Transcription profiling of Aspergillus fumigatus isolates following laboratory culture or initiation of infection in the neutropenic murine lung to understand how metabolic and nutritional factors governing adaptation to the host niche contribute to virulence
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ABSTRACT: To understand how metabolic and nutritional factors governing adaptation to the host niche contribute to the virulence of Aspergillus fumigatus we compared transcriptomes of developmentally matched A.fumigatus isolates following laboratory culture or initiation of infection in the neutropenic murine lung.
Project description:Abstract: Testicular cancers in young adult men derive from an inborn precursor lesion, called carcinoma in situ (CIS) of the testis. CIS cells are believed to arise from primordial germ cells or gonocytes that fail to differentiate into the male germ cell lineage, and show a remarkable resemblance to embryonic stem cells (ESC). With this study we aimed at further elucidating the origin of CIS using microarray analysis of genome-wide gene expression. CIS cells only constitute a small fraction of the tissue and therefore, to reduce contaminating RNA from other cell types, we developed a fast (160 sec) staining procedure specific for CIS and fetal germ cells. Highly enriched cell populations were obtained by laser microdissectioning. The expression profiles of CIS cells were compared to microdissected fetal gonocytes, oogonia and cultured ESC with and without genomic amplifications. The cell type most similar to CIS was the gonocytes indicating malignant transformation at this stage. The enriched cell populations allowed us to find unique expression patterns for the developmentally very related cell types. Analyses of the similarities and differences among the cell types may help us understand when and how the malignant transformation to CIS occurs.
Project description:The presence of Set2-mediated methylation of H3K36 (K36me) correlates with transcription frequency throughout the yeast genome. K36me targets the Rpd3S complex to deacetylate transcribed regions and suppress cryptic transcription initiation at certain genes. Here, using a genome-wide approach, we report that the Set2-Rpd3S pathway is generally required for controlling acetylation at coding regions. When using acetylation as a functional read-out for this pathway, we discovered that longer genes and, surprisingly, genes transcribed at lower frequency exhibit a stronger dependency. Moreover, a systematic screen using high resolution tiling microarrays allowed us to identify a group of genes that rely on Set2-Rpd3S to suppress spurious transcripts. Interestingly, most of these genes are within the group that depend the same pathway to maintain a hypo-acetylated state at coding regions. These data highlight the importance of using the functional readout of histone codes to define the roles of specific pathways.
Project description:Human dermal microvascular endothelial cells (HDMVECs) were irradiated with 2 Gy or non-irradiated. Two methods of RNA isolation were used to comparatively analyze the miRNA status after radiation treatment.
Project description:In order to identify novel regulators of haematopoietic stem cell emergence in the mouse embryonic AGM region, 3 different types of gene expression comparisons were performed. In the first one, whole dorsal aortas from embryonic day (E) 11 embryos and E9 embryos were dissected, tissues from each time point were pooled and their expression profile compared (whole aorta (WA) E9 vs E11). 3 different pools/biological replicas were obtained for each time point, a dye swap was included in each experiment, a technical replicate was performed for each pool (including the dye swap) and 2 self-self hybridisation controls were included. In the second comparison, GFP+ cells were isolated by fluorescence-activated cell sorting from pooled E9 and E11 aortas from transgenic embryos that express GFP under the regulatory elements of the Ly-6A gene. We obtained one pool for each time point and compared their transcription profile (GFP E9 vs E11). This comparison also contained a dye swap and a technical replicate (also of the dye swap). In the third comparison, the E11 aorta was cut into 3 roughly equal parts. Pools were obtained of the middle region ("m") and the two outer regions, rostral and caudal, together ("r+c") and their expression profile compared (11AO m vs r+c). There was one pool of each population, and the experiment included a dye swap and a technical replicate (also of the dye swap) and one self-self hybridisation control. Isolated RNA was amplified by T7-mediated in vitro transcription, with one round for the first and the third comparison and two rounds for the second comparison (sorted GFP+ cells). Probes were labelled during the reverse transcription step. Within array normalisation was achieved with Lowess Smooth and between arrays with Z scores. ANOVA analysis identified differentially expressed genes and K means clustering was used to segregate them into upregulated and downregulated genes.
Project description:Acute kidney Injury (AKI) following open-heart surgery with cardiopulmonary bypass (CPB) is associated with an increased morbidity and mortality. The goal of this study is to explore patterns of gene expression in human AKI following CPB. This prospective observational study was approved by the Institutional Review Board Human Subjects Committee. Design and methods: Peripheral blood RNA samples were collected prospectively from a cohort of 30 patients at Caritas St. Elizabeths Medical Center in Boston, MA, undergoing CPB at time points immediately prior, and two and 24 hours following CPB. Gene expression patterns of four patients who developed AKI (cases) following CPB will be compared with six patients matched for clinical characteristics, who did not develop AKI (controls). AKI was defined by an incremental increase in serum creatinine of 30% within 72 hours following CPB. For peripheral blood RNA sampling, 2.5 ml of venous whole blood was collected into PAXgene Blood RNA Tube collection tubes (PreAnalytiX GmbH, Hombrechtikon, CH). Whole blood RNA was extracted with the PAXgene Blood RNA extraction Kit according to the manufacturers instructions (PAXgene RNA Kit Handbook, 06/2005). All biological samples are stored at 80° C.
Project description:Anaemia negatively affects the prognosis of patients with head and neck squamous cell carcinoma (HNSCC). This study investigates whether anaemia is associated to genetic changes and whether these changes predict the clinical outcome.
Project description:Normal human dermal fibroblasts (NHDF) and human lung microvascular endothelial cells (HMVEC-L) were irradiated with iron ions (0, 0.2, 0.4 and 1 Gy, 1GEv/n) at Brookhaven National Labs (BNL). Aim of the study is to find differentially transcribed genes in dependance of radiation dose/source and cell type.
Project description:Normal human dermal fibroblasts (NHDF) and human lung microvascular endothelial cells (HMVEC-L) were irradiated with protons (0, 0.5, 1 and 2 Gy, 1GEv/n) at Brookhaven National Labs (BNL). Aim of the study is to find differentially transcribed genes in dependence of radiation dose/source and cell type.
Project description:Invasiveness of genetically modified cells is tested. Four cells lines (NIH3T3 untreated control; NIH-Ras positive control which is invasion +; NIH-MKK3actK4 and NIH-MKK3actATN, two constructs with MKK3 gene which shall be investigated for theit invasiveness). Cells on top as well as cells from bottom of separating membrane (those which had "invaded") form all 4 cell lines are collected and expression profiled. Aim is to find genes which are correlated to "invasion".