Motile bacterial cell microarrays were fabricated by attaching Escherichia coli K-12 cells onto predesigned 16-mercaptohexadecanoic acid patterned microarrays, which were covalently functionalized with E. coli antibodies or poly-L-lysine. By utilizing 11-mercaptoundecyl-penta(ethylene glycol) or 11-mercapto-1-undecanol as passivating molecules, nonspecific binding of E. coli was significantly reduced. Microcontact printing and dip-pen nanolithography were used to prepare microarrays for bacterial adhesion, which was studied by optical fluorescence and atomic force microscopy. These data indicate that single motile E. coli can be attached to predesigned line or dot features and binding can occur via the cell body or the flagella of bacteria. Adherent bacteria are viable (remain alive and motile after adhesion to patterned surface features) for more than four hours. Individual motile bacterial cells can be placed onto predesigned surface features that are at least 1.3 microm in diameter or larger. The importance of controlling the adhesion of single bacterial cell to a surface is discussed with regard to biomotor design.
We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC 50 of epidermal growth factor (EGF) ϭ 10.1 Ϯ 0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligandinduced interaction between EGFR and signaling pathwayspecifying adapter proteins.Overexpression and activation of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), plays an important role in the etiology of non-small-cell lung cancer (NSCLC) . Therefore, EGFR is recognized as a key target for the development of NSCLC therapies (Hynes and Lane, 2005). Two drug development strategies focusing on EGFR inhibition are currently pursued: 1) the identification of reversible or irreversible small molecule drugs that inhibit the intracellular tyrosine kinase activity of EGFR by competitively binding to the ATP-binding site of the kinase domain, and 2) the identification of humanized monoclonal antibodies (mAbs) that interact with extracellular EGFR domains interfering with ligand binding (e.g., epidermal growth factor; EGF) or EGFR dimerization (Hynes and Lane, 2005). The reversible small molecule EGFR inhibitors Iressa (gefitinib) (Herbst et al., 2004) and Tarceva (erlotinib) (Minna and Dowell, 2005) and the antibody drug Erbitux (cetuximab; IMC-C225) (Goldberg, 2005) have already been marketed in the United States for NSCLC. However, early clinical studies observed only a 10 to 15% response rate in a U.S. population of unselected NSCLC Article, publication date, and c...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.