bluuebunny/crossref_metadata_2025 huggingface dataset:
A Model of Nonlinear Fatigue-Creep (Dwell) Interactions
Authors: Wu Xijia
Published: February 2009

A nonlinear creep/dwell interaction model is derived based on nucleation and propagation of a surface fatigue crack and its coalescence with creep/dwell damages (cavities or wedge cracks) along its path inside the material, which results in the total damage accumulation rate as given by da/dN=(1+(lc+lz)/λ){(da/dN)f+(da/dN)env}, where (da/dN)f is the pure fatigue crack growth rate, (da/dN)env is the environment-assisted crack growth rate, lc/lz is the cavity/wedge crack size, and λ is the average spacing between the internal cavities or cracks. Since wedge cracks are usually present in the form of dislocation pile-ups at low temperatures and cavitation usually occurs at high temperatures, the model attempts to reconcile the creep-/dwell-fatigue phenomena over a broad temperature range of engineering concern. In particular, the model has been used to explain the dwell fatigue of titanium alloys and high temperature creep-fatigue interactions in Ni-base superalloys under tensile cyclic creep rupture, compressive cyclic creep rupture, and tension/compression-hold strain controlled cyclic test conditions.
Description of a Semi-Independent Time Discretization Methodology for a One-Dimensional Gas Dynamics Model
Authors: Galindo J. , Serrano J. R. , Arnau F. J. , Piqueras P.
Published: February 2009

Modeling has become an essential technique in design and optimization processes of internal combustion engines. As a consequence, the development of accurate modeling tools is, in this moment, an important research topic. In this paper, a gas-dynamics modeling tool is presented. The model is able to reproduce the global behavior of complete engines. This paper emphasizes an innovative feature: the independent time discretization of ducts. It is well known that 1D models solve the flow through the duct by means of finite difference methods in which a stability requirement limits the time step depending on the mesh size. Thus, the use of small ducts in some parts of the engine reduces the speed of the calculation. The model presented solves this limitation due to the independent calculation for each element. The different elements of the engine are calculated following their own stability criterion and a global manager of the model interconnects them. This new structure provides time saving of up to 50% depending on the engine configuration.
Transient Development of Flame and Soot Distribution in Laminar Diffusion Flame With Preheated Air
Authors: Mandal Bijan Kumar , Sarkar Amitava , Datta Amitava
Published: February 2009

A numerical investigation of the transient development of flame and soot distributions in a laminar axisymmetric coflowing diffusion flame of methane in air has been carried out considering the air preheating effect. The gas phase conservation equations of mass, momentum, energy, and species concentrations along with the conservation equations of soot mass concentration and number density are solved simultaneously, with appropriate boundary conditions, by an explicit finite difference method. Average soot diameters are then calculated from these results. It is observed that the soot is formed in the flame when the temperature exceeds 1300 K. The contribution of surface growth toward soot formation is more significant compared with that of nucleation. Once the soot particles reach the high temperature oxygen-enriched zone beyond the flame, the soot oxidation becomes important. During the initial period, when soot oxidation is not contributing significantly, some of the soot particles escape into the atmosphere. However, under steady condition the exhaust product gas is nonsooty. Preheating of air increases the soot volume fraction significantly. This is both due to more number of soot particles and the increase in the average diameter. However, preheating of air does not cause a qualitative difference in the development of the soot-laden zone during the flame transient period.
The Influence of Dump Gap on External Combustor Aerodynamics at High Fuel Injector Flow Rates
Authors: Walker A. Duncan , Carrotte Jon F. , McGuirk James J.
Published: February 2009

The increasing demand to reduce fuel burn, hence CO2 emissions, from the gas turbine requires efficient diffusion to reduce the system pressure loss in the combustor. However, interactions between prediffuser and combustor can have a significant effect on diffuser performance. For example, the consequence of increased fuel injector flow at a dump gap set using conventional design guidelines has been shown (Walker, A. D., Carrotte, J. F., and McGuirk, J. J., 2007. “Compressor∕Diffuser∕Combustor Aerodynamic Interactions in Lean Module Combustors,” ASME Turbo Expo 2007—Power for Land Sea and Air, Paper No. GT2007-27872) to introduce a destabilizing interaction between fuel injector and upstream components. The present paper concentrates on examining the effects of increased dump gap. Dump gap ratios of 0.8, 1.2, and 1.6 were employed, with each test utilizing the same inlet guide vane, compressor rotor, integrated outlet guide vane (OGV)∕prediffuser, and dump geometry. The flow fraction of compressor efflux entering the combustor cowl was set to be representative of lean combustors (50–70%). Measurements were made on a fully annular rig using a generic flame tube with metered cowl and inner∕outer annulus flows. The results demonstrate that, with fixed cowl flow, as the dump gap increases, component interactions decrease. At a dump gap ratio of 0.8, the proximity of the flame tube influences the prediffuser providing a beneficial blockage effect. However, if increased to 1.2, this beneficial effect is weakened and the prediffuser flow deteriorates. With further increase to 1.6, the prediffuser shows strong evidence of separation. Hence, at the dump gaps probably required for lean module injectors, it is unlikely the prediffuser will be influenced beneficially by the flame tube blockage; this must be taken into account in the design. Furthermore, with small dump gaps and high cowl flow fraction, the circumferential variation in cowl flow can feed upstream and cause OGV∕rotor forcing. At larger dump gaps, the circumferential variation does not penetrate upstream to the OGV, and the rotor is unaffected. The optimum dump gap and prediffuser design for best overall aerodynamic system performance from rotor through to feed annuli is a compromise between taking maximum advantage of upstream blockage effects and minimizing any 3D upstream forcing.
Experimental Study on Natural Circulation and Air-Injection Enhanced Circulation With Different Fluids
Authors: Ambrosini W. , Forgione N. , Oriolo F. , Semeraro E. , Tarantino M.
Published: February 2009

This paper reports on an experimental investigation on natural circulation and air-injection enhanced circulation performed adopting different fluids. This work is aimed at providing information on the basic mechanisms proposed in the design of future reactors relying on such circulation mechanisms for core cooling. Though the final objective of the research is the study of heavy metal cooling, the work is here limited to nonmetallic fluids. The working fluid adopted in past analyses was water. Further experimental campaigns were recently performed using the Novec™ HFE-7100 fluid, providing additional information on basic phenomena and the related scaling laws. The new fluid has a greater density and a greater thermal expansion coefficient with respect to water. Air was adopted for gas injection. Both natural circulation and gas-injection enhanced circulation are addressed in this work, drawing quantitative conclusions about the observed parametric trends. A systematic comparison is performed with the results obtained in previous experimental activities using water.
Simulations of Metal Oxidation in Lead Bismuth Eutectic at a Mesoscopic Level
Authors: Tan Taide , Chen Yitung
Published: February 2009

The corrosiveness of lead bismuth eutectic (LBE), as an ideal coolant candidate in reactors and accelerator driven systems (ADSs), presents a critical challenge for safe applications. One of the effective ways to protect the materials is to form and maintain a protective oxide film along the structural material surfaces by active oxygen control technology. The oxidation of metals in LBE environment has been investigated numerically at a mesoscopic scale. A novel stochastic cellular automaton (CA) model has been proposed considering the transport of oxygen along the grain boundaries. The proposed mesoscopic CA model has been mapped with the experimental data. A parametric study was conducted in order to check the importance of the main explicit parameters of the mesoscopic model. The boundary condition at the far end of the specimen has been investigated for the CA model. The model has benchmarked with the analytical solution and with the previous work of a pure diffusion process, and significant agreement has been reached. The developed CA model can be used to solve diffusion problem.
Pressure Load Estimation During Ex-Vessel Steam Explosion
Authors: Leskovar Matjaž
Published: February 2009

An ex-vessel steam explosion may occur when, during a severe reactor accident, the reactor pressure vessel fails and the molten core pours into the water in the reactor cavity. A steam explosion is a fuel-coolant interaction process where the heat transfer from the melt to water is so intense and rapid that the timescale for heat transfer is shorter than the timescale for pressure relief. This can lead to the formation of shock waves and production of missiles that may endanger surrounding structures. A strong enough steam explosion in a nuclear power plant could jeopardize the containment integrity and so lead to a direct release of radioactive material to the environment. In the article, different scenarios of ex-vessel steam explosions in a typical pressurized water reactor cavity are analyzed with the code MC3D, which is being developed for the simulation of fuel-coolant interactions. A comprehensive parametric study was performed by varying the location of the melt release (central and side melt pours), the cavity water subcooling, the primary system overpressure at vessel failure, and the triggering time for explosion calculations. The main purpose of the study was to determine the most challenging ex-vessel steam explosion cases in a typical pressurized water reactor and to estimate the expected pressure loadings on the cavity walls. Special attention was given to melt droplet freezing, which may significantly influence the outcome of the fuel-coolant interaction process. The performed analysis shows that for some ex-vessel steam explosion scenarios much higher pressure loads are predicted than obtained in the OECD program SERENA Phase 1.
Trifurcate Feed-Forward Regulation of Age-Dependent Cell Death Involving
miR164
in
Arabidopsis
Authors: Kim Jin Hee , Woo Hye Ryun , Kim Jeongsik , Lim Pyung Ok , Lee In Chul , Choi Seung Hee , Hwang Daehee , Nam Hong Gil
Published: February 2009

Aging induces gradual yet massive cell death in higher organisms, including annual plants. Even so, the underlying regulatory mechanisms are barely known, despite the long-standing interest in this topic. Here, we demonstrate that ORE1, which is a NAC (NAM, ATAF, and CUC) transcription factor, positively regulates aging-induced cell death in
Arabidopsis
leaves.
ORE1
expression is up-regulated concurrently with leaf aging by
EIN2
but is negatively regulated by
miR164. miR164
expression gradually decreases with aging through negative regulation by
EIN2
, which leads to the elaborate up-regulation of
ORE1
expression. However,
EIN2
still contributes to aging-induced cell death in the absence of
ORE1
. The trifurcate feed-forward pathway involving
ORE1, miR164
, and
EIN2
provides a highly robust regulation to ensure that aging induces cell death in
Arabidopsis
leaves.
Macroscopic 10-Terabit–per–Square-Inch Arrays from Block Copolymers with Lateral Order
Authors: Park Soojin , Lee Dong Hyun , Xu Ji , Kim Bokyung , Hong Sung Woo , Jeong Unyong , Xu Ting , Russell Thomas P.
Published: February 2009

Generating laterally ordered, ultradense, macroscopic arrays of nanoscopic elements will revolutionize the microelectronic and storage industries. We used faceted surfaces of commercially available sapphire wafers to guide the self-assembly of block copolymer microdomains into oriented arrays with quasi–long-range crystalline order over arbitrarily large wafer surfaces. Ordered arrays of cylindrical microdomains 3 nanometers in diameter, with areal densities in excess of 10 terabits per square inch, were produced. The sawtoothed substrate topography provides directional guidance to the self-assembly of the block copolymer, which is tolerant of surface defects, such as dislocations. The lateral ordering and lattice orientation of the single-grain arrays of microdomains are maintained over the entire surface. The approach described is parallel, applicable to different substrates and block copolymers, and opens a versatile route toward ultrahigh-density systems.
Oxide Nanoelectronics on Demand
Authors: Cen Cheng , Thiel Stefan , Mannhart Jochen , Levy Jeremy
Published: February 2009

Electronic confinement at nanoscale dimensions remains a central means of science and technology. We demonstrate nanoscale lateral confinement of a quasi–two-dimensional electron gas at a lanthanum aluminate–strontium titanate interface. Control of this confinement using an atomic force microscope lithography technique enabled us to create tunnel junctions and field-effect transistors with characteristic dimensions as small as 2 nanometers. These electronic devices can be modified or erased without the need for complex lithographic procedures. Our on-demand nanoelectronics fabrication platform has the potential for widespread technological application.
Switching Off Hydrogen Peroxide Hydrogenation in the Direct Synthesis Process
Authors: Edwards Jennifer K. , Solsona Benjamin , N Edwin Ntainjua , Carley Albert F. , Herzing Andrew A. , Kiely Christopher J. , Hutchings Graham J.
Published: February 2009

Hydrogen peroxide (H
2
O
2
) is an important disinfectant and bleach and is currently manufactured from an indirect process involving sequential hydrogenation/oxidation of anthaquinones. However, a direct process in which H
2
and O
2
are reacted would be preferable. Unfortunately, catalysts for the direct synthesis of H
2
O
2
are also effective for its subsequent decomposition, and this has limited their development. We show that acid pretreatment of a carbon support for gold-palladium alloy catalysts switches off the decomposition of H
2
O
2
. This treatment decreases the size of the alloy nanoparticles, and these smaller nanoparticles presumably decorate and inhibit the sites for the decomposition reaction. Hence, when used in the direct synthesis of H
2
O
2
, the acid-pretreated catalysts give high yields of H
2
O
2
with hydrogen selectivities greater than 95%.
A Transferable 20-Kilobase Multiple Drug Resistance-Conferring R Plasmid (pKL0018) from a Fish Pathogen (
Lactococcus garvieae
) Is Highly Homologous to a Conjugative Multiple Drug Resistance-Conferring Enterococcal Plasmid
Authors: Maki Takeshi , Santos Mudjekeewis D. , Kondo Hidehiro , Hirono Ikuo , Aoki Takashi
Published: February 2009

Lactococcus garvieae
, the causative agent of lactococcosis, has evolved strains that are highly resistant to antibiotics. Here, the 20,034-bp sequence of
L. garvieae
conjugative plasmid pKL0018 was determined. It contained two
erm
B genes and one
tet
S gene and a backbone more than 96% identical to that of pRE25, an
Enterococcus faecalis
plasmid from dry sausage.
Formation of Multilayered Photosynthetic Biofilms in an Alkaline Thermal Spring in Yellowstone National Park, Wyoming
Authors: Boomer Sarah M. , Noll Katherine L. , Geesey Gill G. , Dutton Bryan E.
Published: February 2009

In this study, glass rods suspended at the air-water interface in the runoff channel of Fairy Geyser, Yellowstone National Park, WY, were used as a substratum to promote the development of biofilms that resembled multilayered mat communities in the splash zone at the geyser's source. This approach enabled the establishment of the temporal relationship between the appearance of
Cyanobacteria
, which ultimately formed the outer green layer, and the development of a red underlayer containing
Roseiflexus
-like
Chloroflexi
. This is the first study to define time-dependent successional events involved in the development of differently colored layers within microbial mats associated with many thermal features in Yellowstone National Park. Initial (1-month) biofilms were localized below the air-water interface (60 to 70°C), and the majority of retrieved bacterial sequence types were similar to
Synechococcus
and
Thermus
isolates. Biofilms then shifted, becoming established at and above the air-water interface after 3 months. During winter sampling (6 to 8 months), distinct reddish orange microcolonies were observed, consistent with the appearance of
Roseiflexus
-like sequences and bacteriochlorophyll
a
pigment signatures. Additionally, populations of
Cyanobacteria
diversified to include both unicellular and filamentous cell and sequence types. Distinct green and red layers were observed at 13 months.
Planctomycetes
-like sequences were also retrieved in high abundance from final biofilm layers and winter samples. Finally, biomass associated with geyser vent water contained
Roseiflexus
-like sequence types, in addition to other high-abundance sequence types retrieved from biofilm samples, supporting the idea that geothermal water serves as an inoculum for these habitats.
Production of the Phytohormone Indole-3-Acetic Acid by Estuarine Species of the Genus
Vibrio
Authors: Gutierrez Casandra K. , Matsui George Y. , Lincoln David E. , Lovell Charles R.
Published: February 2009

Strains of
Vibrio
spp. isolated from roots of the estuarine grasses
Spartina alterniflora
and
Juncus roemerianus
produce the phytohormone indole-3-acetic acid (IAA). The colorimetric Salkowski assay was used for initial screening of IAA production. Gas chromatography-mass spectroscopy (GC-MS) was then employed to confirm and quantify IAA production. The accuracy of IAA quantification by the Salkowski assay was examined by comparison to GC-MS assay values. Indole-3-acetamide, an intermediate in IAA biosynthesis by the indole-3-acetamide pathway, was also identified by GC-MS. Multilocus sequence typing of concatenated 16S rRNA,
recA
, and
rpoA
genes was used for phylogenetic analysis of environmental isolates within the genus
Vibrio
. Eight
Vibrio
type strains and five additional species-level clades containing a total of 16 environmental isolates and representing five presumptive new species were identified as IAA-producing
Vibrio
species. Six additional environmental isolates similar to four of the
Vibrio
type strains were also IAA producers. To our knowledge, this is the first report of IAA production by species of the genus
Vibrio
or by bacteria isolated from an estuarine environment.
A Novel
Dehalobacter
Species Is Involved in Extensive 4,5,6,7-Tetrachlorophthalide Dechlorination
Authors: Yoshida Naoko , Ye Lizhen , Baba Daisuke , Katayama Arata
Published: February 2009

The purpose of this study was the enrichment and phylogenetic identification of bacteria that dechlorinate 4,5,6,7-tetrachlorophthalide (commercially designated “fthalide”), an effective fungicide for rice blast disease. Sequential transfer culture of a paddy soil with lactate and fthalide produced a soil-free enrichment culture (designated the “KFL culture”) that dechlorinated fthalide by using hydrogen, which is produced from lactate. Phylogenetic analysis based on 16S rRNA genes revealed the dominance of two novel phylotypes of the genus
Dehalobacter
(FTH1 and FTH2) in the KFL culture. FTH1 and FTH2 disappeared during culture transfer in medium without fthalide and increased in abundance with the dechlorination of fthalide, indicating their growth dependence on the dechlorination of fthalide.
Dehalobacter restrictus
TEA is their closest relative, with 97.5% and 97.3% 16S rRNA gene similarities to FTH1 and FTH2, respectively.
Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries
Authors: Waschkowitz Tanja , Rockstroh Stephanie , Daniel Rolf
Published: February 2009

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per μg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing
Escherichia coli
clones to form halos on skim milk-containing agar plates. The screening of 80,000
E. coli
clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus
Xanthomonas
. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (
mprA
, pTW2;
mprB
, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.
Effect of Biowaste Sludge Maturation on the Diversity of Thermophilic Bacteria and Archaea in an Anaerobic Reactor
Authors: Goberna M. , Insam H. , Franke-Whittle I. H.
Published: February 2009

Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined.
Clostridia
(>92%) dominated among bacteria and
Methanoculleus
(>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter
−1
) selected a stable community of halotolerant and halophilic prokaryotes.
Community Composition and Density of Methanogens in the Foregut of the Tammar Wallaby (Macropus eugenii)
Authors: Evans Paul N. , Hinds Lyn A. , Sly Lindsay I. , McSweeney Christopher S. , Morrison Mark , Wright André-Denis G.
Published: February 2009

The composition of the methanogenic archaeal community in the foregut contents of Tammar wallabies (Macropus eugenii) was studied using 16S rRNA and methyl coenzyme reductase subunit A (mcrA) gene clone libraries. Methanogens belonging to theMethanobacterialesand a well-supported cluster of uncultivated archaeon sequences previously observed in the ovine and bovine rumens were found. Methanogen densities ranged from 7.0 × 105and 3.9 × 106cells per gram of wet weight.
Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Randomly Amplified Polymorphic DNA PCR
Authors: Helton Rebekah R. , Wommack K. Eric
Published: February 2009

Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the
Podoviridae
(24%) and
Myoviridae
(22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.
Variation in Bacterial ATP Level and Proton Motive Force Due to Adhesion to a Solid Surface
Authors: Hong Yongsuk , Brown Derick G.
Published: February 2009

Bacterial adhesion to natural and man-made surfaces can be beneficial or detrimental, depending on the system at hand. Of vital importance is how the process of adhesion affects the bacterial metabolic activity. If activity is enhanced, this may help the cells colonize the surface, whereas if activity is reduced, it may inhibit colonization. Here, we report a study demonstrating that adhesion of both
Escherichia coli
and
Bacillus brevis
onto a glass surface resulted in enhanced metabolic activity, assessed through ATP measurements. Specifically, ATP levels were found to increase two to five times upon adhesion compared to ATP levels in corresponding planktonic cells. To explain this effect on ATP levels, we propose the hypothesis that bacteria can take advantage of a link between cellular bioenergetics (proton motive force and ATP formation) and the physiochemical charge regulation effect, which occurs as a surface containing ionizable functional groups (e.g., the bacterial cell surface) approaches another surface. As the bacterium approaches the surface, the charge regulation effect causes the charge and pH at the cell surface to vary as a function of separation distance. With negatively charged surfaces, this results in a decrease in pH at the cell surface, which enhances the proton motive force and ATP concentration. Calculations demonstrated that a change in pH across the cell membrane of only 0.2 to 0.5 units is sufficient to achieve the observed ATP increases. Similarly, the hypothesis indicates that positively charged surfaces will decrease metabolic activity, and results from studies of positively charged surfaces support this finding.
Spontaneous Quinolone Resistance in the Zoonotic Serovar of
Vibrio vulnificus
Authors: Roig Francisco J. , Llorens A. , Fouz B. , Amaro C.
Published: February 2009

This work demonstrates that
Vibrio vulnificus
biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in
gyrA
(substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in
parC
(substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.
Major Histocompatibility Complex Class II Molecule-Human Immunodeficiency Virus Peptide Analysis Using a Microarray Chip
Authors: Gaseitsiwe Simani , Valentini Davide , Ahmed Raija , Mahdavifar Shahnaz , Magalhaes Isabelle , Zerweck Johannes , Schutkowski Mike , Gautherot Emmanuel , Montero Felix , Ehrnst Anneka , Reilly Marie , Maeurer Markus
Published: February 2009

Identification of major histocompatibility complex (MHC) class II binding peptides is a crucial step in rational vaccine design and immune monitoring. We designed a novel MHC class II molecule-peptide microarray binding assay and evaluated 346 peptides from already identified human immunodeficiency virus (HIV) epitopes and an additional set (n= 206) of 20-mer peptides, overlapping by 15 amino acid residues, from HIV type 1B (HIV-1B) gp160 and Nef as a paradigm. Peptides were attached via the N-terminal part to a linker that covalently binds to the epoxy glass slide. The 552 peptides were printed in triplicate on a single peptide microarray chip and tested for stable formation of MHC class II molecule-peptide complexes using recombinant soluble DRB1*0101(DR1), DRB1*1501(DR2), and DRB1*0401(DR4) molecules. Cluster analysis revealed unique patterns of peptide binding to all three, two, or a single MHC class II molecule. MHC class II binding peptides reside within previously described immunogenic regions of HIV gp160 and Nef, yet we could also identify new MHC class II binding peptides from gp160 and Nef. Peptide microarray chips allow the comprehensive and simultaneous screening of a high number of candidate peptide epitopes for MHC class II binding, guided by subsequent quality data extraction and binding pattern cluster analysis.
Seroepidemiological Study after a Long-Distance Industrial Outbreak of Legionnaires' Disease
Authors: Wedege E. , Bergdal T. , Bolstad K. , Caugant D. A. , Efskind J. , Heier H. E. , Kanestrøm A. , Strand B. H. , Aaberge I. S.
Published: February 2009

Following a long-distance outbreak of Legionnaires' disease from an industrial air scrubber in Norway in 2005, a seroepidemiological study measuring levels of immunoglobulin G (IgG) and IgM antibodies to
Legionella pneumophila
was performed with a polyvalent enzyme-linked immunosorbent assay. One year after the outbreak, IgG levels in employees (
n
= 213) at the industrial plant harboring the scrubber and in blood donors (
n
= 398) from the outbreak county were low but significantly higher (
P
≤ 0.002) than those in blood donors (
n
= 406) from a nonexposed county. No differences in IgM levels among the three groups were found after adjustment for gender and age. Home addresses of the seroresponders in the exposed county clustered to the city of the outbreak, in contrast to the scattering of addresses of the seroresponding donors in the nonexposed county. Factory employees who operated at an open biological treatment plant had significantly higher IgG and IgM levels (
P
≤ 0.034) than those working >200 m away. Most of the healthy seroresponders among the factory employees worked near this exposure source. Immunoblotting showed that IgG and IgM antibodies in 82.1% of all seroresponders were directed to the lipopolysaccharide of the
L. pneumophila
serogroup 1 outbreak strain. In conclusion, 1 year after the long-distance industrial outbreak a small increase in IgG levels of the exposed population was observed. The open biological treatment plant within the industrial premises, however, constituted a short-distance exposure source of
L. pneumophila
for factory employees working nearby.
Hog1 Mitogen-Activated Protein Kinase (MAPK) Interrupts Signal Transduction between the Kss1 MAPK and the Tec1 Transcription Factor To Maintain Pathway Specificity
Authors: Shock Teresa R. , Thompson James , Yates John R. , Madhani Hiten D.
Published: February 2009

In
Saccharomyces cerevisiae
, the mating, filamentous growth (FG), and high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) signaling pathways share components and yet mediate distinct responses to different extracellular signals. Cross talk is suppressed between the mating and FG pathways because mating signaling induces the destruction of the FG transcription factor Tec1. We show here that HOG pathway activation results in phosphorylation of the FG MAPK, Kss1, and the MAPKK, Ste7. However, FG transcription is not activated because HOG signaling prevents the activation of Tec1. In contrast to the mating pathway, we find that the mechanism involves the inhibition of DNA binding by Tec1 rather than its destruction. We also find that nuclear accumulation of Tec1 is not affected by HOG signaling. Inhibition by Hog1 is apparently indirect since it does not require any of the consensus S/TP MAPK phosphorylation sites on Tec1, its DNA-binding partner Ste12, or the associated regulators Dig1 or Dig2. It also does not require the consensus MAPK sites of the Ste11 activator Ste50, in contrast to a recent proposal for a role for negative feedback in specificity. Our results demonstrate that HOG signaling interrupts the FG pathway signal transduction between the phosphorylation of Kss1 and the activation of DNA binding by Tec1.
The 3′-to-5′ Exoribonuclease (Encoded by HP1248) ofHelicobacter pyloriRegulates Motility and Apoptosis-Inducing Genes
Authors: Tsao Ming-Yang , Lin Tzu-Lung , Hsieh Pei-Fang , Wang Jin-Town
Published: February 2009

The human gastric pathogenHelicobacter pylorihas many virulence factors involved in pathogenesis, but the mechanisms regulating these virulence factors are not yet fully understood. In this study, we cloned HP1248, which is similar in sequence toEscherichia coli vacB, which was previously shown to be associated with the expression of virulence inShigellaand enteroinvasiveE. coli. E. coli vacBencodes RNase R. RNase R is involved in the posttranscriptional regulation of mRNA stability. By global transcriptional microarray profiling of anH. pyloriHP1248 deletion mutant, we defined six virulence-related genes which were posttranscriptionally downregulated by HP1248, including the motility-related genes HP1192 andflaB, the chemotaxis-related genecheY, and the apoptosis-inducing genes HP0175,cagA, andgtt. In this study, recombinant HP1248 protein expressed inE. colishowed 3′-to-5′ exoribonuclease activity. Motility and apoptosis induction were increased in theH. pyloriHP1248 deletion mutant. We also showed that HP1192 is associated withH. pylorimotility, possibly through HP1248 regulation. Further, we suggested and studied the possible mechanisms of this specific regulation of virulent genes by HP1248. In addition, the expression level of HP1248 mRNA changed dramatically in response to a variety of altered environmental conditions, including pH and temperature. Hence, HP1248 inH. pyloriseems to play a role in environmental sensing and in regulation of virulent phenotypes, such as motility and host apoptosis induction.
Constitutive Mutations in theEscherichia coliAraC Protein
Authors: Dirla Stephanie , Chien John Yeh-Heng , Schleif Robert
Published: February 2009

TheEscherichia coliAraC protein represses and induces thearaBADoperon in response to the absence or presence ofl-arabinose. Constitutive mutations in the AraC gene no longer require the presence ofl-arabinose to convert AraC from its repressing to its inducing state. Such mutations were isolated directly by virtue of their constitutivity or by their resistance to the nonmetabolizable arabinose analog,d-fucose. The majority of the constitutive mutations lie within the same residues of the N-terminal regulatory arm of AraC. Two, however, were found in the core of the dimerization domain. As predicted by the light switch mechanism of AraC, constitutive mutations increase the susceptibility of the N-terminal arms to digestion by trypsin or chymotrypsin, suggesting that these mutations weaken or disrupt the arm structure required for repression by AraC. Fluorescence, circular dichroism, and cysteine reactivity measurements show that the constitutive mutations in the core of the dimerization domain lead to a weakening of the support for the arms and reduce the stability of the minus-arabinose arm structure. These mutations also weaken the interaction between the two-helix bundle and the β-barrel subdomains of the dimerization domain and reduce the structural stability of the β-barrels.
The
Escherichia coli
Common Pilus and the Bundle-Forming Pilus Act in Concert during the Formation of Localized Adherence by Enteropathogenic
E. coli
Authors: Saldaña Zeus , Erdem Ayşen L. , Schüller Stephanie , Okeke Iruka N. , Lucas Mark , Sivananthan Arunon , Phillips Alan D. , Kaper James B. , Puente José L. , Girón Jorge A.
Published: February 2009

Although the bundle-forming pilus (BFP) of enteropathogenic
Escherichia coli
(EPEC) mediates microcolony formation on epithelial cells, the adherence of BFP-deficient mutants is significantly abrogated, but the mutants are still adherent due to the presence of intimin and possibly other adhesins. In this study we investigated the contribution of the recently described
E
.
coli
common pilus (ECP) to the overall adherence properties of EPEC. We found that ECP and BFP structures can be simultaneously observed in the course (between zero time and 7 h during infection) of formation of localized adherence on cultured epithelial cells. These two pilus types colocalized at different levels of the microcolony topology, tethering the adhering bacteria. No evidence of BFP disappearance was found after prolonged infection. When expressed from a plasmid present in nonadherent
E. coli
HB101, ECP rendered this organism highly adherent at levels comparable to those of HB101 expressing the BFP. Purified ECP bound in a dose-dependent manner to epithelial cells, and the binding was blocked with anti-ECP antibodies, confirming that the pili possess adhesin properties. An ECP mutant showed only a modest reduction in adherence to cultured cells due to background expression levels of BFP and intimin. However, isogenic mutants not expressing EspA or BFP were significantly less adherent when the
ecpA
gene was also deleted. Furthermore, a Δ
espA
Δ
ecpA
double mutant (unable to translocate Tir and to establish intimate adhesion) was at least 10-fold less adherent than the Δ
espA
and Δ
ecpA
single mutants, even in the presence of BFP. A Δ
bfp
Δ
espA
Δ
ecpA
triple mutant showed the least adherence compared to the wild type and all the isogenic mutant strains tested, suggesting that ECP plays a synergistic role in adherence. Our data indicate that ECP is an accessory factor that, in association with BFP and other adhesins, contributes to the multifactorial complex interaction of EPEC with host epithelial cells.
Identification and Characterization of Catabolicpara-Nitrophenol 4-Monooxygenase andpara-Benzoquinone Reductase fromPseudomonassp. Strain WBC-3
Authors: Zhang Jun-Jie , Liu Hong , Xiao Yi , Zhang Xian-En , Zhou Ning-Yi
Published: February 2009

Pseudomonassp. strain WBC-3 utilizespara-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase fromPseudomonas putidaU and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase fromEscherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP topara-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependentp-benzoquinone reductase that catalyzes the reduction ofp-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that bothpnpAandpnpBplay essential roles in PNP mineralization in strain WBC-3. Furthermore, thepnpCDEFgene cluster next topnpABshares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) inPseudomonas fluorescensACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol.190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.
σSControls Multiple Pathways Associated with Intracellular Multiplication ofLegionella pneumophila
Authors: Hovel-Miner Galadriel , Pampou Sergey , Faucher Sebastien P. , Clarke Margaret , Morozova Irina , Morozov Pavel , Russo James J. , Shuman Howard A. , Kalachikov Sergey
Published: February 2009

Legionella pneumophilais the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease.L. pneumophilais able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required forLegionellainfection and intracellular replication are poorly understood. Mutation of therpoSgene, which encodes σS, does not affect growth in rich medium but severely decreasesL. pneumophilaintracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of anrpoSmutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that σSaffects distinct groups of genes that contribute toLegionellaintracellular multiplication. We demonstrate thatrpoSmutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that σSaffects the transcription of thecpxRandpmrAgenes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of theL. pneumophilasmall RNAcsrBhomologs,rsmYandrsmZ, introduces a link between σSand the posttranscriptional regulator CsrA. We analyzed the network of σS-controlled genes by mutational analysis of transcriptional regulators affected by σS. One of these, encoding theL. pneumophilaarginine repressor homolog gene,argR, is required for maximal intracellular growth in amoebae. These data show that σSis a key regulator of multiple pathways required forL. pneumophilaintracellular multiplication.
Differential Expression of
sigH
Paralogs during Growth and under Different Stress Conditions in
Mycobacterium smegmatis
Authors: Singh Anirudh K. , Singh Bhupendra N.
Published: February 2009

SigH regulates a transcriptional network that responds to heat and oxidative stress in mycobacteria. Seven
sigH
paralogs are reported to exist in the
Mycobacterium smegmatis
genome. A comprehensive real-time reverse transcriptase PCR analysis during different stages of growth and upon exposure to various stress conditions and antimycobacterial compounds showed differential expression of
sigH
paralogs during stationary phase and severalfold increases in the levels of transcription of
sigH1
,
sigH4
,
sigH5
,
sigH6
, and
sigH7
under specific stress conditions.
Borrelia burgdorferi bba74Is Expressed Exclusively during Tick Feeding and Is Regulated by Both Arthropod- and Mammalian Host-Specific Signals
Authors: Mulay Vishwaroop B. , Caimano Melissa J. , Iyer Radha , Dunham-Ems Star , Liveris Dionysios , Petzke Mary M. , Schwartz Ira , Radolf Justin D.
Published: February 2009

Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in theBorrelia burgdorferitick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA,ospC, andospE) and their proteins. In vitro, transcripts forbba74were expressed at 23°C and further enhanced by a temperature shift (37°C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-typeB. burgdorferigrown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type andrpoSmutant spirochetes, revealed that, likeospA,bba74is transcribed by σ70and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type andrpoSmutant spirochetes was conducted to determine the transcriptional and translational profiles ofbba74during the tick-mouse cycle. Results from these studies revealed (i) thatbba74is transcribed by σ70exclusively during the larval and nymphal blood meals and (ii) that transcription ofbba74is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability ofB. burgdorferito complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation ofospAandospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue asB. burgdorferitransitions between its arthropod vector and mammalian host.
Utility of Multilocus Variable-Number Tandem-Repeat Analysis as a Molecular Tool for Phylogenetic Analysis ofShigella sonnei
Authors: Chiou Chien-Shun , Watanabe Haruo , Wang You-Wun , Wang Wan-Ling , Terajima Jun , Thong Kwai-Lin , Phung Dac Cam , Tung Sheng Kai
Published: February 2009

A panel of 916 isolates, including 703 closely related IST1 isolates, were characterized by inter-IS1spacer typing (IST), pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) to evaluate the utility of MLVA as a molecular tool for the phylogenetic analysis ofShigella sonnei. The global phylogenetic patterns determined by IST, PFGE, and MLVA were concordant. MLVA was carried out using 26 VNTR loci with a range of degrees of variability. MLVA data for the 703 IST1 isolates revealed that diversification among the closely related isolates was attributed mainly to four highly variable loci. The phylogenetic pattern for the closely related isolates determined using MLVA profiles of 8 highly variable loci was in agreement with that determined using the 26-locus profiles. A clustering analysis using the profiles of 18 loci with limited variability established clear phylogenetic relationships among IST clonal groups. Accordingly, MLVA is a useful tool for the phylogenetic analysis ofS. sonnei. Combined VNTR loci with higher variability are useful markers for resolving closely related isolates, whereas combined loci with lower variability are suitable for establishing clear phylogenetic relationships between strains or clones that have evolved over a longer timescale.