Structural statistics for the ensemble of OAIP-1 structures¹.

1<p>All statistics are given as mean ±S.D.</p>2<p>Only structurally relevant restraints, as defined by CYANA, are included.</p>3<p>Two restraints were used per hydrogen bond.</p>4<p>According to MolProbity (<a href="http://molprobity.biochem.duke.edu" target="_blank">http://molprobity.biochem.duke.edu</a>).</p>5<p>Defined as the number of steric overlaps >0.4 Å per thousand atoms.</p

Isolation of an orally active insect toxin from spider venom.

<p>(A) RP-HPLC chromatogram showing fractionation of crude venom from the Australian tarantula <i>Selentypus plumipes</i>. An asterisk highlights the fraction that displayed oral termiticidal activity. (B) Chromatogram from cation exchange fractionation of the active RP-HPLC fraction shown in (A). An asterisk highlights the fraction with oral termiticidal activity. (C). Insecticidal assay of native OAIP-1. The peptide was injected into larvae of the mealworm beetle (<i>Tenebrio molitor</i>) at a dose of 3 pmol/g or fed to termites (<i>Coptotermes acinaciformis</i>) at a dose of 350 nmol/g. Each column represents the mean ±SD of three replicates of 10 insects.</p

Comparison of OAIP-1 with pyrethroid insecticides.

1<p>Pyrethroid-resistant strain BK99R9.</p>2<p>Susceptible strain BK77.</p

Primary structure of OAIP-1.

<p>(A) Sequence of transcript encoding the OAIP-1 prepropeptide precursor isolated from an <i>S. plumipes</i> venom-gland cDNA library. The 3′ and 5′ untranslated region (UTR), signal sequence, propeptide region, and mature toxin are labeled. The “GR” dipeptide sequence at the end of the mature toxin sequence is labeled AS (amidation signal) as it is a signal for C-terminal amidation. (B) Amino acid sequence of OAIP-1 prepropeptide precursor obtained from <i>in silico</i> translation of the cDNA sequence shown in panel (A). (C) Comparison of the amino acid sequence of the mature OAIP-1 toxin obtained from <i>in silico</i> translation of the venom-gland prepropeptide transcript with the N-terminal sequences obtained from Edman degradation of the native toxin at the APAF and APC protein sequencing facilities. (D) Alignment of OAIP-1 primary structure with the two closest hits obtained from a BLAST search against the ArachnoServer database. Identical residues are highlighted by white letters on a black background, while residues that are identical in two of the three sequences are shown on a gray background.</p

Stability of OAIP-1.

<p>(A) Thermal stability of sOAIP-1 over 7 days. Note that the data obtained at −20°C, 22°C, and 30°C overlap completely since OAIP-1 is 100% intact at these temperatures at all time points. OAIP-1 only degrades at temperatures of 37°C and higher. (B) Stability of sOAIP-1 over a range of different pH conditions. The toxin is least stable at alkaline pH. (C) A series of RP-HPLC chromatograms showing fractionation of undiluted hemolymph from <i>H. armigera</i> larvae (cotton bollworms) at various times following addition of 30 µg sOAIP-1 (highlighted in the solid box). Immediately before RP-HPLC fractionation, 30 µg of ω-HXTX-Hv1a (dashed box) was added to each sample for the purposes of quantification. In all experiments shown in panels A–C, intact OAIP-1 was identified using mass spectrometry.</p

Structural homologues of OAIP-1.

<p>Alignment of the structure of OAIP-1 (orange) with the top six structural homologues (all shown in green) as ranked by the Dali server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073136#pone.0073136-Holm1" target="_blank">[43]</a>. The activity of each structural homologue is indicated, as is the <i>Z</i> score and RMSD of the alignment. Disulfide bonds are shown as solid tubes and the N- and C-termini are labeled.</p

Structure of OAIP-1.

<p>(A) Stereoview of the ensemble of 20 OAIP-1 structures. The three disulfide bonds and the N- and C-termini are labeled. (B) Schematic (Richardson) representation of OAIP-1. β-strands are colored blue and disulfide bonds are shown as red tubes. The four intercystine loops (loops 1–4) are labeled. (C) Overlay of OAIP-1 (blue) and the orthologous toxin U₁-TRTX-Pc1a (orange). The intercystine loops and termini are labeled. (D) Overlay of OAIP-1 (blue) and the orthologous toxin U₁-TRTX-Pc1a (orange). Residues Y11 and Y26 in U₁-TRTX-Pc1a (red tubes) interact in such a way that loops 2 and 4 are brought into close proximity. The equivalent residues in OAIP-1, P10 and Y27 (light blue tubes), do not interact and consequently loops 2 and 4 are further apart.</p

Choice test with OAIP-1.

<p>Mortality of <i>T. molitor</i> larvae (mealworms) determined at 48 h after insects were simultaneously offered toxin-treated and untreated agar. The toxin concentration in the treated agar ranged from 1 mmol to 1 pmol, and the data represent the mean and SEM of three replicates of 10 individuals for each dose. The data correlate well with the oral toxicity of sOAIP-1 in a non-choice test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073136#pone-0073136-g003" target="_blank">Fig. 3A</a>); the mortality at the same dose in the choice test is approximately the same as that observed in the non-choice test. Mortality at all but the lowest two doses (10 and 1 pmol) was significantly greater than the untreated agar control (P<0.01). Columns represent the mean ±SD for three replicates of 10 insects for each dose.</p

Image_1_Analysis of insecticides in long-lasting insecticidal nets using X-ray fluorescence spectroscopy and correlation with bioefficacy.jpeg

BackgroundLong-lasting insecticidal nets (LLINs) are a key vector control tool used for the prevention of malaria. Active ingredient (AI) measurements in LLINs are essential for evaluating their quality and efficacy. The main aim of the present study was to determine the utility of X-ray fluorescence (XRF) spectroscopy as a suitable field-deployable tool for total AI quantification in LLINs.MethodsNew and unused LLIN samples containing deltamethrin (PermaNet® 2.0, n = 35) and alpha-cypermethrin (SafeNet®, n = 43) were obtained from batches delivered to Papua New Guinea (PNG) for mass distribution. Insecticides were extracted from the LLINs using a simple extraction technique and quantified using liquid chromatography mass spectrometry (LC-MS). The LC-MS results were correlated with XRF spectroscopy measurements on the same nets. Operators were blinded regarding the type of net. Bioefficacy of the LLIN samples was tested using WHO cone bioassays and test results were correlated with total AI content.ResultsThe results indicate correlation between quantitative XRF and LC-MS. Interestingly, the total AI content was negatively correlated with bioefficacy in PermaNet® 2.0 (especially in recently manufactured nets). In contrast, AI content was positively correlated with bioefficacy in SafeNet®. These results indicate that the chemical content analysis in predelivery inspections does not always predict bioefficacy.ConclusionXRF is a promising field-deployable tool for quantification of both deltamethrin- and alpha-cypermethrin-coated LLINs. Because total AI content is not always a predictor of the efficacy of LLINs to kill mosquitoes, bioefficacy measurements should be included in predelivery inspections.</p

Table_1_Analysis of insecticides in long-lasting insecticidal nets using X-ray fluorescence spectroscopy and correlation with bioefficacy.xlsx

BackgroundLong-lasting insecticidal nets (LLINs) are a key vector control tool used for the prevention of malaria. Active ingredient (AI) measurements in LLINs are essential for evaluating their quality and efficacy. The main aim of the present study was to determine the utility of X-ray fluorescence (XRF) spectroscopy as a suitable field-deployable tool for total AI quantification in LLINs.MethodsNew and unused LLIN samples containing deltamethrin (PermaNet® 2.0, n = 35) and alpha-cypermethrin (SafeNet®, n = 43) were obtained from batches delivered to Papua New Guinea (PNG) for mass distribution. Insecticides were extracted from the LLINs using a simple extraction technique and quantified using liquid chromatography mass spectrometry (LC-MS). The LC-MS results were correlated with XRF spectroscopy measurements on the same nets. Operators were blinded regarding the type of net. Bioefficacy of the LLIN samples was tested using WHO cone bioassays and test results were correlated with total AI content.ResultsThe results indicate correlation between quantitative XRF and LC-MS. Interestingly, the total AI content was negatively correlated with bioefficacy in PermaNet® 2.0 (especially in recently manufactured nets). In contrast, AI content was positively correlated with bioefficacy in SafeNet®. These results indicate that the chemical content analysis in predelivery inspections does not always predict bioefficacy.ConclusionXRF is a promising field-deployable tool for quantification of both deltamethrin- and alpha-cypermethrin-coated LLINs. Because total AI content is not always a predictor of the efficacy of LLINs to kill mosquitoes, bioefficacy measurements should be included in predelivery inspections.</p