Fungal selleck growth after treatment with hydrogen 4EGI-1 peroxide H2O2 (Merck, USA) was added directly to control and TC-treated cultures to final concentrations of 0.005,

0.05 and 0.5 M. Conidia (2 × 103 cells/ml) were incubated in RPMI-1640, for 1 h at 37°C in the presence of the hydrogen peroxide concentrations mentioned above. From each sample, 50 μl were placed in wells of a 24-well plate with 500 μl of CD with 3% agar. The cultures were incubated at 25°C for 10 days. Fungal growth was measured by calculating the relative size of the colonies per well for each condition. Images of the bottom of the plates were digitalised and processed using ImageJ software [40] for the following parameters: (I) gamma correction to ensure adequate brightness and contrast of the image; (II) a threshold to define the interface between the fungal growth (black) and the background (white); and for (III) the inversion to define the background as black (grayscale value = 0) and the area of fungal growth as white (grayscale value = 255). Selleckchem Dinaciclib A constant area with the diameter of a well from a 24-well plate was the template for the measurements of the “”Mean Gray Value”" on the Image J software. Measurements were the sum of the gray values of all pixels in the selection divided by the number of pixels, revealing the area of fungal growth. In this work the values were expressed as the normalised percentage relative to its control

(100% of growth). Fungal growth after incubation with a nitric oxide donor SNAP, a nitric oxide donor, was dissolved in DMSO and added to untreated and TC-treated cultures of conidia (2 × 103 cells/ml) in RPMI-1640 at concentrations of 0.1, 0.3 and 1.0 mM. These cultures were incubated for 24 h at 37°C. From each

condition, 50 μl were plated in one well of a 24-well plate with 500 μl of CD (solid, with 3% agar). Samples were incubated at 25°C for 10 days. The growth area was measured and using the procedure described above. Statistical analysis Graphic and statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, USA). The Student’s t-test was used for experiments with one variable, and results were considered significant if P < 0.0001. ANOVA tests were used for comparing samples in experiments with 4��8C more than one variable; the results were considered significant when P < 0.05. Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). References 1. Lopez Martinez R, Mendez Tovar LJ: Chromoblastomycosis. Clin Dermatol 2007, 25:188–194.PubMedCrossRef 2. Silva JP, de Souza W, Rozental S: Chromoblastomycosis: a retrospective study of 325 cases on Amazonic Region (Brazil). Mycopathologia 1998, 143:171–175.PubMedCrossRef 3. Salgado CG, da Silva JP, da Silva MB, da Costa PF, Salgado UI: Cutaneous diffuse chromoblastomycosis. Lancet Infect Dis 2005, 5:528.