Inter-tester and intra-tester reliability of ultrasound imaging measurements of abdominal muscles in adolescents with and without idiopathic scoliosis: a case-controlled study.

The present study established inter-tester and intra-tester reliabilities of ultrasound imaging and diagnostically differentiated muscle imbalances in lateral abdominal muscle sizes between normal adolescents and adolescents with idiopathic scoliosis (AIS). Fifteen adolescents with AIS were age- and gender-matched with 15 normal adolescents. There was no significant difference between bilateral abdominal muscles in normal adolescents, but there was a significant difference between bilateral abdominal muscles in adolescents with AIS (P<0.05). Overall, inter-tester and intra-tester reliabilities in normal and AIS adolescents ranged from 0.801-0.984. This novel study shows that using ultrasound imaging to measure lateral abdominal muscle thickness is: (1) highly reliable between and within the testers; and (2) capable of distinguishing between subjects with and without pathological muscle morphology due to AIS.

Screening for novel constitutively active CXCR2 mutants and their cellular effects.

Chemokines play an important role in inflammatory, developmental, and homeostatic processes. Deregulation of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the "on" position. This can lead to cellular transformation/tumorigenesis. The CXC chemokine receptor 2 (CXCR2) is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with CXC chemokines induces leukocyte migration, trafficking, leukocyte degranulation, cellular differentiation, and angiogenesis. Activation of CXCR2 can lead to cellular transformation. We hypothesized that CAM CXCR2s may play a role in cancer development. In order to identify CXCR2 CAMs, potential mutant CXCR2 receptors were screened using a modified Saccharomyces cerevisiae high-throughput system. S. cerevisiae has been used successfully to identify GPCR/G-protein interactions and autocrine selection for peptide agonists. The CXCR2 CAMs identified from this screen were characterized in mammalian cells. Their ability to transform cells in vitro was shown using foci formation, soft-agar growth, impedance measurement assays, and in vivo tumor growth following hind flank inoculation into mice. Signaling pathways contributing to cellular transformation were identified using luciferase reporter assays. Studying constitutively active GPCRs is an approach to "capturing" pluridimensional GPCRs in a "locked" activation state. In order to address the residues necessary for CXCR2 activation, we used S. cerevisiae for screening novel CAMs and characterized them using mammalian reporter assays.

A potential proliferative gene, NUDT6, is down-regulated by green tea catechins at the posttranscriptional level.

The main aims of this study were to elucidate the effect of green tea catechins on Nudix-type motif 6 (NUDT6) suppression and to characterize NUDT6's biological activity. Our microarray data showed that the green tea component epicatechin-3-gallate suppressed NUDT6 expression, and this was confirmed by RT-PCR. Subsequently, the use of different catechins showed that the effect of epigallocatechin-3-gallate (EGCG) was stronger than that of other catechins. At the posttranscriptional level, EGCG decreased the RNA stability of NUDT6, indicating it as a potential mechanism of NUDT6 suppression. Further cloning of the 3' untranslated region of human NUDT6 mRNA resulted in reduced luciferase activity by EGCG treatment. This effect was at least, in part, mediated by the extracellular-signal-regulated kinase and p38MAPK pathways. Finally, increased cell proliferation and cell growth in soft agar were observed in NUDT6-overexpressing cells. These findings provide a novel mechanism for the suppression of the proliferative gene NUDT6 by green tea catechins in human colorectal cancer.

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.

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.

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.

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.