Project description:Giardia lamblia is one of most common agents causing persistent abdominal symptoms in developed and developing countries. There are several diagnostic methods for Giardia infection, but none are optimal. In this study our aim was to find a new method based on Giardia microRNA (miRNA) that would contribute to the currently available diagnostic methods of giardiasis. Profiling Giardia small RNAs by deep sequencing revealed that the previously reported putative miR5 and miR6 are expressed in several G. lamblia isolates. These miRNAs were later tested by PCR in duodenal biopsies from 8 patients with positive pathology for giardiasis, while gastric biopsies served as matched negative controls. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. All 8 duodenal samples of patients with histologically proven G. lamblia infection were positive for Giardia miR5 with a mean Ct of 23.7. These results were superior to Ct levels of G. lamblia DNA, which were 26.3 (p=0.004). The miR6 results were close to negative. All 10 gastric biopsies were negative for miR5. Stool studies showed 90% specificity but only 50% sensitivity in diagnosing giardiasis using miR6. The results of miR5 in stool were even less accurate. In conclusion, miR5 testing for Giardia infection in duodenal biopsies, may be a breakthrough method for diagnosis of giardiasis. It seems to be superior to G. lamblia DNA in duodenal biopsies. It would be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies and duodenal aspirates from patients with persistent abdominal symptoms.
Project description:Transcriptional profiling of Giardia lamblia WB comparing control 5'5npac cells with pPMBF1 cells. Multiprotein bridging factor 1 (MBF1) gene family proteins are involved in cell growth and differentiation in various organisms. We asked whether Giardia MBF1-like protein can induce various endogenous genes. We used epitope-tagged system to overexpress Giardia MBF1 by constructing and transfecting pPMBF1, a plasmid for expressing MBF1, to trophozoites. The control cell line is 5'5npac cell line, which expressed only the puromycin selection marker. To understand the effect of MBF1 overexpression on transcription profiles, we performed microarray analysis for pPMBF1 and 5'5npac cell lines.
Project description:To investigate the magnitude of the transcriptional changes occurring during the life cycle of Giardia lamblia we compared the transcriptome of trophozoites and cysts. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription. Six cysts and three trophozoites life cycle stages were analyzed.
Project description:To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the two stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.