Apical localization of PfRH5 in the merozoite.

<p>A Mature Dd2 schizonts were labeled with rabbit anti-PfRH5 IgG and counterstained with FITC-labeled anti-rabbit IgG (green) using immunofluorescence microscopy. B Colocalization studies: Mature Dd2 schizonts were double-labeled with anti-PfRH5 IgG (red) and anti-RhopH3 (green) monoclonal antibody. The merged staining (yellow) by both antibodies indicates their similar location within the parasite. C Immunoelectron microscopy confirming the localization of PfRH5 in the rhoptries within the developing merozoites in the Dd2 schizont. Parasites were fixed with 1% paraformaldehyde and 0.1% glutaraldehyde. Staining was detected with rabbit anti-RH5 and anti-rabbit gold (10 nm). Scale bar represents 500 nm.</p

PfRH5 binds to a ∼32 kDa protein on human erythrocytes.

<p>Normal erythrocyte ghost cells were separated by SDS–PAGE, and the gel was blotted and incubated with GST (the fusion partner of rRH5) or rRH5 or native parasite culture supernatant. After extensive washing, bound protein was detected by rabbit anti-RH5-GST and blots were processed by enhanced chemiluminescence (ECL, Amersham Biosciences). A specific target protein of ∼32 kDA is seen in rRH5 lane (marked with arrow) and Dd2 lanes (marked with arrow). Asterisk denotes a non-specific protein band that appears in control GST and other lanes. Parallel blots were probed with anti-GPA/B or anti GPC/D antibodies to define positions of the glycophorins relative to the 32 kDA band. Pre-immune rabbit sera did not yield any reactivity (PI).</p

Cartoon of PfRH ligands and characterization in the parasite.

<p>A. Schematic depiction of different members of the PfRH family, showing location of the signal peptide, region of homology among the various RH ligands and the trans-membrane region at the C-termini of the proteins. The bar at the bottom of PfRH5 marks the region of PfRH5 that was expressed in <i>E. coli</i>. B. Expression of a recombinant 43-kDa protein of PfRH5 (rRH5), chosen on the basis of homology with putative binding domains of <i>P. vivax</i> reticulocyte-binding protein 1 (PvRBP1) and PfRH4. Arrow indicates rRH5 after purification on GST-agarose column. C. Western Blot and immunoprecipitation analysis of asynchronous Dd2 parasite lysates with anti-rRH5 antibodies (IM). Pre-immune serum (PI) was used as a negative control. Arrow indicates specific 63 kDA RH5 protein seen in lysates.</p

Erythrocyte-binding activity of native PfRH5.

<p>A Binding of the native PfRH5 protein in the HB3 culture supernatant incubated with untreated (WT) erythrocytes, variously enzyme-treated erythrocytes (N: neuraminidase; HT, high trypsin; C, chymotrypsin). The PfRH5 parasite protein was detected in the eluate fractions by immunoprecipitation with anti-RH5 antibodies in WT, N and C lanes but not HT. B The same eluate samples from (A) were used for the detection of EBA-175 by immunoprecipitation. EBA-175 binds to wild type and chymotrypsin-treated erythrocytes but not to neuraminidase and high trypsin-treated erythrocytes C Binding of the native RH5 protein to erythrocytes treated with lower concentrations of trypsin (LT low trypsin and MT: moderate trypsin). D The eluate samples from (C) were used for the detection of EBA-175 by immunoprecipitation. EBA-175 binds to untreated erythrocytes but not to low-trypsin- and moderate-trypsin-treated erythrocytes.</p

Erythrocyte-binding activity of rRH5.

<p>A 0.6 µmoles of rRH5 were used in binding assays with untreated RBCs and RBCs treated with neuraminidase- (N), low-trypsin- (LT), medium-trypsin- (MT), high-trypsin- (HT) and chymotrypsin, followed by elution and immunoprecipitation with anti-RH5 antibodies. Gels containing the immuno-precipitates were blotted and probed with anti-RH5 antibodies. B Antibodies to rRH5 block the erythrocyte binding of the recombinant protein. Total IgG from rabbits immunized with rRH5, blocks erythrocyte binding of the RH5 recombinant protein. 0.6 µmoles of rRH5 was incubated with normal erythrocytes in the presence of purified IgG from rabbit sera at final concentrations of 0–10 µg/100 µl.</p

Treatment of adult female <i>B. malayi</i> with <i>Bm-cpl-1</i>, <i>Bm-cpl-5</i>, and <i>Bm-cpz</i> dsRNAs leads to phenotypic changes in developing embryos.

<p>Intrauterine progeny from individual female worms were examined 2 d after treatment with medium control (A), <i>Ov-cpz-Int2</i> control dsRNA (B), <i>Bm-cpl-5</i> dsRNA (C) and <i>Bm-cpl Pro</i> dsRNA (D).</p

Demonstration of uptake of Cy3-labeled dsRNAs or siRNA by <i>B. malayi</i>.

<p>Adult female <i>B. malayi</i> (2 groups of 4 worms) were soaked in normal culture medium containing Cy3-dsRNA (0.01 mg/ml) for 24–72 h. Uptake was examined for <i>Bm-cpl-5</i> dsRNA (∼800 bp) (A), <i>Bm-cpl-5</i> siRNA (B), <i>Bm-cpl Pro</i> dsRNA (∼400 bp) (C) and <i>Bm-cpl Pro</i> siRNA (D). Fluorescence was visualized using a Zeiss Axiovert fluorescence microscope using the rhodamine filter set, using emission 590 nm.</p

dsRNA-mediated silencing of the <i>B. malayi</i> cathepsin-like genes, <i>Bm-cpl-1</i>, <i>Bm-cpl-5</i>, and <i>Bm-cpz</i> leads to a reduction in microfilaria release from <i>B. malayi in vitro</i>.

<p>Following 24 h culture in normal culture medium, two groups of 3 female <i>B. malayi</i> worms were soaked for 18 h in 2 mg/ml gene-specific dsRNA (<i>Bm-cpl-1</i>, <i>Bm-cpl-5</i>, <i>Bm-cpz</i>, <i>Ov-cpz-Int2</i>) or medium alone (control). Following dsRNA treatment, individual worms were transferred to dsRNA-free medium and cultured for an additional 48 h. Released microfilariae were collected and counted daily. Results are expressed as Mf release before RNAi treatment (A), 24 h (B) and 48 h (C) after treatment. Each graph represents one experiment which is representative of at least 3 separate experiments. <i>P</i> values denote a significant difference between dsRNA-treated worms and either untreated medium controls or negative control (<i>Ov-cpz-Int2</i>) (Mann-Whitney <i>U</i>-test).</p

A brief schematic of the assays for testing the effect of polyanions on HIV-1 infection in the presence of PAP248-286 or SE-F.

<p>Polyanions at graded concentration was mixed with an indicated concentration of PAP248-286 or SE-F at an indicated interval with agitation as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059777#s2" target="_blank">Materials and Methods</a>”. After centrifugation, the supernatants were collected for testing the inhibitory activity of the unbounded polyanions on HIV-1 infection. The pellets containing the PAP248-286 (SEVI) - or SE-F-derived amyloid fibrils were re-suspended in 100 µl medium for testing the enhancing effect on HIV-1 infection.</p

Time courses of PAP248-286 aggregation in the absence or presence of polyanionic candidate microbicides.

<p>2 mg/ml PAP248-286 was agitated at 37°C and 1200 rpm in the presence or absence of various polyanions (30 µg/ml). The status of peptide aggregation is monitored by Thioflavin T staining (A) or Congo red staining (B). The facilitation of the fibrillogenesis of PAP248-286 mediated by cellulose sulfate is dose-dependant as revealed by Thioflavin T staining (C) and Congo red staining (D). The data presented were the median values obtained from one experiment performed in triplicate. Experiments were repeated once that yielded similar trends.</p