Project description:Purpose: The aim was, in the frame of an anti-vector strategy, to use such genes to control Human and/or animal trypanosomiasis. The present objective was to verify whether field tsetse fly gene expression was modified in response to natural infection with trypanosomes as it was when insectary-raised flies were experimentally infected. Method: mRNA from 10 samples of Glossina palpalis (5 non-infected and 5 infected by Trypanosoma congolense s.l.) were sequenced on a high-output flow cell (400M clusters) using the NextSeq® 500/550 High Output v2 150 cycles kit (Illumina), in paired-end 75/75nt mode. The sequenced reads that passed quality filter were mapped onto the genome with the local alignment algorithm subread-align (Liao et al., 2013). Differential analysis was done with the SARTools R package (Varet et al., 2016), which runs separately DESeq2 (Love et al., 2014) and egdeR (Robinson et al., 2010). We identified orthologs between Glossina and Drosophila based on the identification of bidirectional best hits (BBH) using blastp (Altschul et al., 1997), and then made functional enrichment and pathway mapping of these DEGs. Results: Using the RNA-seq approach, differentially expressed genes (DEGs) have been identified in infected versus non-infected tsetse flies, including down-regulated genes and up-regulated genes. Some of the genes, whether down- or up-regulated, were very highly differentially expressed. Down-regulated genes were mainly involved in transcription/translation processes, while up-regulated encoded genes governing amino acid and nucleotide biosynthesis pathways. The data on the molecular cross-talk between the host and the parasite (and the fly microbiome that is always present) recorded when using an experimental biological model have its counterpart in field flies which in turn validates the use of experimental host/parasite couples. Conclusion: This study is the first evaluation of transcriptomic mechanisms related to infection in field tsetse flies. This opens up prospects for vector-based control strategies, and more precisely the blocking of transmission.
Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.
Project description:The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vThe infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic Trapanosoma forms. We used an in vitro system based on expressing the RNA binding protein 6 (RBP6) to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNAseq) in comparison to non-infectious procyclic trypanosomes. This comparison showed that metacyclics are quiescent cells, and we propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector. ector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic Trapanosoma forms. We used an in vitro system based on expressing the RNA binding protein 6 (RBP6) to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNAseq) in comparison to non-infectious procyclic trypanosomes. This comparison showed that metacyclics are quiescent cells, and we propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.
Project description:Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle since the insect stage utilizes the cost-effective oxidative phosphorylation to generate ATP, while bloodstream cells switch to less energetically efficient aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector for biochemical analysis, the dynamics of the parasite´s mitochondrial metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA levels.