Project description:A laboratory colony of Phlebotomus perniciosus sand flies was maintained. Sand flies were infected with cultured Leishmania infantum promastigotes in stationary phase. Ten infected sand flies were dissected after 5 days and promastigotes within the gut pooled. The cells were immediately washed in PBS once and lysed in TRIzol reagent (Life Technologies). RNA isolation was completed according to the manufacturer's instructions, obtaining 63ng. RNA-seq libraries were generated using the spliced leader sequence for second strand synthesis (Cuypers et al., 2017; Haydock et al., 2015), thus allowing for specific amplification of sequences from L. infantum promastigotes, thus avoiding contamination with material from the sand fly gut. Single-end sequencing was performed in an Illumina HiSeq2500 instrument and data analysis was conducted using bowtie2, samtools, featureCounts and Geneious. The main findings are: i) substantial differences in differential gene expression between sand fly-derived (sfPro) and cultured (acPro) promastigotes; and ii) over-expression of genes involved in metacyclogenesis in sfPro vs. acPro, including gp63 genes, autophagy genes, etc.
Project description:Leishmania infantum (Kinetoplastida:Trypanosomatidae) is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The motile promastigote stage infects the hematophagous sand fly vector host and amastigotes survives and multiplies within phagocytes of the mammalian host. Promastigotes are routinely cultured in liquid undefined media and are considered to mimic the environment within the sand fly gut. We have put this to the test by high-throughput gene expression profiling by shotgun DNA microarrays generated in our laboratory. This has been possible thanks to RNA amplification.
Project description:Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil, being domestic and stray dogs the main reservoirs. The development of the parasite involves two stages. The promastigote is extracellular and develops within the sand fly gut. The amastigote survives inside the harsh environment of the phagolysosome of mammalian host phagocytes, where pH is acidic, temperature higher than in the sand fly vector and hydrolytic enzymes act. In addition, the host phagocyte displays the nitric oxide defense mechanism against the amastigote. Promastigotes are also able to withstand NO even when they develop within the sand fly gut. This can be explained with the pre-adaptative hypothesis, which has been supported by us and others elsewhere and consists of preparation of promastigotes in advance for development towards the amastigote stage. For this reason, the comparison of NO-resistant and sensitive promastigotes is valuable. The two-dimension electrophoresis-mass spectrometry (2DE-MS/MS) approach has been performed to study differential protein abundance comparing L. chagasi NO-sensitive and resistant promastigotes. This analysis has revealed differential expression of genes directly and indirectly involved in NO-resistance, highlighting up-regulation of the glucose-6-phosphate dehydrogenase (G6PD) in NO-resistant promastigotes and down-regulation of the glutathione peroxidase (GPX) and the arginase (ARG) in NO-sensitive ones. These data are a starting point in the search of vaccine candidates and/or drug targets.
Project description:Purpose: Using NGD techinque to profile the gene expression in KDR+ hematopietic progenitor cells (HPC) and KDR- HPC during tumor progression. The goal is to study how KDR signaling within hematopieosis respond to tumor progression by analyzing transcriptome of KDR+ HPC Method: Isolate KDR + HPC and KDR- HPC from late stage GL261 tumor mice, and HPC from non tumor mice. Compare their transcriptomes.by deep sequencing, in triplicate. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: Basedl analysis of transcirptome and expression pattern of 11650 genes, KDR+ HPC resprent a different population compared with KDR- HPC in tumor mice or HPC in non-tumor mice.
Project description:Human T-cell leukemia virus type 1 (HTLV-1) is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax oncoprotein regulates viral gene expression and the NF-kB pathway to promote the survival of HTLV-1 infected T cells. In thsi study, we utilize a kinome-wide shRNA screen to identify the tyrosine kinase KDR/VEGFR2 as an essential survival factor of HTLV-1-transformed T cells. Inhibition of KDR induces apoptosis of Tax expressing HTLV-1-transformed cell lines and CD4+ T cells from HAM/TSP patients. Phosphoproteomics analysis of HTLV-1 transformed cells treated with a KDR inhibitor revealed inhibition of the phosphorylation of multiple receptors/cell surface proteins, ubiquitin conjugating systems, proteases, phosphatases, apoptotic regulatory factors, adhesion/extracellular matrix proteins and viral proteins. This work suggests that HTLV-1 Tax has hijacked KDR kinase activity to promote Tax stability and the proliferation and survival of HTLV-1 infected cells.