Protocol II was used only for P. brasiliensis, in which the incubation time was 12 h and the methodology was adapted

from Travassos and collaborators [42]. The peptides P1, P2, P3, and P4 were serially diluted from 16 to 500 μg ml−1 in phosphate buffer saline (PBS, pH 7.2). A 2-fold dilution series (100 μl) were added to 100 μl of 2 × 104 viable cells of the P. brasiliensis in 500 μl plastic tubes. The tubes were incubated at 36 °C in rotatory shaker (100 rpm) during 12 h. After this period, 100 μl of each tube were plated in solid medium Brain Heart Infusion (BHI, Acumedia®, USA) supplemented with 4% (v/v) horse serum (Gibco, USA), 5% (v/v) supernatant of the culture filtrate of the isolate Pb192 and 40 mg l−1 gentamycin (Schering-Plow, USA). The filtrate was prepared according to methodology described previously [42]. The growth of colony-forming units was observed for INCB024360 clinical trial 21 days. The lowest concentration of peptide that completely inhibited growth of the fungi was defined as the minimal inhibitory concentration. The MICs were calculated by the average Osimertinib values obtained in triplicates on

three independent measurements [36]. The experimental controls used in both protocols were amphotericin B (Sigma–Aldrich, USA) and for protocol II the killer peptide (KP) as control was also used. The antibacterial activity was evaluated against human pathogenic bacteria Escherichia coli ATCC8739 and Staphylococcus aureus ATCC25923, both obtained from the American Type Culture Collection (ATCC). Briefly, the bacterial cultures were grown in Lysogeny Broth (LB) medium, pH 7.0, at 37 °C until they reached the exponential phase. The method used to study the antibacterial activity of the peptides was based on the broth microdilution assay. The culture for the assay was prepared by diluting 1:11 the bacteria obtained on the exponential phase. The peptides P1, P2, P3, and P4 were serially diluted from 2 to 256 μg ml−1 in LB medium. A 2-fold dilution series (100 μl) were added to 10 μl of approximately 5 × 106 CFU of bacteria

in each well of a 96-well polypropylene plate. The plates were incubated for 4 h at 37 °C and the peptides antibacterial activities were Adenosine observed in every 30 min by measuring the absorbance in a plate reader (Bio-Rad 680 Microplate Reader) at 595 nm. The controls utilized were distilled water and chloramphenicol 60 μg ml−1. Primaries sequences were obtained from initial selection previously described in this section. All of them being of synthetic peptide amidate. PSI-BLAST was used for templates data mining [48]. For P1 and P2 peptides models, it was possible to obtain templates by homology method (pdb: 2jx6 and 1id3), showing 55 and 88% of identity respectively [45] and [48]. Fifty models for each peptide were constructed by using Modeller v9.8.