Project description:We exposed Salmonella TA100 cells to 3 concentrations of MX that produced a linear concentration-response for mutagenesis with little cell killing. We measured mutagenesis, survival, and global gene expression. We used custom-spotted glass slides as our microarray platform and identified genes whose expressions were altered by MX treatment using three methods: (1) a Bayesian t-test, (2) an operon analysis that assumes if one gene in an operon is differentially expressed then all genes in that operon are differentially expressed, and (3) a monotonic-expression response to increasing doses of MX. The resulting list of genes was analyzed for functional and KEGG pathway representation. Keywords: dose response
Project description:DNA methylation is an epigenetic mark that has a crucial role in regulating gene expression. Aberrant DNA methylation results in severe diseases in humans, such as cancer, autoimmune disease, atherosclerosis, and cardiovascular diseases. Whole-genome bisulfite sequencing and methylated DNA immunoprecipitation are available to study DNA methylation changes, but they are typically used on a few samples at a time. Here, we developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), that can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis.
Project description:Breast cancer is a leading cause of cancer-related deaths among women in the Western world. Anthracyclines, including doxorubicin (DOX), along with taxanes, cyclophosphamide and platinum compounds are the main chemotherapeutic agents applied for breast cancer treatment. However, chemoresistance is the major cause of the disease progression following chemotherapy. Drug resistance can be either inherent or acquired during the treatment, but most possibly the interaction of both mechanisms drives the rapid development of treatment refractory cancer. In the present study, the mechanisms of acquired cellular chemoresistance were studied in breast cancer cell line MX-1 exposed to gradually increasing concentration of the chemotherapeutic compound DOX (MX-1/D). Nongenotoxic phosphorganic compound tetraphenylphosphonium cation (TPP+) – a potent substrate and activator for ABCB1 transporter – was used in the cell line model of intrinsic chemoresistance (MX-1/T). Finally, DOX highly resistant cell subline was derived from ABCB1-activated, TPP+ pretreated cells (MX-1/TD). Chemoresistance in all cell sublines was closely associated with the EMT, and the ABCB1 hyperexpression was a possibly trigger of this process.