<h4>Background</h4>Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. ...[more]
Project description:Each experiment consisted in an artesunate-free culture (control) and an artesunate treated culture. For each time point explored, cDNA from parasites with and without the drug were labeled with different cyanines, mixed and hybridized. To analyse dynamic transcriptome alterations, two pilot experiments were performed in which RNA was harvested at 90 and 180 minutes of incubation with the drug. <br><br> Since artesunate is active on ring stages as well as on older trophozoite stages, we decided to explore different time points along the erythrocytic cycle and search for genes affected at each time point. Therefore, five drug treatment experiments, staggered between 20 hours and 30 hours of parasite development, were performed for the comparison of drug versus no drug at 90 minutes and 3 hours. <br><br> Different expression levels in artesunate-exposed vs. control unexposed cultures could reflect the added effects of the actual response to artesunate together with a possible difference in growth rate/developmental stage. To identify the developmentally regulated genes in the artesunate-free control culture, the 3 hours RNA was hybridized against the time 0 RNA (3 control experiments).
Project description:The aim of the study was to determine the effect of natural killer (NK) cells on the global gene expression in Plasmodium falciparum. FCR3-CSA-infected red blood cells (RBCs) were co-cultured for 24h with NK92. Prior to RNA extraction, the parasitized RBCs were separated via Ficoll from the lymphocyte fraction. Control cultures of parasites without NK cells were cultivated in parallel. In addition, the effect of the separation method was determined via a Ficoll control culture of FCR3-CSA that was passed over a Ficoll gradient. All three treatments were performed in triplicate. The RNA was reverse-transcribed and hybridized onto microarrays.
Project description:Unravel the mechanisms underlying brain aging and Alzheimer´s disease (AD) has been difficult because of complexity of the networks that drive these aging-related changes. Analysis of the gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important research tool that potentially can provide the investigative power needed to address the complexity of brain aging and neurodegenerative processes. One of the reasons that account for the resistance of AD pathogenesis to analysis is that clinically normal subjects may exhibit considerable AD pathology, blurring criteria for distinguishing subjects with normal aging or AD. Here, we analyzed hippocampal and cortex frontal gene expression from 32 subjects separated in individuals presenting, 1) both pathologic and clinical AD (definitive AD); 2) AD pathology and normal clinic (pathologic AD); 3) cognitive impairment, without AD pathology (others dementias); and 4) no cognitive impairment, without AD pathology (normal individuals). Our results show that based on gene expression profile these individuals we could verify similarity between the definitive AD group and the group that only had AD-type pathology (pathologic AD). Specimens of hippocampus and cortex frontal used in this study were obtained at autopsy from 32 subjects. Neuropathology, specifically NFT and NP, was assessed in accordance to the Braak staging and CERAD scores, respectively. Based on pathologic and clinic criteria, subjects were categorized into four groups: 1) nine subjects with AD neuropathologic (Braak = IV / V / VI and CERAD ≠ 0) presenting cognitive impairment (CDR ≥ 1), termed “definitive AD” (dAD); 2) five subjects with AD neuropathologic (Braak = IV / V / VI and CERAD ≠ 0) and without cognitive impairment (CDR = 0), termed “pathologic AD” (pAD); 3) nine subjects without AD neuropathologic (Braak = 0 / I / II and CERAD ≠ C) presenting cognitive impairment (CDR ≥ 1), termed “other dementias” (OD); 4) - nine subjects without AD neuropathologic (Braak = 0 / I / II and CERAD ≠ C) and without cognitive impairment (CDR = 0), termed “normal” (N). RNA isolation and Amplification. From total RNA, a two-round linear amplification procedure (T7-based protocol) was carried out for all samples and for a pool of RNAs obtained from 15 distinct human cell lines used as reference. Labeled cDNA was generated in a reverse transcriptase reaction using amplified RNA (aRNA). Equal amounts of test and reference cDNA reverse color Cy-labeled aRNA targets were competitively hybridized against the cDNA probes in a customized cDNA platform with 4,608 ORESTES representing human genes. Dye-swap was performed for each sample as control for dye bias and used as replicate.
Project description:Gene expression analyses through cDNA microarray of fifteen gastrocnemius muscles from transgenic and wild-type SOD1G93A mouse model by the ages of 40 and 80 days old were performed. We used a customized cDNA array containing the cDNA platform comprised of 2352 spots, 326 of them orthologous to mouse, 1384 additional human cDNA sequences, 496 negative controls (DMSO) and 48 positive controls (the Q gene from λ-phage). Gene expression results for SOD1G93A and WT age matched mice pointed to eight up- (LOXL2, PIK4CA, FZD9, CUL1, CTNND1, SNF1LK, PRKX, DNER) and nine down-regulated genes (PIK3C2A, RIPK4, ID2, C1QDC1, EIF2AK2, RAC3, CDS1, INPPL1, TBL1X) at 40 days and also to one up- (PIK3CA) and five down-regulated genes (CD44, EEF2K, FZD2, CREBBP, PIKI3R1) at 80 days. Based on differentially expressed genes, analyses for gene priorization were performed and used to construct a network of protein-protein interaction. The network based on the genes of 40 and 80 days old mice was composed by 251 and 531 genes, respectively. GRB2 and SRC were identified as central genes of both networks. In conclusion, changes in gene expression of skeletal muscle from transgenic ALS mice in pre-symptomatic periods give further evidence of early neuromuscular abnormalities that precede motor neuron death. We performed gene expression analyses by customized cDNA array, using reference design, of fifteen gastrocnemius muscles from transgenic and wild-type SOD1G93A mouse model by the ages of 40 and 80 days old. These differentially expressed lists were submitted to analyses for gene priorization and used to construct a network of protein-protein interaction.
Project description:Transcription profiling by array using Streptomyces coelicolor oligomerics array to compare the gene expression of streptomyces lividans adpA mutant to the wild type at early stationary phase in YEME medium.
Project description:A green fluorescence protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous control for Apis mellifera RNAi assays by multiple research groups. Its sequence does not share any significant homology with any known honey bee genes. Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene expression, pupal pigmentation or developmental timing have been routinely observed. To better understand the multiple molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression patterns in developing workers. We found that dsRNA-GFP causes large-scale changes in gene expression associated with multiple biological processes. Furthermore, dsRNA-GFP exposure tended to preferentially decrease, rather than increase, expression of genes compared to controls. Gene expression differences were analyzed using a dye-swap design. Second instar worker larvae received 0.5 μg of dsRNA-GFP. Non-treated and dsRNA-GFP treated 7 day-old workers (W7d) were sampled.