Cellular responses to wounding have often been studied at a molecular level after disrupting cell layers by mechanical means. This invariably results in damage to cells at the edges of the wounds, which has been suggested to be instrumental for initiating wound healing. To test this, we devised an alternative procedure to introduce gaps in layers of corneal epithelial cells by casting agarose strips on tissue culture plates. In contrast to mechanical wounding, removal of the strips did not lead to detectable membrane leakage or to activation of the stress-activated kinase JNK. Nonetheless, cells at the edge underwent the typical morphological transition to a highly motile phenotype, and the gaps closed at rates similar to those of mechanically induced wounds. To allow biochemical analysis of cell extracts, a procedure was devised that makes cell-free surface area acutely available to a large proportion of cells in culture. Rapid activation of the epidermal growth factor receptor (EGFR) was detected by immunoblotting, and the addition of an EGFR-blocking antibody completely abolished wound healing. In addition, wound healing was inhibited by agents that block signaling by the heparinbinding epidermal growth factor-like growth factor (HB-EGF). Cells stimulated with cell-free tissue culture surface released a soluble factor that induced activation of the EGFR, which was distinct from HB-EGF. These studies suggest that the triggering event for the induction of motility in corneal epithelial cells is related to the sudden availability of permissive surface area rather than to mechanical damage, and they demonstrate a central role of signaling through HB-EGF.

No abstract

Due to the widespread, antibiotic-resistant nature of biofilm-producing bacteria, there is high demand for research on biofilm formation.  This research serves as a contribution to the existing knowledge of how biofilms form.  Its results could shed light on how biofilm-producing bacteria utilize communication pathways in response to environmental and chemical stimuli.  To determine the effects of glycerol (a sugar alcohol linked to osmotic stress) on biofilm production, Pseudomonas fluorescens strain SBW25 was cultivated within a series of 5 culture tubes containing medias with increasing concentrations of glycerol from 0-10%.  After a week of incubation, a sample from each of the tubes was plated and colony morphology was qualitatively analyzed.  Each of the tubes was then stained with crystal violet and the absorbance of the biofilm could be quantitatively measured at 590nm with a dual-beam spectrophotometer.  The hypothesis is supported with the absorbance and plate data collected over the first and second trials of the experiment.  As glycerol concentration increased, the absorbance (and thus biofilm produced) increased proportionately, and the approximate number of wrinkly mutants on each successive plate increased.  The correlation of the data is significant enough to support the hypothesis, but more trials will need to be conducted in order to conclusively accept it.