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><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.&amp;nbsp; Soluble in DMSO.</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>216763-100ML</td><td>30 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Stabilized.</td></tr><tr><td>Gentamicin</td><td>Gold Bio</td><td>G-400-100</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; 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

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

Genetik olarak kodlanmış Fluorogenic sensörleri kullanarak toksikolojik oksidatif stres ilişkin Değerlendirmeler yaklaşımlar görüntüleme

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Kanser Araştırmaları

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

Miktar hipopigmentasyon aktivitesinin in Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Biyokimya

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Bir Insanlaşmış Fare Modeli epidermal melanin ve Karşı Koruma yanığı Farmakolojik İndüksiyon

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Tıp

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Mühendislik

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Riboflavin-5′- Fosfatın Görünür Işık Fotolizi ile Patojenlerin İnaktivasyonu

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Biyomühendislik

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Uyuyan Güzel Transposon Transposon Transfected İnsan Retina Pigment Epitel Hücrelerinde Oksidatif Stresin İndüksiyonu ve Analizi

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Fare Derisinde Kök Hücre Nişlerinin Uyarılması ve Doku Rejenerasyonu, Değiştirilebilir Protoporfirin IX'a Bağlı Reaktif Oksijen Türlerinin Fotojenerasyonu ile Yerinde

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

Biyoloji

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 Yetişkin Cilt Dokularından İnsan Melanositlerin Verimini Zenginleştirir

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www.jove.com/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Minimum Eritematöz Doz Deneyi: Fare Modellerinde UV Duyarlılığını Ölçmek İçin Bir Yöntem

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Cilt kanseri

Hayvan modeli çalışmaları

A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

Source: Ketelboeter, L. M. et al., <a href="https://app.jove.com/v/53105">Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress.</a> J. Vis. Exp. (2015)
This video demonstrates a spot plate assay tailored to assess the oxidative stress responses of bacterial strains. Some strains produce pyomelanin, an antioxidant pigment. Conversely, strains treated with a test agent, leading to reduced pyomelanin production, exhibit increased susceptibility to oxidative stress, resulting in decreased cell viability compared to pyomelanin-producing strains.

Bakteri Suşlarında Oksidatif Stres Tepkilerini Değerlendirmek için Bir Spot Plaka Testi

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)

	Make Luria-Bertani (LB) broth (1% ...

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

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