Effect of CNS myelin on neurite outgrowth of cortical neurons cultured for 4 days on PLL-P(TMC-CL) substrates coated with CNS myelin.

<p><b>A.</b> Cortical neurons are immunostained for β-III tubulin (green) and nuclei are counterstainned with Hoechst (blue); myelin coating is immunostainned for MBP (green), surfaces were fully covered by myelin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088593#pone.0088593.s002" target="_blank">fig. S2</a> for myelin quantification) <b>B.</b> Effect of myelin on the ability of neurons to extend processes is presented as the % of cells with neurites in relation to the total number of cells. (n = 3 independent studies, mean ± SD; ** for p<0.01).</p

Effect of the PLL coated surfaces on neurite elongation and cellular polarization.

<p><b>A</b>. Fluorescently labeled cortical neurons, immunostained for TAU (green); nuclei are counterstainned with Hoechst (blue); <b>B.</b> Number of primary neurites per cell; <b>C.</b> Total neurite length; <b>D.</b> Average neurite length and <b>E.</b> Length of the longest neurite. (n = 130 cells, mean ± SD, *** for p<0.001).</p

Cortical neuron culture on PLL coated films of P(TMC-CL) and respective homopolymers (2.7×10⁴ viable cells were seeded per sample).

<p><b>A.</b> Number of cortical neurons with and without neurite extensions on polymeric surfaces coated with aqueous solutions at different concentrations of PLL. Glass coated with 24 µg.µl⁻¹ of PLL for 30 minutes was used as control. (n = 3 independent studies, mean ± SD, p<0.05) <b>B.</b> Percentage of PLL covered surface area as a function of the coating conditions. (n = 3, mean ± SD, p<0.05). x = condition not tested, 0 = null value. n.s. = non-significantly different from the control, α = total number of cells not significantly different from the control, β = number of cells with neurite extensions not significantly different from the control and χ = number of cells without extensions not significantly different from the control.</p

Analysis of GSK3β in cortical neurons plated on P(TMC-CL) and effects of GSK3β inhibition on neurite extension.

<p><b>A.</b> Schematic representation of the different phosphorylation forms of GSK3β and their activity status; <b>B.</b> Analysis of the phosphorylated forms of GSK3β by western blot. Representative blots are shown. Expression levels of GSK3β isoforms, β1 and β2, are presented and quantified individually or together. (n = 3 independent studies, average ± SD); <b>C.</b> Morphology of neurons (immunostained for TAU in green and nuclei counterstained in blue) cultured for 24 hours in the presence of DMSO (control) or in the presence of 6-bromoindirubin-3′-acetoxime (BIO) at 30 and 300 nM. Quantifications of the longest neurite, average neurite length and the number of neurites per cell are shown (n = 130 cells, mean ± SD, * for p<0.05, ** for p<0.01 and *** for p<0.001); <b>D.</b> Determination of CRMP4 phosphorylation levels in cortical neurons plated for 4 days on control or P(TMC-CL). Representative western blot is shown and below the quantification (n = 3 independent studies, average ± SD).</p

Characteristics of the synthesized and purified P(TMC-CL) (co)polymers.

a)<p>Determined by 1H NMR on specimens purified by precipitation;</p>b)<p>Determined by GPC at 30°C using chloroform as the eluent.</p

Morphology and mechanical properties of the tested polymeric surfaces.

<p><b>A</b>. Root mean square (RMS) roughness of all polymeric surfaces; <b>B</b>. Representative photographs of the polymeric surfaces before and after nanoindentation; images are color coded, showing elevated areas in bright and lower areas in dark color. <b>C</b>. Representative nanoindentation force-displacement curves; D. Mean hardness values of all polymeric surfaces, calculated for the maximum load and E. Mean stiffness values for all polymeric surfaces. (n = 60 indentations, mean ± SD, *** for p<0.001).</p

Protective effect and liver granuloma size induced by C57BL/6 mice vaccination and challenged with 25 <i>S. mansoni</i> cercariae.

*<p> <b>Statistically significant compared with the control group (p<0.05).</b></p

Release profile of protein SmRho from alginate-coated chitosan nanoparticles in SGF (A) and SIF (B) at 37°C (mean ± SD, n = 3).

<p>Release profile of protein SmRho from alginate-coated chitosan nanoparticles in SGF (A) and SIF (B) at 37°C (mean ± SD, n = 3).</p

Cytokine profiles of mice immunized with coated SmRho-chitosan nanoparticles.

<p>Splenocytes isolated from mice immunized with CH-Rho-Alg, CH-Rho-Cpg-Alg, and CH-Rho-Alg (i.m.) were assayed for IL-10 (A) and INF-γ (B) production in response to <i>in vitro</i> stimulation with SWAP (25 µg/ml), rSmRho (25 µg/ml), SEA (25 µg/ml) or medium alone as control. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001894#s3" target="_blank">Results</a> represent the mean ± SD of each group. *Statistically significant differences between cytokines produced after SWAP, rSmRho or SEA stimulation compared with unstimulated splenocytes (control) (p<0.05).</p

Reactivity of sera from control and <i>S. mansoni</i> infected patient, drug treated or untreated, against SmRho and SWAP observed by ELISA^# (A) and against SmRho, SWAP, SEA and schistosomula extract by Western Blotting (B).

<p>M: Standard molecular weight Page Ruler Unstained Protein Ladder (Fermentas), 1: SWAP; 2: SEA, 3: schistosomula antigens; 4: recombinant protein Rho1-GTPase de <i>S. mansoni</i>. ^#The results are presented as the mean absorbance measured at 450 nm. Statistically significant differences of sera from infected patient with the control group, non infected patient, in each sera dilution evaluated, are indicated by (*) for p<0.05. The molecular weight markers, from top to bottom, are: 116, 66, 45, 35, 25, and 18 kDa.</p