Project description:BACKGROUND: The gene regulation mechanism along the life cycle of the genus Schistosoma is complex. Small non-coding RNAs (sncRNAs) are essential post transcriptional gene regulation elements that affect gene expression and mRNA stability. Preliminary studies indicated that sncRNAs in schistosomal parasites are generated through different pathways, which are developmentally regulated. However, the data of sncRNAs of schistosomal parasites are still fragmental and a complete expression profile of sncRNAs during the parasite development requires a deep investigation. METHODOLOGY/PRINCIPAL FINDINGS: We employed high-throughput genome-wide transcriptome analytic techniques to explore the dynamic expression of microRNAs (miRNAs) and endogenous siRNAs (endo-siRNAs) of Schistosoma japonicum covering the free-living cercarial stage and all stages in the definitive host. This led us to analyze over 70 million clean reads represented both high and low abundance of the small RNA population. Patterns of differential expression of miRNAs and endo-siRNAs were observed. MiRNAs was twice more than endo-siRNAs in cercariae, but gradually decreased along with the development of the parasite. Both small RNA types were presented in equal aboudance in lung-stage schistosomula, while endo-siRNAs accumulated to 6 times more than miRNAs in adult female worms and hepatic eggs. Further, miRNAs were found mainly derived from genes located in the intergenic regions, while endo-siRNAs were mainly generated from transposable elements (TEs). The expression pattern of TE-siRNAs, as well as the pseudogene-derived siRNAs clustered in mRNAs of cytoskeletal proteins, stress proteins, enzymes related to energy metabolism also revealed distinction throughout different developmental stages. Natural antisense transcripts (NATs)-related siRNAs accounted for minor proportion of the endo-siRNAs which were dominantly expressed in cercariae. CONCLUSIONS/SIGNIFICANCE: Our results represented a comprehensive expression profile of sncRNAs in various developmental stages of S. japonicum with high accuracy and coverage. The data would facilitate a deep understanding of the parasite biology and potential discovery of novel targets for the design of anti-parasite drugs.
Project description:We developed an optimized multi-shot proteomics workflow based on high-resolution offline high pH reversed-phase peptide separation of high peptide loads collecting many fractions that were in turn analyzed by short online chromatographic separations and fast peptide sequencing using orbitrap tandem mass spectrometry.
Project description:Primary outcome(s): Concordance rate of both KRAS and NRAS gene exon 2, 3 and 4 mutations between standard genetic testings including sanger sequencing and an established in vitro diagnostic (IVD) kit for KRAS exon2, and a newly developed Luminex-based all RAS assay kit
Project description:BACKGROUND: Schistosomes are trematode parasites of the phylum Platyhelminthes. They are considered the most important of the human helminth parasites in terms of morbidity and mortality. Draft genome sequences are now available for Schistosoma mansoni and Schistosoma japonicum. Non-coding RNA (ncRNA) plays a crucial role in gene expression regulation, cellular function and defense, homeostasis, and pathogenesis. The genome-wide annotation of ncRNAs is a non-trivial task unless well-annotated genomes of closely related species are already available. RESULTS: A homology search for structured ncRNA in the genome of S. mansoni resulted in 23 types of ncRNAs with conserved primary and secondary structure. Among these, we identified rRNA, snRNA, SL RNA, SRP, tRNAs and RNase P, and also possibly MRP and 7SK RNAs. In addition, we confirmed five miRNAs that have recently been reported in S. japonicum and found two additional homologs of known miRNAs. The tRNA complement of S. mansoni is comparable to that of the free-living planarian Schmidtea mediterranea, although for some amino acids differences of more than a factor of two are observed: Leu, Ser, and His are overrepresented, while Cys, Meth, and Ile are underrepresented in S. mansoni. On the other hand, the number of tRNAs in the genome of S. japonicum is reduced by more than a factor of four. Both schistosomes have a complete set of minor spliceosomal snRNAs. Several ncRNAs that are expected to exist in the S. mansoni genome were not found, among them the telomerase RNA, vault RNAs, and Y RNAs. CONCLUSION: The ncRNA sequences and structures presented here represent the most complete dataset of ncRNA from any lophotrochozoan reported so far. This data set provides an important reference for further analysis of the genomes of schistosomes and indeed eukaryotic genomes at large.
Project description:We developed an optimized multi-shot proteomics workflow based on high-resolution offline high pH reversed-phase peptide separation of high peptide loads collecting many fractions that were in turn analyzed by short online chromatographic separations and fast peptide sequencing using orbitrap tandem mass spectrometry.
Project description:Suppression subtractive hybridization(SSH) libraries of Schistosoma japanicum female and male worms were constructed by using Clontech PCR-selectTM cDNA subtraction kit. S.japonicum cDNA microarrays were fabricated using female and male cDNA clones originating from SSH libraries. female-associated and Male-associated differentially expressed gene clones were obtained, Analysis of gender-associated differentially expressed genes helps to determine which genes are important to sexual maturation of schistosome and better understand schistosome biology and host-parasite relationship, facilitate the discovery of novel gene products that could represent targets for the development of new drugs and vaccines to control chitosomiasis. Keywords: gender-associated
Project description:BackgroundSchistosoma japonicum is a parasitic flatworm that causes zoonotic schistosomiasis. The typical outcome of schistosomiasis is hepatic granuloma and fibrosis, which is primarily induced by soluble egg-derived antigens. Although schistosomal eggs represent an important pathogenic stage to the host, the biology of this critical stage is largely unknown. We previously investigated the expression profiles of sncRNAs during different developmental stages of this parasite. However, using small RNA extracted from egg-deposited liver tissues generated limited information about sncRNAs in eggs. Here, we characterized the complete small RNAome in this stage of the parasite after optimization of RNA purification.Methodology and principal findingsA library, SjE, was constructed with the small RNA extracted from S. japonicum eggs and subjected to high-throughput sequencing. The data were depicted by comprehensive bioinformatic analysis to explore the expression features of sncRNAs in the egg stage. MicroRNAs accounted for about one quarter of the total small RNA population in this stage, with a strongly biased expression pattern of certain miRNA family members. Sja-miR-71, sja-miR-71-5p, and sja-miR-36-3p were suggested to play important roles in embryo development. A panel of transfer RNA fragments (tRFs) precisely processed from the 5' end of mature tRNAs was identified for the first time, which represented a strong egg stage-biased expression. The tRNA-Ala derived small RNAs were the most highly expressed Sj-tRFs in eggs. Further, the expression of siRNAs from 29 types of well-defined transposable elements (TEs) was observed to be relatively stable among different developmental stages.Conclusions and significanceIn this study, we characterized the sncRNA profile in the egg stage of S. japonicum. Featured expression of sncRNAs, especially the tRNA-derived small RNAs, was identified, which was further compared with that of other developmental stages. These novel findings would facilitate a deeper understanding of the biology of schistosomal parasites.
Project description:Parasitic flatworms of the genus Schistosoma are the causative agents of schistosomiasis, which afflicts more than 200 million people yearly in tropical regions of South America, Asia and Africa. A promising approach to the control of this and many other diseases involves the application of our understanding of small non-coding RNA function to the design of safe and effective means of treatment. In a previous study, we identified five conserved miRNAs from the adult stage of Schistosoma japonicum. Here, we applied Illumina Solexa high-throughput sequencing methods (deep sequencing) to investigate the small RNAs expressed in S. japonicum schistosomulum (3 weeks post-infection). This has allowed us to examine over four million sequence reads including both frequently and infrequently represented members of the RNA population. Thus we have identified 20 conserved miRNA families that have orthologs in well-studied model organisms and 16 miRNA that appear to be specific to Schistosoma. We have also observed minor amounts of heterogeneity in both 3' and 5' terminal positions of some miRNA as well as RNA fragments resulting from the processing of miRNA precursor. An investigation of the genomic arrangement of the 36 identified miRNA revealed that seven were tightly linked in two clusters. We also identified members of the small RNA population whose structure indicates that they are part of an endogenously derived RNA silencing pathway, as evidenced by their extensive complementarities with retrotransposon and retrovirus-related Pol polyprotein from transposon.