The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

Kültür Ortamı Antibiotics hazırlanması 1. ve 2- [2-nitro-4- (triflorometil) benzoil] -1,3-sikloheksandion (NTBC) <ol><li> Uygun birimlere LB suyu (% 1 Tripton,% 0.5 maya ekstresi,% 0.25 NaCl, H 2 O) ve kısım sağlayın. Otoklav ile sterilize edin. Oda sıcaklığında saklayınız. </li><li> 100 ml LB agar (% 1 tripton,% 0.5 maya ekstresi,% 0.25 NaCl, H2 O% 1.5 ağar) 250 ml&#39;lik şişelere sağlayın. Oda sıcaklığında otoklav ve mağaza tarafından sterilize edin. Agar tabak içine dökülmeden önce eritilir emin olun. Not: 4 plakayı verecektir LB agar 100 ml ihtiva eden şişeler. LB agar miktarı tayini için gereken levha sayısı uygun şekilde değiştirilebilir. </li><li> (137 mM NaCI, 2.7 mM KCI, 10 mM Na-2 HPO 4, 2 mM KH 2 PO 4) 16 PBS sağlayın. Oda sıcaklığında otoklav veya filtrasyon ve mağaza tarafından sterilize edin. </li><li> Gentamisin antibiyotik stok çözümleri hazırlayın, kanamisin, bird tobramisin. <ol><li> P. uygun antibiyotik stok konsantrasyonları hazırlayın 100 mg / ml gentamisin, 30 mg / ml kanamisin ve 10 mg / ml tobramisin ihtiva eden aeruginosa suşu. 4 ° C&#39;de su, filtre ile sterilize (0,2 um) ve deposunda antibiyotik çözülür. Çalışılan bakteriye bağlı antibiyotik ve konsantrasyonları değiştirin. </li></ol></li><li> NTBC stok çözümleri hazırlayın. DMSO 400 ul NTBC 10 mg eritin. Bu 75,9 mM NTBC bir konsantrasyon elde edilir. -20 ° C&#39;de saklayın NTBC stok çözümleri. Oda sıcaklığında çözülme çözümler gerektiği gibi. NOT: NTBC Farklı kaynaklardan çözünürlüğündeki farklılıklar var. Üreticinin önerilerine dayanan NTBC çözülür ve Adım 1.5 buna göre ayarlamak için hangi uygun araç belirleyin. </li></ol> 2. Bakteriyel Suşlarının NTBC Titrasyonları <ol><li> Suşların kurmak gecede kültürleri test edilecek. 2 ml LB suyu 16 x 150 mm test tüpünü ekles (suş başına bir tane) ve her bir suş 1 izole koloni aşılamak. Bir hava kuluçka makinesi içinde bir doku kültürü rotator havalandırma ile 37 ° C&#39;de gece boyunca inkübe edin. </li><li> Ertesi gün, LB suyu NTBC arasında titrasyonları hazırlar. Farklı suşları NTBC duyarlılık farklılıkları beri 0&#39;dan 900 mcM NTBC bir başlangıç ​​aralığı kullanın. <ol><li> Suş başına 4 ila 5 test tüpleri (16 x 150 mm) 1 ml LB suyu ekleyin. </li><li> Bir konsantrasyon aralığında test tüpleri (16 x 150 mm) NTBC stok solüsyonu (75,9 mM) eklenir. LB brot, 1 ml eklemek NTBC konsantrasyonları ve karşılık gelen hazır hacimleri için Tablo 1&#39;e bakınız. </li></ol></li><li> Gece boyunca kültürler OD 600 ölçün. Medyada mevcut pyomelanin ortadan kaldırmak için OD 600 okuma almadan önce kültürleri yıkayın. <ol><li> 2 dakika boyunca 16.000 x g&#39;de bir mikrosantrfuj kültür 1 ml santrifüj edilerek kültürleri yıkayın. Süpernatantı ve herhangi gevşek pelet hücreleri ile kaldırBir mikropipet ve 1 ml LB katı hücre pelletini </li></ol></li><li> OD 600 0.05 titrasyon tüpleri inoküle edin. Tüpleri aşılamak için gerekli yıkanmış kültür miktarını hesaplayın. NOT: Bu olmamalı pyomelanin beri ekimlerde için yıkanmış kültürleri kullanın. </li><li> Havalandırma, bir kuluçka makinesi içinde bir doku kültürü rotator kullanılarak 37 ° C sıcaklıkta yaklaşık 24 saat için titrasyon inkübe edin. </li><li> Titrasyon tüpleri fotoğraf ve MIC ve oksidatif stres testleri için kullanmak NTBC miktarını belirlemek için içinde ve suşları arasında pigment üretimini karşılaştırın. Hücre içermeyen kültür süpernatanında pyomelanin miktarını belirlemek ve hücre yoğunluğu belirlemek için OD 600 değerlendiriniz. Not: hücrelere kültür yüzey maddesinde pyomelanin OD 600 oranı NTBC ile muamele edildikten sonra pyomelanin üretim farklılıkları ölçmek için hesaplanabilir. </li></ol> 3. Antibiyotik Asgari inhibitörlerinin96 oyuklu plakalar içerisinde edici Konsantrasyon (MİK) Deneyi <ol><li> Suşların ayarlama gece boyunca kültürler ile NTBC olmadan LB içinde test edilmelidir. NOT: Bu protokol, 300 mcM NTBC temsilcisi seviyesini kullanarak tarif edilir. Kullanılacak NTBC uygun düzey Aşama 2,6 belirlenir. <ol><li> 2 ml LB 300 uM NTBC ekleme Herhangi bir NTBC durum için 2 ml LB araç (DMSO), eşdeğer bir hacim. </li><li> Steril kürdan kullanarak, bakteri, bir izole edilen bir koloninin Tüpleri inoküle. NTBC ile tek kültür ve her bir suş için NTBC olmadan tek kültür olacak. Havalandırma, bir kuluçka makinesi içinde bir doku kültürü rotator kullanılarak 37 ° C&#39;de gece boyunca inkübe edin. </li></ol></li><li> Ertesi gün, MIC tahlil kurmak için LB + NTBC ve LB + DMSO usta çözümleri yapmak. Aşı ilave edildiğinde, bu da 600 uM bir konsantrasyonda NTBC ekleyin 300 uM&#39;lik bir nihai konsantrasyon verecek şekilde, iki kat seyreltilmiş olacaktır. <ol><li> Bir antibi test etmek içintek zorlanma, 2 ml LB 600 mcM NTBC ekleyebilir ve NTBC ana çözüm yapmak mix otik. 2 ml LB araç (DMSO), eşdeğer bir hacim ilave edin ve herhangi bir NTBC ana çözelti yapmak için karıştırın. Antibiyotik stok solüsyonları oluşturmak için hem de 96-yuvalı plakalar içinde bir seyreltme serisi ayarlamak için bu çözüm kullanın. NOT: Master çözüm formülasyonları pipetleme hataları hesaba ekstra bir çözüm sağlayacaktır. Test antibiyotik ve suşların sayısına bağlı olarak gerektiği gibi usta çözümleri yukarı veya aşağı ölçeklendirilebilir edilebilir. </li></ol></li><li> LB + NTBC veya LB + DMSO usta çözümleri antibiyotik çözümler hazırlayın. Not: Bu çözeltiler antibiyotik konsantrasyonu iki istenen son konsantrasyonu olmalıdır. Yeterli çözelti 96 oyuklu bir plaka içerisinde dört gözeneğe 100 ul aktarma yapılmalıdır. <ol><li> Gentamisin +/- 64 ug / ml NTBC stok çözelti hazırlayın. 450 gentamisin stokunun 0.288 ul (100 mg / ml) ekleyin, bu çözüm yapmakul LB + NTBC veya LB + DMSO ana çözüm. NOT: P. gentamisin maksimum konsantrasyon aeruginosa PAO1 32 ug / ml&#39;dir. </li><li> Kanamisin +/- 256 ug / ml NTBC stok solüsyonu olun. 450 ul LB + NTBC ya da LB + DMSO ana çözümlere kanamisin stokunun 3,84 ul (30 mg / ml) ekleyin, bu çözüm yapmak için. NOT: P. kanamisin maksimum konsantrasyon aeruginosa PAO1 128 ug / ml&#39;dir. </li><li> Tobramisin +/- 8 ug / ml NTBC stok çözelti hazırlayın. Bu çözelti yapmak 450 ul LB + NTBC ya da LB + DMSO ana çözümlere tobramisin stokunun 0,36 ul (10 mg / ml) ekleyin. NOT: P. İçin aeruginosa PAO1 tobramisin maksimum konsantrasyonu 4 ug / ml&#39;dir. Not: Test edilecek bakteriler için antibiyotikler ve konsantrasyonlar ayarlanabilir. </li></ol></li><li> Bir 96-yuvalı plaka içinde dört gözeneğe her 2x antibiyotik çözeltisinin 100 ul ekle. Exampl için satır A&#39;da bu çözümleri yerleştirine, gentamisin A4 boyunca A1 yerleştirilmelidir, kanamisin A8 ile A5 yerleştirilmelidir ve tobramisin A12 ile A9 yerleştirilmelidir. Kurmak 96 oyuklu bir plakanın şeması için Şekil 1A bakınız. Not: Çoklu antibiyotik bir plaka olarak test edilebilir, ancak yalnızca tek bir suş başka türlerden gelen çapraz kirlenme ihtimalinin ortadan kaldırılması için plaka başına test edilmelidir. </li><li> 96 oyuklu plakanın H den satır B LB + NTBC ya da LB + DMSO ana çözeltisi 50 ul ekle. Bu bir tabak olun LB + NTBC ve bir tabak LB + DMSO olduğunu. Şekil 1A bakınız. <ol><li> LB + antibiyotiklerin NTBC LB + NTBC kullanın. LB + DMSO antibiyotikler için LB + DMSO kullanın. </li></ol></li><li> Bir mikropipet pipet ipuçları değiştirmek, B. karıştırın çözüm satır satır A solüsyonu 50 ul aktararak antibiyotik iki kat seri dilüsyonları gerçekleştirmek kullanarak ve satır B solüsyonu 50 ul transferi C. tekrarlayın satır remaining satırlar. Satır G sulandırarak sonra o satır ve artıklar gelen çözelti 50 ul çıkarın. Bakteriyel büyüme için hiçbir antibiyotik kontrol olarak satır H kullanın. Şekil 1B bakınız. NOT: G aracılığıyla her biri iyi satırlarda A şimdi 2X LB + NTBC veya LB + DMSO antibiyotiğin 50 ul nihai istenen konsantrasyon içerir. Satır H hiçbir antibiyotik LB + NTBC veya LB + DMSO içerir. </li><li> Gece boyunca kültürler OD 600 ölçün. Medyada mevcut pyomelanin ortadan kaldırmak için OD 600 okuma almadan önce tüm kültürleri yıkayın. <ol><li> 2 dakika boyunca 16.000 x g&#39;de bir mikrosantrfuj kültür 1 ml santrifüj edilerek kültürleri yıkayın. Bir mikropipetöre ile Süpernatantı ve 1 ml LB hücre pelletini </li></ol></li><li> LB içinde 2.75x10 5 CFU / ml gecede kültürleri sulandırınız NOT: Bir OD 600 adet P. 1x10 9 CFU / ml eşdeğer olduğunu varsayalım aeruginosa. CFU / ml dönüşümleri OD d edilebilirDiğer bakteri ifferent. </li><li> Uygun kuyuya seyreltilmiş bakteri kültürü 50 ul ekleyin. Not: NTBC yetiştirilen kültürler DMSO içinde yetiştirilen NTBC ve kültürler DMSO ihtiva eden oyuklara ilave edilmelidir içeren oyuklara ilave edilmelidir. Şekil 1B bakınız. <ol><li> Her bir suş ve antibiyotik konsantrasyonu için üç kuyu bakteriler ekleyin. Bakteriyel kontaminasyon için bir kontrol olarak hareket etmek dördüncü kuyuya LB 50 ul ekleyin. Şekil 1B bakınız. </li><li> Boşlukların aşılanması için çok kanallı bir micropipettor kullanın. Komşu kuyuların kirlenmesini önlemek için inokulum eklerken ipuçları kuyuların dibine yakın olduğu pipet olun. NOT: kuyulara bakteri kültürü eklemek antibiyotik ve NTBC yoğunluklanyla iki kat sulandırmak olur. </li></ol></li><li> Parafilm, 96 gözlü levhalar kapatılır ve 37 ° C&#39;de yaklaşık 24 saat inkübe edilir. Bir hava kuluçka makinesi içinde, statik 96 oyuklu inkübe edin. </li><li> Incelemekkuyularda bakteriyel büyüme için plakalar. MIC bakteriyel büyüme her suşun her üç kopya için görülebilir olduğu en düşük antibiyotik konsantrasyonu. <ol><li> Görme büyüme için plaka incelemek veya OD 600 plaka okuyucu seti kullanarak okuyun. </li></ol></li></ol> Oksidatif Stres Tepki için 4. Nokta Plaka Testi <ol><li> Aşama 3.1&#39;de tarif edildiği gibi suşların ayarlama gece boyunca kültürler NTBC LB ve olmaksızın test edilecek. </li><li> Sonraki gün, bir oksitleyici zor olarak H2O 2 ihtiva eden LB agar plakaları hazırlamak. 0 ila 1 mM, H bir dizi 2 O 2 konsantrasyonları için iyi bir başlangıç ​​noktasıdır. <ol><li> LB agar şişeler eritin. Oda sıcaklığında, yaklaşık 50 ° C&#39;ye soğutun ortam. </li><li> Arzu edilen konsantrasyonlarda soğutuldu ortam doğrudan O 2 H 2 ekleyin. Girdap şişeler karıştırın. H konsantrasyonları için Tablo 2&#39;ye bakınız 2 O 2ve konsantre H 2 O 2 hacimleri ekleyin. Bu değerler, LB agar 100 ml dayanmaktadır. </li><li> Hemen kabarcıklarını çıkarmak için H 2 O 2 ve alev yüzeyi ekledikten sonra plakaları dökün. Verim LB agar 100 ml başına 4 plakalarının. H 2 O 2 konsantrasyonu ile plakaları işaretleyin. </li><li> Plakalardan fazla nemi çıkarmak için 30 dakika boyunca çalışan fan ile biyolojik akış kaputu ele tabak yerleştirin. NOT: tabak onlar hazırlanır aynı gün kullanın. Bunu yapmamak tutarsız verileri neden olabilir. NOT: paraquat oksidatif stres, bu tahlilde, H2O 2 ikame edilebilir. Diğer oksidatif stres için kullanılan konsantrasyonlar, H2O 2 için kullanılanlardan farklı olabilir. </li></ol></li><li> Yıkayın ve adım 3.7&#39;de açıklandığı gibi OD gecede kültürlerin 600 ölçün. </li><li> Bütün gece boyunca cu OD 600 Normalesuşları seti için en düşük değere ltures denenmektedir. P. aeruginosa genel LB + NTBC ya da LB + DMSO içinde gece boyunca büyütülmüştür, yaklaşık 2.5 OD 600 yer alır. <ol><li> 1 ml &#39;lik bir toplam hacim içinde düşük OD 600 kültürün seyreltilmesi için ihtiyaç duyulan kültür hacminin belirlenmesi. Bir kültür OD 3 600 ve suşlar grubu için en düşük OD 600 yer alır, örneğin, 2.5 olan aşağıdaki hesaplamayı gerçekleştirmek: (2.5) (1 mi) = (3) (x). x = 0.833 mi. Kültür 0,833 ml kadar bir mikrofüj tüpüne yerleştirilir. </li><li> 1 ml kültür hacmi getirmek için gerekli LB + NTBC (300 uM) ya da LB + DMSO miktarını hesaplayın. Aşama 4.4.1 Örneğin, LB + NTBC ya da LB + DMSO miktarı 0,167 ml (- 0,833 ml kültür, 1 ml toplam hacim) olacak kültüre ilave edildi. LB + NTBC ve LB + DMSO stok çözeltileri, tüm suşları seyreltilmesi için gerekli hacme dayanarak bu dilüsyonları için kullandığınızdan emin olun. </li><li> Kültür ve LB + N MixVorteks TBC veya LB + DMSO. </li></ol></li><li> NTBC veya DMSO sabit bir konsantrasyonda kültürleri korumak için, PBS + NTBC veya PBS + DMSO normalize gece boyunca kültürler on kat seri dilüsyonları gerçekleştirmek. <ol><li> PBS + NTBC ve PBS + DMSO stok çözümleri olun. Bir suşu için seyreltileri bir set için, 720 ul bir toplam hacim elde etmek üzere 300 uM NTBC veya PBS ile eşdeğerli bir DMSO hacmi karıştırın. Test kaç suşları bağlı yukarı veya aşağı bu stokları Scale. </li><li> 10 -7 seri seyreltme ile 10 -1 Label mikrofuge&#39;de tüpleri. Uygun tüplere PBS + NTBC veya PBS + DMSO 90 ul ekle. LB + NTBC yetiştirilen suşları için PBS + NTBC kullanımı ve LB + DMSO içinde yetiştirilen suşları için PBS + DMSO kullanılır. </li><li> Uygun 10 -1 seyreltme tüp kültür 10 ul ekleyin. Vorteks karıştırın ve 10 -2 seyreltme tüpüne 10 -1 seyreltme 10 ul aktarın. Tüm seyreltmeler perfo olmuştur kadar tekrarlayınonaylanmamis. Seyreltilerde arasındaki pipet ipuçları değiştirin. </li></ol></li><li> Her bir suş için kopya halinde LB + H 2 O 2 plakaları üzerinde 10 -7 seyreltileri 10 -3 Spot 5 ul. Noktalar kaplanır, en az seyreltilmiş (10 -7 -3 10) sulandırmak itibaren bir pipet ucu kullanın. İpucu veya sıvı plakasına kuruyana kadar plaka eğmeyin. </li><li> Suşuna bağlı olarak 37 ° C&#39;nin (hava kuluçka makinesinde) de 24-48 saat süreyle inkübe edin. NOT: P. aeruginosa PAO1 inkübasyon 24 saat sonra LB iyi ölçekli koloniler olacaktır. Bunlar koloniler PAO1 ile yaklaşık olarak aynı boyuta sahip olana kadar suşları inkübe edin. </li><li> Bir transluminator üstünde bir CCD kamera kullanarak plakaları fotoğrafını. İsteğe bağlı olarak, aynı boyutta aksine ve ekme için fotoğraflarınızı düzenleyebilir. Oksidatif strese duyarlılık değişiklikleri belirlemek için her yerde koloni sayısını sayın. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+(4-Hydroxyphenyl)pyruvate+Dioxygenase+with+the+Specific+Inhibitor+2-[2-Nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.">Interaction of (4-Hydroxyphenyl)pyruvate Dioxygenase with the Specific Inhibitor 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione.</a> Biochemistry. 42, 10238-10245 (2003).</li><li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+minimum+inhibitory+concentrations.">Determination of minimum inhibitory concentrations.</a> J Antimicrob Chemother. 48, 5-16 (2001).</li><li>Nikodinovic-Runic, J., Martin, L. B., Babu, R., Blau, W., O'Connor, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+melanin-overproducing+transposon+mutants+of+Pseudomonas+putida+F6.">Characterization of melanin-overproducing transposon mutants of Pseudomonas putida F6.</a> FEMS Microbiol Lett. 298, 174-183 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Roy-Burman, A., 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=Type+III+protein+secretion+is+associated+with+death+in+lower+respiratory+and+systemic+Pseudomonas+aeruginosa+infections.">Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections.</a> J Infect Dis. 183, 1767-1774 (2001).</li><li>Jacobs, M. A., 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=Comprehensive+transposon+mutant+library+of+Pseudomonas+aeruginosa.">Comprehensive transposon mutant library of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci USA. 100, 14339-14344 (2003).</li><li>Youngchim, S., Pornsuwan, S., Nosanchuk, J. D., Dankai, W., Vanittanakom, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanogenesis+in+dermatophyte+species+in+vitro++during+infection.">Melanogenesis in dermatophyte species in vitro during infection.</a> Microbiology. 157, 2348-2356 (2011).</li><li>Khajo, A., 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=Protection+of+Melanized+Cryptococcus+neoformans+Lethal+Dose+Gamma+Irradiation+Involves+Changes+in+Melanin's+Chemical+Structure+and+Paramagnetism.">Protection of Melanized Cryptococcus neoformans Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism.</a> PLoS ONE. 6, e25092 (2011).</li><li>Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hancock Laboratory Methods. , (2015).</li></ol>

The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td>
			<td>Sigma-Aldrich</td>
			<td>SML0269-50mg</td>
			<td>Also called nitisinone.&#160; Soluble in DMSO.</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td>30 wt. % in H2O.&#160; Stabilized.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gold Bio</td>
			<td>G-400-100</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014-100MG</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
	</tbody>
</table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

NTBC titrasyonları NTBC P. pyomelanin üretimini azaltmak mümkün olsaydı NTBC titrasyonları belirlemek için kullanıldı aeruginosa ve aynı zamanda ortadan kaldırır ya da ek tahlillerinde kullanım için üretim pyomelanin azaltır NTBC konsantrasyonunu belirler. Farklı tekrarlamalı üretilen pyomelanin seviyelerindeki varyasyonlar olabilir, ama genel eğilimler sabit kalabilir. NTBC titrasyon testi, diğer bakterilerde pigment üretiminin etkileyebilecek diğer ...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Yöntemler Bakteriyel Pyomelanin Üretim ønhibe ve Oksidatif Stres Duyarlılık içinde tekabül eden artış belirleme

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

methods-to-inhibit-bacterial-pyomelanin-production-determine

Research

JoVE Journal

Immunology and Infection

8.1K Görüntüleme.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Video: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

İmmünoloji ve Enfeksiyon

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Kaplama Testi kullanarak niteliksel Pnömokok Bakteriyosinlerin Bakteriyel Üretim ve Hassasiyet Tedbir

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Yöntemler sıtma Tek Nükleotid Polimorfizm Genotipleme Erime Yüksek Çözünürlük Duyarlılığının Artırma

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro ve İn Vivo Modeli Gemi Duvar Altında Akış Koşulları Bakteriyel Yapışma Eğitim için

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Kan işleme için hızlı bir yöntem insan kanı plazma peptid düzeylerinin veriminin arttırılması için

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

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

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Metabolik eşleme: Nicel enzim Cytochemistry ve Histochemistry Dehydrogenases hücre ve dokulara etkinliğini belirlemek için

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...