Subcellular localization of PfGDV1.

<p>Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (<i>Upper</i>) expressing GDV1 and a doubly infected erythrocyte (<i>Lower</i>) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (<i>Upper</i>) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (<i>Lower</i>) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (<i>Upper</i>) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (<i>Lower</i>) expressing GDV1 stained with αSir2.</p

<i>In vivo Pfge</i> gene expression profiling.

<p>RNA from 20 gametocytemic patients was analyzed using a <i>P. falciparum</i> whole genome microarray. The color coded cluster analysis of the quantile normalized expression data for the <i>Pfge</i> genes with a G+/G_def ratio>10 is shown with mature gametocyte specific genes <i>Pfs25</i> and <i>Pfs28</i> (Underlined in gray). The gene name and standardized intensity of the color code is indicated below and the patient cluster associated with high gametocytemia is indicated on the right with a vertical green bar. The gametocytemia (G'cyte) and asexual parasitemia (Asex) of the patient samples are represented both by color code and numerically on the right.</p

Expression profile of the <i>Pfge</i> genes through gametocytogenesis.

<p>MACS purified late stage asexual parasite cultures were set up at 6% hematocrit and sorbitol synchronized 2 hours later to remove all but the newly invaded ring stage parasites. Parasitemia was monitored by daily Giemsa-stained smear. Ring (blue), schizont stage parasitemias (red), and stage II gametocytemia (green) are plotted (A & B). The relative abundance, (log2) of the indicated gene in relation to the expression level on day 2 was calculated using the 2^{−ΔΔ<i>C</i>}_T method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Schmittgen1" target="_blank">[43]</a> with seryl tRNA synthetase as the reference and plotted in C and D): <i>Pfaldolase</i> (blue), <i>Pfkahrp</i> (brown), <i>Pfmsp7-5</i> (purple), <i>Pfs16 (</i>green), <i>Pfgdv1</i> (bright red); while <i>Pfge1</i> (orange), <i>Pfge2</i> (pale pink), <i>Pfge3</i> (beige), <i>Pfg27</i> (light blue), <i>Pfge7</i> (dark pink), <i>Pfge8</i> (light green), <i>Pfs47</i>(turquoise) and <i>Pfs16</i> (green) are shown in (E and F). The 3 asexual cycles are indicated by numbers as well as gray dotted lines, and NAG treatment is indicated by the gray box. Representative data from one of three independent experiments is shown. The samples from the different time points were tested in triplicate and the average relative expression is plotted with the error bars representing the range.</p

Pearson correlation analysis of gene expression profiles in <i>in vitro</i>, NAG-treated cultures.

<p>The expression profiles of the relative abundance of indicated genes in relation to seryl tRNA synthetase at day 2 were compared to each other in the NAG treated (NAG) the Pearson correlation coefficient is indicated. Representative data shown is from one of 3 independent experiments.</p

Identification of <i>Plasmodium falciparum</i> gametocyte development 1 gene (<i>Pfgdv1</i>).

<p>A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.G_def clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, <i>Pfgdv1;</i> diagonal stripes, <i>var</i> and <i>rifins</i>. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.G_def parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.G_def, FCR3.G_def, or HB3.G_def gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of <i>Bsa</i>BI-digested gDNA from 3D7.G+ (+), 3D7.G_def (d), 3D7.G_def +<i>Pfgdv1</i> (a), 3D7.G_def +HA.<i>Pfgdv1</i> (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with <i>Pfgdv1</i> (bp 1423–1800) or <i>Pfg27</i> (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.G_def (black triangle), complemented line a, 3D7.G_def +<i>Pfgdv1</i> (Panel D, black inverted triangle) and line b, 3D7.G_def +HA.<i>Pfgdv1</i> (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (<i>P</i><0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.G_def +HA.<i>Pfgdv1</i> (HA.<i>Pfgdv1</i>), and 3D7.G_def (G_def) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.G_def (d), 3D7.G_def<i>+Pfgdv1</i> (a), and 3D7.G_def +HA.<i>Pfgdv1</i> (b) complemented lines were hybridized with probes corresponding to <i>Pfgdv1</i> (<i>gdv1</i>), <i>Pfge</i> genes (<i>ge1–3, 5–11</i>), and merozoite surface protein-1 (<i>msp1</i>) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.G_def+<i>Pfgdv1</i> (<i>a</i>) and 3D7.G_def+HA.<i>Pfgdv1</i> (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a <i>Pfgdv1</i> episomal expression plasmid (WT+HA.<i>Pfgdv1</i>) and the G_def (G_def) line is graphed. The error bars represent the SEM and a significant difference from WT and G_def is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).</p

Model for continuous gametocytogenesis.

<p>During each round of the asexual cycle, a proportion of the schizonts (S_c, light blue) produced are committed to producing merozoites (M_c, light blue) that will differentiate into gametocytes (G_c, G_I–V) after invading a RBC. The expression profile for <i>Pfgdv1</i> is indicated by a line under the corresponding stage.</p

Expression analysis of the 3D7.G+ and 3D7.G_def clones identifies <i>P. falciparum</i>gametocytogenesis early genes (<i>Pfge</i>).

<p>Mean ratios of microarray signals (mean±SEM) obtained from the 3D7.G+ and 3D7.G_def clones at 1.4/0.9% parasitemia (dark gray bar) and at 5.2/5.5% parasitemia (light gray bar), respectively, for the 11 <i>Pfge</i> genes with a ratio of 3D7.G+/3D7.G_def>10 are plotted in descending order. <i>Pfgdv1</i> (PFI1710w) had the fourth highest ratio and is indicated by an ^o. Previously described <i>Pfge</i> genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Young1" target="_blank">[17]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Bruce2" target="_blank">[20]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-vanSchaijk1" target="_blank">[22]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Silvestrini3" target="_blank">[82]</a>, are denoted with an asterisk (*) and a vertical line indicates a gene represented by two oligonucleotides. <i>Pfge</i> number, PlasmoDB ID, and common name are listed on the right. The central panel is a summary of the characteristics of the <i>Pfgdv1</i>-dependent genes. The first row (L) indicates whether the gene is subtelomeric (<150 kb from the telomere) (gray square) or within a region of the chromosome that has synteny with other species (black square) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Kooij1" target="_blank">[83]</a>. The panel on the left indicates whether the gene encodes a secretory signal sequence (S, black square), PEXEL/HTS export domain (E, black square) or transmembrane domain (T, black square).</p

Pearson correlation analysis of gene expression profiles in <i>in vitro</i> untreated cultures.

<p>The expression profiles of the relative abundance of indicated genes in relation to seryl tRNA synthetase at day 2 were compared to each other in the untreated cultures (No NAG) and the Pearson correlation coefficient is indicated. Representative data shown is from one of 3 independent experiments.</p

Additional file 1 of Safety, efficacy, and pharmacokinetics of gremubamab (MEDI3902), an anti-Pseudomonas aeruginosa bispecific human monoclonal antibody, in P. aeruginosa-colonised, mechanically ventilated intensive care unit patients: a randomised controlled trial

Additional file 1: Supplementary Methods, Results, Tables and Figures