Ce of the defensins, we observed that the mutant strain grows approximately 84 less than the wild-type C. albicans (Figure 4). In the presence of PvD1 at 100 g.mL-1, we observed that the mutant strain grew 8.3 and 1.1 more than the control at 6 h and 18 h, respectively. This tendency, observed in the initial hours, increased to 94.4 of additional growth at 24 h with regards to the control (Figure 4B). This result is further analyzed in the Discussion Section. In the presence of PvD1r at the same concentration, we observed a good superposition of the growth curves during the time course assayed (Figure 4D). This result indicates that PvD1r did not present growth inhibition on the C. albicans mutant strain. We further analyzed, through optical microscopy, possible alterations to the morphology of C. albicans cells at the end of the growth inhibition assay for both defensins. First, the wild-type and mutant strain presented contrasting growth patterns. The wild type strain presented single ovoid cells as well as 2-Bromo-1,3-difluoro-4-nitrobenzene long pseudohyphae (Figure 5A). The mutant strains presented aggregated cells with short pseudohyphae and occasionally sparse cells (Figure 5D). With regard to the effect of the defensins on these cells, we noted that in the presence of 100 g.mL-1 of PvD1, the wild-type cells were agglomerated and reduced in number and also presented reductions in the size of the hyphae (Figure 5B). The mutant cells, when treated with PvD1, showed very similar morphology and growth patterns compared to the control mutant cells (Figure 5D,E). Afterde O Mello et al. BMC Biochemistry 2014, 15:7 http://www.biomedcentral.com/1471-2091/15/Page 8 ofA0,B0,0,7 0,6 0,5 0,0,7 0,0,5 0,4 0,3 0,2 0,1 0 0 6 12 18Absorbance, 620 nm0,0,2 0,0 6 12 18Time (h)Time (h)C0,8 0,D0,8 0,7 0,6 0,5 0,Absorbance, 620 nm0,0,5 0,4 0,3 0,0,3 0,2 0,10,0 0 Fmoc-Oic-OH 6 12 18Time (h)Time (h)Figure 4 Effects of PvD1 and PvD1r on the growth of the wild-type and mutant strains of Candida albicans. The absorbance at 62PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8627573 ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 g.mL-1 of PvD1; (C) C. albicans wild-type strain; (D) C. albicans mutant strain; () Control; () 100 g.mL-1 of PvD1r. All of the experiments were performed in triplicate, and the standard errors were omitted for clarity (coefficients of variation were less than 20 ).ABCDEFGFigure 5 Candida albicans cells, wild-type and mutant strains, visualized by light microscopy after the growth inhibition assay. (A) Wild-type control cells; (B) Wild-type cells grown in the presence of PvD1; (C) Wild-type cells grown in the presence of PvD1r; (D) Mutant control cells; (E) Mutant cells grown in the presence of PvD1; (F and G) Mutant cells grown in the presence of PvD1r. Bars = 10 m.de O Mello et al. BMC Biochemistry 2014, 15:7 http://www.biomedcentral.com/1471-2091/15/Page 9 oftreatment with 100 g.mL-1 of PvD1r, wild-type C. albicans cells presented marked reductions in cell number and hyphal size as well as agglomerated cells (Figure 5C). In comparison, the effect of PvD1 and PvD1r on wild-type cells was visually the same. The mutant cells treated with PvD1r presented a very similar morphology and growth pattern when compared to the control mutant cells and the mutant cells treated with PvD1 (Figure 5D,F-G).Optical microscopy with the fluorescent dyes FITC and DAPITo determine the localization of PvD1r in the C. albicans wild-type and mutant strains, 50 g.mL-1 of.