Project description:To examine the comparative transcription profile of WT and ALL1 mutant cells in in vitro conditions to determine genes resposible for hypervirulent phenotype of ALL1 mutant cells. Using transcriptional profiling, we determined that many genes involved in oxidation reduction processes and iron homeostasis were differentially transcribed between the wild type and the mutant strain For arrays, total RNA was isolated from WT and ALL1 mutant using Qiagen RNeasy Kit. For RNA extraction, Wt and ALL1 mutant were grown in two different culture medium conditions including minimal media and YPD broth. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.
Project description:Virulence of Cryptococcus neoformans for mammals was proposed to emerge from evolutionary pressures on its natural environment by protozoan predators, which selected for strategies that allow survival within macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least two-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes common to both groups, we focused on the ORF CNAG_05662, which was potentially related to sugar transport. We constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (Polyol Transporter Protein 1), is involved in the transport of 5- and 6-carbon polyols but its absence had no effect on virulence. Overall, our results are consistent with the hypothesis that mammalian virulence originated from fungal-protozoal interactions and provide a better understanding of how C. neoformans adapts to the mammalian host. Four conditions, pairwise-compared: cells in vegetative growth at 28C versus cells within amoebae at 28C; and cells in vegetative growth at 37C/5% CO2 versus cells within macrophages at 37C/5% CO2. Three biological replicates for each condition. One replicate per array.
Project description:Little is known about transcriptional control of neurite branching or presynaptic differentiation, events that occur relatively late in neuronal development. Using the C. elegans mechanosensory circuit as an in vivo model, we show that SAM-10, an ortholog of mammalian Single-Stranded DNA binding Protein (SSDP), functions cell-autonomously in the nucleus to regulate synaptic differentiation as well as positioning of a single neurite branch. PLM mechanosensory neurons in sam-10 mutants exhibit abnormal placement of the neurite branch point, and defective synaptogenesis, characterized by an overextended synaptic varicosity, underdeveloped synaptic morphology and disrupted co-localization of active zone and synaptic vesicles. SAM-10 functions coordinately with LDB-1 (Lim Domain Binding protein-1), demonstrated by our observations that 1) mutations in either gene show similar defects in PLM neurons; and 2) LDB-1 is required for SAM-10 nuclear localization. SAM-10 regulates PLM synaptic differentiation by suppressing transcription of prk-2, which encodes an ortholog of the mammalian Pim kinase family. PRK-2-mediated activities of SAM-10 are specifically involved in PLM synaptic differentiation, but not other sam-10 phenotypes such as neurite branching. Thus, these data reveal a novel transcriptional signaling pathway that regulates neuronal specification of neurite branching and presynaptic differentiation. Animals were grown on fresh 8P plates (Wormbook.org) until gravid. Eggs were collected by bleaching and hatched in M9 buffer overnight. L1 animals were fed on fresh food for 2 hours at room temperature, harvested and separated from worm debris using a 25μm mesh. RNA was isolated using a standard Trizol protocol. RNA preps were stored at -80C. Quality of RNA preps was first estimated using Agilent 2100 Bioanalyzer. RNA was then reverse-transcribed using the Genisphere Array 350 kit, which generate tagged cDNA. Resulted products are hybridized to the microarrays. Four independent high quality RNA preps (RIN>=7) were chosen for microarray assay using long oligomer-based spotted microarray (Washington University, St. Louis, MO). For dye-swap controls, we ran a technical replicate hybridization for each pair of samples in which the dyes are reversed. Gene spots with “found/good” signals (defined by ScanArray, channel flag 3) in all scans were chosen for gene expression analysis.
Project description:To examine the comparative transcription profile of WT and all1all2Δ mutant cells in in vitro conditions to determine genes resposible for hyporvirulent phenotype of all1all2Δ mutant cells. Using transcriptional profiling, we determined that many genes involved in amino acid transport were differentially transcribed between the wild type and the all1all2Δ mutant strain. The genes differentially expressed in double mutant of ALL1 ALL2 did not overlap with ALL1 and ALL2 comparative transcriptomes with RC2 (WT). For arrays, total RNA was isolated from WT and all1all2Δ mutant using Qiagen RNeasy Kit. For RNA extraction, WT and all1all2Δ mutant were grown in minimal media. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.
Project description:To examine the comparative transcription profile of WT and all2Δ mutant cells in in vitro conditions to determine genes responsible for hypervirulent phenotype of all2Δ cells. Using transcriptional profiling, we determined that many genes involved in transport were differentially transcribed between the wild type and the mutant strain. With the help of further experiments we could rule out that ALL2 has a unique function in maintaining the intracellular pH under acidic conditions. For arrays, total RNA was isolated from WT and all2Δ mutant using Qiagen RNeasy Kit. For RNA extraction, WT and all2Δ mutant were grown in minimal media. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.
Project description:Filarial nematodes are arthropod-borne nematodes that cause a variety of economically important diseases such as onchocerciasis (river blindness), lymphatic filariasis, and heartworm disease. The most widespread filarial disease of humans is lymphatic filariasis, caused by worms in the genera Wuchereria and Brugia. Lymphatic filariasis is an economic and social burden in endemic countries and affects approximately 119 million people worldwide (Michael, 1997). In humans, the worms live in and block the lymph vessels, causing improper flow of lymph, and inflammation of the lymphatic system. The symptoms are fever, swollen limbs and genitals, generalized malaise, and can progress to a debilitating condition known as elephantiasis This research focuses on the transmission of these worms to the disseminating mosquito host, and it is based on the interesting observation that mf must be at least 7 days old to successfully infect the mosquito (de Hollanda, 1982). Newborn mf that have not ‘matured’ cannot successfully penetrate the midgut of the mosquito, and subsequently cannot develop to the L3 stage (Fuhrman, 1987). Previous work done by another group 20 years ago suggests that the molecular makeup of the worm surface changes during this maturation process (Furman, 1983 a and b). We used microarray analysis to characterize changes in gene expression that take place during the mf maturation process. Understanding the gene expression changes that occur as the mf mature will allow us to understand the nature of the philological transition that allows mf to move from the human to the mosquito host. With this information in hand, we can eventually identify parasite molecules that could be targeted to either stop parasite reproduction or prevent transmission of the mf to the mosquito. This would stop parasite transmission in endemic areas. This SuperSeries is composed of the following subset Series: GSE14939: Brugia pahangi mature vs immature microfilariae GSE14940: Brugia malayi mature vs immature microfilariae Refer to individual Series
Project description:Changes in gene expression of serotype D strains JEC21 or 24067 (52D) were examined after incubation with mAb 18B7 or the control MOPC-21 for 1, 2 or 4 hours at 37 C. Analysis used incubation with a non-binding IgG1 mAb as a control for incubation with a protective IgG1 mAb 18B7 at different timepoints.
Project description:Changes in gene expression of the serotype A pre-passage wild-type strain H99W with two mouse-passaged strains were examined during log-phase growth at 37 ºC. Analysis included two replicates of each comparison with a Cy3-Cy5 dye swap
Project description:Doxycycline treatment affects gene expression in Wolbachia and Brugia malayi adult female worms in vivo Two biological replicates of female RNA used for hybridization, in duplicate, to examine the gene expression changes in Wolbachia and Brugia