Showing papers by "Simon C. Watkins published in 2010"

An Autophagy-Enhancing Drug Promotes Degradation of Mutant α1-Antitrypsin Z and Reduces Hepatic Fibrosis

Abstract: In the classical form of alpha1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant alpha1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain-of-toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.

Inhibition and role of let-7d in idiopathic pulmonary fibrosis.

Abstract: Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.Objectives: To determine changes in expression and role of microRNAs in IPF.Methods: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-β. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.Measurements and Main Resu...

AMPK agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells.

Abstract: The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel and epithelial Na(+) channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I(sc)), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I(sc) in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.

Sar1 assembly regulates membrane constriction and ER export

Abstract: The guanosine triphosphatase Sar1 controls the assembly and fission of COPII vesicles. Sar1 utilizes an amphipathic N-terminal helix as a wedge that inserts into outer membrane leaflets to induce vesicle neck constriction and control fission. We hypothesize that Sar1 organizes on membranes to control constriction as observed with fission proteins like dynamin. Sar1 activation led to membrane-dependent oligomerization that transformed giant unilamellar vesicles into small vesicles connected through highly constricted necks. In contrast, membrane tension provided through membrane attachment led to organization of Sar1 in ordered scaffolds that formed rigid, uniformly nonconstricted lipid tubules to suggest that Sar1 organization regulates membrane constriction. Sar1 organization required conserved residues located on a unique C-terminal loop. Mutations in this loop did not affect Sar1 activation or COPII recruitment and enhanced membrane constriction, yet inhibited Sar1 organization and procollagen transport from the endoplasmic reticulum (ER). Sar1 activity was directed to liquid-disordered lipid phases. Thus, lipid-directed and tether-assisted Sar1 organization controls membrane constriction to regulate ER export.

Identification of a cardiac specific protein transduction domain by in vivo biopanning using a M13 phage peptide display library in mice.

Abstract: Background A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library.

On Low Altitude Flight through the Atmospheric Boundary Layer

Abstract: Major challenges to low speed micro flight are the transient and time-averaged velocities arising from the Atmospheric Boundary Layer (ABL), particularly turbulence a few metres above the ground In this paper, existing data from meteorologists and wind engineers are reviewed and measurements dedicated to understanding the spatial and temporal velocity fields that MAVs experience are briefly described Data from a wide variety of terrains are analysed, with the majority of data obtained in relatively well mixed turbulent flow (ie away from local effects such as buildings) and for conditions of nominally neutral stability Spectra for data well removed from local effects exhibited the expected 5/3rds Kolmogorov law Transient flow pitch angles were investigated (obtained from four small laterally displaced probes), in order to understand the possible roll and pitch inputs to MAVs It was noted that for all data obtained the variation with lateral separation decreased relatively slowly with reducing separation down to the closest inter-probe spacing of 14mm This effect is thought to explain the increasing piloting difficulties experienced in maintaining good roll control for decreasing scales of craft when any appreciable atmospheric winds are present

Ectopic Matrix Metalloproteinase 9 Expression in Human Brain Tumor Cells Enhances Oncolytic HSV Vector Infection

Abstract: Oncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.

Characterization of an lhx1a transgenic reporter in zebrafish.

Abstract: The LIM-domain containing transcription factor, Lhx1, is involved in the regulation of early gastrulation cell movements, kidney organogenesis and other processes in vertebrate model organisms. To follow the expression of this gene in live embryos, we created transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of lhx1a regulatory regions. Tg(lhx1a:EGFP) pt303 recapitulates the expression of endogenous lhx1a beginning at early gastrula stages through 72 hours of development with only few exceptions. In addition, over-expression of the Nodal ligand, ndr1, results in the concomitant expansion of the transgene and endogenous lhx1a expression. Treatment of Tg(lhx1a:EGFP) pt303 embryos with the small molecule SB-431542, an inhibitor of Nodal signaling, results in the loss of both transgene and endogenous lhx1a expression. These experiments suggest that Tg(lhx1a:EGFP) pt303 is regulated in a manner similar to endogenous lhx1a. Therefore, this reporter can be utilized not only for monitoring lhx1a expression, but also for numerous applications, including chemical genetics screening.

An overview of experiments on the dynamic sensitivity of MAVs to turbulence

Abstract: Aspects of the turbulent wind environment Micro Air Vehicles (MAVs) experience when flying outdoors were replicated in a large wind tunnel. An overview of the facility, instrumentation and initial flight tests is given. Piloting inputs and aircraft accelerations were recorded on fixed and rotary wing MAVs and for some tests, measurements of the approach flow (u,v,w sampled at 1,250Hz at four laterally disposed upstream locations) were made. The piloting aim was to hold straight and level flight in the 12m wide × 4m high × ~50m long test section, while flying in a range of turbulent conditions. The Cooper-Harper rating system showed that a rotary craft was less sensitive to the effects of turbulence compared to the fixed wing craft and that while the fixed wing aircraft was relatively easy to fly in smooth air, it became extremely difficult to fly under high turbulence conditions. The rotary craft, while more difficult to fly per. se., did not become significantly harder to fly in relatively high turbulence levels. However the rotary craft had a higher mass and MOI than the fixed wing craft and further work is planned to understand the effects of these differences.

Dynamic Sensitivity to Atmospheric Turbulence of Unmanned Air Vehicles with Varying Configuration

Abstract: DOI: 10.2514/1.46860 Air vehicle flight in turbulence is generally treated as an anomalous part of the flying environment. Aircraft geometries and flight-control systems are often designed and tested for calm atmospheric conditions, where both steady winds and gusts are minor. As a result, the flight performance of small aircraft deteriorates in the presence of atmospheric turbulence, where gust disturbances can be large relative to the flying speeds. A better approach is needed in the aircraft and control system design process that specifically accounts for the effects of turbulence and providesameansofmitigatingdisturbancestoimprovethemissioneffectivenessofmicrounmannedairvehiclesand small unmanned air vehicles. The current research considers untethered flight tests of a small unmanned air vehicle in a large wind engineering tunnel that can be configured to replicate turbulence levels expected from urban and suburban environments. Systematic changes to the configuration of the fixed-wing aircraft are made to evaluate the roleofmetrics,suchasc.g.,mass,momentofinertia,wingspan,andwingloadingtoturbulencesensitivity.Estimates of the force and moment disturbances indicate that some parameters, such as moment of inertia, have simple and expectedinfluencesontheresponsetoturbulence. Conversely,wingareaandmasshaveconflictingeffectsduetothe compounded influences on the aircraft response. The sensitivity of the various aircraft configurations to turbulence are presented as control equivalent turbulence disturbances, which equate forces and moments acting on the airframe to control deflections. This method normalizes the aircraft responses with respect to the ability to suppress disturbances with actuated controls.

DC expressing transgene Foxp3 are regulatory APC

Abstract: Tolerogenic DC and suppressive Foxp3(+) Treg play important roles in preventing autoimmunity and allograft rejection. We report that (adenovirus mediated) ectopic expression of Foxp3 in human DC (i.e. DC.Foxp3) yields an APC that severely limits T-cell proliferation and type-1 immune responses from the naive, but not memory, pool of responder T cells in vitro. In marked contrast, the frequencies of type-2 and Treg responses were dramatically increased after stimulation of naive T cells with DC.Foxp3 versus control DC. DC.Foxp3-induced CD4(+)CD25(+) Treg cells potently suppressed the proliferation of, and IFN-gamma production from, CD4(+) and CD8(+) responder T cells. Notably, the immunosuppressive biology of DC.Foxp3 was effectively normalized by addition of 1-methyl-tryptophan or neutralizing anti-TGF-beta1 Ab during the period of T-cell priming. These data suggest the potential utility of regulatory DC.Foxp3 and/or DC.Foxp3-induced CD4(+)CD25(+) Treg as translational agents for the amelioration or prevention of pathology in the setting of allograft transplantation and/or autoimmunity.

Numerical Simulation of an Adaptive Airfoil System using SMA Actuators

Abstract: Shape memory alloy (SMA) is lightweight, produces high force and large deflection which makes it a suitable candidate for actuator in the adaptive airfoil system design. The deflection of a variable cambered wing is controlled by means of resistive heating of SMA actuator and cooling in the surrounding air. An experimental wing model was developed using ABS to test the application of the SMA actuators. The SMA actuators were fixed underneath the wing skin. The heating of the wires caused them to contract, creating a force and generating a moment which deflects the wing. A wind tunnel test was performed to investigate the change in lift to drag ratio of the wing when the actuator is switched on and off. The results proved that the use of ABS skin and SMA actuators in the wing model is reliable as significant change in lift to drag ratio was detected when the wing was morphed.

Testing of adaptive airfoil for uav using shape memory alloy actuators

Abstract: Adaptive airfoil control system can potentially improve flight performance by optimizing the maximum lift-to-drag ratio throughout all flight regimes. Improved flight performance translates into weight and fuel savings.

Effects of Vehicle Add-Ons on Aerodynamic Performance

Abstract: Most modern passenger cars use various add-ons including roof-rack, ski-rack, bicycle rack, advertising signboard, police and ambulance siren, portable ladder and taxi signs for commercial and professional reasons. As over 80% of the required total vehicle power is essential to overcome the aerodynamic resistance (and the remaining power is used for rolling resistance), any add-ons causes extra drag to the total system hence the extra fuel cost. The primary objectives of this study were to experimentally measure the aerodynamic drag generated by various vehicle add-ons under a range of vehicle operating speeds. The study was conducted using a reduced scale (25%) detailed model of a production large family size passenger car manufactured in Australia. The aerodynamic drag coefficient was related to fuel consumption and a detailed analysis of fuel savings was performed.

Reduction of roof rack noise and vibration

Abstract: The noise and vibration design of automotive add-on component roof rack was investigated. Different modification options were evaluated which include mounting location alteration, mounting damping change and material revision. Results from experimental modal analysis and sound pressure spectrum measurement in a wind tunnel indicate the direction of design modification for reduction of the roof rack noise and vibration. The modal analysis and physical tests of wind tunnel proved to be good tools for evaluating and ranking roof rack noise and vibration performance.

Mitigating the Effects of Atmospheric Turbulence: Towards More Useful Micro Air Vehicles

Abstract: : This report is the research summary of flying MAV experiments in turbulent flow, utilizing prior research measuring and reproducing aspects of the outdoor environment. Turbulence was replicated in a large wind engineering tunnel and was well mixed, thus replicating atmospheric turbulence under neutrally stable conditions far removed from local effects. Current studies are focused on using increasingly small fixed-wing and flapping-wing aircraft with IMU video tracking, and upstream flow measurements to correlate measured turbulence with vehicle disturbances. Rate, acceleration, force and pressure sensors are being evaluated to determine candidates for providing phase-advanced measurement of incipient turbulence, which can allow the aircraft to preemptively move the control surfaces to suppress attitude and position disturbances.

Effects of Wing Design on Aerodynamics of MAVs in Turbulent Flow

Abstract: Micro air vehicles have the potential to complete useful missions in regions of dense obstacles near the Earth’s surface. However, the airflow profile in these regions is characterized by a high level of turbulence. Additionally, the characteristics of micro air vehicles make them much more susceptible to the effects of turbulence. To be effective in their missions, this new class of vehicles must be stable in the face of turbulence yet controllable and agile to perform the necessary maneuvers in an obstacle rich environment. This paper characterizes the impact of standard wing design parameters and turbulence intensity on the stability derivatives of micro air vehicles. Data show that turbulence has a significant impact on the lift, drag, and pitching moment on a micro air vehicle, of which some of the effects appear to be nonlinear. However, the potential for wing design to be used to counteract the effects of turbulence is still unclear.

NLRP3 inflammasome-dependent HMGB1 release induced by a bacterial pore-forming toxin (136.15)

Abstract: The NLRP3 inflammasome is a caspase-1-containing complex involved in the maturation of pro-inflammatory cytokines IL-1β and IL-18. NLRP3, independent of its inflammasome functions, also plays a role in a cathepsin B-dependent, caspase-1-independent necrotic cell death process known as pyronecrosis. We have previously shown that the cholesterol-dependent, pore-forming toxin tetanolysin O (TLO) triggers IL-1β release from LPS-primed murine bone marrow-derived macrophages (BMDM) in a NLRP3 inflammasome- and cathepsin B-dependent manner. The work contained herein supports an additional role for TLO in the necrosis of LPS-primed BMDM. Both a low and a high TLO dose induce release of lactate dehydrogenase (LDH) and high mobility group box 1 (HMGB1) and this release is NLRP3-dependent for the low toxin dose. Low dose TLO-induced HMGB1 release is also dependent on the activities of caspase-1 and cathepsin B, suggesting a novel form of cell death that is distinct from caspase-1-independent pyronecrosis. We also observe via fixed cell microscopy that TLO causes HMGB1 to move from the nucleus to the cytoplasm in LPS-primed BMDM and that HMGB1 release is dependent on NLRP3 activity. To our knowledge, this is the first study to visualize HMGB1 localization in cells lacking NLRP3. These results provide information on the regulation of HMGB1 release and could eventually lead to new therapeutic approaches to target HMGB1, which contributes to many inflammatory diseases.

BiochemicalIsolationandCharacterizationofthe TubulovesicularLC3-positiveAutophagosomalCompartment * □

Abstract: Autophagosomes and their precursors are best defined by electron microscopy but may also be traced in living cells based on the distribution of specific autophagy molecules. LC3, the most commonly examined autophagy marker in mammalian cells, labels structures that are frequently manifested as dots or rings using light microscopy; however, the nature of these structures is not entirely clear. We reported here a novel approach to examine the LC3-positive compartment in cell-free lysates, which revealed that they were actually tubulovesicular structures with considerable heterogeneity. Using affinity purification, we isolated these membranes for electron microscopy, which indicated that they possessed ultrastructural features consistent with autophagosomal membranes at various maturation stages. Further biochemical and proteomics analyses demonstrated the presence of multiple autophagy-related and other functional molecules. The different distribution patterns of Atg5, Atg16, Atg9, and p62/SQSTM1 on the LC3-positive compartment provided new clues on how these molecules might be involved in the dynamics of the autophagosomal membranes. Finally, several morphologically unique groups of LC3-positive membranes were categorized. Their topological configurations suggested that double-membrane vesicles could be derived from single membrane compartments via different means, including tubule-to-vesicle conversion, whose presence was supported by live cell imaging. These findings thus provide new information on the dynamics of the autophagosomal compartment.