We have demonstrated that these peptides exert broad-spectrum activity against both gram-positive and gram-negative bacteria, and thus could be useful in the treatment of patients with polymicrobial wounds infections [6, 7]. Methods 5.1 Bacterial strains and media S.

aureus (ATCC 25923, American Type Culture Collection, Manassas, VA) was grown in Nutrient Broth (Difco Laboratories, Detroit, Mich.) at pH 7, 37°C, 24 h with shaking at 200 rpm. The overnight culture was frozen with 20% glycerol and stored find more at -80°C. The frozen stock was enumerated (CFU/ml) by dilution plating and growth on Nutrient Agar plates. 5.2 Peptides and Anti-microbial assays The sequences and net charges of the peptides are shown in Table 1. The molecular weight reported here for each peptide reflects the trifluoroacetic acid (TFA) salt form of the peptides. NA-CATH, NA-CATH:ATRA1-ATRA1, ATRA-1, ATRA-1A, ATRA-2 peptides (86.1 and 89.7, 97.2, 94.5, and 88.2%, respectively) (Genscript, Piscataway, NJ), LL-37 (95% purity) (AnaSpec 61302) and D-LL-37 (92.0% purity) (Lifetein, South Plainfield, NJ) were synthesized commercially. The anti-microbial activity of the NA-CATH and NA-CATH:ATRA1-ATRA1, the variations

on the ATRA peptides LL-37 and D-LL-37 against S. aureus were determined as previously described, with some modification [26, 29]. For anti-microbial assays, frozen enumerated aliquots were thawed and gently mixed immediately before use. In a 96-well plate (BD Falcon 353072), 1 × 105 CFU per well bacteria were incubated with different peptide concentrations (in serial dilutions of 1:10 across the plate) in a solution of buffer containing H 89 in vitro sterile 10 mM sodium phosphate (pH 7.4) and incubated (3 h, 37°C). Negative control wells contained bacteria with no peptide. Serial dilutions were then carried out in sterile 1x PBS (Fisher Scientific) (pH 7) and plated in triplicate on Nutrient Agar plates, incubated (37°C, 24 h) and counted. Bacterial survival at each peptide concentration was calculated as previously described [25, 26] based on the

mafosfamide percentage of colonies in each experimental plate relative to the average number of colonies observed for assay cultures lacking peptide. The EC50 was calculated as previously described [26, 47]. Each experiment was repeated at least twice, and a representative experiment is shown, for clarity. Errors were reported based on the standard deviation from the mean of the log10 EC50 values [19]. 95% confidence intervals were used to determine whether points were statistically different at p = 0.05. 5.3 CD Spectroscopy Circular dichroism (CD) spectra of the peptides were collected using Jasco J-815 spectropolarimeter. Samples were allowed to equilibrate (10 min, 25°C) prior to data collection in a 0.1 cm path length cuvette, with a chamber temperature 25°C throughout each scan. Spectra were collected from 190 to 260 nm using 0.