MAPPINGS v1.0, a tool for network analysis of large phospho-signalling datasets: application to host erythrocyte response to Plasmodium infection

Phosphorylation-based signalling implicates a complex and intertwined series of pathways and is critical to all domains of life. The interconnectivity between pathways results in the emergence of complex networks whose elucidation present a serious challenge. Large datasets of phosphorylation interactions through the activity of kinases on their numerous substrates are constantly being generated, however deciphering the complex network structure hidden in these datasets remains challenging. Many phosphorylation interactions occurring in human cells have been identified and constitute the basis for the known phosphorylation interaction network. We overlayed onto this network phosphorylation datasets obtained from an antibody microarray approach aimed at determining changes in phospho-signalling of host erythrocytes to infection with the malaria parasite Plasmodium falciparum. To analyse the datasets now mapped into the interaction network, we designed a pathway analysis tool denoted MAPPINGS that uses random walks to identify chains of phosphorylation events occurring much more or much less frequently than expected. MAPPINGS highlights pathways of phosphorylation that work synergistically, providing a rapid interpretation of the most critical pathways in each dataset. MAPPINGS confirmed several signalling interactions previously shown to be modulated by infection, and revealed additional interactions which could form the basis of numerous future studies. The MAPPINGS analysis strategy described here is widely applicable to comparative phosphorylation datasets in any context (e.g. response of cells to infection, treatment, or comparison between differentiation stages of any cell populations) and provides a rapid and reliable analysis to guide validation studies.

CXCR6-CXCL16 Axis Promotes Breast Cancer by Inducing Oncogenic Signaling

Precise mechanisms underlying breast cancer (BrCa) metastasis are undefined, which becomes a challenge for effective treatments. Chemokine signaling instigates the trafficking of cancer cells in addition to leukocytes. This study aimed to ascertain the clinical and biological significance of the CXCR6/CXCL16 signaling axis in the pathobiology of BrCa. Our data show a higher expression of CXCR6 in BrCa cell lines and tissues. Stage-III BrCa tissues express significantly higher CXCR6 compared to stage-II tissues. The ligand, CXCL16, could remain tethered to the cell surface, and, after proteolytic shedding of the ectodomain, the N-terminal fragment is released, converting it to its oncogenic, soluble form. Like CXCR6, N-terminal CXCL16 and ADAM-10 were significantly higher in stage-III than stage-II, but no significant difference was observed in the C-terminal fragment of CXCL16. Further, stimulation of the CXCR6/CXCL16 axis activated Src, FAK, ERK1/2, and PI3K signaling pathways, as per antibody microarray analysis, which also underlie CXCL16-induced F-actin polymerization. The CXCR6/CXCL16 axis induces cytoskeleton rearrangement facilitating migration and invasion and supports BrCa cell survival by activating the PI3K/Akt pathway. This study highlights the significance of the CXCR6/CXCL16 axis and ADAM10 as potential therapeutic targets for advanced-stage BrCa.

Antibody microarray analysis of the amniotic fluid proteome for predicting the outcome of rescue cerclage in patients with cervical insufficiency

Little is known about the biomarkers that can identify patient candidates suitable for rescue cerclage procedure. The purpose of the study was to identify novel biomarkers in amniotic fluid (AF) that can predict the outcome of rescue cerclage in patients with cervical insufficiency by using an antibody microarray. This case-control study was conducted using AF samples collected from singleton pregnant women who underwent rescue cerclage following a diagnosis of cervical insufficiency (19–25 weeks). Patients were divided into case (n=20) and control (n=20) groups based on the occurrence of spontaneous preterm delivery (SPTD) at <34 weeks of gestation after cerclage placement. The AF proteomes were analyzed using an antibody microarray for biomarker discovery work. Ten candidate biomarkers of interest were validated by ELISA. Thirty-one of the molecules studied showed significant intergroup differences (≥ 2-fold change in signal intensity). Validation by ELISA confirmed significantly higher levels of APRIL, S100 A8/A9, TIMP-1, MIP-1α, and IL-8 in women who had SPTD at <34 weeks. Of these, AF S100 A8/A9 and TIMP-1 levels were independent of other potentially confounding factors (e.g., cervical dilatation). S100 A8/A9 had the highest area under the curve at 0.857. Using protein–antibody microarray technology, we identified differentially expressed proteins and several novel biomarkers (APRIL, IL-8, MIP-1α, S100 A8/A9, and TIMP-1) in AF from women who had SPTB at <34 weeks after cerclage for cervical insufficiency. These data can provide an insight into the molecular mechanisms underlying SPTD after rescue cerclage in patients with cervical insufficiency.

Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

The tissue proteome of dorsal root ganglia in Friedreich ataxia

Dorsal root ganglia (DRG) at all levels of the spinal cord are a prominent target of Friedreich ataxia (FA). The lesions include hypoplasia of neurons, proliferation of satellite cells, infiltration by IBA- 1-reactive monocytes, and formation of residual nodules. Paucity and smallness of DRG neurons account for the lack of large myelinated axons in dorsal roots and sensory peripheral nerves. The lack of myelin in dorsal roots can be attributed to low levels of neuregulin 1 type III (NRG1[III]). Lysates of 13 DRG of genetically confirmed FA patients were analyzed by antibody microarray with 878 different validated antibodies that target structural and signaling proteins, and by Western blots. KIT and mTOR, two proteins involved in cellular proliferation, were significantly upregulated in the DRG of FA. KIT is a transmembrane receptor that dimerizes when it binds two molecules of stem cell factor (SCF) in its extracellular domain and becomes activated as protein tyrosine kinase. Immunohistochemistry with anti-KIT generated reaction product in satellite cells of normal DRG and prominent labeling of these cells in FA that co-localized with SCF on double- label immunofluorescence; SCF was present in S100-positive satellite cells rather than monocytes. Immunohistochemical reaction product of mTOR and other mTOR complex proteins, such as hamartin (TSC1), tuberin (TSC2), raptor (mTOR complex 1) and rictor (mTOR complex 2) was also present in satellite cells of normal DRG and DRG of FA. Antibodies to two downstream proteins that are considered to be indicators of mTOR activity, P70 S6K and 4E-binding protein 1, revealed no reaction product in DRG of FA. TSC1, TSC2, and mTOR are best known from their roles in tuberous sclerosis, but expression of these proteins, and KIT, in DRG may contribute to signaling cascades underlying the proliferation of satellite cells in FA.LEARNING OBJECTIVESThis presentation will enable the learner to: 1.Discuss cellular proliferation in the pathogenesis of the DRG lesion in Friedreich ataxiaCONFLICT OF INTERESTAHK is a consultant to PTC Therapeutics of South Plainfield, NJ USA. SP and CS are majority owners of Kinexus.

Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

Enterotoxigenic Escherichia coli (ETEC) F4ac is a major constraint to the development of the pig industry, which is causing newborn and post-weaning piglets diarrhea. Previous studies proved that FaeG is the major fimbrial subunit of F4ac E. coli and efficient for bacterial adherence and receptor recognition. Here we show that the faeG deletion attenuates both the clinical symptoms of F4ac infection and the F4ac-induced intestinal mucosal damage in piglets. Antibody microarray analysis and the detection of mRNA expression using porcine neonatal jejunal IPEC-J2 cells also determined that the absence of FaeG subunit alleviated the F4ac promoted apoptosis in the intestinal epithelial cells. Thus, targeted depletion of FaeG is still beneficial for the prevention or treatment of F4ac infection.

A Multiplex Immunosensor for Detecting Perchlorate-Reducing Bacteria for Environmental Monitoring and Planetary Exploration

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration.