Oocysts numbers in four different generations (13–16) of control (C) and exposed lines (A and B) of after infection with

<p><b>Copyright information:</b></p><p>Taken from "Continuous exposure to results in decreased susceptibility and transcriptomic divergence of the immune system"</p><p>http://www.biomedcentral.com/1471-2164/8/451</p><p>BMC Genomics 2007;8():451-451.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2234432.</p><p></p> : Individual values (each dot represents the oocysts number in the midgut of each mosquito). : Average and standard error of each group of individual values for generations 13–16. Asterisks indicate statistically significant differences (Mann-Whitney test) with respect to control values (*< 0.05). (see Additional File Table)

Experimental Design and Global Gene Expression Patterns at the Different Conditions of Infection

<div><p>(A) Model outlining the experimental design for assessing responses to the invading ookinetes (indicated as <i>P.f.</i> ookinete [<i>P.f.</i> o.] and <i>P.b</i>. ookinete [<i>P.b.</i> o.] in (B) and (C) by comparing transcription between mosquitoes fed on blood infected with a wt <i>Plasmodium</i> strain and those fed on blood infected with the invasion-incapable mutant <i>Plasmodium</i> strain CTRP⁻. Responses to infected blood (indicated as <i>P.f</i>. blood in [B] and [C]) in the absence of ookinete invasion were assessed by comparing gene expression between mosquitoes fed on blood infected with the P. falciparum invasion-incapable mutant and mosquitoes fed on noninfected (no <i>Plasmodium</i>) blood.</p><p>(B) Gene regulation in midgut and carcass tissues triggered by P. falciparum ookinete invasion (<i>P.f.</i> ookinete), P. berghei ookinete invasion (<i>P.b.</i> ookinete), and P. falciparum strain CTRP⁻-infected blood lacking invasive ookinetes (<i>P.f.</i> blood). Colored arrows indicate genes that are up- or down-regulated in the various unique and overlapping sections.</p><p>(C) Proportions and numbers of genes belonging to distinct functional classes which were up- or down-regulated by the various stimuli in the gut and carcass tissues. DIV: diverse; R/T/T: replication, transcription, translation; MET: metabolism; TRP: transport; CY/ST: cytoskeletal, structural; PR/DI: proteolysis, digestion; MIT: mitochondrial; RE/ST: oxidoreductive, stress-related; APO: apoptosis; P/A: putative immunity and apoptosis. Gene functions were predicted based on Gene Ontology data and manual sequence homology searches.</p><p>(D) Same as in (C), but also including genes of diverse functions (DIV) and unknown functions (UKN).</p></div

Comparison of Anti-<i>Plasmodium</i> and Antibacterial Activities for 16 Selected Immune Genes

<div><p>(A) Effect of gene silencing on P. berghei development, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020052#ppat-0020052-st006" target="_blank">Table S6</a>. For ease of comparison, only the mosquito portions with the highest P. berghei oocyst numbers (>200) are presented. The effect of gene silencing on bacterial infection is presented as the mosquito survival at d 6 after challenge with E. coli and <i>S. aureus.</i> After 6 d, the survival rates stabilized and did not change significantly until age-related mortality ensued. The baseline survival rate was set to that of the challenged GFP dsRNA-treated control mosquitoes (~70%). Standard error bars with asterisks indicate the results of two-way analysis of variance, with <i>p</i> < 0.05 considered statistically significant. The gene names are numbered for ease of comparison.</p><p>(B) Mosquito survival rates for each silenced gene after challenge with E. coli and <i>S. aureus.</i> The numbers in parenthesis correspond to the numbers in (A). Open squares, <i>dsGFP</i> control-treated mosquitoes challenged with E. coli; solid squares, <i>dsGFP</i> control–treated mosquitoes challenged with <i>E. coli;</i> open triangles, gene-silenced mosquitoes challenged with <i>E. coli;</i> and solid triangles, gene-silenced mosquitoes challenged with <i>S. aureus.</i> Standard error bars from three replicate experiments are included for each time point.</p></div

Effects of Gene Silencing of 11 Selected Putative Immune Genes on P. falciparum and P. berghei Infection

<p>The gene silencing efficiency values (KD %) are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020052#ppat-0020052-st006" target="_blank">Table S6</a>. The frequency distribution of oocysts pooled from three independent assays is displayed, with bars indicating the percentile of mosquitoes with the corresponding oocyst number in the range indicated on the <i>x</i>-axis. Equal numbers of midguts from all three experiments in each dataset were pooled. Bars with asterisks indicate the statistically significant differences at the 95% confidence level, based on the <i>p</i> value from two independent probability tests, the KS and Mann-Whitney tests (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020052#ppat-0020052-st005" target="_blank">Tables S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020052#ppat-0020052-st006" target="_blank">S6</a>). n: total midguts assayed; MI: mean intensity of infection (oocysts number); S.E.: standard error of mean intensity; <i>p</i> value: from Mann-Whitney test.</p

AgMDL Gene Family

<div><p>(A) A. gambiae MD2-like genes encode proteins ranging from 130 to 162 amino acids and include signal peptides and an ML lipid recognition domain. Alignment of AgMDL1 with the human homologues MD1, MD2, and Npc2, the mite allergen Der-P2, and the Bombyx mori promotor protein (BmPP). Two conserved cysteines, Cys95 and Cys105, that are essential for binding to TLR4 are indicated with asterisks.</p><p>(B) Phylogenetic tree of MD2-like proteins from <i>A. gambiae, D. melanogaster, B. mori,</i> and humans. 1:1 orthologs and ortholog groups are highlighted with filled circles. Ag, <i>Anopheles gambiae;</i> Dm, D. melanogaster. The accession numbers for these genes are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020052#ppat-0020052-st007" target="_blank">Table S7</a>.</p></div

Sensitivity, specificity and accuracy of internationally accepted cut-off values of iron markers to identify iron stores deficiency using bone marrow iron content as “gold standard”.

1<p>By C reactive protein (CRP): <12 ng/ml if CRP<1 mg/dl, and <30 ng/ml if CRP≥1 mg/dl.</p>2<p>By age: <50 ng/ml in children 3–5 months of age, and <7 ng/ml in children >5 months of age.</p>3<p>By CRP: >1.5 if CRP<1 mg/dl, and >0.8 if CRP≥1 mg/dl.</p>4<p>By age: <70 fl in children<2 years of age, and <73 fl in children ≥2 years of age.</p><p>Abbreviations: MCHC, mean cell haemoglobin concentration; MCV, mean cell volume; Neg, negative; Pos, positive; sTfR, soluble transferrin receptor; TfR-F index, transferrin-ferritin index; TIBC, total iron binding capacity.</p

Demographic and clinical characteristics of the study participants.

*<p>Arithmetic Mean (SD).</p><p>N = 180 and results expressed as n (%) unless otherwise indicated.</p><p>Abbreviations: HAZ, height for age Z score; Hb, haemoglobin; HIV, human immunodeficiency virus; WAZ, weight for age Z score.</p

Proportion of children classified as iron deficient using internationally accepted cut-off values of iron markers.

<p>Abbreviations: CRP, C reactive protein; MCHC, mean cell haemoglobin concentration; MCV, mean cell volume; Obs, observations; sTfR, soluble transferrin receptor; TfR-F index, transferrin-ferritin index; TIBC, total iron binding capacity.</p

Receiver operating characteristic curves of the iron markers in the identification of iron stores deficiency.

<p>Cut-off values for sTfR and TfR-F index with the highest sensitivity to detect iron deficiency maintaining the specificity≥50% are indicated with arrows. Abbreviations: Sat. Transf., transferrin saturation; sTfR, soluble transferrin receptor; TfR-F index, transferrin-ferritin index; TIBC, total iron binding capacity.</p

Epidemiologic and demographic characteristics of study participants stratified by RT-PCR, IgM and IgG results.

<p>Epidemiologic and demographic characteristics of study participants stratified by RT-PCR, IgM and IgG results.</p