Systemic and mucosal immune responses induced by mucosal immunization of AFCo2 or PLc in BALB/c mice.

<p>AFCo2 or PLc (100 µg) was administered by i.n or i.g route in BALB/c mice. Results of anti-PLc IgA in saliva and faeces and anti-PLc IgG in sera are expressed as the logarithm of the mean ± SEM. The ratio of IgG subclasses IgG2a/IgG1 is also reported. Vibriocidal activity of sera from immunized mice is also expressed as logarithm of the mean ± SEM. Statistical significance of the variance between multiple groups of experiments were calculated with one-way ANOVA, followed by a Tukey's multiple comparison test.</p>a,b,c<p>Different letters on the same column mean that values are significantly different (p<0.05); - no response detected.</p

Scanning electron micrographs of AFCo2.

<p>(A) Tubular and multilamellar structures observed at ×1400 [multilamellarity is indicated by white arrows], (B) the open end of the structure magnified ×3300.</p

Systemic and mucosal anti-OVA IgA and IgG response induced by intranasal immunization of AFCo2+ OVA or PLc+OVA in BALB/c mice.

<p>AFCo2 or PLc (100 µg) was admixed with 20 µg of OVA and administered by i.n route to BALB/c mice. Results are expressed as the logarithm of the mean ± SEM. Statistical significance of the variance between multiple groups of experiments were calculated with one-way ANOVA, followed by a Tukey's multiple comparison test.</p>a,b,c<p>Different letters on the same column mean that values are significantly different (p<0.05); - no response detected.</p

Labeling AFCo2 and PLc with OVA-TR.

<p>(A) AFCo2 admixed with OVA-TR. The images were obtained with a coupled-device camera linked to an epi-fluorescence microscope. After sonication and ultrafiltration AFCo2+OVA-TR was analyzed by light microscopy as shown in (B). The broken structure is a consequence of the sonication process. (C) The same AFCo2 structure observed by epi-fluorescence showing the distribution of the Ova-TR in AFCo2. (D) PLc+OVA-TR observed by epi-fluorescence showing a lack of signal.</p

AFCo2 unfolding with EDTA.

<p>The micrographs represent stills of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046461#pone.0046461.s001" target="_blank">Video S1</a> (supporting information) that shows the unfolding process of AFCo2 under the action of EDTA chelating agent (25 mM). AFCo2 starts to shrinks in the first 15 seconds and completely disappears at 25 seconds of exposure with EDTA.</p

AFCo2 formation.

<p>(A) White milky suspension (AFCo2) obtained after calcium interaction with a solution of PLc, (B) AFCo2 observed by light microscopy with an Opton Standard 25 microscope (magnification ×400), (C) distribution percentage (82.6%) of AFCo2 with a length of 16.3±4.6 µm measured using a graduated scale on the ocular lens of the microscope.</p

Freeze fracture electron micrographs of AFCo2.

<p>(A) Cylindrical shape of AFCo2 and black arrows point to the multiple layers forming the structure, (B) Overlapping layers can be observed with a curvature and disposition similar to the classic spiral pattern characteristic of cochleate structures.</p

AFCo2 unfolding in the presence of EDTA.

<p>AFCo2 was resuspended at a final protein concentration of 0.05 mg/mL with buffer containing Tris 30 mM and EDTA ranging from 1 to 50 mM. The results show the number of particles observed in 64 fields in a Neubauer chamber using light microscopy. EDTA concentrations from 25 to 50 mM were suitable for completely unfolding the AFCo2.</p><p>+++ more than 100 particles observed per view, ++ 40–80 particles, −/+ 10–40 particles, - no particles.</p

Activity of samples P1-P12 against <i>P</i>.<i>falciparum</i> (n = 3).

<p>Activity of samples P1-P12 against <i>P</i>.<i>falciparum</i> (n = 3).</p

OPLS plot of observed against predicted activity against <i>P</i>. <i>falciparum</i> based on five compounds (A-E).

<p>OPLS plot of observed against predicted activity against <i>P</i>. <i>falciparum</i> based on five compounds (A-E).</p