel pollame e associata a gravi malattie umane negli Stati Uniti. Le coppie di primer PCR sono stati mirati a geni chiave o sequenze uniche ad un sierotipo di Salmonella specifiche e destinate a produrre un amplicone con dimensioni specifiche per quel gene o allele. Salmonella sierotipo viene assegnato a un isolato basata sulla combinazione dei risultati dei test PCR per LPS specifico e flagellina alleli del gene. La PCR multiplex descritto in questo articolo sono specifici per la rilevazione di S. enterica sottospecie I sierotipi Enteritidis, Hadar, Heidelberg, e Typhimurium. Qui mostriamo come utilizzare il PCR multiplex per identificare un sierotipo di Salmonella isolare.

Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Descriviamo un multiplex PCR per la rilevazione rapida di Salmonella enterica sierotipi Enteritidis, Hadar, Heidelberg, e Typhimurium. Sierotipi di Salmonella specifici possono essere identificati prendendo di mira un multiplex PCR di geni e sequenze uniche per l&#39;O-antigene grappolo biosintesi e flagellina di un sierotipo dato. Sierotipo viene assegnato poi a isolare Salmonella in base alla comparsa di specifiche, ampliconi dimensioni (prodotto PCR) corrispondente al allele di destinazione.

Un Allelotyping PCR per l&#39;identificazione Salmonella enterica Sierotipi Enteritidis, Hadar, Heidelberg, e Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

an-allelotyping-pcr-for-identifying-salmonella-enterica-serovars

Research

JoVE Journal

Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

21.6K Views.  University of Georgia. Descriviamo un multiplex PCR per la rilevazione rapida di Salmonella enterica sierotipi Enteritidis, Hadar, Heidelberg, e Typhimurium. Sierotipi di Salmonella specifici possono essere identificati prendendo di mira un multiplex PCR di geni e sequenze uniche per l'O-antigene grappolo biosintesi e flagellina di un sierotipo dato. Sierotipo viene assegnato poi a isolare Salmonella in base alla comparsa di specifiche, ampliconi dimensioni (prodotto PCR) corrispondente al ...

Video: An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificare i geni disregolazione indotta da sarcoma di Kaposi associato Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Ricerca

Immunologia e infezioni

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Un<em&gt; In vitro</em&gt; Modello per studiare eterogeneità di differenziazione dei macrofagi umani e polarizzazione

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Crescita di misura e di espressione genica Dinamica di tumore-mirata<em&gt; S. Typhimurium</em&gt; Batteri

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedure per l&#39;identificazione Infettive prioni dopo il passaggio attraverso l&#39;apparato digerente di una specie aviaria

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Un protocollo Infettare<em&gt; Caenorhabditis elegans</em&gt; Con<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

High-throughput Assay per Fenotipo<em&gt; Salmonella enterica</em&gt; Typhimurium Associazione, Invasion, e replica nei macrofagi

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Creazione di una configurazione high-throughput screening per piccole molecole che modulano c-di-GMP Segnalazione di<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

La genotipizzazione di<em&gt; Staphylococcus aureus</em&gt; Per ribosomiale Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una piattaforma di High-throughput per lo Screening della Salmonella spp /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...