bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Lock-In Infrared Thermography Techniques for Quantitative Nondestructive Evaluations
Authors: Sakagami Takahide , Kubo Shiro
Published: August 2009

In this paper, lock-in thermography techniques for quantitative nondestructive evaluations developed by the present authors are reviewed. Self-reference lock-in thermography was developed for remote nondestructive testing of fatigue cracks. This technique is based on the measurement of thermoelastic temperature change due to stress change. Cracks can be identified from significant temperature change observed at crack tips due to the stress singularity. For accurate measurement of the thermoelastic temperature change under random loading, a self-reference lock-in data processing technique was developed, in which a reference signal was constructed by using the temperature data simultaneously taken at a remote area. Thermoelastic temperature change in a region of interest was correlated with that at the area for reference signal construction. It enabled us to measure the relative stress distribution under random loading without using any external loading signal. The self-reference lock-in thermography was applied for fatigue crack identification in welded steel plate specimens and actual steel structures. It was found that significant temperature change was observed at the crack tip in the self-reference lock-in thermal image, demonstrating the feasibility of the proposed technique. Lock-in thermography technique was also applied to quantitative nondestructive evaluation of material loss defects. Transient temperature data under pulse or step heating were measured by infrared thermography. Temperature data were processed by the lock-in analysis scheme based on the Fourier series expansion, in which Fourier coefficients synchronizing with sine and cosine waves were correlated with defect parameters. Experimental investigations were conducted using steel samples with artificial material loss defects. It was found that the defect parameters can be quantitatively determined from the Fourier coefficients, demonstrating the feasibility of the proposed technique.
Stochastic Meshfree Method for Computational Fracture Mechanics
Authors: Rao B. N. , Arun C. O. , Siva Kumar M. S.
Published: August 2009

In the stochastic mechanics community, the need to account for uncertainty has long been recognized as key to achieving the reliable design of structural and mechanical systems. It is generally agreed that advanced computational tools must be employed to provide the necessary computational framework for describing structural response. A currently popular method is the stochastic finite element method (SFEM), which integrates probability theory with the standard finite element method (FEM). However, SFEM requires a structured mesh to perform the underlying finite element analysis. It is generally recognized that the creation of workable meshes for complex geometric configurations can be difficult, time consuming, and expensive. This discrepancy is further exacerbated when solving solid mechanics problems characterized by a continuous change in the geometry of the domain under analysis. The underlying structures of these methods, which rely on a mesh, are cumbersome in treating moving cracks or mesh distortion. Consequently, the only viable option when applying FEM is to remesh during each discrete step of the model’s evolution. This creates numerical difficulties, even for deterministic analysis, and often leads to degradation in solution accuracy, complexity in computer programming, and a computationally intensive environment. Consequently, there is considerable interest in eliminating or greatly simplifying the meshing task. In recent years, a class of Galerkin-based meshfree or meshless methods have been developed that do not require a structured mesh to discretize the problem, such as the element-free Galerkin method, and the reproducing kernel particle method. These methods employ a moving least-squares approximation method that allows resultant shape functions to be constructed entirely in terms of arbitrarily placed nodes. Meshless discretization presents significant advantages for modeling fracture propagation. By sidestepping remeshing requirements, crack-propagation analysis can be dramatically simplified. Since mesh generation of complex structures can be far more time-consuming and costly than the solution of a discrete set of linear equations, the meshless method provides an attractive alternative to FEM. However, most of the development in meshless methods to date has focused on deterministic problems. Research into probabilistic meshless analysis has not been widespread and is only now gaining attention. Due to inherent uncertainties in loads, material properties and geometry, a probabilistic meshless model is ultimately necessary. Hence, there is considerable interest in developing stochastic meshless methods capable of addressing uncertainties in loads, material properties and geometry, and of predicting the probabilistic response of structures. This paper presents a new stochastic meshless method for predicting probabilistic structural response of cracked structures i.e., mean and variance of the fracture parameters.
Concurrent Load Design Criteria for Buried High Density Polyethylene Pipe in ASME BPVC Section III, Division 1, Applications: Part III — Sample Problem
Authors: Thomas George G. , Spanner Jack R. , Adams Timothy M. , Hall Siegrid
Published: August 2009

The commercial Light Water Reactors operating within the United States have been in service from about 20 to 35 years. These plants include buried Service Water piping systems primarily made from low carbon steel. This piping has been subject to aging over the years, resulting in degradation and corrosion that will require replacement of the piping. Due to the advantageous cost and durability of High Density Polyethylene (HDPE) piping (as demonstrated in other commercial industries), the industry has expressed interest in replacing steel buried Service Water Piping in Nuclear Power Stations with HDPE piping. To assist in this effort EPRI has funded and supported the work summarized in this paper to develop design criteria for HPDE Pipe. This paper provides an example problem demonstrating the application of recently developed design criteria for HDPE piping. The technical bases of these criteria are presented in separate papers and are not repeated in this discussion.
Toward the Development of a Consensus Materials Database for Pressure Technology Applications
Authors: Swindeman R. W. , Ramirez J. , Ren W.
Published: August 2009

The ASME construction code books specify materials and fabrication procedures that are acceptable for pressure technology applications. However, with few exceptions, the materials properties provided in the ASME code books provide no statistics or other information pertaining to material variability. Such information is central to the prediction and prevention of failure events. Many sources of materials data exist that provide variability information but such sources do not necessarily represent a consensus of experts with respect to the reported trends that are represented. Such a need has been identified by the ASME Standards Technology, LLC and initial steps have been taken to address these needs: however, these steps are limited to project-specific applications only, such as the joint DOE-ASME project on materials for Generation IV nuclear reactors. In contrast to light-water reactor technology, the experience base for the Generation IV nuclear reactors is somewhat lacking and heavy reliance must be placed on model development and predictive capability. The database for model development is being assembled and includes existing code alloys such as alloy 800H and 9Cr-1Mo-V steel. Ownership and use rights are potential barriers that must be addressed.
The Researches on Toughness Index of Reheated High Grade Linepipe Bend Pipes
Authors: Zhang Xiaoli , Liu Yinglai , Feng Yaorong , Zhao Wenzhen , Huo Chunyong , Zhuang Chuanjing , Ji Lingkang , Li Weiwei
Published: August 2009

In the development of high grade pipeline steels, the technologies on bend pipes are key points. Most reports are mainly on the cold bending techniques which allow a small deflection angle usually less than 10°. And induction heat is then introduced for higher deflection angle bend pipes. So it is very important to know the toughness index of reheated high grade linepipes. In current used standard in China for West-East Gas project, Charpy toughness is required to be ≥120J at −20°C, and corresponding shearing area is required to be ≥85%. Based on a large number of experimental data on high grade linepipes with heat treatment, it was found in the present study that the shearing area and Charpy toughness have a linear relationship and the slope coefficient is 0.34. As a result, the when shearing area is 85%, the corresponding Charpy toughness is 250J. and This is different from that of mother pipe. To find the reasons, the fractures and microstructure of these reheated linepipes are analysed and compared with that of mother pipes.
Estimation of Point Vibration Loads From Field Measurements for Industrial Piping
Authors: Saha S.
Published: August 2009

Estimation of point loads on a span for a vibrating pipe has been done with the help of boundary measurement of slope data. The approach is based on the theory of inverse force identification for hyperbolic systems. Depending on the form of the forcing function, which also satisfies some continuity conditions an exact determination, is possible. Though restrictive in its formulation some applications are still possible. A typical case for harmonic excitation of a pulsating reciprocating compressor has been shown as an illustration.
Safety Requirements for the Second High-Pressure and Large-Diameter West-East Gas Pipeline
Authors: Zhuang Chuanjing
Published: August 2009

The Second West-East Gas Pipeline (WEGP) is planed be constructed trans-China within recent years. The Pipeline is about 7,000 kilometers with outside diameter 1219mm, operation pressure 12MPa, and steel grade API-X80 [1], all of which is the first time in China. Both safety and reliability of the Pipeline are the most important issues which should be thought over by the gas company. In this paper, the method of limit state analysis is used to study the effect of strength, toughness and allowable flaw size on the safety and reliability of the Pipeline. The calculation results suggest that the overmatched weld has the advantages of improving limit load and maximum allowable defect sizes of the pipelines. When Ms (Ms is the ratio of yield strength of weld σSW over that of base metal σSB, i.e. MS = σSW / σSB) is lower than 0.9, there is much possibility for the happening of fracture initiation in the weld zone. When Ms is higher than 1.2, the limit load will not increase with the increase of weld strength. Although the Second WEGP will transmit sweet gas, there is still possibility that sour gas releases to the Pipeline in case of operation accident. As to the high-grade pipeline, in order to reduce the sensitivity of cold crack, hydrogen induced crack (HIC) and stress induced corrosion crack (SCC), etc., However, chemical composition and hardness of both base and weld metal can influence the resistance to HIC and SCC. It can be concluded that the best range of strength mismatched ratios Ms is 0.9 ∼ 1.2 for the Second WEGP. The critical defect sizes proposal is 10.0mm of maximum allowable defect length and 0.95mm of maximum allowable defect depth at the weld mismatch.
An Understanding of Bending and Pressure Fatigue of Composite Pipes
Authors: Gerrard D. P. , Srivatsan T. S. , Scavuzzo R. J. , Miner T. S. , Olabisi O.
Published: August 2009

In this technical paper is presented a comparative study of the fatigue strength of high-pressure composite pipes and high-pressure composite pipes containing joints. The test specimens used in this experimental investigation were exposed to cyclic bending stresses and to cyclic bending stresses in combination with constant or cyclic internal pressures generated by water, brine, and crude oil. The extrinsic influence of elevated temperature on fatigue performance was also examined. A new Four-Point Bending Fatigue Machine was developed at the University of Akron to accomplish the testing. For each test specimen, two types of failure were distinctly observed. After a number of repeated cycles the fluid under internal pressure began to gradually leak through the fine microscopic cracks in the matrix. The fine microscopic cracks were oriented in the circumferential direction of the pipe. However, despite the occurrence of “weeping failure” of the pipe, internal pressure could be easily maintained. After about 10 to 100 times the number of cycles required for “weeping”, the fiber reinforcements of the pipe gradually fractured and the internal pressure could no longer be maintained. The loss of pressure is referred to as “pipe failure”. In these tests, the primary parameter controlling failure was orientation of the fiber reinforcements in the body of the pipe. Those fibers aligned along the pipe axis revealed a substantial improvement in cyclic fatigue resistance. The existence of a joint in the test specimen showed secondary importance, while contributing to degradation of the fatigue resistance of the specimen. Both types of failure were found to be dependant on temperature, over the range tested (22°–66° C), and also dependent on the type of internal liquid used. The specimens tested using crude oil at 66° C as the internal fluid revealed the lowest fatigue resistance.
Status of Fitness-for-Service Assessment of Defected Pipeline in China
Authors: Zhao Xinwei , Luo Jinheng , Dong Baosheng , Zhang Hua , Zhang Guangli
Published: August 2009

Fitness-for-service assessment of defected pipeline is an important part of oil and gas pipeline integrity assessment and management. Fitness-for-service assessment comprises a residual strength evaluation and a remaining life prediction for a damaged pipeline. In the present paper, the objectives and methods for fitness-for-service assessment are classified scientifically, and their worldwide research status has been reviewed. Several novel fitness-for-service assessment criteria proposed in China are presented. Challenging problems and key technologies for fitness-for-service assessment of pipelines are outlined.
A New Ultrasonic Inspection Technique for Flaw Depth Sizing Based on Short Path of Diffraction
Authors: Lin Shan , Fukutomi Hiroyuki , Ogata Takashi
Published: August 2009

This paper describes a new tip diffraction technique, short path of diffraction (SPOD), for accurate depth sizing of flaws on the surface opposite to the inspection surface in which a set of an angle beam and a longitudinal 0-degree transducers is used as a transmitter and a receiver. In this technique, flaw depth is just calculated from the time difference between two echoes of a longitudinal diffracted wave at the tip of a flaw, which arises after an incident wave from the angle beam transducer impinges on the flaw. One echo is related to a wave traveling directly from the tip to the receiver, and the other is a wave which is reflected at the back wall and subsequently travels to the receiver. The two echoes are detected by the longitudinal 0-degree transducer located right above the flaw. Incident angles and incident points have no effect on determining flaw depth in this technique, therefore, more accurate and efficient flaw sizing becomes possible for the proposed technique. The new technique will be demonstrated by applying to depth sizing of slits and stress corrosion cracks which are machined into welded specimens made from austenitic stainless steel.
The Effect of Throughwall Axial Crack on In-Plane Collapse Moment of Pipe Bend
Authors: Chattopadhyay J. , Venkatramana W. V. , Dutta B. K. , Kushwaha H. S.
Published: August 2009

A throughwall axial crack may develop in an elbow or pipe bend due to service related degradation mechanism. It is very important to know the plastic collapse moment (PCM) of an elbow in the presence of a throughwall axial crack. The existing PCM equations of throughwall axially cracked (TAC) elbows are either too conservative or inadequate to correctly quantify the weakening effect due to the presence of the crack. Further, they do not differentiate between closing and opening modes of bending although deformation characteristics under these two modes are completely different. Therefore, the present study has been undertaken to investigate through 3-D elastic-plastic finite element analysis. A total of 84 elbows with various sizes of axial cracks (a/Dm = 0–1), different wall thickness (R/t = 5 — 20), different elbow bend radii (Rb/R = 2,3) and two different bending modes, namely closing and opening have been considered in the analysis. Elastic-perfectly plastic stress-strain response of material has been assumed. Both geometric and material non-linearity are considered in the analysis. Crack closing is observed in most of the cases. To capture the crack closure effect, contact analysis has been performed. Plastic collapse moments have been evaluated from moment — end rotation curves by twice-elastic slope method. From these results, closed-form equations are proposed to evaluate plastic collapse moments of elbows under closing and opening mode of bending moment. The predictions of these proposed equations are compared with the test data available in the literature. Matching between predictions and experimental results is found to be satisfactory.
A Tribute to Dr Spencer Bush (1920-2005): On Behalf of the Institution of Mechanical Engineers, UK
Authors: Darlaston John
Published: August 2009

This is not a technical paper but simply a tribute to Dr Spencer Bush. Through recollections in various areas of activity, some of the contributions made by him are identified.
Bolted Flange Joints Under External Moments: An Analysis Using the Compound Gasket Approach for Spiral Wound Gaskets
Authors: Seipp Trevor G. , Reichert Christopher , Messer Barry
Published: August 2009

It is common to rate a piping system to its weakest component to maximize flexibility for future operations. In many situations, the bolted flange joint is the lowest rated component. Rating a system for its full flange rating reduces the flange’s capacity to carry external bending moments. In the past, moments on flanged joints have been evaluated by using the concept of equivalent pressure, first presented in the Kellogg Design of Piping Systems. Operating moments are converted to an equivalent pressure. This equivalent pressure is added to the design pressure and compared against a limit. According to conventional practices, the design pressure plus the equivalent pressure must not exceed the rating pressure. Consequently, designing up to the flange rating pressure presents an issue, since no margin is left for the effects of external moments on flange joints. Depending on the circumstances, many designers have compensated by permitting the combined design pressure and equivalent pressure to be as high as twice the flange rating. In this paper, the authors demonstrate a robust methodology to define an appropriate limit for operating moments on bolted flange joints. Using the calculation methodologies of EN-1591-1, the authors calculate the maximum external moment that various classes of standard ASME B16.5 flanges (for Group 1.1 materials) can tolerate over a range of temperatures and present a representative sample. Conclusions are drawn about appropriate limits for moments on flanges and are compared to results using the equivalent pressure method.
Applicability of (K, T-Stress) Methodology to Analyze RPV Under Thermal-Hydraulic Transients
Authors: Yin Shengjun , Williams Paul T. , Dickson Terry L. , Bass B. Richard
Published: August 2009

The (K, T-stress) methodology developed by Gao and Dodds [1] is being utilized to introduce crack front plasticity with constraint effects when plastic deformation occurs in structures, for example, when the Reactor Pressure Vessels (RPVs) are subjected to thermal-hydraulic loadings. One crucial step in this procedure is to quantify combinations of flaw geometries and loading conditions (transient sequences) that illustrate the limits of applicability of the two-parameter (K, T-stress) advanced fracture methodology relevant to integrity analyses of RPVs subjected to normal and emergency operating conditions. Numerical analyses were conducted to determine the limits of applicability of (K, T-stress) advanced fracture technology for RPV under thermal-hydraulic loadings. The numerical results indicate that the (K, T-stress) methodology captures the constraint condition of the RPV with typical embedded flaws under a postulated dominant thermal-hydraulic transient.
Robotic Comanipulation With Active Impedance Control
Authors: Cortesa˜o Rui , Zenowich Brian , Arau´jo Rui , Townsend William
Published: August 2009

The paper presents active impedance control for robotic comanipulation tasks, enabling virtual contact interactions. Computed torque control in the task space powered by multiple-output active observers (AOBs) is proposed, enhancing haptic perception. Forces and force derivatives are artificially measured from position data around an equilibrium point that can move with time. Control techniques to deal with critical impedances are introduced, taking into account the noise distribution along the system. Stochastic design is discussed. A dynamic model of the redundant lightweight 7-DOF WAM™ arm is derived and evaluated, playing a key role in the control design. Experiments for small and high impedances are presented, highlighting merits and limitations of the approach. A comparative study between active and non-active impedance control is made.
Enacting Actions in Simulated Environments
Authors: Pierce Devin , Lu Shulan , Harter Derek
Published: August 2009

The past decade has witnessed incredible advances in building highly realistic and richly detailed simulated worlds. We readily endorse the common-sense assumption that people will be better equipped for solving real-world problems if they are trained in near-life, even if virtual, scenarios. The past decade has also witnessed a significant increase in our knowledge of how the human body as both sensor and as effector relates to cognition. Evidence shows that our mental representations of the world are constrained by the bodily states present in our moment-to-moment interactions with the world. The current study investigated whether there are differences in how people enact actions in the simulated as opposed to the real world. The current study developed simple parallel task environments and asked participants to perform actions embedded in a stream of continuous events (e.g., cutting a cucumber). The results showed that participants performed actions at a faster speed and came closer to incurring injury to the fingers in the avatar enacting action environment than in the human enacting action environment.
Enhancement of operation temperature of InAs∕GaAs quantum-dot infrared photodetectors with hydrogen-plasma treatment
Authors: Lin Wei-Hsun , Tseng Chi-Che , Chao Kuang-Ping , Lin Shih-Yen , Wu Meng-Chyi
Published: August 2009

Postprocess hydrogen treatment is performed over fabricated ten-period InAs∕GaAs quantum-dot infrared photodetectors. While keeping similar spectral responses at the same applied voltage, a reduced dark current is observed for the H-plasma-treated device, which is attributed to the suppression of surface leakage-current induced by surface damage during device processing. The significant reduction in dark current also enhanced the operation temperature of the device up to 100K. Also observed are the smoothed-out mesa edges after the H-plasma treatment, which results in trapezoidal mesa edges. In this case, a longer propagation length in the device of the reflected incident light at the mesa edge would enhance the normal-incident absorption ratio of the H-plasma-treated device.
Great Teachers for STEM
Authors: Singer Maxine
Published: August 2009

Many countries are striving to develop the human resources required to advance science and technology. Success requires a system of education that prepares young people for life in today's complex societies. The Obama administration has recently stressed the need to strengthen science, technology, engineering, and mathematics (STEM) education in the United States. U.S. Secretary of Education Arne Duncan clearly understands that the quality of STEM teaching is of singular importance to the success of students, requiring “great teachers, who know the content.”
*
Herein lies a major challenge: How to develop and cultivate great STEM teachers?
Comparison of Methods To Test Antibiotic Combinations against Heterogeneous Populations of Multiresistant
Pseudomonas aeruginosa
from Patients with Acute Infective Exacerbations in Cystic Fibrosis
Authors: Foweraker Juliet E. , Laughton Christian R. , Brown Derek F. , Bilton Diana
Published: August 2009

Multiresistant
Pseudomonas aeruginosa
isolates can chronically infect patients with cystic fibrosis. Acute infective exacerbations are treated with combinations of two antipseudomonal antibiotics. Patients may respond clinically even if the bacteria are resistant, possibly due to antimicrobial synergy. The challenge for testing for synergy in vitro is that there is no standardized method, and the antibiotic susceptibility in a population of
P. aeruginosa
isolates in a single sputum sample can vary. We therefore compared (i) antibiotic combinations with different examples of resistant bacteria from the same sputum sample and (ii) the results of synergy testing by different methods. Antibiotic synergy was tested by using resistant
P. aeruginosa
isolates recovered from sputum samples taken just before the start of treatment for an acute infective exacerbation. Several examples of each morphotype of
P. aeruginosa
were tested by cidal checkerboard, time-kill curve, and multiple-combination bactericidal testing. The isolates were typed by pulsed-field gel electrophoresis (PFGE). The results were compared with the clinical and microbiological responses to 14 days of antibiotic treatment. Forty-four resistant isolates from nine patients were tested. Some
P. aeruginosa
isolates with the same morphotype and PFGE pulsotype had different results by synergy testing. There was a poor correlation between the results of the different methods of synergy testing, and no one method would have predicted the response to treatment in all patients. The in vitro effects of antibiotic combinations against different isolates from the same sputum sample can vary, and the results depend on the methodology used. The role of combination testing for the treatment of antibiotic-resistant
P. aeruginosa
in acute exacerbations of chronic infection in patients with cystic fibrosis needs to be reviewed.
Penetration of Colistin into Cerebrospinal Fluid
Authors: Markantonis S. L. , Markou N. , Fousteri M. , Sakellaridis N. , Karatzas S. , Alamanos I. , Dimopoulou E. , Baltopoulos G.
Published: August 2009

Colistin penetration into the cerebrospinal fluid (CSF) was studied in five critically ill adult patients receiving colistin methanesulfonate for infections by multiresistant gram-negative bacilli. Colistin concentrations were determined in paired serum and CSF samples, with the latter taken by lumbar puncture, with the exception of one patient with an external ventriculostomy. CSF-to-serum ratios (0.051 to 0.057) for all study patients coincided at all sampling times. The low level (5%) of penetration suggests inadequate bactericidal colistin concentrations in the CSF.
Tooth-Binding Micelles for Dental Caries Prevention
Authors: Chen Fu , Liu Xin-Ming , Rice Kelly C. , Li Xue , Yu Fang , Reinhardt Richard A. , Bayles Kenneth W. , Wang Dong
Published: August 2009

Maintenance of the effective local concentration of antimicrobials on the tooth surface is critical for the management of cariogenic bacteria in the oral cavity. We report on the design of a simple tooth-binding micellar drug delivery platform that would effectively bind to tooth surfaces. To achieve tooth-binding ability, the chain termini of biocompatible Pluronic copolymers were modified with a biomineral-binding moiety (i.e., alendronate). The micelles formulated with this polymer were shown to be able to swiftly (<1 min) bind to hydroxyapatite (HA; a model tooth surface) and gradually release the encapsulated model antimicrobial (farnesol). These tooth-binding micelles were negatively charged and had an average effective hydrodynamic diameter of less than 100 nm. In vitro biofilm inhibition studies demonstrated that the farnesol-containing tooth-binding micelles were able to provide significantly stronger inhibition ofStreptococcus mutansUA159 biofilm formation on HA discs than the untreated blank control micelles (P< 0.0001). Upon further optimization, this delivery platform could provide an effective tool for caries prevention and treatment.
IS
CR2
, Another Vehicle for
bla
VEB
Gene Acquisition
Authors: Poirel Laurent , Mugnier Pauline D. , Toleman Mark A. , Walsh Timothy R. , Rapoport Melina J. , Petroni Alejandro , Nordmann Patrice
Published: August 2009

The expanded-spectrum β-lactamase (ESBL) gene
bla
VEB-1
, identified worldwide in
Enterobacteriaceae
and
Pseudomonas aeruginosa
, is associated with either class 1 integrons or repeated elements. We report here the first association of
bla
VEB-1a
with the insertion sequence IS
CR2
in six
Acinetobacter
species isolates recovered from Argentina. That genetic structure was likely at the origin of the mobilization of this ESBL gene.
A Ser29Leu Substitution in the Cytosine Deaminase Fca1p Is Responsible for Clade-Specific Flucytosine Resistance in
Candida dubliniensis
Authors: McManus Brenda A. , Moran Gary P. , Higgins Judy A. , Sullivan Derek J. , Coleman David C.
Published: August 2009

The population structure of the opportunistic yeast pathogen
Candida dubliniensis
is composed of three main multilocus sequence typing clades (clades C1 to C3), and clade C3 predominantly consists of isolates from the Middle East that exhibit high-level resistance (MIC
50
≥ 128 μg/ml) to the fungicidal agent flucytosine (5FC). The close relative of
C. dubliniensis
,
C. albicans
, also exhibits clade-specific resistance to 5FC, and resistance is most commonly mediated by an Arg101Cys substitution in the
FUR1
gene encoding uracil phosphoribosyltransferase. Broth microdilution assays with fluorouracil (5FU), the toxic deaminated form of 5FC, showed that both 5FC-resistant and 5FC-susceptible
C. dubliniensis
isolates exhibited similar 5FU MICs, suggesting that the
C. dubliniensis
cytosine deaminase (Fca1p) encoded by
C. dubliniensis FCA1
(Cd
FCA1
) may play a role in mediating
C. dubliniensis
clade-specific 5FC resistance. Amino acid sequence analysis of the Cd
FCA1
open reading frame (ORF) identified a homozygous Ser29Leu substitution in all 12 5FC-resistant isolates investigated which was not present in any of the 9 5FC-susceptible isolates examined. The tetracycline-inducible expression of the Cd
FCA1
ORF from a 5FC-susceptible
C. dubliniensis
isolate in two separate 5FC-resistant clade C3 isolates restored susceptibility to 5FC, demonstrating that the Ser29Leu substitution was responsible for the clade-specific 5FC resistance and that the 5FC resistance encoded by
FCA1
genes with the Ser29Leu transition is recessive. Quantitative real-time PCR analysis showed no significant difference in Cd
FCA1
expression between 5FC-susceptible and 5FC-resistant isolates in either the presence or the absence of subinhibitory concentrations of 5FC, suggesting that the Ser29Leu substitution in the Cd
FCA1
ORF is the sole cause of 5FC resistance in clade C3
C. dubliniensis
isolates.
New Approach for Drug Susceptibility Testing: Monitoring the Stress Response of Mycobacteria
Authors: Rieder Ronald J. , Zhao Zhihui , Zavizion Boris
Published: August 2009

Methods currently used for in vitro drug susceptibility testing are based on the assessment of bacterial growth-related processes. This reliance on cellular reproduction leads to prolonged incubation times, particularly for slowly growing organisms such as mycobacteria. A new rapid phenotypic method for the drug susceptibility testing of mycobacteria is described. The method is based on the detection of the physiological stress developed by susceptible mycobacterial cells in the presence of an antimicrobial compound. The induced stress was quantified by differential monitoring of the dielectric properties of the bacterial suspension, an easily measurable electronic property. The data presented here characterize the stress developed by
Mycobacterium tuberculosis
cells treated with rifampin (rifampicin), isoniazid, ethambutol, and pyrazinamide. Changes in the dielectric-based profiles of the drug-treated bacteria revealed the respective susceptibilities in near real time, and the susceptibilities were well correlated with conventional susceptibility test data.
Pulmonary Epithelial Lining Fluid Concentrations after Use of Systemic Amphotericin B Lipid Formulations
Authors: Weiler Stefan , Falkensammer Gerda , Hammerer-Lercher Angelika , Anliker Markus , Vogelsinger Helene , Joannidis Michael , Dunzendorfer Stefan , Stein Markus , Bellmann Romuald
Published: August 2009

Amphotericin B (AMB) concentrations were determined in pulmonary epithelial lining fluid (ELF) of 44 critically ill patients, who were receiving treatment with liposomal AMB (LAMB) (
n
= 11), AMB colloidal dispersion (ABCD) (
n
= 28), or AMB lipid complex (ABLC) (
n
= 5). Mean AMB levels (± standard errors of the means) in ELF amounted to 1.60 ± 0.58, 0.38 ± 0.07, and 1.29 ± 0.71 μg/ml in LAMB-, ABCD-, and ABLC-treated patients, respectively (differences are not significant).
Radiolabeled Antibodies to
Bacillus anthracis
Toxins Are Bactericidal and Partially Therapeutic in Experimental Murine Anthrax
Authors: Rivera Johanna , Nakouzi Antonio S. , Morgenstern Alfred , Bruchertseifer Frank , Dadachova Ekaterina , Casadevall Arturo
Published: August 2009

Bacillus anthracis
is a powerful agent for use in biological warfare, and infection with the organism is associated with a high rate of mortality, underscoring the need for additional effective therapies for anthrax. Radioimmunotherapy (RIT) takes advantage of the specificity and affinity of the antigen-antibody interaction to deliver a microbicidal radioactive nuclide to a site of infection. RIT has proven therapeutic in experimental models of viral, bacterial, and fungal infections; but it is not known whether this approach can successfully employ toxin binding monoclonal antibodies (MAbs) for diseases caused by toxigenic bacteria. Indirect immunofluorescence studies with MAbs to protective antigen (MAbs 7.5G γ2b and 10F4 γ1) and lethal factor (MAb 14FA γ2b) revealed the surface expression of toxins on bacterial cells. Scatchard analysis of MAbs revealed high binding constants and numerous binding sites on the bacterial surface. To investigate the microbicidal properties of these MAbs, our group radiolabeled MAbs with either
188
Re or
213
Bi. In vitro,
213
Bi was more efficient than
188
Re in mediating microbicidal activity against
B. anthracis
. The administration of MAbs [
213
Bi]10F4 γ1 and [
213
Bi]14FA γ2b prolonged the survival of A/JCr mice infected with
B. anthracis
Sterne bacterial cells but not
B. anthracis
Sterne spores. These results indicate that RIT with MAbs that target
B. anthracis
toxin components can be used to treat experimental anthrax infection and suggest that toxigenic bacteria may be targeted with radiolabeled MAbs.
Kinetics of the Natural, Humoral Immune Response toSalmonella entericaSerovar Typhi in Kathmandu, Nepal
Authors: Pulickal Anoop S. , Gautam Samir , Clutterbuck Elizabeth A. , Thorson Stephen , Basynat Buddha , Adhikari Neelam , Makepeace Katherine , Rijpkema Sjoerd , Borrow Ray , Farrar Jeremy J. , Pollard Andrew J.
Published: August 2009

Typhoid fever is a major public health problem in developing countries, conservatively estimated to occur in 17 million cases and be responsible for 200,000 deaths annually. We investigated the acquisition of natural immunity toSalmonella entericaserovar Typhi in a region where typhoid is endemic by testing sera from an age-stratified sample of 210 healthy participants in Kathmandu, Nepal, for bactericidal activity towardS. Typhi and for anti-Vi capsular polysaccharide antibodies. Bactericidal titers in children were significantly lower than those in newborns and adults (P< 0.0001). Anti-S. Typhi bactericidal geometric mean titers were age dependent, increasing 10-fold during childhood. Anti-Vi polysaccharide antibody geometric mean concentrations were also lower in children than in adults. Data presented here indicate the possibility of a relationship between low levels of bactericidal activity towardS. Typhi in serum and susceptibility to disease, as observed for other polysaccharide-encapsulated bacteria. Bactericidal antibody may be a marker of protective immunity againstS. Typhi.
Nondividing but Metabolically Active Gamma-IrradiatedBrucella melitensisIs Protective against VirulentB. melitensisChallenge in Mice
Authors: Magnani D. M. , Harms J. S. , Durward M. A. , Splitter G. A.
Published: August 2009

Brucellaspp. are gram-negative bacteria that cause the most frequent zoonotic disease worldwide, with more than 500,000 human infections yearly; however, no human vaccine is currently available. As with other intracellular organisms, cytotoxic mechanisms against infected cells are thought to have an important role in controlling infection and mediating long-term immunity. Live attenuated strains developed for use in animals elicit protection but retain unacceptable levels of virulence. Thus, the optimal design for a brucellosis vaccine requires a nonliving vaccine that confers effective immunity. Historically, inactivation methods such as chemical or heat treatment successfully impairBrucellareproductive capacity; nevertheless, metabolically inactive vaccines (subunit or killed) present very limited efficacy. Hence, we hypothesized that bacterial metabolism plays a major role in creating the proper antigenic and adjuvant properties required for efficient triggering of protective responses. Here, we demonstrate that inactivation ofBrucella melitensisby gamma-irradiation inhibited its replication capability and yet retained live-Brucellaprotective features. IrradiatedBrucellapossessed metabolic and transcriptional activity, persisted in macrophages, generated antigen-specific cytotoxic T cells, and protected mice against virulent bacterial challenge, without signs of residual virulence. In conclusion, pathogen metabolic activity has a positive role in shaping protective responses, and the generation of inactivated and yet metabolically active microbes is a promising strategy for safely vaccinating against intracellular organisms such asB. melitensis.
Francisella tularensis
Induces Extensive Caspase-3 Activation and Apoptotic Cell Death in the Tissues of Infected Mice
Authors: Wickstrum Jason R. , Bokhari Sirosh M. , Fischer Jeffrey L. , Pinson David M. , Yeh Hung-Wen , Horvat Rebecca T. , Parmely Michael J.
Published: August 2009

Although
Francisella tularensis
subsp.
tularensis
is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A
F. tularensis
, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other
Francisella
subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A
F. tularensis
did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A
F. tularensis
-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A
F. tularensis
, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A
F. tularensis
-infected caspase-3-deficient mice showed much less death among their F4/80
+
spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A
F. tularensis
induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.
Mouse Eosinophils Possess Potent Antibacterial Properties In Vivo
Authors: Linch Stefanie N. , Kelly Ann M. , Danielson Erin T. , Pero Ralph , Lee James J. , Gold Jeffrey A.
Published: August 2009

Eosinophils are best known as the predominant cellular infiltrate associated with asthma and parasitic infections. Recently, numerous studies have documented the presence of Toll-like receptors (TLRs) on the surfaces of eosinophils, suggesting that these leukocytes may participate in the recognition and killing of viruses and bacteria. However, the significance of this role in the innate immune response to bacterial infection is largely unknown. Here we report a novel role for eosinophils as antibacterial defenders in the host response. Isolated mouse eosinophils possessed antipseudomonal properties in vitro. In vivo, interleukin-5 transgenic mice, which have profound eosinophilia, demonstrated improved clearance ofPseudomonas aeruginosaintroduced into the peritoneal cavity. The findings of improved bacterial clearance following adoptive transfer of eosinophils, and impaired bacterial clearance in mice with a congenital eosinophil deficiency, established that this effect was eosinophil specific. The data presented also demonstrate that eosinophils mediate this antibacterial effect in part through the release of cationic secondary granule proteins. Specifically, isolated eosinophil granules had antibacterial properties in vitro, and administration of eosinophil granule extracts significantly improved bacterial clearance in vivo. These data suggest a potent yet underappreciated antibacterial role for eosinophils in vivo, specifically for eosinophil granules. Moreover, the data suggest that the administration of eosinophil-derived products may represent a viable adjuvant therapy for septic or bacteremic patients in the intensive care unit.
Atypical Roles for
Campylobacter jejuni
Amino Acid ATP Binding Cassette Transporter Components PaqP and PaqQ in Bacterial Stress Tolerance and Pathogen-Host Cell Dynamics
Authors: Lin Ann E. , Krastel Kirsten , Hobb Rhonda I. , Thompson Stuart A. , Cvitkovitch Dennis G. , Gaynor Erin C.
Published: August 2009

Campylobacter jejuni
is a human pathogen causing severe diarrheal disease; however, our understanding of the survival of
C. jejuni
during disease and transmission remains limited. Amino acid ATP binding cassette (AA-ABC) transporters in
C. jejuni
have been proposed as important pathogenesis factors. We have investigated a novel AA-ABC transporter system, encoded by cj0467 to cj0469, by generating targeted deletions of cj0467 (the membrane transport component) and cj0469 (the ATPase component) in
C. jejuni
81-176. The analyses described here have led us to designate these genes
paqP
and
paqQ
, respectively (pathogenesis-associated glutamine [
q
] ABC transporter permease [
P
] and ATPase [
Q
]). We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Altered resistance to a series of environmental and in vivo stresses was also observed: both mutants were hyperresistant to aerobic and organic peroxide stress, and while the Δ
paqP
mutant was also hyperresistant to heat and osmotic shock, the Δ
paqQ
mutant was more susceptible than the wild type to the latter two stresses. The Δ
paqP
and Δ
paqQ
mutants also displayed a surprising but statistically significant increase in recovery from macrophages and epithelial cells in short-term intracellular survival assays. Annexin V, 4′,6-diamidino-2-phenylindole (DAPI), and Western blot analyses revealed that macrophages infected with the Δ
paqP
or Δ
paqQ
mutant exhibited transient but significant decreases in cell death and extracellular signal-regulated kinase-mitogen-activated protein kinase activation compared to levels in wild-type-infected cells. The Δ
paqP
mutant was not defective in either short-term or longer-term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation of an AA-ABC transporter with bacterial stress tolerances and host cell responses to pathogen infection.
Comprehensive Evaluation of
Streptococcus sanguinis
Cell Wall-Anchored Proteins in Early Infective Endocarditis
Authors: Turner Lauren Senty , Kanamoto Taisei , Unoki Takeshi , Munro Cindy L. , Wu Hui , Kitten Todd
Published: August 2009

Streptococcus sanguinis
is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by
S. sanguinis
surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by
S. sanguinis
and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified—a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (∼2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in
S. sanguinis
infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention.