From microarray to biology: an integrated experimental, statistical and in silico analysis of how the extracellular matrix modulates the phenotype of cancer cells

A statistically robust and biologically-based approach for analysis of microarray data is described that integrates independent biological knowledge and data with a global F-test for finding genes of interest that minimizes the need for replicates when used for hypothesis generation. First, each microarray is normalized to its noise level around zero. The microarray dataset is then globally adjusted by robust linear regression. Second, genes of interest that capture significant responses to experimental conditions are selected by finding those that express significantly higher variance than those expressing only technical variability. Clustering expression data and identifying expression-independent properties of genes of interest including upstream transcriptional regulatory elements (TREs), ontologies and networks or pathways organizes the data into a biologically meaningful system. We demonstrate that when the number of genes of interest is inconveniently large, identifying a subset of "beacon genes" representing the largest changes will identify pathways or networks altered by biological manipulation. The entire dataset is then used to complete the picture outlined by the "beacon genes." This allow construction of a structured model of a system that can generate biologically testable hypotheses. We illustrate this approach by comparing cells cultured on plastic or an extracellular matrix which organizes a dataset of over 2,000 genes of interest from a genome wide scan of transcription. The resulting model was confirmed by comparing the predicted pattern of TREs with experimental determination of active transcription factors

Elevated AKR1C3 expression promotes prostate cancer cell survival and prostate cell-mediated endothelial cell tube formation: implications for prostate cancer progressioan

<p>Abstract</p> <p>Background</p> <p>Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR1C enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we propose a novel pathological function of AKR1C3 in tumor angiogenesis and its potential role in promoting PCa progression.</p> <p>Methods</p> <p>To recapitulate elevated AKR1C3 expression in cancerous prostate, the human PCa PC-3 cell line was stably transfected with an AKR1C3 expression construct to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analysis were performed to identify AKR1C3-mediated pathways of activation and their potential biological consequences in PC-3 cells. Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and an <it>in vitro </it>Matrigel angiogenesis assays were applied to validate the pro-angiogenic activity of PC3-AKR1C3 transfectants identified by bioinformatics analysis.</p> <p>Results</p> <p>Microarray and bioinformatics analysis suggested that overexpression of AKR1C3 in PC-3 cells modulates estrogen and androgen metabolism, activates insulin-like growth factor (IGF)-1 and Akt signaling pathways, as well as promotes tumor angiogenesis and aggressiveness. Levels of IGF-1 receptor (IGF-1R) and Akt activation as well as vascular endothelial growth factor (VEGF) expression and secretion were significantly elevated in PC3-AKR1C3 transfectants in comparison to PC3-mock transfectants. PC3-AKR1C3 transfectants also promoted endothelial cell (EC) tube formation on Matrigel as compared to the AKR1C3-negative parental PC-3 cells and PC3-mock transfectants. Pre-treatment of PC3-AKR1C3 transfectants with a selective IGF-1R kinase inhibitor (AG1024) or a non-selective phosphoinositide 3-kinases (PI3K) inhibitor (LY294002) abolished ability of the cells to promote EC tube formation.</p> <p>Conclusions</p> <p>Bioinformatics analysis followed by functional genomics demonstrated that AKR1C3 overexpression promotes angiogenesis and aggressiveness of PC-3 cells. These results also suggest that AKR1C3-mediated tumor angiogenesis is regulated by estrogen and androgen metabolism with subsequent IGF-1R and Akt activation followed by VEGF expression in PCa cells.</p

Efficacy of the InvictDetectTM Immunostrip® to Taxonomically Identify the Red Imported Fire Ant, Solenopsis invicta, Using A Single Worker Ant

The early detection and identification of the red imported fire ant Solenopsis invicta are crucial to intercepting and preventing it from becoming established in new areas. Unfortunately, the visual identification of fire ants to species is difficult and ant samples must often be couriered to an expert for positive identification, which can delay control interventions. A lateral flow immunoassay that provides a rapid and portable method for the identification of S. invicta ants was developed and commercialized, and it is available from Agdia, Inc. under the trade name InvictDetectTM. While the test was 100% accurate when using the recommended minimum sample of three ant workers, InvictDetectTM was field tested for the first time while using homogenates prepared from single S. invicta workers to determine the effectiveness of the method under these non-recommended conditions. Disregarding social form, the false negative rate was 25.5% for an initial single worker ant test and 10% after a repeat test was performed. The InvictDetectTM false negative response was independent of worker weight. Though InvictDetectTM requires a minimum of three worker ants, the test improves upon current identification methods because it can be conducted in the field, be completed in 10&ndash;30 min, and requires no special training or expertise

Winter-summer Variations of Calcite and Aragonite Saturation in the Northeast Pacific

New carbonate data obtained on February-March and June-July cruises in the northeast Pacific during 1985 were utilized to describe processes affecting seasonal variations of calcite and aragonite saturation. Large gradients in saturation state occur in the region between the Subtropical and the Subarctic Fronts in the north-south direction. These gradients are a function of largescale mixing and biological processes in the North Pacific. The saturation values in the upper kilometer of the water column were observed to be significantly lower in winter than in summer. These decreases were due to a number of processes including: (i) the seasonal decrease in the temperature of the water column; (ii) the seasonal increase in vertical mixing causing CO2-enriched deep waters to be upwelled; and (iii) the seasonal enhancement of respiration over photosynthesis. Seasonal changes in total CO2 (TCO2) concentrations appeared to have the greatest overall effect on the saturation state. Aragonite dissolution rate experiments were conducted during the June-July cruise to provide an independent verification of the saturation calculations. In all cases, significant increases in aragonite dissolution were observed below the 100% saturation depth. In the northeast Pacific, shallow undersaturation horizons provide for significant dissolution rates at depths below 400 m

Urothelial expression of neuropilins and VEGF receptors in control and interstitial cystitis patients

Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies