This work was supported by the Lehigh University Start-up Package. We thank Dr. Walter Fast for providing the GST-PAD4 plasmid for protein expression.

1. PAD4 Dönüşüm, Expression ve Arıtma <ol><li> PAD4 Dönüşüm için, kimyasal olarak, yeterli E. içine PAD4 GSTfüzyonunu içeren pGEX plazmid dönüşümü Aşağıdaki prosedür kullanılarak protein ifadesi için E. coli (E. coli türü BL21 (DE3) &#39;) hücreleri. Kimyasal yetkili (kalsiyum klorür) hazırlayın E. Standart protokollere göre coli (E. coli türü BL21 (DE3) &#39;) hücreleri. <ol><li> Önceden hazırlanan kimyasal yetkili BL21 (DE3) hücreleri buz üzerinde 50 ul çözülme ve 5 ml kültür tüpü içinde PAD4 genini içeren pGEX plazmidin 1 ul ile karıştırılır. Yavaşça her 2 dakikada çalkalayarak 10 dakika süreyle buz üzerinde inkübe karışımı. </li><li> Isı 40 sn için 42 ° C su banyosu içinde karışımın yerleştirerek hücreleri şok. Hemen hücrelerinin iyileşmesi için verilen 2 dakika geri buz üzerinde hücreye plazmid karışımı yerleştirin. Karışıma steril LB et suyu 1 ml ilave edilir ve 1 dakika boyunca buz üzerine yerleştirin. </li><li> 1 250 rpm&#39;de çalkalanarak, 37 ° C&#39;de hücreler şok ısı inkübehr. Pipet 75 bir ampisilin dayanıklı agar plakası üzerinde dönüştürülmüş hücrelerin ul ve 15 saat boyunca 37 ° C&#39;de inkübe edin. 4 ° C&#39;de muhafaza plaka. </li></ol></li><li> PAD4 ifadesi. <ol><li> 1x ampisilin içeren LB 5 ml ampisilin agar plaka ve yerden BL21 (DE3) hücreleri 1 koloni seçin. Küvöze ve 37 ° C&#39;de O / N sallayın. </li><li> 1x amfisilin trihidrat içeren steril LB (MW 403,45 g / Mol) 1 L LB (başlangıç) ve 5 ml aktarın. 37 ° C&#39;de çalkalama inkübatöründe yer büyüme. Büyüme OD 600 izler. Büyüme 600 0.3 bir OD ulaştığında, inkübatör sallayarak 16 ° C içine büyüme taşıyın. </li><li> OD 0.6 600, 0.3 mM isopropylthiagalactoside (IPTG, MW 238,30 g / Mol) ile hücrelerin uyarılması ulaştıktan sonra. Hücreler 16 ° C&#39;de 15 saat boyunca çalkalanır izin verin. </li><li> 0 ° C&#39;de 20 dakika boyunca 4000 x g&#39;de santrifüj ile hasat hücreleri. -80 ° C&#39;de süpernatant ve mağaza pelet kapalı dökün. </li></ol></li><li> PAD4 saflaştırma <ol><li> Yeniden askıya 50 mM NaCI (MA 58.44 g / Mol), bir tampon maddesi içinde hücreleri BL21 (DE3) &#39;de ifade edilen PAD4 içeren pelet, 300 mM NaH 2 PO 4 (MW 119,98 g / Mol), 10 mM imidazol (MW 68,077 g / mol), 0.1 mM fenilmetilsülfonil flüorür (PMSF, MW 174,94 g / Mol) ve 1 mM ditiyotretol (DTT, 154,25 g / Mol), pH = 8.0. </li><li> 4 ° C&#39;de 15 dakika süreyle sese tabi tutularak ile lizleyin hücreleri. Sonikasyonun ardından, 0 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de hücre lizat santrifüj. Dökün ve süpernatant kaydedin. Toplu, oda sıcaklığında 30 dakika boyunca glutatyon (GSH) agaroz boncuklar ile yüzer. </li><li> Gravite ile GST boncuk / sütundan süpernatant boşaltın. Oda sıcaklığında 1 x PBS&#39;de (fosfat tamponlu tuz çözeltisi, pH = 8.0) ve 4 x 25 ml boncuk yıkayın. </li><li> Zehir PAD4 2 x 10 mi elüsyon tamponu ile, 50 mM tris (hidroksimetil) aminometan (Tris baz, MW 121.14 g / mol), 10 mM glutatyon (GSH, MW 307,32 g / Mol), pH = 8.0. </li><li> 100k MW kesme o kullanarak PAD4 Konsantreff santrifüj tüpleri ve santrifüj 4 ° C&#39;de 20 dakika boyunca 4000 x g&#39;de. -80 ° C&#39;de kısım 1.0 ml mikrosantrifüj tüpleri içinde 200 ul hacim içinde, protein saklayın. </li></ol></li></ol> Tamponlar 2. hazırlanması Stok Çözümleri <ol><li> Sodyum klorür (NaCI, MA 58.44 g / mol) tartıldı ve 2M solüsyon hazırlanır. Berrak kadar çözüm karıştırın. </li><li> Tris (hidroksimetil) aminometan (Tris baz, MW 121,14 g / Mol) tartın ve = 8.0, 2 M çözelti, pH hazırlar. Berrak kadar çözüm karıştırın. </li><li> Kalsiyum klorür dihidrat (CaCl2 2H 2, O, MW 147,01 g / Mol) tartılır ve 500 mM&#39;lik çözelti hazırlayın. Berrak kadar çözüm karıştırın. </li><li> Tris (2-karboksietil) fosfin (TCEP, MW 250,19 g / Mol) tartılır ve 200 mM&#39;lik çözelti hazırlayın. -20 ° C&#39;de berrak ve mağaza kadar çözüm karıştırın. </li><li> Ditiyotretol (DTT, MW 154,25 g / mol) tartıldı ve 1 M solüsyon hazırlanır. -20 ° C&#39;de berrak ve mağaza kadar çözüm karıştırın. </li><li> HazırlamakTriton X-100% 0.5 çözeltisi. </li><li> Etilendiamintetraasetik asit (EDTA, MW 292,24 g / Mol) tartılır ve 100 mM&#39;lik çözelti hazırlayın. Berrak kadar çözüm karıştırın. </li><li> Hidroklorür (ZRcoum MW 621,69 g / Mol) metilkumarin Z Arg- Arg- -7- amido- 4- tartın ve dimetil sülfoksit (DMSO) içinde 10 mM&#39;lik bir çözelti hazırlayın. </li></ol> 37 ° C&#39;de 3 PAD4 Deneyi <ol><li> 10 mM ZRcoum stoklar kaynaktan su içinde ZRcoum bir 125 uM solüsyon hazırlanır. Bu 125 uM ZRcoum çözeltisi Çözelti A olacak </li><li> 62.5 mM NaCI, 62.5 mM Tris, 12.5 mM CaCI2, 6.25 mM DTT tamponu ve 5 uM PAD4 (pH = 8.0) hazırlayın. Bu çözelti olacak B. </li><li> 62.5 mM NaCI, 62.5 mM Tris, 12.5 mM CaCI2, ve 6.25 mM DTT (pH = 8.0) &#39;in bir tamponu hazırlayın. Bu çözelti C olacak </li><li> (Büyükbaş hayvan pankreasından) tripsin, kristalin tartılır ve 100 mM EDTA içinde 10 mg / ml hazırlar. Berrak kadar çözüm karıştırın ve -20 ° C&#39;de saklayın° C. Bu çözelti, D olacak </li><li> 96 oyuklu bir plaka elde edilir ve bir kontrol için kuyu numarasına (PAD4) ve test kuyuları (+ PAD4) işaret etmektedir. </li><li> Pipet testi kuyular ve pipet kontrol kuyulara Çözüm C 160 ul içine Solüsyon B 160 ul. 37 ° C&#39;de 15 dakika boyunca inkübe edin. </li><li> Pipet 40, tüm oyuklara Eriyik A ul ve 45 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Test kuyularının kontrol kuyuları yarısı ve yarısı dH 2 O Pipet 10 ul. Kontrol bölümlerinin ve test kontrolleri, bir pipet, 37 ° C&#39;de 10 dakika boyunca Çözelti D inkübe 10 ul diğer yarısına. </li><li> 345 nm&#39;lik bir eksitasyon dalga boyu ve 465 nm&#39;lik bir emisyon dalga boyunda bir çok modlu okuyucu üzerinde okuma bir floresan elde edilir. </li></ol> Oda sıcaklığında 4 PAD4 Deneyi <ol><li> 10 mM ZRcoum stoklar kaynaktan su içinde ZRcoum bir 50 uM solüsyon hazırlanır. Bu 50 uM ZRcoum çözeltisi Çözelti A olacak </li><li> 100 mM Na bir tampon hazırlayınCI, 100 mM Tris, 20 mM CaCI2, 2 mM TCEP,% 0.02 Triton X-100 ve 8 uM PAD4 (pH = 8.0). Bu çözelti olacak B. </li><li> 100 mM NaCI, 100 mM Tris, 20 mM CaCI2, 2 mM TCEP,% 0.02 Triton X-100 (pH = 8.0) &#39;in bir tamponu hazırlayın. Bu çözelti C olacak </li><li> (Büyükbaş hayvan pankreasından) tripsin, kristalin tartılır ve 100 mM EDTA içinde 10 mg / ml hazırlar. -20 ° C&#39;de berrak ve mağaza kadar çözüm karıştırın. Bu çözelti, D olacak </li><li> 384 oyuklu plaka elde edilir ve kontrol (hiçbir PAD4) ve test deliklerine (+ PAD4) için kuyu işaret etmektedir. </li><li> Pipet testi kuyular ve pipet kontrol kuyulara Çözüm C 25 ul içine Solüsyon B 25 ul. Oda sıcaklığında 20 dakika oyuklarda kuluçkalanması PAD4 olmadan / tampon izin verir. </li><li> Tüm kuyulara C çözeltisi pipetle 25 ul ve oda sıcaklığında 45 dakika inkübe edilir. </li><li> Test kuyularının kontrol kuyuları yarısı ve yarısı dH 2 O Pipet 10 ul. Kontrol bölümlerinin ve test kontrollerin diğer yarısı, pipett için10 dakika boyunca oda sıcaklığında Çözelti D inkübe e 10 ul. </li><li> 345 nm&#39;lik bir eksitasyon dalga boyu ve 465 nm&#39;lik bir emisyon dalga boyunda bir çok modlu okuyucu üzerinde okuma bir floresan elde edilir. </li></ol> 37 ° C&#39;de ve oda sıcaklığında 5. PAD4 saat Sahası Tahliller <ol><li> 10 mM ZRcoum stoklar kaynaktan su içinde ZRcoum bir 125 uM solüsyon hazırlanır. Bu 125 uM ZRcoum çözeltisi Çözelti A olacak </li><li> 62.5 mM NaCI, 62.5 mM Tris, 12.5 mM CaCI2, 6.25 mM DTT tamponu ve 5 uM PAD4 (pH = 8.0) hazırlayın. Bu çözelti olacak B. </li><li> (Büyükbaş hayvan pankreasından) tripsin, kristalin tartılır ve 100 mM EDTA içinde 10 mg / ml hazırlar. -20 ° C&#39;de berrak ve mağaza kadar çözüm karıştırın. Bu çözelti C olacak </li><li> (Kaç zaman noktalarında bağlıdır kuyuların #) 2 96-iyi plakaları ve pipet kuyuların # miktarda içine Solüsyon B 160 ul edinin. </li><li> 37 ° C&#39;de bir 96 oyuklu plaka inkübe edin. Oda sıcaklığında, diğer 96 plaka inkübe edin. </li><li> Ölmekette hemen 40 hem 96 oyuklu plakalar içinde, tüm oyuklara Eriyik A ul ve t = 0 dakika kuyulara çözelti C, 10 ul pipetle. Tüm kuyuları söndürüldü edilinceye kadar belirli zaman noktalarında Çözüm C 10 ul ekleyerek devam edin. </li><li> 345 nm&#39;lik bir eksitasyon dalga boyu ve 465 nm&#39;lik bir emisyon dalga boyunda bir çok modlu okuyucu üzerinde okuma bir floresan elde edilir. </li></ol> RT&#39;de 6 PAD4 Engelleme Deneyi: <ol><li> 10 mM ZRcoum stoktan, su ZRcoum bir 83 uM solüsyon hazırlanır. Bu 83 uM ZRcoum çözeltisi Çözelti A olacak </li><li> Cı-amidin (MW 424,8 g / Mol) tartıldı ve 100 uM stok solüsyonu hazırlanır. Bu çözelti olacak B. </li><li> 100 mM NaCI, 100 mM Tris, 20 mM CaCI2, 2 mM TCEP,% 0.02 Triton X-100 ve tampon ve 8 uM PAD4 (pH = 8.0) hazırlayın. Bu çözelti C olacak </li><li> 100 mM NaCI, 100 mM Tris, 20 mM CaCI2, 2 mM TCEP,% 0.02 Triton X-100 (pH = 8.0) &#39;in bir tamponu hazırlayın. ThÇözüm D. olacak olan </li><li> (Büyükbaş hayvan pankreasından) tripsin, kristalin tartılır ve 100 mM EDTA içinde 10 mg / ml hazırlar. -20 ° C&#39;de berrak ve mağaza kadar çözüm karıştırın. Bu çözelti, E. olacak </li><li> 384 oyuklu plaka elde edilir ve kontroller için kuyu (Resim PAD4) Test oyuklarını (+ PAD4) ve inhibisyon kuyu (PAD4 + CI-amidin) işaret etmektedir. </li><li> Pipet testi kuyular ve pipet kontrol kuyulara Çözüm D 25 ul içine Çözüm C 25 ul. Oda sıcaklığında 20 dakika oyuklarda kuluçkalanması PAD4 olmadan / tampon izin verir. </li><li> Inhibisyon kuyulara Çözelti B&#39;nin pipetle 10 ul ve RTE&#39;nin 20 dakika süreyle inkübe edin. </li><li> Tüm kuyulara Çözelti A&#39;nın Pipet 15 ul ve oda sıcaklığında 45 dakika inkübe edilir. </li><li> Pipet tüm kuyulara Çözüm E 10 ul. 10 dakika boyunca oda sıcaklığında inkübe edin. </li><li> 345 nm&#39;lik bir eksitasyon dalga boyu ve 465 nm&#39;lik bir emisyon dalga boyunda bir çok modlu okuyucu üzerinde okuma bir floresan elde edilir. </li></ol> 7. ham hücre lizatının DeneyiRT&#39;de <ol><li> 50 mM Tris, 50 mM NaCI, 0.1 mM fenilmetilsülfonil flüorür (PMSF, MW 174,94 g / Mol) ve 1 mM ditiyotretol (DTT, 154,25 g / Mol), pH, 10 ml PAD4 1 L gelişme gelen süspansiyon hücreleri hücrelerin hasat edilmesinden sonra, = 8.0. </li><li> 4 ° C&#39;de 15 dakika süreyle sese tabi tutularak ile lizleyin hücreleri. 0 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de hücre lizatı santrifüjleyin. 100k MA kesme santrifüj tüpleri kullanılarak 4 ° C&#39;de 4,000 rpm&#39;de santrifüjleme ile yarım hacme berrak bir aktif madde konsantre edilir. </li><li> 10 mM ZRcoum stoklar kaynaktan su içinde ZRcoum bir 250 uM solüsyon hazırlanır. Bu 250 uM ZRcoum çözeltisi Çözelti A olacak </li><li> 100 mM NaCI, 100 mM Tris, 20 mM CaCI2, ve 2 mM TCEP (pH = 8.0) &#39;in bir tamponu hazırlayın. Bu çözelti olacak B. </li><li> (Büyükbaş hayvan pankreasından) tripsin, kristalin tartılır ve 100 mM EDTA içinde 10 mg / ml hazırlar. -20 ° C&#39;de berrak ve mağaza kadar çözüm karıştırın. Bu çözelti C olacak </li><li> Siyah, 384 oyuklu plaka ve tasarım elde edilirkontroller (hiçbir hücre dağılma) ve test kuyuları (+ hücre dağılma) için kuyu yedik. </li><li> Pipet testi kuyulara bütün kuyuları ve hücre lisatından pipet 60 ul içine Çözüm B 100 ul. Oda sıcaklığında 20 dakika boyunca inkübe edin. </li><li> Tüm kuyulara Çözelti A&#39;nın Pipet 40 ul ve oda sıcaklığında 45 dakika inkübe edilir. </li><li> Pipet tüm kuyulara Çözüm C 10 ul. 10 dakika boyunca oda sıcaklığında inkübe edin. </li><li> 345 nm&#39;lik bir eksitasyon dalga boyu ve 465 nm&#39;lik bir emisyon dalga boyunda olan çok modlu okuyucu üzerinde okuma bir floresan elde edilir. </li></ol>

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Sci. 7, 769-781 (2011).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+PADI4+cooperates+with+Elk-1+to+activate+c-Fos+expression+in+breast+cancer+cells.">Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells.</a> PLoS Genet. 7, (2011).</li><li>Tanikawa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+histone+modification+and+chromatin+structure+by+the+p53-PADI4+pathway.">Regulation of histone modification and chromatin structure by the p53-PADI4 pathway.</a> Nat. Commun. 3, 676 (2012).</li><li>Cui, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+microRNA-16+in+colon+cancer+cells+by+protein+arginine+deiminase+inhibition+causes+a+p53-dependent+cell+cycle+arrest.">The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest.</a> PLos One. 8, (2013).</li><li>Jones, J. E., Causey, C. P., Knuckley, B., Slack-Noyes, J. L., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+deiminase+4+(PAD4):+Current+understanding+and+future+therapeutic+potential.">Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic potential.</a> Curr. Opin. Drug Discov. Devel. 12, 616-627 (2009).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+p53+target+gene+expression+by+peptidylarginine+deiminase+4.">Regulation of p53 target gene expression by peptidylarginine deiminase 4.</a> Mol. Cell Biol. 28, 4745-4758 (2008).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+peptidylarginine+deiminase+(PAD)+inhibitors+regulate+the+autophagy+flux+and+the+mammalian+target+of+rapamycin+complex+1+activity.">Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.</a> J. Biol. Chem. 287, 25941-25953 (2012).</li><li>Slack, J. L., Causey, C. P., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+deiminase+4:+a+target+for+an+epigenetic+cancer+therapy.">Protein arginine deiminase 4: a target for an epigenetic cancer therapy.</a> Cell. Mol. Life Sci. 68, 709-720 (2011).</li><li>Luo, Y., Knuckley, B., Lee, Y. H., Stallcup, M. R., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluoroacetamidine-based+inactivator+of+protein+arginine+deiminase+4:+design,+synthesis,+and+in+vitro+and+in+vivo+evaluation.">A fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis, and in vitro and in vivo evaluation.</a> J. Am. Chem. Soc. 128, 1092-1093 (2006).</li><li>Knuckley, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+specificity+and+kinetic+studies+of+PADs.">Substrate specificity and kinetic studies of PADs.</a> Biochemistry. 1, 4852-4863 (2010).</li><li>Knipp, M., Vasak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+96-well+microtiter+plate+assay+for+the+determination+of+enzymatically+formed+citrulline.">A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline.</a> Anal. Biochem. 286, 257-264 (2000).</li><li>Knuckley, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluopol-ABPP+HTS+assay+to+identify+PAD+inhibitors.">A fluopol-ABPP HTS assay to identify PAD inhibitors.</a> Chem. Commun. 46, 7175-7177 (2010).</li><li>Bicker, K. L., Subramanian, V., Chumanevich, A. A., Hofseth, L. J., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+citrulline:+development+of+a+phenylglyoxal-based+probe+to+visualize+protein+citrullination.">Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.</a> J. Am. Chem. Soc. 134, 17015-17018 (2012).</li><li>Wang, Q., Priestman, M. A., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+protein+arginine+deiminase+activity+by+using+fluorescence+quenching:+multicolor+visualization+of+citrullination.">Monitoring of protein arginine deiminase activity by using fluorescence quenching: multicolor visualization of citrullination.</a> Angew. Chem. Int. Ed. Engl. 52, 2323-2325 (2013).</li><li>Jones, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+screening+of+a+haloacetamidine+containing+library+to+identify+PAD4+selective+inhibitors.">Synthesis and screening of a haloacetamidine containing library to identify PAD4 selective inhibitors.</a> ACS Chem. Biol. 7, 160-165 (2012).</li><li>Dreyton, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor. , (2010).</li><li>Shimoyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deimination+stabilizes+histone+H2A/H2B+dimers+as+revealed+by+electrospray+ionization+mass+spectrometry.">Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry.</a> J. Mass Spectrom. 45, 900-908 (2010).</li><li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+and+inactivators+of+protein+arginine+deiminase+4:+functional+and+structural+characterization.">Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.</a> Biochemistry. 45, 11727-11736 (2006).</li></ol>

The authors have nothing to disclose.

Herein, a fluorescence based assay was successfully developed that monitors the activity of PAD4. The assay has proven to be incredibly robust in a number of high-throughput conditions and is also compatible with whole cell lysates37.
The affinity between the histone proteins and the DNA strands surrounding it loosens due the neutralization of the positive charge on the arginine side chain upon citrullination. It was envisioned that the neutralization of the arginine side-chain coul...

Memeli proteinlerinin bir çok sayıda ağır ribozom tarafından proteinlerin sentezi, aşağıdaki enzimlerin etkimesi ile modifiye edilir. Bu post-translasyonel modifikasyonlar, (PTMS) büyük ölçüde protein 1-3 arasında (diğer özellikleri yanı sıra) boyut, yük, yapı ve oligomerizasyon durumunu değiştirerek Proteome fonksiyonel çeşitliliği artırabilir. Bunun bir sonucu olarak, protein yapısındaki bir değişiklik, örneğin protein bozulması, hücre farklılaşması, iletimde, gen ekspresyonunda modülasyonu ve protein-protein etkileşimleri gibi fizyolojik sonuçlara yol açabilir. Bu değişiklikler tüm insan proteinlerinin büyük bir yüzdesi görülürken histon proteinler kovalent modifikasyonlar 4 alışılmadık derecede yüksek sayıda geçmesi üzerine, terminal biter. Histon proteinler genomik DNA yoğunlaştırılmasını kolaylaştırır, yapısal proteinlerin bir ailesidir. Yapılandırılmamış histon kuyrukları Kovalent modifikasyonlar bir seride tarafından yürütülen ve düzenlenir, artıkları (yazarlar) ve kovalent modifikasyonunu katalize aynı değişiklikleri (silgiler), ters ve değişiklikler arasında ayrım enzimlerin s histon kuyrukları (okuyucu) 5-7 üzerine basılmış olan. Aslında, bilinen PTMS en metilasyon, fosforilasyon, asetilasyon, sumoylation, ubikitinasyon ve sitrülinlenmesine 8 de dahil olmak üzere histonun bu kısa segmenti içinde görülmektedir. Sitrülinlenmesine olmayan T RNA kodlanmış peptidil-sitrulin (Şekil 1A) peptidil-argininin dönüştürülmesini kapsar. Bu yan zinciri nötrleştirme sorumlu proteinleri, PAD üyesi olan kalsiyum-bağımlı enzimler olması her biri. 9,10, protein ailesini (s rginine D eiminase peptit). Bugüne kadar, PAD ailesinin beş üyesi (PAD1, PAD2, PAD3, PAD4 ve PAD6) &#39;de tarif edilmiştir. Bu ailenin her üyesi farklı hücresel proteinleri hedef görünür ve aynı zamanda uni görüntülerque doku dağılımı profilleri. PAD4 bir nükleer lokalizasyon dizisi 11 ile çekirdek içinde lokalize olduğu bilinmektedir, bu protein ailesinin tek üyesidir. Bu duruma göre, histon H2A arginin kalıntısının 3, PCR&#39;ın N-terminal kuyrukları arginin yan zincirler (H2R3), H + 3 (H3R2, H3R17 ve H3R26) ve H4 (H4R3) 12 nükler hedeflerin bir dizi deiminate gösterilmiştir, 13. PAD izozimlerinin, her özel ve kritik fizyolojik işlevlere sahip olsa da, PAD4 dolayı hastalıklı ve sağlıklı hücrelerinin her ikisinde de, insan gibi bir dizi işlem rolünü önemli ölçüde daha fazla ilgi çekmektedir. Son zamanlarda, PAD4 Pluripotency transkripsiyonel ağı 14 üye olduğu gösterilmiştir. Her iki PAD4 sentezleme seviyeleri ve aktivite, farelerde yeniden programlama ve taban durumu, pluripotent durumlarında yüksek olduğu gösterilmiştir. Kök hücre genlerin düzenlenmesini kontrol edilerek, PAD4 hücre yeniden programlama veriminde önemli bir rol tutabilirler. PAD4 de implike edilmiştirüzerine patojenlerin bağlanmasını nötrofil hücre dışı tuzakları, oluşumu kendi sistem açıklığı sağlar. PAD4 histon proteinleri hypercitrullination dolayısıyla bakteriyel enfeksiyonların 15,16 sakınma, hücre dışı tutucusu ile, patojen bakterilerin kapsüllenmesi için taban malzemesi olarak hizmet eden kromatin yoğunluk azaltılmasından neden olur. Buna ek olarak, PAD4 insan hastalıklarının bir dizi aktif bir rol oynadığı tespit edilmiştir. Daha önce PAD4 anormal ekspresyon romatoid arteritin başlamasında ve şiddeti ile ilişkili olduğu gösterilmiştir, Alzheimer, Parkinson hastalığı ve multipl skleroz 17. Aslında, bir anti-sitrülin proteinin antikorların varlığı romatoid artrit 18 arasında en güvenilir ve kesin bir teşhis ve prognostik biyolojik biridir. Benzer şekilde, bozulmuş PAD4 aktivitesi en son yumurtalık, meme, akciğer, bir de dahil olmak üzere insan kanserlerinin bir dizi gözlenmiştird özofagus kanserleri 19-22. PAD4 ve kanser arasındaki bağlantı, p53 tümör bastırıcı bir protein 22,23 ELK1 onkogen üzerinden veya yoluyla aracılık ettiği gösterilmiştir ve önceki iş PAD4 bir yeni anti-kanser terapötik hedef 17,24,25 olabileceğini öne sürdü. Ilke çalışmanın bir kanıtı olarak, kolorektal kanser hücre çizgisi, HCT116 olarak shRNA ile PAD4 tükenmesi apoptoz ve hücre döngü yakalanmasını 26 uyarılması için yeterli olduğu bulundu. Son yıllarda geliştirilmiş olan geri dönüşü olmayan bir PAD4 inhibitörünün farelerde 27 tümör kütlesinde bir yetmiş oranında azalmaya yol açmıştır. Oldukça önemli ölçüde, PAD4 inhibisyon dönüştürülmemiş hücreleri ayırarak, kanser hücrelerinin seçici ölümüyle sonuçlanan bir hedefe yönelik bir tedavi olarak hareket ortaya çıktı. PAD4 işlevini kapatmak için küçük moleküllerin kullanımı, kanser hücrelerini hedef veya mevcut kanser, kemoterapi 28 artırmak için yeni ve güçlü bir strateji olduğu ispat edebilir. Ne yazık ki, POÇadır geri dönüşümlü PAD4 inhibitörü henüz keşfedilmeyi etti. Kovalent önleyicileri bir dizi alt-tabakanın arginin 27,29,30 taklit ile sağlıklı ve hastalıklı durumu hücrelerinin her ikisinde de PAD4 rolünü anlamak için pratik araçlar olduğu kanıtlanmıştır kloro / floro dj pirimidin kolu kullanarak geliştirilmiştir. Bununla birlikte, bu molekül, benzer etki ile, aktif pedlerin her inhibe eder. Bu nedenle, PAD4 aktivitesine ilişkin raporlar, basit bir deneme için olan ihtiyaç çok önemlidir. Bugüne kadar, PAD4 deneyler, kolorimetrik bir okuma 31 ile reaksiyondan amonyağın serbest bağlantı tarif edilmiştir, floresan polarizasyon tahlili için 32 flüoresan chloroamidine alt-tabaka analog kullanmaktadır glioksal ve sitrulline 33 arasındaki asit yardımlı reaksiyon üzerine dayanır ve bağlanma aşamasının 34 dequenching bir floresan PAD4 faaliyet. Bunlardan sadece kovalent değiştirici haloacetamidine stratejisi, yüksek verimli Scre ile uyumlu olduğu kanıtlanmıştırsürmesi gelen platformlar 32,35,36. Biz güvenilir PAD4 aktivitesini ölçen basit floresan dayalı tahlil açıklar. Güçlü bir sinyal-gürültü oranı, analiz hızı ve ölçüm sağlamlık gösterir deneyi, gerçekten kuvvetli ve seçici PAD4 inhibitörü keşfetmek için potansiyele sahiptir.

<table border="0" cellpadding="0" cellspacing="0" width="716">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:192px;">Name of the Material/Equipment</td>
			<td style="width:123px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:267px;">Comments/ Description</td>
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		<tr height="66">
			<td height="66" style="height:66px;width:192px;">LB Broth, Miller (LURIA-BERTANI)</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:135px;">J106-2KG</td>
			<td style="width:267px;"><a href="http://www.amresco-inc.com/">http://www.amresco-inc.com</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td>BP902-25&#160;</td>
			<td style="width:267px;"><a href="http://www.fishersci.com/">http://www.fishersci.com</a></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:192px;">Isopropylthiagalactonse (IPTG)</td>
			<td style="width:123px;">Life Technogolies</td>
			<td>15529-019</td>
			<td style="width:267px;"><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com</a></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:192px;">All Buffer Salts&#160;</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:192px;">Protino Glutathione (GSH) agarose&#160;</td>
			<td style="width:123px;">Machery-Nagel</td>
			<td style="width:135px;">745500.10</td>
			<td style="width:267px;">Store at 4 &#176;C; http://www.mn-net.com/</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Z-&#8203;Arg-&#8203;Arg-&#8203;7-&#8203;amido-&#8203;4-&#8203;methylcoumarin hydrochloride (Zcoum)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>C5429</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:192px;">Chloroamidine&#160;</td>
			<td style="width:123px;">Cayman Chemical&#160;</td>
			<td style="width:135px;">10599</td>
			<td style="width:267px;">Store at -20 &#176;C; https://www.caymanchem.com/app/template/Home.vm</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:192px;">Tris(2-&#8203;carboxyethyl)&#8203;phosphine hydrochloride (TCEP HCl)</td>
			<td style="width:123px;">Thermo Scientifc</td>
			<td style="width:135px;">20490</td>
			<td style="width:267px;"><a href="http://www.piercenet.com/">http://www.piercenet.com</a></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:192px;">Triton X-100</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:135px;">X100-100ML</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Corning/Nunclon MicroWell plates (96 and 384)</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Purchase from Corning; Sigma-Aldrich</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Tecan Infinite 200 / Tecan i-control microplate reader software</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.tecan.com/">www.tecan.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Europium Ex. 340/40 Em. 475/15 filter</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.perkinelmer.com/">http://www.perkinelmer.com</a></td>
		</tr>
	</tbody>
</table>

fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

Başlangıçta ZRcoum 96 oyuklu bir plaka biçiminde, PAD4 aktivitesi rapor göstermiştir edilmiştir. Wells, alt-tabaka ZRcoum ve PAD4 varlığında / yokluğunda inkübe edildi. 475/15 nm filtre (Şekil 2A), - 37 ° C &#39;de 45 dakikalık bir inkübasyon döneminden sonra, floresan, bir 340/40 ile ölçüldü. Beklendiği gibi, floresans seviyesi ZRcoum (veya sitrülin ZRcoum) içindeki florofor olarak düşük kalmıştır kilitli ...

Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Pro-floresan alt madde analog ile PAD4 Faaliyet flüoresan&#39;a bağlı İzleme

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

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Research

JoVE Journal

Chemistry

9.8K Görüntüleme.  Lehigh University. PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Video: Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Floresan Bazlı Buz Düzlemi Benzeşimi ile Antifriz Proteinlerinin Buz Bağlayıcı Düzlemlerinin Belirlenmesi

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

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Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

Bir Rapid Süperkritik Ekstraksiyon Yöntemi ile Silika AEROJEL Monoliths hazırlanıyor

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

Sürekli akış İşleme kullanma Gerçekleştirilen Reaksiyonları Gerçek zamanlı izleme: Bir Örnek olarak 3-Acetylcoumarin Hazırlanması

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Boc-Ala-Ala-Asp-S-Bzl hidrolizi, A Kolorimetrik Özellikle Granzim B proteolitik aktivitesini ölçer Deneyi

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

Bir Yöntem Yüzey Oksidasyon ve İndirgeme yoluyla Sıvı Metal Yüzey Gerilim işleyin

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Eş zamanlı ölçüm HDAC1 ve HDAC6 faaliyete HeLa hücreleri kullanarak UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

İmalat ve katalitik Aerogels hızlı süperkritik ayıklama hazırlanan test

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Substrat bağlı Au Nanowires bir aktif yüzey büyüme mekanizması yoluyla sentezi

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

Esterleri bir yeşil Steglich esterleşme Asetonitril içinde yolu ile sentezi

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR tabanlı aktivite bileşik Inhibisyonu belirlenmesi için Assays, ıC50 değerleri, Artifactual aktivite, ve Nükrenoside Ribohidolases tüm hücre aktivitesi

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...