bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Flow Behavior for Clearance Brush Seals
Authors: Dogu Yahya , Aksit Mahmut F. , Demiroglu Mehmet , Dinc Osman Saim
Published: January 2008

The industrial applications of brush seals have been increasing due to their superior sealing performance. Advances in the understanding of seal behavior have been pushing the design limits to higher-pressure load, temperature, surface speed, and rotor excursion levels. The highest sealing performance can be achieved when the bristle pack maintains contact with the rotor surface. However, due to many design and operational constraints, most seals operate with some clearance. This operating clearance cannot be avoided due to rotor runouts, transient operating conditions, or excessive bristle wear. In some applications, a minimum initial clearance is required to ensure a certain amount of flow rate for component cooling or purge flow. Typically, brush seal failure occurs in the form of degraded sealing performance due to increasing seal clearance. The seal performance is mainly characterized by the flow field in close vicinity of the bristle pack, through the seal-rotor clearance, and within the bristle pack. This work investigates the flow field for a brush seal operating with some bristle-rotor clearance. A nonlinear form of the momentum transport equation for a porous medium of the bristle pack has been solved by employing the computational fluid dynamics analysis. The results are compared with prior experimental data. The flow field for the clearance seal is observed to have different characteristics compared to that for the contact seal. Outlined as well are the flow features influencing the bristle dynamics.
Validating the Phenomenological Smoke Model at Different Operating Conditions of DI Diesel Engines
Authors: Aghav Y. V. , Lakshminarayanan P. A. , Babu M. K. G. , Uddin Azeem , Dani A. D.
Published: January 2008

A new phenomenological model that was published in Aghav et al. (2005, “Phenomenology of Smoke From Direct Injection Diesel Engines,” Proceedings of ICEF2005, ASME Paper No. 1350) encompasses the spray and the wall interaction by a simple geometrical consideration. The current study extends this earlier work with investigations made on 16 different engines from six-engine families of widely varying features, applied to off-highway as well as on-road duty. A dimensionless factor was introduced to take care of the nozzle hole manufactured by hydroerosion, as well as the conical shape of the nozzle hole (k factor) in the case of valve-closed-orifice type of nozzles. The smoke emitted from the wall spray formed after wall impingement is the major contributor to the total smoke at higher loads. As the fuel spray impinges upon the walls of the combustion chamber, its velocity decreases. This low-velocity jet contributes to the higher rate of the smoke production. Therefore, the combustion bowl geometry along with injection parameters play a significant role in the smoke emissions. The new model is one dimensional and based on the recent phenomenological description of spray combustion in a direct injection diesel engine. The satisfactory comparison of the predicted and observed smoke over the wide range of engine operation demonstrated applicability of the model in simulation study of combustion occurring in direct injection (DI) diesel engines.
Improved High Cycle Fatigue Damage Tolerance of Turbine-Engine
Compressor Components by Low Plasticity Burnishing
Authors: Prevéy Paul S. , Jayaraman N. , Ravindranath Ravi A. , Shepard Michael
Published: January 2008

Significant progress has been made in the application of low plasticity burnishing (LPB) technology to military engine components, leading to orders of magnitude improvement in damage tolerance. Improved damage tolerance can facilitate inspection, reduce inspection frequency, and improve engine operating margins, all leading to improved military readiness at significantly reduced total costs. Basic understanding of the effects of the different LPB process parameters has evolved, and finite element based compressive residual stress distribution design methodologies have been developed. By incorporating accurate measurement of residual stresses to verify and validate processing, this combined technology leads to a total solution approach to solve damage problems in engine components. An example of the total solution approach to develop LPB processing of a first stage Ti-6Al-4V compressor vane to improve the foreign object damage tolerance from 0.002in.to0.025in. is presented. The LPB process, tooling, and control systems are described, including recent developments in real-time process monitoring for quality control. Performed on computer numerical control (CNC) machine tools, LPB processing is easily adapted to overhaul and manufacturing shop operations with quality assurance procedures meeting military and industry standards, facilitating transition to military depots and manufacturing facilities.
Effect of Fuel System Impedance Mismatch on Combustion Dynamics
Authors: Richards Geo A. , Robey Edward H.
Published: January 2008

Combustion dynamics are a challenging problem in the design and operation of premixed gas turbine combustors. In premixed combustors, pressure oscillations created by the flame dynamic response can lead to damage. These dynamics are typically controlled by designing the combustor to achieve a stable operation for planned conditions, but dynamics may still occur with minor changes in ambient operating conditions or fuel composition. In these situations, pilot flames or adjustment to fuel flow splits can be used to stabilize the combustor, but often with a compromise in emission performance. As an alternative to purely passive design changes, prior studies have demonstrated that adjustment to the fuel system impedance can be used to stabilize combustion. Prior studies have considered just the response of an individual fuel injector and combustor. However, in practical combustion systems, multiple fuel injectors are used. In this situation, individual injector impedance can be modified to produce a different dynamic response from individual flames. The resulting impedance mismatch prevents all injectors from strongly coupling to the same acoustic mode. In principle, this mismatch should reduce the amplitude of dynamics and may expand the operating margin for stable combustion conditions. In this paper, a 30kW laboratory combustor with two premixed fuel injectors is used to study the effect of impedance mismatch on combustion stability. The two fuel injectors are equipped with variable geometry resonators that allow a survey of dynamic stability while changing the impedance of the individual fuel systems. Results demonstrate that a wide variation in dynamic response can be achieved by combining different impedance fuel injectors. A base line 7% rms pressure oscillation was reduced to less than 3% by mismatching the fuel impedance.
Thermomechanical Fatigue Life Prediction of Cylinder Heads in Combustion Engines
Authors: Trampert Stefan , Gocmez Taner , Pischinger Stefan
Published: January 2008

While the deformation and damage behavior of aluminum cylinder heads under complex thermal mechanical loading has been the subject of numerous studies in the past, cast iron cylinder heads have been in the focus of thermomechanical fatigue (TMF) only to a minor extent. In this paper, a feasible procedure is presented to set up material models and estimate service life of cast iron cylinder heads under variable thermomechanical loading conditions by the use of computer-aided engineering tools. In addition, the influence of thermal load and mechanical constraints on TMF life span is shown. A specimen model is used for parameter identification in material model setup and a cylinder head model is used for correlation with cracking phenomena. Investigation of different thermomechanical load influences is conducted on the cylinder head model. The principal strain and energy based fatigue criteria are used in assessment of TMF lifetime for the cast iron family and material specific evaluation procedures are pointed out. The results highlight the importance of exact definitions of the boundary conditions and underline the sensitivity of TMF lifespan of cast iron cylinder heads with respect to the defined boundary conditions. Considering this sensitivity, an approach conforming to the engine development requirements is proposed. It is shown that both the crack location and fatigue lifetime are predicted with high accuracy.
Measurement and Simulation of Instantaneous Emissions of a Heavy Truck Diesel Engine During Transients
Authors: Tauzia Xavier , Chesse Pascal , Hetet Jean-François , Thouvenel Nicolas
Published: January 2008

During the last decades, pollutant emissions from internal combustion engines used for transportation have become a major concern. Today, not only steady state emissions but also emissions during transients are regulated and have to be studied in order to be reduced. In this paper, we describe a new methodology developed to measure the instantaneous level of gaseous emissions from a internal combustion engine during transients, using an analyzer initially designed for steady state operation. Moreover, a new phenomenological thermodynamical combustion model is proposed in order to compute emissions during transients. The results of these two methods are compared on various transients. The measurement method seems to give good results (except for hydrocarbon (HC) measurements), as long as the speed and load variations are not too fast. Otherwise, the frequency of the analyzer which was used becomes the limiting factor. The new combustion heat release developed to simulate transients, coupled with an existing two-zone model for emission calculations, leads to satisfactory results for CO2 and O2 concentrations and NOx emissions. The agreement with measurements is good for smooth transients and seems promising for faster dynamics. The initial goal was reached, although some improvements are still necessary concerning HC measurements and the fastest transients. These results could be helpful when trying to reduce the amount of pollutant emissions at the exhaust during transients, directly or with after treatment devices.
Experimental studies of Ge1−xCx and Ge1−x−yCxAly thin films
Authors: Soukup R. J. , Huguenin-Love J. L. , Ianno N. J. , Thompson D. W.
Published: January 2008

Experimental results on thin films of the materials GexC1−x, and Ge1−x−yCxAly deposited by a unique multiplasma hollow cathode sputtering technique, are presented. These films have been characterized by several techniques, the most important being in situ and ex situ spectroscopic ellipsometry, Auger electron spectroscopy (AES), and x-ray diffraction (XRD). The spectroscopic ellipsometry measurements of the films indicate that this material is a very strong absorber of photons. The XRD measurements reveal that the carbon is entering the lattice in substitutional sites, to a certain extent, and the AES depth profiling measurements determine the concentration and location of the different constituents. The materials appear to grow such that the concentration of the different elements is uniform throughout the films. The photon absorption inferred from the ellipsometry measurements corroborated the fact that GeC has a photon absorption efficiency much greater than crystalline Si or Ge and even more than amorphous Si in the wavelength range most useful for photovoltaic applications. In fact, the addition of aluminum increased this absorption efficiency substantially.
Oxygen adsorption on Ag∕Si(111)-7×7 surfaces
Authors: Zhang Zhen , Jiao Jian , Jiang Zhiquan , Tan Dali , Fu Qiang , Bao Xinhe , Liu Xi , Jia Jinfeng , Xue Qikun
Published: January 2008

The growth of Ag clusters on Si(111)-7×7 surfaces was studied by scanning tunneling microscopy (STM), ultraviolet photoelectron spectroscopy, and x-ray photoelectron spectroscopy (XPS). A shift in the Ag 3d binding energy and a noticeable change in the valence-band structure reveal a significant modification of the electronic states of the Si(111)-7×7 surface and the dispersed Ag clusters, which had a strong dependence on the coverage of Ag. Furthermore, these Ag clusters deposited on the Si surface alter the behavior of oxygen adsorption on the Si(111)-7×7 surface. As evidenced by XPS, the presence of Ag inhibits the adsorbed surface oxygen species, the ins-ins and ad-ins oxygen, in which “ad” denotes an O atom bonding on top of the Si adatom and “ins” denotes an O atom inserted into a Si adatom back bond. The STM and high-resolution electron energy loss spectroscopy results show that the ins-ins oxygen species are more significantly suppressed by the Ag clusters compared to the ad-ins oxygen.
Quantitative determination of reaction products by in-line thermal desorption spectroscopy: The system methanol∕Pd(111)
Authors: Demirci E. , Winkler A.
Published: January 2008

An evaluation procedure for the quantitative determination of thermal desorption products based on in-line mass spectrometry is described in detail. This procedure is, in particular, applicable for species which are pumped with high speed and, therefore, yield small signals in conventional mass spectrometry. The evaluation method was applied to the adsorption system methanol∕Pd(111). After dosing a Pd(111) surface at 140K with methanol up to saturation of the chemisorbed layer, desorption of methanol, hydrogen, carbon monoxide, and water can be observed. The amount of all of the reaction products was determined quantitatively. The total number of adsorbed methanol was found to be 2.6×1014MeOH∕cm2, which is a lower limit, because coadsorbed water and hydrogen partially block adsorption sites on the surface. While 62% of the adsorbed methanol desorbs associatively, 38% of the adsorbed methanol dehydrogenate, resulting in desorption of hydrogen and carbon monoxide. No indication for C–O bond scission could be observed when methanol was adsorbed on the clean Pd(111) surface.
Surface roughening of silicon, thermal silicon dioxide, and low-k dielectric coral films in argon plasma
Authors: Yin Yunpeng , Sawin Herbert H.
Published: January 2008

The surface roughness evolutions of single crystal silicon, thermal silicon dioxide (SiO2), and low dielectric constant film coral in argon plasma have been measured by atomic force microscopy as a function of ion bombardment energy, ion impingement angle, and etching time in an inductively coupled plasma beam chamber, in which the plasma chemistry, ion energy, ion flux, and ion incident angle can be adjusted independently. The sputtering yield (or etching rate) scales linearly with the square root of ion energy at normal impingement angle; additionally, the angular dependence of the etching yield of all films in argon plasma followed the typical sputtering yield curve, with a maximum around 60°–70° off-normal angle. All films stayed smooth after etching at normal angle but typically became rougher at grazing angles. In particular, at grazing angles the rms roughness level of all films increased if more material was removed; additionally, the striation structure formed at grazing angles can be either parallel or transverse to the beam impingement direction, which depends on the off-normal angle. More interestingly, the sputtering caused roughness evolution at different off-normal angles can be qualitatively explained by the corresponding angular dependent etching yield curve. In addition, the roughening at grazing angles is a strong function of the type of surface; specifically, coral suffers greater roughening compared to thermal silicon dioxide.
Angular etching yields of polysilicon and dielectric materials in Cl2∕Ar and fluorocarbon plasmas
Authors: Yin Yunpeng , Sawin Herbert H.
Published: January 2008

The angular etching yields of polysilicon in Cl2∕Ar plasmas, and dielectric materials (thermal silicon dioxide and low-k dielectric coral) in fluorocarbon plasmas, have been characterized in an inductively coupled plasma beam apparatus. The effects of ion energy, feed gas composition, and plasma source pressure are studied. The experimental results showed that these etching parameters had a significant impact on the resulting angular etching yield curve. In particular, the angular etching yield curve was more sputteringlike at low plasma source pressure and/or low effective gas percentage (Cl2 and C4F8), with a peak around 60°–70° off-normal ion incident angle. In contrast, ion-enhanced-etching-like angular curves, which dropped gradually with off-normal angle, were formed at high plasma source pressure and/or high effective gas percentage. Further analysis indicated that the effective neutral-to-ion flux ratio reaching the surface was the primary factor influencing the angular etching yield curve. More specifically, the angular etching yield curve had physical sputtering characteristics at low neutral-to-ion flux ratios; while etching process was really dominated by ion-enhanced etching at high ratios and the angular curve was ion-enhanced-etching-like. The polymer deposition effects are also discussed in this article.
Two-stage flow-dividing system for the calibration of vacuum gauges
Authors: Yoshida Hajime , Arai Kenta , Akimichi Hitoshi , Hirata Masahiro
Published: January 2008

A two-stage flow-dividing system was developed for calibrating an ionization gauge (IG) and residual gas analyzer (RGA). This system generates a stable high and ultrahigh vacuum from 8×10−3to2×10−7Pa by adjusting the pressure in the first chamber using N2, Ar, He, and H2. The calibration pressure in the third chamber is calculated from the pressure in the second chamber using their linear relation in molecular flow. The uncertainty of the generated pressure was comparable to or several times larger than that of the continuous-expansion system. However, this system has a simple configuration and is easy to operate compared with the continuous-expansion system because it has no moving parts. Results of the calibration of IG and RGA showed that the two-stage flow-dividing system is useful for a routine calibration of practical vacuum gauges in high and ultrahigh vacuum.
Characterization of gaseous species in scanning atmospheric rf plasma with transmission infrared spectroscopy
Authors: Kim Seong H. , Kim Jeong Hoon , Kang Bang-Kwon
Published: January 2008

A scanning atmospheric radio-frequency (rf) plasma was analyzed with transmission infrared (IR) spectroscopy. The IR analyses were made for the plasmas used for hydrophobic coating deposition and superhydrophobic coating deposition processes. Since the rf plasma was generated in a small open space with a high gas flow rate in ambient air, the density of gas-phase molecules was very high and the plasma-generated reactive species seemed to undergo various reactions in the gas phase. So, the transmission IR spectra of the scanning atmospheric rf plasma were dominated by gas-phase reaction products, rather than plasma-generated intermediate species. In the CH4∕He plasma used for hydrophobic coating deposition, C2H6, C2H2, and a small amount of C2H4 as well as CO were detected in transmission IR. The intensities of these peaks increased as the rf power increased. The CO formation is due to the activation of oxygen and water in the air. In the CF4∕H2∕He plasma used for deposition of superhydrophobic coatings, C2F6, CF3H, COF2, and HF were mainly detected. When the H2∕CF4 ratio was ∼0.5, the consumption of CF4 was the highest. As the H2∕CF4 ratio increased higher, the C2F6 production was suppressed while the CF3H peak grew and the formation of CH4 were detected. In both CH4∕He and CF4∕H2∕He plasma systems, the undissociated feed gas molecules seem to be highly excited vibrationally and rotationally. The information on plasma-generated reactive species and their reactions was deduced from the distribution of these gas-phase reaction products.
Lithium phosphorous oxynitride films synthesized by a plasma-assisted directed vapor deposition approach
Authors: Kim Yoon Gu , Wadley H. N. G.
Published: January 2008

A plasma-assisted directed vapor deposition approach has been explored for the synthesis of lithium phosphorous oxynitride (Lipon) thin films. A Li3PO4 source was first evaporated using a high voltage electron beam and the resulting vapor entrained in a nitrogen-doped supersonic helium gas jet and deposited on a substrate at ambient temperature. This approach failed to incorporate significant concentrations of nitrogen in the films. A hollow cathode technique was then used to create an argon plasma that enabled partial ionization of both the Li3PO4 vapor and nitrogen gas just above the substrate surface. The plasma-enhanced deposition process greatly increased the gas phase and surface reactivity of the system and facilitated the synthesis and high rate deposition of amorphous Lipon films with the N∕P ratios between 0.39 and 1.49. Manipulation of the plasma-enhanced process conditions also enabled control of the pore morphology and significantly affected the ionic transport properties of these films. This enabled the synthesis of electrolyte films with lithium ion conductivities in the 10−7–10−8S∕m range. They appear to be well suited for thin-film battery applications.
Real-Time PCR Approach for Detection of Environmental Sources of
Campylobacter
Strains Colonizing Broiler Flocks
Authors: Ridley Anne M. , Allen Vivien M. , Sharma Meenaxi , Harris Jill A. , Newell Diane G.
Published: January 2008

Reducing colonization of poultry flocks by
Campylobacter
spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the
flaA
short variable region (SVR) of
Campylobacter jejuni
or
C
.
coli
. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different
C
.
jejuni
strains, to be 1.3 × 10
2
to 3.7 × 10
2
CFU/ml in bacterial cultures and 6.6 × 10
2
CFU/ml in spiked fecal samples. The specificity of the SVR for
C
.
jejuni
and
C
.
coli
was confirmed by using a panel of strains comprising other
Campylobacter
species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of
flaA
, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.
Novel Microarray Design Strategy To Study Complex Bacterial Communities
Authors: Huyghe Antoine , Francois Patrice , Charbonnier Yvan , Tangomo-Bento Manuela , Bonetti Eve-Julie , Paster Bruce J. , Bolivar Ignacio , Baratti-Mayer Denise , Pittet Didier , Schrenzel Jacques
Published: January 2008

Assessing bacterial flora composition appears to be of increasing importance to fields as diverse as physiology, development, medicine, epidemiology, the environment, and the food industry. We report here the development and validation of an original microarray strategy that allows analysis of the phylogenic composition of complex bacterial mixtures. The microarray contains ∼9,500 feature elements targeting 16S rRNA gene-specific regions. Probe design was performed by selecting oligonucleotide sequences specific to each node of the seven levels of the bacterial phylogenetic tree (domain, phylum, class, order, family, genus, and species). This approach, based on sequence information, allows analysis of the bacterial contents of complex bacterial mixtures to detect both known and unknown microorganisms. The presence of unknown organisms can be suspected and mapped on the phylogenetic tree, indicating where to refine analysis. Initial proof-of-concept experiments were performed on oral bacterial communities. Our results show that this hierarchical approach can reveal minor changes (≤1%) in gingival flora content when samples collected in individuals from similar geographical origins are compared.
Bacterial Communities of
Bartonella
-Positive Fleas: Diversity and Community Assembly Patterns
Authors: Jones Ryan T. , McCormick Katherine F. , Martin Andrew P.
Published: January 2008

We investigated the bacterial communities of nine
Bartonella
-positive fleas (
n
= 6
Oropsylla hirsuta
fleas and
n
= 3
Oropsylla montana
fleas), using universal primers, clone libraries, and DNA sequencing. DNA sequences were used to classify bacteria detected in a phylogenetic context, to explore community assembly patterns within individual fleas, and to survey diversity patterns in dominant lineages.
The Chemolithoautotroph
Acidithiobacillus ferrooxidans
Can Survive under Phosphate-Limiting Conditions by Expressing a C-P Lyase Operon That Allows It To Grow on Phosphonates
Authors: Vera Mario , Pagliai Fernando , Guiliani Nicolas , Jerez Carlos A.
Published: January 2008

The chemolithoautotrophic bacterium
Acidithiobacillus ferrooxidans
is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (P
i
), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is P
i
, some bacteria are also able to metabolize P
i
esters (with a C-O-P bond) and phosphonates (with a very inert C-P bond). By using bioinformatic analysis of genomic sequences of the type strain of
A. ferrooxidans
(ATCC 23270), we found that as part of a Pho regulon, this bacterium has a complete gene cluster encoding C-P lyase, which is the main bacterial enzyme involved in phosphonate (Pn) degradation in other microorganisms.
A. ferrooxidans
was able to grow in the presence of methyl-Pn or ethyl-Pn as an alternative phosphorus source. Under these growth conditions, a great reduction in inorganic polyphosphate (polyP) levels was seen compared with the level for cells grown in the presence of P
i
. By means of reverse transcription-PCR (RT-PCR), DNA macroarrays, and real-time RT-PCR experiments, it was found that
A. ferrooxidans phn
genes were cotranscribed and their expression was induced when the microorganism was grown in methyl-Pn as the only phosphorus source. This is the first report of phosphonate utilization in a chemolithoautotrophic microorganism. The existence of a functional C-P lyase system is a clear advantage for the survival under P
i
limitation, a condition that may greatly affect the bioleaching of ores.
Detection of Viable but Nonculturable
Escherichia coli
O157:H7 Bacteria in Drinking Water and River Water
Authors: Liu Yanming , Gilchrist Ainslie , Zhang Jing , Li Xing-Fang
Published: January 2008

A sensitive method for specific detection of viable
Escherichia coli
O157:H7 cells, including viable but nonculturable (VBNC) cells, in water samples was developed. This method involved capture of the bacterial cells on a low-protein-binding membrane and direct extraction and purification of RNA followed by reverse transcription-PCR and electronic microarray detection of the
rfbE
and
fliC
genes of
E. coli
O157:H7. It detected as few as 1 CFU of
E. coli
O157:H7 in diluted cultures, 3 to 4 CFU/liter in tap water, 7 CFU/liter in river water, and 50 VBNC cells in 1 liter of river water, demonstrating the best limit of detection reported to date for VBNC cells in environmental water samples.
Emergence of Secretion-Defective Sublines of
Pseudomonas aeruginosa
PAO1 Resulting from Spontaneous Mutations in the
vfr
Global Regulatory Gene
Authors: Fox Áine , Haas Dieter , Reimmann Cornelia , Heeb Stephan , Filloux Alain , Voulhoux Romé
Published: January 2008

Pseudomonas aeruginosa
undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene
lasR
. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the
vfr
gene, which is known to control positively the expression of the
lasR
gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the
vfr
mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a
vfr
mutant of PAO1, we observed that the
vfr
mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous
vfr
emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to
P. aeruginosa
under certain environmental conditions.
Trm1p, a Zn(II)
2
Cys
6
-Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast
Candida boidinii
Authors: Sasano Yu , Yurimoto Hiroya , Yanaka Mikiko , Sakai Yasuyoshi
Published: January 2008

The methylotrophic yeasts are commonly used as hosts for heterologous gene expression. In this study, we describe a novel gene,
TRM1
, in
Candida boidinii
, responsible for the transcriptional activation of several methanol-inducible promoters. The encoded protein, Trm1p, is a Zn(II)
2
Cys
6
-type zinc cluster protein. Deletion of
TRM1
completely inhibits growth on methanol but causes no growth defect on glucose or other nonfermentative carbon sources, glycerol, ethanol, or oleate. Trm1p is responsible for transcriptional activation of five methanol-inducible promoters tested, but not for peroxisome assembly or peroxisomal protein transport. Expression of the
TRM1
gene was constitutive, and Trm1p localizes to the nuclei regardless of the carbon source. Two
cis
-acting methanol response elements (MREs), MRE1 and MRE2 are present in the promoter of the dihydroxyacetone synthase gene. Trm1p is shown to be required for MRE1-dependent methanol-inducible gene expression. Chromatin immunoprecipitation assays reveal that Trm1p binds to five methanol-inducible promoters upon methanol induction but does not bind in glucose-grown cells. Thus, the
TRM1
gene encodes a master transcriptional regulator responsible for methanol-specific gene activation in the methylotrophic yeasts.
TheXanthomonas oryzaepv. oryzae PhoPQ Two-Component System Is Required for AvrXA21 Activity,hrpGExpression, and Virulence
Authors: Lee Sang-Won , Jeong Kyu-Sik , Han Sang-Wook , Lee Seung-Eun , Phee Bong-Kwan , Hahn Tae-Ryong , Ronald Pamela
Published: January 2008

The rice pathogen recognition receptor, XA21, confers resistance toXanthomonas oryzaepv. oryzae strains producing the type one system-secreted molecule, AvrXA21.X. oryzaepv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eightrax(required forAvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in araxRgene knockout strain. This survey led to the identification of thephoPgene encoding a response regulator that is up-regulated in theraxRknockout strain. Next we generated aphoPknockout strain and found it to be impaired inX. oryzaepv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca2+. The impaired virulence of thephoPknockout strain can be partially complemented by constitutive expression ofhrpG, indicating that PhoP controls a key aspect ofX. oryzaepv. oryzae virulence through regulation ofhrpG. A gene encoding the cognate putative histidine protein kinase,phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in aphoQknockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that theX. oryzaepv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH.
Proteomic, Microarray, and Signature-Tagged Mutagenesis Analyses of AnaerobicPseudomonas aeruginosaat pH 6.5, Likely Representing Chronic, Late-Stage Cystic Fibrosis Airway Conditions
Authors: Platt Mark D. , Schurr Michael J. , Sauer Karin , Vazquez Gustavo , Kukavica-Ibrulj Irena , Potvin Eric , Levesque Roger C. , Fedynak Amber , Brinkman Fiona S. L. , Schurr Jill , Hwang Sung-Hei , Lau Gee W. , Limbach Patrick A. , Rowe John J. , Lieberman Michael A. , Barraud Nicolas , Webb Jeremy , Kjelleberg Staffan , Hunt Donald F. , Hassett Daniel J.
Published: January 2008

Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogenPseudomonas aeruginosain their airways. During chronic late-stage CF,P. aeruginosais known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of ∼6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant “anaerobiome” or “proteogenome” ofP. aeruginosawas assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO3−and NO2−are those encoding Pf1 bacteriophage. With anaerobic NO2−, the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library ofP. aeruginosawas constructed to further screen genes required for both NO3−and NO2−respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr,dnr,nar,nir, andnuo), thecysGanddksAgenes were found to be required for both anaerobic NO3−and NO2−respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO3−and NO2−respiration and offers clues as to how we might disrupt such pathways inP. aeruginosato limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.
Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the
Bacillus megaterium
Spore Germinant Receptor
Authors: Christie Graham , Lazarevska Milena , Lowe Christopher R.
Published: January 2008

The extreme metabolic dormancy and resistance properties of spores formed by members of the
Bacillus
and
Clostridium
genera are lost upon exposure to a variety of small-molecule germinants. Germinants are known to interact in an as yet undefined manner with cognate receptor complexes that reside in the inner membrane that surrounds the spore protoplast. The receptor itself is a complex of at least three proteins, and in this study we identify amino acid residues, predicted to lie in loop regions of GerVB on the exterior aspect of the membrane, that influence the
Bacillus megaterium
spore germination response. Three consecutive residues adjacent to putative transmembrane domain 10 (TM10) were demonstrated to mediate to various degrees the proline germinative response while also influencing germination in response to leucine, glucose, and inorganic salts, suggesting that this region may be part of a ligand binding pocket. Alternatively, substitutions in this region may affect the conformation of associated functionally important TM regions. Leucine- and KBr-mediated germination was also influenced by substitutions in other outer loop regions. These observations, when considered with accompanying kinetic analyses that demonstrate cooperativity between germinants, suggest that binding sites for the respective germinants are in close spatial proximity in the receptor but do not overlap. Additionally, proline recognition was conferred to a chimeric receptor when TM regions associated with the putative binding loop were present, indicating that residues in TM9 and/or TM10 of GerVB are also of functional importance in the proline-induced germinative response.
Structure-Function Relationships in the HAMP and Proximal Signaling Domains of the Aerotaxis Receptor Aer
Authors: Watts Kylie J. , Johnson Mark S. , Taylor Barry L.
Published: January 2008

Aer, the
Escherichia coli
aerotaxis receptor, faces the cytoplasm, where the PAS (
P
er-
A
RNT-
S
im)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (
h
istidine kinase,
a
denylyl cyclase,
m
ethyl-accepting protein, and
p
hosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from
Archaeoglobus fulgidus
was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.
High-intensity ultraendurance promotes early release of muscle injury markers
Authors: Bessa A , Nissenbaum M , Monteiro A , Gandra P G , Nunes L S , Bassini-Cameron A , Werneck-de-Castro J P S , de Macedo D Vaz , Cameron L-C
Published: January 2008

Objective:

To evaluate the impact of high-intensity ultraendurance (HIU) cycling, using it as a possible way to understand muscle injury kinetics and blood immune cells’ release during high-intensity prolonged exercise

Design:

Male amateur triathletes enrolled during a cycling race of the International Bike Championship 800 km cycling relay (∼23 h). Each athlete alternately cycled 20–25 minutes until exhaustion and performed a total of approximately 200 km.

Results:

Creatine kinase levels in blood reached a 300% rise in a sigmoidal pattern, while lactate dehydrogenase levels increased by 30–40% following a hyperbolic pattern. Aspartate aminotransferase and alanine aminotransferase levels increased by up to 250% and 140%, respectively. Liver injury markers such as alkaline phosphatase and γ-glutamyltransferase remained stable. Platelets increased by 20–30% from pre-exercise, and there was no change in haematocrit during the race. White blood cells rose by nearly 200%. Leucocytes rose 210% during the race, with a major component coming from neutrophils, which increased more than 300%. Triacylglycerol levels were decreased at the finish and total cholesterol levels remained unchanged. Urate increased (by up to 35%) during the first half of the race, and urea levels increased with a different pattern, increasing by 45% in the second half.

Conclusions:

This study showed the blood appearance kinetics of muscle injury markers and some metabolites. It is suggested that the increase in these enzymes came primarily from muscle damage, rather than liver damage, and that white blood cells are selectively mobilised independently of haemoconcentration. The early appearance of muscle injury markers in this kind of exercise was also shown.
Is the association between job strain and carotid intima-media thickness attributable to pre-employment environmental and dispositional factors? The Cardiovascular Risk in Young Finns Study
Authors: Hintsa T , Kivimäki M , Elovainio M , Vahtera J , Hintsanen M , Viikari J S A , Raitakari O T , Keltikangas-Järvinen L
Published: January 2008

Objectives:

Most previous studies of job strain and cardiovascular risk have been limited to adult data. It remains unclear whether this association might be explained by factors already present before entering the labour market. This study examined whether pre-employment family factors and participants’ own dispositional factors contribute to the relationship between job strain and carotid intima-media thickness (CIMT) among male employees.

Methods:

The sample consisted of 494 men from the Cardiovascular Risk in Young Finns Study. Parental socioeconomic position and parental life dissatisfaction were assessed at 9–21 years of age and components of type A behaviour (Hunter-Wolf) were assessed at 12–24 years of age before the participants had entered the labour market. Job strain, education and CIMT were assessed at 27–39 years of age when all participants were employed.

Results:

There was an association between higher job strain and increased CIMT in adulthood (mean 0.59 mm; 95% CI 0.42 to 0.76) which was only slightly affected on adjustment for parental socioeconomic position and parental life dissatisfaction as well as participants’ education. However, the job strain/CIMT relationship attenuated by 17% to non-significant after taking into account the effect of the participants’ type A behaviour components.

Conclusions:

In this contemporary cohort of men, lack of leadership (a type A behaviour component) contributed to the association between job strain and CIMT 15 years later, whereas pre-employment family factors had only a modest effect on this association.
Role of comorbidities in a cohort of patients with COPD undergoing pulmonary rehabilitation
Authors: Crisafulli E , Costi S , Luppi F , Cirelli G , Cilione C , Coletti O , Fabbri L M , Clini E M
Published: January 2008

Background:

Chronic obstructive pulmonary disease (COPD) is often associated with other chronic diseases. These patients are often admitted to hospital based rehabilitation programmes.

Objectives:

To determine the prevalence of chronic comorbidities in patients with COPD undergoing pulmonary rehabilitation and to assess their influence on outcome.

Design:

Observational retrospective cohort study.

Setting:

A single rehabilitation centre.

Patients:

2962 inpatients and outpatients with COPD (73% male, aged 71 (SD 8) years, forced expiratory volume in 1 s (FEV1) 49.3 (SD 14.8)% of predicted), graded 0, 1 or ⩾2 according to the comorbidity categories and included in a pulmonary rehabilitation programme.

Measurements:

The authors analysed the number of self-reported comorbidities and recorded the Charlson Index. They then calculated the percentage of patients with a predefined positive response to pulmonary rehabilitation (minimum clinically important difference (MCID)), as measured by improvement in exercise tolerance (6 min walking distance test (6MWD)), dyspnoea (Medical Research Council scale) and/or health related quality of life (St George’s Respiratory Questionnaire (SGRQ)).

Results:

51% of the patients reported at least one chronic comorbidity added to COPD. Metabolic (systemic hypertension, diabetes and/or dyslipidaemia) and heart diseases (chronic heart failure and/or coronary heart disease) were the most frequently reported comorbid combinations (61% and 24%, respectively) among the overall diseases associated with COPD. The prevalence of patients with MCID was different across the comorbidity categories and outcomes. In a multiple categorical logistic regression model, the Charlson Index (OR 0.72 (96% CI 0.54 to 0.98) and 0.51 (96% CI 0.38 to 0.68) vs 6MWD and SGRQ, respectively), metabolic diseases (OR 0.57 (96% CI 0.49 to 0.67) vs 6MWD) and heart diseases (OR 0.67 (96% CI 0.55 to 0.83) vs SGRQ) reduced the probability to improve outcomes of rehabilitation.

Conclusions:

Most patients with COPD undergoing pulmonary rehabilitation have one or more comorbidities. Despite the fact that the presence of comorbidities does not preclude access to rehabilitation, the improvement in exercise tolerance and quality of life after rehabilitation may be reduced depending on the comorbidity.