Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles

Wu, Ying; Kievit, Peggy de; Vahlkamp, Lars; Pijnenburg, Dirk; Smit, Maarten H. de; Dankers, Martijn; Melchers, Diana; Stax, Martijn J.; Boender, Piet J.; Ingham, Colin J.; Bastiaensen, Niek; Wijn, Rik de; Alewijk, Dirk van; Damme, Henk van; Raap, Anton K.; Chan, Alan; Beuningen, Rinie van

doi:10.1093/nar/gnh118

Cited by 49 publications

(35 citation statements)

References 25 publications

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“…Two strains of P. mirabilis were allowed to swarm on different sides of a sheep blood agar plate separated by a sterile barrier embedded in the agar, a highly porous Anopore membrane that was permeable to most proteins and macromolecules but not to bacteria. Both untreated and poly-L-lysine-treated Anopore strips were tested (33). To prevent bacteria from bypassing the membrane, amoxicillin-clavulanate tablets (Rosco, Taastrup, Denmark) were placed at each end of the 36-mm-long barrier.…”

Section: Methodsmentioning

confidence: 99%

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

et al. 2009

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When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.The gram-negative bacterium Proteus mirabilis is well known for its ability to differentiate into hyperflagellated, motile, and elongated swarmer cells that rapidly spread over a surface. When cultured on a nutrient agar plate, a strain of P. mirabilis typically is able to colonize the whole plate within 24 h. This phenomenon is both of interest in terms of the differentiation and survival strategy of the organism and of practical importance, as contamination of agar plates by swarming P. mirabilis is a common problem in diagnostic microbiology. When two different strains of P. mirabilis swarm on the same agar plate, a visible demarcation line with lower cell density forms at the intersection, and this line is known as a Dienes line (5, 6). A Dienes line is seen when both strains are swarming; it is not a property of the smaller vegetative cells (5). When two identical isolates meet, the swarming edges merge without formation of a Dienes line. This phenomenon has been used in epidemiological typing of clinical isolates (4,20,23,28) and raises interesting questions concerning its mechanism and biological importance. Dienes typing in the clinical environment suggests that the number of Dienes types is large; 81 types were found in one study alone (25).

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Section: Methodsmentioning

confidence: 99%

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

et al. 2009

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“…The PamGene platform has been used frequently and in various iterations to demonstrate gene expression 41 , biochemical mechanisms 42 , rational personalized therapy selection and design [43][44][45] , drug target discovery 46,47 , and microbe species detection and identification 48,49 . Many of these studies have involved cancer signaling, treatment, and target identification and characterization.…”

Section: Discussionmentioning

confidence: 99%

Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer

Ferguson

Kanteti

Tan

et al. 2014

International Journal of Radiation Oncology*Biology*Physics

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“…This integrated microarray platform allows automated running of samples including incubation by pumping the sample up and down through the three-dimensional porous chip. The automated run includes data capturing by real time imaging (fluorescence signal development is read out by charge-coupled device imaging) and washing (17)(18)(19)(20). A PamChip consists of four identical peptide microarrays, which run four samples in parallel during an experiment.…”

Section: Methodsmentioning

confidence: 99%

Endogenous Phosphotyrosine Signaling in Zebrafish Embryos

Lemeer

Ruijtenbeek

Pinkse

et al. 2007

Molecular & Cellular Proteomics

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In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms. Molecular & Cellular Proteomics 6:2088 -2099, 2007.Embryonic development is tightly regulated, and numerous developmental processes like cell growth, differentiation, and migration are controlled by phosphotyrosine signaling, which is mediated by protein-tyrosine kinases and protein-tyrosine phosphatases. Improved knowledge about the identity of tyrosine phosphorylated proteins, their interacting proteins, and their involvement in signaling will improve our understanding of biological pathways and critical cellular processes. A first step toward elucidation of the critical signaling pathways is the identification of tyrosine phosphorylated proteins in the developing embryo.In recent years the availability of selective antibodies (1-4), targeted at tyrosine phosphorylated proteins, facilitated more global purification of these proteins and their binding partners, allowing identification by very sensitive LC-MS-based analysis of the proteins tryptic digests (1, 4, 5). Such approaches have been used for instance to study elegantly the temporal, global response to receptor stimulation. However, all of these data have been obtained from well defined cell systems under stimulated conditions. Although these data contribute well to our understanding of complex tyrosine phosphorylation-mediated signaling pathways, it is still unclear how they relate to in vivo processes. Therefore, in vivo studies are essential (6). Here we explored whether the above described technologies may be extended to perform a global analysis of in vivo signaling processes involved in zebrafish development. Zebrafish is n...

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Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles

Cited by 49 publications

References 25 publications

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer

Endogenous Phosphotyrosine Signaling in Zebrafish Embryos

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