Insect Biochemistry Goes to School .

Many arthropods feed on vertebrate blood and eventually transmit diseases to the human being. Ordinary citizens are continuously exposed to vectors of major diseases and thus prone to contamination. In the present study we have evaluated a new approach regarding the basic biochemistry mechanism that regulates thebiology of Triatominaes, well known as vectors of Chagas Disease. We have used a comic book "Carlos Chagas: 100 years of the discovery of a hero", produced by researchers at our laboratory. The main concepts presented include: neurochemistry of insect olfaction, hemostasis, blood digestion, redox balance to mention some. The material was applied between 60 pupils of theseventh year of elementary school. The work is divided into two phases: an assessment of the concepts that thestudents bring their experiences into the classroom by producing conceptual maps followed by the intervention with the comic and production ofnew conceptual maps under the influence of activity with the material. Analysis of 18conceptual maps obtained from 06 different groups at three different points revealedthat the average score 07 at the beginning of theevaluation went to an average of 45 at the end of the study. This result suggests that a change in the approach of content can give encouragement to students to better develop the theme of contributing to a proper process of conceptual change

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

<div><p>A group of peptides from the salivary gland of the tick <i>Hyalomma marginatum rufipes</i>, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, <i>Haemaphysalis longicornis</i>. We have evaluated the anti-serine protease activity of one of these <i>H</i>. <i>marginatum</i> peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.</p></div

Hyalomin-1 does not inhibit or interact with γ-thrombin.

<p>(A) Steady state kinetic analysis of γ-thrombin in the absence (filled circles) or presence (filled squares) of 600 nM hyalomin-1. (B) γ-Thrombin (black circles) and α-thrombin (black squares)-catalyzed hydrolysis S2238 (50 μM) in the presence of hyalomin-1. (C) Binding of γ-thrombin and α-thrombin to immobilized hyalomin-1 measured by SPR. Buffer alone (1), 50 nM γ-thrombin (2), 100 nM γ-thrombin (3), 50 nM α-thrombin (4), 100 nM α-thrombin (5).</p

Alignment of hyalomins from <i>H</i>. <i>marginatum rufipes</i> with the madanins from <i>H</i>. <i>longicornis</i> and similar sequences from <i>D</i>. <i>andersoni</i>.

<p>Signal peptide sequences have been removed, and regions of conservation are highlighted in black. Accession numbers from the Genbank database are: GI:307006449, GI:307006483, GI:307006445 and GI:307006427 for hyalomins 1–4, GI:30025562 and GI:30025564 for madanins 1 and 2, GI:67906166 and GI:67968369 for madanin-like peptides 1 and 2. D. ander 76, 310, 82 and 240 sequences are found in Francischetti et al, 2009 support material.</p

Sulfation of hyalomin-1 (13–44) truncated form results in a modest increase in potency.

<p>(A) Conversion of fibrinogen to fibrin by thrombin in the presence of different hyalomin-1 truncated forms (5 μM) as indicated by time in seconds for increase in absorbance of 0.01 at 650 nm. Bars represent mean with SE. Full hyalomin-1 (01–59) completely inhibited fibrinogen clotting during the time assayed. (B) Same assay as in A but at different concentrations of hyalomin-1 (13–44) sulfated. (C) APTT and (D) PT assay procedures for coagulation time of human plasma in the presence of different concentrations of hyalomin -1 (13–44) sulfated (dashed lines), or hyalomin-1 (13–44) solid lines.</p

Hyalomin-1 is a competitive fast binding classical inhibitor of thrombin that is also cleaved by thrombin.

<p>(A) Kinetics of S2238 hydrolysis by thrombin in the presence of increasing concentrations of hyalomin-1 at a sodium chloride concentration of 150 mM. Inhibitor concentrations: 0 nM (filled circles), 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles), 200 nM (filled diamonds), 400 nM (open circles), 600 nM (open squares). (B) Experiment performed as in panel A but at a sodium chloride concentration of 50 mM. Hyalomin-1 concentrations: 0 nM (filled circles), 1.25 nM (filled squares), 2.5 nM (filled triangles), 5 nM (filled inverted triangles), 10 nM (filled diamonds), 25 nM (open circles), 50 nM (open squares), 100 nM (open triangles). (C) Progress curves of thrombin activity in the presence of hyalomin-1: 0 nM (filled circles), 50 nM (filled squares), 100 nM (filled triangles), 200 nM (filled inverted triangles), 400 nM (filled diamonds), 600 nM (open circles). (D) Measurement of thrombin binding to immobilized hyalomin-1 by SPR. Sensograms were obtained by injection of thrombin at concentrations of 50 nM (1), 25 nM (2), 12.5 nM (3), 6.25 nM (4), 3.125 nM (5) and 1.563 nM (6). Kinetic constants are indicated in the text. (E) Mass spectral analysis of hyalomin-1 cleavage products after incubation with thrombin for 2 h at 37°C. The mass values on the graph correspond to cleavage at the Arg₄₁-Leu₄₂ peptide bond (peptides 01–41 and 42–59 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133991#pone.0133991.g005" target="_blank">Fig 5A</a>). (F) Effect of incubation time at 37°C on the inhibition of thrombin (0.5 nM) by hyalomin-1 (400 nM) in the presence of 50 μM S2238. The activity of thrombin in the absence of hyalomin-1 is shown as filled circles, while activity in the presence of hyalomin-1 is shown as filled squares.</p

Hyalomin-1 exhibits antithrombotic activity in vivo.

<p>(A) Occlusion of the carotid artery in BALB/c mice induced by FeCl₃ and measured by laser Doppler flowmetry after injection of vehicle alone or hyalomin-1 at doses of 1 and 2.5 mg/kg. (B) Bleeding from the tail of BALB/c mice after injection of vehicle alone or vehicle plus hyalomin-1 at a dose of 2.5mg/kg. Statistical significance levels: * P < 0.05, ** P < 0.001.</p