Apoptotic signaling pathways triggered by LB-piVe infection.

<p>Pathway analysis was performed with the Pathway Studio 9 software. Using the PCR Array data, a network was generated in which the genes and gene products are represented by nodes. The shapes of the nodes represent the protein’s primary function and the connection lines indicate the type of interaction (colored lines: direct regulation; dotted gray lines: indirect regulation). Color intensity reflects the level of gene expression in LB-piVe infected cells versus control if the fold-change ≥1.2 (red: up-regulated; blue: down-regulated; grey: no change in expression).</p

LB-piVe infection up-regulates active caspase-3 expression.

<p><b>(A)</b> Huh7.5 cells were UV-LB-piVe-infected (left panel), LB-piVe infected (right panel) or treated with Staurosporine (center panel). At day 3 post-infection, cells were treated (right panel) with Z-VAD-FMK at 20 μM for 16 hr at 37°C. The following day, cells were fixed/ permeabilized, stained with FITC Rabbit Anti-Active Caspase-3 antibodies or FITC mouse isotype control and analyzed by flow cytometry. Data were processed with the FloJo 7.6.3 software. Numbers indicate the percentage of caspase-3 positive and negative cells. <b>(B)</b> Representation of the frequency of caspase-3 positive and negative cells from <b>(A).</b></p

Ultrastructural changes in LB-piVe infected Huh7.5 cells.

<p><b>(A)</b> Huh7.5 cells were infected with LB-piVe (center) or with mock infection, UV-LB-piVe (left). Four days post-infection, cells were fixed, permeabilized, stained with anti-HCV NS5A, developed with anti-mouse antibodies conjugated with Alexa Fluor 488, and analyzed by flow cytometry. Numbers indicate the percentage of HCV NS5A-positive and -negative cells. <b>(B)</b> Light microscopy of LB-piVe and UV-LB-piVe infected cells demonstrating that CPE was an effect of live virus infection. MOI: multiplicity of infection. <b>(C-G)</b> LB-piVe infected or <b>(H)</b> UV-LB-piVe treated cells were fixed at 4 days post-infection, embedded in Epon resin and deposited on grids coated with collodion membrane for examination under a Jeol 1230 transmission electron microscope (TEM). Mitochondria (M); Nucleolus (N); Nuclear Envelope (NE); Filopodia (F); Endoplasmic Reticulum (ER); Lipid droplet (LD); Multiple-membrane vesicles (MMV). Green arrows denote markers of apoptosis.</p

Effect of LB-piVe infection on apoptosis-related genes expression.

<p>50μg of total protein samples were resolved by SDS-PAGE in Novex 4–20% Tris-glycine polyacrylamide gels and then transferred to Hybond ECL membranes. Blots were probed with anti-caspase-3, anti-cleaved PARP1, anti-BIK, and anti- Bcl-xL. Horse radish peroxidase-conjugated secondary antibodies were used for detection and developed with the Femto Chemiluminescence Substrate kit. GAPDH was used as internal loading control. Negative controls: naïve Huh7.5 cells (C), and Huh7.5 cells treated with UV-LB-piVe (UV).</p

LB-piVe infection triggers exposure of phosphatidylserine on the surface of infected cells.

<p>(A) UV-LB-piVe (left panel) or LB-piVe infected (right panel) cells and Staurosporine-treated (center panel) were incubated with PE Annexin V in a buffer containing 7-Amino-Actinomycin D (7-AAD), and analyzed by flow cytometry. Numbers in the quadrants indicate the percentage of cells in the corresponding areas. (B) Representation of the frequency of live, early apoptotic, late apoptotic and necrotic cells from (A).</p

Expression profiles of Caspase and Caspase Regulator Genes.

<p>Expression profiles of Caspase and Caspase Regulator Genes.</p

LB-piVe infection up-regulates Fas expression.

<p><b>(A)</b> Huh7.5 cells were treated with UV-LB-piVe (left panel), infected with LB-piVe (right panel) or treated with Staurosporine as a positive control for apoptosis (center panel). Four days post-infection, cells were fixed, permeabilized, and then stained with a PE-Mouse anti-human CD95 antibody or a PE Mouse IgG1 Kappa Isotype Control. Stained cells were analyzed using a FACS Calibur cytometer. Data were processed with the FlowJo 7.6.3 software. Numbers indicate the percentage of Fas-positive and Fas-negative cells. <b>(B)</b> Representation of the frequency of Fas-positive and Fas-negative cells from <b>(A).</b> Frequency values are means ± SD, representative of 2 different experiments.</p

LB-piVe infection is associated with caspase activation.

<p><b>(A)</b> Intracellular caspase detection was performed by staining live Huh7.5 cells with FITC-conjugated ApoStat and analyzing by flow cytometry. Cells were evaluated at 4 days post-infection or treatment. UV-LB-piVe-treated and Staurosporine-treated cells were used as negative and positive controls, respectively. Data were processed with the FlowJo 7.6.3 software. Numbers indicate the percentage of caspase positive and negative cells. <b>(B)</b> Representation of the frequency of caspase positive and negative cells from <b>(A)</b>. Frequency values are means ± SD, representative of 2 different experiments.</p

Selecting targets for the diagnosis of <i>Schistosoma mansoni</i> infection: An integrative approach using multi-omic and immunoinformatics data

<div><p>In order to effectively control and monitor schistosomiasis, new diagnostic methods are essential. Taking advantage of computational approaches provided by immunoinformatics and considering the availability of <i>Schistosoma mansoni</i> predicted proteome information, candidate antigens of schistosomiasis were selected and used in immunodiagnosis tests based on Enzime-linked Immunosorbent Assay (ELISA). The computational selection strategy was based on signal peptide prediction; low similarity to human proteins; B- and T-cell epitope prediction; location and expression in different parasite life stages within definitive host. Results of the above-mentioned analysis were parsed to extract meaningful biological information and loaded into a relational database developed to integrate them. In the end, seven proteins were selected and one B-cell linear epitope from each one of them was selected using B-cell epitope score and the presence of intrinsically disordered regions (IDRs). These predicted epitopes generated synthetic peptides that were used in ELISA assays to validate the rational strategy of <i>in silico</i> selection. ELISA was performed using sera from residents of areas of low endemicity for <i>S</i>. <i>mansoni</i> infection and also from healthy donors (HD), not living in an endemic area for schistosomiasis. Discrimination of negative (NEG) and positive (INF) individuals from endemic areas was performed using parasitological and molecular methods. All infected individuals were treated with praziquantel, and serum samples were obtained from them 30 and 180 days post-treatment (30DPT and 180DPT). Results revealed higher IgG levels in INF group than in HD and NEG groups when peptides 1, 3, 4, 5 and 7 were used. Moreover, using peptide 5, ELISA achieved the best performance, since it could discriminate between individuals living in an endemic area that were actively infected from those that were not (NEG, 30DPT, 180DPT groups). Our experimental results also indicate that the computational prediction approach developed is feasible for identifying promising candidates for the diagnosis of schistosomiasis and other diseases.</p></div

Calculation of Maturation Index Ratios for the numerical determination of cellularity in the bone marrow.

<p>M indicates myeloid; E, erythroid; I, immature; M, mature; I:Mg, immature: mature (granulopoiesis); MMI, myeloid maturation index; I:Me, immature: mature (erythropoiesis); EMI, erythroid maturation index.</p