NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases

The new generation of PowerPC VMEbus front end computers for the CERN SPS and LEP accelerators system

The CERN SPS and LEP PowerPC project is aimed at introducing a new generation of PowerPC VMEbus processor modules running the LynxOS real-time operating system. This new generation of front end computers using the state-of-the-art microprocessor technology will first replace the obsolete XENIX PC based systems (about 140 installations) successfully used since 1988 to control the LEP accelerator. The major issues addressed in the scope of this large scale project are the technical specification for the new PowerPC technology, the re-engineering aspects, the interfaces with other CERN wide projects, and the set up of a development environment. This project offers also support for other major SPS and LEP projects interested in the PowerPC microprocessor technology

Evidence of chitosanase involvement in the protection of bacteria against the antimicrobial activity of the chitosan

Chitosan, a biopolymer composed of [béta]-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine residues has multiple industrial applications. Recently, chitosan has gained great interest due to its antimicrobial activity. Chitosan has antimicrobial activity against a wide range of target organisms such as bacteria, fungi and viruses. This antimicrobial activity is based on its cationic character, and is mediated by the chitosan's positively charged amino groups interactions with negatively charged residues in the bacterial cell wall. Enzymes with chitosanase activity catalyzing the hydrolysis of glycoside linkages in chitosan are found in many organisms, including bacteria, fungi, and plants. In the last three decades, chitosanases have been intensively studied as tools for biotechnological transformation of chitosan. However, less is known about their physiological functions in chitosanase-producing microorganisms. Previous reports have characterized chitosanases as metabolic enzymes allowing bacteria to use chitosan as carbon and nitrogen sources. The aim of this research project was to examine chitosanases significance as possible resistance factors against the antimicrobial effect of chitosan. Our work, as well as previous studies realized in our laboratory, showed that expression of a heterologous chitosanase gene in the Gram-negative bacterium Escherichia coli (naturally devoid of chitosanase activity) increases the level of resistance against chitosan. Interestingly, the resistance level to chitosan was influenced by the relative activity of the heterologous chitosanase. The expression of inactive heterologous chitosanase did not confer any resistance to chitosan supporting our hypothesis that chitosanases may have a role in the protection against the antimicrobial effect of chitosan. In order to obtain more direct evidence sustaining our hypothesis, we inactivated the chitosanase gene from Streptomyces lividans TK24. Hence, we developed a new system for gene disruption and replacement in Streptomyces with cytosine deaminase as negative selection marker. The disruption of the chitosanase gene in S. lividans TK24 resulted in an increased susceptibility of the mutant strain towards the toxic effect of chitosan. Our in vivo experiments showed that, in the presence of chitosan, growth of this mutant strain as well as its ability for xylose uptake were impaired compared to the wildtype strain. This represents the first genetical proof for the protective role of a chitosanase against the bactericidal effect of chitosan. In our quest to discover chitosanases with new characteristics, we determined the biochemical properties of the chitosanase CsnA from Streptomyces coelicolor A3(2). Our studies revealed that CsnA was, in many aspects, very similar to the chitosanase CsnN174 from Streptomyces sp. N174. An interesting feature of the CsnA is its secretion. The signal peptide of the CsnA has a Tat-dependent motif. The CsnA is the first studied chitosanase to be secreted via the Tat pathway. These studies also contributed to a better understanding of the chitosanase secretion. Evidence concerning the role of chitosanases in the protection of bacteria against the bactericidal effect of chitosan was also brought by the study of cell localization of the exo-[béta]-D-glucosaminidase (CsxA) from Amycolatopsis orientalis. CsxA has a carbohydrate-binding module (CBM35) with an unusual affinity This module appended to CsxA recognizes as substrate glucuronic acid, a component of the Gram-positive bacterie cell wall. Thereby, we analyzed by epifluorescence and confocal microscopy the cellular localization of the CsxA-CBM35 in Amycolatopsis orientalis cells grown in the presence of chitosan

Acoustic transmission properties of pressurised and pre-stressed composite structures

this work was focused on the examination of the effect of the pre-stress, namely tension and pressure, on the wave propagation and acoustic behaviour of composite laminates. The dispersion characteristics of two dimensional layered and sandwich structures were predicted using Wave Finite Element Method (WFEM). The structures were examined in non-stressed and pre-stressed scenarios. After extracting the mass and stiffness matrix of a small periodic segment of the structure using commercially available Finite Elements software, a polynomial eigenvalue problem was formed, the solutions of which consisted of the propagation constants of the waves of the structure. This way the wavenumbers and eigenvectors of the out of plane structural displacements were extracted. These wave propagation magnitudes were then used to calculate important Statistical Energy Analysis (SEA) quantities, such as modal density and radiation efficiency. The effect of pre-stress on these quantities, along with its effect on loss factor of the structure were examined

Computing the broadband vibroacoustic response of arbitrarily thick layered panels by a wave finite element approach

A robust procedure for the prediction of the dynamic response of layered panels within a SEA wave-context approach is proposed hereby. The dispersion characteristics of two dimensional composite orthotropic structures are predicted using a Wave Finite Element method. By manipulating the mass and stiffness matrices of the modelled structural segment a polynomial eigenvalue problem is formed, the solutions of which correspond to the propagation constants of the waves travelling within the structure. The wavenumbers and group velocities for waves comprising out of plane structural displacements can then be calculated. Using the numerically extracted wave propagation data the most important SEA quantities of the structure, namely the modal density and the radiation efficiency of each wave type are calculated. The vibroacoustic response of the structure under a broadband diffused excitation is then computed within a SEA approach. The impact of the symmetric and the antisymmetric vibrational motion of the panel on its sound transmission loss is exhibited and the approach proves robust enough for thin as well as for thick layered structures

Re-Engineering of the CERN Accelerators and Services Control System based on VMEbus and PowerPC Technology

A new generation of PowerPC VMEbus front-end computers is being introduced in the CERN accelerators and services control system infrastructure. This new technology is aimed at replacing existing PC based front-end computers and at offering a high performance microprocessor platform for present and future engineering developments for the LHC era. This paper describes the re-engineering strategy and the core architecture of the new systems. Special performance issues are also addressed in this paper

Numerical and experimental investigation of the design of a piezoelectric de-icing system for small rotorcraft part 1/3 : development of a flat plate numerical model with experimental validation

The objective of this research project is divided in four parts: (1) to design a piezoelectric actuator-based de-icing system integrated to a flat plate experimental setup and develop a numerical model of the system with experimental validation, (2) use the experimental setup to investigate actuator activation with frequency sweeps and transient vibration analysis, (3) add ice layer to the numerical model and predict numerically stresses for different ice breaking with experimental validation, and (4) bring the concept to a blade structure for wind tunnel testing. This paper presents the first objective of this study. First, preliminary numerical analysis was performed to gain basic guidelines for the integration of piezoelectric actuators in a simple flat plate experimental setup for vibration-based de-icing investigation. The results of these simulations allowed to optimize the positioning of the actuators on the structure and the optimal phasing of the actuators for mode activation. A numerical model of the final setup was elaborated with the piezoelectric actuators optimally positioned on the plate and meshed with piezoelectric elements. A frequency analysis was performed to predict resonant frequencies and mode shapes, and multiple direct steady-state dynamic analyses were performed to predict displacements of the flat plate when excited with the actuators. In those steady-state dynamic analysis, electrical boundary conditions were applied to the actuators to excite the vibration of the plate. The setup was fabricated faithful to the numerical model at the laboratory with piezoelectric actuator patches bonded to a steel flat plate and large solid blocks used to mimic perfect clamped boundary condition. The experimental setup was brought at the National Research Council Canada (NRC) for testing with a laser vibrometer to validate the numerical results. The experimental results validated the model when the plate is optimally excited with an average of error of 20% and a maximal error obtained of 43%. However, when the plate was not efficiently excited for a mode, the prediction of the numerical data was less accurate. This was not a concern since the numerical model was developed to design and predict optimal excitation of structures for de-icing purpose. This study allowed to develop a numerical model of a simple flat plate and understand optimal phasing of the actuators. The experimental setup designed is used in the next phase of the project to study transient vibration and frequency sweeps. The numerical model is used in the third phase of the project by adding ice layers for investigation of vibration-based de-icing, with the final objective of developing and integrating a piezoelectric actuator de-icing system to a rotorcraft blade structure

Numerical and experimental investigation of the design of a piezoelectric de-icing system for small rotorcraft part 2/3 : investigation of transient vibration during frequency sweeps and pptimal piezoelectric actuator excitation

The objective of this research project is divided in four parts: (1) to design a piezoelectric actuator based de-icing system integrated to a flat plate experimental setup, develop a numerical model of the system and validate experimentally; (2) use the experimental setup to investigate actuator activation with frequency sweeps and transient vibration analysis; (3) add an ice layer to the numerical model, predict numerically stresses at ice breaking and validate experimentally; and (4) implement the concept to a blade structure for wind tunnel testing. This paper presents the second objective of this study, in which the experimental setup designed in the first phase of the project is used to study transient vibration occurring during frequency sweeps. Acceleration during different frequency sweeps was measured with an accelerometer on the flat plate setup. The results obtained showed that the vibration pattern was the same for the different sweep rate (in Hz/s) tested for a same sweep range. However, the amplitude of each resonant mode increased with a sweep rate decrease. Investigation of frequency sweeps performed around different resonant modes showed that as the frequency sweep rate tends towards zero, the amplitude of the mode tends toward the steady-state excitation amplitude value. Since no other transient effects were observed, this signifies that steady-state activation is the optimal excitation for a resonant mode. To validate this hypothesis, the flat plate was installed in a cold room where ice layers were accumulated. Frequency sweeps at high voltage were performed and a camera was used to record multiple pictures per second to determine the frequencies where breaking of the ice occur. Consequently, the resonant frequencies were determined from the transfer functions measured with the accelerometer versus the signal of excitation. Additional tests were performed in steady-state activation at those frequencies and the same breaking of the ice layer was obtained, resulting in the first ice breaking obtained in steady-state activation conditions as part of this research project. These results confirmed the conclusions obtained following the transient vibration investigation, but also demonstrated the drawbacks of steady-state activation, namely identifying resonant modes susceptible of creating ice breaking and locating with precision the frequencies of the modes, which change as the ice accumulates on the structure. Results also show that frequency sweeps, if designed properly, can be used as substitute to steady-state activation for the same results

Numerical and experimental investigation of the design of a piezoelectric de-icing system for small rotorcraft part 3/3 : numerical model and experimental validation of vibration-based de-icing of a flat plate structure

The objective of this research project is divided in four parts: (1) to design a piezoelectric actuator-based de-icing system integrated to a flat plate experimental setup and develop a numerical model of the system with experimental validation, (2) use the experimental setup to investigate actuator activation with frequency sweeps and transient vibration analysis, (3) add an ice layer to the numerical model and predict numerically stresses at ice breaking with experimental validation, and (4) bring the concept to a blade structure for wind tunnel testing. This paper presents the third part of the investigation in which an ice layer is added to the numerical model. Five accelerometers are installed on the flat plate to measure acceleration. Validation of the vibration amplitude predicted by the model is performed experimentally and the stresses calculated by the numerical model at cracking and delamination of the ice layer are determined. A stress limit criteria is then defined from those values for both normal stress at cracking and shear stress at delamination. As a proof of concept, the numerical model is then used to find resonant modes susceptible to generating cracking or delamination of the ice layer within the voltage limit of the piezoelectric actuators. The model also predicts a voltage range within which the ice breaking occurs. The experimental setup is used to validate positively the prediction of the numerical model