bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells
Authors: Matsuoka Hidetada , Harada Keita , Endo Yutaka , Warashina Akira , Doi Yoshiaki , Nakamura Jun , Inoue Masumi
Published: October 2008

GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre‐like structures immunoreactive to an anti‐GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The α1, α3, β2/3, γ2 and δ subunits of GABAAreceptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA‐induced Cl−currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only γ2‐, but also δ‐containing GABAAreceptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABAAreceptors. Increases in Ca2+signal in AM cells evoked trans‐synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA‐induced increase in conductance. Overall Ca2+responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only γ2‐, but also δ‐GABAAreceptors.
An Inverse Method for the Determination of Thermal Stress-Intensity Factors Under Arbitrary Thermal-Shocks
Authors: Meeker J. , Segall A. E. , Gondar E.
Published: October 2008

The analysis of stress-intensity factors is of immense importance when designing vessels, pipes, and end-caps as well as supporting structures and plates seen in high-temperature applications. Given this importance and the difficulty of measuring actual thermal boundary conditions, a generalized series based on a new and infinitely differentiable polynomial was employed to inversely determine the transient temperature distribution in a semi-infinite slab using only a single temperature history. These temperature distributions were in turn used to find the potential crack-opening stresses throughout the body. Using the found stresses and a weight-function approach, stress-intensity factors were then determined for both edge and semi-elliptical cracks under an arbitrary thermal-shock. When compared to other methods for various thermal scenarios, the method showed good agreement for both edge- and semi-elliptical surface cracks.
Study of Plastic Limit Load on Pressurized Cylinders With Lateral Nozzle
Authors: Li N. , Sang Z. F. , Widera G. E. O.
Published: October 2008

The objective of this work is to study the plastic limit load of cylindrical vessels with different lateral angles θ under increasing internal loadings. Three full size test vessels with different structure dimensions were fabricated for testing. A three-dimensional, nonlinear, finite element numerical simulation was also performed. The approximate plastic limit load was obtained using a twice-elastic-slope criterion. The plastic deformation characteristics for the analysis model are discussed. The results show that distinct deformation characteristics occurred on the three experimental vessels. The intersection area shrank in the longitudinal section of the cylinder, while bulges appeared in the transverse section. The angle between the axis of lateral and the cylinder increased. The plastic limit loads determined by experiment and numerical simulation methods are in good agreement.
Finite Element Analysis of the Effect of Brazed Residual Stress on Creep for Stainless Steel Plate-Fin Structure
Authors: Jiang Wen-Chun , Gong Jian-Ming , Chen Hu , Tu S. T.
Published: October 2008

This paper presented a finite element analysis of the effect of brazed residual stress on creep for stainless steel plate-fin structure using finite element code ABAQUS. The as-brazed residual stress distribution generated during the brazing process was obtained. Two cases, which are denoted Cases 1 and 2, were analyzed and compared to discuss the effect of as-brazed residual stress on creep. Case 1 was to carry out creep analysis just at the internal operating pressure. Case 2 was to perform the creep analysis considering the internal operating pressure in conjunction with as-brazed residual stress. The results show that due to the mechanical property mismatch between filler metal and base metal, large residual stress is generated in the brazed joint, which has a great influence on creep for stainless steel plate-fin structure. The creep strain and stress distribution of the overall plate-fin structure is obtained. The position that is most likely to fail is the fillet for the plate-fin structure at high temperature. Especially in the fillet interface, the creep strain and stress distribution are discontinuous and uncoordinated, which have great effect on creep failure.
Effect of High-Pressure Hydrogen Gas on Fracture of Austenitic
Steels
Authors: San Marchi C. , Balch D. K. , Nibur K. , Somerday B. P.
Published: October 2008

Applications requiring the containment and transportation of hydrogen gas at pressures greater than 70MPa are anticipated in the evolving hydrogen economy infrastructure. Since hydrogen is known to alter the mechanical properties of materials, data are needed to guide the selection of materials for structural components. The objective of this study is to characterize the role of yield strength, microstructural orientation, and small concentrations of ferrite on hydrogen-assisted fracture in two austenitic stainless steels: 21Cr–6Ni–9Mn (21-6-9) and 22Cr–13Ni–5Mn (22-13-5). The testing methodology involves exposure of tensile specimens to high-pressure hydrogen gas at elevated temperature in order to precharge the specimens with hydrogen, and subsequently testing the specimens in laboratory air to measure strength and ductility. In all cases, the alloys remain ductile despite precharging to hydrogen concentrations of ∼1at.%, as demonstrated by reduction in area values between 30% and 60% and fracture modes dominated by microvoid processes. Low concentrations of ferrite and moderate increases in yield strength do not exacerbate hydrogen-assisted fracture in 21-6-9 and 22-13-5, respectively. Microstructural orientation has a pronounced effect on ductility in 22-13-5 due to the presence of aligned second-phase particles.
The Assessment of Residual Stress Effects on Ductile Tearing Using Continuum Damage Mechanics
Authors: Sherry Andrew H. , Wilkes Mark A. , Sharples John K. , Budden Peter J.
Published: October 2008

This paper presents the results of a numerical study undertaken to assess the influence of residual stresses on the ductile tearing behavior of a high strength low toughness aluminum alloy. The Gurson–Tvergaard model was calibrated against conventional fracture toughness data using parameters relating to void nucleation, growth, and coalescence. The calibrated model was used to predict the load versus ductile tearing behavior of a series of full-scale and quarter-scale wide-plate tests. These center-cracked tension tests included specimens that contained a self-balancing residual stress field that was tensile in the region of the through-wall crack. Analyses of the full-scale wide-plate tests indicated that the model provides a good prediction of the load versus the ductile tearing behavior up to approximately 3mm of stable tearing. The influence of residual stress on the load versus the crack growth behavior was accurately simulated. Predictions of the load versus the crack growth behavior of full-scale wide-plate tests for crack extensions greater than 3mm and of the quarter-scale tests were low in terms of predicted load at a given amount of tearing. This was considered to result from (i) the “valid” calibration range in terms of specimen thickness and crack extension, (ii) the development of shear lips, and (iii) the differences in the micromechanism of ductile void formation under plane strain and under plane stress conditions.
General Variable Material Property Formulation for the Solution of Autofrettaged Thick-Walled Tubes With Constant Axial Strains
Authors: Farrahi G. H. , Hosseinian E. , Assempour A.
Published: October 2008

In this paper a general variable material property (VMP) formulation for the solution of thick-walled tubes with constant axial strains was developed and compared with the alternative VMP method that is called the Hencky program. The VMP method was initially developed for the analysis of plane stress and plane strain states. However, the actual autofrettage process is under constant axial strain, i.e., open-end and closed-end conditions. Results indicate very good agreement with the Hencky program. Our method is simple, accurate, and very efficient, so that the number of iterations for convergence reduces approximately to one-tenth of Hencky program iterations. The solution algorithm for plane strain, open-end, and closed-end conditions is the same.
Study of 193nm photoresist degradation during short time fluorocarbon plasma exposures. II. Plasma parameter trends for photoresist degradation
Authors: Sumiya M. , Bruce R. , Engelmann S. , Weilnboeck F. , Oehrlein G. S.
Published: October 2008

The mechanisms of modified layer formation and the effect of surface condition on the roughness formation of 193nm photoresist (PR) materials during short time fluorocarbon plasma exposures were investigated. The authors employed a shutter approach to minimize the plasma transient effects on processing of PR surfaces. Photoresist etching behavior and surface roughness formation during the initial etch period depend strongly on ion energy, pressure, and stage temperature, and reflect the degree of surface fluorination for these process conditions. The authors found that a high etch rate for low fluorination conditions leads to rougher surfaces as compared to etching of highly fluorinated PRs. The comparison of x-ray photoelectron spectroscopy and atomic force and scanning electron microscopy data for PR materials processed using different plasma operating conditions showed that smooth surfaces resulted when during the initial period of plasma-polymer interaction the fluorine content of the PR surface was large relative to the oxygen content of the unexposed polymer. One interpretation of this observation is that a larger amount of fluorine on the PR surface relative to the bulk oxygen content of the PR material reduces the microscopic etch nonuniformity, e.g., due to selective etching of ester groups. The grain size distribution of PR roughness was determined through Fourier transformation of the atomic force microscopy data. The results indicate that a process dependent grain size distribution is introduced initially (approximately several seconds) and subsequently only grows in amplitude as the root mean square roughness increases. The authors interpret this observation as surface roughness being formed at the beginning of the etch process and that subsequently the height of the grains grows by the roughness transfer into the underlying polymer material.
Nanofabrication by mechanical and electrical processes using electrically conductive diamond tip
Authors: Miyake Shojiro , Zheng Haifeng , Kim Jongduk , Wang Mei
Published: October 2008

The authors report on a method of nanofabrication involving mechanical or/and electrical processes and atomic force microscopy (AFM). Nanostructures were formed on an oxidized silicon wafer by applying a load and/or electrical voltage using an electrically conductive diamond tip (approximately 45nm radius) and the features of these nanostructures were investigated. AFM investigation showed that the height and morphology of the nanostructures were determined by the load and voltage. I∕V Schottky curves were used to clarify the electric properties of the processed regions, which were shown to depend on the extent of local oxidation by mechanochemical and electrochemical reactions generated by tip sliding during the mechanical and/or electrical processing. In particular, the complex process including both mechanical and electrical processes enhances the local oxidation at a high load and voltage.
Oxidation behavior of In95Sn5 solid solution
Authors: Ramasamy S. , Sabarinathan V. , Agarwal Nipun , Smith David J.
Published: October 2008

Nanocrystalline indium-tin alloy particles, having the composition of In95Sn5, were prepared by the inert-gas condensation technique and then examined for the possible formation of a core-shell structure after oxidation and different heat treatments. The crystalline structure and phase(s) of the as-prepared and heat-treated samples were investigated by x-ray diffraction, differential scanning calorimetry, small-angle x-ray scattering, and high-resolution electron microscopy. The results showed that the oxidation treatment led to highly faceted indium-tin oxide nanoparticles and indium-tin alloy grains with sizes in the nanometer range. No particles having a core-shell structure consisting of InSn alloy coated with oxide were detected.
ArF resist-friendly etching technology
Authors: Hayashi Toshio , Morikawa Yasuhiro , Suu Koukou , Ishikawa Michio
Published: October 2008

ArF resist has very weak plasma resistivity, so linewidth roughness (LWR) and line-edge roughness (LER) generally occur under the conventional etching conditions using perfluorocarbon gases. The main cause of the roughness generation has been thought to be high energy ions. However, decreasing the pressure to 0.4Pa, the roughness was suppressed considerably. So, the origin of the roughness is not depend solely on ion energy, but on the amount of fluorine atoms. Therefore, iodoperfluorocarbon gas was used to decrease the flux density of fluorine atoms. Iodine atoms react with fluorine atoms and interhalogen compounds are formed. The resultant etched surface was very smooth without LWR and LER, even at 1.3Pa.
Nanochannels fabricated in polydimethylsiloxane using sacrificial electrospun polyethylene oxide nanofibers
Authors: Bellan Leon M. , Strychalski Elizabeth A. , Craighead Harold G.
Published: October 2008

The authors have used electrospun polyethylene oxide nanofibers as sacrificial templates to form nanofluidic channels in polydimethylsiloxane (PDMS). By depositing fibers on silicon templates incorporating larger structures, the authors demonstrate that these nanochannels can be integrated easily with microfluidics. They use fluorescence microscopy to image channels filled with dye solution. The utility of the hybrid micro- and nanofluidic PDMS structures for single molecule observation and manipulation was demonstrated by introducing single molecules of λ-DNA into the channels. This nanofabrication technique allows the simple construction of integrated micro- and nanofluidic PDMS structures without lithographic nanofabrication techniques.
Correlation of the growth rate for selective epitaxial growth of silicon and oxide thickness and oxide coverage in a reduced pressure chemical vapor deposition pancake reactor
Authors: Lee Kung-Yen
Published: October 2008

The selective epitaxial growth (SEG) rate of silicon for wafers with various oxide coverages (OCs) (the ratio of oxide area to silicon area on a silicon wafer) and oxide thicknesses, ranging from 0% to 90% and 0to500nm, respectively, in a rf heated cold-wall reduced-pressure chemical vapor deposition (RPCVD) pancake reactor, using the SiH2Cl2–HCl–H2 gas system, was investigated at a chamber pressure of 40Torr and a temperature of 970°C. The highest growth rate is about 175nm∕min, occurred at the oxide coverage of 90% and oxide thickness of 200nm; the lowest growth rate is about 112nm∕min, occurred at the oxide coverage of 30% and oxide thickness of 500nm. The growth rate of a control sample (a bare silicon wafer) is 110nm∕min. According to these data, a semiempirical expression for the SEG rate of undoped wafers in an RPCVD pancake reactor system is developed. The growth rate is exponentially dependent on the oxide coverage and a function of oxide thickness. This expression is suitable for the in situ p-type doped wafer with 60% OC and the boron concentration of 1.0×1017cm−3 as well.
Comparative study of ZrN and Zr–Ge–N thin films as diffusion barriers for Cu metallization on Si
Authors: Leu L. C. , Sadik P. , Norton D. P. , McElwee-White L. , Anderson T. J.
Published: October 2008

The diffusion barrier properties of Zr–Ge–N and ZrN thin films for Cu metallization on Si have been examined. Both Zr–Ge–N and ZrN thin films deposited at room temperature via reactive magnetron sputtering are amorphous. X-ray diffraction of annealed Cu/nitride/Si structures indicates that a 50nm thick ZrN film is an effective diffusion barrier for annealing at 600°C for 1h. In contrast, Zr–Ge–N failed as a diffusion barrier upon annealing at 600°C as evidenced by the appearance of copper silicide diffraction peaks. Cross-section transmission electron microscopy also showed the formation of Cu3Si crystallites at the annealed Zr–Ge–N barrier interfaces. For ZrN barriers annealed at 600°C, the integrity of the nitride/Si interface is preserved and the energy-dispersive spectrometry line scan showed no Cu diffusion through the barrier into Si substrate. The result indicated that ZrN is superior to Zr–Ge–N as a copper diffusion barrier on Si.
Programmable proximity aperture lithography with MeV ion beams
Authors: Puttaraksa Nitipon , Gorelick Sergey , Sajavaara Timo , Laitinen Mikko , Singkarat Somsorn , Whitlow Harry J.
Published: October 2008

A novel MeV ion beam programmable proximity aperture lithography system has been constructed at the Accelerator Laboratory of the University of Jyväskylä, Finland. This facility can be used to fabricate three dimensional microstructures in thick (<100μm) polymer resist such as polymethylmethacrylate. In this method, MeV ion beams from the 1.7 MV pelletron and K130 cyclotron accelerators are collimated to a beam spot of rectangular shape. This shape is defined by a computer-controlled aperture made of a pair of L-shaped Ta blades which are in close proximity to the sample to minimize the penumbra broadening. Here the authors report on development of the system, the controlling software, the calibration procedures, investigations of multiple scattering effects, and present illustrative results using 3MeV He2+4 ion beams for lithography and 56MeV N3+14 ion beams for creating patterns of regions with ion tracks.
Comparison of selective Ge growth in SiO2 trenches on Si(001) and on blanket Si(001) substrates: Surface roughness and doping
Authors: Park J.-S. , Curtin M. , Hydrick J. M. , Carroll M. , Fiorenza J. G. , Lochtefeld A. , Novak S.
Published: October 2008

The authors investigate growth behavior, morphology, and doping of Ge grown selectively on silicon in submicron SiO2 trenches, in comparison with growth on blanket Si. Based on the surface morphology of Ge in trenches, it appears that lateral transport of Ge-containing species over the SiO2 surface extends on the order of ∼100μm from the trench. For growth at 600°C, preferential nucleation at trench edges leads to significant surface roughness. Contrary to behavior on blanket Si, reduction in growth temperature to 400°C leads to increased Ge roughness in trenches. Finally, with very low temperature of 350°C a very smooth surface in the trenches is achieved. Growth on blanket Si at this temperature is impractically slow, but is appreciable in the trenches; a discrepancy arising from the dominant supply of Ge-containing species migrating from the oxide. For boron and phosphorus-doped Ge inside the trenches, boron and phosphorus doping levels inside the trenches were about a half of those on blanket.
Overcoming material challenges for replication of “motheye lenses” using step and flash imprint lithography for optoelectronic applications
Authors: Kettle J. , Hoyle R. T. , Perks R. M. , Dimov S.
Published: October 2008

In this article, the fabrication and characterization of two-dimensional (2D) and three-dimensional (3D) structures using focused ion beam milling onto templates for step and flash imprint lithography (S-FIL) is discussed. It has been discovered that the 2D linewidth and height are closely related to the ion dose. At low doses (∼1pC∕μm2), the surface of the quartz template swells, thus, affecting the shape of the subsequent imprint. Furthermore, it has been shown that during UV curing of the S-FIL resist, the polymeric resist layer contracts as it solidifies, resulting in a dimensionally reduced replication of the original structure. The authors introduce a method to overcome the problem for 3D patterns, using a “multilayer” imprinting technique and apply this technique to the fabrication of “motheye” lenses. With respect to the imprinted replica, they show that the feature profile using this approach has a high fidelity in comparison to the template structure, and thus motheye lenses can be consistently replicated employing the S-FIL technology.
Large areas of periodic nanoholes perforated in multistacked films produced by lift-off
Authors: Wu Wei , Dey Dibyendu , Katsnelson Alex , Memis Omer G. , Mohseni Hooman
Published: October 2008

The authors report a low-cost and high-throughput method—nanosphere photolithography, for generating periodic subwavelength holes in metals/dielectrics. By combining the self-assembled and focus properties of micro-/nanospheres, the authors utilized the sphere arrays as lenses to produce large areas of nanopillars with a strong undercut in negative photoresist. Using lift-off with the nanopillars of photoresist, the authors demonstrate a large area of uniform nanoholes of as small as 50 nm in diameter at the bottom of ∼160 nm thick metal. The authors also show that the nanohole arrays can be generated in multistacked layers of different materials and these nanoholes can be processed with different sidewall shapes. The technique promises to be an alternative nanopatterning method that is simple, economical, fast, and flexible.
Optical super-resolution with aperture-function engineering
Authors: Ramsay E. , Serrels K. A. , Waddie A. J. , Taghizadeh M. R. , Reid D. T.
Published: October 2008

We demonstrate optical super-resolution (resolution better than conventional diffraction-limited resolution) in a simple optical configuration by using annular apertures to manipulate the pupil function of the system. The theoretical basis of the technique is described, and it is shown how good agreement between theory and experiment can be achieved by suitable selection of the principal system parameters. The ready implementation and suitability for theoretical interpretation makes the demonstration a good candidate for an undergraduate laboratory experiment in classical optics.
Blogging in the physics classroom: A research-based approach to shaping students’ attitudes toward physics
Authors: Duda Gintaras , Garrett Katherine
Published: October 2008

Although there has been much research on how to help students learn physics, students still come away without a deep understanding of how physics has transformed the world around them and almost all leave with decreased expectations and a more negative attitude toward physics. We discuss an experiment to address this problem: a course weblog which discusses real-world applications of physics and engages students in discussion and thinking outside of class. Students’ attitudes toward the value of physics and its applicability to the real-world were probed using a 26-question Likert scale survey over the course of four semesters in an introductory physics course. We found that students who did not participate in the blog generally exhibited a deterioration in attitude toward physics as seen previously. Students who read, commented, and were involved with the blog maintained their initially positive attitudes toward physics. Student response to the blog was overwhelmingly positive, with students claiming that the blog made the subjects studied in the classroom come alive for them and seem more relevant.
Photorealistic visualization of imaging in canonical optical resonators
Authors: Nelson John , Courtial Johannes , Whyte Graeme
Published: October 2008

We investigate the capability of open source freeware ray tracing rendering software to demonstrate the imaging properties of canonical optical resonators. This software can directly visualize the imaging properties of canonical resonators. We can also demonstrate effects such as the trapping of light rays in geometrically stable resonators and the formation of fractal intensity patterns in the eigenmodes of geometrically unstable resonators.
Time asymmetry in a dynamical model of the one-dimensional ideal gas
Authors: Boozer A. D.
Published: October 2008

We present a simple dynamical model of the one-dimensional ideal gas and show how it can be used to introduce a number of fundamental ideas in statistical mechanics. We use the model to illustrate the role of initial conditions in explaining time asymmetry and show that although the dynamical model is time-reversal invariant, the macroscopic behavior of the gas can be time-asymmetric if the initial conditions are chosen properly.
Peer instruction: From Harvard to the two-year college
Authors: Lasry Nathaniel , Mazur Eric , Watkins Jessica
Published: October 2008

We compare the effectiveness of a first implementation of peer instruction (PI) in a two-year college with the first PI implementation at a top-tier four-year research institution. We show how effective PI is for students with less background knowledge and what the impact of PI methodology is on student attrition in the course. Results concerning the effectiveness of PI in the college setting replicate earlier findings: PI-taught students demonstrate better conceptual learning and similar problem-solving abilities than traditionally taught students. However, not previously reported are the following two findings: First, although students with more background knowledge benefit most from either type of instruction, PI students with less background knowledge gain as much as students with more background knowledge in traditional instruction. Second, PI methodology is found to decrease student attrition in introductory physics courses at both four-year and two-year institutions.
Detection of Staphylococcal Cassette Chromosome
mec
-Associated DNA Segments in Multiresistant Methicillin-Susceptible
Staphylococcus aureus
(MSSA) and Identification of
Staphylococcus epidermidis ccrAB4
in both Methicillin-Resistant
S. aureus
and MSSA
Authors: Shore Anna C. , Rossney Angela S. , O'Connell Brian , Herra Celine M. , Sullivan Derek J. , Humphreys Hilary , Coleman David C.
Published: October 2008

Methicillin-susceptible
Staphylococcus aureus
(MSSA) can arise from methicillin-resistant S.
aureus
(MRSA) following partial or complete excision of staphylococcal cassette chromosome
mec
(SCC
mec)
. This study investigated whether multiresistant MSSA isolates from Irish hospitals, where MRSA has been endemic for decades, harbor SCC
mec
DNA. Twenty-five multiresistant MSSA isolates recovered between 2002 and 2006 were tested for SCC
mec
DNA by PCR and were genotyped by multilocus sequence typing and
spa
typing. All isolates lacked
mecA
. Three isolates (12%) harbored SCC
mec
DNA; two of these (genotype ST8/t190) harbored a 26-kb SCC
mec
IID (II.3.1.2) remnant that lacked part of
mecI
and all of
mecR1
,
mecA
, and IS
431
; the third isolate (ST8/t3209) harbored the SCC
mec
region from
dcs
to
orfX
. All three isolates were detected as MRSA using the BD GeneOhm and Cepheid's Xpert MRSA real-time PCR assays. Six isolates (ST8/t190,
n
= 4; ST5/t088,
n
= 2), including both isolates with the SCC
mec
IID remnant, harbored
ccrAB4
with 100% identity to
ccrAB4
from the
Staphylococcus epidermidis
composite island SCC-CI. This
ccrAB4
gene was also identified in 23 MRSA isolates representative of ST8/t190-MRSA with variant SCC
mec
II subtypes IIA to IIE, which predominated previously in Irish hospitals.
ccrAB4
was located 5,549 bp upstream of the left SCC
mec
junction in both the MRSA and MSSA isolates with SCC
mec
elements and remnants and 5,549 bp upstream of
orfX
in the four MSSA isolates with
ccrAB4
only on an SCC-CI homologous region. This is the first description of a large SCC
mec
remnant with
ccr
and partial
mec
genes in MSSA and of the
S. epidermidis
SCC-CI and
ccrAB4
genes in
S. aureus
.
Development and Qualification of a Pharmacodynamic Model for the Pronounced Inoculum Effect of Ceftazidime against
Pseudomonas aeruginosa
Authors: Bulitta Jürgen B. , Ly Neang S. , Yang Jenny C. , Forrest Alan , Jusko William J. , Tsuji Brian T.
Published: October 2008

Evidence is mounting in support of the inoculum effect (i.e., slow killing at large initial inocula [CFUo]) for numerous antimicrobials against a variety of pathogens. Our objectives were to (i) determine the impact of the CFUo of
Pseudomonas aeruginosa
on ceftazidime activity and (ii) to develop and validate a pharmacokinetic/pharmacodynamic (PKPD) mathematical model accommodating a range of CFUo. Time-kill experiments using ceftazidime at seven concentrations up to 128 mg/liter (MIC, 2 mg/liter) were performed in duplicate against
P. aeruginosa
PAO1 at five CFUo from 10
5
to 10
9
CFU/ml. Samples were collected over 24 h and fit by candidate models in NONMEM VI and S-ADAPT 1.55 (all data were comodeled). External model qualification integrated data from eight previously published studies. Ceftazidime displayed approximately 3 to 4 log
10
CFU/ml net killing at 10
6.2
CFUo and concentrations of 4 mg/liter (or higher), less than 1.6 log
10
CFU/ml killing at 10
7.3
CFUo, and no killing at 10
8.0
CFUo for concentrations up to 128 mg/liter. The proposed mechanism-based model successfully described the inoculum effect and the concentration-independent lag time of killing. The mean generation time was 28.3 min. The effect of an autolysin was assumed to inhibit successful replication. Ceftazidime concentrations of 0.294 mg/liter stimulated the autolysin effect by 50%. The model was predictive in the internal cross-validation and had excellent in silico predictive performance for published studies of
P. aeruginosa
ATCC 27853 for various CFUo. The proposed PKPD model successfully described and predicted the pronounced inoculum effect of ceftazidime in vitro and integrated data from eight literature studies to support translation from time-kill experiments to in vitro infection models.
1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside Inhibitors
Authors: Nyanguile Origène , Pauwels Frederik , Van den Broeck Walter , Boutton Carlo W. , Quirynen Ludo , Ivens Tania , van der Helm Liesbet , Vandercruyssen Geneviève , Mostmans Wendy , Delouvroy Frédéric , Dehertogh Pascale , Cummings Maxwell D. , Bonfanti Jean-Francois , Simmen Kenneth A. , Raboisson Pierre
Published: October 2008

The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.
Impact of Secondary Structure of Toll-Like Receptor 9 Agonists on Interferon Alpha Induction
Authors: Yu Dong , Putta Mallikarjuna R. , Bhagat Lakshmi , Dai Meiru , Wang Daqing , Trombino Anthony F. , Sullivan Tim , Kandimalla Ekambar R. , Agrawal Sudhir
Published: October 2008

Oligodeoxynucleotides containing a CpG motif and double- or multistranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of interferon alpha (IFN-α) in addition to other Th1-type cytokines. In the present study, we evaluated three highly effective IFN-α-inducing agonists of TLR9 to determine the type of duplex structures formed and the agonist's ability to induce immune responses, including IFN-α induction, in human cell-based assays and in vivo in mice and nonhuman primates. Thermal melting studies showed that two of the agonists evaluated had a single melting transition with similar hyperchromicity in both heating and cooling cycles, suggesting the formation of intermolecular duplexes. A third agonist showed a biphasic melting transition in the heating cycle and a monophasic melting transition with lower hyperchromicity during the cooling cycle, suggesting the formation of both intra- and intermolecular duplexes. All three agonists induced the production of Th1-type cytokines and chemokines, including high levels of IFN-α, in human peripheral blood mononuclear cell and plasmacytoid dendritic cell cultures. Subcutaneous administration of the two intermolecular duplex-forming agonists, but not the intramolecular duplex-forming agonist, induced cytokine secretion in mice. In nonhuman primates, the two agonists that formed intermolecular duplexes induced IFN-α and IP-10 secretion. On the contrary, the agonist that formed an intramolecular duplex induced only low levels of cytokines in nonhuman primates, suggesting that this type of structure formation is less immunostimulatory in vivo than the other structure. Taken together, the present results suggest that oligonucleotide-based agonists of TLR9 that form intermolecular duplexes induce potent immune responses in vivo.
Lysine-Enriched Cecropin-Mellitin Antimicrobial Peptides with Enhanced Selectivity
Authors: Sato Hiromi , Feix Jimmy B.
Published: October 2008

Lysine-enriched analogs of the cecropin-mellitin hybrid peptide, CA1-7M2-9(designated CM15), designed with optimized amphipathicity, retained antimicrobial activities similar to that of wild-type CM15 and had substantially reduced levels of hemolytic activity and cytotoxicity toward cultured macrophages, resulting in enhanced selectivity. These lysine-enriched analogs provide templates for improved CM15 peptide or peptidomimetic antibiotics.
Inhibition of Herpesvirus Replication by Hexadecyloxypropyl Esters of Purine- and Pyrimidine-Based Phosphonomethoxyethyl Nucleoside Phosphonates
Authors: Prichard Mark N. , Hartline Caroll B. , Harden Emma A. , Daily Shannon L. , Beadle James R. , Valiaeva Nadejda , Kern Earl R. , Hostetler Karl Y.
Published: October 2008

Patients infected with human immunodeficiency virus (HIV) often suffer from herpesvirus infections as a result of immunosuppression. These infections can occur while patients are receiving antiretroviral therapy, and additional drugs required to treat their infection can adversely affect compliance. It would be useful to have antivirals with a broader spectrum of activity that included both HIV and the herpesviruses. We reported previously that alkoxyalkyl ester prodrugs of cidofovir are up to 3 orders of magnitude more active against herpesvirus replication and may be less toxic than the unmodified drug. To determine if this strategy would be effective for certain phosphonomethoxyethyl nucleoside phosphonates which are also active against HIV infections, the hexadecyloxypropyl (HDP) esters of 1-(phosphonomethoxyethyl)-cytosine, 1-(phosphonomethoxyethyl)-5-bromo-cytosine (PME-5BrC), 1-(phosphonomethoxyethyl)-5-fluoro-cytosine, 9-(phosphonomethoxyethyl)-2,6-diaminopurine (PME-DAP), and 9-(phosphonomethoxyethyl)-2-amino-6-cyclopropylaminopurine (PME-cPrDAP) were evaluated for activity against herpesvirus replication. The HDP esters were substantially more active than the unmodified acyclic nucleoside phosphonates, indicating that esterification with alkoxyalkyl groups increases the antiviral activity of many acyclic nucleoside phosphonates. The most interesting compounds included HDP-PME-cPrDAP and HDP-PME-DAP, which were 12- to 43-fold more active than the parent nucleoside phosphonates against herpes simplex virus and cytomegalovirus, and HDP-PME-cPrDAP and HDP-PME-5BrC which were especially active against Epstein-Barr virus. The results presented here indicate that HDP-esterified acyclic nucleoside phosphonates with antiviral activity against HIV also inhibit the replication of some herpesviruses and can extend the spectrum of activity for these compounds.
Gene Therapy Using Adeno-Associated Virus Vectors
Authors: Daya Shyam , Berns Kenneth I.
Published: October 2008

The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack of apparent pathogenicity. Gene transfer studies using AAV have shown significant progress at the level of animal models; clinical trials have been noteworthy with respect to the safety of AAV vectors. No proven efficacy has been observed, although in some instances, there have been promising observations. In this review, topics in AAV biology are supplemented with a section on AAV clinical trials with emphasis on the need for a deeper understanding of AAV biology and the development of efficient AAV vectors. In addition, several novel approaches and recent findings that promise to expand AAV's utility are discussed, especially in the context of combining gene therapy ex vivo with new advances in stem or progenitor cell biology.
Detection of Respiratory Viruses by Molecular Methods
Authors: Mahony James B.
Published: October 2008

Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.
Identification of a Surrogate Marker for Infection in the African Green Monkey Model of Inhalation Anthrax
Authors: Rossi Cynthia A. , Ulrich Melanie , Norris Sarah , Reed Douglas S. , Pitt Louise M. , Leffel Elizabeth K.
Published: October 2008

In 2001, a bioterrorism attack involving
Bacillus anthracis
spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to
B. anthracis
spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of
B. anthracis
therapeutic development.
A Conserved C-Terminal 13-Amino-Acid Motif of Gap1 Is Required for Gap1 Function and Necessary for the Biogenesis of a Serine-Rich Glycoprotein ofStreptococcus parasanguinis
Authors: Zhou Meixian , Peng Zhixiang , Fives-Taylor Paula , Wu Hui
Published: October 2008

Adhesion ofStreptococcus parasanguinisto saliva-coated hydroxyapatite (SHA), an in vitro tooth model, is mediated by long peritrichous fimbriae. Fap1, a fimbria-associated serine-rich glycoprotein, is required for fimbrial assembly. Biogenesis of Fap1 is controlled by an 11-gene cluster that containsgly,nss,galT1and -2,secY2,gap1to -3,secA2, andgtf1and -2. We had previously isolated a collection of nine nonadherent mutants using random chemical mutagenesis approaches. These mutants fail to adhere to the in vitro tooth model and to form fimbriae. In this report, we further characterized these randomly selected nonadherent mutants and classified them into three distinct groups. Two groups of genes were previously implicated in Fap1 biogenesis. One group has a mutation in a glycosyltransferase gene,gtf1, that is essential for the first step of Fap1 glycosylation, whereas the other group has defects in thefap1structural gene. The third group mutant produces an incompletely glycosylated Fap1 and exhibits a mutant phenotype similar to that of a glycosylation-associated protein 1 (Gap1) mutant. Analysis of this new mutant revealed that a conserved C-terminal 13-amino-acid motif was missing in Gap1. Site-directed mutagenesis of a highly conserved amino acid tryptophan within this motif recapitulated the deletion phenotype, demonstrating the importance of the Gap1 C-terminal motif for Fap1 biogenesis. Furthermore, the C-terminal mutation does not affect Gap1-Gap3 protein-protein interaction, which has been shown to mediate Fap1 glycosylation, suggesting the C-terminal motif has a distinct function related to Fap1 biogenesis.
Evidence for Pore Formation in Host Cell Membranes by ESX-1-Secreted ESAT-6 and Its Role in
Mycobacterium marinum
Escape from the Vacuole
Authors: Smith Jennifer , Manoranjan Joanna , Pan Miao , Bohsali Amro , Xu Junjie , Liu Jun , McDonald Kent L. , Szyk Agnieszka , LaRonde-LeBlanc Nicole , Gao Lian-Yong
Published: October 2008

The ESX-1 secretion system plays a critical role in the virulence of
M. tuberculosis
and
M. marinum
, but the precise molecular and cellular mechanisms are not clearly defined. Virulent
M. marinum
is able to escape from the
Mycobacterium
-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine
M. marinum
ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in
M. marinum
escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that
M. marinum
induces membrane pores ∼4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by
M. marinum
ESX-1 could play a direct role in producing pores in MCV membranes, facilitating
M. marinum
escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.
The Early Phagosomal Stage of
Francisella tularensis
Determines Optimal Phagosomal Escape and
Francisella
Pathogenicity Island Protein Expression
Authors: Chong Audrey , Wehrly Tara D. , Nair Vinod , Fischer Elizabeth R. , Barker Jeffrey R. , Klose Karl E. , Celli Jean
Published: October 2008

Francisella tularensis
is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway,
F. tularensis
rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the
Francisella
intracellular cycle, we have characterized early trafficking events of the
F. tularensis
subsp.
tularensis
strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early
Francisella
-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further,
Francisella
pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.