L&#39;assistenza tecnica in grado di Anthony Thomas Si ringrazia.

Procedura per l&#39;elaborazione campioni di urina <ol><li> Scongelare il campione di urina in un bagno d&#39;acqua a 37 ° C. Decantare in un nuovo contenitore se l&#39;originale è compromessa. </li><li> Prendete un aliquota massima di 13 ml di campione e conservarlo a -20 ° C in una provetta per centrifuga coniche. </li><li> Misurare e registrare la densità ottica del campione mettendo un paio di gocce di urina nel rifrattometro. </li><li> Filtrare il campione attraverso un filtro da 0,2 um. </li><li> Misurare il volume del campione al di sotto in base alla densità ottica <table border="0"><tbody><tr><td> 1,000-1,009 </td><td> 1,00 ml </td></tr><tr><td> 1,010-1,019 </td><td> 0,50 ml </td></tr><tr><td> 1,020-1,050 </td><td> 0,25 ml + 0,25 ml di H 2 O </td></tr></tbody></table></li><li> Il volume specificato non è poi trasferito in un Reactivial contenente le seguenti norme interne: 500 nanomoli (nmoli) 3 Creatina, 10 nmoli d 3 acido metilmalonico, 100 nmoli ciascuno dei seguenti 13 C 3 lattato, 13 C 3 piruvato, 13 C 2 15 N glicina, serina d 3, d 5 fenilalanina, D 11 hexanoylglycine, 15 N 2 orotato, d 4 acido sebacico, 13 C 6 glucosio, d 6 inositolo e d 5 triptofano. </li><li> 20 microlitri (microlitri) di un 7,5 Unità / ul di soluzione di ureasi (Calzyme catalogo Laboratori no. 116A0100) viene aggiunto al campione, che viene poi lavata e sigillato in CO 2 attraverso un setto inerte. </li><li> Il campione è a 37 ° C per 30 minuti con il gas di biossido di carbonio aggiunto a intervalli di 15 minuti per mantenere la pressione. </li><li> 20 l più della soluzione di ureasi si aggiunge, il flacone viene lavata con l&#39;anidride carbonica, e il campione mantenuto a 37 ° C per altri 15 minuti. </li><li> 500 ml di acetone 30:70: metanolo viene aggiunto, il setto di gomma viene sostituito con un setto rivestito in teflon, e il campione viene raffreddato a -20 ° C per 15 minuti. </li><li> Solidi vengono rimossi mediante centrifugazione a 1500 rpm x 10 minuti, poi travasata in un ambiente pulito 2,0 cc Reactivial (Supelco / Sigma) </li><li> Aggiungi trifluoroacetato triethylammonium (TEA / TFA) (Sigma) come segue: <ul><li> 20 l per 1,00 ml campioni </li><li> 40 microlitri per 0,5 ml, o meno, i campioni </li></ul></li><li> Top fuori con acetonitrile e di luogo in un flusso di azoto a 70 ° C fino a volume costante si ottiene (TEA / TFA rimarrà) (~ 15 minuti). </li><li> Ripetere il passaggio 13, fino a 4 volte fino a quando si forma un precipitato (~ 10 minuti ciascuno). </li><li> Lasciate raffreddare per circa 2 minuti. Top fuori con cloruro di metilene, facendo attenzione di ebollizione, e secco (~ 4:00 minuti). </li><li> Ripetere il punto 15. </li><li> Aggiungi MSTFA (N-metil-N-trimethylsilyltrifluoroacetamide) (termica scientifico) alle seguenti tariffe: <ul><li> 150 microlitri per 1,00 ml campioni </li><li> 200 ul per 0,50 ml, o meno, i campioni </li></ul></li><li> Cap in atmosfera di azoto e incubare a 70 ° C per 1 ora. </li><li> Trasferimento a microprovette, in atmosfera di azoto, per l&#39;analisi dei gas cromatografo / spettrometro di massa. </li><li> Microprovette posto per l&#39;iniezione automatizzato dal 5975 Agilent GC / MS: le temperature dello strumento sono: iniettore 200 ° C, l&#39;interfaccia di 250 ° C, forno a 80 ° C per 1 minuto; rampa a 4 ° C / minuto 80-130 ° C, rampa 6 ° C / minuto 130-200 ° C, rampa a 12 ° C / minuto 200-285 ° C, mantenere per 10 minuti. Colonna: 25 m, 320 micron ID, spessore 0.5 micron DB-5. Mass Spec: fonte 230 ° C, quad 150 ° C, Scansione 50-650 amu a 2,46 scansioni / sec. Solvente pausa di 3,5 minuti. </li></ol> Rappresentante Risultati Si prega di <a href="http://www.jove.com/files/ftp_upload/2014/ShoemakerResultHawkinsinuria.pdf">cliccare qui</a> per vedere i risultati rappresentativi.

<ol>
	<li>Shoemaker, J. D., Elliott, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+of+urine+samples+for+carbohydrates,+organic+and+amino+acids+after+treatment+with+urease.">Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease.</a> J. Chromatogr. 562 (1-2), 125-138 (1991).</li><li>Kuhara, T., Shinka, T., Inoue, Y., Ohse, M., Zhen-wei, X., Yoshida, I., Inokuchi, T., Yamaguchi, S., Takayanagi, M., Matsumoto, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+study+of+gas+chromatographic-mass+spectrometric+screening+of+newborn+urine+for+inborn+errors+of+metabolism+after+treatment+with+urease.">Pilot study of gas chromatographic-mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease.</a> J Chromatogr B Biomed Sci Appl. 731 (1), 141-147 (1999).</li><li>Fu, X., Iga, M., Kimura, M., Yamaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+screening+for+organic+acidemia+using+GC/MS+and+dried+urine+filter+paper:+a+study+on+neonatal+mass+screening.">Simplified screening for organic acidemia using GC/MS and dried urine filter paper: a study on neonatal mass screening.</a> Early Hum Dev. 58 (1), 41-55 (2000).</li><li>Kuhara, T., Ohdoi, C., Ohse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+gas+chromatographic-mass+spectrometric+procedure+for+diagnosing+pyrimidine+degradation+defects+for+prevention+of+severe+anticancer+side+effects.">Simple gas chromatographic-mass spectrometric procedure for diagnosing pyrimidine degradation defects for prevention of severe anticancer side effects.</a> J Chromatogr B Biomed Sci Appl. 758 (1), 61-74 (2001).</li><li>Kuhara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+inborn+errors+of+metabolism+using+filter+paper+urine,+urease+treatment,+isotope+dilution+and+gas+chromatography-mass+spectrometry.">Diagnosis of inborn errors of metabolism using filter paper urine, urease treatment, isotope dilution and gas chromatography-mass spectrometry.</a> J Chromatogr B Biomed Sci Appl. 758, 3-25 (2001).</li><li>Iga, M., Kimura, M., Ohura, T., Kikawa, Y., Yamaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+simplified+and+sensitive+method+for+screening+fructose-1,6-diphosphatase+deficiency+by+analyzing+urinary+metabolites+in+urease/direct+preparations+and+gas+chromatography-mass+spectrometry+in+the+selected-ion+monitoring+mode.">Rapid, simplified and sensitive method for screening fructose-1,6-diphosphatase deficiency by analyzing urinary metabolites in urease/direct preparations and gas chromatography-mass spectrometry in the selected-ion monitoring mode.</a> J Chromatogr B Biomed Sci Appl. 746 (1), 75-82 (2000).</li><li>Kuhara, T., Ohse, M., Ohdoi, C., Ishida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+diagnosis+of+homocystinuria+by+urease+treatment,+isotope+dilution+and+gas+chromatography-mass+spectrometry.">Differential diagnosis of homocystinuria by urease treatment, isotope dilution and gas chromatography-mass spectrometry.</a> J Chromatogr B Biomed Sci Appl. 742 (1), 59-70 (2000).</li><li>Young, V., Lo, S., Shoemaker, J., Thomas, A., Rhead, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+Extension+of+the+Urease+Method+for+Urine+Organic+and+Amino+Acid+Analysis.">Validation and Extension of the Urease Method for Urine Organic and Amino Acid Analysis.</a> , (2008).</li><li>Smith, E. M., Hoi, J. T., Eissenberg, J. C., Shoemaker, J. D., Neckameyer, W. S., Ilvarsonn, A. M., Harshman, L. G., Schlegel, V. L., Zempleni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+Drosophila+a+biotin-deficient+diet+for+multiple+generations+increases+stress+resistance+and+lifespan+and+alters+gene+expression+and+histone+biotinylation+patterns.">Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns.</a> J Nutr. 137 (9), 2006-2012 (2007).</li><li>Brown, S. M., Shoemaker, J. D., Lodge, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Importance+of+NADP+-Dependent+Isocitrate+Dehydrogenase+in+Resistance+to+Nitrosative+Stress+and+the+Inessentiality+of+Glucose-6-Phosphate+Dehydrogenase+for+Oxidative+Stress+Resistance+in+Cryptococcus+neoformans.">The Importance of NADP+-Dependent Isocitrate Dehydrogenase in Resistance to Nitrosative Stress and the Inessentiality of Glucose-6-Phosphate Dehydrogenase for Oxidative Stress Resistance in Cryptococcus neoformans.</a> Eukaryotic Cell. , (2010).</li><li>Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+Screening+in+Humans+by+Gas+Chromatography-Mass+Spectrometry+of+Urine+Metabolites+After+Substrate+Loading.">Nutritional Screening in Humans by Gas Chromatography-Mass Spectrometry of Urine Metabolites After Substrate Loading.</a> , (1992).</li><li>Baggot, P. J., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproate-induced+biochemical+abnormalities+in+pregnancy+corrected+by+vitamins:+A+Case+Report.">Valproate-induced biochemical abnormalities in pregnancy corrected by vitamins: A Case Report.</a> Epilepsia. 40 (44), 512-515 (1999).</li><li>Baggot, P. J., Eliseo, A. J., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+folate-dependent+metabolite+in+amniotic+fluid+from+pregnancies+with+normal+or+trisomy+21+chromosomes.">A folate-dependent metabolite in amniotic fluid from pregnancies with normal or trisomy 21 chromosomes.</a> Fetal Diagn Ther. 21 (1), 148-152 (2006).</li><li>Baggot, P. J., Eliseo, A. J., DeNicola, N. G., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+acid+concentrations+in+amniotic+fluid+found+in+normal+and+Down+syndrome+pregnancies.">Organic acid concentrations in amniotic fluid found in normal and Down syndrome pregnancies.</a> Fetal Diagn Ther. 23 (3), 245-248 (2008).</li><li>Baggot, P. J., Eliseo, A. J., DeNicola, N. G., Kalamarides, J. A., Shoemaker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyridoxine-related+metabolite+concentrations+in+normal+and+Down+syndrome+amniotic+fluid.">Pyridoxine-related metabolite concentrations in normal and Down syndrome amniotic fluid.</a> Fetal Diagn Ther. 23 (4), 254-257 (2008).</li></ol>

Il Laboratorio di Screening metabolica è una CLIA con licenza laboratori clinici di proprietà della Saint Louis University, un&#39;organizzazione non-profit. Dr. Shoemaker non un beneficio diretto dal fatturato di laboratorio, ma può beneficiare indirettamente la produttività del laboratorio. Nessuno dei metodi, tecniche, risultati, gamma normale, macro, il software, le librerie o le conclusioni sono considerati di proprietà e sono liberamente a disposizione degli interessati.

Il metodo di ureasi (1) è stato citato 62 volte nella letteratura medica con varie modifiche. Gruppo di Matsumoto (2,3) ha semplificato la procedura per l&#39;high-throughput screening neonatale e riportato i risultati da 16000 pazienti. Kuhara e altri (4-7) hanno riportato l&#39;utilizzo del metodo in diversi casi di diagnosi degli errori congeniti e follow-up. Rhead (8) ha confermato l&#39;utilità del metodo per la diagnosi clinica e follow-up di errori congeniti. Il metodo è stato applicato a urina di orsi, topi knock-out, elefanti e omogenati di mosche frutti interi e le loro larve (9). Terreni di coltura da Cryptococcus prima e dopo la mutagenesi sito-diretta sono stati analizzati, senza il passaggio ureasi (10). Il metodo è stato applicato per la valutazione dei nutrizionale studenti di medicina, pazienti con sindrome di Down e anziani dementi veterani dopo aver caricato i soggetti con dosi orali degli aminoacidi triptofano, metionina ed isoleucina (11). Tutti gli otto vitamine del gruppo B sono stati valutati da quantificare il prodotti di degradazione dei tre aminoacidi che, tra i quali, che tutte le 8 vitamine ad un certo punto della loro degradazione. Gli effetti tossici dei farmaci e la loro mitigazione da supplementazione di vitamina è stata riportata (12). Campioni di liquido amniotico da gravidanze normali e sindrome di Down sono stati analizzati e riportati (13-15).

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MSD 5975</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>&nbsp;</td>
<td>GC/MS/Computer</td>
</tr>
<tr>
<td>MSD 5973</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>&nbsp;</td>
<td>GC/MS/Computer</td>
</tr>
<tr>
<td>Urease</td>
<td>&nbsp;</td>
<td>Calzyme</td>
<td>116A0100</td>
<td>Also Sigma C3</td>
</tr>
<tr>
<td>Stable Isotope Standards</td>
<td>&nbsp;</td>
<td>CDN Isotopes, Isotec, Cambridge Isotope Lab</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Solvents</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Analytes</td>
<td>&nbsp;</td>
<td>Sigma, Universidad Autonoma de Madrid, Ernesto Brunet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>GC Columns</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>123-5026</td>
<td>&nbsp;</td>
<tr>
<td>TEA/TFA</td>
<td>&nbsp;</td>
<td>Fluka/Sigma</td>
<td>09747</td>
<td>&nbsp;</td>
<tr>
<td>Vials</td>
<td>&nbsp;</td>
<td>Supelco/Sigma</td>
<td>Z115088/12EA</td>
<td>&nbsp;</td>
<tr>
<td>Merlin Microseal</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5181-8815</td>
<td>&nbsp;</td>
<tr>
<td>MSTFA</td>
<td>&nbsp;</td>
<td>Thermal Scientific</td>
<td>48913</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

one-step metabolomics: carbohydrates, organic and amino acids quantified in a single procedure

Ogni bambino nato negli Stati Uniti è ora proiettato fino a 42 malattie rare genetiche chiamate &quot;errori congeniti del metabolismo&quot;. Il metodo di screening si basa sulla spettrometria di massa tandem e quantifica acilcarnitine come schermo per acidemias organici e misure anche aminoacidi. Tutti gli stati anche eseguire il test enzimatico per i disturbi di carboidrati come la galattosemia. Perché i risultati possono essere non specifici, test di follow-up dei risultati positivi è necessario utilizzare un metodo più definitiva. La presente relazione descrive il metodo &quot;ureasi&quot; di preparazione dei campioni per lo screening errore congenito. Enzima ureasi cristallino è utilizzato per rimuovere l&#39;urea da fluidi corporei che permette maggior parte degli altri metaboliti solubili in acqua da disidratare e derivatizzati per gascromatografia in un unico procedimento. Disidratazione per evaporazione in corrente di azoto è facilitato con l&#39;aggiunta di acetonitrile e cloruro di metilene. Poi, trimethylsilylation avviene in presenza di un unico catalizzatore, trifluoroacetato triethylammonium. Iniezione automatizzato e cromatografia è seguito da macro-driven quantificazione usanza di 192 metaboliti e semi-quantificazione di ogni componente principale utilizzando librerie specializzate di spettri di massa di TMS derivatizzati composti biologici. L&#39;analisi può essere eseguita sul ampiamente utilizzato piattaforma Chemstation utilizzando le macro e librerie disponibili da parte dell&#39;autore. Nel nostro laboratorio, oltre 16.000 campioni di pazienti sono stati analizzati utilizzando il metodo con una resa diagnostica di circa il 17% - vale a dire il 17% dei campioni risultati rivelano che i risultati dovrebbero essere attuate da parte del medico di ordinazione. Inclusi in queste oltre 180 sono gli errori confermata congeniti, di cui circa il 38% non avrebbe potuto essere diagnosticata con metodi precedenti.

Watch this Scientific Journal Video about One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure at JoVE.com

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Il metodo ureasi di preparazione dei campioni per GC / MS dei metaboliti di intermediazione è presentato dal suo inventore. Il metodo permette di one-step di follow-up di screening neonatale per errori congeniti mediante spettrometria di massa tandem dai carboidrati quantificazione, organici e aminoacidi tutti in un unico processo.

One-step Metabolomica: carboidrati, acidi organici e aminoacidi quantificati in una procedura unica

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

one-step-metabolomics-carbohydrates-organic-amino-acids-quantified

Research

JoVE Journal

Biology

13.0K Views.  Saint Louis University School of Medicine. Il metodo ureasi di preparazione dei campioni per GC / MS dei metaboliti di intermediazione è presentato dal suo inventore. Il metodo permette di one-step di follow-up di screening neonatale per errori congeniti mediante spettrometria di massa tandem dai carboidrati quantificazione, organici e aminoacidi tutti in un unico processo.

Video: One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murino procedura trapianto renale

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Ricerca

Biologia

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

Procedura di trapianto epiteliali mammarie

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Percorso di conferma metaboliche e scoperta attraverso 13 C-etichettatura dei proteinogenici Aminoacidi

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Una procedura di screening chimico per glucocorticoidi segnalazione con un reporter del sistema Zebrafish Larva Luciferase

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Una strategia per sensibili, su larga scala quantitative Metabolomica

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

One-step purificazione di proteine ​​Twin-Strep-taggate e dei loro complessi su Strep-Tactin resina reticolato con Bis (Sulfosuccinimidil) suberato (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

L&#39;incorporazione specifici residuo di non canonici amminoacidi in proteine ​​modello utilizzando un<em&gt; Escherichia coli</em&gt; Trascrizione-traduzione sistema non cellulare

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...