Gli autori desidera ringraziare Mandy Loebert e Tina Czajka per supporto tecnico eccellente. Ringraziamo Flora Luo (Harvard Medical School, Boston, MA, USA) per la lingua modifica Guida.

1. preparazione di costrutti pcDNA3.1/GLA e pcDNA3.1/GAA mutante
Nota: Le strategie di clonazione per il GLA e GAA codifica di sequenze (cds) sono stati segnalati precedenti15,18.
<ol>
	<li>Mutagenesi sito-diretta mediante mutagenesi sito-diretta
	<ol>
		<li>Uso il riferimento sequenze NM_000169.2 e NM_000152.4 come modelli per la mutagenesi dei geni GLA e GAA , rispettivamente. Avere un set di primer gratuito sale ad alta purezza (25-37-mers) sintetizzato da un provider commerciale, con gli iniettori di senso e antisenso che trasportano uno delle modifiche rispettiva sequenza centrale alla loro lunghezza di introdurre individualmente la mutazione. Utilizzare lo strumento di disegno di primer gratuito per sostenere il disegno di primer19.</li>
		<li>Per la miscela di reazione, utilizzare le condizioni standard per la soluzione di reazione e le condizioni PCR fornite dal produttore.<ol>
			<li>Mix 5 µ l di tampone di reazione, 10 10x ng modello double-stranded del DNA del plasmide (pcDNA3.1/GLA o pcDNA3.1/GAA), 125 ng di ogni primer, 1 µ l della miscela dei dNTP fornito, 3 µ l di reagente di DMSO in un volume adeguato di acqua deionizzata (volume di reazione finale : 50 Μ L). Infine, aggiungere 2,5 U di DNA polimerasi e Miscelare pipettando su e giù. Come controllo negativo, portare con sé un campione no-primer.</li>
		</ol>
		</li>
		<li>Avviare la PCR utilizzando il seguente programma: passo 1: 95 ° C per 1 min, passo 2: 95 ° C per 50 s, passo 3: 60 ° C per 50 s, passo 4: 68 ° C per 8 min, ripetere il passaggio 2-4 18 volte e passo 5: 68 ° C per 10 min.<ol>
			<li>Dopo la PCR, aggiungere 1 µ l di Dpnho degli enzimi di limitazione (10 U / µ l) e ulteriormente Incubare la fiala di reazione a 37 ° C per 1 h.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Trasformazione e lo Screening per il Clone desiderato
	<ol>
		<li>Trasformare un'aliquota di cellule ultracompetent in conformità con le raccomandazioni del produttore. Utilizzo medio SOC (tryptone 2% (p/v), lievito estratto di 0,5% (p/v), 10mm di NaCl, KCl 2.5 mM, sterilizzare a 121 ° C e quindi aggiungere soluzioni sterili filtrato di MgCl2 e il glucosio fino a concentrazioni finali di 10 e 20 mM, rispettivamente) invece del produttore medio. Dopo la procedura, piastra 250 µ l della mutagenesi di esempio su un LB piastra contenente 100 ampicillina di µ g/mL e incubare a 37 ° C per 18 ore.</li>
		<li>Assicurare che il numero dei transformants è &#62; 10 e la reazione produce almeno tre volte come molte colonie come la reazione di controllo no-primer, per esempio, usando un piatto luminoso per facilitare il conteggio di Colonia. Poi scegli 3 colonie e preparare 3ml culture durante la notte nel mezzo di libbra di brodo.</li>
		<li>Il giorno successivo, svolgere Miniprep con un kit standard e analizzare l'intera sequenza usando T7 (5ʹ-TAA TAC GAC TCA CTA TAG GG-3ʹ) e BGHr (5ʹ-TAG AAG GCA CAG TCG AGG-3ʹ) primer tramite standard Sanger sequenziamento.</li>
		<li>Utilizzare uno strumento adatto biologia molecolare per analizzare la sequenza. Quando viene rilevata la mutazione desiderata e nessun ulteriore sequenza anomalia rispetto al NM_000169.2 (α-galattosidasi A) o NM_000152.4 (α-glucosidasi acida) è visto, selezionare il clone per la purificazione del plasmide di transfezione-grado.</li>
		<li>Determinare la purezza del DNA misurando l'assorbanza a uno spettrofotometro. Nota: Consenti solo i preparativi che producono una purezza del plasmide con un rapporto di assorbanza 260/280 di &#62; 1.8 per cella esperimenti della coltura.</li>
	</ol>
	</li>
</ol>
2. coltivazione di cellule HEK293H
<ol>
	<li>Mantenere le cellule HEK293H in alto glucosio (4,5 g/L) Dulbecco´s Modified Eagle Medium (DMEM) supplementato con 10% siero bovino fetale (FBS) e 1% di penicillina/streptomicina. Mantenere le cellule un incubatore a 37 ° C sotto un 5% CO2 ambiente di pluritubulare.</li>
	<li>Coltivare le cellule ad una densità di 80-90%.</li>
	<li>Il mezzo di aspirare e lavare una volta utilizzando fosfato tampone salino (PBS) senza Ca2 + e Mg2 +.</li>
	<li>Passaggio con l'aggiunta di 0.05% tripsina-EDTA e incubare per 5 min a 37 ° C e 5% CO2.</li>
	<li>Dividere le celle 01:15 in mezzo fresco e il seme in una nuova staffa T75 per mantenere la cultura permanente. Non usare celle con più di 25 passaggi.</li>
</ol>
3. pcDNA3.1/GLA e pcDNA3.1/GAA plasmide transfezione e trattamento di HEK293H
<ol>
	<li>24 h prima la transfezione, lavare le cellule HEK293H in un matraccio di cultura T75 cella una volta con PBS con Ca2 +, Mg2 +. Raccogliere le cellule con 0,05% tripsina-EDTA come indicato sopra e cellule5 seme 1.5 x 10 nelle cavità di una piastra di coltura ben 24 utilizzando 500 µ l DMEM supplementato con 10% FBS senza antibiotici.</li>
	<li>Eseguire un protocollo di transfezione secondo il manuale del produttore. In genere, è possibile utilizzare una miscela di 1 µ g di DNA plasmidico e 2,5 µ l di reagente di transfezione in 100 µ l di DMEM privo di siero. Incubare per 20 minuti a temperatura ambiente e aggiungere alle celle in modo sospensione da allora in poi.</li>
	<li>Rimuovere il supporto contenente il reagente di transfezione dopo un periodo di 4 h a 37 °C/5% CO2 e aggiungere 500 µ l di fresco DMEM con 10% FBS / 1% di penicillina/streptomicina. Nota: Durante questa fase, 1-Deoxygalactonojirimycin cloridrato (DGJ) o 1-Deoxynojirimycin cloridrato (DNJ) potrebbe essere aggiunto al terreno di coltura dove previsto (utilizzare una soluzione acquosa di stock di 10 mM per ottenere una concentrazione finale di 20 µM DGJ e DNJ ). Fresco DGJ o DNJ è stato aggiunto 42h dopo trasfezione di plasmide.</li>
</ol>
4. cellula raccolto e α-galattosidasi A o la misurazione dell'attività α-glucosidasi acida
<ol>
	<li>Colture di cellule
	<ol>
		<li>Il giorno della raccolta, rimuovere le cellule dall'incubatrice ed aspirare il mezzo. Lavare accuratamente le cellule 2 volte con PBS con Ca2 + e Mg2 +. Nota: Questo passaggio è fondamentale perché DGJ e DNJ sono potenti inibitori della α-galattosidasi e α-glucosidasi, rispettivamente, ed eventuali rimanenze invaliderebbe il test.</li>
		<li>Aggiungere 200 µ l di acqua deionizzata direttamente sopra le cellule. Sciacquare le cellule dalla piastra e trasferirli in una provetta di reazione da 1,5 mL.</li>
	</ol>
	</li>
	<li>Omogeneizzazione di congelamento e scongelamento
	<ol>
		<li>Mettere i campioni in un rack di schiuma appropriata e vortexare per 5 s per rendere più efficiente la lisi. Mettere i campioni alternati in azoto liquido per 10 s e in un bagno di acqua temperatura ambiente fino a quando lo scongelamento era completa (5 min).</li>
		<li>Ripetere questa procedura 5 volte e poi girare i campioni per 5 min a 10.000 x g.</li></ol></li></ol>Conservare il surnatante e pipetta in un nuovo tubo di reazione.
	
	
	<li>Determinazione di concentrazione nella proteina usando bicinconinico acido (BCA) analisi
	<ol>
		<li>Preparare una nuova provetta per ciascun campione contenente 40 µ l di deionizzata H2O e aggiungere 10 µ l di campione. Mescolare la soluzione nel Vortex brevemente e trasferire 10 µ l in una cavità di una piastra ben 96 (ogni campione in triplice copia). Diluire una soluzione madre di albumina di siero bovino (BSA) 2 mg/mL in deionizzata di H2O come indicato di seguito per ottenere una curva standard: 50 µ l di H2O/50 BSA µ l; 60 µ l H2O/40 µ l BSA; µ L 70 H2O/30 µ l BSA; BSA a 80 µ l H2O/20 µ l; µ L 90 H2O/10 µ l BSA; 100 µ l di H2O.</li>
		<li>Iniziare la reazione aggiungendo 200 µ l di reagente BCA (Reagente A e il reagente B mescolati ad un rapporto di 50: 1) e incubare per 1 h al buio a 37 ° C sotto agitazione leggera su agitatore orbitale (300 giri/min). Misurare l'assorbanza a 560 nm in un lettore di piastra. Nota: I campioni contengono in genere tra 1 e 1,5 µ g di proteine per µ l.</li>
	</ol>
	</li>
	<li>Misurazione dell'attività enzimatica con substrati artificiali 4-Methylumbelliferyl (4-MUG)
	<ol>
		<li>Diluire la quantità calcolata di ogni campione e dispensare nelle provette di reazione fresco 1,5 mL per ottenere 0,5 (per α-glucosidasi acida) o 0.05 (per α-galattosidasi A) µ g proteine / µ l soluzioni. Vortice i campioni per 5 s nuovo e dispensare 10 µ l di questa diluizione in un 96 ben piatto (ogni campione in duplicato).</li>
		<li>Iniziare la reazione aggiungendo 20 µ l di soluzione substrato rispettivi: Per α-galattosidasi r: 2 mM 4-Methylumbelliferyl-α-D-galattopiranoside (4-MU-gal) in tampone citrato di 0,06 M fosfato, pH 4,7. Per α-glucosidasi acida: 2 mM 4-methylumbelliferyl α-D-glucopyranoside (4-MU-glu) in acetato di sodio 0,025 M, pH 4,0.</li>
		<li>Incubare le reazioni enzimatiche per 1 h al buio a 37 ° C sotto agitazione leggera su agitatore orbitale (300 giri/min). Terminare la reazione con l'aggiunta di 200 µ l di 1,0 M, tampone a pH 10,5 regolato glicina-NaOH.</li>
		<li>Preparare una curva standard di 4-methylumbelliferone (4-MU) da uno stock di 0,01 mg/mL come segue: 100 µ l di H2O / no 4-MU; 80 µ l di H2O / 20 µ l 4-MU; 60 µ l H2O / 40 µ l 4-MU; 40 µ l di H2O / 60 µ l 4-MU; 20 µ l H2O / 80 µ l 4-MU; No di H2O / 100 µ l 4-MU, dispensare 10 µ l di ciascuna diluizione nel pozzo 96 piastra (in duplicato) e aggiungere 200 µ l di buffer di glicina-NaOH 1,0 M in ciascun pozzetto al fine di regolare il volume e il pH.</li>
		<li>Misura l'attività dell'enzima in un lettore di fluorescenza con il set di filtri appropriati e analizzare i dati utilizzando il software appropriato per la periferica del lettore di fluorescenza. Nota: Entrambi i substrati 4-MUG sono ridotti a 4-MU durante l'esposizione al α-galattosidasi A o α-glucosidasi acida. Rilasciato 4-MU è un fluorocromo, che può essere misurato a 360 e 465 nm come lunghezze d'onda di eccitazione e di emissione, rispettivamente, utilizzando un lettore di micropiastre fluorescenza.</li>
	</ol>
	</li>

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Gli autori dichiarano di non avere nessun concorrenti interessi finanziari.

Il protocollo descritto nel presente documento fornisce risultati affidabili per la valutazione dei danni degli enzimi in malattie lisosomiali ereditarie del metabolismo. Questo manoscritto è che un emendamento al protocollo pubblicato precedenti15. Le modifiche più cruciale coinvolgono stringenza (cioè, nel processo di preparazione del costrutto di mutante vettoriale), standardizzazione del protocollo di cultura di cellule (cioè, condizioni di manutenzione e di transfezione ...

Ci sono oltre una dozzina i disordini da accumulo lisosomiale (LSD) legati alla disfunzione di glycosidase come conseguenza delle mutazioni di gene primario. In Fabry (OMIM #301500) e malattia di Pompe (OMIM #232300), più di 500 e 200 mutazioni di senso sbagliato sono stati segnalati1,2,3 , rispettivamente, che corrisponde a circa il 60% del conteggio totale mutazione. Numerose nuove varianti del gene ancora stanno identificandi, molte delle quali hanno ignoto significato. I vasti studi biochimici hanno rivelato che alcuni genotipi non conducono ad una completa perdita di funzione del gene GLA (OMIM * 300644) nella malattia di Fabry, ma causare l'enzima corrispondente a non riuscire a raggiungere uno stato termodinamicamente favorito pieghevole4 . Ciò provoca ritenzione di ER e degradazione prematura dell'enzima altrimenti funzionale. Simili conclusioni sono state tratte in altre malattie da accumulo lisosomiale, tra cui Pompe malattia5. Inoltre, caratterizzazione molecolare delle varianti di enzima può facilitare l'interpretazione clinica delle mutazioni al momento della diagnosi6, suggerendo che la progressione di LSD è un processo individuale sulla base della natura della mutazione. Di conseguenza, la classificazione convenzionale in diversi tipi clinici in genere 2 o 3 dovrebbe essere rivalutata al fine di ottimizzare la consulenza clinica e le decisioni terapeutiche.
Enzimatica sostitutiva (ERT) è disponibile per entrambe le malattie. ERT, tuttavia, ha limitato l'efficacia nei tessuti/organi colpiti come il cervello e muscolo scheletrico. Inoltre, ERT può suscitare una risposta immunogenica che compromette i suoi benefici terapeutici. Chaperoni farmacologici (pz) sono un'alternativa attraente di trattamento per i pazienti con mutazioni reattive cosiddette. Pz servire come un'impalcatura molecolare per la piegatura della proteina corretta e stabilizzazione che a sua volta impedisce la ritenzione del reticolo endoplasmatico (ER) ed ER-collegata di degradazione dell'enzima. Inoltre, il PC può essere somministrato per via orale e sono potenzialmente in grado di attraversare la barriera ematoencefalica. Di conseguenza, PCT potrebbe essere un'opzione più praticabile per il trattamento di pazienti con determinati genotipi. Per un'ampia rassegna su applicazione PC nelle malattie da accumulo lisosomiale, consultare l'eccellente recensione di Parenti7.
La scoperta di centinaia di malattia che causa gli alleli del mutante sfida test anti-droga pre-clinica e richiede una valutazione semplice, veloce e altamente standardizzata dei pazienti suscettibili di un approccio di medicina personalizzata. Al fine di valutare gli effetti nocivi delle mutazioni di gene di LSD e di testare le mutazioni candidato per predire i pazienti suscettibili per PCT, è stato un sistema altamente standardizzati over-espressione in cellule di HEK293H che consente la misurazione dell'attività dell'enzima veloce e affidabile sviluppato. Sistemi simili di sovra-espressione precedentemente sono stati descritti per la malattia di Fabry e Pompe utilizzando COS-78,9,10,11, HeLa cellule12o HEK29313 ,14,15,16 celle per il gene di glicosidasi.
Un metodo molto simile è stato brevettato anche come un "metodo per predire la risposta al trattamento farmacologico Chaperone di malattie"17 che indica la pertinenza di una cella cultura sistema in grado di essere integrati nella pratica clinica.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuikChange II XL Site-Directed Mutagenesis Kit&#160;</td>
			<td>Stratagene, La Jolla, CA, USA</td>
			<td>#200522</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-frw: 5&#180;- CTA ATG ACC TCC AAC ACA TCA GCC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-rev: 5&#180;- GGG CTG ATG TGT TGG AGG TCA TTA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-rev: 5&#180;-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A143T]-rev: 5&#180;-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-frw: 5&#180;-CTG CCG GGA GCT TCA GGC CCT ACG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-rev: 5&#180;-TCG TAG GGC CTG AAG CTC CCG GCA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-frw: 5&#180;-CAC CCT ACG TGC TTG GGG TGG TTG G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-CCA ACC ACC CCA AGC ACG TAG GGT G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[L552P]-frw: 5&#180;-TTG GGG GGA CCC CCC AGG CGG CCA C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-GTG GCC GCC TGG GGG GTC CCC CCA A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer T7: 5&#180;-TAA TAC GAC TCA CTA TAG GG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer BGHrev: 5&#180;-TAG AAG GCA CAG TCG AGG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>103,753</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,049,360,500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,058,330,250</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+)-Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,083,422,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,980,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>3908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X863.3</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium hydrogen phosphate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,065,860,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,062,640,050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X969.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep Kit</td>
			<td>ZymoResearch, Freiburg, Germany</td>
			<td>D4020</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAfilter Plasmid Midi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12245</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293H cell line</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11631-017&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium&#160;</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum&#160;</td>
			<td>GE Healthcare, South Logan, Utah, USA</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Standard Cell Culture Flasks, Greiner Bio One</td>
			<td>VWR International GmbH, Hannover, Germany</td>
			<td>82050-854</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (24 well)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>831,836</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 96 well plate</td>
			<td>Greiner bio one, Frickenhausen, Germany</td>
			<td>655185</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tubes, 1,5mL&#160;</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9641</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Toronto Research Chemicals, Toronto, Canada</td>
			<td>D236500</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxynojirimycin&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9305</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Calcium, w/o Magnesium</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-galactopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M7633</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-glucopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M9766</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>incubator type T6</td>
			<td>Heraeus Instruments, Hanau, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminous Plate Gr. 2 E</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>P265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems, Darmstadt, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GFL-3032 bacterial shaker</td>
			<td>GFL, Burgwedel, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-25 centrifuge</td>
			<td>Beckman/Coulter, Krefeld, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraspec 3100 pro Spectrophotometer</td>
			<td>Amersham Biosciences, Buckinghamshire, United Kingdom</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>water-jacket incubator&#160;</td>
			<td>Binder, Tuttlingen, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 1 touch mixer</td>
			<td>Scientific Industries, Bohemia, NY, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Z 233 MK-2 refrigerated microtube centrifuge</td>
			<td>Hermle, Tuttlingen, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboStar ultrapure water device</td>
			<td>Siemens, Berlin, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker PST-60HL-4 orbital shaker</td>
			<td>Biosan, Riga, Latvia</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan GENios Reader</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>HI 223 Microprocessor pH Meter</td>
			<td>HANNA Instruments, Arvore, Portugal</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>CASY 
			Cell Counter + Analyser System 
			Model TT</td>
			<td>Innovatis AG, Cham/Zug, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magellan data analysis software v6.6</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.04</td>
			<td>GraphPad Software, Inc., La Jolla, CA; USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft Corporation, Redmond, WA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>BioEdit Alignment Editor, V7.0.1</td>
			<td>http://www.mbio.ncsu.edu/bioedit/bioedit.html</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Abbreviations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>frw = forward; rev = reverse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n.a. = not applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease

L'uso della medicina personalizzata per il trattamento di malattie monogeniche rare come disturbi connessi all'accumulo lisosomiale (LSD) è sfidato da disegni di trial clinici complessi, costi elevati e basso numero di pazienti. Centinaia di alleli mutanti è implicati nella maggior parte delle malattie da accumulo lisosomiale. Le malattie sono in genere classificate in 2 o 3 tipi clinici differenti secondo la severità. Inoltre, caratterizzazione molecolare del genotipo può aiutare a predire gli esiti clinici e informare la cura del paziente. Di conseguenza, abbiamo sviluppato un saggio di cultura cellulare semplice basato su cellule HEK293H heterologously che sovraesprimono le mutazioni identificate nella malattia di Fabry e Pompe. Un dosaggio simile recentemente è stato introdotto come un test preclinico per identificare mutazioni favorevoli per la terapia Chaperone farmacologici (PCT) nella malattia di Fabry. Questo manoscritto descrive un'analisi di cultura cellulare modificato che consente la rapida valutazione fenotipica delle varianti alleliche nella malattia di Fabry e Pompe per identificare i pazienti eleggibili per PCT e può essere di aiuto nello sviluppo del romanzo pharmacochaperones.

La procedura di mutagenesi Per valutare l'efficienza di mutagenesi gene GLA , le mutazioni sono state classificate in una delle categorie seguenti. Questo approccio per generare mutazioni ha rivelato che circa il 66,5% delle mutazioni GLA sono stati ottenuti nel primo tentativo. Un ulteriore 25% potrebbe essere ottenuto dopo una PCR seconda leggermente modificata.
Categoria 1: La mutagenesi ...

Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

C'è una domanda per fare pre-clinica test per una nuova classe di farmaci "orfani" chiamato chaperoni farmacologici riproducibile, veloce ed efficiente. Abbiamo sviluppato un test cellulari semplice, altamente standardizzate e versatile cultura alla schermata per i pazienti eleggibili come pure droghe romanzo chaperoni farmacologici.

In Vitro Misurazione di enzima per Test farmacologici Chaperone reattività in Fabry e malattia di Pompe

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

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In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

8.0K Views.  University Rostock Medical Center. C'è una domanda per fare pre-clinica test per una nuova classe di farmaci "orfani" chiamato chaperoni farmacologici riproducibile, veloce ed efficiente. Abbiamo sviluppato un test cellulari semplice, altamente standardizzate e versatile cultura alla schermata per i pazienti eleggibili come pure droghe romanzo chaperoni farmacologici.

Video: In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

Making Sense di Ascolto: La batteria di test IMAP

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

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Medicina

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Infarto acuto del miocardio in topi

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Una strategia per identificare le mutazioni de novo nei disturbi comuni come autismo e schizofrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La misurazione e trattamento di soppressione in Ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un metodo semplice di intubazione mouse polmone

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Misurazione ripetuta di attività muscolare respiratoria e ventilazione in modelli murini di malattie neuromuscolari

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Neonatal Murine Cochlear Explant Technique come un<em&gt; In vitro</em&gt; Strumento di screening nella ricerca acustica

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Utilizzo di una biopsia chimica per la valutazione della qualità dell'innesto

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Campionamento ed estrazione in loco di tumori cerebrali per l'analisi metabolomica e lipidomica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Un modello murino BALB/c neonatale di enterocolite necrotizzante

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...