Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials.

Pseudomonas aeruginosa causes chronic pulmonary infections in patients with cystic fibrosis (CF). P. aeruginosa mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both in vitro and in vivo Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H2O2). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H2O2 stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on lys, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of P. aeruginosa mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung.IMPORTANCEP. aeruginosa mucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities of P. aeruginosa demonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversified P. aeruginosa communities in vivo, as these variants can differentially shield one another from components of the host response.

Electrical impedance measurements predict cellular transformation.

Cellular transformation is the first step in cancer development. Two features of cellular transformation are proliferation in reduced serum and loss of contact inhibition. Electronic Cell-Substrate Impedance Sensing (ECIS) measurements have been used to measure cellular proliferation, cytotoxicity, apoptosis, and attachment. We have used impedance measurements to distinguish normal cells from cells transformed with a constitutively active chemokine receptor, CXCR2. CXCR2, a member of the G-protein coupled receptor (GPCR) family, is normally involved in cellular activation and migration, but a single amino acid substitution leads to constitutive activity. NIH3T3 cells were transformed with a constitutively active CXCR2 (D143V_CXCR2) and growth in reduced serum and foci formation were measured using established biological assays and compared to data from ECIS. The results of this study show that impedance measurements provide a quick and reliable way of measuring cellular transformation and provide real time assessment of transformed cellular parameters. Use of the ECIS system could allow a rapid screening of anti-cancer drugs that alter cellular transformation.

Surface elasticity and charge concentration-dependent endothelial cell attachment to copolymer polyelectrolyte hydrogel.

The surface micromechanical properties of 2-hydroxyethyl methacrylate (HEMA) and 2-methacryloxyethyl trimethyl ammonium chloride (MAETAC) copolymer hydrogels are probed using atomic force microscopy. HEMA-MAETAC polyelectrolyte hydrogels with increasing positive charge concentrations ranging from 0 to 400mM in increments of 40mM, are fabricated using different proportions of HEMA and MAETAC monomers. Increasing proportions of positively charged MAETAC monomers produce hydrogels with increasingly swollen states and correspondingly decreasing measures of stiffness, or Young's modulus. Increasing the relative proportion of charged monomers also increases the hysteresis in the approaching and retracting components of the force spectroscopy curves. When these hydrogels are equilibrated in cell-culture media without fetal bovine serum and a pH-controlled CO(2) environment, precipitation reactions increase the variability of the Young's modulus estimates. Adding a buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, maintains physiological pH without the use of a CO(2) environment, and thus reduces salt precipitation reactions and the variability of the Young's modulus. The attachment of porcine pulmonary artery endothelial cells increases with increasing prepared hydrogel charge concentration and decreasing elasticity.

ESE-1/EGR-1 pathway plays a role in tolfenamic acid-induced apoptosis in colorectal cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to prevent colorectal tumorigenesis. Although antitumor effects of NSAIDs are mainly due to inhibition of cyclooxygenase activity, there is increasing evidence that cyclooxygenase-independent mechanisms may also play an important role. The early growth response-1 (EGR-1) gene is a member of the immediate-early gene family and has been identified as a tumor suppressor gene. Tolfenamic acid is a NSAID that exhibits anticancer activity in a pancreatic cancer model. In the present study, we investigated the anticancer activity of tolfenamic acid in human colorectal cancer cells. Tolfenamic acid treatment inhibited cell growth and induced apoptosis as measured by caspase activity and bioelectric impedance. Tolfenamic acid induced EGR-1 expression at the transcription level, and analysis of the EGR-1 promoter showed that a putative ETS-binding site, located at -400 and -394 bp, was required for activation by tolfenamic acid. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed that this sequence specifically bound to the ETS family protein epithelial-specific ETS-1 (ESE-1) transcription factor. Tolfenamic acid also facilitated translocation of endogenous and exogenous ESE-1 to the nucleus in colorectal cancer cells, and gene silencing using ESE-1 small interfering RNA attenuated tolfenamic acid-induced EGR-1 expression and apoptosis. Overexpression of EGR-1 increased apoptosis and decreased bioelectrical impedance, and silencing of endogenous EGR-1 prevented tolfenamic acid-induced apoptosis. These results show that activation of ESE-1 via enhanced nuclear translocation mediates tolfenamic acid-induced EGR-1 expression, which plays a critical role in the activation of apoptosis.

Multicontrast microscopy technique to dynamically fingerprint live-cell focal contacts during exposure and replacement of a cytotoxic medium.

Multicontrast microscopy techniques were used to comprehensively and dynamically map the cellular contact area adhering to a substrate. The natural fringe patterns observed with interference reflection contrast microscopy were used to map the dynamic fingerprint of a porcine pulmonary artery endothelial cell's ventral surface and to examine the focal and/or close contacts to the substrate when exposed to a toxic agent Cytochalasin D. In addition, differential interference contrast microscopy sequentially imaged the overall cellular morphological responses to the agent. It was observed that focal contacts, which are tightly attached to the substrate, are strongly resistant to even high doses of the cytotoxic agent and that they also form the basis of cellular recovery after replacement of the cytotoxic medium with fresh medium.

Cellular electrical micro-impedance parameter artifacts produced by passive and active current regulation.

This study analyzes the cellular microelectrode voltage measurement errors produced by active and passive current regulation, and the propagation of these errors into cellular barrier function parameter estimates. The propagation of random and systematic errors into these parameters is accounted for within a Riemannian manifold framework consistent with information geometry. As a result, the full non-linearity of the model parameter state dependence, the instrumental noise distribution, and the systematic errors associated with the voltage to impedance conversion, are accounted for. Specifically, cellular model parameters are treated as the coordinates of a model space manifold that inherits a Riemannian metric from the data space. The model space metric is defined in terms of the pull back of an instrumental noise-dependent Fisher information metric. Additional noise sources produced by the evaluation of the cell-covered electrode model that is a function of a naked electrode random variable are also included in the analysis. Based on a circular cellular micro-impedance model in widespread use, this study shows that cellular barrier function parameter estimates are highly model state dependent. Systematic errors produced by coaxial lead capacitances and circuit loading can also lead to significant and model state-dependent parameter errors and should, therefore, be either reduced or corrected for analytically.

Opto-Electric Cellular Biosensor Using Optically Transparent Indium Tin Oxide (ITO) Electrodes.

Indium tin oxide (ITO) biosensors are used to perform simultaneous optical and electrical measurements in order to examine the dynamic cellular attachment, spreading, and proliferation of endothelial cells (ECs) as well as cytotoxic effects when exposed to cytochalasin D. A detailed description of the fabrication of these sensors is provided and their superior optical characteristics are qualitatively shown using four different microscopic images. Differential interference contrast microscopy (DICM) images were acquired simultaneously with micro-impedance measurements as a function of frequency and time. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. In addition, cytotoxicity effects, produced by the toxic agent cytochalasin D, were examined using micro-impedance measurements, confocal microscopy images of stained actin-filaments, and interference reflection contrast microscopy (IRCM) capable of examining the bottom morphology of a cell. The results of this study show (1) the dynamic optical and electrical cellular characteristics using optically thin ITO biosensors; (2) qualitative agreement between cell-covered electrode area and electrical impedance during cellular attachment; (3) in vitro cytotoxicity detection of ECs due to 3 mM cytochalasin D. The present opto-electric biosensor system is unique in that a simultaneous and integrated cellular analysis is possible for a variety of living cells.

Equilibrium and non-equilibrium charge-dependent quantification of endothelial cell hydrogel scaffolds.

Using equilibrium swelling and non-equilibrium membrane potential measurements, this study assesses the charge density in two representative series of polyelectrolyte hydrogels and examines the morphological and proliferative responses of endothelial cells as a function of the prepared charge offset. The neutral monomers 2-hydroxyethylmethacrylate (HEMA) and poly(ethylene glycol) dimethacrylate (n = 1,000) (PEGDMA) were copolymerized with either the acidic monomer 2-sulfoethyl methacrylate (SEMA) or the basic monomer methacryloxy ethyltrimethylammonium chloride (MAETAC) to make membranes with pregelation charge offset concentrations varying from 0 to +/-200 mM. A thermodynamic analysis of swelling and membrane potential measurements quantified the hydrogel charge density state following equilibration at different ion strengths. Porcine pulmonary artery endothelial cells were seeded on samples of each HEMA and PEGDMA copolymer and the amount of cell coverage was measured over a 4-day period. Cellular attachment and proliferation increased with increasing proportions of charged monomers and showed a threshold pattern of attachment and growth on the positively charged HEMA-MAETAC copolymer hydrogels with increasing proportions of initially prepared charge. The series of PEGDMA copolymer hydrogels remained relatively resistant to cellular attachment and proliferation over the range of prepared charges considered in this study.

A Riemannian manifold analysis of endothelial cell monolayer impedance parameter precision.

Endothelial cell adhesion and barrier function play a critical role in many biological and pathophysiological processes. The decomposition of endothelial cell adhesion and barrier function into cell-cell and cell-matrix components using frequency dependent cellular micro-impedance measurements has, therefore, received widespread application. Few if any studies, however, have examined the precision of these model parameters. This study presents a parameter sensitivity analysis of a representative cellular barrier function model using a concise geometric formulation that includes instrumental data acquisition settings. Both model state dependence and instrumental noise distributions are accounted for within the framework of Riemannian manifold theory. Experimentally acquired microimpedance measurements of attached endothelial cells define the model state domain, while experimentally measured noise statistics define the data space Riemannian metric based on the Fisher information matrix. The results of this analysis show that the sensitivity of cell-cell and cell-matrix impedance components are highly model state dependent and several well defined regions of low precision exist. The results of this study further indicate that membrane resistive components can significantly reduce the precision of the remaining parameters in these models.

Optoelectric biosensor using indium-tin-oxide electrodes.

The use of an optically thin indium-tin-oxide (ITO) electrode is presented for an optoelectric biosensor simultaneously recording optical images and microimpedance to examine time-dependent cellular growth. The transmittance of a 100 nm thick ITO electrode layer is approximately the same as the transmittance of a clean glass substrate, whereas the industry-standard Au(47.5 nm)/Ti(2.5 nm) electrode layer drops the transmittance to less than 10% of that of the glass substrate. The simultaneous optoelectric measurements permit determining the correlation of the cell-covered area increase with the microimpedance increase, and the example results obtained for live porcine pulmonary artery endothelial cells delineate the quantitative and comprehensive nature of cellular attachment and spreading to the substrate, which has not been clearly perceived before.

Endothelial cell electrical impedance parameter artifacts produced by a gold electrode and phase sensitive detection.

Frequency dependent cellular micro-impedance estimates obtained from a gold two-electrode configuration using phase sensitive detection have become increasingly used to evaluate cellular barrier model parameters. The results of this study show that cellular barrier function parameter estimates optimized using measurements obtained from this biosensor are highly susceptible to both time dependent and systematic instrumental artifacts. Based on a power spectral analysis of experimentally measured microelectrode voltages, synchronous, 60 Hz, and white Gaussian noise were identified as the most significant time dependent instrumental artifacts. The reduction of these artifacts using digital filtering produced a corresponding reduction in the optimized model parameter fluctuations. Using a series of instrumental circuit models, this study also shows that electrode impedance voltage divider effects and circuit capacitances can produce systematic deviations in cellular barrier function parameter estimates. Although the implementation of an active current source reduced the voltage divider effects, artifacts produced by coaxial cable and other circuit capacitive elements at frequencies exceeding 1 kHz still remained. Reducing time dependent instrumental fluctuations and systematic errors produced a significant reduction in cellular model barrier parameter errors and improved the model fit to experimental data.

An endothelial cell compatible biosensor fabricated using optically thin indium tin oxide silicon nitride electrodes.

This study describes the fabrication and performance of an endothelial cell compatible, optically thin, indium tin oxide (ITO) microimpedance biosensor. The biosensor was constructed by sputtering a thin insulating layer of silicon nitride (Si(3)N(4)) onto a 100 nm thick ITO layer. Indium tin oxide electrodes were formed by chemically etching 250 or 500 microm diameter holes through the Si(3)N(4) insulating layer. The exposed ITO electrode was electrically connected to an ITO counter electrode, approximately 2 cm(2) in area, via a 400 microL well containing cell culture media. A lock-in amplifier circuit monitored the impedance of porcine pulmonary artery endothelial cells (PPAECs) cultivated on the electrodes as a function of frequency, between 10 and 100 kHz, and as a function of time, at 5.62 kHz. The ITO-Si(3)N(4) microelectrodes provided consistent and repeatable impedance measurements to the attachment and spreading of PPAECs. In addition, the ITO-Si(3)N(4) electrodes were recyclable, robust, resistant to ethanol sterilization, and had a high optical transmittance. Most importantly, the ITO-Si(3)N(4) electrodes allowed optical access for dynamic cellular attachment imaging. The 5.62 kHz time dependent cellular impedance response to the drug Cytochalasin D further demonstrated the feasibility of using this electrode configuration for dynamic cellular impedance studies.

Simultaneous dynamic optical and electrical properties of endothelial cell attachment on indium tin oxide bioelectrodes.

This study quantifies the dynamic attachment and spreading of porcine pulmonary artery endothelial cells (PPAECs) on optically thin, indium tin oxide (ITO) biosensors using simultaneous differential interference contrast microscopy (DICM) and electrical microimpedance spectroscopy. A lock-in amplifier circuit monitored the impedance of PPAECs cultivated on the transparent ITO bioelectrodes as a function of frequency between 10 Hz and 100 kHz and as a function of time, while DICM images were simultaneously acquired. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. The results of this study show that the fraction of the cell-covered electrode area is in qualitative agreement with the electrical impedance during the attachment phase following the cell settling on the electrode surface. The possibility of several distinctly different states of electrode coverage and cellular attachment giving rise to similar impedance signals is discussed.