Flagellin Acting Via TLR5 is the Major Activator of Key Signaling Pathways Leading to NF-kappa B and Proinflammatory Gene Program activation in intestinal epithelial cells

BACKGROUND: Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-kappa B is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-kappa B during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-kappa B activation in all the cells; invasion of cells by the bacteria is not required to activate NF-kappa B. RESULTS: Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and I kappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-kappa B via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-kappa B activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-kappa B activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both. CONCLUSION: These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-kappa B and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition

Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

BACKGROUND: Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-κB is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-κB during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-κB activation in all the cells; invasion of cells by the bacteria is not required to activate NF-κB. RESULTS: Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and Ikappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-κB via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-κB activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-κB activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both. CONCLUSION: These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-κB and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition

The Low-Resolution Structure of Nascent High Density Lipoprotein Reconstituted with DMPC With and Without Cholesterol Reveals A Mechanism for Particle Expansion

High density lipoproteins (HDL) are athero-protective particles under investigation as potential therapeutic agents for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation to obtain the low resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidyl choline (DMPC), apoA1:DMPC (1:80, mol:mol). The overall shape of the entire particle is discoidal, with low resolution architecture of apoA1 visualized as an open, contorted, and slightly out of plane structure with three arms, while the low resolution shape of the lipid phase is an oblate ellipsoid that fits well within the protein shape. Modeling studies incorporating the SANS data indicate that apoA1 within the lipoprotein is folded onto itself, making a hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry analyses. The open conformation of apoA1 observed coupled with the lipid shape indicate that the lipid is predominantly a bilayer with a small micelle domain between the open apoA1 arms. Collectively, these studies demonstrate that full length apoA1 retains an open architecture that is dictated by its lipid cargo. This configuration may help accommodate potential changing lipid cargo content of the particle by quantized expansion of hairpin structures in apoA

Angiotensin-(1–7) and the G Protein-Coupled Receptor Mas Are Key Players in Renal Inflammation

Angiotensin (Ang) II mediates pathophysiologial changes in the kidney. Ang-(1–7) by interacting with the G protein-coupled receptor Mas may also have important biological activities

Nano-electromechanical oscillators (NEMOs) for RF technologies.

Nano-electromechanical oscillators (NEMOs), capacitively-coupled radio frequency (RF) MEMS switches incorporating dissipative dielectrics, new processing technologies for tetrahedral amorphous carbon (ta-C) films, and scientific understanding of dissipation mechanisms in small mechanical structures were developed in this project. NEMOs are defined as mechanical oscillators with critical dimensions of 50 nm or less and resonance frequencies approaching 1 GHz. Target applications for these devices include simple, inexpensive clocks in electrical circuits, passive RF electrical filters, or platforms for sensor arrays. Ta-C NEMO arrays were used to demonstrate a novel optomechanical structure that shows remarkable sensitivity to small displacements (better than 160 fm/Hz {sup 1/2}) and suitability as an extremely sensitive accelerometer. The RF MEMS capacitively-coupled switches used ta-C as a dissipative dielectric. The devices showed a unipolar switching response to a unipolar stimulus, indicating the absence of significant dielectric charging, which has historically been the major reliability issue with these switches. This technology is promising for the development of reliable, low-power RF switches. An excimer laser annealing process was developed that permits full in-plane stress relaxation in ta-C films in air under ambient conditions, permitting the application of stress-reduced ta-C films in areas where low thermal budget is required, e.g. MEMS integration with pre-existing CMOS electronics. Studies of mechanical dissipation in micro- and nano-scale ta-C mechanical oscillators at room temperature revealed that mechanical losses are limited by dissipation associated with mechanical relaxation in a broad spectrum of defects with activation energies for mechanical relaxation ranging from 0.35 eV to over 0.55 eV. This work has established a foundation for the creation of devices based on nanomechanical structures, and outstanding critical research areas that need to be addressed for the successful application of these technologies have been identified

Fundamental study of CO2-H2O-mineral interactions for carbon sequestration, with emphasis on the nature of the supercritical fluid-mineral interface.

In the supercritical CO2-water-mineral systems relevant to subsurface CO2 sequestration, interfacial processes at the supercritical fluid-mineral interface will strongly affect core- and reservoir-scale hydrologic properties. Experimental and theoretical studies have shown that water films will form on mineral surfaces in supercritical CO2, but will be thinner than those that form in vadose zone environments at any given matric potential. The theoretical model presented here allows assessment of water saturation as a function of matric potential, a critical step for evaluating relative permeabilities the CO2 sequestration environment. The experimental water adsorption studies, using Quartz Crystal Microbalance and Fourier Transform Infrared Spectroscopy methods, confirm the major conclusions of the adsorption/condensation model. Additional data provided by the FTIR study is that CO2 intercalation into clays, if it occurs, does not involve carbonate or bicarbonate formation, or significant restriction of CO2 mobility. We have shown that the water film that forms in supercritical CO2 is reactive with common rock-forming minerals, including albite, orthoclase, labradorite, and muscovite. The experimental data indicate that reactivity is a function of water film thickness; at an activity of water of 0.9, the greatest extent of reaction in scCO2 occurred in areas (step edges, surface pits) where capillary condensation thickened the water films. This suggests that dissolution/precipitation reactions may occur preferentially in small pores and pore throats, where it may have a disproportionately large effect on rock hydrologic properties. Finally, a theoretical model is presented here that describes the formation and movement of CO2 ganglia in porous media, allowing assessment of the effect of pore size and structural heterogeneity on capillary trapping efficiency. The model results also suggest possible engineering approaches for optimizing trapping capacity and for monitoring ganglion formation in the subsurface