Structure of the CFA/III major pilin subunit CofA from human enterotoxigenic Escherichia coli determined at 0.90 Å resolution by sulfur-SAD phasing.

CofA, a major pilin subunit of colonization factor antigen III (CFA/III), forms pili that mediate small-intestinal colonization by enterotoxigenic Escherichia coli (ETEC). In this study, the crystal structure of an N-terminally truncated version of CofA was determined by single-wavelength anomalous diffraction (SAD) phasing using five sulfurs in the protein. Given the counterbalance between anomalous signal strength and the undesired X-ray absorption of the solvent, diffraction data were collected at 1.5 Å resolution using synchrotron radiation. These data were sufficient to elucidate the sulfur substructure at 1.38 Å resolution. The low solvent content (29%) of the crystal necessitated that density modification be performed with an additional 0.9 Å resolution data set to reduce the phase error caused by the small sulfur anomalous signal. The CofA structure showed the αß-fold typical of type IVb pilins and showed high structural homology to that of TcpA for toxin-coregulated pili of Vibrio cholerae, including spatial distribution of key residues critical for pilin self-assembly. A pilus-filament model of CofA was built by computational docking and molecular-dynamics simulation using the previously reported filament model of TcpA as a structural template. This model revealed that the CofA filament surface was highly negatively charged and that a 23-residue-long loop between the α1 and α2 helices filled the gap between the pilin subunits. These characteristics could provide a unique binding epitope for the CFA/III pili of ETEC compared with other type IVb pili.

Immune response in diarrheal patients and asymptomatic carrier with CS6-producing enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of diarrhea in children and travelers in developing countries. ETEC colonization factors (CFs) are virulence determinants considered as protective antigens and major targets for vaccine development against ETEC infections. One of the most prevalent CFs, coli surface antigen 6 (CS6), a non-fimbrial polymeric protein consisting of two major subunits, CssA and CssB, is produced by approximately 25-35% of ETEC worldwide. We could isolate only CS6-producing ETEC strains from two diarrheal patients and one asymptomatic carrier, but we could not detect CssA- or CssB-specific antibodies in the feces and blood of two patients convalescing from natural ETEC infection and of an asymptomatic carrier using western blotting. Therefore, in order to protect against infection with CS6-producing ETEC, protective levels of CS6 immunity should be incorporated in any future vaccines against ETEC.

Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients.

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Stochastic boundary approaches to many-particle systems coupled to a particle reservoir.

Stochastic boundary conditions for interactions with a particle reservoir are discussed in many-particle systems. We introduce the boundary conditions with the injection rate and the momentum distribution of particles coming from a particle reservoir in terms of the pressure and the temperature of the reservoir. It is shown that equilibrium ideal gases and hard-disk systems with these boundary conditions reproduce statistical-mechanical properties based on the corresponding grand canonical distributions. We also apply the stochastic boundary conditions to a hard-disk model with a steady particle current escaping from a particle reservoir in an open tube, and discuss its nonequilibrium properties such as a chemical potential dependence of the current and deviations from the local equilibrium hypothesis.

Dynamics of strongly localized Lyapunov vectors in many-hard-disk systems.

The dynamics of the localized region of the Lyapunov vector for the largest Lyapunov exponent is discussed in quasi-one-dimensional hard-disk systems at low density. We introduce a hopping rate to quantitatively describe the movement of the localized region of the Lyapunov vector, and show that it is a decreasing function of the hopping distance, implying a spatial correlation of the localized regions. This behavior is explained quantitatively by a brick accumulation model derived from hard-disk dynamics in the low density limit, in which hopping of the localized Lyapunov vector is represented as the movement of the highest brick position. We also give an analytical expression for the hopping rate, which is obtained as a sum of probability distributions for brick height configurations between two separated highest brick sites. The results of these simple models are in good agreement with the simulation results for hard-disk systems.

Time-dependent mode structure for Lyapunov vectors as a collective movement in quasi-one-dimensional systems.

The time-dependent mode structure of the Lyapunov vectors associated with the stepwise structure of the Lyapunov spectra and its relation to the momentum autocorrelation function are discussed in quasi-one-dimensional many-hard-disk systems. We obtain the complete mode structures (Lyapunov modes) for all components of the Lyapunov vectors, including the longitudinal and transverse components of both the spatial and momentum parts, and their phase relations. These mode structures are suggested by the form of the Lyapunov vectors for the zero-Lyapunov exponents. The spatial node structures of these modes are explained by the reflection properties of the hard walls used in the models. Our main result is that the largest time-oscillating period of the Lyapunov modes is twice as long as the time-oscillating period of the longitudinal momentum autocorrelation function. This relation is satisfied irrespective of the number of particles and the boundary conditions. A simple explanation for this relation is given based on the form of the time-dependent Lyapunov mode.

Inactivation of Foot-and-Mouth Disease Virus by Commercially Available Disinfectants and Cleaners.

Foot-and-mouth disease virus (FMDV) is an animal pathogen of great concern. It is contagious to cloven-hoofed animals and affects animals in extensive areas worldwide. In general, the primary eradication strategies for foot-and-mouth disease (FMD) in Japan are stamping out the disease and restriction of movement. It is also important to completely disinfect the infected area to prevent the spread of FMDV, including vehicles and people as well. However, there is no report on the effect of commercially available disinfectants against FMDV in a short contact time. In this study, we evaluated the virucidal effect of thirteen commercially available products, and got the following results: acidic ethanol disinfectants, alkaline cleaners and sodium hypochlorite had great effect (>3.0 log10 reduction in titer) against FMDV. On the other hand, neutral ethanol disinfectants, hand soaps, and quaternary ammonium compound sanitizers did not show great effect against FMDV. Therefore, it is presumed that acidic ethanol disinfectants are effective for human use and alkaline cleaners are effective for use in the infected environment for the control of a FMD outbreak.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

Stereoselectivity of superacid-catalyzed Pictet-Spengler cyclization reactions.

[reaction: see text] High stereoselectivities were found in a wide range of superacid-catalyzed Pictet-Spengler cyclization reactions. Particularly in the cases of 2-alkyl-N-benzylidene-2-phenethylamines, an enhanced stereoselectivity was observed under the superacid conditions as compared with the corresponding weak acid (TFA)-catalyzed (monocationic) cyclization reaction of the N-benzylidene-2-(3',4'-dimethoxy)phenethylamines that bear electron-donating groups on the cyclizing aromatic ring. The computational study also supported the energetic favorability of the cyclization of the N,N-diprotonated imine and revealed a significantly early transition-state structure.

Steady shear flow thermodynamics based on a canonical distribution approach.

A nonequilibrium steady-state thermodynamics to describe shear flow is developed using a canonical distribution approach. We construct a canonical distribution for shear flow based on the energy in the moving frame using the Lagrangian formalism of the classical mechanics. From this distribution, we derive the Evans-Hanley shear flow thermodynamics, which is characterized by the first law of thermodynamics dE=TdS-Qdgamma relating infinitesimal changes in energy E, entropy S, and shear rate gamma with kinetic temperature T. Our central result is that the coefficient Q is given by Helfand's moment for viscosity. This approach leads to thermodynamic stability conditions for shear flow, one of which is equivalent to the positivity of the correlation function for Q. We show the consistency of this approach with the Kawasaki distribution function for shear flow, from which a response formula for viscosity is derived in the form of a correlation function for the time-derivative of Q. We emphasize the role of the external work required to sustain the steady shear flow in this approach, and show theoretically that the ensemble average of its power W must be non-negative. A nonequilibrium entropy, increasing in time, is introduced, so that the amount of heat based on this entropy is equal to the average of W. Numerical results from nonequilibrium molecular-dynamics simulation of two-dimensional many-particle systems with soft-core interactions are presented which support our interpretation.

Localized behavior in the Lyapunov vectors for quasi-one-dimensional many-hard-disk systems.

We introduce a definition of a "localization width" whose logarithm is given by the entropy of the distribution of particle component amplitudes in the Lyapunov vector. Different types of localization widths are observed, for example, a minimum localization width where the components of only two particles are dominant. We can distinguish a delocalization associated with a random distribution of particle contributions, a delocalization associated with a uniform distribution, and a delocalization associated with a wavelike structure in the Lyapunov vector. Using the localization width we show that in quasi-one-dimensional systems of many hard disks there are two kinds of dependence of the localization width on the Lyapunov exponent index for the larger exponents: one is exponential and the other is linear. Differences due to these kinds of localizations also appear in the shapes of the localized peaks of the Lyapunov vectors, the Lyapunov spectra, and the angle between the spatial and momentum parts of the Lyapunov vectors. We show that the Krylov relation for the largest Lyapunov exponent lambda approximately -rho ln rho as a function of the density rho is satisfied (apart from a factor) in the same density region as the linear dependence of the localization widths is observed. It is also shown that there are asymmetries in the spatial and momentum parts of the Lyapunov vectors, as well as in their x and y components.

Boundary effects in the stepwise structure of the Lyapunov spectra for quasi-one-dimensional systems.

Boundary effects in the stepwise structure of the Lyapunov spectra and corresponding wavelike structure of the Lyapunov vectors are discussed numerically in quasi-one-dimensional systems of many hard disks. Four different types of boundary conditions are constructed by combinations of periodic boundary conditions and hard-wall boundary conditions, and each leads to different stepwise structures of the Lyapunov spectra. We show that for some Lyapunov exponents in the step region, the spatial y component of the corresponding Lyapunov vector deltaq(yj), divided by the y component of momentum p(yj), exhibits a wavelike structure as a function of position q(xj) and time t. For the other Lyapunov exponents in the step region, the y component of the corresponding Lyapunov vector deltaq(yj) exhibits a time-independent wavelike structure as a function of q(xj). These two types of wavelike structure are used to categorize the type and sequence of steps in the Lyapunov spectra for each different type of boundary condition.

Stepwise structure of Lyapunov spectra for many-particle systems using a random matrix dynamics.

The structure of the Lyapunov spectra for the many-particle systems with a random interaction between the particles is discussed. The dynamics of the tangent space is expressed as a master equation, which leads to a formula that connects the positive Lyapunov exponents and the time correlations of the particle interaction matrix. Applying this formula to one- and two-dimensional models we investigate the stepwise structure of the Lyapunov spectra that appear in the region of small positive Lyapunov exponents. Long range interactions lead to a clear separation of the Lyapunov spectra into a part exhibiting stepwise structure and a part changing smoothly. The part of the Lyapunov spectrum containing the stepwise structure is clearly distinguished by a wave-like structure in the eigenstates of the particle interaction matrix. The two-dimensional model has the same step widths as found numerically in a deterministic chaotic system of many hard disks.

Master equation approach to the conjugate pairing rule of Lyapunov spectra for many-particle thermostated systems.

The master equation approach to Lyapunov spectra for many-particle systems is applied to nonequilibrium thermostated systems to discuss the conjugate pairing rule. We consider iso-kinetic thermostated systems with a shear flow sustained by an external restriction, in which particle interactions are expressed as a Gaussian white randomness. Positive Lyapunov exponents are calculated by using the Fokker-Planck equation to describe the tangent vector dynamics. We introduce another Fokker-Planck equation to describe the time-reversed tangent vector dynamics, which leads to the calculation of the negative Lyapunov exponents. Using the Lyapunov exponents provided by these two Fokker-Planck equations we show the conjugate pairing rule is satisfied for thermostated systems with a shear flow in the thermodynamic limit which allow us to replace the friction coefficient with a constant number. We also give an explicit form to connect the Lyapunov exponents with the time correlation of the interaction matrix in a thermostated system with a color field.

Escape dynamics of many hard disks.

Many-particle effects in escapes of hard disks from a square box via a hole are discussed in a viewpoint of dynamical systems. Starting from N disks in the box at the initial time, we calculate the probability P_{n}(t) for at least n disks to remain inside the box at time t for n=1,2,...,N. At early times, the probabilities P_{n}(t),n=2,3,...,N-1, are described by superpositions of exponential decay functions. On the other hand, after a long time the probability P_{n}(t) shows a power-law decay ∼t^{-2n} for n≠1, in contrast to the fact that it decays with a different power law ∼t^{-n} for cases without any disk-disk collision. Chaotic or nonchaotic properties of the escape systems are discussed by the dynamics of a finite-time largest Lyapunov exponent, whose decay properties are related with those of the probability P_{n}(t).

Chaos and ergodicity of two hard disks within a circular billiard.

We investigate dynamical properties of the system of two interacting hard disks within a circular billiard numerically in the case of zero total angular momentum. Varying the radius of two identical disks, we examine chaotic irregularity and ergodicity of the system. Single-particle configuration and velocity distributions are obtained from dynamical trajectory calculations and compared with those in the microcanonical ensemble. We also analyze properties of trajectories by calculating the finite-time maximum Lyapunov exponent and clarify the existence of sticky motions around Kolmogorov-Arnold-Moser (KAM) tori even for small radii of disks. It is shown that the present system is almost ergodic in spite of the existence of tori for small radii of disks since the ratio of tori to the whole phase space is extremely small. On the other hand, a number of tori increase abruptly as the radius of disks increases beyond some value and tori prevent trajectories to run over the phase space uniformly, which makes the ergodicity of the system broken down.

Rapid identification of Mycobacterium tuberculosis in BACTEC MGIT960 cultures by in-house loop-medicated isothermal amplification.

Definitive diagnosis of tuberculosis (TB) by conventional culture, followed by bacterial identification based on biochemical tests is time-consuming and tedious. Simple loop-mediated isothermal amplification (LAMP) specific for Mycobacterium tuberculosis complex, targeting the M. tuberculosis 16S ribosomal RNA gene, termed TB-LAMP, was evaluated as an alternative for rapid culture confirmation. TB-LAMP was assessed for its ability to detect M. tuberculosis complex in BACTEC MGIT 960-positive cultures. Of the 103 cultures evaluated, 100 were identified to contain M. tuberculosis complex by TB-LAMP and had concordant results with standard biochemical tests of niacin accumulation, nitrate reductase, lack of heat-stable catalase, and susceptibility to para-nitrobenzoic acid. These results indicate that TB-LAMP in combination with BACTEC MGIT 960 is a specific, reliable, and technically feasible method for rapid and accurate identification of M. tuberculosis complex.

Escape behavior of quantum two-particle systems with Coulomb interactions.

Quantum escapes of two particles with Coulomb interactions from a confined one-dimensional region to a semi-infinite lead are discussed by using the probability of finding all particles within the confined region, that is, the survival probability, in comparison with free particles. By taking into account the quantum effects of two identical particles, such as the Pauli exclusion principle, it is shown analytically that for two identical free fermions (bosons), the survival probability decays asymptotically in power ~t(-10) (~t(-6)) as a function of time t, although it decays in power ~t(-3) for one free particle. On the other hand, for two particles with attractive Coulomb interactions it is shown numerically that the survival probability decays in power ~t(-3) after a long time. Moreover, for two particles with repulsive Coulomb interactions it decays exponentially in time ~exp (-αt) with a constant α, which is almost independent of the initial energy of particles.

A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples.

Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the ctxA gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the "gold standard" culture method, while the polymerase chain reaction-based method failed to detect V. cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V. cholerae carrying the ctxA gene in the clinical laboratory and in the field.

Time-oscillating Lyapunov modes and the momentum autocorrelation function.

The Lyapunov vectors corresponding to the steps of Lyapunov spectra for many-particle systems show time-oscillating behavior in two types of Lyapunov modes, one associated with time-translational invariance and the other with spatial translational invariance. Our result is that, for each coordinate direction, the longest period of the Lyapunov modes is twice as long as the period of the momentum autocorrelation function. A simple explanation for this relation is proposed and we argue that this result is generally true for many-particle systems. This gives the first quantitative connection between the Lyapunov modes and an experimentally accessible quantity.