LPC inhibits LPS-induced ERK activation.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of 1 µg/mL LPS or in the presence or absence of the indicated concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere (<b>A</b>, <b>D</b>, <b>E</b>). In parallel HEK 293A cells with TLR constructs as indicated (<b>B</b>, <b>C</b>). Each group received expression constructs for TLR4 (<b>B</b>) or both TLR2 and TLR1 (<b>C</b>), as well as MD-2, CD14 and CD36 plasmids. The cells were then incubated in the absence or presence of 100 ng/mL LPS or 1 nM Pam3CSK4 (P3C) and 10 or 100 µM of LPC, for 40 min at 37 °C in a 5% CO₂ atmosphere. After incubation either macrophages or HEK cells were homogenized, the protein levels was determined and samples evaluated by Western blot with the use of antibodies against p-ERK (<b>A</b>, <b>B</b>, <b>C</b>), p-JNK (<b>D</b>) and p-P38 (<b>E</b>). Loading controls were run with the use of antibodies raised towards actin. Experiments were performed at least two times with different animals and samples.</p

LPC triggers NF-қB activation through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected in three different groups. Groups A and B received expression constructs for TLR4 (<b>A</b>) or TLR2 and TLR1 (<b>B</b>). Both also received MD-2, CD14, and CD36 constructs and the ELAM-1-firefly luciferase and β-actin-<i>Renilla</i> luciferase reporter plasmids. The third group (<b>C</b>) received only the empty vector pDisplay and the luciferase reporter plasmids. Groups A and B were separately stimulated with 0.1, 1, 10, 100 and 200 µM of different types of LPC (Sigma; C14:0, C16:0, C18:0, and C18:1), 100 ng/mL of LPS and 1 nM of Pam3CSK4 (P3C). Group C was stimulated with LPS, Pam3Cys or 0.1, 1, 10 and 100 µM of LPC (C16:0). The agonists were diluted in DMEM medium with 10% bovine fetal serum. After 4 h of incubation, luciferase activity was measured and expressed as the ratio of NF-қB-dependent firefly luciferase activity to the control <i>Renilla</i> luciferase activity. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits NF-қB translocation, iNOS expression, and NO production in LPS-stimulated macrophages.

<p>Peritoneal macrophages from BALB/cmice were incubated in the absence or presence of 1 µg/mL LPS and different concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere. After 1 h of incubation, NF-қB translocation (<b>A</b>) was assayed by Western blot analysis. After 24 hours, NO production (<b>B</b>) was assayed by measuring the amount of nitrite in the culture supernatant using the Griess reagent, and iNOS expression (<b>C</b>) was determined by Western blot analysis followed by densitometry (lower panel). Data is the mean ± S.D. of three different experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC triggers IL-8 production through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected and stimulated as described on Figure 1. After 20 hours of incubation, IL-8 production was measured by the ELISA assay. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC activates JNK and p38, but not ERK, in macrophages.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of different concentrations of LPC mix (Sigma) for 20 min at 37 °C in a 5% CO₂ atmosphere, and the cytoplasm content was homogenized and assayed as follows. The Phospho-MAPK array was used for analysis of enzymatic activation (<b>A</b>). The reaction was visualized with the enhanced chemiluminescent system and subjected to densitometric analysis (***, p< 0.001, ANOVA). Protein levels of the phosphorylated MAPKs JNK (<b>B</b>), p38 (<b>C</b>) and ERK (<b>D</b>) were determined by Western blot. Data is the mean ± S.E. of two different experiments.</p

Effect of PTP inhibitors and silencing action of the AAEL001919 gene on egg formation in <i>Aedes aegypti</i>.

<p>(A) Five- and seven-day old females were artificially fed with rabbit blood supplemented with the PTP inhibitor vanadate or the PTP substrate Difmup. Following a blood meal, fat bodies were dissected from females at the indicated times, and the expression of Vg mRNA was measured by qPCR and normalized against Rp49 gene expression. (B) Females from the experiments shown in A were placed in individual tubes, and the eggs laid were quantified on subsequent days after the blood meal. (C) One- and two-day old females (5 insects) were injected with either 140 ng of AAEL001919 RNAi or dsMal. AAEL001919 expression levels were quantified three days after RNAi injection as shown. (D) Injected mosquitoes from the experiment shown in panel C were naturally blood-fed three days later, and the number of eggs laid following the blood meal was quantified. Normalized data from four experiments were analyzed by One-way ANOVA (panels A and B) and Student's t-test (Panel D) with significance levels set at p<0.05. Groups assigned with the same letter (a, b or c) indicate that they do not show statistically significant difference among them. Different letters in different groups indicate that such groups show significant differences among themselves (Panels A and B). Asterisks indicate (Panel B) indicate a significant difference among groups (student T- test, p<0.05).</p

Molecular Analysis of <i>Aedes aegypti</i> Classical Protein Tyrosine Phosphatases Uncovers an Ortholog of Mammalian PTP-1B Implicated in the Control of Egg Production in Mosquitoes

<div><p>Background</p><p>Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control.</p><p>Methodology/Principal Findings</p><p>We conducted an analysis to identify the ortholog of mammalian PTP1B in the <i>Aedes aegypti</i> genome. We identified eight genes coding for classical PTPs. <i>In silico</i> structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production.</p><p>Conclusions/Significance</p><p>The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.</p></div

Expression levels of classical PTPs in <i>Aedes</i> tissues.

<p>Female mosquitoes (20–30 insects) were maintained on 10% sucrose <i>ad libitum</i> and were collected at four and seven days after emergence from the pupal stage. They were dissected, and RNA was extracted from different tissues. Expression levels of PTPs were measured using qPCR. The following tissues were evaluated: (A) Ovaries; (B) Fat body; (C) Midgut and (D) Head. Expression levels of the following genes were evaluated: AAEL003108, AAEL001919, AAEL008528, AAEL005492. The latter were normalized against the expression of the mosquito Rp49 gene. Data are the means ± S.E.M of three different experiments.</p

Analysis of PTP catalytic sequences.

<p>Analysis of PTP catalytic sequences.</p

Evolutionary analysis of <i>Aedes</i> PTP sequences with the well-characterized PTPs from <i>H. sapiens</i> and <i>D. melanogaster</i>.

<p>PTP domains from <i>A. aegypti</i> and from sequences derived from human and fly (names starting with “h” and “dm”) were clustered genomes. The <i>A. aegypti</i> active PTP domains are highlighted in red, while the closest human and fly PTPs are highlighted in blue and green, respectively.</p