Project description:The interaction of animals with microbes relies on the specific recognition of microbial-derived molecules by receptors of the immune system. Sponges (phylum Porifera), as sister group of the Eumetazoa, provide insights into conserved mechanisms for animal-microbe crosstalk, but empirical data is limited. Here we aimed to characterize the immune response of sponges upon microbial stimuli by RNA-Seq. Two sponges species from the Mediterranean Sea, Aplysina aerophoba and Dysidea avara, were challenged with microbial-associated molecular patterns (lipopolysaccharide and peptidoglycan) or sterile artificial seawater (control) in aquarium experiments. Sponge tissue samples were collected 1h, 3h, and 5h after treatment. The response of the sponges to the treatments was assessed by differential gene expression analysis of RNA-Seq data. For each species, we compared the transcriptomic profiles of the samples in MAMP treatment to control within each time point.
Project description:In order to compare sponge and eumetazoan (higher animal) body plans, we identified and studied expression of a broad range of eumetazoan developmental regulatory genes in Sycon ciliatum (Calcispongiae). In this species, embryonic development is semi-synchronous within a population, synchronous within individuals, and oocytes and embryos occupy a significant fraction of the volume of the sponges during the reproductive period. RNASeq libraries representing non-reproductive (somatic) tissue slices along the body axis, as well as oocytes, embryos and free swimming larvae were generated from material obtained by sampling throughout the life cycle.
Project description:In order to compare sponge and eumetazoan (higher animal) body plans, we identified and studied expression of a broad range of eumetazoan developmental regulatory genes in Sycon ciliatum (Calcispongiae). In this species, embryonic development is semi-synchronous within a population, synchronous within individuals, and oocytes and embryos occupy a significant fraction of the volume of the sponges during the reproductive period. RNASeq libraries representing non-reproductive (somatic) tissue slices along the body axis, as well as oocytes, embryos and free swimming larvae were generated from material obtained by sampling throughout the life cycle.
Project description:MicroRNA (miRNA) sponges containing miRNA complementary binding sites constitute a potentially useful strategy for miRNA-inhibition therapeutics in cancer patients. Recently, naturally occurring circular RNAs (circRNAs) have been revealed to function as efficient microRNA sponges. We hypothesized that synthetic circRNA sponges targeting oncomiRs could be constructed and used to achieve potentially therapeutic microRNA loss of function. In this study, linear RNA molecules containing five miR-21 binding sites were transcribed in vitro. After dephosphorylation by calf intestinal phosphatase and phosphorylation by T4 polynucleotide kinase, circRNA sponges were circularized using 5’-3’ end ligation by T4 RNA ligase 1. Synthetic circular sponge stability was assayed in the presence of RNase R or fetal bovine serum. Luciferase reporter and cell proliferation assays were performed to assess competitive inhibition of miR-21 activity by circRNA sponges in NCI-N87 gastric cancer cells. Tandem Mass Tag (TMT) labeling proteomics analysis and Western blotting were performed to delineate effects of circRNA sponges on miR-21 downstream targeted proteins. Our experiments revealed that artificial circRNA sponges can be synthesized using enzymatic ligation. These synthetic circRNA sponges are more resistant than their linear RNA counterparts to nuclease degradation in vitro. They effectively suppress the activity of miR-21 on its downstream protein targets, including the important cancer protein DAXX. Finally, they also inhibit gastric cancer cell proliferation. Our results suggest that synthetic circRNA sponges represent a rapid, effective, convenient strategy to achieve loss of miRNA function in vitro, with potential future therapeutic application in vivo.
Project description:Sponges (Porifera) are early-branching Metazoa who do not posess muscles or neurons, however are able to undergo a whole-body movement that involves the closure of their canal system and collapse of an epithelial tent. In this study we profile proteomic responses of the freshwater sponge Spongilla lacustris during agitation induced movements to elucidate the early evolution of coordination. Results from Thermal Proteome Profiling (TPP) experiments of agitated sponges suggest active secretion during the movement. Here, we use tandem mass tag (TMT) labeling-based quantification of proteins in the medium to systematically measure quantitative differences of secretion before and after agitation induced movement. In total, 146 proteins were detected in the medium, 47 of which were significantly upregulated during the movement.