The cell growth was

monitored by turbidimetry absorbance at 600 nm using the Elisa Espectra Max 190 (Molecular Devices). In order to determine the bactericidal or bacteriostatic action of Pg-AMP1, 20 μL of each treatment was re-inoculated in 1 mL of liquid TSB and incubated for 16 h at 37 °C under 100 rpm and the cell growth was measured by turbidimetry absorbance at 600 nm using the Elisa Espectra Max 190 (Molecular Devices). Hemolytic activity assays were performed as described Gefitinib research buy by Jang et al. [15]. Three mL of fresh human red blood cells (RBCs) was washed with 9 mL of sterile isotonic phosphate-buffered saline, pH 7.4 (PBS), until the color of the supernatant turned clear. The washed RBCs were then diluted to final volume of 20 mL with the PBS buffer and 10 μL of different solutions of Pg-AMP1 PBS diluted (200, 100 and 50 μg mL−1) was added to 190 μL of the cell suspension in 0.5 mL microfuge tubes. Following the gentle mixing, the tubes were incubated at 37 °C for 30 min and then centrifuged at 4000 × g for 5 min. One hundred microliter of supernatant was taken, diluted to 1 mL with PBS, and 100 μL were removed and placed in a microplate to be read in Varioskan (Thermo) under 567 nm absorbance and the released hemoglobin Cyclopamine indicated RBC membrane damage. Zero hemolysis and 100% hemolysis

consisted of RBC suspended in PBS and 0.2% Triton X-100, respectively. The percentage of hemolysis was determined as follows: Hemolysis %=As−A0A100−A0×100 As corresponds to the absorbance of the treatment, A100 corresponds

to the absorbance of completely lysed RBC in 0.2% Triton X-100, and A0 corresponds to the absorbance of zero hemolysis in PBS. The highest concentration of peptide that did not induce hemolysis was defined as the ‘minimum hemolytic concentration’ (MHC). Sequences of Pg-AMP1 and its recombinant form were submitted to Local Meta-Threading-Server (LOMETS) [43]. However, no significant templates were found. Therefore, Monte-Carlo simulations were performed by QUARK Ab initio server [45] in order to create an initial structure. Based on this initial structure, Modeller 9.9 [6] was used to generate 100 novel structures through loop-refinement sub-routine and structural information from Psi-Pred [13] and Protein DisOrder DOK2 prediction System (PrDOS) [25]. Ten models with minor discrete optimized protein energy (DOPE score) for each sequence were selected and analyzed on PROCHECK [20] and protein structure analysis (ProSA) [42]. Models were visualized on PyMOL (The PyMOL Molecular Graphics System, Version 1.4.1, Schrödinger, LLC). The expression of recombinant Pg-AMP1 peptide in BL21 (DE3) after purification yielded 2 mg L−1 and the highest expression level was obtained after 4 h induction with 0.5 mM IPTG (data not shown). The Pg-AMP1 was fused to a histidine tag producing a 6.983 kDa peptide that showed a predicted pI of 8.01(http://expasy.org/cgi-bin/pi_tool).