Investigation of the roles of CheD in the Bacillus subtilis chemotactic signal transduction

Abstract The two-component based chemotaxis signal transduction system allows flagellated bacteria to sense their surrounding chemical environment and move towards more favorable conditions. The attractant signals can be sensed by transmembrane chemoreceptors, and then transmitted to the histidine kinase CheA. Once activated, CheA interacts with the response regulator CheY through phosphorelay, which causes a change in the rotation of the flagella. The direction of flagella rotation determines whether a cell swims straight or just tumbles. Cells also need adaptation to respond to a change in chemical concentrations, and return to their prestimulated level. Adaptation in the B. subtilis chemotaxis system is achieved by three coordinated systems: the methylation system, the CheC/CheD/CheY-p system and the CheV system. CheD, the previously identified receptor deamidase, was shown to be critical to the ability of B. subtilis to perform chemotaxis and is the main focus of this study. This study started from characterization of the enzymatic mechanism of CheD. Results showed that CheD deamidase uses a cysteine hydrolase mechanism. The catalytic triad consisting of Cys33-His50-Thr27, and Ser27 is essential for receptor recognition and binding. In addition, in this study CheC was found to inhibit CheD’s deamidase activity. Through mutant screening, Phe102 on CheD was found to be the essential site to interact with CheC. Furthermore, the CheD/CheC interaction is necessary for the robust chemotaxis in vivo as demonstrated by the cheD (F102E) mutant, which lacks the ability to swim on swarm plates. Despite its deamidase activity, we hypothesized that CheD’s main role is its involvement in the CheD-CheC-CheY-p negative feedback pathway during adaptation. In particular, CheD is likely to help stabilize the transient kinase-activating state through binding to receptors. When CheY-p level is increased, CheC-CheY-p complex may attract CheD away from receptors. In this study, CheC-CheD binding kinetics with CheY or CheYp presence was successfully obtained by a series of SPR experiments. The increased affinity of CheD for CheC in presence of CheYp but not CheY makes likely the hypothesis that CheC-CheD-CheY interact as part of a negative feedback pathway during adaptation. Last, the interaction between CheD and chemoreceptor McpC was studied in order to better understand the role of CheD in adaptation. Results showed that Q304 and Q305 on McpC are essential to recruit CheD. Additionally, the reduced levels of CheD in mcpC (Q304A) or (Q305A) mutants suggested that the dynamic interaction between CheD and receptors is vital to maintain the normal CheD level. These findings suggest more complicated roles of CheD than its previously identified function as a receptor deamidase, and will lead to a clearer picture of the coordination of the three adaptational systems in the B. subtilis chemotactic sensory transduction system.

Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells -- and only those cells -- to be labeled with azides. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affnity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins -- including proteins with previously unknown expression patterns -- expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.

Quantum Monte Carlo study of effective masses in a polarized unitary Fermi gas

Ultracold gases provide an ideal platform for quantum simulations of many-body systems. Here we are interested in a particular system which has been the focus of most experimental and theoretical works on ultracold fermionic gases: the unitary Fermi gas. In this work we study with Quantum Monte Carlo simulations a two-component gas of fermionic atoms at zero temperature in the unitary regime. Specifically, we are interested in studying how the effective masses for the quasi-particles of the two components of the Fermi liquid evolve as the polarization is progressively reduced from full to lower values. A recent theoretical work, based on alternative diagrammatic methods, has indeed suggested that such effective masses should diverge at a critical polarization. To independently verify such predictions, we perform Variational Monte Carlo (VMC) calculations of the energy based on Jastrow-Slater wavefunctions after adding or subtracting a particle with a given momentum to a full Fermi sphere. In this way, we determine the quasi-particle dispersions, from which we extract the effective masses for different polarizations. The resulting effective masses turn out to be quite close to the non-interacting values, even though some evidence of an increase for the effective mass of the minority component appears close to the predicted value for the critical polarization. Preliminary results obtained for the majority component with the Fixed-node Diffusion Monte Carlo (DMC) method seem to indicate that DMC could lead to an increase of the effective masses in comparison with the VMC results. Finally, we point out further improvements of the trial wave-function and boundary conditions that would be necessary in future simulations to draw definite conclusions on the effective masses of the polarized unitary Fermi gas.

Template-based fluoroethylenepropylene piezoelectrets with tubular channels for transducer applications

We describe the concept, the fabrication, and the most relevant properties of a piezoelectric-polymer system: Two fluoroethylenepropylene (FEP) films with good electret properties are laminated around a specifically designed and prepared polytetrafluoroethylene (PTFE) template at 300 degrees C. After removing the PTFE template, a two-layer FEP film with open tubular channels is obtained. For electric charging, the two-layer FEP system is subjected to a high electric field. The resulting dielectric barrier discharges inside the tubular channels yield a ferroelectret with high piezoelectricity. d(33) coefficients of up to 160 pC/N have already been achieved on the ferroelectret films. After charging at suitable elevated temperatures, the piezoelectricity is stable at temperatures of at least 130 degrees C. Advantages of the transducer films include ease of fabrication at laboratory or industrial scales, a wide range of possible geometrical and processing parameters, straightforward control of the uniformity of the polymer system, flexibility, and versatility of the soft ferroelectrets, and a large potential for device applications e.g., in the areas of biomedicine, communications, production engineering, sensor systems, environmental monitoring, etc.

Defects in vesicle core induced by Escherichia coli dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 1 0-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.

Gastroenterology training in Latin America

Latin America is characterized by ethnic, geographical, cultural, and economic diversity; therefore, training in gastroenterology in the region must be considered in this context. The continent's medical education is characterized by a lack of standards and the volume of research continues to be relatively small. There is a multiplicity of events in general gastroenterology and in sub-disciplines, both at regional and local levels, which ensure that many colleagues have access to information. Medical education programs must be based on a clinical vision and be considered in close contact with the patients. The programs should be properly supervised, appropriately defined, and evaluated on a regular basis. The disparity between the patients' needs, the scarce resources available, and the pressures exerted by the health systems on doctors are frequent cited by those complaining of poor professionalism. Teaching development can play a critical role in ensuring the quality of teaching and learning in universities. Continuing professional development programs activities must be planned on the basis of the doctors' needs, with clearly defined objectives and using proper learning methodologies designed for adults. They must be evaluated and accredited by a competent body, so that they may become the basis of a professional regulatory system. The specialty has made progress in the last decades, offering doctors various possibilities for professional development. The world gastroenterology organization has contributed to the speciality through three distinctive, but closely inter-related, programs: Training Centers, Train-the-Trainers, and Global Guidelines, in which Latin America is deeply involved. (C) 2011 Baishideng. All rights reserved.

Localized modes of binary mixtures of Bose-Einstein condensates in nonlinear optical lattices

The properties of the localized states of a two-component Bose-Einstein condensate confined in a nonlinear periodic potential (nonlinear optical lattice) are investigated. We discuss the existence of different types of solitons and study their stability by means of analytical and numerical approaches. The symmetry properties of the localized states with respect to nonlinear optical lattices are also investigated. We show that nonlinear optical lattices allow the existence of bright soliton modes with equal symmetry in both components and bright localized modes of mixed symmetry type, as well as dark-bright bound states and bright modes on periodic backgrounds. In spite of the quasi-one-dimensional nature of the problem, the fundamental symmetric localized modes undergo a delocalizing transition when the strength of the nonlinear optical lattice is varied. This transition is associated with the existence of an unstable solution, which exhibits a shrinking (decaying) behavior for slightly overcritical (undercritical) variations in the number of atoms.

NMR analog of Bell's inequalities violation test

In this paper, we present an analog of Bell's inequalities violation test for N qubits to be performed in a nuclear magnetic resonance (NMR) quantum computer. This can be used to simulate or predict the results for different Bell's inequality tests, with distinct configurations and a larger number of qubits. To demonstrate our scheme, we implemented a simulation of the violation of the Clauser, Horne, Shimony and Holt (CHSH) inequality using a two-qubit NMR system and compared the results to those of a photon experiment. The experimental results are well described by the quantum mechanics theory and a local realistic hidden variables model (LRHVM) that was specifically developed for NMR. That is why we refer to this experiment as a simulation of Bell's inequality violation. Our result shows explicitly how the two theories can be compatible with each other due to the detection loophole. In the last part of this work, we discuss the possibility of testing some fundamental features of quantum mechanics using NMR with highly polarized spins, where a strong discrepancy between quantum mechanics and hidden variables models can be expected.

Design of 1D and 2D molecule-based magnets with the ligand 4,5-dimethyl-1,2-phenylenebis(oxamato)

Three new bimetallic oxamato-based magnets with the proligand 4,5-dimethyl-1,2-phenylenebis-(oxamato) (dmopba) were synthesized using water or dimethylsulfoxide (DMSO) as solvents. Single crystal X-ray diffraction provided structures for two of them: [MnCu(dmopba)(H(2)O)(3)]n center dot 4nH(2)O (1) and [MnCu(dmopba)(DMSO)(3)](n center dot)nDMSO (2). The crystalline structures for both 1 and 2 consist of linearly ordered oxamato-bridged Mn(II)Cu(II) bimetallic chains. The magnetic characterization revealed a typical behaviour of ferrimagnetic chains for 1 and 2. Least-squares fits of the experimental magnetic data performed in the 300-20 K temperature range led to J(MnCu) = -27.9 cm(-1), g(Cu) = 2.09 and g(Mn) = 1.98 for 1 and J(MnCu) = -30.5 cm(-1), g(Cu) = 2.09 and g(Mn) = 2.02 for 2 (H = -J(MnCu)Sigma S(Mn, i)(S(Cu, i) + S(Cu, i-1))). The two-dimensional ferrimagnetic system [Me(4)N](2n){Co(2)[Cu(dmopba)](3)}center dot 4nDMSO center dot nH(2)O (3) was prepared by reaction of Co(II) ions and an excess of [Cu(dmopba)](2-) in DMSO. The study of the temperature dependence of the magnetic susceptibility as well as the temperature and field dependences of the magnetization revealed a cluster glass-like behaviour for 3.

Robust STATCOM control for the stabilisation of fixed-speed wind turbines during low voltages

This paper presents a robust voltage control scheme for fixed-speed wind generators using a static synchronous compensator (STATCOM) controller. To enable a linear and robust control framework with structured uncertainty, the overall system is represented by a linear part plus a nonlinear part that covers an operating range of interest required to ensure stability during severe low voltages. The proposed methodology is flexible and readily applicable to larger wind farms of different configurations. The performance of the control strategy is demonstrated on a two area test system. Large disturbance simulations demonstrate that the proposed controller enhances voltage stability as well as transient stability of induction generators during low voltage ride through (LVRT) transients and thus enhances the LVRT capability. (C) 2011 Elsevier Ltd. All rights reserved.

Constitutive model for long term municipal solid waste mechanical behavior

This paper presents some improvements in the model proposed by Machado et al. [Machado SL, Carvalho MF, Vilar OM. Constitutive model for municipal solid waste. J Geotech Geoenviron Eng ASCE 2002; 128(11):940-51] now considering the influence of biodegradation of organic matter in the mechanical behavior of municipal solid waste. The original framework considers waste as composed of two component groups; fibers and organic paste. The particular laws of behavior are assessed for each component group and then coupled to represent waste behavior. The improvements introduced in this paper take into account the changes in the properties of fibers and mass loss due to organic matter depletion over time. Mass loss is indirectly calculated considering the MSW gas generation potential through a first order decay model. It is shown that as the biodegradation process occurs the proportion of fibers increases, however, they also undergo a degradation process which tends to reduce their ultimate tensile stress and Young modulus. The way these changes influence the behavior of MSW is incorporated in the final framework which captures the main features of the MSW stress-strain behavior under different loading conditions. (C) 2007 Elsevier Ltd. All rights reserved.

Influence of organic shock loads in an ASBBR treating synthetic wastewater with different concentration levels

Safe application of the anaerobic sequencing biofilm batch reactor (ASBBR) still depends on deeper insight into its behavior when faced with common operational problems in wastewater treatments such as tolerance to abrupt variations in influent concentration, so called shock loads. To this end the current work shows the effect of organic shock loads on the performance of an ASBBR, with a useful volume of 5 L, containing 0.5-cm polyurethane cubes and operating at 30 degrees C with mechanical stirring of 500 rpm. In the assays 2 L of two types of synthetic wastewater were treated in 8-h cycles. Synthetic wastewater I was based on sucrose-amide-cellulose with concentration of 500 mg COD/L and synthetic wastewater II was based on volatile acids with concentration ranging from 500 to 2000 mg COD/L. Organic shock loads of 2-4 times the operation concentration were applied during one and two cycles. System efficiency was monitored before and after application of the perturbation. When operating with concentrations from 500 to 1000 mg COD/L and shock loads of 2-4 times the influent concentration during one or two cycles the system was able to regain stability after one cycle and the values of organic matter, total and intermediate volatile acids, bicarbonate alkalinity and pH were similar to those prior to the perturbations. At a concentration of 2000 mg COD/L the reactor appeared to be robust, regaining removal efficiencies similar to those prior to perturbation at shock loads twice the operation concentration lasting one cycle and stability was recovered after two cycles. However, for shock loads twice the operation concentration during two cycles and shock loads four times the operation concentration during one or two cycles filtered sample removal efficiency decreased to levels different from those prior to perturbation, on an average of 90-80%, approximately, yet the system managed to attain stability within two cycles after shock application. Therefore, this investigation envisions the potential of full scale application of this type of bioreactor which showed robustness to organic shock loads, despite discontinuous operation and the short times available for treating total wastewater volume. (c) 2007 Elsevier Ltd. All rights reserved.

Thermal inactivation of polyphenoloxidase and peroxidase in green coconut (Cocos nucifera) water

P>Coconut water is an isotonic beverage naturally obtained from the green coconut. After extracted and exposed to air, it is rapidly degraded by enzymes peroxidase (POD) and polyphenoloxidase (PPO). To study the effect of thermal processing on coconut water enzymatic activity, batch process was conducted at three different temperatures, and at eight holding times. The residual activity values suggest the presence of two isoenzymes with different thermal resistances, at least, and a two-component first-order model was considered to model the enzymatic inactivation parameters. The decimal reduction time at 86.9 degrees C (D(86.9 degrees C)) determined were 6.0 s and 11.3 min for PPO heat labile and heat resistant fractions, respectively, with average z-value = 5.6 degrees C (temperature difference required for tenfold change in D). For POD, D(86.9 degrees C) = 8.6 s (z = 3.4 degrees C) for the heat labile fraction was obtained and D(86.9 degrees C) = 26.3 min (z = 6.7 degrees C) for the heat resistant one.

Flatness-based control of a single qubit gate

This work considers the open-loop control problem of steering a two-level quantum system from any initial to any final condition. The model of this system evolves on the state space X = SU(2), having two inputs that correspond to the complex amplitude of a resonant laser field. A symmetry preserving flat output is constructed using a fully geometric construction and quaternion computations. Simulation results of this flatness-based open-loop control are provided.

Modelling the Distribution of Ischaemic Stroke-Specific Survival Time Using an EM-based Mixture Approach with Random Effects Adjustment

A two-component survival mixture model is proposed to analyse a set of ischaemic stroke-specific mortality data. The survival experience of stroke patients after index stroke may be described by a subpopulation of patients in the acute condition and another subpopulation of patients in the chronic phase. To adjust for the inherent correlation of observations due to random hospital effects, a mixture model of two survival functions with random effects is formulated. Assuming a Weibull hazard in both components, an EM algorithm is developed for the estimation of fixed effect parameters and variance components. A simulation study is conducted to assess the performance of the two-component survival mixture model estimators. Simulation results confirm the applicability of the proposed model in a small sample setting. Copyright (C) 2004 John Wiley Sons, Ltd.