bluuebunny/crossref_metadata_2025 huggingface dataset:
An Experimental Study on the Effects of Compressive Stress on the
Fatigue Crack Growth of Low-Alloy Steel
Authors: Jones D. P. , Hoppe R. G. , Hechmer J. L. , James B. A.
Published: June 2008

A series of fatigue crack growth rate tests was conducted in order to study the effects of negative stress ratio upon the fatigue crack growth rate of low-alloy steel in air environment. The tests used four-point bend specimens in order to simulate linear stress distributions typical of many pressure vessel applications. This type of testing adds to the knowledge on negative stress ratio effects for low-alloy steels that in the past have been obtained from uniform tension-compression tests. Additionally, the applied bending stress range was varied from low values of applied stress to high values of applied stress over twice the yield strength. Load control was used for tests for which the stress range was less than twice the yield strength and deflection control was used for the higher stress range tests. The crack geometries involved were both short and long fatigue cracks started at notches and tight fatigue cracks for which crack closure could occur over the full crack face. The results are presented in terms of the stress intensity factor ratio R = KMIN/KMAX. The negative R-ratio test results were correlated to an equation of the form da/dN = C[ΔK/(A−R)]n where A, C, and n are curve-fitting parameters. It was found that the effects of negative R-ratio on the fatigue crack growth rates for even the high stress range tests could be bounded by correlating the foregoing equation to only positive R-ratio test results and extending the resulting equation into the negative R-ratio regime.
Validation of Creep Crack Growth Life Estimation Methodology/Hot
Reheat Steam Pipes
Authors: Bloom J. M.
Published: June 2008

This paper documents a validation study of a creep crack growth life estimation methodology developed by Riedel and Rice, Saxena, and Bassani which has been implemented into a Babcock & Wilcox computer code. This computer code called PCCREEP was developed for estimation of remaining lives of fossil power plant components with both postulated and in-service determined defects. The initial validation was performed through comparisons with other computer codes. However, these comparisons were limited to continuous operating conditions (steady-state creep) and bulk creep deformation properties for secondary creep only. Even with the independent comparison with other available life estimation codes, the question still remained of how well can the creep crack growth methodology estimate actual field lives? This paper presents the results of a study of a life estimation scheme for hot reheat steam pipes having defects found in service. The initial survey was conducted by the Electric Power Research Institute (EPRI) of U.S. electric utility problems with hot reheat steam pipes. The primary reason for that study was three catastrophic failures of hot reheat steam pipes in 1979, 1985, and 1986. These pipes were seam-welded 1-1/4 Cr-1/2 Mo and 2-1/4 Cr-1 Mo. All these ruptures initiated from flaws (near the weld fusion line) which grew by a creep mechanism. This EPRI data base consisted of twelve pipes with flaws found from inspection. While several assumptions relating to material properties and operating conditions were required (due to insufficient information), predictions demonstrated that life estimation is possible provided material property data representative of the material condition for the component in question is available. Discussion is given regarding the significance of the postulated flaw location, constraint (stress triaxiality) effects for buried flaws, and the importance of weld metal, heat-affected zone, and base metal properties to the accuracy of the life estimations.
The Treatment of Residual Stress in Fracture Assessment of Pressure Vessels
Authors: Green D. , Knowles J.
Published: June 2008

The treatment of weld residual stress in the fracture assessment of cylindrical pressure vessels is considered through partitioning the stress into membrane, bending, and self-balancing through-wall components. The influence of each on fracture behavior is discussed. Stress intensity factor solutions appropriate to each type of stress are presented. Short-range, medium-range, and long-range stress categories are identified according to simple rules relating the effect of increasing crack length to stress intensity factor and ligament net stress. Proposals are made on how the stress intensity factor from these stress types may be incorporated into a Kr, Lr-based fracture assessment.
A New Approach to Stress Analysis of Various Wound Vessels
Authors: Huang P. S. , Friedrich C. R.
Published: June 2008

As the width of windings of wound pressure vessels increases, the vessels are categorized as filament-wound, ribbon-wound, thin plate-wound, and finally the conventional multilayered vessels. The filament-wound vessel is one of the wound vessels with small winding width. A multilayered vessel can be regarded as one where the winding width is very great. Based on winding width, the other types of wound vessels are structurally situated between the filament-wound vessel and the multilayered vessel. As the winding width increases, the stress state in the windings gradually changes from purely biaxial to triaxial, and the windings are able to carry more axial load. Therefore, the windings and the inner shell of any wound vessel should mechanically behave in a state between that of the filament-wound and nonwound multilayered vessels, depending on winding width. By utilizing an analysis technique, analogous to mathematical interpolation, a new approach for calculating stresses for various wound vessels has been developed. The technique uses the stresses in the filament-wound vessel and of the multilayered vessel with the same dimensions, and compensates for the winding width through the use of a weighting factor. Comparisons between theoretical analyses and experiments are given for flat steel ribbon-wound vessels. The results show that the calculated stresses at the inside surface of inner shell are in very good agreement with published experimental data.
Optimization of a Compound Vessel Supported by the Wound Ribbon Cylinder
Authors: Vrbka J. , Sucha´nek M.
Published: June 2008

The paper deals with the optimal strength design of the belt chamber compound vessel of mixed construction, consisting of the matrix made of sintered carbide, supporting steel ring, and outer wound supporting cylinder. The maximum stress of all components being equal to the design stresses, the effective stress distribution in wound cylinder is uniform. Outer radius of the wound part is taken as an objective function. The set of transcendental equations has been derived to calculate all necessary geometrical parameters and their discussion was carried out.
Weight Functions and Stress Intensity Factors for Axial Cracks in Hollow Cylinders
Authors: Ma C.-C. , Huang J.-I. , Tsai C.-H.
Published: June 2008

In this study, stress intensity factors for axial cracks in hollow cylinders subjected to mechanical and thermal loadings are determined by using the weight function method. The weight function is a universal function for a given cracked body and can be obtained from any arbitrary loading system. The weight function may be thought of as Green’s function for the stress intensity factor of cracked bodies. Once the weight function for a cracked body is determined, the stress intensity factor for any arbitrary loading can be simply and efficiently evaluated through the integration of the product of the loading and weight function. A numerical method for the determination of weight functions relevant to cracked bodies with finite dimensions is used. Results for weight functions covering a wide range of hollow cylinder geometries are presented in functional or graphical form. The explicit crack face weight functions for applying mechanical loadings are obtained by using the least-squares fitting procedure. As a demonstration, some examples of special loading problems are solved by the weight function method, and the results are compared with available results in the published literature.
The Effect of Multiaxiality and Follow-Up on Creep Damage
Authors: Seshadri R.
Published: June 2008

A simple approximate method for estimating creep damage in pressure components experiencing multiaxial relaxation and follow-up is presented. The theoretical formulation essentially relates the relaxation process that accounts for multiaxiality and follow-up to the uniaxial relaxation model. The effect of follow-up is determined by studying the relaxation response on the Generalized Local Stress Strain (GLOSS) diagram. A procedure for partitioning creep damage into load and deformation-controlled fractions is also discussed. GLOSS analysis is applied to several component configurations that exhibit follow-up potential.
Limit Loads of Buried Pipelines With Asymmetric Initial Imperfections
Authors: Koh C. G. , Quek S. T.
Published: June 2008

The effect of asymmetric imperfection on the limit-load response of pipelines buried in shallow trenches is investigated. The pipeline is modeled as a long beam resting on a rigid foundation and a small strain, large displacement formulation is used. Three different asymmetric imperfection shapes for the beam are considered and the corresponding limit loads are compared with that for a symmetric imperfection. It is found that the shape of initial imperfection plays an important role. The difference between limit loads based on a symmetric imperfection and a nonsymmetric imperfection can be quite significant.
Improve the Stability of Rotor Subjected to Fluid Leakage by Optimum Diameters Design
Authors: Wang J. H. , Shih F. M.
Published: June 2008

For high performance turbomachinery, the fluid leakage in the shroud of the blades and the labyrinth seal may cause instability and limit the output rating. In this work, an optimization technique has been used to find the optimum diameters of shaft elements so that the optimized rotor can sustain maximum fluid leakage excitation. The results show that, even without the increase of total mass of the rotor system, the threshold performance of rotor-bearing systems can be significantly improved by slight modification of the shaft diameters.
Dynamics of a Radially Rotating Beam With Impact, Part 1: Theoretical and Computational Model
Authors: Yigit A. S. , Ulsoy A. G. , Scott R. A.
Published: June 2008

A model is presented for the dynamics of a radially rotating beam with impact. The model uses a momentum balance method and a coefficient of restitution, and enables one to predict the rigid body motion as well as the elastic motion before and after impact. A computational algorithm is also developed to implement the model. In Part 2 simulation results will be compared with experimental results to investigate the validity of the model.
Candidate Reduced Order Models for Structural Parameter Estimation
Authors: Friswell M. I.
Published: June 2008

This paper considers the reduction of high order models generated by a finite element analysis. The aim is to provide a low order model which retains the effect of parameter changes and so may be used to update the unknown physical structural parameters of the modelled system in the time or frequency domains. After reviewing the methods of structural parameter identification, the available order reduction algorithms and their properties are introduced. Modal truncation is determined to be the most suitable method and the standard algorithm is extended to accommodate unknown parameters. Finally the effect of the reduction process on the modelled receptances is demonstrated.
A Linear Time-Domain Method for Structural Dynamics Identification
Authors: Fassois S. D. , Eman K. F. , Wu S. M.
Published: June 2008

A two-stage, time-domain structural dynamics identification method, that circumvents some of the main drawbacks of current techniques, and combines good noise rejection properties with a low computational load and accurate modal parameter extraction, is proposed. In the first stage of the method a discrete model is obtained through a linear estimation scheme and a systematic and effective modeling strategy. In the second stage the modal parameters are evaluated according to a new procedure that is capable of detecting and correcting large errors that may be introduced into the modal parameter estimates through the discrete-to-continuous transformation. The proposed method yields consistent modal parameter estimates at a low computational load, and is well-suited for microcomputer implementation. Its effectiveness is finally demonstrated with both simulated and actual experimental data.
An Electroviscous Damper for Rotor Applications
Authors: Nikolajsen J. L. , Hoque M. S.
Published: June 2008

A new type of vibration damper for rotor systems has been developed and tested. The damper contains electroviscous fluid which solidifies and provides Coulomb-type friction damping when an electric voltage is imposed across the fluid. The damping capacity is controlled by the voltage. The damper has been incorporated in a flexible rotor system and found to be able to reduce high levels of unbalance excited vibrations. Other proven advantages include controllability, simplicity, and no requirement for oil supply. The anticipated capabilities to circumvent the critical speeds and to suppress rotor instabilities are still unconfirmed.
Theoretical and Experimental Studies on Squeeze Film Stabilizers for Flexible Rotor-Bearing Systems Using Newtonian and Viscoelastic Lubricants
Authors: Halder B. , Mukherjee A. , Karmakar R.
Published: June 2008

A combination of a squeeze film damper and a plane journal bearing is studied as a stabilizing scheme. The damper is made to play the role of a stabilizer to postpone the instability threshold speeds of flexible rotors. Both Newtonian and viscoelastic fluids are used in the rotor-bearing system. Dynamics of the system is theoretically analyzed using bond graphs. Analysis reveals that the use of a Newtonian fluid in the stabilizer largely improves the high speed stability range. However, viscoelastic stabilizing fluid has a detrimental effect on highly flexible rotors. Experimental investigations, conducted on a flexible rotor (natural frequency, 30 Hz), confirm the theoretical findings. In addition, experiments indicate that though the use of viscoelastic stabilizing fluids leads to instability in flexible rotors, the growth of large amplitude whirl is postponed to very high speeds.
Application of Learning Control to Active Damping of Forced Vibration for Periodically Time Variant Systems
Authors: Hac´ A. , Tomizuka M.
Published: June 2008

This paper deals with discrete time learning controllers for systems with periodically varying parameters. Several control rules are proposed which use a basic principle of learning control that is to utilize the information from the most recent cycle to improve the system performance in the next cycle. Convergence properties of proposed learning control algorithms are examined. These algorithms can easily be implemented without additional measurements and without modification of existing feedback/feedforward controller. In a numerical example learning controllers are applied to eliminate the forced vibrations of a magnetically suspended rotor with nonsymmetric stiffness properties. The vibrations result from unbalanced inertia forces. Simulation results show that the learning controller is effective in absorbing periodic disturbances.
Nonlinear Forced Oscillations Caused by Quartic Nonlinearity in a Rotating Shaft System
Authors: Ishida Y. , Ikeda T. , Yamamoto T.
Published: June 2008

This paper deals with nonlinear forced oscillations in a rotating shaft system which are caused by quartic nonlinearity in a restoring force. These oscillations are theoretically analyzed by paying attention to the nonlinear components represented by the polar coordinates. It is clarified which kind of nonlinear component has an influence on each oscillation. In experiments it was shown that, when the shaft was supported by double-row angular contact ball bearings, the restoring force had nonlinear spring characteristics involving quartic nonlinearity in addition to quadratic and cubic ones. Experimental results were compared with the theoretical results regarding the probability of occurrence and the shapes of the resonance curves.
A Modified Conical Beam Element Based on Finite Element Analysis: Experimental Correlations
Authors: Vest T. A. , Darlow M. S.
Published: June 2008

A portion of the results from a previous investigation into the structural behavior of hollow conical sections has been used to develop a method of correcting the conical beam element used in modern rotordynamic programs. The modification is made by altering the local value of the Young’s modulus so that the equations used in the conical element produce a bending flexibility which corresponds to that determined from detailed, three-dimensional finite element models. The use of this modification produces substantial improvement in the prediction of the first five natural frequencies of a hollow nonrotating shaft containing two opposed conical sections, for a range of wall thicknesses. The modification exhibits a length dependency which appears to be a main cause of the remaining discrepancies, though it is pointed out that the procedure is only a partial implementation of a more complete hybrid element to be presented later. Static experiments are also discussed, and the verification of a unique behavior characteristic of steep tapered sections is reported.
Dynamic Modeling and Analysis of a High Speed Precision Drilling Machine
Authors: Lin Yih-Hwang , Trethewey M. W. , Reed H. M. , Shawley J. D. , Sager S. J.
Published: June 2008

This paper describes the modeling and dynamic analysis of a high speed precision circuit board drilling machine. The drilling machine has two banks of movable drill spindles supported between parallel granite beams on an air bearing suspension. Each drill spindle is programmed to move laterally between the beams under the position control of a servo-feedback system. The drill spindles must complete a nominal 3.048 mm (0.12 in.) lateral move and a vertical drilling sequence every 200 ms. while maintaining an absolute positioning accuracy within 0.01778 mm (0.0007 in.). In order to meet these demanding specifications the dynamic behavior of the complete drill spindle/air bearing/granite beam system must be well understood. A specialized finite element model (FEM) is used to examine the dynamic responses of the critical components of this machine. The FEM code is capable of analyzing a sprung two degrees of freedom system moving on an elastic beam in an arbitrary fashion. The discussion concentrates on the engineering modeling considerations and compromises which were necessary to analyze the drilling machine with this computer code. The modeling rationale and use of experimental data to form an effective computer model capable of analyzing the dynamic behavior of the drilling machine are emphasized. Typical results from the finite element model are presented to illustrate the obtainable level of accuracy, detail and the limitations for modeling a system of this nature.
Elastic Wave Propagation from Point Excitations on Thin-Walled Cylindrical Shells
Authors: Pierce A. D. , Kil Hyun-Gwon
Published: June 2008

The circular cylindrical shell is described by equations that govern wave propagation in a two-dimensional homogeneous, but anisotropic and dispersive, medium. The idealization of an unbounded medium is applicable if the source is replaced by a periodic array of forces, the repetition distance being the cylinder circumference. Analytical expressions and calculations are presented for wavefront patterns, amplitude distributions, polarizations, and phase velocities for waves on the cylinder surface. The analysis includes all three possible directions of the exciting force and gives fundamental results that can be superimposed to predict vibration fields resulting from arbitrary excitations.
Natural Variability of Greenland Climate, Vegetation, and Ice Volume During the Past Million Years
Authors: de Vernal Anne , Hillaire-Marcel Claude
Published: June 2008

The response of the Greenland ice sheet to global warming is a source of concern notably because of its potential contribution to changes in the sea level. We demonstrated the natural vulnerability of the ice sheet by using pollen records from marine sediment off southwest Greenland that indicate important changes of the vegetation in Greenland over the past million years. The vegetation that developed over southern Greenland during the last interglacial period is consistent with model experiments, suggesting a reduced volume of the Greenland ice sheet. Abundant spruce pollen indicates that boreal coniferous forest developed some 400,000 years ago during the “warm” interval of marine isotope stage 11, providing a time frame for the development and decline of boreal ecosystems over a nearly ice-free Greenland.
Strong Limit on a Variable Proton-to-Electron Mass Ratio from Molecules in the Distant Universe
Authors: Murphy Michael T. , Flambaum Victor V. , Muller Sébastien , Henkel Christian
Published: June 2008

The Standard Model of particle physics assumes that the so-called fundamental constants are universal and unchanging. Absorption lines arising in molecular clouds along quasar sightlines offer a precise test for variations in the proton-to-electron mass ratio, μ, over cosmological time and distance scales. The inversion transitions of ammonia are particularly sensitive to μ as compared to molecular rotational transitions. Comparing the available ammonia spectra observed toward the quasar B0218+357 with new, high-quality rotational spectra, we present the first detailed measurement of μ with this technique, limiting relative deviations from the laboratory value to |Δμ/μ| < 1.8 × 10
–6
(95% confidence level) at approximately half the universe's current age—the strongest astrophysical constraint to date. Higher-quality ammonia observations will reduce both the statistical and systematic uncertainties in these observations.
Single-Cycle Nonlinear Optics
Authors: Goulielmakis E. , Schultze M. , Hofstetter M. , Yakovlev V. S. , Gagnon J. , Uiberacker M. , Aquila A. L. , Gullikson E. M. , Attwood D. T. , Kienberger R. , Krausz F. , Kleineberg U.
Published: June 2008

Nonlinear optics plays a central role in the advancement of optical science and laser-based technologies. We report on the confinement of the nonlinear interaction of light with matter to a single wave cycle and demonstrate its utility for time-resolved and strong-field science. The electric field of 3.3-femtosecond, 0.72-micron laser pulses with a controlled and measured waveform ionizes atoms near the crests of the central wave cycle, with ionization being virtually switched off outside this interval. Isolated sub-100-attosecond pulses of extreme ultraviolet light (photon energy ∼ 80 electron volts), containing ∼0.5 nanojoule of energy, emerge from the interaction with a conversion efficiency of ∼10
–6
. These tools enable the study of the precision control of electron motion with light fields and electron-electron interactions with a resolution approaching the atomic unit of time (∼24 attoseconds).
Ankyrin Repeat Proteins Comprise a Diverse Family of Bacterial Type IV Effectors
Authors: Pan Xiaoxiao , Lührmann Anja , Satoh Ayano , Laskowski-Arce Michelle A. , Roy Craig R.
Published: June 2008

Specialized secretion systems are used by many bacteria to deliver effector proteins into host cells that can either mimic or disrupt the function of eukaryotic factors. We found that the intracellular pathogens
Legionella pneumophila
and
Coxiella burnetii
use a type IV secretion system to deliver into eukaryotic cells a large number of different bacterial proteins containing ankyrin repeat homology domains called Anks. The
L. pneumophila
AnkX protein prevented microtubule-dependent vesicular transport to interfere with fusion of the
L. pneumophila
-containing vacuole with late endosomes after infection of macrophages, which demonstrates that Ank proteins have effector functions important for bacterial infection of eukaryotic host cells.
Bovine Colostrum Contains Immunoglobulin G Antibodies against Intimin, EspA, and EspB and Inhibits Hemolytic Activity Mediated by the Type Three Secretion System of Attaching and Effacing
Escherichia coli
Authors: Vilte Daniel A. , Larzábal Mariano , Cataldi Ángel A. , Mercado Elsa C.
Published: June 2008

Enterohemorrhagic
Escherichia coli
(EHEC) is the main cause of hemolytic-uremic syndrome, an endemic disease in Argentina which had an incidence in 2005 of 13.9 cases per 100,000 children younger than 5 years old. Cattle appear to be a major reservoir of EHEC, and a serological response to EHEC antigens has been demonstrated in natural and experimental infections. In the current study, antibodies against proteins implicated in EHEC's ability to form attaching and effacing lesions, some of which are exported to the host cell via a type three secretion system (TTSS), were identified in bovine colostrum by Western blot analysis. Twenty-seven (77.0%) of the 35 samples examined contained immunoglobulin G (IgG) antibodies against the three proteins assayed in this study: EspA, EspB, and the carboxy-terminal 280 amino acids of γ-intimin, an intimin subtype associated mainly with O157:H7 and O145:H- serotypes. Every colostrum sample was able to inhibit, in a range between 45.9 and 96.7%, the TTSS-mediated hemolytic activity of attaching and effacing
E. coli
. The inhibitory effect was partially mediated by IgG and lactoferrin. In conclusion, we found that early colostrum from cows contains antibodies, lactoferrin, and other unidentified substances that impair TTSS function in attaching and effacing
E. coli
strains. Bovine colostrum might act by reducing EHEC colonization in newborn calves and could be used as a prophylactic measure to protect non-breast-fed children against EHEC infection in an area of endemicity.
Use of Phenotypic and Molecular Serotype Identification Methods To Characterize Previously Nonserotypeable Group B Streptococci
Authors: Kong Fanrong , Lambertsen Lotte Munch , Slotved Hans-Christian , Ko Danny , Wang Hui , Gilbert Gwendolyn L.
Published: June 2008

Among 1,762 isolates of
Streptococcus agalactiae
(group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning
cpsH
to
cpsM
). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the
cpsE
-
cpsF
-
cpsG
region of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the
cpsE
-
cpsF
-
cspG
region of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516;
P
< 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.
Optimal Probe Length and Target Location for Electrochemical Detection of Selected Uropathogens at Ambient Temperature
Authors: Mastali Mitra , Babbitt Jane T. , Li Yang , Landaw Elliot M. , Gau Vincent , Churchill Bernard M. , Haake David A.
Published: June 2008

We have previously demonstrated the clinical validity of the rapid detection of uropathogens by use of a DNA biosensor. This assay involves the hybridization of capture and detector probe pairs with bacterial 16S rRNA target molecules to form a DNA-RNA sandwich on the sensor surface. Horseradish peroxidase-conjugated antibody binds to the detector probe to enzymatically amplify the hybridization signal. These previous studies involved the hybridization of bacterial 16S rRNA target sequences with 35-mer oligonucleotide probe pairs at 65°C. Achievement of point-of-care technology will be greatly facilitated by ambient-temperature detection. The purpose of this study was to examine the effects of probe length and target location on signal intensity using hybridization temperatures of 20 to 25°C. Signal intensity was found to vary dramatically with hybridization location in the species-specific bulge region of 16S rRNA helix 18. Probe pairs of as short as 10 nucleotides in length were able to produce a significant electrochemical signal, and signal intensity was correlated with probe length for probes of 10 to 20 nucleotides in length. The sensitivity of the
Escherichia coli
-specific 15-mer probe pairs was approximately 330 cells. These shorter probes allowed differentiation of
Klebsiella pneumoniae
from
Proteus mirabilis
16S rRNA target sequences differing by a single nucleotide. A panel of oligonucleotide probe pairs ranging from 11 to 23 nucleotides in length was able to distinguish among seven groups of urinary tract pathogens. In conclusion, we have developed short oligonucleotide probe pairs for the species-specific identification of uropathogens at ambient temperature by use of an electrochemical sensor.
Universal Primer Set for Amplification and Sequencing of HA
0
Cleavage Sites of All Influenza A Viruses
Authors: Gall Astrid , Hoffmann Bernd , Harder Timm , Grund Christian , Beer Martin
Published: June 2008

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA
0
is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA
0
cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA
0
cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay—followed by cycle sequencing—can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.
Streptobacillus moniliformis
as the Causative Agent in Spondylodiscitis and Psoas Abscess after Rooster Scratches
Authors: Dubois Damien , Robin Frédéric , Bouvier Damien , Delmas Julien , Bonnet Richard , Lesens Olivier , Hennequin Claire
Published: June 2008

We report a case of
Streptobacillus moniliformis
spondylodiscitis accompanied by a psoas abscess in an 80-year-old man scratched by a rooster.
S. moniliformis
was identified from abscess fluid by use of 16S rRNA gene sequencing. After 18 weeks of antimicrobial therapy, the clinical condition of the patient improved.
Geographic Distribution and Antifungal Susceptibility of the Newly Described Species
Candida orthopsilosis
and
Candida metapsilosis
in Comparison to the Closely Related Species
Candida parapsilosis
Authors: Lockhart Shawn R. , Messer Shawn A. , Pfaller Michael A. , Diekema Daniel J.
Published: June 2008

Candida orthopsilosis
and
Candida metapsilosis
are recently described species, having previously been grouped with the more prevalent species
Candida parapsilosis
. Current literature contains very little data pertaining to the distributions and antifungal susceptibilities of these
Candida
species. We determined the species and antifungal susceptibilities of 1,929 invasive clinical isolates from the ARTEMIS antifungal surveillance program collected between 2001 and 2006 and identified as
C. parapsilosis
using Vitek and conventional methods. Of the 1,929 isolates of presumed
C. parapsilosis
tested, 117 (6.1%) were identified as
C. orthopsilosis
and 34 (1.8%) as
C. metapsilosis
. The percentage of presumed
C. parapsilosis
isolates found to be
C. orthopsilosis
varied greatly by region, with the highest percentage (10.9%) from South America and the lowest (0.7%) from Africa. The MIC distributions of the
C. orthopsilosis
and
C. metapsilosis
isolates were statistically significantly lower than those of
C. parapsilosis
for all drugs except fluconazole, for which they were significantly higher (
P
< 0.001 for all). No
C. orthopsilosis
or
C. metapsilosis
isolates were fluconazole resistant, and all were susceptible to caspofungin, anidulafungin, and micafungin.
Effect of Swab Composition and Use of Swabs versus Swab-Containing Skim Milk-Tryptone-Glucose-Glycerol (STGG) on Culture- or PCR-Based Detection of
Streptococcus pneumoniae
in Simulated and Clinical Respiratory Specimens in STGG Transport Medium
Authors: Rubin Lorry G. , Rizvi Atqia , Baer Aryeh
Published: June 2008

To detect
Streptococcus pneumoniae
colonization, the nasopharynx is sampled using a swab placed in skim milk-tryptone-glucose-glycerol (STGG) transport medium, and then the swab specimen or STGG medium is cultured or subjected to PCR. We evaluated the effect of swab composition and compared the sensitivities of detection of culture and PCR using swabs and swab-containing medium. Calcium alginate, Dacron polyester, or rayon-tipped swabs were inoculated with pneumococci or were immersed in nasal wash specimens from children and then placed in STGG medium. Swabs and medium inoculated with pneumococci were cultured. Swabs grew significantly more colonies than medium. The number of colonies cultured from rayon swabs or medium was significantly higher than the number cultured from the calcium alginate swab or medium. The number of colonies from both the Dacron polyester swabs and medium were significantly lower than with either calcium alginate or rayon swabs. When DNA was separately extracted from the calcium alginate swab and medium and subjected to PCR for pneumococcal detection from either
S. pneumoniae
-inoculated swabs or clinical specimens that grew
S. pneumoniae
, the sensitivity was at least 10 times higher using the swab. With Dacron polyester or rayon-tipped swabs, there was no consistent difference between the sensitivity of PCR using swabs and that of PCR using medium. Thus, calcium alginate swabs may be superior to STGG medium for the culture and PCR-based detection of
S. pneumoniae
. For culture, rayon swabs are superior and Dacron polyester swabs are inferior. The sensitivity of the swab and swab-containing medium for culture or PCR detection of
S. pneumoniae
varies with swab composition.
Structure of the Main Protease from a Global Infectious Human Coronavirus, HCoV-HKU1
Authors: Zhao Qi , Li Shuang , Xue Fei , Zou Yilong , Chen Cheng , Bartlam Mark , Rao Zihe
Published: June 2008

The newly emergent human coronavirus HKU1 (HCoV-HKU1) was first identified in Hong Kong in 2005. Infection by HCoV-HKU1 occurs worldwide and causes syndromes such as the common cold, bronchitis, and pneumonia. The CoV main protease (M
pro
), which is a key enzyme in viral replication via the proteolytic processing of the replicase polyproteins, has been recognized as an attractive target for rational drug design. In this study, we report the structure of HCoV-HKU1 M
pro
in complex with a Michael acceptor, inhibitor N3. The structure of HCoV-HKU1 provides a high-quality model for group 2A CoVs, which are distinct from group 2B CoVs such as severe acute respiratory syndrome CoV. The structure, together with activity assays, supports the relative conservation at the P1 position that was discovered by sequencing the HCoV-HKU1 genome. Combined with structural data from other CoV M
pro
s, the HCoV-HKU1 M
pro
structure reported here provides insights into both substrate preference and the design of antivirals targeting CoVs.
Intramembrane Processing by Signal Peptide Peptidase Regulates the Membrane Localization of Hepatitis C Virus Core Protein and Viral Propagation
Authors: Okamoto Kiyoko , Mori Yoshio , Komoda Yasumasa , Okamoto Toru , Okochi Masayasu , Takeda Masatoshi , Suzuki Tetsuro , Moriishi Kohji , Matsuura Yoshiharu
Published: June 2008

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala
191
by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe
177
by mass spectrometry. Mutations introduced into two residues (Ile
176
and Phe
177
) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.
Effects of Hemagglutinin-Neuraminidase Protein Mutations on Cell-Cell Fusion Mediated by Human Parainfluenza Type 2 Virus
Authors: Tsurudome Masato , Nishio Machiko , Ito Morihiro , Tanahashi Shunsuke , Kawano Mitsuo , Komada Hiroshi , Ito Yasuhiko
Published: June 2008

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.
Genetic Characterization of Human Immunodeficiency Virus Type 1 in Elite Controllers: Lack of Gross Genetic Defects or Common Amino Acid Changes
Authors: Miura Toshiyuki , Brockman Mark A. , Brumme Chanson J. , Brumme Zabrina L. , Carlson Jonathan M. , Pereyra Florencia , Trocha Alicja , Addo Marylyn M. , Block Brian L. , Rothchild Alissa C. , Baker Brett M. , Flynn Theresa , Schneidewind Arne , Li Bin , Wang Yaoyu E. , Heckerman David , Allen Todd M. , Walker Bruce D.
Published: June 2008

Despite reports of viral genetic defects in persons who control human immunodeficiency virus type 1 (HIV-1) in the absence of antiviral therapy, the extent to which such defects contribute to the long-term containment of viremia is not known. Most previous studies examining for such defects have involved small numbers of subjects, primarily focused on subjects expressing HLA-B57, or have examined single viral genes, and they have focused on cellular proviral DNA rather than plasma viral RNA sequences. Here, we attempted viral sequencing from 95 HIV-1 elite controllers (EC) who maintained plasma viral loads of <50 RNA copies/ml in the absence of therapy, the majority of whom did not express HLA-B57. HIV-1 gene fragments were obtained from 94% (89/95) of the EC, and plasma viral sequences were obtained from 78% (61/78), the latter indicating the presence of replicating virus in the majority of EC. Of 63 persons for whomnefwas sequenced, only three cases ofnefdeletions were identified, and gross genetic defects were rarely observed in other HIV-1 coding genes. In a codon-by-codon comparison between EC and persons with progressive infection, correcting for HLA bias and coevolving secondary mutations, a significant difference was observed at only three codons in Gag, all three of which represented the historic population consensus amino acid at the time of infection. These results indicate that the spontaneous control of HIV replication is not attributable to shared viral genetic defects or shared viral polymorphisms.
Nuclear Import of the Varicella-Zoster Virus Latency-Associated Protein ORF63 in Primary Neurons Requires Expression of the Lytic Protein ORF61 and Occurs in a Proteasome-Dependent Manner
Authors: Walters Matthew S. , Kyratsous Christos A. , Wan Shilin , Silverstein Saul
Published: June 2008

Varicella-zoster virus (VZV) open reading frame (ORF) 63 protein (ORF63p) is one of six VZV ORFs shown to be transcribed and translated in latently infected human dorsal root ganglia. ORF63p accumulates exclusively in the cytoplasm of latently infected sensory neurons, whereas it is both nuclear and cytoplasmic during lytic infection and following reactivation from latency. Here, we demonstrate that infection of primary guinea pig enteric neurons (EN) with an adenovirus expressing ORF63p results in the exclusive cytoplasmic localization of the protein reminiscent of its distribution during latent VZV infection in humans. We show that the addition of the simian virus 40 large-T-antigen nuclear localization signal (NLS) results in the nuclear import of ORF63p in EN and that the ORF63p endogenous NLSs are functional in EN when fused to a heterologous protein. These data suggest that the cytoplasmic localization of ORF63p in EN results from the masking of the NLSs, thus blocking nuclear import. However, the coexpression of ORF61p, a strictly lytic VZV protein, and ORF63p in EN results in the nuclear import of ORF63p in a proteasome-dependent manner, and both ORF63p NLSs are required for this event. We propose that the cytoplasmic localization of ORF63p in neurons results from NLS masking and that the expression of ORF61p removes this block, allowing nuclear import to proceed.
Development of Reverse Genetics Systems for Bluetongue Virus: Recovery of Infectious Virus from Synthetic RNA Transcripts
Authors: Boyce Mark , Celma Cristina C. P. , Roy Polly
Published: June 2008

Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.
Differential Requirements of the C Terminus of Nbs1 in Suppressing Adenovirus DNA Replication and Promoting Concatemer Formation
Authors: Lakdawala Seema S. , Schwartz Rachel A. , Ferenchak Kevin , Carson Christian T. , McSharry Brian P. , Wilkinson Gavin W. , Weitzman Matthew D.
Published: June 2008

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.