Enriched gene interaction network detected with similar expression pattern in PZQ-treated paired males and females.

<p>The gene interaction network is related to carbohydrate metabolism, molecular transport and small molecule biochemistry. The shapes of elements correspond to different types of molecules, as indicated in the inset box. Arrows indicate the relationship between the elements: dashed or solid lines indicate indirect or direct interactions, respectively. The color intensity is proportional to expression value, computed as log2 [PZQ/Control]; red corresponds to positive log-ratios, <i>i</i>.<i>e</i>. genes up-regulated in paired males and females treated with PZQ when compared with their respective no-drug controls, grey corresponds to those genes present in the analysis but not differentially expressed. In humans, one gene homolog in this network encodes a protein that is a known drug target and the corresponding drugs (Rx) are indicated.</p

Global transcriptional changes driven by PZQ on <i>S</i>. <i>mansoni</i> adult worms.

<p>Overall summary with the names of the associated functional interaction networks identified as significantly enriched with differentially expressed <i>S</i>. <i>mansoni</i> genes in different comparisons: (A) genes affected by PZQ in opposite directions in paired or unpaired mature females, (B) all genes affected by PZQ in paired females, (C) all genes affected by PZQ in unpaired mature females, (D) all genes affected by PZQ in paired males, (E) genes affected in common in paired males and females. Color arrows indicated the direction of change in expression of the majority of the genes in each network: green implies a down-regulated expression of the majority of the genes in PZQ-treated worms, and red an up-regulated expression of the majority of the genes in PZQ-treated worms when compared with their respective no-drug controls. Rx corresponds to the identification of new candidate drugs known to act on the encoded protein products of the homolog human genes present in the indicated network, as described in the main text.</p

Synergy of Omeprazole and Praziquantel <i>In Vitro</i> Treatment against <i>Schistosoma mansoni</i> Adult Worms

<div><p>Background</p><p>Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.</p><p>Methodology</p><p>We used a custom-designed <i>Schistosoma mansoni</i> expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against <i>S</i>. <i>mansoni</i> adult worms with a survival <i>in vitro</i> assay.</p><p>Principal Findings</p><p>We identified sets of genes that were affected by PZQ in paired and unpaired mature females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired mature females), indicating that PZQ effects are heavily influenced by the mating status. We also identified genes that were similarly affected by PZQ in males and females. Functional analyses of gene interaction networks were performed with parasite genes that were differentially expressed upon PZQ treatment, searching for proteins encoded by these genes whose human homologs are targets of different drugs used for other diseases. Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested. Sublethal doses of PZQ combined with OMP significantly increased worm mortality <i>in vitro</i> when compared with PZQ or OMP alone, thus evidencing a synergistic effect.</p><p>Conclusions</p><p>Functional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against <i>S</i>. <i>mansoni</i> adult worms <i>in vitro</i> when compared with either drug alone.</p></div

General information about gene expression of paired or unpaired mature females exposed to PZQ compared with no-drug controls.

<p>^a Number of genes expressed in at least one condition, <i>i</i>.<i>e</i>. in no-drug control or in treated worms of either the paired or the unpaired worm groups.</p><p>^b q-value < 5%</p><p>^c Either predicted in <i>S</i>. <i>japonicum</i>, present in other species, or detected only in <i>S</i>. <i>mansoni</i> without close homologs in other species (no match).</p><p>General information about gene expression of paired or unpaired mature females exposed to PZQ compared with no-drug controls.</p

General information about gene expression in paired males exposed to PZQ compared with no-drug controls.

<p>^a Number of genes expressed in at least one condition, <i>i</i>.<i>e</i>. in control or in treated paired male adult worms.</p><p>^b q-value < 5%</p><p>^c Either predicted in <i>S</i>. <i>japonicum</i>, present in other species, or detected only in <i>S</i>. <i>mansoni</i> without close homologs in other species (no match).</p><p>General information about gene expression in paired males exposed to PZQ compared with no-drug controls.</p

Kaplan-Meier survival curves for adult <i>S</i>. <i>mansoni</i> worms treated <i>in vitro</i> with PZQ+OMP, PZQ or OMP.

<p>Adult worm couples were treated with a synergistic combination of PZQ+OMP (blue line), with PZQ alone (red line) or with OMP alone (black line). The assays were performed with paired adult worm couples (20 couples for each of the three conditions in each of the two biological replicates) and the fraction of surviving worms was recorded after 2, 24, 48, 72, 96 and 120 hours; the Kaplan-Meier survival curve was calculated using all the events of the two biological replicas together, and the Log-rank Mantel-Cox statistical test was used to calculate the significance of the Hazard Ratio between OMP+PZQ and PZQ alone. The effect of the synergistic combination was analyzed for each gender separately: (A) for scoring males percent survival, couples were incubated in the presence of 150 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone; and (B) for scoring females percent survival, couples were incubated in the presence of 532 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone.</p

Most significantly enriched interaction network of differentially expressed genes in PZQ-treated paired males.

<p>The gene interaction network is related to cellular assembly and organization, cell-to-cell signaling and interaction. The shapes of elements correspond to different types of molecules, as indicated in the inset box. Arrows indicate the relationship between the elements: dashed or solid lines indicate indirect or direct interactions, respectively. The color intensity is proportional to expression value, computed as log2 [PZQ/Control]; red corresponds to positive log-ratios, <i>i</i>.<i>e</i>. genes up-regulated in paired males treated with PZQ when compared with no-drug controls, green corresponds to negative log-ratios, <i>i</i>.<i>e</i>. down-regulated genes, and grey corresponds to those genes present in the analysis but not differentially expressed. Two genes from this network encode human proteins that are known drug targets and the corresponding drugs (Rx) are indicated.</p

Validation by RT-qPCR of expression changes induced by PZQ treatment in <i>S</i>. <i>mansoni</i> adult worms.

<p>The expression levels of genes were measured by RT-qPCR in parasites treated with PZQ (black bars) relative to the levels in the respective no-drug control parasites (gray bars). Genes were measured (A) in paired females; (B) in unpaired mature females; (C) in paired males; and (D) in paired females. Genes with significant difference (t-test, p < 0.05) are marked with asterisk. Next to each gene name, the gene expression fold-change induced by PZQ and detected by microarray is shown for comparison.</p

Enriched gene interaction network detected with opposite expression patterns in PZQ-treated paired or unpaired mature females.

<p>This gene interaction network is significantly enriched (p-value = 10⁻⁵²) with differentially expressed genes related to cellular development and drug metabolism whose expression was affected by PZQ in paired as well as in unpaired mature females (with opposite patterns). The shapes of elements correspond to different types of molecules, as indicated in the inset box. Arrows indicate the relationship between the elements: dashed or solid lines indicate indirect or direct interactions, respectively. The color intensity is proportional to the expression value, computed as log2 [PZQ/Control]; red corresponds to positive log-ratios, <i>i</i>.<i>e</i>. these genes were up-regulated in paired females treated with PZQ when compared with the no-drug controls; grey corresponds to a gene present in the analysis but not differentially expressed. In humans, a number of gene homologs in this network encode proteins that are known drug targets and the corresponding drugs (Rx) are indicated.</p

Single nucleotide variants and InDels identified from whole-genome re-sequencing of Guzerat, Gyr, Girolando and Holstein cattle breeds

<div><p>Whole-genome re-sequencing, alignment and annotation analyses were undertaken for 12 sires representing four important cattle breeds in Brazil: Guzerat (multi-purpose), Gyr, Girolando and Holstein (dairy production). A total of approximately 4.3 billion reads from an Illumina HiSeq 2000 sequencer generated for each animal 10.7 to 16.4-fold genome coverage. A total of 27,441,279 single nucleotide variations (SNVs) and 3,828,041 insertions/deletions (InDels) were detected in the samples, of which 2,557,670 SNVs and 883,219 InDels were novel. The submission of these genetic variants to the dbSNP database significantly increased the number of known variants, particularly for the indicine genome. The concordance rate between genotypes obtained using the Bovine HD BeadChip array and the same variants identified by sequencing was about 99.05%. The annotation of variants identified numerous non-synonymous SNVs and frameshift InDels which could affect phenotypic variation. Functional enrichment analysis was performed and revealed that variants in the olfactory transduction pathway was over represented in all four cattle breeds, while the ECM-receptor interaction pathway was over represented in Girolando and Guzerat breeds, the ABC transporters pathway was over represented only in Holstein breed, and the metabolic pathways was over represented only in Gyr breed. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Gyr, Girolando, Guzerat and Holstein breeding programs.</p></div