Use of an ABP to identify a DPAP1-selective substrate in parasite lysates.

<p><b>A.</b> Structure and reaction mechanism of the (Pro-Arg)₂-Rho substrate. <b>B.</b> Measurement of (Pro-Arg)₂-Rho apparent <i>K</i>_m in trophozoite lysates (circles) and with recombinant DPAP1 (triangle). Turnover rates at increasing concentrations of substrate were fitted to a Michaelis-Menten equation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011985#s4" target="_blank">methods</a> section. <b>C.</b> Labeling of DPAP1 activity in parasite lysates with FY01. Trophozoite lysates were incubated for 1 h with increasing concentrations of FY01. Labeling was stopped by boiling the sample in SDS-PAGE loading buffer. DPAP1 activity was measured using a flatbed fluorescent scanner. <b>D.</b> DPAP1 labeling correlates with substrate turnover inhibition. An aliquot of the samples treated for 1 h with FY01 was diluted in assay buffer containing 10 µM of (Pro-Arg)₂-Rho, and the initial turnover rate was measured in a 96-well plate (circles). This turnover rate is plotted with the labeling quantified in C.</p

Glutathione-Triggered Formation of a Fmoc-Protected Short Peptide-Based Supramolecular Hydrogel

<div><p>A biocompatible method of glutathione (GSH) catalyzed disulfide bond reduction was used to form Fmoc-short peptide-based supramolecular hydrogels. The hydrogels could form in both buffer solution and cell culture medium containing 10% of Fetal Bovine Serum (FBS) within minutes. The hydrogel was characterized by rheology, transmission electron microscopy, and fluorescence emission spectra. Their potential in three dimensional (3D) cell culture was evaluated and the results indicated that the gel with a low concentration of the peptide (0.1 wt%) was suitable for 3D cell culture of 3T3 cells. This study provides an alternative candidate of supramolecular hydrogel for 3D cell culture and cell delivery.</p></div

Cat C-specific fluorogenic assay in rat liver lysates.

<p><b>A.</b> Labeling of Cat C with FY01. Rat liver extract extracts were treated with increasing concentrations of FY01 for 1 h and labeled proteins analyzed by SDS-PAGE followed by scanning of the gel using a flatbed laser scanner. The location of labeled Cat C is indicated. <b>B.</b> Inhibition of substrate turnover specifically correlates with Cat C labeling. The cleavage of (Pro-Arg)₂-Rho substrate was measured prior to analysis of FY01 labeling shown in part A. Quantification of the indicated labeled proteins relative to DMSO control is shown. <b>C.</b> Cat C-specific HTS assay in rat liver extracts. Rat liver lysates were treated for 30 min with either DMSO or JCP410 (10 µM) followed by the addition of 10 µM of (Pro-Arg)₂-Rho. The turnover rate was continuously measured for 5 min in a 384-well plate. Z’ factor, S/N, and % CV of the negative control are shown.</p

Development of a DPAP1-specific HTS assay.

<p><b>A.</b> Continuous assay. The assay was carried out in 384-well plates using 1% of parasite lysates. Substrate turnover was continuously measured for 5 min. JCP410 (10 µM) was used as a positive inhibition control. Z’ factor, S/N, and % CV of the negative control are shown. <b>B.</b> End-point assay for HTS. The reaction described in A was quenched after 10 min by addition of 0.5 M acetic acid. The final concentration of rhodamine product was quantified by fluorescence.</p

Emission spectra of PBS solution of the pro-gelator and the gel (excitation wavelength = 265 nm).

<p>Emission spectra of PBS solution of the pro-gelator and the gel (excitation wavelength  = 265 nm).</p

Single Dose of Protein Vaccine with Peptide Nanofibers As Adjuvants Elicits Long-Lasting Antibody Titer

Self-assembling materials based on peptides have shown great potential as vaccine adjuvants. In our previous work, we have demonstrated that nanofibers based on D-peptide Nap-G^DF^DF^DY are good candidates for vaccine adjuvants. Here we further found that supramolecular hydrogels based on positively charged D-peptide Nap-G^DF^DF^DY^DK as vaccine adjuvants could induce stronger immune response. We designed and synthesized two D-peptide derivatives, one with a positive charge (Nap-G^DF^DF^DY^DK) and the other with a negative charge (Nap-G^DF^DF^DY^DE). Both of them could form the hydrogels constructed by nanofibers. The nanofibers formed by Nap-G^DF^DF^DY^DK promoted the more powerful immune response in mice against the antigen chicken egg albumin (OVA) than peptides Nap-G^DF^DF^DY and Nap-G^DF^DF^DY^DE. Through cell experiments, we demonstrated that the main reason was that nanofibers formed by Nap-G^DF^DF^DY^DK could enhance the uptake of OVA by primary antigen presenting cells. Most importantly, it was intriguing that the nanofibers based on Nap-G^DF^DF^DY^DK could evoke long-lasting antibody titers for 28 weeks at a single dose of protein vaccine. Our study demonstrated that supramolecular hydrogels based on positively charged D-peptide were promising vaccine adjuvants and might be very useful for antibody production and vaccine development

A cryo-transmission electron microscopy (cryo-TEM) image of the gel from a PBS solution containing 0.3 wt% of the pro-gelator with 4 equiv. of GSH.

<p>A cryo-transmission electron microscopy (cryo-TEM) image of the gel from a PBS solution containing 0.3 wt% of the pro-gelator with 4 equiv. of GSH.</p

Optical images of cells at day 5 in gels at A) 0.3 wt%, B) 0.2 wt%, and C) 0.1 wt% (scale bars represent 10 µm).

<p>Optical images of cells at day 5 in gels at A) 0.3 wt%, B) 0.2 wt%, and C) 0.1 wt% (scale bars represent 10 µm).</p

A representative rheological measurement with the mode of dynamic sweep at the frequency of 1 rad/s and the strain of 1% for a PBS solution containing 0.3 wt% of the pro-gelator and 4 equiv. of GSH (A) and rheological measurements with the mode of dynamic frequency sweep at the strain of 1% for gels from PBS solutions containing different concentrations of the pro-gelator (B) (filled symbols: G′ and open symbols: G″, diamonds: 0.3 wt%, triangles: 0.2 wt%, and squares: 0.1 wt%).

<p>A representative rheological measurement with the mode of dynamic sweep at the frequency of 1 rad/s and the strain of 1% for a PBS solution containing 0.3 wt% of the pro-gelator and 4 equiv. of GSH (A) and rheological measurements with the mode of dynamic frequency sweep at the strain of 1% for gels from PBS solutions containing different concentrations of the pro-gelator (B) (filled symbols: G′ and open symbols: G″, diamonds: 0.3 wt%, triangles: 0.2 wt%, and squares: 0.1 wt%).</p

Chemical structure of the pro-gelator (Fmoc-FFE-ss-EE) and the resulting gelator (Fmoc-FFE-s) and an optical image of a gel formed by treating a DMEM solution containing 0.3 wt% of the pro-gelator with 4 equiv. of GSH.

<p>Chemical structure of the pro-gelator (Fmoc-FFE-ss-EE) and the resulting gelator (Fmoc-FFE-s) and an optical image of a gel formed by treating a DMEM solution containing 0.3 wt% of the pro-gelator with 4 equiv. of GSH.</p