The definition of Echinococcus multilocularis differentially expressed molecules using deep sequencing

Echinococcus multilocularis has recently been developed as a model for basic and applied studies on trematodes and cestodes. Deep understanding of molecular biology of this parasite is urgently required for efficient and extensive applications such as functional gene probing and anti-helminth drug screening. In our project, we aimed to unveil E. multilocularis miRNA repertoires and the identification of differentially expressed molecules from the mRNA and miRNA transcriptomes using next-generation sequencing technology. Furthermore, one family of E. multilocularis molecules identified, fatty acid-binding proteins that are involved in lipid metabolisms, was phylogenetically and functionally characterized. Our data presented genome-widely developmental expression profiles of E. multilocularis miRNAs. Sixty-five potential miRNAs were predicted with nineteen of them being novel. The majority of differentially expressed miRNAs such as emu-miR-1, 16 and 71 were found to be significantly down-regulated in the primary neoblast-rich cells derived from the vesicles. Comparative analysis revealed that the miR-71/2 cluster was conserved in the platyhelminths. In addition, seven homologues to miRNAs likely linked to planarian neoblasts were present in E. multilocularis with the intact seed regions. Although their expression in E. multilocularis neoblasts remains to be fully certified, these miRNAs are plausible biomarkers for the germline stem cells. For mRNA profiling, we firstly originated an enzymatic approach for the elimination of mitochondrial transcripts to increase the depth of mRNA transcriptomes. In comparison with 2.7% in the control, the frequency of the sequences mapped to the mitochondria was dramatically reduced to 0.04~0.1% in the treated samples without significantly adverse effects on the targets. A number of differentially expressed genes were inferred from E. multilocularis mRNA transcriptomes and FABP-encoding genes were further phylogenetically and functionally characterised. E. multilocularis encoded at least four distinct FABPs and comparative analysis revealed that the current gene set of FABPs may have emerged before speciation of E. multilocularis and E. granulosus. The apparent divergence between Echinococcus and vertebrate FABPs in genomic organizations prompted us to further explore phylogenetically FABP genes across thirty-five invertebrates. The results demonstrated that FABP genes were organized in diverse ways, with a predominant pattern that is commonly present in vertebrates. Moreover, both gene duplication and alternative splicing might be most likely responsible for variety of invertebrate FABP functions. Four E. multilocularis FABPs were developmentally regulated and FABP3 was highly expressed in the vesicles and secreted or released into the hydatid fluid. These proteins appeared to be able to weakly interact with cis-parinaric acid but not a fluorescent fatty acid DAUDA and retinol. In vitro analysis indicated that FABP3 was capable of the induction of cytokine secretion by bone marrow-derived macrophages and dendritic cells probably via Toll-like receptor 2. Additionally, FABP3 was also demonstrated to activate macrophage small reactive molecule production together with a ligand for TLR 2