A Theorem on Matroid Homomorphism

This note generalizes a result contained in a previous paper [ J. Sanders, Circuit preserving edge maps II, J. Combin. Theory Ser. B 42 (1987), 146-155].Comment: 5 pages, 0 figure

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

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

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

Optimization of a Protease Activated Probe for Optical Surgical Navigation

Molecularly targeted optical contrast agents have the potential to enable surgeons to visualize specific molecular markers that can help improve surgical precision and thus outcomes. Fluorescently quenched substrates can be used to highlight tumor lesions by targeting proteases that are highly abundant in the tumor microenvironment. However, the majority of these and other molecularly targeted optical contrast agents are labeled with reporter dyes that are not ideally matched to the properties of clinical camera systems, which are typically optimized for detection of indocyanine-green (ICG). While a wide range of near-infrared (NIR) dyes are suitable for use with highly sensitive and highly tunable research-focused small animal imaging systems, most have not been evaluated for use with commonly used clinical imaging systems. Here we report the optimization of a small molecule fluorescently quenched protease substrate probe 6QC-ICG, which uses the indocyanine green (ICG) dye as its optical reporter. We evaluated dosing and kinetic parameters of this molecule in tumor-bearing mice and observed optimal tumor over background signals in as little as 90 min with a dose of 2.3 mg/kg. Importantly, the fluorescence intensity of the probe signal in tumors did not linearly scale with dose, suggesting the importance of detailed dosing studies. Furthermore, when imaged using the FDA approved <i>da Vinci</i> Si surgical system with Firefly detection, signals were significantly higher for the ICG probe compared to a corresponding probe containing a dye with similar quantum yield but with a slightly shifted excitation and emission profile. The increased signal intensity generated by the optimal dye and dose of the ICG labeled probe enabled detection of small, flat lesions that were less than 5 mm in diameter. Therefore, 6QC-ICG is a highly sensitive probe that performs optimally with clinical imaging systems and has great potential for applications in optical surgical navigation

Stage-Specific Expression of Falstatin

<p>Extracts from highly synchronized parasites were collected every 8 h, separated by SDS-PAGE, and evaluated by immunoblotting with anti-falstatin antibodies. Each sample of early-ring, late-ring, early-trophozoite, late-trophozite, early-schizont, or late-schizont extracts corresponded to 1.3 × 10⁷ parasitized cells. The positions of molecular weight markers (kDa) are indicated. ER, early-ring; LR, late-ring; ET, early-trophozite; LT, late-trophozite; ES, early-schizont; LS, late-schizont.</p

Inhibition of Falstatin Function by Anti-Falstatin Antibodies

<p>Hydrolysis of the peptide substrate Z-Leu-Arg-AMC by falcipain-2 (FP2; 19.8 nM), falcipain-3 (FP3; 27.1 nM), or trophozoite extract (TE; corresponding to 5.5 × 10⁶ parasites per reaction) was evaluated in the absence or presence of falstatin (31 nM) and the indicated quantities of anti-falstatin antibodies in 350 μl of 100 mM sodium acetate, 8 mM DTT (pH 6.0). Reaction components were incubated for 15 min before addition of substrate, and activity was measured as arbitrary fluorescence units over time (FU/min). Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p

Inhibitor Competition

<p>The indicated amounts of falstatin and anti-falstatin antibody were incubated with lysates from asynchronous parasite cultures before addition of [¹²⁵I] DCG04, electrophoresis, and analysis by autoradiography. Results with increasing concentrations of falstatin (A), increasing concentrations of antibody (B), and increasing falstatin in the presence of antibody (C) are shown. Labels above the gels represent concentrations of falstatin and antibody (μg/ml). Proteins are labeled based on known migration patterns that were previously confirmed by mass spectrometry. FP, falcipain; DPAP1, dipeptidyl aminopeptidase1[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020117#ppat-0020117-b028" target="_blank">28</a>].</p

Activity of Falstatin against Different Classes of Proteases

<p>Equal amounts (4 μg) of proteases (FP2, falcipain-2; FP3, falcipain-3; trypsin; α-chymo, α-chymotrypsin; pepsin; renin; collagenase; MM-2, matrix-metalloprotease-2) were mixed with 350 μl of appropriate buffers containing falstatin (1.5 μg) for 15 min, FITC-casein (20 μg) was added, and hydrolysis of the substrate with and without falstatin was compared for each protease. Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p

Immunolocalization of Falstatin

<div><p>(A) Immunofluorescence microscopy. Erythrocytes infected with synchronized 3D7 or W2 parasites were collected every 8 h, stained with DAPI and anti-falstatin antibodies and FITC-second antibody, and then evaluated by immunofluorescence microscopy.</p><p>(B) Immunoelectron microscopy. Late-schizont stage parasites were incubated with anti-falstatin antibodies and gold-conjugated second antibody and then evaluated by electron microscopy. Labels show individual merozoites (M) and erythrocyte cytosol (EC).</p></div