bluuebunny/crossref_metadata_2025 huggingface dataset:
Fuzzy Logic Based Drilling Force Control in a Network-Based Application
Authors: Haber Rodolfo E. , Haber-Haber Rodolfo , Alique Angel , Jime´nez Agusti´n
Published: March 2009

In order to improve efficiency of high-performance drilling processes while preserving tool life, the current study focuses on the design and implementation of an optimal fuzzy-control system for drilling force. The main topic of this study is the design and implementation of a networked fuzzy controller. The control algorithm is connected to the process through a multipoint interface (MPI) bus, a proprietary programming and communication interface for peer-to-peer networking that resembles the PROFIBUS protocol. The output (i.e., feed-rate) signal is transmitted through the MPI; therefore, network-induced delay is unavoidable. The optimal tuning of the fuzzy controller using a maximum known delay is based on the integral time absolute error (ITAE) criterion. In this study, a step in the force reference signal is considered a disturbance, and the goal is to assess how well the system follows set-point changes using the ITAE criterion. The main advantage of the approach presented herein is the design of an optimal fuzzy controller using a known maximum allowable delay to deal with uncertainties and nonlinearities in the drilling process and delays in the network-based application. In order to suppress the cutting-force increase, the feed rate is decreased gradually as the drilling depth increases, and the cutting force is quite well regulated at the given setpoint. The good transient response is verified by improvements in the integral time absolute error (11.77), integral time square error (2.912) and integral of absolute error (12.81) performance indices. Moreover, the experimental results without oscillations and overshoot corroborate that increases and fluctuations in force drilling can be suppressed despite an increase in drilling depth. Thus, the drilling process can be stabilized and the risk of drill failure can be greatly reduced through a fuzzy-control system.
Damage Behavior and Tribo-Characteristics of DLC-Si Coating for Forming Die
Authors: Tsuchiya Yoshinari , Kubota Hiroyuki , Dohda Kuniaki , Kitamura Kazuhiko
Published: March 2009

Improvement of not only lubricity but also the quality of die materials is desired in the fields where the difficult-to-form materials such as aluminum, stainless steel and high tensile steel sheets are formed. Aluminum is also difficult-to-form material because of its seizure property. Recently DLC (Diamond Like Carbon) which has quite low friction is used and VC coating by TRD (Thermo-reactive Deposition and Diffusion) process is reevaluate in forming high intensity materials. But it is entirely inadequate to adopt the evaluating methods for characteristics of exfoliation resistance. The method used most widely is the scratch test. However, practical limit is not clear to use for judging method of selecting coatings. Therefore the damage behavior accompanies the scratch of various coatings is observed and evaluated in conditions of some kinds of base metal, difference of hardness, the coating thickness and lubricating. Through these observations the DLC-Si coating, which Toyota Central R and D Labs., Inc. developed, is evaluated to have superior adhesion performance. And the ironing type tribo-test and the ball penetration test are conducted. The results of these tests are as follows; 1) DLC-Si coating shows low friction under dry lubricating condition ironed high-tensile strength steel sheets. 2) It shows low forming load in the case of using mineral oil without additives. There are less gallings on tool and work metal.
Monitoring and Diagnosis of Assembly Fixture Faults Using Modified Multivariate Control Charts and Surface Scanning Content
Authors: Rickli Jeremy , Camelio Jaime
Published: March 2009

Recent advances in process monitoring technology have introduced an influx of exceptionally large data sets containing information on manufacturing process health. Recorded data sets are comprised of numerous parameters for which multivariate statistical process control (MSPC) methodologies are required. Current multivariate control charts are ideal for monitoring data sets with a minimal amount of parameters, however, new monitoring devices such as surface scanning cameras increase the number of parameters by two orders of magnitude in some cases. This paper proposes a modified form of the original multivariate Hotelling T2 chart possessing the capability to monitor manufacturing processes containing a large number of parameters and a fault diagnosis procedure incorporating least squares analysis in conjunction with univariate control charts. A case study considering surface scanning of compliant sheet metal components and comparisons to processes utilizing Optical CMM’s is presented as verification of the proposed assembly fixture fault diagnosis methodology and modified Hotelling T2 multivariate control chart.
Concurrent Design of Product Families and Assembly Systems
Authors: Bryan A. , Hu S. J. , Koren Y.
Published: March 2009

In order to gain competitive advantage, manufacturers require cost effective methods for developing a variety of products within short time periods. Product families, reconfigurable assembly systems and concurrent engineering are frequently used to achieve this desired cost effective and rapid supply of product variety. The independent development of methodologies for product family design and assembly system design has led to a sequential approach to the design of product families and assembly systems. However, the designs of product families and assembly systems are interdependent and efficiencies can be gained through their concurrent design. There are no quantitative concurrent engineering techniques that address the problem of the concurrent design of product families and assembly systems. In this paper, a non-linear integer programming formulation for the concurrent design of a product family and assembly system is introduced. The problem is solved with a genetic algorithm. An example is used to demonstrate the advantage of the concurrent approach to product family and assembly system design over the existing sequential methodology.
The Impact of Surface Integrity by Hard Turning vs. Grinding on Rolling Contact Fatigue
Authors: Warren A. W. , Guo Y. B.
Published: March 2009

Hard turning and grinding are finishing processes for the manufacture of precision components such as bearings, gears, and cams. However, the effects of distinct surface integrity by hard turning vs. grinding on rolling contact life are poorly understood. Four representative surface types were prepared: as-turned, as-ground, turned and polished, and ground and polished. Surface integrity was characterized by surface topography, microstructure, and micro/nanohardness. Fatigue tests were performed with an acoustic emission sensor and the signal processing software. The amplitude of acoustic emission signal is the most stable and sensitive signal to fatigue failure. The turned surface may have a longer life (>84%) than the ground one with equivalent surface finish.
Manufacturing System Design Considering Generational Product Evolution and Task Recurrence
Authors: Ko Jeonghan , Hu S. Jack
Published: March 2009

The classical studies on manufacturing system design and line balancing have often focused on a single generation of products, thus leading to new design or re-balancing when a new generation of products is introduced. As the life-cycles of product models become shorter and shorter, this ‘new products then new system-design’ approach is becoming increasingly ineffective due to too frequent production interruption. Therefore, effective solutions to system-design problems should consider products of multiple generations. This paper presents new methods to design manufacturing systems for products evolving over several generations. Mixed integer programming models are developed for (1) designing system configurations that are cost-effective for uncertain product evolution, and (2) maximizing the recurrences of a task on the same machine throughout product generations. A decomposition-based solution procedure is also proposed to reduce computational complexity. These new methods can provide a system design solution enabling quick product launches with less line change-over for new products.
Effect of Micro-Manufacturing Processes on Surface Behavior
Authors: Davis Tiffany , Cao Jian , Chen Wei , Wang Q. Jane , Xia Cedric , Talwar Raj , Lederich Richard
Published: March 2009

Surface texturing has become a valuable technique for reducing friction and wear in contacting parts; laser surface texturing is one such method used to create micro-dimples on the interface surface. This work investigates the surface material property variation caused by laser surface texturing. The hardness and modulus of elasticity of a steel laser surface texture sample were evaluated near the dimples and away from the dimpled zone through nano-indentation. Resulting data shows that no significant difference exists between the material properties from the two positions. An alternate technique for surface texture generation was also explored, involving the use of micro-punches to create surface features in a metal sample. Computational simulations were performed using a second material underneath a thin copper sheet. The second material was present to serve as a support and to allow extensive deformation of the top material. The choice of the support material and ratio of material thicknesses was optimized to minimize pile up. Trials were conducted for three base supporting materials: PTFE, PMMA, and aluminum. Results show that PMMA performed better than the other materials. Positive deflection was minimized when the PMMA thickness was at least fifteen times that of the copper sheet. Physical experiments were completed with a thin copper sheet to verify the results. An array of micro-indentations was also created in a bulk steel sample. In order to assess the effect of dimpling via micro-forming, nano-indentation was performed near and far from the deformed material of the dimples. Similar to the laser textured sample, no significant differences were found between the two locations.
PVA-Based Scaffolds for the Repair of Musculoskeletal Soft Tissue
Authors: Serino L. P. , Cascone M. G. , Lazzeri L. , Torzilli P. A. , Maher S. A.
Published: March 2009

The objective of this study was to design a partly-degradable scaffold to repair cartilage defects. The scaffold, based on poly(vinyl alcohol), PVA, was intended to maintain long-term mechanical integrity and to facilitate cell proliferation via bioactive agent release from contained microparticles, made from either alginate, ALG or poly(lactic-co-glycolic acid), PLGA. The aim of this study was to characterize the morphological features and mechanical behaviour of composite scaffolds as a function of microparticle type and percent content. Our hypothesis was that the dynamic mechanical properties (Dynamic Modulus and Phase Angle) of the composite scaffold would not be affected by microparticle type, but that Dynamic Modulus would increase as a function of increased microparticle content. Scanning Electron Microscopy confirmed that the manufacturing process homogenously dispersed microspheres within the scaffolds. For pure PVA samples Dynamic Modulus ranged from 66±3 kPa at 0.01 Hz to 83±3 kPa at 50 Hz. As ALG microsphere content increased from 25% to 75%, Dynamic Modulus ranged from 92±5 kPa at 0.01 Hz to 153±19 kPa at 50 Hz. As the microsphere content increased from 25% to 75% PLGA, Dynamic Modulus ranged from 85±9 kPa at 0.01 Hz, to 157±16 kPa at 50 Hz. As expected, Dynamic Modulus increased with increasing test frequencies. For pure PVA specimens Phase Angle ranged from 4.3±0.8 degrees at 0.01 Hz to 12±1.2 degrees at 50 Hz. Phase Angle was not affected by microsphere content. In conclusion, the addition of microspheres affected the dynamic mechanical behavior, in particular Dynamic Modulus, of PVA scaffolds. However, the dynamic mechanical properties were not affected by the polymer from which the microspheres were manufactured. These findings suggest that microsphere type can be chosen to optimize the inclusion of bioactive factors, without detrimentally affecting the mechanical properties of the composite scaffold. It also suggests that % content of included microspheres can be used to modulate the mechanical properties of the scaffold at time zero.
Characteristic Evaluation of Silicon-Based MEMS Acoustic/Acceleration Sensor
Authors: Chao Ru-Min , Li Sie-Yu , Hsu Chih-Chao , Liang Steven Y.
Published: March 2009

Due to its smallness in size and economic-saving in batch fabrication, the acoustic micro sensor shows many potential applications in the field of manufacturing and machining monitoring system if it can help to pick up information of sound and vibration signals at specific range. In this paper, brief discussion for the design of the micro-sensor and its fabrication issue will be made. Fundamental frequency testing results by the piezo-electric shaker and acoustic measurement within the water tank are given. A 6-inch silicon wafer fabrication process and sensor design having resonant frequency in the range of 20k–60k Hz are presented and discussed. By selecting appropriate thickness of the structure layer on a SOI wafer, it is possible to customer-make a micro-sensor to sense within a given-range of interest and to apply it in a manufacturing monitoring system.
Estimation of Metal Hardening Models at Large Strains
Authors: Cai Wayne W. , Carsley John E. , Hayden Daniel B. , Hector Louis G. , Stoughton Thomas B.
Published: March 2009

Simulation accuracy of large strain deformation of sheet metals, such as that which occurs during hemming and vehicle crash situations, is limited because existing hardening laws (true stress vs. true strain relationships) are extrapolated from uniform elongation data and applied for post-uniform deformation. In this paper, a reverse-engineering method was developed to predict metal hardening laws at large strains beyond uniform elongation for sheet metals. The method required a standard uniaxial tensile test and finite element analyses (FEA), and was implemented as a custom computer code called GMSS (General Motors Stress-Strain). The true stress vs. true strain data pairs are determined when the load and displacement history of a tensile test specimen matches the FEA results using GMSS. Test cases showed that the true stress vs. true strain relationships at very large strains (75% for AA6111 aluminum, and 85% for DP600 steel) could be automatically generated using GMSS. This reverse-engineering method will provide General Motors with an easy-to-use tool for generating very accurate metal hardening laws for post-uniform deformation that can greatly improve the accuracy of FEA for formability (including hemming), and crashworthiness simulations.
Augmented Reality Enhanced Nanomanipulation by Atomic Force Microscopy With Local Scan
Authors: Li Guangyong , Liu Lianqing , Xi Ning
Published: March 2009

Atomic Force Microscope (AFM) has been used to manipulate nano-objects for more than a decade. However, it is still in the infant stage to serve as a manufacturing tool for fabrication of nanodevices. The major hindrance is the low efficiency due to the absence of visual feedback, positioning errors, and losing objects during manipulation. The lack of visual feedback can be solved by integrating an augmented reality interface into an AFM based nano-robotic system. Through the augmented reality interface, the operator can manipulate the nano-objects and simultaneously observe the real-time changes of the nano-environment. Position errors caused by thermal drift and nonlinearity of piezoactuators often lead the AFM tip to a wrong position and in turn miss the nano-objects. Due to the small touching area between AFM tip and the object, the tip often slips over or slips aside the nano-object during manipulation. All these problems can be solved by introducing a local scan mechanism to the AFM based robotic system. The local scan strategies will improve the reliability of the visual feedback, therefore, significantly improve the efficiency of AFM based nano-manipulation. In this paper, the augmented reality interface is briefly introduced. And then the local scan strategies are proposed to eliminate the positioning errors, relocate the actual position of nano-objects, and find back the nano-objects if they are lost during manipulation. The paper finally demonstrates that single carbon nanotube (CNT) based nano-sensors can be fabricated by the AFM based nano-robotic system assisted by local scan.
E-Collaboration in a Multiple Engineering Projects Environment: A Facilities Development Approach
Authors: Pereda Francisco J. , Penaranda Nicolas , Jimenez Guillermo , Molina Arturo
Published: March 2009

With the development of Information Systems (IS) for project lifecycle management, there is a new competitive scenario where organizations, suppliers (contractors or manufacturers) and clients could collaborate in facility development projects. An e-collaboration environment is a set of integrated information technologies that are required to create an infrastructure for a simultaneous and concurrent multiple projects management in distributed networks. Some companies have failed in the implementation of a complete e-collaboration environment due to the lack of a holistic perspective. In this paper a framework to integrate an e-collaboration environment for multiple projects management is presented. Important issues for a multi-project management as human resources, projects organization, projects activities, technologies and methodologies, are described. Recommendations about the implications for the implementation of an e-collaborative environment are presented, from a current case in an Original Equipment Manufacturing (OEM) company.
Applications of Micro Sensors for Fundamental Study of Manufacturing Processes
Authors: Cheng Xudong , Choi Hongseok , Li Xiaochun
Published: March 2009

Fundamental understanding of many multiscale manufacturing processes can be significantly improved with better sensing and instrumentation techniques. In light of the recent advances in micro senor technologies, this paper first reviews the recent research conducted at University of Wisconsin-Madison in developing methods to integrate micro thin film sensor into harsh environment manufacturing processes. The study demonstrates how thermomechanical data obtained with distributed micro sensors to improve the understanding of laser micromachining and mesoscale ultrasonic metal welding. The paper then presents the current study in an effort to extend the method to more manufacturing process, the feasibility of embedded micro sensors in metallic material using ultrasonic welding and diffusion bonding was investigated.
Study on Mechanical Properties and Microstructure of Magnesium/SiC Nanocomposites Fabricated by Ultrasonic Cavitation Based Solidification Processing
Authors: Cao Guoping , Konishi Hiromi , Li Xiaochun
Published: March 2009

Magnesium, the lightest structural metal, is of significance to improve energy efficiency in various applications. Mg/SiC nanocomposites were successfully fabricated by ultrasonic cavitation based dispersion of SiC nanoparticles in Mg melts. As compared to pure magnesium, the mechanical properties including tensile strength and yield strength of the Mg/SiC nanocomposites were improved significantly, while the good ductility of pure Mg casting was retained. The grain size of the pure magnesium was refined when SiC nanoparticles were added. In the microstructure of Mg/SiC nanocomposites, there are still quite some SiC clusters, but in the areas free of large clusters, the SiC nanoparticles were dispersed very well. TEM study of the interface between SiC nanoparticles and magnesium matrix indicates a good bonding, but no chemical reaction between SiC nanoparticles and magnesium matrix.
Effect of Friction on Ultrasonic Consolidation
Authors: Zhang Chunbo , Li Leijun
Published: March 2009

Friction and plastic flow at the bond interface are believed to be the most fundamental phenomena in ultrasonic consolidation. This paper focuses on friction’s contribution to bonding mechanism in ultrasonic welding. Based on a 3-D thermo-mechanical dynamic FEM model, the distributions of temperature and plastic deformation caused by the friction stress are presented. Temperature and plastic deformation are found to vary with vibration cycles. A saturation phenomenon is identified that after certain number of vibration cycles, the plastic deformation at the bond interface reaches a “steady-state”.
Comparison of Tangential Force Models for Feedrate Selection in Milling
Authors: Yalcin Cuneyt , Fussell Barry K. , Jerard Robert B.
Published: March 2009

Several mechanistic force models are investigated for use in a feedrate selection system that uses average spindle cutting power for model calibration. Strategies for feedrate selection using a spindle power sensor are discussed. Five different flat end mill force models are considered, some with an edge effect component and some with a size effect component. The models are evaluated for average tangential force prediction accuracy and ease of calibration using a variety of experiments with different flat end mills and workpiece materials. A method of showing all the experimental data points and tangential force model predictions on one plot is developed. The models are shown to be much more accurate than a simple volumetric power model in predicting average tangential force. The performances of all the mechanistic force models are similar, with the linear edge effect model and the nonlinear size effect model showing the most potential for feedrate selection.
An Innovative Quasi-Continuous Power Transmission System
Authors: Nasr Asghar , Jafari Jozani Yasser , Zoroufi Mehrdad
Published: March 2009

Conventional stepped power transmission systems exhibit abundant energy dissipation, complicated handling and costly maintenance. On the other hand, continuously-variable power transmissions (CVTs), which are recently considered to be used in the industry, despite their high capabilities, face a number of drawbacks including limited torque transmission capacity, high-precision manufacturing and installation requirements, low cost effectiveness and relatively modest power transmission efficiencies. Therefore, innovative power transmission systems that intend to resolve or lessen one or more of these disadvantages are critical in power transmission from pinion to wheel in electric traction motors of both diesel and electric locomotives; especially when active and advanced control of traction effort and adhesion is of high importance and are going to be welcomed by rail industries. In this research, an innovative quasi-continuous power transmission (QCPT) system is introduced. In this system, a fully-automatic gear box including six pairs of engaging gears is considered where only one pair of gears is engaged in any operating moment. The main components of the QCPT are the input and output shafts each having six engaging gears, speed regulating sensor, electrical module and intelligent pins. The governing parameter in this design is output shaft rotating speed or output torque of the system. When high output torque is needed, the system automatically transfers power to lower gears, and in the need of high output speed, higher gears are assigned. The proposed system is simple and cost-effective, while having high reliability and efficiency.
Increase of Railway Transportation Safety by a New Method of Determination of Wheel Pair and Rail Wear and Damage
Authors: Mgebrishvili Nikoloz , Tatanashvili Marina , Nadiradze Tengiz , Kekelia Ketevan
Published: March 2009

As a result of interaction between the wheel pairs and rails during rolling stock movement, lateral, longitudinal and torsional creep forces are created. Over the long term these forces cause wear and damage of the wheel pairs and rails. Accordingly railway safety decreases and the probability of derailment increases. Development of modern railways largely depends on controlling the proper functionality of wheel pairs and rails. It is impossible to ensure safety and increase the speed of railway traffic without providing such control. Detection of the wheel pair and rail wear damage is one of the key problems of railway science. Many scientific studies have been performed and a number of devices have been developed in this field, but the mentioned problem still remains urgent. In order to increase railway safety, the authors of this paper have proposed a conceptual design of a mobile device for detection of wheel pair and rail wear and damage. Such a device does not currently exist. By installation of the proposed device on each wheel pair of each railcar, the automatic control of wheel pair and rail condition will be achieved. Namely: • Detection of the worn out wheel pair and determination of the degree of wear; • Detection of the damaged wheel pair; • Identification of the worn out or damaged wheel pair. Also possible, on the basis of elaborated mathematical model: • Detection of the worn out rail; • Detection of the damaged rail; • Identification of location of the worn out or damaged rail. Information obtained from the device would be constantly connected to the locomotive computer system. Therefore, for checking rolling stock, there could no longer be a need to build expensive stationary systems or to move trains great distances for their inspection. As a result, the time lost due to equipment stoppage will be saved. Also, with the help of the proposed mobile device, railway safety could improve, while at the same time expenses for detection of wheel pair wear and damage could potentially decrease. This could result in significant economic savings.
Graphene at the Edge: Stability and Dynamics
Authors: Girit Çağlar Ö. , Meyer Jannik C. , Erni Rolf , Rossell Marta D. , Kisielowski C. , Yang Li , Park Cheol-Hwan , Crommie M. F. , Cohen Marvin L. , Louie Steven G. , Zettl A.
Published: March 2009

Although the physics of materials at surfaces and edges has been extensively studied, the movement of individual atoms at an isolated edge has not been directly observed in real time. With a transmission electron aberration–corrected microscope capable of simultaneous atomic spatial resolution and 1-second temporal resolution, we produced movies of the dynamics of carbon atoms at the edge of a hole in a suspended, single atomic layer of graphene. The rearrangement of bonds and beam-induced ejection of carbon atoms are recorded as the hole grows. We investigated the mechanism of edge reconstruction and demonstrated the stability of the “zigzag” edge configuration. This study of an ideal low-dimensional interface, a hole in graphene, exhibits the complex behavior of atoms at a boundary.
Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses
Authors: Hübner Wolfgang , McNerney Gregory P. , Chen Ping , Dale Benjamin M. , Gordon Ronald E. , Chuang Frank Y. S. , Li Xiao-Dong , Asmuth David M. , Huser Thomas , Chen Benjamin K.
Published: March 2009

The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized “buttons” containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.
Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding
Authors: Aller Stephen G. , Yu Jodie , Ward Andrew , Weng Yue , Chittaboina Srinivas , Zhuo Rupeng , Harrell Patina M. , Trinh Yenphuong T. , Zhang Qinghai , Urbatsch Ina L. , Chang Geoffrey
Published: March 2009

P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of ∼6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.
Critical Population Density Triggers Rapid Formation of Vast Oceanic Fish Shoals
Authors: Makris Nicholas C. , Ratilal Purnima , Jagannathan Srinivasan , Gong Zheng , Andrews Mark , Bertsatos Ioannis , Godø Olav Rune , Nero Redwood W. , Jech J. Michael
Published: March 2009

Similarities in the behavior of diverse animal species that form large groups have motivated attempts to establish general principles governing animal group behavior. It has been difficult, however, to make quantitative measurements of the temporal and spatial behavior of extensive animal groups in the wild, such as bird flocks, fish shoals, and locust swarms. By quantifying the formation processes of vast oceanic fish shoals during spawning, we show that (i) a rapid transition from disordered to highly synchronized behavior occurs as population density reaches a critical value; (ii) organized group migration occurs after this transition; and (iii) small sets of leaders significantly influence the actions of much larger groups. Each of these findings confirms general theoretical predictions believed to apply in nature irrespective of animal species.
Tracking the Emerging Human PathogenPseudallescheria boydiiby Using Highly Specific Monoclonal Antibodies
Authors: Thornton Christopher R.
Published: March 2009

Pseudallescheria boydiihas long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis ofP. boydiiis problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such asAspergillus fumigatusandScedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 κ-light chain monoclonal antibodies (MAbs) specific toP. boydiiand certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls ofP. boydiiandScedosporium apiospermum. The MAbs do not react withS. prolificans,Scedosporium dehoogii, or a large number of clinically relevant fungi, includingA. fumigatus,Candida albicans,Cryptococcus neoformans,Fusarium solani, andRhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiateP. boydiifrom other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.
Good Performance of Immunoglobulin M Assays in Diagnosing Genotype 3 Hepatitis E Virus Infections
Authors: Legrand-Abravanel Florence , Thevenet Isabelle , Mansuy Jean-Michel , Saune Karine , Vischi Françoise , Peron Jean-Marie , Kamar Nassim , Rostaing Lionel , Izopet Jacques
Published: March 2009

We have evaluated three anti-hepatitis E virus (anti-HEV) immunoglobulin M (IgM) assays, the EIAgen HEV IgM assay (Adaltis), the HEV IgM enzyme-linked immunosorbent assay 3.0, and the Assure HEV IgM rapid test (MP Diagnostics), for the routine detection of acute genotype 3 HEV. Their sensitivities were fairly good (90%, 88%, and 82%), and their specificities were excellent (100%, 99.5%, and 100%).
Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the
Staphylococcus aureus
Iron Surface Determinant B Antigen
Authors: Raedler Michael D. , Heyne Samantha , Wagner Erica , Shalkowski Sheri K. , Secore Susan , Anderson Annaliesa S. , Cook James , Cope Leslie , McNeely Tessie , Retzlaff Mary , Shanter Jon , Rubinstein Leonard J. , Green Tina , Kartsonis N. , Esser Mark T.
Published: March 2009

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the
Staphylococcus aureus
iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational
Saccharomyces cerevisiae
-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with
S. cerevisiae
-derived IsdB, an
Escherichia coli
-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to μg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 μg/ml with a dynamic range of 2.1 to 10,625 μg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is −4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.
Early Diagnosis of Extrapulmonary Tuberculosis by a New Procedure Combining Broth Culture and PCR
Authors: Noussair Latifa , Bert Frédéric , Leflon-Guibout Véronique , Gayet Nadine , Nicolas-Chanoine Marie-Hélène
Published: March 2009

The diagnosis of extrapulmonary tuberculosis is difficult because of the paucibacillary nature of these infections. We developed a culture-enhanced PCR assay combining a preliminary step of broth culture in BacT/Alert MP bottles with the subsequent detection of
Mycobacterium tuberculosis
using the GenoType Mycobacteria Direct test. First, the procedure was applied to 10-fold-diluted suspensions of
M. tuberculosis
prepared in vitro. These experiments showed that a 15-day incubation time was required to detect bacilli in the suspension, with the lowest inoculum size yielding a single colony on Lowenstein-Jensen slants. The efficacy of culture-enhanced PCR at day 15 was subsequently evaluated with 225 nonrespiratory specimens from 189 patients with suspected tuberculosis. All these specimens were smear negative, and 31 (13.8%) from 27 patients were culture positive. The result of culture-enhanced PCR at day 15 was consistent with final culture results in all specimens tested. Compared to culture results, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%. Four patients with a negative culture and a negative PCR result were diagnosed as having tuberculosis on the basis of histological findings or therapeutic response. When using a positive diagnosis of tuberculosis as a gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value were 88.6%, 100%, 100%, and 97.9%, respectively. These results indicate that culture-enhanced PCR is a highly sensitive and specific method for the early detection of
M. tuberculosis
in extrapulmonary specimens.
Use of Dried Serum Spots for Serological and Molecular Detection of Hepatitis A Virus
Authors: Desbois Delphine , Roque-Afonso Anne-Marie , Lebraud Patricia , Dussaix Elisabeth
Published: March 2009

We assessed the feasibility of using dried serum spots (DSS) for the serological and molecular diagnosis of hepatitis A virus (HAV) infection. Sixty-eight sera spotted onto filter papers (Whatman International Ltd., United Kingdom) were used for detection of total anti-HAV antibodies, and 64 sera were used for detection of immunoglobulin M antibody to HAV. DSS were stored at 4°C, room temperature, and 37°C for 1, 2, and 4 weeks. Sensitivity and specificity of the serological assays were 100% regardless of temperature and storage duration. To assess the stability of HAV RNA, we performed qualitative and quantitative reverse transcription-PCRs (RT-PCRs) with human plasma spiked with serial dilutions of cultured HAV spotted on Flinders Technology Associates filter paper cards (Whatman International Ltd.). Filter papers were stored at room temperature and processed for RT-PCR assays. No reduction of viral load was observed after 5, 15, and 30 days of storage. The ∼10-fold reduction of sensitivity from DSS was attributable to a smaller sample input in DSS samples. This method was further evaluated using 35 frozen sera. HAV RNA amplification showed 100% specificity and 92.3% sensitivity, and sequence analysis from DSS and sera provided identical results. HAV RNA can be accurately recovered from DSS for molecular epidemiology purposes, and we confirm the reliability of blotted samples in the serological diagnosis of HAV infection. The DSS method facilitates storage and shipment of samples from routine laboratories to reference centers for further investigations and large epidemiological studies.
Dried-Plasma Transport Using a Novel Matrix and Collection System for Human Immunodeficiency Virus and Hepatitis C Virus Virologic Testing
Authors: Lloyd R. M. , Burns D. A. , Huong J. T. , Mathis R. L. , Winters M. A. , Tanner M. , De La Rosa A. , Yen-Lieberman B. , Armstrong W. , Taege A. , McClernon D. R. , Wetshtein J. L. , Friedrich Brian M. , Ferguson Monique R. , O'Brien William , Feorino P. M. , Holodniy M.
Published: March 2009

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log10copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log10copies/ml, depending on assay type (P< 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.
Molecular Identification of T4 and T5 Genotypes in Isolates from
Acanthamoeba
Keratitis Patients
Authors: Ledee D. R. , Iovieno A. , Miller D. , Mandal N. , Diaz M. , Fell J. , Fini M. E. , Alfonso E. C.
Published: March 2009

Acanthamoeba
keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the
Rns
genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.
Surveillance of Antibiotic Susceptibility Patterns among
Shigella sonnei
Strains Isolated in Belgium during the 18-Year Period 1990 to 2007
Authors: Vrints M. , Mairiaux E. , Van Meervenne E. , Collard J.-M. , Bertrand S.
Published: March 2009

This study was conducted to determine the frequency and pattern of antimicrobial susceptibility of
Shigella sonnei
, the predominant species causing shigellosis in Belgium. Between 1990 and 2007, a total of 7,307 strains, mainly (98.2%) isolated from stools, were diagnosed by peripheral laboratories before being confirmed as
Shigella
strains by serotyping by the National Reference Center of
Salmonella
and
Shigella
. A significant increase in resistances to tetracycline, streptomycin, trimethoprim, sulfonamides, and cotrimoxazole (i.e., trimethoprim in combination with sulfonamides) was observed during this period. Since 1998, resistance to nalidixic acid also increased to reach a peak (12.8%) of resistant isolates in 2004. Concomitantly, multidrug resistance (MDR) in this species emerged in 2007, with 82% of total isolates being MDR. However, during this 18-year period, all isolates remained fully susceptible to ciprofloxacin and gentamicin. The work includes the molecular characterization of mechanisms of resistance to ampicillin, tetracycline, chloramphenicol, and cotrimoxazole and class 1 and class 2 integrons.
S. sonnei
acquired antimicrobial resistance to traditional antibiotics (ampicillin and tetracycline) by horizontal gene transfer, while the genetic stability of transposons was responsible for a high (89%) proportion of resistance to a commonly prescribed antibiotic (cotrimoxazole). Therefore, cotrimoxazole should no longer be considered appropriate as empirical therapy for treatment of shigellosis in Belgium when antibiotics are indicated. Rates of resistance to nalidixic acid should also be attentively monitored to detect any shift in fluoroquinolone resistance, because it represents the first line among antibiotics used in the treatment of shigellosis.
Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication
Authors: Nichols Gena J. , Schaack Jerome , Ornelles David A.
Published: March 2009

Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.
Effect of Ribavirin on the Mutation Rate and Spectrum of Hepatitis C Virus In Vivo
Authors: Cuevas José M. , González-Candelas Fernando , Moya Andrés , Sanjuán Rafael
Published: March 2009

Their extremely error-prone replication makes RNA viruses targets for lethal mutagenesis. In the case of hepatitis C virus (HCV), the standard treatment includes ribavirin, a base analog with an in vitro mutagenic effect, but the in vivo mode of action of ribavirin remains poorly understood. Here, we test the mutagenic effects of ribavirin plus interferon treatment in vivo using a new method to estimate mutation rates based on the analysis of nonsense mutations. We apply this methodology to a large HCV sequence database containing over 15,000 reverse transcription-PCR molecular clone sequences from 74 patients infected with HCV. We obtained an estimate of the spontaneous mutation rate of ca. 10−4substitutions per site or lower, a value within the typically accepted range for RNA viruses. A roughly threefold increase in mutation rate and a significant shift in mutation spectrum were observed in samples from patients undergoing 6 months of interferon plus ribavirin treatment. This result is consistent with the known in vitro mutagenic effect of ribavirin and suggests that the antiviral effect of ribavirin plus interferon treatment is at least partly exerted through lethal mutagenesis.