Cell migration and invasion are processes that offer rich targets for intervention in key physiologic and pathologic phenomena such as wound healing and cancer metastasis. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screening process. This article will review the effective use of several principle formats for studying cell motility: scratch assays, transmembrane assays, microfluidic devices and cell exclusion zone assays.

Rapid identification of both species and even specific strains of human pathogenic bacteria grown on standard agar have been achieved from the volatiles they produce using a disposable colorimetric sensor array in a Petri dish imaged with an inexpensive scanner. All ten strains of bacteria tested, including E. faecalis and S. aureus and their antibiotic resistant forms, were identified with 98.8% accuracy within 10 h, a clinically important timeframe. Furthermore, the colorimetric sensor arrays also prove useful as a simple research tool for the study of bacterial metabolism and as an easy method for the optimization of bacterial production of fine chemicals or other fermentation processes.

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

Objective. In order to investigate potential regulatory mechanisms for the increased production of prostaglandin E2 (PGE2) in interleukin‐1β (IL‐1β)–stimulated rheumatoid synovial fibroblasts (RSF), this study examined the induction of phospholipase A2 (PLA2) and prostaglandin H synthase (PGHS) enzymes and the correlation of these events with PGE2 production in IL‐1β–stimulated RSF. Methods. Protein and messenger RNA (mRNA) levels of cytosolic PLA2 (cPLA2) and PGHS‐2 enzymes in IL‐1β–stimulated RSF were measured by Western and Northern blotting, respectively, using specific antisera and complementary DNA probes. Enzymatic activity of cPLA2 was determined in cell‐free reaction mixtures utilizing mixed micelles of 14C‐phosphatidylcholine and Triton X‐100 as the substrate. PGE2 levels were quantitated using a commercial enzyme immunoassay kit. Results. Incubation of RSF with IL‐1β increased the mRNA and protein levels for the high molecular weight cPLA2 as well as for the mitogen/growth factor–responsive PGHS (PGHS‐2). The IL‐1 receptor antagonist completely abolished the induction of these two enzymes and the stimulation of PGE2 production by IL‐1β in RSF. In contrast, levels of the other known forms of these enzymes, i.e., the 14‐kd secretory group II PLA2 (sPLA2) and the constitutive form of PGHS (PGHS‐1), were unaffected by IL‐1β treatment. Conclusion. These are the first data to demonstrate the coordinate induction by IL‐1 of cPLA2 and PGHS‐2 in RSF. The time‐course for the induction of these enzymes suggests that their increase contributes to the increased production of PGE2 in IL‐1–treated RSF, and may help explain the capacity of RSF to produce large amounts of PGE2.

Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.