Il lavoro di autori FCO, DF, JN, DJH e GDS di cui sopra è finanziato dall&#39;Ufficio Chief Scientist, un numero di concessione: ETM37 e sostenuto dal Centro di Cancer Research UK Medicina sperimentale sul cancro. Il lavoro di AL è finanziato dal Royal College of Surgeons di Edimburgo Robertson, il trust Melville per la cura e la cura del cancro e del Medical Research Council. IO è sostenuto da una borsa di studio della Royal Society di Edimburgo governo scozzese cofinanziato Azioni Marie Curie e il Medical Research Council britannico. Gli autori desiderano ringraziare SCOTRRCC co-candidati e collaboratori per le loro discussioni utili su alcuni degli argomenti trattati in questo documento.

1. Identificazione di eterogeneità morfologica e molecolare del tumore <ol><li> Tumori rimossi dal congelatore -80 ° C e mantenuto in ghiaccio secco. </li><li> Dividere i tumori in sezioni di circa 1 cm 3. Mappare la posizione originale di ogni sezione tumore rispetto all&#39;altro ed etichetta con un nome univoco. Conservare i campioni in cryovials individuali a -80 ° C fino al momento dell&#39;uso. </li><li> Campioni Coat in ottobre e tagliare in un criostato a -22 ° C. </li><li> I campioni colorati con ematossilina eosina e il metodo di contrasto. </li><li> Analisi al microscopio di sezioni congelate per garantire che siano di natura CCRcc, tumore vitale e per la classificazione (di basso grado, di alto grado o misti alta / bassa qualità, difficile da differenziare gradi Fuhrman 1 a 4 su sezioni congelate). Figura 1 a &amp; b (H &amp; E colorazione di alta qualità bassa e mista). </li><li> Selezionare fino a 4 campioni da ogni regione morfologicamente differenti all&#39;interno di ogni tumore per l&#39;estrazione delle proteine. </li></ol> 2. Estrazione di proteine ​​da campioni tumorali <ol><li> Tagliare campione del tumore da ottobre </li><li> Luogo 50-75 mg di tessuto in 2 ml provette con 990 pl di tampone di lisi [50 mM Tris-HCl (pH 7,5), 5 mM EGTA (pH 8,5), 150 mM NaCl] supplementato con aprotinina (Sigma A6279) (10 mg / ml), fosfatasi inibitore cocktail 2 (Sigma, P5716), cocktail di inibitori delle fosfatasi 3 (Sigma, P0044) e un cocktail di inibitori delle proteasi (Roche, 11.836.153,001 mila). </li><li> Aggiungere un singolo 5 sfera in acciaio mm a ciascuna provetta e omogeneizzare a 50Hz per 5 minuti due volte utilizzando un TissueLyser, il controllo del livello di omogeneizzazione dopo ogni periodo di 5 minuti. </li><li> Trasferimento campione omogeneizzato al nuovo 2 ml tubetto con pipetta lasciando la sfera d&#39;acciaio dietro. </li><li> Aggiungere 10 ml di Triton X-100 per ciascun campione prima centrifugazione a 13.000 xg per 30 min a 4 ° C. </li><li> Trasferimento surnatanti ai tubi microcentrifuga freschi. </li><li> Determinare la concentrazione di proteina mediante saggio BCA (Figura 2). </li><li>Normalizzare le concentrazioni di proteine ​​a 1 mg / ml. </li></ol> 3. Anticorpo di convalida <ol><li> Preparare campioni proteici (estratto da linee cellulari o di tessuto appropriati) per western blot ed eseguito su un 10% SDS-PAGE gel. </li><li> Campioni di trasferimento su membrana di nitrocellulosa notte a 4 ° C. </li><li> Bloccare la membrana in Li-Cor Buffer Odyssey blocco (50:50 diluito in PBS) per 1 ora a temperatura ambiente. </li><li> Diluire gli anticorpi primari in Li-Cor Buffer Odyssey Blocco (50:50 diluito in PBS) presso il produttore consiglia di diluizione in genere di 1 su 1000. </li><li> Incubare membrana in anticorpi primari overnight a 4 ° C. </li><li> Make up 0,1% PBS-Tween20 (PBS-T, 1 ml Tween20 / 1L PBS). </li><li> Lavare membrana in PBS-T a temperatura ambiente per 5 min (x3). </li><li> Diluire fluorescente marcato anticorpi secondari nel tampone Odyssey blocco (50:50 diluito in PBS) 0,01% SDS a 1:10.000 diluizione (1,5 μl/15 ml). </li><li> Incubare membrana secondariaanticorpi a temperatura ambiente per 45 minuti con agitazione delicata - è importante proteggere la membrana dalla luce fino a quando è stato finalmente scansione. </li><li> Lavare membrana in PBS-T a temperatura ambiente per 5 min (x3), mantenendo la membrana al buio. </li><li> Lavare membrana in PBS a temperatura ambiente per 5 min (x3) per rimuovere residui Tween20, mantenendo ancora la membrana al buio. </li><li> Lie membrana piatta su un pezzo di carta da filtro al buio e lasciare asciugare all&#39;aria - permettendo la membrana ad asciugare può migliorare il segnale e ridurre il fondo, ma renderlo inutile per mettere a nudo e ri-sondaggio. </li><li> Scansione della membrana sullo scanner Odyssey licor. Mantenere la membrana al buio fino a quando non è stato analizzato. </li><li> Solo selezionare anticorpi che generano una singola banda predominante il peso molecolare corretta. Figura 3 mostra esempi di anticorpi accettabili e inaccettabili per l&#39;utilizzo con RPPA. </li></ol> 4. RPPA Printing <ol><li> Lisati proteici sono stati avvistati su nitrocellulosa rivestite con lastre di vetro (Whatman Fastslides-) con un microgrid II robot spotter. Le slide utilizzate conteneva 2 pastiglie su cui sono stati avvistati i campioni. Ogni pad è stata vista con campioni identici, in questo caso 100 campioni. Altri formati disponibili sono 1, 8 e 16 slide pad. Maggiore è il numero di pastiglie minore è il numero dei campioni che possono essere macchiati su ciascuna. </li><li> Una serie di cinque 2-diluizioni sono state effettuate da ciascun campione e ciascun stato poi notato in triplicato (per un totale di 15 punti per campione). </li></ol> 5. RPPA proteine ​​di rilevamento <ol><li> Vetrini bagnati nel buffer Licor eccesso di blocco (50:50 diluito in PBS). Incubare a temperatura ambiente per 1 ora su una piattaforma oscillante. </li><li> Preparare 800 ml di anticorpi primari nel tampone Licor blocco (50:50 diluito in PBS), alle concentrazioni desiderate e tenerla in ghiaccio. </li><li> Montare i vetrini in (i) la clip singolo chip corniceo (ii) l&#39;&#39;FastFrame&#39; quattro portavetrini baia modo che una tenuta stagna è formata tra la slitta e la camera di incubazione. </li><li> Rimuovere i residui di tampone da pozzi e aggiungere 600 microlitri anticorpo primario rispettivi pozzetti. </li><li> Posizionare il vetrino e la camera in una scatola sigillata bagnato e incubare in ballo piattaforma per una notte a 4 ° C. </li><li> Costituiscono 0,1% PBS-Tween20 (PBS-T; 100 pl Tween 20/100 ml PBS). </li><li> Togliere i vetrini dalla camera fredda e rimuovere con attenzione gli anticorpi primari da ogni pozzetto. </li><li> Aggiungere 600 pl di PBS-T e lavare i vetrini sulla piattaforma oscillante a temperatura ambiente per 5 min (X3). </li><li> Preparare fluorescente marcati anticorpi secondari diluendo nel tampone Odyssey blocco (50:50 diluito in PBS) 0,01% SDS a 1:2.000 diluizione (1 pl / 2 ml) in prima istanza. </li><li> Rimuovere tampone da pozzi e aggiungere 600 microlitri fluorescente-anticorpi marcati secondarie rispettivi pozzetti. Anticorpi secondari incubare a temperatura ambiente per 45 minuti con agitazione moderata - èè importante proteggere la membrana dalla luce fino a quando non è stato definitivamente scansione. </li><li> Rimuovere anticorpi secondari da lavare bene e brevemente (x3) in 600 microlitri di PBS-T a temperatura ambiente. Rimuovere il vetrino dal vettore, trasferire in un contenitore adatto e lavare in eccesso PBS-T per 15 minuti, mantenendo la membrana al buio. </li><li> Rimuovere PBS-T e membrana ulteriore lavaggio con PBS a temperatura ambiente per 15 minuti per rimuovere residui di Tween-20, mantenendo ancora la membrana al buio. </li><li> Asciugare il Fastslide forno in 50 ° C per 10 minuti e quindi eseguire la scansione sul Li-Cor scanner Odyssey. Tenere il vetrino al buio fino a quando non è stato analizzato. </li><li> Scansione del vetrino a 680 nm e / o 800 nm in funzione del anticorpo secondario / anticorpi utilizzati. Per i due colori di rilevamento utilizzare sempre altamente anticorpi secondari cross-adsorbite per ridurre al minimo di reattività crociata. Accurata selezione di anticorpi primari e secondari è necessario per due colori rilevamento. Di primaria importanza è la selezione didifferenti specie (es. coniglio e mouse) per i due anticorpi primari. In questo modo la discriminazione da anticorpi secondari anti-coniglio e anti-topo che sono contrassegnati con colorante con spettri di emissione facilmente distinguibili. </li><li> I file di immagine vengono salvati come file TIFF.. Figura 4 (immagine di file sottoposti a scansione). </li></ol> 6. Analisi dei dati <ol><li> Lanciare il software MicroVigene (VigeneTech, Carlisle, MA, USA). </li><li> Aprire file di immagine. TIFF contenente la scansione della diapositiva RPPA. </li><li> Selezionare un file modello predefinito che avrà una griglia per sovrapporre l&#39;immagine della diapositiva RPPA. </li><li> Fare clic sul definire le regioni di interesse (ROI) il pulsante, per visualizzare la griglia. </li><li> Posizionare la griglia sopra le macchie RPPA. Figura 6a (immagine della griglia sopra l&#39;immagine). </li><li> Fare clic sul pulsante Seleziona tutto per evidenziare tutto il ROI. </li><li> Fare clic su Trova tutti. MicroVigene si AUTOMticamente trovare il ROI, trovare i punti, sottrarre il fondo, rimuovere la polvere e quantificare i punti. </li><li> Fare clic sul pulsante Visualizza Curve di diluizione per far apparire i risultati per tutti i campioni della diapositiva RPPA. </li><li> Fare clic su Salva dati di diluizione. </li><li> Dato che ciascun campione è stampato in 5 punti ciascuna diluizione in triplicato ci sono 15 punti da analizzare, che riduce il rischio di errori e migliora la qualità del montaggio di curva. MicroVigene produce un 4 parametri logistic-log modello di algoritmo &quot;Supercurve&quot; (Figura 6b), che incorpora tutti i punti di produrre una curva sigmoide di antigene-anticorpo cinetica di legame. Il presupposto è che lo stesso anticorpo-antigene cinetica di legame si svolge in ogni punto del campione, anche nei diversi campioni, quindi prendendo tutti i punti su un array per adattarsi a una curva di risposta comune può aumentare la fiducia del raccordo curva 10,11 Y = a + ((ba) / (1 + e (c * d-ln (x))) dove x è il dilution fattore e Y rappresenta l&#39;intensità del segnale. I campioni possono essere comparativamente analizzati utilizzando il valore y0, che nella nostra analisi corrispondeva al valore y nel punto medio dei valori di x dopo la mappatura quelli sul supercurve. </li><li> Esportare i dati in Microsoft Excel e trama y0 come in figura 7. </li><li> Varianza proteina intratumorale è stato calcolato separatamente per i pazienti trattati non trattati e di quelli trattati in un quadro ANOVA. Distribuzioni varianza combinando i dati di tutte le proteine ​​analizzati sono stati confrontati con un test Mann-Whitney (MWT). Varianze intratumorale di singole proteine ​​sono stati confrontati con un F-test in cui le ipotesi di normalità e omoschedasticità tenuto, rispettivamente, valutati secondo i Lillefours e le prove Fligner; tasso di scoperta false (FDR) la correzione è stata applicata 12. Proteina espressione differenziale tra i campioni trattati e trattato è stato testato per ogni proteina usando studente t-test dove normalità e assunzione omoschedasticitàs sono state soddisfatte, altrimenti MWT è stata eseguita; FDR è stato applicato su valori combinati t-test e MWT 12. Significato dell&#39;espressione proteica e varianza di correlazione di Pearson per le proteine ​​sono state stimate utilizzando un approccio standard [R di riferimento] e FDR applicata 12. </li></ol>

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Nessun conflitto di interessi dichiarati.

Il metodo RPPA qui presentato rappresenta una valida alternativa ad alta velocità per il ampiamente utilizzato, ma relativamente basso tecnica blot velocità occidentale di analisi delle proteine. Il metodo permette centinaia di campioni da semi-quantitativamente analizzato e confrontato simultaneamente consentendo confronto diretto delle proteine ​​chiave attraverso un&#39;ampia selezione di linee cellulari e campioni di tessuto. Multiplexing con diverse specie anticorpali aumenta ulteriormente la potenza della te...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> aprotinina </td><td> Sigma </td><td> A6279 </td></tr><tr><td> cocktail di inibitori delle fosfatasi 2 </td><td> Sigma </td><td> P5726 </td></tr><tr><td> cocktail di inibitori delle fosfatasi 3 </td><td> Sigma </td><td> P0044 </td></tr><tr><td> inibitore della proteasi cocktail </td><td> Roche </td><td> 11836153001 </td></tr><tr><td> Triton X-100 </td><td> Triton-X </td><td> T8787 </td></tr><tr><td> Li-Cor Odyssey tampone di bloccaggio </td><td> Li-Cor </td><td> 927-40000 </td></tr><tr><td> TissueLyser </td><td> Qiagen </td><td> 85600 </td></tr><tr><td> Microgrid II robot spotter </td><td> Biorobotica </td><td></td></tr><tr><td> FastFrame &#39;quattro vani diapositive, </td><td> Whatman </td><td> 10486001 </td></tr><tr><td> Scorrimento veloce - 2-Pad </td><td> Whatman </td><td> 10485317 </td></tr><tr><td> IRDye 680LT capra anti-topo IgG </td><td> Licor </td><td> 926-68020 </td></tr><tr><td> IRDye 800CW di capra anti-IgG di coniglio </td><td> Licor </td><td> 926-32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

Attualmente non esiste alcun trattamento curativo per carcinoma a cellule chiare metastatico a cellule renali, il più comune variante della malattia. Un fattore chiave in questa resistenza trattamento è pensato per essere la complessità molecolare della malattia 1. La terapia mirata come l&#39;inibitore della tirosin-chinasi (TKI), sunitinib sono stati utilizzati, ma solo il 40% dei pazienti risponde, con la stragrande maggioranza di questi pazienti recidivanti entro 1 anno 2. Come tale la questione della resistenza intrinseca e acquisita nei pazienti con carcinoma renale delle cellule è molto importante 3. Al fine di studiare la resistenza a TKIs, con l&#39;obiettivo finale di sviluppare efficaci, trattamenti personalizzati, tessuti sequenziale dopo un determinato periodo di terapia mirata è necessario, un approccio che ha avuto successo in quattro mieloide cronica leucemia. Tuttavia l&#39;applicazione di tale strategia nel carcinoma renale è complicata dal livello di bOTH eterogeneità inter-e intratumorale, che è una caratteristica del carcinoma renale 5,6 e altri tumori solidi 7. Eterogeneità Intertumoral a causa delle differenze trascrittomica e genetico è ben definito anche nei pazienti con presentazione simile, stadio e grado del tumore. Inoltre, è chiaro che vi è grande morfologica (intratumorale) eterogeneità in RCC, che rischia di rappresentare ancora maggiore eterogeneità molecolare. Mappatura dettagliata e classificazione dei tumori RCC di combinata analisi morfologica e classificazione Fuhrman consente la selezione di aree rappresentative per l&#39;analisi proteomica. Analisi a base di proteine ​​del RCC 8 è interessante per la sua ampia disponibilità in laboratori di patologia, ma la sua applicazione può essere problematico a causa della limitata disponibilità di anticorpi specifici 9. A causa della natura dot blot delle matrici Reverse proteina di fase (RPPA), specificità anticorpale deve be pre-validato, in quanto tale controllo di qualità rigoroso di anticorpi utilizzati è di fondamentale importanza. Nonostante questa limitazione, il formato dot blot non consentono miniaturizzazione dosaggio, consentendo la stampa di centinaia di campioni su un vetrino nitrocellulosa singola. Diapositive stampate possono poi essere analizzate in modo simile ad analisi Western con l&#39;uso di anticorpi specifici obiettivi primari e contrassegnati in modo fluorescente anticorpi secondari, consentendo multiplexing. Proteina espressione differenziale in tutti i campioni di una diapositiva possono essere analizzati simultaneamente confrontando il livello relativo di fluorescenza in un modo più conveniente e ad alta produttività.

Un esempio di una diapositiva digitalizzata RPPA può essere visto in Figura 4 (i) sia con 680 e 800 nm canali visualizzati. Separate le immagini dalla lunghezza d&#39;onda, la figura 4 (ii) permette a ciascun pad sul vetrino RPPA da analizzare e proteina espressione individuale determinato Figura 4 (iii). Come si può vedere nella Figura 4 (iii) l&#39;espressione di proteine ​​individuali attraverso i campioni è unico con Gelsolin ha un alto livel...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA consente l&#39;espressione della proteina di centinaia di campioni, stampati su vetrini nitrocellulosa da interrogare simultaneamente, utilizzando fluorescente anticorpi. Questa tecnica è stata applicata per studiare l&#39;effetto di eterogeneità farmaco nel trattamento carcinoma a cellule renali chiare.

L&#39;uso di matrici inverse proteina di fase (RPPA) per esplorare Espressione Variazione proteine ​​all&#39;interno dei singoli tumori a cellule renali

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

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The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

33.5K Views.  University of Edinburgh. RPPA consente l'espressione della proteina di centinaia di campioni, stampati su vetrini nitrocellulosa da interrogare simultaneamente, utilizzando fluorescente anticorpi. Questa tecnica è stata applicata per studiare l'effetto di eterogeneità farmaco nel trattamento carcinoma a cellule renali chiare.

Video: The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

L&#39;uso di un Hanging-peso di sistema per isolati di occlusione dell&#39;arteria renale

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

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Medicina

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Mappatura eterogeneità di espressione delle proteine ​​nei tumori mediante immunofluorescenza quantitativa

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Produzione e di rilevamento di specie reattive dell&#39;ossigeno (ROS) in Cancro

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Purificazione e aggregazione della proteina precursore dell&#39;amiloide dominio intracellulare

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Microarray identificazione dei singoli HERV Loci Expression: Domanda di Biomarker Discovery in Cancro alla prostata

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Three-Dimensional Cultura Assay per esplorare Cancer Cell invasività e formazione di tumori Satellite

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Un metodo efficiente di setacciatura per isolare i Glomeruli intatti dal rene di ratto adulto.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Implementazione di precisione dei test di dose minima di erithema (MED) per valutare la variazione individuale nella risposta infiammatoria umana

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

Consegna diretta di farmaci ai reni attraverso l'arteria renale

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

L'uso di matrici inverse proteina di fase (RPPA) per esplorare Espressione Variazione proteine ​​all'interno dei singoli tumori a cellule renali

L'uso di matrici inverse proteina di fase (RPPA) per esplorare Espressione Variazione proteine all'interno dei singoli tumori a cellule renali