HHPred identification of novel F_O subunit a based on conserved structural features.

<p>(A) Representation of the pairwise sequence alignments generated by HHPred [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref046" target="_blank">46</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref047" target="_blank">47</a>] for the putative F_O subunit a from <i>T</i>. <i>gondii</i> and 4 other F_O subunit a proteins for which the structure is known. The table provides details of the amino acid length and a probability score for the prediction from the hit alignments. The red lines indicated the 3 transmembrane domains present in the <i>T</i>. <i>gondii</i> protein. (B) Protein sequence alignments and secondary structure information were made using the Clustal Omega [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref048" target="_blank">48</a>] and ESPript [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref049" target="_blank">49</a>] software, and only the C-terminal portion of respective proteins is shown. The names of alveolate species are highlighted in blue. For the nonalveolate species included in the alignment, the F_O subunit a is either readily identified from sequence (<i>Scer</i>, <i>Hsap</i>, <i>Atha</i>, <i>Ecol</i>, and <i>Pten</i>) or has been experimentally determined (<i>Crei</i> and <i>Poly</i>). Positions with similar amino acids are highlighted in red in the alignment. The arginine and glutamine residues, highlighted in yellow, are conserved in all species and important for function. The helices (α9, α10, and α12) shown are from the structure of <i>Poly</i> F-type ATP synthase. Species names: <i>Tgon</i>, <i>T</i>. <i>gondii</i>; <i>Hham</i>, <i>Hammondia hammondi</i>; <i>Pfal</i>, <i>P</i>. <i>falciparum</i>; <i>Pviv</i>, <i>P</i>. <i>vivax</i>; <i>Tann</i>, <i>Theileria annulata</i>; <i>Bbov</i>, <i>B</i>. <i>bovis</i>; <i>Cmur</i>, <i>C</i>. <i>muris</i>; <i>Cvel</i>, <i>C</i>. <i>velia</i>; <i>Crei</i>, <i>C</i>. <i>reinhardtii</i>; <i>Poly</i>, <i>Polytomella</i> sp.; <i>Scer</i>, <i>S</i>. <i>cerevisiae</i>; <i>Hsap</i>, <i>Homo sapiens</i>; <i>Atha</i>, <i>A</i>. <i>thaliana</i>; <i>Ecol</i>, <i>Escherichia coli</i>; <i>Pden</i>, <i>Paracoccus denitrificans</i>.</p

Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from <i>Toxoplasma gondii</i>

<div><p>The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In <i>T</i>. <i>gondii</i> and other apicomplexan parasites, many subunit components necessary for proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of <i>T</i>. <i>gondii</i> F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key F_O subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.</p></div

Identification of novel <i>T</i>. <i>gondii</i> F-type ATP synthase subunits from LC-MS/MS analysis.

<p>(A) The Venn diagram shows shared identification of proteins following BNP, SEC, and IP sample preparation. Total number of proteins identified in each technique is given within brackets outside the Venn diagram. The numbers shown in white font and within brackets are the final set of proteins assigned as subunit components of <i>T</i>. <i>gondii</i> F-type ATP synthase. (B) List of all genes identified in this work by mass spectrometry analysis. Gene ID and product description details are from Toxodb.org (release 36). The entries shown in bold indicate that the protein was detected with high confidence from only BNP and IP samples. Asterisk indicates that the corresponding peptides were detected in only one of the replicate runs for SEC sample. BNP data are a combination of experiments done with both <i>Tg</i>ATPβ-YFP-HA–and <i>Tg</i>ATPOSCP-YFP-HA–expressing transgenic parasites. SEC and IP data are from <i>Tg</i>ATPβ-YFP-HA and <i>Tg</i>ATPOSCP-YFP-HA expressing transgenic parasites, respectively. BNP, blue native PAGE; IP, immunoprecipitation; LC-MS/MS, liquid chromatography–tandem mass spectrometry; OSCP, oligomycin sensitivity–conferring protein; SEC, size exclusion chromatography; <i>Tg</i>ATPβ, <i>T</i>. <i>gondii</i> ATP synthase β subunit; <i>Tg</i>ATPOSCP, <i>T</i>. <i>gondii</i> ATP synthase OSCP subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.</p

Identifying the dimer and monomer forms of <i>T</i>. <i>gondii</i> F-type ATP synthase by BNP analysis.

<p>(A) Mitochondria lysates were prepared from tachyzoites stage transgenic parasites expressing <i>Tg</i>ATPβ-YFP-HA protein were separated by BNP. Lane M, native molecular weight markers; lane A, Coomassie blue staining of BNP gel; lane B, western blotting of BNP gel using α-HA antibodies. The dimer and monomer forms are indicated by arrows. The boxed regions in lane A correspond to the excised gel pieces, which were processed for LC-MS/MS analysis. (B) In-gel ATPase activity assays following BNP separation confirms that the dimer and monomer forms of F-type ATP synthase are functionally intact. BNP, blue native PAGE; HA, hemagglutinin; LC-MS/MS, liquid chromatography–tandem mass spectrometry; <i>Tg</i>ATP-β, <i>T</i>. <i>gondii</i> ATP synthase β subunit; <i>Tg</i>ATP-OSCP, <i>T</i>. <i>gondii</i> ATP synthase OSCP subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.</p

Phylogenetic profile of the alveolate infrakingdom for all <i>T</i>. <i>gondii</i> F-type ATP synthase subunits.

<p>Alveolate clades are highlighted with a gray background, and their expected phylogenetic relationship is indicated by a tree structure above. Gray and white boxes indicate the presence and absence of the corresponding ortholog, respectively. The hatched boxes represent the presence of the ortholog in <i>C</i>. <i>muris</i> only and absence in <i>C</i>. <i>parvum</i> and <i>C</i>. <i>hominis</i>. The table on the left lists the gene ID for all ASAPs, along with their annotation, essentiality phenotypes (phenotype score from CRISPR/Cas9 knockout study [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006128#pbio.2006128.ref050" target="_blank">50</a>]), and protein localization. The key for localization annotation is given below the table. ASAP, ATP synthase–associated protein; Cas9, CRISPR-associated 9; CRISPR, clustered regularly interspaced short palindromic repeat; OSCP, oligomycin sensitivity–conferring protein.</p

Detail of F-type ATP synthase subunits missing in <i>T</i>. <i>gondii</i> and other apicomplexan parasites.

<p>(A) Schematic representation of core subunit composition of F-type ATP synthase. The different subunits are color coded and annotated. Middle, the complete set of subunits of the core enzyme; left and right, subunits identified and not identified by in silico methods in apicomplexan genome. (B) Table showing the presence/absence of orthologs of yeast and bovine F-type ATP synthase in <i>Apicomplexa</i> and other alveolate species. +/−, presence/absence of ortholog; blue, diverged (no clear orthologs) functional equivalent; light blue, experimentally identified species-specific novel subunits (numeric values indicate the total number of such novel subunits). Species names: <i>Pfal</i>, <i>P</i>. <i>falciparum</i>; <i>Pviv</i>, <i>Plasmodium vivax</i>; <i>Tpar</i>, <i>Theileria parva</i>; <i>Bbov</i>, <i>Babesia bovis</i>; <i>Tgon</i>, <i>T</i>. <i>gondii</i>; <i>Ncan</i>, <i>Neospora caninum</i>; <i>Cmur</i>, <i>Cryptosporidium muris</i>; <i>Cpar</i>, <i>Cryptosporidium parvum</i>; <i>Chom</i>, <i>Cryptosporidium hominis</i>; <i>Cvel</i>, <i>Chromera velia</i>; <i>Pmar</i>, <i>Perkinsus marinus</i>; <i>Tthe</i>, <i>T</i>. <i>thermophila</i>; <i>Scer</i>, <i>Saccharomyces cerevisiae</i>; <i>Btau</i>, <i>Bos taurus</i>; <i>Atha</i>, <i>Arabidopsis thaliana</i>; <i>Crei</i>, <i>Chlamydomonas reinhardtii</i>; <i>Tbru</i>, <i>T</i>. <i>brucei</i>. IF1, inhibitory factor 1; OSCP, oligomycin sensitivity–conferring protein.</p

Partial purification of dimer and monomer forms of <i>T</i>. <i>gondii</i> F-type ATP synthase by chromatography.

<p>(A) Ion exchange (DEAE sepharose) separation of mitochondrial lysates prepared from <i>Tg</i>ATPβ-YFP-HA expressing transgenic parasites. Absorbance at 280 nm (filled circles) and NaCl concentration (open circles) are plotted for each fraction. Fractions 5 and 18 are marked with arrows. Bottom panel shows SDS-PAGE western blotting for fractions 5–18 to find out the elution profile of <i>Tg</i>ATPβ-YFP-HA. (B) Size exclusion profile of the pooled fractions from ion exchange chromatography. The absorbance at 280 nm is plotted for each fraction. The size exclusion column was calibrated with the following native markers: thyroglobulin (labeled “T,” 660 kDa), ferritin (labeled “F,” 440 kDa), conalbumin (labeled “C,” 75 kDa), Ovalbumin (labeled “O,” 45 kDa). Peak elution volume for each marker is indicated by arrow. Fractions 1–3, 4–6, and 7–9 were pooled, concentrated, and subject to SDS-PAGE western blotting to detect <i>Tg</i>ATPβ-YFP-HA, as shown in bottom panel. DEAE, diethylaminoethanol; OD, optical density; <i>Tg</i>ATPβ, <i>T</i>. <i>gondii</i> ATP synthase β subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.</p

Srebrenica as a symbol in international politics

The bachelor thesis deals with the massacre in Srebrenica which took place during the war in Bosnia and Herzegovina in 1995. In the theoretical part is explained the concept of symbol in international relations. Furthermore ethnic development as well as events which led to the Srebrenica massacre. In the practical part the thesis tries to answer the question whether the massacre on the basis of media became a symbol and if it has special meaning

Resistance to <i>B</i>. <i>malayi</i> is dominant and sex-linked, and it impairs early stages of <i>B</i>. <i>malayi</i> development.

<p>Female progeny from a backcross inherited the resistance phenotype of their maternal grandparent, whether resistance was measured as A) the proportion of mosquitoes infected with L3 stage parasites or B) the number of L3 stage parasites at 10 days after infection. S♀ x R♂ (referred to as susceptible from this point forward) were created by crossing an LVP^S female with an LVP^R male, followed by backcrossing the F₁ progeny to LVP^S. R♀ x S♂ (referred to as resistant) were created in the same way except an LVP^R female was crossed with an LVP^S male in the parental generation. By 24 hours after infection, many <i>B</i>. <i>malayi</i> microfilariae have migrated from the midgut to the thoracic tissues in both C) susceptible and D) resistant hosts. At 48 hours after infection, microfilariae are molting into the non-feeding L1 developmental stage in E) susceptible hosts, whereas growth is arrested in F) resistant hosts. By 72 hours after infection, nearly all surviving <i>B</i>. <i>malayi</i> are in the L1 stage in G) susceptible hosts, whereas they are dead or dying in H) resistant hosts. I) Genome-wide gene expression levels of <i>B</i>. <i>malayi</i> are comparable in resistant and susceptible hosts at 12 hours after infection, whereas by 48 hours, gene expression, and presumably growth, of <i>B</i>. <i>malayi</i> is higher in susceptible mosquitoes. <i>B</i>. <i>malayi</i> gene expression is estimated by dividing the total number of RNA-seq reads mapping to the <i>B</i>. <i>malayi</i> genome by the total number of reads mapping to the <i>Ae</i>. <i>aegypti</i> genome.</p

Assembly of the Genome of the Disease Vector <i>Aedes aegypti</i> onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

<div><p>The mosquito <i>Aedes aegypti</i> transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of <i>Ae. aegypti</i>, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes <i>Ae. aegypti</i> resistant to the human parasite <i>Brugia malayi</i>, and generated a list of candidate genes that could be affecting the trait.</p></div