bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Trace Layers and Joule Heating on the Temperature Distribution of Printed Circuit Boards: A Computational Study
Authors: Dogruoz M. Baris , Nagulapally Manoj K.
Published: November 2009

A printed circuit board (PCB) is generally a multilayered board made of dielectric material and several layers of traces and vias. Performing detailed system-level computational fluid dynamics (CFD) simulations of PCBs including meshed trace and via geometries for each of the layers is impractical. In the present approach, the effects of the trace and via geometry are accurately modeled in the physical model by importing electronics computer aided-design data consisting of the trace and via layout of the board and computing locally varying orthotropic conductivity (kx, ky, and kz) on the printed circuit board using a background mesh. The spatially varying orthotropic conductivity is then mapped from the background mesh to the CFD mesh and used in a system-level simulation of the PCB with a minimal increase in the overall computational cost. On the other hand, as PCB component densities increase, the current densities increase thereby leading to regions of hot spots due to Joule heating. Hence, it is essential that the computational heat transfer simulations account for the heating due to the high current carrying traces. In order to accurately model the Joule heating of traces and vias, it is of essence to solve for the conservation of current in each of these traces. In this study, the effects of both trace layer nonhomogeneity and Joule heating are examined on a sample PCB with several components attached to it. The results are then compared with those from the conventional modeling techniques. It is demonstrated that there is considerable difference in the location of the hot spots and temperature values between two different methods.
Total Cost Minimization of a High-Pressure Natural Gas Network
Authors: Tabkhi F. , Pibouleau L. , Azzaro-Pantel C. , Domenech S.
Published: November 2009

This paper deals with a high-pressure gas pipeline optimization, where the problem is to find the design properties of the pipelines and necessary compressor stations to satisfy customer requirements, using available supply gas and storage capacities. The considered objective function is the total annualized cost, including the investment and operating costs. The binary variables used to represent the flow direction of pipelines lead to a mixed integer nonlinear programming problem, solved by using the standard branch and bound solver in GAMS. The optimization strategy provides the main design parameters of the pipelines (diameters, pressures, and flow rates) and the characteristics of compressor stations (location, suction pressure, pressure ratio, station throughput, fuel consumption, and station power consumption) to satisfy customer requirements.
Performance Evaluation of a Pump-Assisted, Capillary Two-Phase Cooling Loop
Authors: Park Chanwoo , Vallury Aparna , Zuo Jon
Published: November 2009

A hybrid (pump-assisted and capillary) two-phase loop (HTPL) is experimentally investigated to characterize its thermal performance under stepwise heat input conditions. An integration of mechanical pumping with capillary pumping is achieved by using planar evaporator(s) and a two-loop design separating liquid and vapor flows. The evaporator(s) use a sintered copper grooved wick bonded with a liquid screen artery. No active flow control of the mechanical pumping is required because of the autonomous capillary pumping due to the self-adjusting liquid menisci to variable heat inputs of the evaporators. Unlike other active two-phase cooling systems using liquid spray and microchannels, the HTPL facilitates a passive phase separation of liquid from vapor in the evaporator using capillary action, which results in a lower flow resistance of the single-phase flows than two-phase mixed flows in fluid transport lines. In this work, a newly developed planar form-factor evaporator with a boiling heat transfer area of 135.3 cm2 is used aiming for the power electronics with large rectangular-shaped heat sources. This paper presents the experimental results of the HTPLs with a single evaporator handling a single heat source and dual evaporators handling two separate heat sources, while using distilled water as the working fluid for both cases. For the single evaporator system, the temperature results show that the HTPL does not create a big temperature upset under a stepwise heat load with sudden power increases and decreases. The evaporator thermal resistance is measured to be as low as 0.5 K cm2/W for the maximum heat load of 4.0 kW. A cold-start behavior characterized by a big temperature fluctuation was observed at the low heat inputs around 500 W. The HTPL with dual evaporators shows a strong interaction between the evaporators under an asymmetric heat load of the total maximum heat input of 6.5 kW, where each evaporator follows a different heat input schedule. The temperatures of the dual-evaporator system follow the profile of the total heat input, while the individual heat inputs determine the relative level of the temperatures of the evaporators.
Numerical Modeling of Heat Transfer and Pressure Losses for an Uncooled Gas Turbine Blade Tip: Effect of Tip Clearance and Tip Geometry
Authors: El-Gabry Lamyaa A.
Published: November 2009

A computational study has been performed to predict the heat transfer distribution on the blade tip surface for a representative gas turbine first stage blade. Computational fluid dynamics (CFD) predictions of blade tip heat transfer are compared with test measurements taken in a linear cascade, when available. The blade geometry has an inlet Mach number of 0.3 and an exit Mach number of 0.75, pressure ratio of 1.5, exit Reynolds number based on axial chord of 2.57×106, and total turning of 110 deg. Three blade tip configurations were considered; a flat tip, a full perimeter squealer, and an offset squealer where the rim is offset to the interior of the tip perimeter. These three tip geometries were modeled at three tip clearances of 1.25%, 2.0%, and 2.75% of the blade span. The tip heat transfer results of the numerical models agree well with data. For the case in which side-by-side comparison with test measurements in the open literature is possible, the magnitude of the heat transfer coefficient in the “sweet spot” matches data exactly and shows 20–50% better agreement with experiment than prior CFD predictions of this same case.
Alloy liquid metal ion source for carbon focused ion beams
Authors: Mazarov P. , Wieck A. D. , Bischoff L. , Pilz W.
Published: November 2009

A carbon-cerium alloy liquid metal ion source (LMIS) with unintentional aluminum content is presented for generating focused ion beams of carbon ions, as well as ionized clusters with sizes of 2, 4, and 8 atoms. Emission-current-dependent measurements were carried out for the mass spectra and energy spread of all species, but focused on the carbon monomer ions and clusters. The full width at half maximum of the energy distribution was determined to be 6.5eV for the monomer carbon ion and 14eV for the light clusters at an emission current of 5μA. The source showed good beam performance when used with a mass-separated focused ion beam column. Applications to graphene structures, organic matter, and other carbon-containing materials are promising tasks for the new carbon-containing alloy LMIS.
Selective etching and polymer deposition on InP surface in reactive ion etching with a mixture of methane and hydrogen
Authors: Yamamoto Norio
Published: November 2009

In a reactive ion etching with a mixture of methane and hydrogen, the authors observed selectivity in an etched InP surface or polymeric surface in a wafer, on which SiO2 mask was patterned into a 2-μm-wide grating, when the hydrogen flow rate was 5SCCM (SCCM denotes cubic centimeter per minute at STP) at a methane flow rate of 40SCCM. The polymers deposited on the InP surface were increased when the width of the grating pattern was increased. The InP surface configuration, such as etching or polymer deposition selectivity in unpatterned or patterned surfaces, was found to vary with hydrogen flow rate. They consider this to result from hydrogen radicals diffusing from the mask to the window in the direction across the grating pattern. The window is then loaded with the hydrogen radicals to a concentration sufficient to etch InP in the patterned surface.
Concept and operation of Schottky emitter without suppressor electrode
Authors: Dokania A. K. , Kruit P.
Published: November 2009

The Schottky electron emitter is the most frequently used electron source in electron microscopes. A suppressor electrode around the emitter is usually employed to prevent emission from the shank of the cathode. A concept of operating the Schottky emitter without the suppressor electrode is proposed with the aim of lowering the potential of the extractor electrode. Simulation results show that if the suppressor electrode is removed, then the same field as for the standard configuration can be obtained at the tip apex at an extraction voltage of 2265 V instead of 5000 V. The total emission from the shank region is calculated by estimating the emission area of the shank, taking into the account the different work functions of the crystal facets. The total emission for typical operating parameters is calculated to rise from 500 to 668 μA. The total emission from the shank and the filament of the Schottky emitter is measured experimentally in two different configurations, which match with the simulated results. The measured total emission of 450–750 μA confirms the idea that a Schottky emitter can be operated without suppressor, all the more so because the power at the extractor aperture is even reduced as a result of the lower acceleration voltage.
Thermodynamically stable nanotips of Au–Mo alloy
Authors: Nomura K. , Nagao T. , Cho B. L. , Katsuda H. , Matsumura T. , Oshima C.
Published: November 2009

The authors propose a simple new method for fabricating stable nanotips using sharpened Au–Mo alloy tips. By annealing at 1000–1200K in UHV, Au atoms segregate to the alloy surface to form a Au film of one physical monolayer thickness, resulting in formation of nanopyramids on the (111) surface. Although field ion microscopy images of the nanotips show no single-atom termination similar to the previous cases of Au-deposited W nanotips, energy spectra of the emitted electrons strongly suggest the realization of a single-atom termination just after annealing.
Emission site density depending on surface area and morphology of nanotube film emitters
Authors: Liu Huarong , Kato Shigeki , Saito Yahachi
Published: November 2009

The influence of emitter surface morphology on emission site density (ESD) is investigated for carbon nanotube films. The authors show that the ESD varies with cathode-anode distance for rough surface emitters while it is almost invariant for smooth ones. In addition, it has been revealed that the ESD decreases with the increase in the film area, resulting in low emission current density for large-area films. The present study suggests that the high ESD is more important to achieve high emission current density and long lifetime for film emitters than the high field enhancement factor.
Protein patterning on the micro- and nanoscale by thermal nanoimprint lithography on a new functionalized copolymer
Authors: Merino S. , Retolaza A. , Trabadelo V. , Cruz A. , Heredia P. , Alduncín J. A. , Mecerreyes D. , Fernández-Cuesta I. , Borrisé X. , Pérez-Murano F.
Published: November 2009

The localized depositions such as DNA or proteins at the micron or submicron scale on solids supports are a crucial step in the fabrication of advanced biochips and laboratory on chip devices. The present work shows a new approach: A new biofunctionalized copolymer based on 80% benzyl methacrylate and 20% succinimydil methacrylate is used as a printable polymer with a great affinity to proteins, leading to combined areas covered by proteins and areas with resistance to protein adsorption in the submicron scale. Based on this approximation, an immunoassay is proposed as a proof of concept to detect the presence of the rabbit IgG protein up to concentrations of 200 ng/ml.
Carbon nanotube pillar structures for human neural cell culture
Authors: Lee Jin Woo , Lee Kyong Soo , Ju Byeong Kwon , Cho Hyun Jin , Lee Nae Sung , Kim Min Young , Kim Eun Hye , Lee Kyu Back
Published: November 2009

Human neuroblastoma cells were cultured and differentiated over patterned (dot, dash, and square pattern) pillar structures of multiwalled carbon nanotubes vertically grown on a SiO2 film-coated quartz substrate to observe cytoskeletal responses to the nanotube-based scaffold, especially filopodia activities. The cells over the square pattern formed a colony and showed short and thick filopodia protrusion as if the surface adhesion was unstable, whereas the cells over the dot or dash patterns showed long extensions of filopodia and unique serial branching onto the sidewall or over the tips of nanotube pillars. The results are important for the investigation of neural network regeneration using carbon nanotubes.
Fabrication of large-area gallium arsenide nanowires using silicon dioxide nanoparticle mask
Authors: Wang Ding-Shin , Chao Jiun-Jie , Hung Shih-Che , Lin Ching-Fuh
Published: November 2009

Large-area GaAs nanowires are fabricated using SiO2 nanoparticles as the etching mask. SiO2 nanoparticle monolayer is spin coated on the GaAs substrate. To obtain a uniform monolayer of SiO2 nanoparticles across the substrate, raised temperature, adequate solution concentration, and the substrate treated with a solvent for interface activation are required. With the monolayer of SiO2 nanoparticles as the etching mask, the GaAs substrate is etched by induced-coupled plasma reactive ion etcher (ICP-RIE) to form GaAs nanowires with a high aspect ratio. The diameter and length of GaAs nanowires are 70nm and 1.2μm, respectively. The diameter and length of GaAs nanowires can be controlled by the size of SiO2 nanoparticles and etching time of ICP-RIE.
Researching Genetic Versus Nongenetic Determinants of Disease: A Comparison and Proposed Unification
Authors: Ioannidis John P. A. , Loy En Yun , Poulton Richie , Chia Kee Seng
Published: November 2009

Current research designs that dissociate genetic and nongenetic
measurements are reaching their limits.
Cognitive Enhancement Therapy for a Model of Down Syndrome
Authors: Wiseman Frances K.
Published: November 2009

Successful drug treatment of learning deficits in an animal model of
Down syndrome (DS) highlights the norepinephrine-ergic system and
suggests possible therapeutic options for people with DS.
Block Copolymers Have Differing Adjuvant Effects on the Primary Immune Response Elicited by Genetic Immunization and on Further Induced Allergy
Authors: Adel-Patient Karine , Pothelune Laetitia , Ah-Leung Sandrine , Wal Jean-Michel , Créminon Christophe , Chatel Jean-Marc
Published: November 2009

Block copolymers were recently used to promote gene delivery in various tissues. Using a plasmid encoding a food allergen, bovine β-lactoglobulin (BLG), we studied the effects of block copolymers on gene expression levels and primary immune response and on further induced allergy. Block copolymers (i.e., Tetronic 304, 908, and 1107) and various quantities of DNA were injected into the tibialis muscles of BALB/c mice. The BLG levels in injected muscle and the BLG-specific induced immune response were analyzed after injection. DNA-immunized mice were further experimentally sensitized with BLG, and the effects of block copolymer and DNA doses on allergic sensitization and elicitation were compared. Tetronic 304 induced a 12-fold increase in BLG production, while Tetronic 1107 increased the duration of BLG expression. Different Th1 primary specific immune responses were observed, either strong humoral and cellular (304), only cellular (1107), or weak cellular and humoral (908) responses. After BLG sensitization, increased BLG-specific IgG2a production was observed in all groups of mice independently of the presence and nature of the block copolymer. Increased BLG-specific IgG1 production was also detected after sensitization, except with Tetronic 1107. Compared with naked DNA, Tetronic 304 was the only block polymer that decreased BLG-specific IgE concentrations. However, after allergen challenge, Tetronic 1107 was the only block copolymer to reduce eosinophils and Th2 cytokines in bronchoalveolar lavage (BAL) fluid. Tetronic 304 amplified local inflammation. Each block copolymer elicited a different immune response, although always Th1 specific, in BALB/c mice.
Removal of Species Constraints in Antibody Detection
Authors: Basile Alison Jane , Biggerstaff Brad J. , Kosoy Olga L. , Junna Shilpa R. , Panella Nicholas A. , Powers Ann M. , Stark Lillian M. , Nemeth Nicole M.
Published: November 2009

Serum antibodies from myriad species, particularly birds, can provide key information regarding the transmission and the expansion of the territory of emerging pathogens. Expedient antibody analysis is constrained by a lack of species-specific reagents, a deficiency potentially highlighted by the recent swine-origin influenza A virus (H1N1) outbreak. Available methodologies present difficulties that discourage thorough serologic monitoring of potential disease vectors or hosts. Rapid high-throughput procedures that combined serum amine labeling
via
biotinylation, contaminant removal, and microsphere-based immunoassays for antibodies to three arboviruses were developed. Agent-specific adaptations of this simple format should facilitate expanded surveillance and diagnostic capabilities regarding pathogens of human and veterinary importance.
Adjuvant Effects for Oral Immunization Provided by RecombinantLactobacillus caseiSecreting Biologically Active Murine Interleukin-1β
Authors: Kajikawa Akinobu , Masuda Kazuya , Katoh Mitsunori , Igimi Shizunobu
Published: November 2009

Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1β (IL-1β), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinantLactobacillus caseiproducing IL-1β as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, including a mature IL-1β gene from mouse. The biological activity of the cytokine was confirmed by IL-8 production from Caco-2 cells. In response to the recombinantL. caseisecreting IL-1β, expression of IL-6 was detectedin vivousing a ligated-intestinal-loop assay. The release of IL-6 from Peyer's patch cells was also detectedin vitro. Intragastric immunization with heat-killedSalmonella entericaserovar Enteritidis (SE) in combination with IL-1β-secreting lactobacilli resulted in relatively high SE-specific antibody production. In this study, it was demonstrated that recombinantL. caseisecreting bioactive murine IL-1β provided adjuvant effects for intragastric immunization.
Evaluation of a Whole-Blood Cytokine Release Assay for Use in Measuring Endotoxin Activity of Group B
Neisseria meningitidis
Vaccines Made from Lipid A Acylation Mutants
Authors: Stoddard Mark B. , Pinto Valerian , Keiser Paul B. , Zollinger Wendell
Published: November 2009

Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The
Limulus
amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other
in vitro
assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B
Neisseria meningitidis
. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-α assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.
Identification of Western Equine Encephalitis Virus Structural Proteins That Confer Protection after DNA Vaccination
Authors: Gauci Penelope J. , Wu Josh Q. H. , Rayner George A. , Barabé Nicole D. , Nagata Leslie P. , Proll David F.
Published: November 2009

DNA vaccines encoding different portions of the structural proteins of western equine encephalitis virus were tested for the efficacy of their protection in a 100% lethal mouse model of the virus. The 6K-E1 structural protein encoded by the DNA vaccine conferred complete protection against challenge with the homologous strain and limited protection against challenge with a heterologous strain.
Asymptomatic
Leishmania infantum
Infection in an Area of Northwestern Italy (Piedmont Region) Where Such Infections Are Traditionally Nonendemic
Authors: Biglino Alberto , Bolla Cesare , Concialdi Erika , Trisciuoglio Anna , Romano Angelo , Ferroglio Ezio
Published: November 2009

The prevalence of
Leishmania infantum
-specific antibodies and asymptomatic infection was assessed in a randomized sample of 526 healthy adults from a continental area of Northwestern Italy where
L. infantum
is not endemic and where autochthonous cases of visceral leishmaniasis (VL) were recently reported.
L. infantum
-specific antibodies were detected by Western blotting (WB) in 39 subjects (7.41%), while
L. infantum
kinetoplast DNA was amplified from buffy coat in 21 out of 39 WB-positive subjects, confirming asymptomatic infection in 53.8% of seropositives. Risk factors significantly associated with WB positivity were uninterrupted residence since childhood in a local rural environment (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.7 to 7.3), daily contact with animals though not exclusively with dogs (OR, 3.7; 95% CI, 1.3 to 10.7), older age (OR, 2.31; 95% CI, 1.2 to 4.5), and agricultural/other outdoor activities (OR, 3.8; 95% CI, 0.99 to 3.7.) Logistic regression analysis showed that uninterrupted residence in a local rural environment and an age of >65 years were the only independent predictors of seropositivity assessed by WB. Follow-up at 24 months did not show evidence of VL in either seropositive or PCR-positive subjects. The detection of a high seroprevalence rate, confirmed as asymptomatic infection by PCR in more than half of the cases, among healthy residents in a continental area of northwestern Italy makes local
L. infantum
transmission very likely. In a region where VL is considered nonendemic, these findings warrant further epidemiological investigations as well as interventions with respect to both the canine reservoir and vectors, given the possible risks for immunosuppressed patients.
Evaluation of
tcdB
Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic
Clostridium difficile
Authors: Larson Ann M. , Fung Angela M. , Fang Ferric C.
Published: November 2009

Clostridium difficile
is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic
C. difficile
remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the
tcdB
gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for
C. difficile
glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified “gold standard” that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic
C. difficile
was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic
C. difficile
, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic
C. difficile
that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.
Sputum
Mycobacterium tuberculosis
mRNA as a Marker of Bacteriologic Clearance in Response to Antituberculosis Therapy
Authors: Li L. , Mahan C. S. , Palaci M. , Horter L. , Loeffelholz L. , Johnson J. L. , Dietze R. , Debanne S. M. , Joloba M. L. , Okwera A. , Boom W. H. , Eisenach K. D.
Published: November 2009

mRNA is a marker of cell viability. Quantifying
Mycobacterium tuberculosis
mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of
M. tuberculosis
viability.
Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Echinocandins and
Candida
spp
Authors: Pfaller M. A. , Boyken L. , Hollis R. J. , Kroeger J. , Messer S. A. , Tendolkar S. , Jones R. N. , Turnidge J. , Diekema D. J.
Published: November 2009

We tested a global collection of
Candida
sp. strains against anidulafungin, caspofungin, and micafungin, using CLSI M27-A3 broth microdilution (BMD) methods, in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2003 to 2007, 8,271 isolates of
Candida
spp. (4,283
C. albicans
, 1,236
C. glabrata
, 1,238
C. parapsilosis
, 996
C. tropicalis
, 270
C. krusei
, 99
C. lusitaniae
, 88
C. guilliermondii
, and 61
C. kefyr
isolates) were obtained from over 100 centers worldwide. The modal MICs (in μg/ml) for anidulafungin, caspofungin, and micafungin, respectively, for each species were as follows:
C. albicans
, 0.03, 0.03, 0.015;
C. glabrata
, 0.06, 0.03, 0.015;
C. tropicalis
, 0.03, 0.03, 0.015;
C. kefyr
, 0.06, 0.015, 0.06;
C. krusei
, 0.03, 0.06, 0.06;
C. lusitaniae
, 0.05, 0.25, 0.12;
C. parapsilosis
, 2, 0.25, 1; and
C. guilliermondii
, 2, 0.5. 05. The ECVs, expressed in μg/ml (percentage of isolates that had MICs that were less than or equal to the ECV is shown in parentheses) for anidulafungin, caspofungin, and micafungin, respectively, were as follows: 0.12 (99.7%), 0.12 (99.8%), and 0.03 (97.7%) for
C. albicans
; 0.25 (99.4%), 0.12 (98.5%), and 0.03 (98.2%) for
C. glabrata
; 0.12 (98.9%), 0.12 (99.4%), and 0.12 (99.1%) for
C. tropicalis
; 0.25(100%), 0.03 (100%), and 0.12 (100%) for
C. kefyr
; 0.12 (99.3%), 0.25 (96.3%), and 0.12 (97.8%) for
C. krusei
; 2 (100%), 0.5 (98.0%), and 0.5 (99.0%) for
C. lusitaniae
; 4 (100%), 1 (98.6%), and 4 (100%) for
C. parapsilosis
; 16 (100%), 4 (95.5%), and 4 (98.9%) for
C. guilliermondii
. These WT MIC distributions and ECVs will be useful in surveillance for emerging reduced echinocandin susceptibility among
Candida
spp. and for determining the importance of various
FKS1
or other mutations.
Diagnosis of Congenital Toxoplasmosis by Using a Whole-Blood Gamma Interferon Release Assay
Authors: Chapey Emmanuelle , Wallon Martine , Debize Gisèle , Rabilloud Muriel , Peyron François
Published: November 2009

Congenital toxoplasmosis in newborns is generally subclinical, but infected infants are at risk of developing ocular lesions. Diagnosis at birth relies mainly on serological tests. Cell-mediated immunity plays the major role in resistance to infection but is not routinely investigated for diagnostic purposes. Here, we describe a simple test based on the gamma interferon (IFN-γ) response after stimulation of whole blood by crude parasitic antigens. One milliliter of heparinized blood was centrifuged; plasma was kept for routine serological tests, and pellets were resuspended in culture medium. After 24 h of culture in the presence of crude
Toxoplasma gondii
antigen, the cells were centrifuged and the supernatant was assayed for IFN-γ. For 62 infants under 1 year of age born to mothers who were infected during pregnancy, the sensitivity and specificity of the test were 94% (with positive results for 16 of 17 infected infants) and 98% (with negative results for 44 of 45 uninfected infants), respectively. The false-negative result was for a treated baby who gave positive results after the withdrawal of treatment. The false positive was obtained for a 3-month-old baby. For a cohort of 124 congenitally infected patients between 1 and 30 years of age, the sensitivity of the assay was 100%. We present a simple test based on IFN-γ secretion to assess cell-mediated immunity in toxoplasmosis. As only 1 ml of blood is required to investigate humoral and cellular immunity, our assay is well adapted for the study of congenital toxoplasmosis in infants. Using purified antigens or recombinant peptides may improve the test performance.
Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes
Authors: Oldak Monika , Maksym Radoslaw B. , Sperling Tanya , Yaniv Moshe , Smola Hans , Pfister Herbert J. , Malejczyk Jacek , Smola Sigrun
Published: November 2009

The papillomavirus life cycle parallels keratinocyte differentiation in stratifying epithelia. We have previously shown that the human papillomavirus type 8 (HPV8) E2 protein downregulates β4-integrin expression in normal human keratinocytes, which may trigger subsequent differentiation steps. Here, we demonstrate that the DNA binding domain of HPV8 E2 is sufficient to displace a cellular factor from the β4-integrin promoter. We identified the E2-displaceable factor as activator protein 1 (AP-1), a heteromeric transcription factor with differentiation-specific expression in the epithelium. β4-Integrin-positive epithelial cells displayed strong AP-1 binding activity. Both AP-1 binding activity and β4-integrin expression were coregulated during keratinocyte differentiation suggesting the involvement of AP-1 in β4-integrin expression. In normal human keratinocytes the AP-1 complex was composed of JunB and Fra-1 subunits. Chromatin immunoprecipitation assays confirmed that JunB/Fra-1 proteins interactin vivowith the β4-integrin promoter and that JunB/Fra-1 promoter occupancy is reduced during keratinocyte differentiation as well as in HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the β4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 effect. In summary, we identified a novel mechanism of human β4-integrin regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle.
Early Events in Kaposi's Sarcoma-Associated Herpesvirus Infection of Target Cells
Authors: Chandran Bala
Published: November 2009

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently identified member of the herpesvirus family, infects a variety of target cells in vitro and in vivo. This minireview surveys current information on the early events of KSHV infection, including virus-receptor interactions, involved envelope glycoproteins, mode of entry, intracellular trafficking, and initial viral and host gene expression programs. We describe data supporting the hypothesis that KSHV manipulates preexisting host cell signaling pathways to allow successful infection. The various signaling events triggered by infection, and their potential roles in the different stages of infection and disease pathogenesis, are summarized.
Evidence for an Activation Domain at the Amino Terminus of Simian Immunodeficiency Virus Vpx
Authors: Gramberg Thomas , Sunseri Nicole , Landau Nathaniel R.
Published: November 2009

Vpx and Vpr are related lentiviral accessory proteins that enhance virus replication in macrophages and dendritic cells. Both proteins are packaged into virions and mediate their effects in the target cell through an interaction with an E3 ubiquitin ligase that contains DCAF1 and DDB1. When introduced into primary macrophages and dendritic cells in viruslike particles, Vpx can enhance the efficiency of a subsequent infection. Here, we confirm the ability of Vpx to enhance simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) infection of macrophages up to 100-fold by using single-cycle reporter viruses and by pretreatment of the cells with Vpx-containing viruslike particles. Vpx was also active in differentiated THP-1 cells but not in other cell lines. Induction of an antiviral state in macrophages with type I interferon significantly magnified the effect of Vpx on HIV-1 infection, suggesting that Vpx helps the virus to overcome an inducible intracellular restriction. Quantitative PCR quantitation of SIV and HIV-1 reverse transcripts in newly infected macrophages showed that the block was at an early step in reverse transcription. In spite of its structural similarity, Vpr was inactive. This difference allowed us to map the functional domains of Vpx with a panel of Vpr/Vpx chimeras. Analysis of the chimeras demonstrated that the amino-terminal domain of Vpx is important for the enhancement of infection. Fine mapping of the region indicated that amino acids at positions 9, 12, and 15 to 17 were required. Although the mutants failed to enhance infection, they retained their ability to interact with DCAF1. These findings suggest that the Vpx amino terminus contains an activation domain that serves as the binding site for a cellular restriction factor.
In Vitro
and
In Vivo
Studies of the RNA Conformational Switch in
Alfalfa Mosaic Virus
Authors: Chen Shih-Cheng , Olsthoorn René C. L.
Published: November 2009

The 3′ termini of
Alfalfa mosaic virus
(AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme)
in vitro
is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the
in vitro
and
in vivo
data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.
Characterization of T-Cell Responses in Macaques Immunized with a Single Dose of HIV DNA Vaccine
Authors: Arrode-Brusés Géraldine , Sheffer Darlene , Hegde Ramakrishna , Dhillon Sukbir , Liu Zhengian , Villinger François , Narayan Opendra , Chebloune Yahia
Published: November 2009

The optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIVKU2(Δ4SHIVKU2) induced long-lasting, potent, and polyfunctional HIV-specific CD8+T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol.178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Δ4SHIVKU2DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4+and CD8+T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8+T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.
Antibody to Varicella-Zoster Virus Immediate-Early Protein 62 Augments Allodynia in Zoster via Brain-Derived Neurotrophic Factor
Authors: Hama Yuka , Shiraki Kimiyasu , Yoshida Yoshihiro , Maruyama Atsushi , Yasuda Makoto , Tsuda Masaaki , Honda Mariko , Takahashi Michiaki , Higuchi Hiroshi , Takasaki Ichiro , Daikoku Tohru , Tsumoto Tadaharu
Published: November 2009

Varicella-zoster virus (VZV) expresses immediate-early protein 62 (IE62), and zoster is associated with neuropathic pain. Brain-derived neurotrophic factor (BDNF) is involved in the neuronal mechanism underlying pain hypersensitivity. Zoster is associated with prodrome and the robust production of booster antibody to VZV. We hypothesized that the intrathecal production of antibody to IE62 cross-reacting with BDNF and the nerve injury by skin lesions may augment allodynia in zoster by enhancing BDNF activity. One of three monoclonal antibodies against the 268-556 peptide of IE62 recognized BDNF. Immunological cross-reactivity between IE62 and BDNF and the effects of anti-IE62 monoclonal antibody (anti-IE62 MAb) cross-reactivity with BDNF on BDNF activity in cultured neurons were examined. Anti-IE62 MAb and anti-BDNF MAbs recognized the 414-429 peptide of IE62 and the BDNF dimer. Anti-IE62 MAb significantly augmented BDNF-related transcription in neurons and the morphological development of spinal dorsal horn neurons. Sera from patients recognized IE62 and BDNF and enhanced BDNF activity in neurons. The effect of anti-IE62 antibody on mechanical allodynia was characterized by the threshold of allodynia using von Frey filaments in a spinal nerve injury (SNI) in mice. The administration of anti-IE62 MAb to or immunization with cross-reacting IE62 protein to mice significantly enhanced mechanical allodynia on the side with SNI but not on the uninjured side. Anti-IE62 antibody augmented BDNF activity in neurons and allodynia in mice with SNI. The intrathecal production of anti-IE62 antibody augmenting BDNF activity and peripheral nerve injury by zoster may participate in the pathogenesis of allodynia in zoster.
Infection with Strains of
Citrus Tristeza Virus
Does Not Exclude Superinfection by Other Strains of the Virus
Authors: Folimonova Svetlana Y. , Robertson Cecile J. , Shilts Turksen , Folimonov Alexey S. , Hilf Mark E. , Garnsey Stephen M. , Dawson William O.
Published: November 2009

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of
Citrus tristeza virus
(CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.
Phosphorylation of Serine 177 of the Small Hepatitis Delta Antigen Regulates Viral Antigenomic RNA Replication by Interacting with the Processive RNA Polymerase II
Authors: Hong Shiao-Ya , Chen Pei-Jer
Published: November 2009

Recent studies revealed that posttranslational modifications (e.g., phosphorylation and methylation) of the small hepatitis delta antigen (SHDAg) are required for hepatitis delta virus (HDV) replication from antigenomic to genomic RNA. The phosphorylation of SHDAg at serine 177 (Ser
177
) is involved in this step, and this residue is crucial for interaction with RNA polymerase II (RNAP II), the enzyme assumed to be responsible for antigenomic RNA replication. This study demonstrated that SHDAg dephosphorylated at Ser
177
interacted preferentially with hypophosphorylated RNAP II (RNAP IIA), which generally binds at the transcription initiation sites. In contrast, the Ser
177
-phosphorylated counterpart (pSer
177
-SHDAg) exhibited preferential binding to hyperphosphorylated RNAP II (RNAP IIO). In addition, RNAP IIO associated with pSer
177
-SHDAg was hyperphosphorylated at both the Ser
2
and Ser
5
residues of its carboxyl-terminal domain (CTD), which is a hallmark of the transcription elongation isoform. Moreover, the RNAP II CTD kinase inhibitor 5,6-dichloro-1-β-
d
-ribofuranosyl-benzimidazole (DRB) not only blocked the interaction between pSer
177
-SHDAg and RNAP IIO but also inhibited HDV antigenomic replication. Our results suggest that the phosphorylation of SHDAg at Ser
177
shifted its affinity toward the RNA RNAP IIO isoform and thus is a switch for HDV antigenomic RNA replication from the initiation to the elongation stage.
Regulation on Energy Metabolism and Protection on Mitochondria ofPanax GinsengPolysaccharide
Authors: Li Xing-Tai , Chen Rui , Jin Li-Ming , Chen Hui-Ying
Published: November 2009

Panax ginseng C A Meyer (PG) is one of the most popular qi-invigorating herbal medicine and has been used to promote health, vitality, and longevity in China. Although PG has been used in traditional Chinese medicine for millennia, its qi-invigorating activities still lack convincing evidence. We investigated the effects of Panax ginseng polysaccharide (PGP) on energy metabolism and mitochondrial protection. The chronic hypoxia model was set up. Lipid peroxidation product malondialdehyde (MDA) was assayed by thiobarbituric acid (TBA) colorimetry. Mice liver mitochondria were isolated by differential centrifugation. The spectrophotometric method was used to measure the swelling of mitochondria. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in liver cells were determined by reversed-phase high performance liquid chromatography (RP-HPLC), adenylate energy charge (AEC), total adenylate pool (TAP), ATP/ADP and ATP/AMP ratio were calculated. The creatine kinase (CK) activities in mice skeletal muscle were determined by a commercial monitoring kit. The result showed that PGP could inhibit mitochondrial injury and swelling induced by Fe2+-L-Cys in a concentration-dependent manner. PGP which was administered by oral gavage daily for 10 days could inhibit the formation of MDA in mice brain, increase levels of ATP, ADP, TAP and AEC, ratio of ATP/ADP and ATP/AMP in liver cells, increase CK activities in mice skeletal muscle under chronic hypoxia condition. These results indicate that PGP protect mitochondria by inhibiting mitochondrial swelling, and improving energy metabolism. PGP functions as a preventive antioxidant by increasing CK activities. Therefore, PGP had the pharmaceutical activities of antihypoxia, antioxidation and improving energy status.