bluuebunny/crossref_metadata_2025 huggingface dataset:
Burr-Free Microblanking Using Piezoelectric Actuator
Authors: Aoki Isamu , Sasada Masahiro
Published: February 2005

Burr-free blanking requirements are strong; however, the thinner the product, the more difficult the burr-free blanking will be, due mainly to the control of the punch penetration depth. In this study, the principle of accompanying burr-free blanking problems, and the driving mechanism of methods for realizing burr-free blanking of foil were realized. The conditions for realizing successful burr-free blanking are the clearance of step 1 and punch penetration depth. Use of the piezoelectric actuator element and the design method for this was proposed as the punch drive mechanism. Good results were obtained by using this press machine for burr-free blanking of 50 μm copper foil.
The Mechanics of Machining at the Microscale: Assessment of the Current State of the Science
Authors: Liu X. , DeVor R. E. , Kapoor S. G. , Ehmann K. F.
Published: February 2005

This paper provides a comprehensive review of the literature, mostly of the last 10–15 years, that is enhancing our understanding of the mechanics of the rapidly growing field of micromachining. The paper focuses on the mechanics of the process, discussing both experimental and modeling studies, and includes some work that, while not directly focused on micromachining, provides important insights to the field. Experimental work includes the size effect and minimum chip thickness effect, elastic-plastic deformation, and microstructure effects in micromachining. Modeling studies include molecular dynamics methods, finite element methods, mechanistic modeling work, and the emerging field of multiscale modeling. Some comments on future needs and directions are also offered.
The Drilling Vibration Behavior of a Twisted Microdrill
Authors: Huang Bo-Wun
Published: February 2005

The dynamic characteristics of a microdrill in the drilling process were investigated in this study. The trend toward higher density printed circuit boards requires smaller holes, down to 0.3 mm in diameter, to be drilled through the board layers. To improve quality, produce a higher production rate, and avoid drill breakage, the dynamic characteristics of the microdrilling process must be studied. A stepped pretwisted beam is used to simulate the microdrill. A moving Winkler-Type elastic foundation is used to approximate the drilling process. A time-dependent vibration model for drilling is presented. The rotating speed, pretwisted angle, and thrust force effects of the microdrill are considered. The numerical analysis indicates that the natural frequency is reduced suddenly as the microdrill moves into a workpiece.
The Influence of Material Models on Finite Element Simulation of Machining
Authors: Shi Jing , Liu C. Richard
Published: February 2005

In literature, four models incorporating strain rate and temperature effects are able to generalize material test results of HY-100 steel. This study compares the four models, namely, Litonski-Batra, power law, Johnson-Cook, and Bodner-Partom, in finite element modeling of orthogonal machining of this material. Consistency is found in cutting forces, as well as in stress and temperature patterns in all but the Litonski-Batra model. However, the predicted chip curls are inconsistent among the four models. Furthermore, the predicted residual stresses are substantially sensitive to the selection of material models. The magnitudes, and even the sign of the residual stresses in machined surfaces, vary with different models.
A Static Model of Chip Formation in Microscale Milling
Authors: Kim Chang-Ju , Mayor J. Rhett , Ni Jun
Published: February 2005

This paper discusses the development of an enhanced, static model of chip formation in micromilling processes that is able to describe the intermittency of the chip formation observed at low feeds per tooth due to the dominance of the minimum chip thickness effect. Experimental analyses demonstrate the validity of the proposed model by verifying the level of periodicity in the cutting forces present at various feeds per tooth. A key finding of this study is the identification of a local maximum in the radial thrust forces in the micromilling process during the noncutting regime, at feeds per tooth that are of the order of the minimum chip thickness. To overcome the challenges in the direct measurement of the minimum chip thickness, this paper presents a method for estimating the minimum chip thickness of various combinations of tools and workpiece materials based on easily attainable cutting-force data. A discussion on the selection of process parameters to avoid intermittent chip formation is also presented.
A Simulation-based Correlation of Cross-Sectional Area of the Thin Film Produced by Laser Chemical Vapor Deposition With a Moving Laser Beam
Authors: Zhang Yuwen
Published: February 2005

A parametric study on shape and cross-sectional area of the thin film produced by Laser Chemical Vapor Deposition (LCVD) with a moving laser beam is presented. The problem is formulated in the coordinate system that moves with the laser beam, and, therefore, the problem is a quasi-steady state. The effects of laser scanning velocity, laser power, and radius of the laser beam on the shapes of the deposited film are investigated. A simulation-based correlation of the cross-sectional area is proposed based on the simulation results.
Scaling Issues in Miniaturization of Injection Molded Parts
Authors: Yao Donggang , Kim Byung
Published: February 2005

Miniaturization of injection molded parts causes changes in the relative contribution of relevant design and process parameters. As a result, scaling-related size effects occur. Size effects can be either of the first order or of the second order. First-order size effects can be predicted using standard modeling, while second order ones cannot. This paper deals with first-order size effects encountered in injection molding miniaturized parts. Through the scaling analysis of the heat transfer and flow process in injection molding, the size effects on the change of molding characteristics including viscous heating, freezing time, capillary force effect, and moldability were identified. Strategies were consequently proposed to alleviate or eliminate the scaling-related molding difficulties in molding ultrathin-wall parts and microparts. Particularly, a scalable filling process was presented, with experimental verification.
Mechanistic Cutting Process Calibration via Microstructure-Level Finite Element Simulation Model
Authors: Park Sunghyuk , Kapoor S. G. , DeVor R. E.
Published: February 2005

A methodology for mechanistic cutting force model calibration via microstructure-level finite element cutting process simulation is presented and applied to ferrous materials, including ductile and gray irons and carbon steels. The methodology combines graphite, ferrite, and pearlite grains to produce ductile iron, gray iron, and carbon steel microstructures and obtains cutting forces via orthogonal machining simulations. The simulated forces are used to perform the cutting force model calibration. The cutting forces for the turning process are predicted based on the calibration thusly obtained and experimentally shown to be in good agreement with actual cutting force data.
Novel Sensor Fabrication Using Direct-Write Thermal Spray and Precision Laser Micromachining
Authors: Chen Q. , Tong T. , Longtin J. P. , Tankiewicz S. , Sampath S. , Gambino R. J.
Published: February 2005

This work presents a direct-write technique for rapid prototyping and manufacturing of sensors onto structural and functional components for many diverse applications. The technique combines thermal spray (an additive process), which produces blanket depositions of films and coatings, with ultrafast laser micromachining (a subtractive process) to produce functional sensors. Microheaters and strain gauges have been successfully fabricated in this work to demonstrate the feasibility and advantages of the proposed technique. Electrical and thermal characterization of the sensors is also performed. With minor modifications to the pattern design and processing procedures, additional sensing structures and electronic components, for example, precision resistors and interdigitated capacitors, can be fabricated using the presented technique.
Study of 3D Micro-Ultrasonic Machining
Authors: Yu Z. Y. , Rajurkar K. P. , Tandon A.
Published: February 2005

Many manufacturing processes, such as lithography, etching, laser, electrical discharge machining (EDM), and electrochemical machining (ECM), are being applied to produce the meso- and microscale parts and products. Materials such as silicon, glass, quartz crystal, and ceramics are being increasingly used in microelectromechanical system (MEMS) devices. Ultrasonic machining (USM) offers an attractive alternative to machine some of the hard and brittle materials. However, the tool wear in micro-ultrasonic machining adversely affects the machining accuracy. Therefore, it is necessary to account for and to compensate the tool wear during machining. This paper reports the feasibility of applying the uniform wear method developed for micro electrical discharge machining and its integration with CAD/CAM to microultrasonic vibration process for generating accurate three-dimensional (3D) microcavities. Experimental results show that the tool shape remains unchanged and the tool wear has been compensated.
Microfactory—Concept, History, and Developments
Authors: Okazaki, Yuichi , Mishima, and Nozomu , Ashida Kiwamu
Published: February 2005

This paper reviews the new concept “microfactory” and related developments. Machined parts are becoming progressively smaller, so production machinery that remains a conventional size is often inappropriate for such products. The term “microfactory” represents an entirely new approach to design and manufacture that minimizes production systems to match the size of the parts they produce. It leads to conservation of space and energy, and the reduction of investment and operational costs, as well as the reduction of emissions and the load on operators. Furthermore, it provides a system with dynamic reconfigurability, aiming at a light and agile manufacturing system optimized for current manufacturing needs in a borderless and highly competitive market. In Japan, research institutes, research consortia, and the private sector have carried out targeted research and development aimed at this concept for over a decade. Some systems are past the research stage and in daily use. Outside Japan, the philosophy and advantages of microfactory have reached an appreciative audience in the U.S., Europe, and Asia. Microfactory is at the cutting edge of competitive manufacturing in the 21st century, ushering in a multidimensional paradigm shift. Here we also briefly examine some future tasks.
A Mechanical Model for Collagen Fibril Load Sharing in Peripheral Nerve of Diabetic and Nondiabetic Rats
Authors: Layton B. E. , Sastry A. M.
Published: February 2005

Peripheral neuropathy affects approximately 50% of the 15 million Americans with diabetes. It has been suggested that mechanical effects related to collagen glycation are related to the permanence of neuropathy. In the present paper, we develop a model for load transfer in a whole nerve, using a simple pressure vessel approximation, in order to assess the significant of stiffening of the collagenous nerve sheath on endoneurial fluid pressure. We also develop a fibril-scale mechanics model for the nerve, to model the straightening of wavy fibrils, producing the toe region observed in nerve tissue, and also to interrogate the effects of interfibrillar crosslinks on the overall properties of the tissue. Such collagen crosslinking has been implicated in complications in diabetic tissues. Our fibril-scale model uses a two-parameter Weibull model for fibril strength, in combination with statistical parameters describing fibril modulus, angle, wave-amplitude, and volume fraction to capture both toe region and failure region behavior of whole rat sciatic nerve. The extrema of equal and local load-sharing assumptions are used to map potential differences in diabetic and nondiabetic tissues. This work may ultimately be useful in differentiating between the responses of normal and heavily crosslinked tissue.
Freezing by a Flat, Circular Surface Cryoprobe of a Tissue Phantom With an Embedded Cylindrical Heat Source Simulating a Blood Vessel
Authors: Massalha Loay , Shitzer Avraham
Published: February 2005

The effects of a thermally-significant blood vessel, simulated by an embedded acrylic tube, 4.8 mm outer diameter, on the freezing field caused by a surface cryoprobe were studied experimentally in a tissue phantom. The flat, 15 mm diameter, circular cryoprobe was operated at a constant cooling rate of −8°C/min by liquid nitrogen down to −160°C. Water flow rates of 30 and 100 ml/min, at a constant temperature of 32.5°C, were maintained in the embedded tube. The latter flow rate is typical to the lower range of blood flows in large arteries in the human body. The phase changing medium (PCM) used was a 30/70% by volume mashed potatoes flakes–water solution. Temperature measurements inside the PCM were performed in one plane perpendicular to the embedded tube, relative to which the cryoprobe was placed at 5 locations in separate experiments. This novel experimental method reduced the perturbation caused by the thermocouple junctions while facilitating rather detailed measurements of the temperature fields developing in the PCM. Results show the development of two hump-like formations on either side of the embedded tube. Freezing was retarded in the region away from the surface cryoprobe and under the tube. This accentuated the dominance of the axial effects, due to the embedded tube, over the radial ones due to the cryoprobe. Results of this study should be considered in designing protocols of cryosurgical procedures performed in the vicinity of thermally-significant blood vessels.
Cartilage Interstitial Fluid Load Support in Unconfined Compression Following Enzymatic Digestion
Authors: Basalo Ines M. , Mauck Robert L. , Kelly Terri-Ann N. , Nicoll Steven B. , Chen Faye H. , Hung Clark T. , Ateshian Gerard A.
Published: February 2005

Interstitial fluid pressurization plays an important role in cartilage biomechanics and is believed to be a primary mechanism of load support in synovial joints. The objective of this study was to investigate the effects of enzymatic degradation on the interstitial fluid load support mechanism of articular cartilage in unconfined compression. Thirty-seven immature bovine cartilage plugs were tested in unconfined compression before and after enzymatic digestion. The peak fluid load support decreased significantly p<0.0001 from 84±10% to 53±19% and from 80±10% to 46±21% after 18-hours digestion with 1.0 u/mg-wet-weight and 0.7 u/mg-wet-weight of collagenase, respectively. Treatment with 0.1 u/ml of chondroitinase ABC for 24 hours also significantly reduced the peak fluid load support from 83±12% to 48±16%p<0.0001. The drop in interstitial fluid load support following enzymatic treatment is believed to result from a decrease in the ratio of tensile to compressive moduli of the solid matrix.
Local, Three-Dimensional Strain Measurements Within Largely Deformed Extracellular Matrix Constructs
Authors: Roeder Blayne A. , Kokini Klod , Robinson J. Paul , Voytik-Harbin Sherry L.
Published: February 2005

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However, the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. “Self-assembled” collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both 1D and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.
Effect of Surrounding Tissue on Vessel Fluid and Solid Mechanics
Authors: Zhang Wei , Herrera Carly , Atluri Satya N. , Kassab Ghassan S.
Published: February 2005

There is no doubt that atherosclerosis is one of the most important health problems in the Western Societies. It is well accepted that atherosclerosis is associated with abnormal stress and strain conditions. A compelling observation is that the epicardial arteries develop atherosclerosis while the intramural arteries do not. Atherosclerotic changes involving the epicardial portion of the coronary artery stop where the artery penetrates the myocardium. The objective of the present study is to understand the fluid and solid mechanical differences between the two types of vessels. A finite element analysis was employed to investigate the effect of external tissue contraction on the characteristics of pulsatile blood flow and the vessel wall stress distribution. The sequential coupling of fluid-solid interaction (FSI) revealed that the changes of flow velocity and wall shear stress, in response to cyclical external loading, appear less important than the circumferential stress and strain reduction in the vessel wall under the proposed boundary conditions. These results have important implications since high stresses and strains can induce growth, remodeling, and atherosclerosis; and hence we speculate that a reduction of stress and strain may be atheroprotective. The importance of FSI in deformable vessels with pulsatile flow is discussed and the fluid and solid mechanics differences between epicardial and intramural vessels are highlighted.
A Multiaxial Computer-Controlled Organ Culture and Biomechanical Device for Mouse Carotid Arteries
Authors: Gleason R. L. , Gray S. P. , Wilson E. , Humphrey J. D.
Published: February 2005

Much of our understanding of vascular mechanotransduction has come from studies using either cell culture or in vivo animal models, but the recent success of organ culture systems offers an exciting alternative. In studying cell-mediated vascular adaptations to altered loading, organ culture allows one to impose well-controlled mechanical loads and to perform multiaxial mechanical tests on the same vessel throughout the culture period, and thereby to observe cell-mediated vascular adaptations independent of neural and hormonal effects. Here, we present a computer-controlled perfused organ culture and biomechanical testing device designed for small caliber (50–5000 micron) blood vessels. This device can control precisely the pulsatile pressure, luminal flow, and axial load (or stretch) and perform intermittent biaxial (pressure–diameter and axial load–length) and functional tests to quantify adaptations in mechanical behavior and cellular function, respectively. Device capabilities are demonstrated by culturing mouse carotid arteries for 4 days.
Direct Numerical Simulations of Micro-Bubble Expansion in Gas Embolotherapy
Authors: Ye Tao , Bull Joseph L.
Published: February 2005

We are currently developing a novel gas embolotherapy technique that involves the selective, acoustic vaporization of liquid perfluorocarbon droplets in or near a tumor as a possible treatment for cancer. The resulting bubbles can then stick within the tumor vasculature to occlude blood flow and “starve” the tumor. The potential development of high stresses during droplet vaporization is a major concern for safe implementation of this technique. No prior study, either experimentally or theoretically, addresses this important issue. In this work, the acoustic vaporization procedure of the therapy is investigated by direct numerical simulations. The nonlinear, multiphase, computational model is comprised of an ideal gas bubble surrounded by liquid inside a long tube. Convective and unsteady inertia, viscosity, and surface tension affect the bubble dynamics and are included in this model, which is solved by a novel fixed-grid, sharp-interface, moving boundary method. We assess the potential for flow-induced wall stresses to rupture the vessel or damage the endothelium during vaporization under a range of operating conditions by varying dimensionless parameters—Reynolds, Weber, and Strouhal numbers, inertial energy and initial droplet size. It is found that the wall pressure is typically highest at the start of the bubble expansion, but the maximum wall shear stress occurs at a later time. Smaller initial bubble diameters, relative to the vessel diameter, result in lower wall stresses.
A Quasi-Linear, Viscoelastic, Structural Model of the Plantar Soft Tissue With Frequency-Sensitive Damping Properties
Authors: Ledoux William R. , Meaney David F. , Hillstrom Howard J.
Published: February 2005

Little is known about the structural properties of plantar soft-tissue areas other than the heel; nor is it known whether the structural properties vary depending on location. Furthermore, although the quasi-linear viscoelastic (QLV) theory has been used to model many soft-tissue types, it has not been employed to model the plantar soft tissue. The structural properties of the plantar soft tissue were quantified via stress relaxation experiments at seven regions (subcalcaneal, five submetatarsal, and subhallucal) across eight cadaveric feet. The cadaveric feet were 36.9±17.4 (mean±S.D.) years of age, all free from vascular diseases and orthopedics disorders. All tests were performed at a constant environmental temperature of 35°C. Stress relaxation experiments were performed; different loads were employed for different areas based on normative gait data. A modification of the relaxation spectrum employed within the QLV theory allowed for the inclusion of frequency-sensitive relaxation properties in addition to nonlinear elastic behavior. The tissue demonstrated frequency-dependent damping properties that made the QLV theory ill suited to model the relaxation. There was a significant difference between the elastic structural properties (A) of the subcalcaneal tissue and all other areas p=0.004, and a trend p=0.067 for the fifth submetatarsal to have less viscous damping c1 than the subhallucal, or first, second, or third submetatarsal areas. Thus, the data demonstrate that the structural properties of the foot can vary across regions, but careful consideration must be given to the applied loads and the manner in which the loads were applied.
Stabilizing PID Controllers for a Single-Link Biomechanical Model with Position, Velocity, and Force Feedback
Authors: Iqbal Kamran , Roy Anindo
Published: February 2005

In this paper we address the problem of PID stabilization of a single-link inverted pendulum-based biomechanical model with force feedback, two levels of position and velocity feedback, and with delays in all the feedback loops. The novelty of the proposed model lies in its physiological relevance, whereby both small and medium latency sensory feedbacks from muscle spindle (MS), and force feedback from Golgi tendon organ (GTO) are included in the formulation. The biomechanical model also includes active and passive viscoelastic feedback from Hill-type muscle model and a second-order low-pass function for muscle activation. The central nervous system (CNS) regulation of postural movement is represented by a proportional-integral-derivative (PID) controller. Pade´ approximation of delay terms is employed to arrive at an overall rational transfer function of the biomechanical model. The Hermite–Biehler theorem is then used to derive stability results, leading to the existence of stabilizing PID controllers. An algorithm for selection of stabilizing feedback gains is developed using the linear matrix inequality (LMI) approach.
An Algorithm for Estimating Acceleration Magnitude and Impact Location Using Multiple Nonorthogonal Single-Axis Accelerometers
Authors: Crisco Joseph J. , Chu Jeffrey J. , Greenwald Richard M.
Published: February 2005

Accelerations of the head are the likely cause of concussion injury, but identifying the specific etiology of concussion has been difficult due to the lack of a valid animal or computer model. Contact sports, in which concussions are a rising health care concern, offer a unique research laboratory environment. However, measuring head acceleration in the field has many challenges including the need for large population sampling because of the relatively low incidence of concussions. We report a novel approach for calculating linear acceleration that can be incorporated into a head-mounted system for on-field use during contact sports. The advantages of this approach include the use of single-axis linear accelerometers, which reduce costs, and a nonorthogonal arrangement of the accelerometers, which simplifies the design criteria for a head-mounted and helmet compatible system. The purpose of this study was to describe the algorithm and evaluate its accuracy for measuring linear acceleration magnitude and impact location using computer simulation and experimental tests with various accelerometer configurations. A 10% error in magnitude and a 10 deg error in impact location were achieved using as few as six single-axis accelerometers mounted on a hemispherical headform.
Analysis of Nonlinear Responses of Adherent Epithelial Cells Probed by Magnetic Bead Twisting: A Finite Element Model Based on a Homogenization Approach
Authors: Ohayon Jacques , Tracqui Philippe , Fodil Redouane , Fe´re´ol Sophie , Laurent Vale´rie M. , Planus Emmanuelle , Isabey Daniel
Published: February 2005

An original homogenization method was used to analyze the nonlinear elastic properties of epithelial cells probed by magnetic twisting cytometry. In this approach, the apparent rigidity of a cell with nonlinear mechanical properties is deduced from the mechanical response of the entire population of adherent cells. The proposed hyperelastic cell model successfully accounts for the variability in probe-cell geometrical features, and the influence of the cell–substrate adhesion. Spatially distributed local secant elastic moduli had amplitudes ranging from 10 to 400 Pa. The nonlinear elastic behavior of cells may contribute to the wide differences in published results regarding cell elasticity moduli.
Insect Sex-Pheromone Signals Mediated by Specific Combinations of Olfactory Receptors
Authors: Nakagawa Takao , Sakurai Takeshi , Nishioka Takaaki , Touhara Kazushige
Published: February 2005

We describe two male-specific olfactory receptors (ORs) in the silk moth,
Bombyx mori
, that are mutually exclusively expressed in a pair of adjacent pheromone-sensitive neurons of male antennae: One is specifically tuned to bombykol, the sex pheromone, and the other to bombykal, its oxidized form. Both pheromone ORs are coexpressed with an OR from the highly conserved insect OR subfamily. This coexpression promotes the functional expression of pheromone receptors and confers ligand-stimulated nonselective cation channel activity. The same effects were also observed for general ORs. Both odorant and pheromone signaling pathways are mediated by means of a common mechanism in insects.
RNA Polymerase IV Directs Silencing of Endogenous DNA
Authors: Herr A. J. , Jensen M. B. , Dalmay T. , Baulcombe D. C.
Published: February 2005

Plants encode subunits for a fourth RNA polymerase (Pol IV) in addition to the well-known DNA-dependent RNA polymerases I, II, and III. By mutation of the two largest subunits (NRPD1a and NRPD2), we show that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3. The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.
Laser-Initiated Shuttling of a Water Molecule Between H-Bonding Sites
Authors: Clarkson Jasper R. , Baquero Esteban , Shubert V. Alvin , Myshakin Evgeniy M. , Jordan Kenneth D. , Zwier Timothy S.
Published: February 2005

The two-step laser excitation scheme of stimulated emission pumping (SEP) induces shifts of a single water molecule between two remote hydrogen bonding sites on
trans
-formanilide. This reaction can be initiated by selective excitation of either isomer (C

O-bound or NH-bound) with different SEP excitation wavelengths. Energy (
E
) thresholds for isomerization in both directions have been measured [796 wave numbers ≤
E
(C

O→NH) ≤ 988 wave numbers and 750 wave numbers ≤
E
(NH→C

O) ≤ 988 wave numbers], and the energy difference D
E
between the C

O-bound and NH-bound isomers was extracted (–238 wave numbers ≤ D
E
≤ +192 wave numbers).
Chaperone Activity of Protein
O
-Fucosyltransferase 1 Promotes Notch Receptor Folding
Authors: Okajima Tetsuya , Xu Aiguo , Lei Liang , Irvine Kenneth D.
Published: February 2005

Notch proteins are receptors for a conserved signaling pathway that affects numerous cell fate decisions. We found that in
Drosophila
, Protein
O
-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor–like domains of Notch, also has a distinct Notch chaperone activity. OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. Thus, a glycosyltransferase can bind its substrate in the endoplasmic reticulum to facilitate normal folding.
Pyogenic Liver Abscess Due to
Rhodococcus equi
in an Immunocompetent Host
Authors: Napoleão Fátima , Vieira Damasco Paulo , Ferreira Camello Thereza Cristina , Damasceno do Vale Márcio , Braga de Andrade Arnaldo Feitosa , Hirata Raphael , de Mattos-Guaraldi Ana Luíza
Published: February 2005

A case of pyogenic liver abscess (PLA) due to
Rhodococcus equi
in an immunocompetent individual was successfully treated by combining surgery and antibiotics. The
R. equi
-targeted antimicrobial agents erythromycin and rifampin were used only after surgical resection of the lesion and identification of the infective organism.
Multiple Genetically Distinct Groups Revealed among Clinical Isolates Identified as Atypical
Aspergillus fumigatus
Authors: Katz Margaret E. , Dougall Annette M. , Weeks Kerry , Cheetham Brian F.
Published: February 2005

To investigate whether genetic variants of
A. fumigatus
are found among clinical isolates, four isolates that were originally identified as poorly sporulating strains of
Aspergillus fumigatus
were subjected to molecular analysis. DNA sequence analysis of the alkaline protease genes of these isolates showed that each is genetically distinct and each shows substantial variation (7 to 11%) from the
A. fumigatus
nucleotide sequence. Subsequent morphological examination suggested that all of the isolates could be classified as
Aspergillus viridinutans
. To clarify the taxonomic status of these four clinical isolates and of two previously identified as atypical
A. fumigatus
isolates, partial β-tubulin and 18S rRNA gene sequences were determined. Each of the six atypical strains had a unique β-tubulin sequence, whereas the sequences of three standard isolates of
A. fumigatus
, which were included as controls, were identical to the published
A. fumigatus
β-tubulin sequence. The very low level of DNA sequence variation detected in standard isolates of
A. fumigatus
compared with other isolates from members of
Aspergillus
section
Fumigati
suggests that it may be a relatively recently evolved species. The 18S rRNA gene of two of the atypical isolates differed from that of
A. fumigatus
at a single nucleotide position. Phylogenetic analyses do not support the classification of all of these isolates as
A. viridinutans
. Thus, some of these isolates represent new species which are potential opportunistic pathogens.
The Pneumoplex Assays, a Multiplex PCR-Enzyme Hybridization Assay That Allows Simultaneous Detection of Five Organisms,
Mycoplasma pneumoniae
,
Chlamydia
(
Chlamydophila
)
pneumoniae
,
Legionella pneumophila
,
Legionella micdadei
, and
Bordetella pertussis
, and Its Real-Time Counterpart
Authors: Khanna M. , Fan J. , Pehler-Harrington K. , Waters C. , Douglass P. , Stallock J. , Kehl S. , Henrickson K. J.
Published: February 2005

Respiratory disease caused by atypical bacteria remains an important cause of morbidity and mortality for adults and children, despite the widespread use of effective antimicrobials agents. Culture remains the “gold standard” for the detection of these agents. However, culture is labor-intensive, takes several days to weeks for growth, and can be very insensitive for the detection of some of these organisms. Newer singleplex PCR diagnostic tests are sensitive and specific, but multiple assays would be needed to detect all of the common pathogens. Therefore, we developed the Pneumoplex assays, a multiplex PCR-enzyme hybridization assay (the standard assay) and a multiplex real-time assay to detect the most common atypical pathogens in a single test. Primer and probe sequences were designed from conserved regions of specific genes for each of these organisms. The limits of detection were as follows: for
Bordetella pertussis
, 2 CFU/ml; for
Legionella pneumophila
(serotypes 1 to 15) and
Legionella micdadei
, 9 and 80 CFU/ml, respectively; for
Mycoplasma pneumoniae
, 5 CFU/ml; and for
Chlamydia
(
Chlamydophila
)
pneumoniae
, 0.01 50% tissue culture infective doses. Recombinant DNA controls for each of these organisms were constructed, and the number of copies for each DNA control was calculated. The Pneumoplex could detect each DNA control down to 10 copies/ml. The analytical specificity demonstrated no cross-reactivity between 23 common respiratory pathogens. One hundred twenty-five clinical bronchoalveolar lavage fluid samples tested by the standard assay demonstrated that the Pneumoplex yielded a sensitivity and a specificity of 100 and 98.5%, respectively. This test has the potential to assist clinicians in establishing a specific etiologic diagnosis before initiating therapy, to decrease hospital costs, and to prevent inappropriate antimicrobial therapy.
Impact of Patient Characteristics on Performance of Nucleic Acid Amplification Tests and DNA Probe for Detection of
Chlamydia trachomatis
in Women with Genital Infections
Authors: Marrazzo Jeanne M. , Johnson Robert E. , Green Timothy A. , Stamm Walter E. , Schachter Julius , Bolan Gail , Hook Edward W. , Jones Robert B. , Martin David H. , St. Louis Michael E. , Black Carolyn M.
Published: February 2005

The performance of nucleic acid amplified tests (NAAT) for
Chlamydia trachomatis
at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical
C. trachomatis
over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women ≥20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of
C. trachomatis
in diverse clinical populations of women.
Description of
Campylobacter curvus
and
C. curvus
-Like Strains Associated with Sporadic Episodes of Bloody Gastroenteritis and Brainerd's Diarrhea
Authors: Abbott Sharon L. , Waddington Michael , Lindquist David , Ware Jim , Cheung Wendy , Ely Janet , Janda J. Michael
Published: February 2005

Campylobacter curvus
is a rarely encountered
Campylobacter
species in human, animal, and environmental samples. During the course of two investigations, one involving a search for possible bacterial agents causing bloody gastroenteritis and a second concerning a small outbreak of Brainerd's diarrhea in northern California, 20 strains of
C. curvus
or
C. curvus
-like organisms were isolated by a microfiltration technique and prolonged incubation. The results suggest that
C. curvus
may be an underappreciated
Campylobacter
that may be involved in sporadic and outbreak cases of bloody or chronic diarrhea in humans.
Detection and Identification of
Enterocytozoon bieneusi
and
Encephalitozoon
Species in Stool and Urine Specimens by PCR and Differential Hybridization
Authors: Notermans Daan W. , Peek Ron , de Jong Menno D. , Wentink-Bonnema Ellen M. , Boom René , van Gool Tom
Published: February 2005

Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals.
Enterocytozoon bieneusi
and
Encephalitozoon intestinalis
are most commonly associated with chronic diarrhea. All
Encephalitozoon
species, including
E. intestinalis
,
E. hellem
, and
E. cuniculi
, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and
Vittaforma corneae
as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three
Encephalitozoon
species and
V. corneae
. An analytical sensitivity of 3.5 × 10
2
to 3.5 × 10
3
spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different
Encephalitozoon
species.
Production of Herpes B Virus Recombinant Glycoproteins and Evaluation of Their Diagnostic Potential
Authors: Perelygina Ludmila , Patrusheva Irina , Hombaiah Soumya , Zurkuhlen Holley , Wildes Martin J. , Patrushev Nikolai , Hilliard Julia
Published: February 2005

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n= 40) and -positive (n= 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.
Isolation ofCandida famatafrom a Patient with Acute Zonal Occult Outer Retinopathy
Authors: Carrasco Luis , Ramos Marta , Galisteo Rebeca , Pisa Diana , Fresno Manuel , González María Eugenia
Published: February 2005

The etiology of a number of retinopathies, such as acute zonal occult outer retinopathy (AZOOR), remains undetermined.Candida famatawas isolated from conjunctival exudates of a patient diagnosed with AZOOR. This yeast was very abundant, particularly in the more affected eye, while no other pathogens or fungal species were in evidence. Immunological tests revealed the presence of antigen-specific T lymphocytes by usingC. famataas a challenge. Moreover, enzyme-linked immunosorbent assay analysis showed the presence of specific antibodies against this yeast in the patient's blood. Delayed hypersensitivity by use of a skin test was also positive. Finally, antifungal treatments led to improvements in several clinical symptoms, including funduscopic analysis. However, despite prolonged treatment with fluconazole and itraconazole,C. famatastill appeared in the conjunctival exudates. The new antifungal voriconazole may represent a better choice for treatment.
Identifying Sources of Human Exposure to Plague
Authors: Lowell Jennifer L. , Wagner David M. , Atshabar Bakyt , Antolin Michael F. , Vogler Amy J. , Keim Paul , Chu May C. , Gage Kenneth L.
Published: February 2005

Yersinia pestis
, the etiologic agent of plague, has shaped the course of human history, killing millions of people in three major pandemics. This bacterium is still endemic in parts of Asia, Africa, and the Americas, where it poses a natural disease threat to human populations.
Y. pestis
has also recently received attention as a possible bioterrorism agent. Thus, rapid methods to distinguish between bioterrorism and naturally occurring plague infections are of major importance. Our study is the first to demonstrate that variable-number tandem repeats (VNTRs) in the
Y. pestis
genome can link human case isolates to those obtained from suspected environmental sources of infection. We demonstrate the valuable utility of VNTR markers in epidemiological investigations of naturally occurring plague and the forensic analysis of possible bioterrorism events.
Use of PCR Targeting of Internal Transcribed Spacer Regions and Single-Stranded Conformation Polymorphism Analysis of Sequence Variation in Different Regions of rRNA Genes in Fungi for Rapid Diagnosis of Mycotic Keratitis
Authors: Kumar Manish , Shukla P. K.
Published: February 2005

The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. Immunological methods of diagnosis have not been found to be satisfactory, and recent research has been diverted to the use of PCR for the sensitive and early diagnosis at the molecular level. In the present study we targeted different regions of the rRNA gene to diagnose cases of mycotic keratitis and identify the causal agents. Six fungus-specific primers (primers ITS1, ITS2, ITS3, ITS4, invSR1R, and LR12R) were used, and the amplified products were analyzed by single-stranded conformation polymorphism (SSCP) analysis. Dendrograms of these SSCP patterns, prepared on the basis of Jaccard's coefficient, indicated that the PCR products obtained with primer pair ITS1 and ITS2 were the best for the identification of fungi. The results were confirmed by sequencing of the PCR products, and the approach was successfully tested experimentally for the detection of mycotic keratitis caused by
Aspergillus fumigatus
and was used for the diagnosis of fungal corneal ulcers in patients.
Molecular Characterizations of Human and Animal Group A Rotaviruses in The Netherlands
Authors: van der Heide R. , Koopmans M. P. G. , Shekary N. , Houwers D. J. , van Duynhoven Y. T. H. P. , van der Poel W. H. M.
Published: February 2005

To gain more insight into interspecies transmission of rotavirus group A, human and animal fecal samples were collected between 1997 and 2001 in The Netherlands. A total of 110 human stool samples were successfully P and G genotyped by reverse transcriptase PCR. All strains belonged to the main human rotavirus genotypes G1 to G4, G9, [P4], [P6], [P8], and [P9]. [P8]G1 was predominant, and 5.5% belonged to the G9 genotype. Eleven percent of all P[8] genotypes could be genotyped only by a recently published modified primer. Rotavirus-positive fecal samples from 28 calf herds were genotyped by DNA sequencing. Genotypes G6 and G10 predominated; G6 and G10 were detected in 22 (78.6%) and 16 (57.1%) of the rotavirus-positive calf herds, respectively. In 12 (42.9%) calf herds, we found mixed infections. Genotype G8 was not found. Genotype G6 bovine rotaviruses were divided into three clusters: UK-like, VMRI-29-like, and Hun4-like. DNA sequencing of a part of the VP7 gene was shown to be useful as a quick determination of uncommon or novel strains of which the genotyping cannot be done by genotyping PCR. Of equine strains, both VP4 and VP7 genes could be used for genotyping: two [P12]G3 and four [P12]G14 equine rotaviruses were determined. We did not find indications for rotavirus interspecies transmissions, although the recently published human G6-Hun4 is genetically related to our G6 bovine isolates. All bovine, porcine, and equine rotaviruses were within genotypes previously reported for these animal species.