Search results

Now showing 1 - 10 of 216
  • Publication
    Photoacoustic effect for multiply scattered light
    (2007-09-25) Fisher, Andrew R; Schissler, Andrew J; Schotland, John C
    We consider the photoacoustic effect for multiply scattered light in a random medium. Within the accuracy of the diffusion approximation to the radiative transport equation, we present a general analysis of the sensitivity of a photoacoustic wave to the presence of one or more small absorbing objects. Applications to tumor detection by photoacoustic imaging are suggested.
  • Publication
    Cultured Alveolar Epithelial Cells From Septic Rats Mimic in Vivo Septic Lung
    (2010-01-01) Cohen, Taylor Sitarik; Lawrence, Gladys Gray; Margulies, Susan S
    Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200–300g) were sacrificed 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A) and MAPk (JNK, ERK, an p38) signaling activation, or barrier function was examined by measuring transepithelial resistance (TER) or the flux of two molecular tracers (5 and 20 Å). Inhibitors of JNK (SP600125, 20 µM) and ERK (U0126, 10 µM) were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm2, respectively), however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique preparation for probing the mechanisms by which sepsis alters alveolar epithelium.
  • Publication
    Calpain mediates proteolysis of the voltage-gated sodium channel alpha-subunit
    (2009-08-19) von Reyn, Catherine R; Spaethling, Jennifer M; Mesfin, Mahlet N; Ma, Marek; Neumar, Robert W; Smith, Douglas H; Siman, Robert; Meaney, David F
    Alterations in the expression, molecular composition, and localization of voltage-gated sodium channels play major roles in a broad range of neurological disorders. Recent evidence identifies sodium channel proteolysis as a key early event after ischemia and traumatic brain injury, further expanding the role of the sodium channel in neurological diseases. In this study, we investigate the protease responsible for proteolytic cleavage of voltage-gated sodium channels (NaChs). NaCh proteolysis occurs after protease activation in rat brain homogenates, pharmacological disruption of ionic homeostasis in cortical cultures, and mechanical injury using an in vitro model of traumatic brain injury. Proteolysis requires Ca2+ and calpain activation but is not influenced by caspase-3 or cathepsin inhibition. Proteolysis results in loss of the full-length {alpha}-subunits, and the creation of fragments comprising all domains of the channel that retain interaction even after proteolysis. Cell surface biotinylation after mechanical injury indicates that proteolyzed NaChs remain in the membrane before noticeable evidence of neuronal death, providing a mechanism for altered action potential initiation, propagation, and downstream signaling events after Ca2+ elevation.
  • Publication
    MicroRNA-10a Regulation of Proinflammatory Phenotype in Athero-susceptible Endothelium in Vivo and in Vitro
    (2010-07-27) Fang, Yun; Shi, Congzhu; Manduchi, Elisabetta; Civelek, Mete; Davies, Peter F
    A chronic proinflammatory state precedes pathological change in arterial endothelial cells located within regions of susceptibility to atherosclerosis. Thepotential contributions of regulatory microRNAs to this disequilibrium were investigated by artery site-specific profiling in normal adult swine. Expression of endothelial microRNA10a (miR-10a) was lower in the athero-susceptible regions of the inner aortic arch and aorto-renal branches than elsewhere. Expression of Homeobox A1 (HOXA1), a known miR-10a target, was up-regulated in the same locations. Endothelial transcriptome microarray analysis of miR-10a knockdown in cultured human aortic endothelial cells (HAEC) identified IκB/NF-κB–mediated inflammation as the top category of up-regulated biological processes. Phosphorylation of IκBα, a prerequisite for IκBα proteolysis and NF-κB activation, was significantly up-regulated in miR-10a knockdown HAEC and was accompanied by increased nuclear expression of NF-κB p65. The inflammatory biomarkers monocyte chemotactic protein 1 (MCP-1), IL-6, IL-8, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were elevated following miR-10a knockdown. Conversely, knockin of miR- 10a (a conservative 25-fold increase) inhibited the basal expression of VCAM-1 and E-selectin in HAEC. Two key regulators of IκBα degradation— mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and β-transducin repeat-containing gene (βTRC)—contain a highly conserved miR-10a binding site in the 3′ UTR. Bothmolecules were up-regulated by miR-10a knockdown and suppressed by miR-10a knockin, and evidence of direct miR-10a binding to the 3′ UTRwas demonstrated by luciferase assay. Comparative expression studies of endothelium located in athero-susceptible aortic arch and athero-protected descending thoracic aorta identified significantly up-regulated MAP3K7, βTRC, phopho-IκBα, and nuclear p65 expression suggesting that the differential expression of miR-10a contributes to the regulation of proinflammatory endothelial phenotypes in athero-susceptible regions in vivo.
  • Publication
    A Microcantilever Device to Assess the Effect of Force on the Lifetime of Selectin-Carbohydrate Bonds
    (2001-02-01) Tees, Davis F. J.; Waugh, Richard E; Hammer, Daniel A
    A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-μm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewisx, was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), rf. From the distribution of forces at failure, the average force was determined and plotted as a function of ln rf. The slope and intercept of the plot yield the unstressed reverse reaction rate, kro , and a parameter that describes the force dependence of reverse reaction rates, ro. The ligand was titrated so adhesion occurred in ~30% of tests, implying that >80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are ro = 0.048 and 0.016 nm and kro = 0.72 and 2.2 s-1 for loading rates in the ranges 200–1000 and 1000–5000 pN s-1, respectively. Levenberg-Marquardt fitting across all values of rf gives ro = 0.034 nm and kro = 0.82 s-1. The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations.
  • Publication
    Dynamic Computation in a Recurrent Network of Heterogeneous Silicon Neurons
    (2006-05-01) Merolla, Paul; Boahen, Kwabena
    We describe a neuromorphic chip with a two-layer excitatory-inhibitory recurrent network of that exhibits localized clusters of neural activity. Unlike other recurrent networks, the clusters in our network are pinned to certain locations due to transistor mismatch introduced in fabrication. As described in previous work, our pinned clusters respond selectively to oriented stimuli and the neurons' preferred orientations are distributed similar to the visual cortex. Here we show that orientation computation is rapid when activity alternates between layers (staccato-like), dislodging pinned clusters, which promotes fast cluster diffusion.
  • Publication
    Effect of oxidatively damaged DNA on the active site preorganization during nucleotide incorporation in a high fidelity polymerase from Bacillus stearothermophilus
    (2007-12-07) Venkatramani, Ravindra; Radhakrishnan, Ravi
    We study the effect of the oxidative lesion 8-oxoguanine (8oxoG) on the preorganization of the active site for DNA replication in the closed (active) state of the Bacillus fragment (BF), a Klenow analog from Bacillus stearothermophilus. Our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of BF bound to correct dCTP/incorrect dATP opposite guanine (G) and 8oxoG bases in DNA suggest that the lesion introduces structural and energetic changes at the catalytic site to favor dATP insertion. Despite the formation of a stable Watson- Crick pairing in the 8oxoG:dCTP system, the catalytic geometry is severely distorted to possibly slow down catalysis. Indeed, our calculated free energy landscapes associated with active site preorganization suggest additional barriers to assemble an efficient catalytic site, which need to be overcome during dCTP incorporation opposite 8oxoG relative to that opposite undamaged G. In contrast, the catalytic geometry for the Hoogsteen pairing in the 8oxoG:dATP system is highly organized and poised for efficient nucleotide incorporation via the "two-metal-ion" catalyzed phosphoryl transfer mechanism. However, the free energy calculations suggest that the catalytic geometry during dATP incorporation opposite 8oxoG is considerably less plastic than that during dCTP incorporation opposite G despite a very similar, well organized catalytic site for both systems. A correlation analysis of the dynamics trajectories suggests the presence of significant coupling between motions of the polymerase fingers and the primary distance for nucleophilic attack (i.e., between the terminal primer O3′ and the dNTP Pα atoms) during correct dCTP incorporation opposite undamaged G. This coupling is shown to be disrupted during nucleotide incorporation by the polymerase with oxidatively damaged DNA/dNTP substrates. We also suggest that the lesion affects DNA interactions with key polymerase residues, thereby affecting the enzymes ability to discriminate against noncomplementary DNA/dNTP substrates. Taken together, our results provide a unified structural, energetic, and dynamic platform to rationalize experimentally observed relative nucleotide incorporation rates for correct dCTP/incorrect dATP insertion opposite an undamaged/oxidatively damaged template G by BF.
  • Publication
    The Rho Pathway Mediates Transition to an Alveolar Type I Cell Phenotype During Static Stretch of Alveolar Type II Cells
    (2010-06-01) Foster, Cherie D; Varghese, Linda S; Gonzales, Linda W; Margulies, Susan S; Guttentag, Susan H
    Stretch is an essential mechanism for lung growth and development. Animal models in which fetal lungs have been chronically over or underdistended demonstrate a disrupted mix of type II and type I cells, with static overdistention typically promoting a type I cell phenotype. The Rho GTPase family, key regulators of cytoskeletal signaling, are known to mediate cellular differentiation in response to stretch in other organs. Using a well-described model of alveolar epithelial cell differentiation and a validated stretch device, we investigated the effects of supraphysiologic stretch on human fetal lung alveolar epithelial cell phenotype. Static stretch applied to epithelial cells suppressed type II cell markers (SP-B and Pepsinogen C, PGC), and induced type I cell markers (Caveolin-1, Claudin 7 and Plasminogen Activator Inhibitor-1, PAI-1) as predicted. Static stretch was also associated with Rho A activation. Furthermore, the Rho kinase inhibitor Y27632 decreased Rho A activation and blunted the stretch-induced changes in alveolar epithelial cell marker expression. Together these data provide further evidence that mechanical stimulation of the cytoskeleton and Rho activation are key upstream events in mechanotransduction-associated alveolar epithelial cell differentiation.
  • Publication
    Potential for head injuries in infants from low-height falls: Laboratory investigation
    (2008-11-01) Coats, Brittany; Margulies, Susan S
    Object. Falls are the most common accident scenario in young children as well as the most common history provided in child abuse cases. Understanding the biomechanics of falls provides clinicians with objective data to aid in their diagnosis of accidental or inflicted trauma. The objective of this study was to determine impact forces and angular accelerations associated with low-height falls in infants. Methods. An instrumented anthropomorphic infant surrogate was created to measure the forces and 3D angular accelerations associated with falls from low heights (0.3–0.9 m) onto a mattress, carpet pad, or concrete. Results. Although height significantly increased peak angular acceleration (αp), change in peak-to-peak angular velocity, time duration associated with the change in velocity, and peak impact force (Fp) for head-first drops onto a carpet pad or concrete, none of these variables were significantly affected by height when dropped onto a mattress. The αp was not significantly different for drops onto a carpet pad and concrete from 0.6 or 0.9 m due to compression of the carpet pad. Surprisingly, sagittal αp was equaled or surpassed by axial αp. Conclusions. These are the first 3D angular acceleration and impact force data available for head impact in infants from low-height falls. A future study involving a computational model of the infant head will use the loads measured in this study to predict the probability of occipital skull fracture on impact from head-first low-height falls. Together, these studies will provide data that will aid clinicians in the evaluation of accidental and inflicted head injuries, and will contribute to the design of safer environments for children. (DOI: 10.3171/PED.2008.2.11.321)
  • Publication
    E-cadherin engagement stimulates proliferation via Rac1
    (2006-05-08) Liu, Wendy F; Nelson, Celeste M; Pirone, Dana M; Chen, Christopher S.
    E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK- 52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120- catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell–cell contact may trigger or inhibit epithelial cell proliferation in different settings.