Integration of global SNP-based mapping and expression arrays reveals key regions, mechanisms, and genes important in the pathogenesis of multiple myeloma.

Multiple myeloma is characterized by genomic alterations frequently involving gains and losses of chromosomes. Single nucleotide polymorphism (SNP)-based mapping arrays allow the identification of copy number changes at the sub-megabase level and the identification of loss of heterozygosity (LOH) due to monosomy and uniparental disomy (UPD). We have found that SNP-based mapping array data and fluorescence in situ hybridization (FISH) copy number data correlated well, making the technique robust as a tool to investigate myeloma genomics. The most frequently identified alterations are located at 1p, 1q, 6q, 8p, 13, and 16q. LOH is found in these large regions and also in smaller regions throughout the genome with a median size of 1 Mb. We have identified that UPD is prevalent in myeloma and occurs through a number of mechanisms including mitotic nondisjunction and mitotic recombination. For the first time in myeloma, integration of mapping and expression data has allowed us to reduce the complexity of standard gene expression data and identify candidate genes important in both the transition from normal to monoclonal gammopathy of unknown significance (MGUS) to myeloma and in different subgroups within myeloma. We have documented these genes, providing a focus for further studies to identify and characterize those that are key in the pathogenesis of myeloma.

Connectivity Mapping (ssCMap) To Predict A20 Inducing Drugs: Anti-inflammatory Action In Cystic Fibrosis

Cystic Fibrosis (CF) lung disease is characterised by a chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in CFTR (cystic fibrosis transmembrane conductance regulator), there is still an unmet need to also normalise the inflammatory response. The prolonged and heightened inflammatory response in CF is in part mediated by a lack of intrinsic downregulation of the pro-inflammatory NF-kB pathway. We have previously identified reduced expression of the NF-kB down-regulator A20 in CF as a key target to normalise the inflammatory response. Here we have used publically available gene array expression data together with sscMap (statistically significant connections’map)to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NFkB (p65) expression (mRNA) as well as pro-inflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells (PNECs) from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with TNFAIP3 (A20) knockdown (siRNA). We also show that the A20 inducing effect of ikarugamycin and quercetin is lower in CF derived airway epithelial cells than in non-CF cells.

Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2"