bluuebunny/crossref_metadata_2025 huggingface dataset:
The ‘neuroadrenergic hypothesis’ in hypertension: current evidence
Authors: Grassi Guido , Seravalle Gino , Quarti‐Trevano Fosca
Published: December 2009

Data collected in experimental animals and in humans support the hypothesis that sympathetic neural mechanisms are involved in the development and progression of hypertension. Direct approaches to assess human adrenergic cardiovascular drive have shown that sympathetic activation occurs in hypertensive patients, the magnitude of which is proportional to the degree of elevation of the blood pressure. Evidence has also been obtained that sympathetic activation participates in the development of hypertension‐related target organ damage, such as left ventricular diastolic dysfunction, left ventricular hypertrophy and arterial remodelling and hypertrophy. Despite the large amount of information collected on the main features of the hypertension‐related neurogenic abnormality, the causes of the sympathetic activation remain undefined, although alterations in the reflex modulation of adrenergic drive and/or participation of metabolic factors are likely candidates. This paper will provide background information on the behaviour of the sympathetic nervous system in experimental hypertension, followed by a review of the main features, mechanisms and effects of the sympathetic overdrive in human hypertension. Finally, the new frontiers of research in the area of therapeutic intervention aimed at reducing the adrenergic overdrive will be highlighted.
Enhanced survival of vascular smooth muscle cells accounts for heightened elastin deposition in arteries of neonatal spontaneously hypertensive rats
Authors: Arribas Silvia M. , Hermida Carmen , González M. Carmen , Wang Yanting , Hinek Aleksander
Published: December 2009

Abnormal stiffening and narrowing of arteries are characteristic features of spontaneously hypertensive rats (SHR). In this strain, we have previously demonstrated an increased elastin content and abnormal organization of lamellae in conduit and resistance arteries from neonatal rats that preceded the impending inward remodelling, increased vascular stiffness and development of hypertension. The aim of this study was to assess the mechanism responsible for such excessive and aberrant elastin deposition in SHR vessels during perinatal development. We compared elastin, collagen and fibronectin production (inmunocytochemistry and quantitative assay of metabolically labelled insoluble elastin), DNA content as well as cell proliferation (proliferative cellular nuclear antigen, bromodeoxyuridine incorporation) and death rates (propidium iodide exclusion test, terminal transferase nick and labeling (TUNEL) assay) in cultures of vascular smooth muscle cells (VSMC) derived from neonatal SHR and Wistar–Kyoto (WKY) control rats. Cultures of VSMC derived from neonatal SHR exhibited hypertrophy, produced more elastin, collagen and fibronectin and contained more DNA than equally plated WKY counterparts. Further analysis revealed that the higher net DNA content in SHR‐derived cultures was due to increased diploidy, but not to a heightened cell multiplication. The SHR‐derived VSMC also exhibited lower rates of cell death and apoptosis, which were associated with increased levels of the anti‐apoptotic protein, survivin. We therefore conclude that the peculiar heightened survival of matrix‐producing VSMC in neonatal SHR is responsible for accumulation of hard‐wearing elastin and other extracellular matrix elements in the growing arteries, thereby contributing to the subsequent development of systemic hypertension.
Resolution and total blur: Correlation and focus dependencies in e-beam lithography
Authors: Keil K. , Hauptmann M. , Kretz J. , Constancias C. , Pain L. , Bartha J.-W.
Published: December 2009

The resolution limit in e-beam lithography is dependent on the resist process, the proximity effect, and the e-beam writer itself. However, it is difficult to distinguish these contributions just by comparing critical dimensions (CDs) of isolated and dense lines. Since the calculation of the parameter α of the point-spread function is usually based on the CD measurement of resist structures, α should include all mentioned contributions. Therefore, the total blur αtotal can be defined as the square root of the squared sums of αprocess, αforwardscattering, and αbeamblur. With keeping αprocess constant, the authors compared Gaussian beam (GB) and variable shaped beam (VSB) writers via the isofocal-dose method and proved that the isofocal dose is independent of the beam profile. For further comparison, they enhanced the method to the linewidth roughness and the total blur. For the latter, a special frequency modulated pattern was applied, which enables the determination of αtotal in dependency on the focus. The analysis of the total blur in best focus and the resolution of dense lines, exposed in a negative chemically amplified resist on GB and VSB writers, indicates their proportionality, which is supported by the same analysis with a nonchemically amplified resist. The resolution of the VSB writer cannot be directly compared to the GB writers’ resolution since the beam blur of the former changes with the shot size.
Scanning-helium-ion-beam lithography with hydrogen silsesquioxane resist
Authors: Winston D. , Cord B. M. , Ming B. , Bell D. C. , DiNatale W. F. , Stern L. A. , Vladar A. E. , Postek M. T. , Mondol M. K. , Yang J. K. W. , Berggren K. K.
Published: December 2009

A scanning-helium-ion-beam microscope is now commercially available. This microscope can be used to perform lithography similar to, but of potentially higher resolution than, scanning electron-beam lithography. This article describes the control of this microscope for lithography via beam steering/blanking electronics and evaluates the high-resolution performance of scanning helium-ion-beam lithography. The authors found that sub-10nm-half-pitch patterning is feasible. They also measured a point-spread function that indicates a reduction in the micrometer-range proximity effect typical in electron-beam lithography.
Real time scatterometry for profile control during resist trimming process
Authors: El Kodadi M. , Soulan S. , Besacier M. , Schiavone P.
Published: December 2009

In situ and real time control of the different process steps in semiconductor device manufacturing becomes a critical challenge, especially for the lithography and plasma etching processes. Dynamic scatterometry is among the few solutions able to meet the requirement for in line monitoring. In this article, the authors demonstrate that dynamic scatterometry can be used as a real time monitoring technique during the resist trimming process. Different process parameters, such as chemistries and bias power, were used in the experiments for the demonstration; they discuss the influence of these different parameters on the measurement. For validation purposes, the dynamic scatterometry measurements are compared to three dimensional atomic force microscopy measurements made in the same process conditions. The agreement between both is excellent.
Automatic measurement of electron beam size by beam metrology technique using 20nm test pattern
Authors: Peroz C. , Babin S. , Machin M. , Anderson E. , Cabrini S. , Dhuey S. , Harteneck B.
Published: December 2009

A robust operator independent measurement of electron beam sizes in two coordinates is demonstrated by using beam metrology (BEAMETR) technique. This method involves software associated with a specially designed pattern sample. The fabrication of this sample was done using 100keV electron beam lithography and lift-off of metal. A proximity correction was applied to improve pattern quality. The minimum feature size of the fabricated BEAMETR patterns was 20nm; this allowed for the measuring of beam size down to 2nm. Beam size and shape measurements were done using three scanning electron microscopes; their operating conditions (voltage, aperture, and astigmatism) were varied. Repeatability and test pattern dependence were also studied, which demonstrated a good consistency of the results.
The Lorentz transformation: Simplification through complexification
Authors: Newman Ezra T. , Price Richard H.
Published: December 2009

The general Lorentz transformation, the relation between observations made in different frames, involves the three components of the relative velocity of the two frames and three parameters describing the relative orientation of the frames. A simpler description is known in which the six parameters are encoded in four complex numbers subject to a single algebraic constraint. This description is tied to the transformation of the directions of light propagation and underscores how closely related special relativity is to light propagation. This form of the transformation, which is particularly useful for dealing with the aberration of light is usually introduced through advanced mathematical formulations. We show that the basic idea, in addition to being useful, is quite simple.
A cost-efficient frequency domain fluorescence lifetime measurement system
Authors: Yuan Baohong , McClellan Samantha R. , Al-Mifgai Bandar F. , Growney Emily A. , Komolafe Olamide A.
Published: December 2009

A cost-efficient frequency domain system for measuring nanosecond fluorescence lifetime has been designed and tested. In this design, function generators are adopted to directly modulate a light-emitting diode at radio frequencies. A heterodyne detection technique is used to reduce the bandwidth of the system and to improve the signal-to-noise ratio. A fluorescence lifetime close to 2 ns was measured and verified by comparison with the results from a broadband lock-in amplifier. The total cost of the system is reasonable for undergraduate laboratories, which enables students to gain hands-on experience in designing and constructing a nanosecond fluorescence lifetime measurement system. Some suggestions for conducting the measurement with a limited budget are provided.
A simple model of a Slinky walking down stairs
Authors: Hu Ai-Ping
Published: December 2009

The motion of a Slinky walking down a set of stairs is modeled by a simple dynamical system with two degrees of freedom undergoing inelastic collisions. Numerical integration of the model’s equations of motion shows that the model’s behavior is similar to observations of the motion of an actual Slinky. In particular, it is found that the model’s motion exhibits a periodic gait, although subject to more restrictive launch conditions than an actual Slinky.
Determining the muon mass in an instructional laboratory
Authors: Brau Benjamin , May Christopher , Ormond Robert , Essick John
Published: December 2009

An instructional laboratory experiment to measure the muon mass is described. Using coincidence-anticoincidence detection, the decay of a cosmic-ray muon into an electron (or positron) is observed in a multiplate spark chamber, and recorded with a triggered CCD detector. The energy of the charged decay-product particle is quantified by counting the number of spark gaps ns it traverses before being stopped by the chamber’s aluminum plates. By running this apparatus under computer control for several hours, the number of product particles N(ns) with various ns-values is obtained. The muon mass is obtained by a least-squares fit of the experimentally observed N(ns) to simulation values predicted for this distribution by the Fermi description of muon decay via the weak interaction. We present our results for the muon mass and discuss the simulation we developed to account for the observed skewing of N(ns) due to the various directions the spark-producing product particles move as well as the escape of some of the higher-energy particles from the chamber.
The EPR paradox, Bell’s inequality, and the question of locality
Authors: Blaylock Guy
Published: December 2009

Most physicists agree that the Einstein–Podolsky–Rosen–Bell paradox exemplifies much of the strange behavior of quantum mechanics, but argument persists about what assumptions underlie the paradox. To clarify what the debate is about, we employ a simple and well-known thought experiment involving two correlated photons to help us focus on the logical assumptions needed to construct the EPR and Bell arguments. The view presented in this paper is that the minimal assumptions behind Bell’s inequality are locality and counterfactual definiteness but not scientific realism, determinism, or hidden variables as are often suggested. We further examine the resulting constraints on physical theory with an illustration from the many-worlds interpretation of quantum mechanics—an interpretation that we argue is deterministic, local, and realist but that nonetheless violates the Bell inequality.
A model of peak production in oil fields
Authors: Abrams Daniel M. , Wiener Richard J.
Published: December 2009

We developed a model for oil production on the basis of simple physical considerations. The model provides a basic understanding of Hubbert’s empirical observation that the production rate for an oil-producing region reaches its maximum when approximately half the recoverable oil has been produced. According to the model, the oil production rate at a large field must peak before drilling peaks. We use the model to investigate the effects of several drilling strategies on oil production. Despite the model’s simplicity, predictions for the timing and magnitude of peak production match data on oil production from major oil fields throughout the world.
A comparison of the temporal weighting of annoyance and loudness
Authors: Dittrich Kerstin , Oberfeld Daniel
Published: December 2009

The influence of single temporal portions of a sound on global annoyance and loudness judgments was measured using perceptual weight analysis. The stimuli were 900-ms noise samples randomly changing in level every 100 ms. For loudness judgments, Pedersen and Ellermeier [J. Acoust. Soc. Am. 123, 963–972 (2008)] found that listeners attach greater weight to the beginning and ending than to the middle of a stimulus. Qualitatively similar weights were expected for annoyance. Annoyance and loudness judgments were obtained from 12 listeners in a two-interval forced-choice task. The results demonstrated a primacy effect for the temporal weighting of both annoyance and loudness. However, a significant recency effect was observed only for annoyance. Potential explanations of these weighting patterns are discussed. Goodness-of-fit analysis showed that the prediction of annoyance and loudness can be improved by allowing a non-uniform weighting of single temporal portions of the signal, rather than assuming a uniform weighting as in measures like the energy-equivalent level (Leq). A second experiment confirmed that the listeners were capable of separating annoyance and loudness of the stimuli. Noises with the same Leq but different amplitude modulation depths were judged to differ in annoyance but not in loudness.
Use of the matching pursuit algorithm with a dictionary of asymmetric waveforms in the analysis of transient evoked otoacoustic emissions
Authors: Jedrzejczak W. Wiktor , Kwaskiewicz Konrad , Blinowska Katarzyna J. , Kochanek Krzysztof , Skarzynski Henryk
Published: December 2009

Transiently evoked otoacoustic emissions (TEOAEs) are normally modeled as the sum of asymmetric waveforms. However, some previous studies of TEOAEs used time-frequency (TF) methods to decompose the signals into symmetric waveforms. This approach was justified mainly as a means to reduce the complexity of the calculations. The present study extended the dictionary of numeric functions to incorporate asymmetric waveforms into the analysis. The necessary calculations were carried out using an adaptive approximation algorithm based on the matching pursuit (MP) numerical technique. The classic MP dictionary uses Gabor functions and consists of waveforms described by five parameters, namely, frequency, latency, time span, amplitude, and phase. In the present investigation, a sixth parameter, the degree of asymmetry, was added in order to enhance the flexibility of this approach. The effects of expanding the available functions were evaluated by means of both simulations using synthetic signals and authentic TEOAEs. The resulting analyses showed that the contributions of asymmetric components in the OAE signal are appreciable. In short, the expanded analysis method brought about important improvements in identifying TEOAE components including the correct detection of components with long decays, which are often related to spontaneous OAE activity, the elimination of a “dark energy” effect in TF distributions, and more reliable estimates of latency-frequency relationships. The latter feature is especially important for correct estimation of latency-frequency data, which is a crucial factor in investigations of OAE-generation mechanisms.
Authors’ Response to Strasberg’s “Comment on ‘Measurement of the frequency dependence of the ultrasonic parametric threshold amplitude for a fluid-filled cavity’ ” [J. Acoust. Soc. Am. 125, 1857 (2009)]
Authors: Teklu A. , McPherson Michael S. , Breazeale M. A. , Hasse Roger D. , Declercq Nico F.
Published: December 2009

This letter is a response to Strasberg’s recent paper, “Comment on ‘Measurement of the frequency dependence of the ultrasonic parametric threshold amplitude for a fluid-filled cavity.’” The authors dispute the conclusions of Strasberg regarding the effect observed by Teklu et al. [J. Acoust. Soc. Am. 120, 657–660 (2006)] published previously in JASA.
High-rate synthetic aperture communications in shallow water
Authors: Song H. C. , Hodgkiss W. S. , Kuperman W. A. , Akal T. , Stevenson M.
Published: December 2009

Time reversal communication exploits spatial diversity to achieve spatial and temporal focusing in complex ocean environments. Spatial diversity can be provided easily by a vertical array in a waveguide. Alternatively, spatial diversity can be obtained from a virtual horizontal array generated by two elements, a transmitter and a receiver, due to relative motion between them, referred to as a synthetic aperture. This paper presents coherent synthetic aperture communication results from at-sea experiments conducted in two different frequency bands: (1) 2–4kHz and (2) 8–20kHz. Case (1) employs binary-phase shift-keying modulation, while case (2) involves up to eight-phase shift keying modulation with a data rate of 30kbits∕s divided by the number of transmissions (diversity) to be accumulated. The receiver utilizes time reversal diversity combining followed by a single channel equalizer, with frequent channel updates to accommodate the time-varying channel due to coupling of space and time in the presence of motion. Two to five consecutive transmissions from a source moving at 4kts over 3–6km range in shallow water are combined successfully after Doppler compensation, confirming the feasibility of coherent synthetic aperture communications using time reversal.
Automatic estimation of position and orientation of an acoustic source by a microphone array network
Authors: Nakano Alberto Yoshihiro , Nakagawa Seiichi , Yamamoto Kazumasa
Published: December 2009

A method which automatically provides the position and orientation of a directional acoustic source in an enclosed environment is proposed. In this method, different combinations of the estimated parameters from the received signals and the microphone positions of each array are used as inputs to the artificial neural network (ANN). The estimated parameters are composed of time delay estimates (TDEs), source position estimates, distance estimates, and energy features. The outputs of the ANN are the source orientation (one out of four possible orientations shifted by 90° and either the best array which is defined as the nearest to the source) or the source position in two dimensional/three dimensional (2D/3D) space. This paper studies the position and orientation estimation performances of the ANN for different input/output combinations (and different numbers of hidden units). The best combination of parameters (TDEs and microphone positions) yields 21.8% reduction in the average position error compared to the following baselines and a correct orientation ratio greater than 99%. Position localization baselines consist of a time delay of arrival based method with an average position error of 34.1cm and the steered response power with phase transform method with an average position error of 29.8cm in 3D space.
Effects of voicing in the recognition of concurrent syllables
Authors: Vestergaard Martin D. , Patterson Roy D.
Published: December 2009

This letter reports a study designed to measure the benefits of voicing in the recognition of concurrent syllables. The target and distracter syllables were either voiced or whispered, producing four combinations of vocal contrast. Results show that listeners use voicing whenever it is present either to detect a target syllable or to reject a distracter. When the predictable effects of audibility were taken into account, limited evidence remained for the harmonic cancellation mechanism thought to make rejecting distracter syllables more effective than enhancing target syllables.
Resonant ultrasound studies of the layered perovskite system Ca2−xSrxRuO4
Authors: Luan Yanbing , Keppens Veerle , Jin Rongying , Mandrus David
Published: December 2009

The elastic response of the layered perovskite system Ca2−xSrxRuO4 (0.2≤x≤2) has been studied as a function of temperature and doping concentration x using resonant ultrasound spectroscopy. The elastic constants c11 and c44 have been obtained for three polycrystalline samples (x=1.0, 0.5, and 0.3) and show a softening trend with increasing Ca-content. In addition, the temperature-dependence of the elastic response of five single-crystals (x=2.0, 1.9, 0.5, 0.3, and 0.2) has been measured. For 2.0≥x≥0.5, a dramatic softening over a wide temperature range is observed upon cooling, which is attributed to the rotational instability of RuO6 octahedra (for x=2.0 and 1.9) and the static rotation of the octahedra (for x=0.5). For the Ca-rich samples (x=0.3 and 0.2), the softening occurs in a very narrow temperature range, corresponding to the structural phase transition from high-temperature tetragonal to low-temperature orthorhombic symmetry.
Direct Link between Toluene Degradation in Contaminated-Site Microcosms and a
Polaromonas
Strain
Authors: Sun Weimin , Xie Shuguang , Luo Chunling , Cupples Alison M.
Published: December 2009

Stable isotope probing (SIP) was used to identify the aerobic toluene-degrading microorganism in soil microcosms. Several approaches (terminal restriction fragment length polymorphism, 16S rRNA gene sequencing, and quantitative PCR) provided evidence that the microorganism responsible was a member of the genus
Polaromonas
and could grow on toluene. This microorganism also transformed benzene, but not
m
-xylene or
cis
-dichloroethene.
Correlation of
Dehalococcoides
16S rRNA and Chloroethene-Reductive Dehalogenase Genes with Geochemical Conditions in Chloroethene-Contaminated Groundwater
Authors: van der Zaan Bas , Hannes Fredericke , Hoekstra Nanne , Rijnaarts Huub , de Vos Willem M. , Smidt Hauke , Gerritse Jan
Published: December 2009

Quantitative analysis of genes that code for
Dehalococcoides
16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions.
Dehalococcoides
16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 10
6
gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in
Dehalococcoides
16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast,
vcrA
gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly,
bvcA
and especially
tce
A were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of
vcrA
and/or
bvcA
gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of
Dehalococcoides
16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby
tceA
and
bvcA
correlated to more oxidized conditions than
Dehalococcoides
16S rRNA and
vcrA
. Because the variation in VC-reductase gene numbers was not directly correlated to variation in
Dehalococcoides
spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than
Dehalococcoides
spp.
Bacteria Associated with Immunoregulatory Cells in Mice
Authors: Presley Laura L. , Wei Bo , Braun Jonathan , Borneman James
Published: December 2009

This study examined bacteria-immune interactions in a mouse model possessing microbiota-dependent immune regulatory features similar to those occurring in human atopy, colitis, and immune regulation. Associations between the abundance of several bacterial phylotypes and immunoregulatory target cell types were identified, suggesting that they may play a role in these phenotypes.
Reductive Debromination of Polybrominated Diphenyl Ethers by Anaerobic Bacteria from Soils and Sediments
Authors: Lee Lip Kim , He Jianzhong
Published: December 2009

Polybrominated diphenyl ethers (PBDEs) have attracted attention recently due to their proven adverse effects on animals and their increasing concentrations in various environmental media and biota. To gain insight into the fate of PBDEs, microcosms established with soils and sediments from 28 locations were investigated to determine their debromination potential with an octa-brominated diphenyl ether (octa-BDE) mixture consisting of hexa- to nona-BDEs. Debromination occurred in microcosms containing samples from 20 of the 28 locations when they were spiked with octa-BDE dissolved in the solvent trichloroethene (TCE), which is a potential cosubstrate for stimulating PBDE debromination, and in microcosms containing samples from 11 of the 28 locations when they were spiked with octa-BDE dissolved in nonane. Debromination products ranging from hexa- to mono-BDEs were generated within 2 months. Notably, the toxic tetra-BDEs accounted for 50% of the total product. In sediment-free culture C-N-7* amended with the octa-BDE mixture and nonane (containing 45 nM nona-BDE, 181 nM octa-BDEs, 294 nM hepta-BDE, and 19 nM hexa-BDE) there was extensive debromination of the parent compounds, which produced hexa-BDE (56 nM), penta-BDEs (124 nM), and tetra-BDEs (150 nM) within 42 days, possibly by a metabolic process. A 16S rRNA gene-based analysis revealed that
Dehalococcoides
species were present in 11 of 14 active microcosms. However, unknown debrominating species in some of the microcosms debrominated the octa-BDE mixture in the absence of other added halogenated electron acceptors (such as TCE). These findings provide information that is useful for assessing microbial reductive debromination of higher brominated PBDEs to less-brominated congeners, a possible source of the more toxic congeners (e.g., penta- and tetra-BDEs) detected in the environment.
Diversity and Evolution of the Phenazine Biosynthesis Pathway
Authors: Mavrodi Dmitri V. , Peever Tobin L. , Mavrodi Olga V. , Parejko James A. , Raaijmakers Jos M. , Lemanceau Philippe , Mazurier Sylvie , Heide Lutz , Blankenfeldt Wulf , Weller David M. , Thomashow Linda S.
Published: December 2009

Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera
Pseudomonas
,
Burkholderia
,
Pectobacterium
,
Brevibacterium
, and
Streptomyces
. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (
phzF
) and housekeeping (
rrs
,
recA
,
rpoB
,
atpD
, and
gyrB
) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in
Pseudomonas
spp. to horizontal gene transfer in
Burkholderia
spp. and
Pectobacterium
spp. DNA extracted from cereal crop rhizospheres and screened for the presence of
phzF
contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.
Key Process Conditions for Production of C
4
Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered
Saccharomyces cerevisiae
Strain
Authors: Zelle Rintze M. , de Hulster Erik , Kloezen Wendy , Pronk Jack T. , van Maris Antonius J. A.
Published: December 2009

A recent effort to improve malic acid production by
Saccharomyces cerevisiae
by means of metabolic engineering resulted in a strain that produced up to 59 g liter
−1
of malate at a yield of 0.42 mol (mol glucose)
−1
in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO
3
, was required for efficient C
4
dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO
3
dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)
−1
, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered
S. cerevisiae
strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO
2
, calcium, and O
2
). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)
−1
was obtained in bioreactors, a 19% increase over yields in shake flask experiments.
PrhG, a Transcriptional Regulator Responding to Growth Conditions, Is Involved in the Control of the Type III Secretion System Regulon in
Ralstonia solanacearum
Authors: Plener Laure , Manfredi Pablo , Valls Marc , Genin Stéphane
Published: December 2009

The ability of
Ralstonia solanacearum
to cause disease in plants depends on its type III secretion system (T3SS). The expression of the T3SS and its effector substrates is coordinately controlled by a regulatory cascade, at the bottom of which is HrpB. Transcription of the
hrpB
gene is activated by a plant-responsive regulator named HrpG, which is a master regulator of a wide array of pathogenicity functions in
R
.
solanacearum
. We have identified in the genome of strain GMI1000 a close paralog of
hrpG
(83% overall similarity at the protein level) that we have named
prhG
. Despite this high similarity, the expression pattern of
prhG
is remarkably different from that of
hrpG
:
prhG
expression is activated after growth of bacteria in minimal medium but not in the presence of host cells, while
hrpG
expression is specifically induced in response to plant cell signals. We provide genetic evidence that
prhG
is a transcriptional regulator that, like
hrpG
, controls the expression of
hrpB
and the
hrpB
-regulated genes under minimal medium conditions. However, the regulatory functions of
prhG
and
hrpG
are distinct:
prhG
has no influence on
hrpB
expression when the bacteria are in the presence of plant cells, and transcriptomic profiling analysis of a
prhG
mutant revealed that the PrhG and HrpG regulons have only one pathogenicity target in common,
hrpB
. Functional complementation experiments indicated that PrhG and HrpG are individually sufficient to activate
hrpB
expression in minimal medium. Rather surprisingly, a
prhG
disruption mutant had little impact on pathogenicity, which may indicate that
prhG
has a minor role in the activation of T3SS genes when
R
.
solanacearum
grows parasitically inside the plant. The cross talk between pathogenicity regulatory proteins and environmental signals described here denotes that an intricate network is at the basis of the bacterial disease program.
Identification of Conserved Amino Acid Residues of the
Salmonella
σ
S
Chaperone Crl Involved in Crl-σ
S
Interactions
Authors: Monteil Véronique , Kolb Annie , D'Alayer Jacques , Beguin Pierre , Norel Françoise
Published: December 2009

Proteins that bind σ factors typically attenuate the function of the σ factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor σ
S
(RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of σ
S
-dependent genes. Analyses of sequenced bacterial genomes revealed that
crl
is less widespread and less conserved at the sequence level than
rpoS.
Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the
crl
gene from
Salmonella enterica
serovar Typhimurium and complementation of a Δ
crl
mutant of
Salmonella
indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-σ
S
interaction and that residues 1 to 71 in σ
S
are dispensable for Crl binding. In
Escherichia coli
, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses
crl
transcription. However, the
Salmonella
Crl and Fur proteins did not interact in the BACTH system. In addition, a
fur
mutation did not have any significant effect on the expression level of Crl in
Salmonella.
These results suggest that the relationship between Crl and Fur is different in
Salmonella
and
E. coli
.
The Cyclic Nucleotide Monophosphate Domain ofXanthomonas campestrisGlobal Regulator Clp Defines a New Class of Cyclic Di-GMP Effectors
Authors: Tao Fei , He Ya-Wen , Wu Dong-Hui , Swarup Sanjay , Zhang Lian-Hui
Published: December 2009

The widely conserved second messenger cyclic diguanosine monophosphate (c-di-GMP) plays a key role in quorum-sensing (QS)-dependent production of virulence factors inXanthomonas campestrispv. campestris. The detection of QS diffusible signal factor (DSF) by the sensor RpfC leads to the activation of response regulator RpfG, which activates virulence gene expression by degrading c-di-GMP. Here, we show that a global regulator in theX. campestrispv. campestris QS regulatory pathway, Clp, is a c-di-GMP effector. c-di-GMP specifically binds to Clp with high affinity and induces allosteric conformational changes that abolish the interaction between Clp and its target gene promoter. Clp is similar to the cyclic AMP (cAMP) binding proteins Crp and Vfr and contains a conserved cyclic nucleotide monophosphate (cNMP) binding domain. Using site-directed mutagenesis, we found that the cNMP binding domain of Clp contains a glutamic acid residue (E99) that is essential for c-di-GMP binding. Substituting the residue with serine (E99S) resulted in decreased sensitivity to changes in the intracellular c-di-GMP level and attenuated bacterial virulence. These data establish the direct role of Clp in the response to fluctuating c-di-GMP levels and depict a novel mechanism by which QS links the second messenger with theX. campestrispv. campestris virulence regulon.
Virulence Factors Encoded by
Legionella longbeachae
Identified on the Basis of the Genome Sequence Analysis of Clinical Isolate D-4968
Authors: Kozak Natalia A. , Buss Meghan , Lucas Claressa E. , Frace Michael , Govil Dhwani , Travis Tatiana , Olsen-Rasmussen Melissa , Benson Robert F. , Fields Barry S.
Published: December 2009

Legionella longbeachae
causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of
L. longbeachae
-specific diagnostic tests.
L. longbeachae
displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to
Legionella pneumophila
, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems,
L. longbeachae
is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an
L. longbeachae
clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of
L. pneumophila
. The results revealed that the D-4968 genome is larger than the
L. pneumophila
genome and has a gene order that is different from that of the
L. pneumophila
genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding
L. pneumophila
Icm/Dot substrates have been found in the D-4968 genome.
L. longbeachae
encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the
L. longbeachae
Icm/Dot secretion system. In contrast to the
L. pneumophila
genome, the
L. longbeachae
D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of
L. longbeachae
to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of
L. longbeachae
may reflect adaptation of this species to life in soil.
Impact of Silver(I) on the Metabolism of
Shewanella oneidensis
Authors: Wang Hui , Law Nicholas , Pearson Geraldine , van Dongen Bart E. , Jarvis Roger M. , Goodacre Royston , Lloyd Jonathan R.
Published: December 2009

Anaerobic cultures of
Shewanella oneidensis
MR-1 reduced toxic Ag(I), forming nanoparticles of elemental Ag(0), as confirmed by X-ray diffraction analyses. The addition of 1 to 50 μM Ag(I) had a limited impact on growth, while 100 μM Ag(I) reduced both the doubling time and cell yields. At this higher Ag(I) concentration transmission electron microscopy showed the accumulation of elemental silver particles within the cell, while at lower concentrations the metal was exclusively reduced and precipitated outside the cell wall. Whole organism metabolite fingerprinting, using the method of Fourier transform infrared spectroscopy analysis of cells grown in a range of silver concentrations, confirmed that there were significant physiological changes at 100 μM silver. Principal component-discriminant function analysis scores and loading plots highlighted changes in certain functional groups, notably, lipids, amides I and II, and nucleic acids, as being discriminatory. Molecular analyses confirmed a dramatic drop in cellular yields of both the phospholipid fatty acids and their precursor molecules at high concentrations of silver, suggesting that the structural integrity of the cellular membrane was compromised at high silver concentrations, which was a result of intracellular accumulation of the toxic metal.
Temporal and Spatial Regulation of the Four Transcription Start Sites of
hetR
from
Anabaena
sp. Strain PCC 7120
Authors: Rajagopalan Ramya , Callahan Sean M.
Published: December 2009

The filamentous cyanobacterium
Anabaena
sp. strain PCC 7120 forms nitrogen-fixing heterocysts in a periodic pattern in response to combined-nitrogen limitation in the environment. The master regulator of heterocyst differentiation, HetR, is necessary for both pattern formation and commitment of approximately every 10th cell of a filament to differentiation into a heterocyst. In this study, the individual contributions of four transcriptional start points (tsps) in regulation of transcription of
hetR
were assessed, and the effects of the regulatory genes
patS
,
hetN
, and
patA
on transcription from the tsps were determined. The tsp located at nucleotide −271 relative to the translational start site (−271 tsp) was the most tightly regulated tsp, and fluorescence from a −271 tsp-green fluorescent protein (GFP) reporter fusion was observed initially in groups of two cells and later in single cells arranged in a spatial pattern that mimicked the pattern of heterocysts that emerged. Conversely, the fluorescence from the −184 and −728/−696 tsp-GFP reporter fusions was uniform throughout filaments. Transcription from the −271 tsp was severely downregulated in a strain in which the
patA
gene, which encodes a positive regulator of differentiation, was deleted, and it was not detectable in strains overexpressing the genes encoding the negative regulators of differentiation,
patS
and
hetN
. In strains lacking the −271 tsp of
hetR
, pattern formation, the timing of commitment to differentiation, and the number of cells that differentiated into heterocysts were affected. Taken together, these results demonstrate the role of regulation of the −271 tsp of
hetR
in the genetic network that governs heterocyst pattern formation and differentiation.
Extracytoplasmic PAS-Like Domains Are Common in Signal Transduction Proteins
Authors: Chang Changsoo , Tesar Christine , Gu Minyi , Babnigg Gyorgy , Joachimiak Andrzej , Pokkuluri P. Raj , Szurmant Hendrik , Schiffer Marianne
Published: December 2009

We present the crystal structure of the extracytoplasmic domain of the
Bacillus subtilis
PhoR sensor histidine kinase, part of a two-component system involved in adaptation to low environmental phosphate concentrations. In addition to the PhoR structure, we predict that the majority of the extracytoplasmic domains of
B. subtilis
sensor kinases will adopt a fold similar to the ubiquitous PAS domain.
An unusual hadrosaurid braincase from the Dinosaur Park Formation and the biostratigraphy of Parasaurolophus (Ornithischia: Lambeosaurinae) from southern Alberta
Authors: Evans David C. , Bavington Rebecca , Campione Nicolás E.
Published: December 2009

The lambeosaurine hadrosaurid Parasaurolophus is known from rare occurrences in Campanian deposits of western North America. A previously undescribed large hadrosaurid braincase from the Dinosaur Park Formation (Alberta, Canada) is assigned to the genus Parasaurolophus on the basis of several derived characters associated with the frontal–nasal articulation at the base of the crest. This identification is supported by two separate phylogenetic analyses, in which the specimen clusters with other more completely known Parasaurolophus exemplars. If correctly identified, the specimen represents the third and largest cranial specimen of the genus from the Late Cretaceous of Alberta. The specimen occurs in the same deposits as the holotype specimen of Parasaurolophus walkeri and may represent a late ontogenetic stage of this taxon. As opposed to a small frontal dome in the holotype of P. walkeri , the external contribution of the frontal to the skull roof is obliterated in the new specimen. If these hypothesized ontogenetic changes in the skull roof correlate with the size and posterodorsal development of the crest, as in other lambeosaurines, it suggests that the crest had not reached its full expression in the holotype. When placed into a detailed biostratigraphic context for the first time, the limited Parasaurolophus material from the Belly River Group is distributed in the lower half of the Dinosaur Park Formation at Dinosaur Provincial Park. This suggests that Parasaurolophus may be associated with the lower Centrosaurus – Corythosaurus assemblage zone and may have preferred more inland environments than other hadrosaurids, such as Lambeosaurus and Prosaurolophus .
Can the data from at-sea observer surveys be used to make general inferences about catch composition and discards?
Authors: Benoît Hugues P. , Allard Jacques
Published: December 2009

Some fishery characteristics such as total discards are often inferred from data collected by at-sea observers on a subset of fishing trips. Such inference is predicated on the assumption that observed and unobserved trips are statistically exchangeable. There are two principal reasons why this may not be so. A deployment effect results from nonrandom distribution of observers among sampling units. An observer effect results from changes in fishing practice or location when observers are present. Both effects can impact the precision and accuracy of fishery-level inferences drawn from observer data, though this is rarely addressed quantitatively. We found evidence for deployment and observer effects in Gulf of St. Lawrence fisheries. The impact of deployment bias was further quantified by resampling from fisheries data collected with 100% observer coverage. We conclude that the nature of the effects observed in our study preclude merely correcting observer-collected catch data for possible biases and imprecision. Furthermore, regulatory compliance monitoring by observers in the existing program may not be completely effective. Modifications to program structure would therefore be beneficial and some suggestions are evaluated in this paper.
A three-dimensional model for analyzing the effects of salmon redds on hyporheic exchange and egg pocket habitat
Authors: Tonina Daniele , Buffington John M.
Published: December 2009

A three-dimensional fluid dynamics model is developed to capture the spatial complexity of the effects of salmon redds on channel hydraulics, hyporheic exchange, and egg pocket habitat. We use the model to partition the relative influences of redd topography versus altered hydraulic conductivity (winnowing of fines during spawning) on egg pocket conditions for a simulated pool–riffle channel with a redd placed at the pool tail. Predictions show that altered hydraulic conductivity is the primary factor for enhancing hyporheic velocities and dissolved oxygen content within the egg pocket. Furthermore, the simulations indicate that redds induce hyporheic circulation that is nested within that caused by pool–riffle topography and that spawning-related changes in hyporheic velocities and dissolved oxygen content could create conditions suitable for incubation in locations that otherwise would be unfavorable (reinforcing the notion that salmonids actively modify their environment in ways that may be beneficial to their progeny).