bluuebunny/crossref_metadata_2025 huggingface dataset:
Residual Stress Distribution in Hard-Facing of Pressure Relief Valve Seat
Authors: Ai Li , Yu Xinhai , Jiang Wenchun , Woo Wanchuck , Ze Xiaofeng , Tu Shan-Tung
Published: March 2014

In this study, for the hard-facing of spring-loaded pressure relief valve seats, the residual stress distributions after the tungsten inert gas welding, (TIG) postwelded heat treatment and subsequent surface turning were investigated. The heat input parameters of welding were calibrated using an infrared imaging and thermocouples. The residual stress distributions were computed using three-dimensional finite element model. The neutron diffraction approach was employed to verify the finite element calculation. It is found that, the surface temperature during hard-facing welding shows a double ellipsoidal shape with the highest value of around 1570 °C. The high residual stress zones are located exactly under the welded joint except a slight deviation in the hoop direction. The magnitudes of tensile residual stresses in the three directions increase with their corresponding locations from the root of the joint into the base metal. The residual stresses in all of the three directions decrease significantly after the heat treatment. After surface turning, the residual stresses are tensile except for those close to the inner surface that are compressive in axial and radial directions.
Standing Gravity Waves in a Horizontal Circular Eccentric Annular Tank
Authors: Nezami Mohammad , Oveisi Atta , Mehdi Mohammadi Mohammad
Published: March 2014

Standing gravity waves in half-full horizontal cylindrical containers with eccentric tube are analyzed using the linear theory of water waves. The problem solution is obtained by the method of conformal coordinate transformation, leading to standard truncated matrix Eigen-value problem from which fluid motion characteristics (Eigen-frequencies and wave modes) are calculated. The effects of tube eccentricity and radius ratio upon the three lowest antisymmetric and symmetric sloshing frequencies and the associated hydrodynamic pressure mode shapes are examined. Also, convergence of the adopted approach with respect to the eccentricity condition, and radius ratio is discussed. Accuracy of the present analysis is checked by comparison with the known results of the limiting cases.
Reliability Assessment of the Water Distributing Valve of a Hydrostatic Sediment Corer
Authors: Qin Huawei , Hu Hangmin , Ye Wei , Wang Jianjun , Cai Zhen , Chen Ying
Published: March 2014

The hydrostatic corer is designed for sampling sediment from the seafloor. Its operation relies on a water distributing valve which converts the potential pressure difference between ambient seawater and a built-in chamber with atmospheric pressure to the dynamic energy as the driving power. As the valve is exposed to the ambient water, the deformation of its components may exceed their fitting tolerance under the high pressure and low temperature on seafloor, and thus cause the failure of the valve. Three possible failure modes have been taken into account, representing the positions where interference of fitting tolerance is likely to occur. Corresponding models are then created considering the coupled effects of pressure and temperature on the valve. Based on the model results and the reliability requirement of the corer, one failure mode is selected to calculate the reliability of the valve and is used as guidance for the future improvement of the design.
Evaluation of the Integrity of Steam Generator Tubes Subjected to Flow Induced Vibrations
Authors: Hassan Marwan , Riznic Jovica
Published: March 2014

Flow-induced vibrations (FIV) continue to affect the operations of nuclear power plant components such as heat exchanger tube bundles. The negative effect of FIV is in the form of tube fatigue, cracking, and fretting wear at the supports. Fretting wear at the supports is the result of tube/support impact and friction. Fluidelastic and turbulence forces are the two main excitation mechanisms that feed energy into the system causing these violent vibrations. To minimize this effect all support clearances must be kept at a very small value. This paper investigates the consequences of losing the effectiveness of a particular support as a result of corrosion or excessive fretting wear. A full U-bend tube subjected to both fluidelastic and turbulence forces is utilized in this work. The performance of countermeasures such as the installation of additional flat bars in the U-bend region is thoroughly investigated. The investigation utilized both deterministic and probabilistic techniques.
Influence of Injection and Cavity Pressure on the Demoulding Force in Micro-Injection Moulding
Authors: Griffiths C. A. , Dimov S. S. , Scholz S. G. , Tosello G. , Rees A.
Published: March 2014

The paper reports an experimental study that investigates part demoulding behavior in micro-injection moulding with a focus on the effects of pressure and temperature on the demoulding forces. In particular, the demoulding performance of a representative microfluidics part was studied as a function of four process parameters, melt temperature, mould temperature, holding pressure, and injection speed, employing the design of experiment approach. In addition, the results obtained using different combinations of process parameters were analyzed to identify the best processing conditions in regards to demoulding behavior of microparts when utilizing a COC polymer to mould them.
A dosimetric characterization of a novel linear accelerator collimator
Authors: Thompson C. M. , Weston S. J. , Cosgrove V. C. , Thwaites D. I.
Published: March 2014

Purpose:

The aim of this work is to characterize a new linear accelerator collimator which contains a single pair of sculpted diaphragms mounted orthogonally to a 160 leaf multileaf collimator (MLC). The diaphragms have “thick” regions providing full attenuation and “thin” regions where attenuation is provided by both the leaves and the diaphragm. The leaves are mounted on a dynamic leaf guide allowing rapid leaf motion and leaf travel over 350 mm.

Methods:

Dosimetric characterization, including assessment of leaf transmission, leaf tip transmission, penumbral width, was performed in a plotting tank. Head scatter factor was measured using a mini‐phantom and the effect of leaf guide position on output was assessed using a water phantom. The tongue and groove effect was assessed using multiple exposures on radiochromic film. Leaf reproducibility was assessed from portal images of multiple abutting fields.

Results:

The maximum transmission through the multileaf collimator is 0.44% at 6 MV and 0.52% at 10 MV. This reduced to 0.22% and 0.27%, respectively, when the beam passes through the dynamic leaf guide in addition to the MLC. The maximum transmission through the thick part of the diaphragm is 0.32% and 0.36% at 6 and 10 MV. The combination of leaf and diaphragm transmission ranges from 0.08% to 0.010% at 6 MV and 0.10% to 0.14% depending on whether the shielding is through the thick or thin part of the diaphragm. The off‐axis intertip transmission for a zero leaf gap is 2.2% at 6 and 10 MV. The leaf tip penumbra for a 100 × 100 mm field ranges from 5.4 to 4.3 mm at 6 and 10 MV across the full range of leaf motion when measured in the AB direction, which reduces to 4.0–3.4 mm at 6 MV and 4.5–3.8 mm at 10 MV when measured in the GT direction. For a 50 × 50 mm field, the diaphragm penumbra ranges from 4.3 to 3.7 mm at 6 MV and 4.5 to 4.1 mm at 10 MV in the AB direction and 3.7 to 3.2 mm at 6 MV and 4.2 to 3.7 mm when measured in the GT direction. The tongue and groove effect observed from exposure of a radiochromic film to two abutting fields is an underdose of 25%. The head scatter factor at both 6 and 10 MV is similar to that from the MLCi2 collimator to within 0.8%. The uncertainty in the leaf position reproducibility is 0.05 mm (2σ).

Conclusions:

The Agility collimator is a low leakage, high definition collimator where both the MLC and the sculpted diaphragm have been optimized for dynamic treatments.
Occurrence of an Environmental Acinetobacter baumannii Strain Similar to a Clinical Isolate in Paleosol from Croatia
Authors: Hrenovic Jasna , Durn Goran , Goic-Barisic Ivana , Kovacic Ana
Published: March 2014

Over the past decade, bacteria of the genus
Acinetobacter
have emerged as a leading cause of hospital-acquired infections. Outbreaks of
Acinetobacter
infections are considered to be caused exclusively by contamination and transmission in hospital environments. The natural habitats of clinically important multiresistant
Acinetobacter
spp. remain to be defined. In this paper, we report an incidental finding of a viable multidrug-resistant strain of
Acinetobacter baumannii
, related to clinical isolates, in acid paleosol from Croatia. The environmental isolate of
A. baumannii
showed 87% similarity to a clinical isolate originating from a hospital in this geographic area and was resistant to gentamicin, trimethoprim-sulfamethoxazole, ciprofloxacin, and levofloxacin. In paleosol, the isolate was able to survive a low pH (3.37), desiccation, and a high temperature (50°C). The probable source of
A. baumannii
in paleosol is illegally disposed waste of external origin situated in the abandoned quarry near the sampling site. The bacteria could have been leached from waste by storm water and thus infiltrated the paleosol.
Establishment of Intestinal Microbiota during Early Life: a Longitudinal, Explorative Study of a Large Cohort of Danish Infants
Authors: Bergström Anders , Skov Thomas Hjort , Bahl Martin Iain , Roager Henrik Munch , Christensen Line Brinch , Ejlerskov Katrine Tschentscher , Mølgaard Christian , Michaelsen Kim F. , Licht Tine Rask
Published: March 2014

Fecal samples were obtained from a cohort of 330 healthy Danish infants at 9, 18, and 36 months after birth, enabling characterization of interbacterial relationships by use of quantitative PCR targeting 31 selected bacterial 16S rRNA gene targets representing different phylogenetic levels. Nutritional parameters and measures of growth and body composition were determined and investigated in relation to the observed development in microbiota composition. We found that significant changes in the gut microbiota occurred, particularly from age 9 to 18 months, when cessation of breastfeeding and introduction of a complementary feeding induce replacement of a microbiota characterized by lactobacilli, bifidobacteria, andEnterobacteriaceaewith a microbiota dominated byClostridiumspp. andBacteroidesspp. Classification of samples by a proxy enterotype based on the relative levels ofBacteroidesspp. andPrevotellaspp. showed that enterotype establishment occurs between 9 and 36 months. Thirty percent of the individuals shifted enterotype between 18 and 36 months. The composition of the microbiota was most pronouncedly influenced by the time of cessation of breastfeeding. From 9 to 18 months, a positive correlation was observed between the increase in body mass index and the increase of the short-chain-fatty-acid-producing clostridia, theClostridum leptumgroup, andEubacterium hallii. Considering previously established positive associations between rapid infant weight gain, early breastfeeding discontinuation, and later-life obesity, the corresponding microbial findings seen here warrant attention.
Metabolic Changes in Klebsiella oxytoca in Response to Low Oxidoreduction Potential, as Revealed by Comparative Proteomic Profiling Integrated with Flux Balance Analysis
Authors: Zhu Yan , Li Dan , Bao Guanhui , Wang Shaohua , Mao Shaoming , Song Jiangning , Li Yin , Zhang Yanping
Published: March 2014

Oxidoreduction potential (ORP) is an important physiological parameter for biochemical production in anaerobic or microaerobic processes. However, the effect of ORP on cellular physiology remains largely unknown, which hampers the design of engineering strategies targeting proteins associated with ORP response. Here we characterized the effect of altering ORP in a 1,3-propanediol producer,
Klebsiella oxytoca
, by comparative proteomic profiling combined with flux balance analysis. Decreasing the extracellular ORP from −150 to −240 mV retarded cell growth and enhanced 1,3-propanediol production. Comparative proteomic analysis identified 61 differentially expressed proteins, mainly involved in carbohydrate catabolism, cellular constituent biosynthesis, and reductive stress response. A hypothetical oxidoreductase (HOR) that catalyzes 1,3-propanediol production was markedly upregulated, while proteins involved in biomass precursor synthesis were downregulated. As revealed by subsequent flux balance analysis, low ORP induced a metabolic shift from glycerol oxidation to reduction and rebalancing of redox and energy metabolism. From the integrated protein expression profiles and flux distributions, we can construct a rational analytic framework that elucidates how (facultative) anaerobes respond to extracellular ORP changes.
Effects of Leachate from Crumb Rubber and Zinc in Green Roofs on the Survival, Growth, and Resistance Characteristics of Salmonella enterica subsp. enterica Serovar Typhimurium
Authors: Crampton Mollee , Ryan Allayna , Eckert Cori , Baker Katherine H. , Herson Diane S.
Published: March 2014

The use of green roofs is a growing practice worldwide, particularly in densely populated areas. In an attempt to find new methods for recycling crumb rubber, incorporation of crumb rubber into artificial medium for plant growth in green roofs and similar engineered environments has become an attractive option for the recycling of waste tires. Though this approach decreases waste in landfills, there are concerns about the leaching of zinc and other heavy metals, as well as nutrient and organic compounds, into the environment. The present study analyzed the impact of leachate from crumb rubber and zinc on the growth and viability of
Salmonella enterica
subsp.
enterica
serovar Typhimurium. Zinc was chosen for further studies since it has been previously implicated with other biological functions, including biofilm formation, motility, and possible cross-resistance to antimicrobial agents. The study showed that
Salmonella
can colonize crumb rubber and that crumb rubber extract may provide nutrients that are usable by this bacterium.
Salmonella
strains with reduced susceptibility (SRS) to zinc were obtained after subculturing in increasing concentrations of zinc. The SRS exhibited differences in gene expression of flux pump genes
zntA
and
znuA
compared to that of the parent when exposed to 20 mM added zinc. In biofilm formation studies, the SRS formed less biofilm but was more motile than the parental strain.
Abiotic Factors Affecting the Persistence of Avian Influenza Virus in Surface Waters of Waterfowl Habitats
Authors: Keeler Shamus P. , Dalton Melinda S. , Cressler Alan M. , Berghaus Roy D. , Stallknecht David E.
Published: March 2014

Avian influenza (AI) virus can remain infectious in water for months, and virus-contaminated surface water is considered to be a source of infection within wild waterfowl populations. Previous work has characterized the effects of pH, salinity, and temperature on viral persistence in water, but most of that work was done with modified distilled water. The objective of this study was to identify the abiotic factors that influence the duration of AI virus persistence in natural surface water. Surface water samples were collected from 38 waterfowl habitats distributed across the United States. Samples were submitted to the U.S. Geological Survey National Water Quality Laboratory for chemical analysis and the University of Georgia for viral reduction time analysis. Samples were filtered with 0.22-μm filters, and the durations of persistence of three wild-bird-derived influenza A viruses within each water sample at 10, 17, and 28°C were determined. The effects of the surface water physicochemical factors on the duration of AI viral persistence in laboratory experiments were evaluated by multivariable linear regression with robust standard errors. The duration of AI virus persistence was determined to be longest in filtered surface water with a low temperature (<17°C), a neutral-to-basic pH (7.0 to 8.5), low salinity (<0.5 ppt), and a low ammonia concentration (<0.5 mg/liter). Our results also highlighted potential strain-related variation in the stability of AI virus in surface water. These results bring us closer to being able to predict the duration of AI virus persistence in surface water of waterfowl habitats.
Adaptation of Escherichia coli to Elevated Sodium Concentrations Increases Cation Tolerance and Enables Greater Lactic Acid Production
Authors: Wu Xianghao , Altman Ronni , Eiteman Mark A. , Altman Elliot
Published: March 2014

Adaptive evolution was employed to generate sodium (Na+)-tolerant mutants ofEscherichia coliMG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na+concentrations. The isolates also showed increased tolerance of K+, although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters ofE. coliMG1655 and ALS1187, the isolate with the greatest Na+tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes,emrR,hfq,kil,rpsG, andsspA, all of which could potentially affect the ability ofE. colito tolerate Na+. Two of these genes,hfqandsspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na+tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations,E. coliALS1187pflBgenerated 76.2 g/liter lactate compared to MG1655pflB, which generated only 56.3 g/liter lactate.
Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli
Authors: Wong Matthew S. , Li Mai , Black Ryan W. , Le Thao Q. , Puthli Sharon , Campbell Paul , Monticello Daniel J.
Published: March 2014

Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by
Escherichia coli
that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes
dhaKLM
,
gldA
, and
glpK
, as well as the ethanol pathway gene
adhE
. To eliminate the cost of inducers and antibiotics from the fermentation, we used the
adhE
and
icd
promoters from
E. coli
in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an
E. coli
-based glycerol-to-ethanol process.
Genetic Diversity Analysis Reveals that Geographical Environment Plays a More Important Role than Rice Cultivar in Villosiclava virens Population Selection
Authors: Wang Fei , Zhang Shu , Liu Mei-Gang , Lin Xian-Song , Liu Hui-Jiang , Peng You-Liang , Lin Yang , Huang Jun-Bin , Luo Chao-Xi
Published: March 2014

Rice false smut caused by
Villosiclava virens
is an economically important disease of grains worldwide. The genetic diversity of 153 isolates from six fields located in Wuhan (WH), Yichang Wangjia (YCW), Yichang Yaohe (YCY), Huanggang (HG), Yangxin (YX), and Jingzhou (JZ) in Hubei province of China were phylogenetically analyzed to evaluate the influence of environments and rice cultivars on the
V. virens
populations. Isolates (43) from Wuhan were from two rice cultivars, Wanxian 98 and Huajing 952, while most of the other isolates from fields YCW, YCY, HG, YX, and JZ originated from different rice cultivars with different genetic backgrounds. Genetic diversity of isolates was analyzed using random amplified polymorphic DNA (RAPD) and single-nucleotide polymorphisms (SNP). The isolates from the same cultivars in Wuhan tended to group together, indicating that the cultivars had an important impact on the fungal population. The 110 isolates from individual fields tended to cluster according to geographical origin. The values of Nei's gene diversity (
H
) and Shannon's information index (
I
) showed that the genetic diversity among isolates was higher between than within geographical populations. Furthermore, mean genetic distance between groups (0.006) was higher than mean genetic distance within groups (0.0048) according to MEGA 5.2. The pairwise population fixation index (
F
ST
) values also showed significant genetic differentiation between most populations. Higher genetic similarity of isolates from individual fields but different rice cultivars suggested that the geographical factor played a more important role in the selection of
V. virens
isolates than rice cultivars. This information could be used to improve the management strategy for rice false smut by adjusting the cultivation measures, such as controlling fertilizer, water, and planting density, in the rice field to change the microenvironment.
Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase
Authors: Inoue Kengo , Usami Yusuke , Ashikawa Yuji , Noguchi Haruko , Umeda Takashi , Yamagami-Ashikawa Aiko , Horisaki Tadafumi , Uchimura Hiromasa , Terada Tohru , Nakamura Shugo , Shimizu Kentaro , Habe Hiroshi , Yamane Hisakazu , Fujimoto Zui , Nojiri Hideaki
Published: March 2014

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from
Janthinobacterium
sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.
Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry
Authors: Fagerquist Clifton K. , Zaragoza William J. , Sultan Omar , Woo Nathan , Quiñones Beatriz , Cooley Michael B. , Mandrell Robert E.
Published: March 2014

We have analyzed 26 Shiga toxin-producing
Escherichia coli
(STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)–tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (
stx
2
) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.
Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity
Authors: Maitre Magali , Weidmann Stéphanie , Dubois-Brissonnet Florence , David Vanessa , Covès Jacques , Guzzo Jean
Published: March 2014

Malolactic fermentation in wine is often carried out by
Oenococcus oeni
. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in
O. oeni
. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from
O. oeni
grown under optimal growth conditions (here, control liposomes), one was prepared from
O. oeni
grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from
Bacillus subtilis
or
Lactococcus lactis
. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from
O. oeni
suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of
O. oeni
. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.
Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase
Authors: Ushimaru Kazunori , Motoda Yoko , Numata Keiji , Tsuge Takeharu
Published: March 2014

In this study, we performed
in vitro
and
in vivo
activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from
Aeromonas caviae
(PhaC
Ac
). In
in vitro
assays, among the three PhaCs tested, PhaC
Ac
was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaC
Ac
), whereas the prepolymerization PhaC
Re
(derived from
Ralstonia eutropha
) and PhaC
Da
(
Delftia acidovorans
) showed reduced activity with PhaPs. The PhaP-activated PhaC
Ac
showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C
6
). PhaP
Ac
also activated PhaC
Ac
when it was added during polymerization (polymer-elongating PhaC
Ac
), while this effect was not observed for PhaC
Re
. In an
in vivo
assay using
Escherichia coli
TOP10 as the host strain, the effect of PhaP
Ac
expression on PHA synthesis by PhaC
Ac
or PhaC
Re
was examined. As PhaP
Ac
expression increased, PHA production was increased by up to 2.3-fold in the PhaC
Ac
-expressing strain, whereas it was slightly increased in the PhaC
Re
-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaC
Ac
both
in vitro
and
in vivo
but do not activate PhaC
Re
. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaC
Ac
.
Analysis of the Microbial Community Structure by Monitoring an Hg Methylation Gene (
hgcA
) in Paddy Soils along an Hg Gradient
Authors: Liu Yu-Rong , Yu Ri-Qing , Zheng Yuan-Ming , He Ji-Zheng
Published: March 2014

Knowledge of the diversity of mercury (Hg)-methylating microbes in the environment is limited due to a lack of available molecular biomarkers. Here, we developed novel degenerate PCR primers for a key Hg-methylating gene (
hgcA
) and amplified successfully the targeted genes from 48 paddy soil samples along an Hg concentration gradient in the Wanshan Hg mining area of China. A significant positive correlation was observed between
hgcA
gene abundance and methylmercury (MeHg) concentrations, suggesting that microbes containing the genes contribute to Hg methylation in the sampled soils. Canonical correspondence analysis (CCA) showed that the
hgcA
gene diversity in microbial community structures from paddy soils was high and was influenced by the contents of total Hg, SO
4
2−
, NH
4
+
, and organic matter. Phylogenetic analysis showed that
hgcA
microbes in the sampled soils likely were related to
Deltaproteobacteria
,
Firmicutes
,
Chloroflexi
,
Euryarchaeota
, and two unclassified groups. This is a novel report of
hgcA
diversity in paddy habitats, and results here suggest a link between Hg-methylating microbes and MeHg contamination
in situ
, which would be useful for monitoring and mediating MeHg synthesis in soils.
Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions
Authors: Grimm Verena , Gleinser Marita , Neu Caroline , Zhurina Daria , Riedel Christian U.
Published: March 2014

Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria have hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonization of, and interaction with the host. In the present study, a range of molecular tools were developed that will allow the closing of some of the gaps in functional analysis of bifidobacteria. A number of promoters were tested for transcriptional activity in
Bifidobacterium bifidum
S17 using pMDY23, a previously published promoter probe vector. The promoter of the
gap
gene (P

gap

) of
B. bifidum
S17 yielded the highest promoter activity among the promoters tested. Thus, this promoter and the pMDY23 backbone were used to construct a range of vectors for expression of different fluorescent proteins (FPs). Successful expression of cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry could be shown for three strains representing three different
Bifidobacterium
spp. The red fluorescent
B. bifidum
S17/pVG-mCherry was further used to demonstrate application of fluorescent bifidobacteria for adhesion assays and detection in primary human macrophages cultured
in vitro
. Furthermore, pMGC-mCherry was cloned by combining a chloramphenicol resistance marker and expression of the FP mCherry under the control of P

gap

. The chloramphenicol resistance marker of pMGC-mCherry was successfully used to determine gastrointestinal transit time of
B. bifidum
S17. Moreover,
B. bifidum
S17/pMGC-mCherry could be detected in fecal samples of mice after oral administration.
Purification and Characterization of Phosphonoglycans from Glycomyces sp. Strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338
Authors: Yu Xiaomin , Price Neil P. J. , Evans Bradley S. , Metcalf William W.
Published: March 2014

Two related actinomycetes,
Glycomyces
sp. strain NRRL B-16210 and
Stackebrandtia nassauensis
NRRL B-16338, were identified as potential phosphonic acid producers by screening for the gene encoding phosphoenolpyruvate (PEP) mutase, which is required for the biosynthesis of most phosphonates. Using a variety of analytical techniques, both strains were subsequently shown to produce phosphonate-containing exopolysaccharides (EPS), also known as phosphonoglycans. The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The EPS from the
Glycomyces
strain has a mass of 40 to 50 kDa and is composed of galactose, xylose, and five distinct partially
O
-methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal, 2,4-linked 3-MeGal, 2,3-linked Gal, 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the
Stackebrandtia
strain is comprised of glucose, galactose, xylose, and four partially
O
-methylated galactose residues. Isotopic labeling indicated that the
O
-methyl groups in the
Stackebrandtia
phosphonoglycan arise from
S
-adenosylmethionine. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP) by
31
P nuclear magnetic resonance (NMR) and mass spectrometry following strong acid hydrolysis of the purified molecules. Partial acid hydrolysis of the purified EPS from
Glycomyces
yielded 2-HEP in ester linkage to the
O
-5 or
O
-6 position of a hexose and a 2-HEP mono(2,3-dihydroxypropyl)ester. Partial acid hydrolysis of
Stackebrandtia
EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl)ester. Examination of the genome sequences of the two strains revealed similar
pepM
-containing gene clusters that are likely to be required for phosphonoglycan synthesis.
Mycobacterium tuberculosis Latin American-Mediterranean Family and Its Sublineages in the Light of Robust Evolutionary Markers
Authors: Mokrousov Igor , Vyazovaya Anna , Narvskaya Olga
Published: March 2014

Mycobacterium tuberculosis
has a clonal population structure, and the Latin American-Mediterranean (LAM) family is one of the largest and most widespread within this species, showing evidence for remarkable pathobiology and a confusing phylogeny. Here, we applied robust phylogenetic markers to study the evolution of the LAM family and its major sublineages circulating in Russia and neighboring countries. A total of 250
M. tuberculosis
isolates were confirmed to belong to the LAM family based on the analysis of the LAM-specific single-nucleotide polymorphisms (SNPs) in the
Rv3062
and
Rv0129c
genes. At this stage, the family status was rectified for 121 isolates misleadingly assigned by CRISPR spoligotyping to non-LAM families (T1- or T5-RUS1). Consequently, the reestimated LAM prevalence rate increased 2-fold in Russia and Kazakhstan and 4-fold in Belarus. The majority (91.8 to 98.7%) of the LAM isolates from all three countries belonged to the LAM-RUS sublineage. In contrast, the Ibero-American LAM RD-Rio sublineage was identified in only 7 Russian isolates. Taken together, our findings and further analyses suggest a monophyletic origin of LAM-RUS: at a historically distant time, in Russia, in a small founding bacterial/human population. Its dissemination pattern and high prevalence rate in Northern Eurasia may indicate a long-term coexistence of the LAM-RUS sublineage and local human populations hypothetically leading to coadaptation and reduced pathogenicity of the relatively more ancient clones, such as spoligotype international type 254 (SIT254), compared to the more recent SIT252 and SIT266 clones. In contrast, rare LAM RD-Rio isolates were likely brought to Russia through occasional human contact. The spread of RD-Rio strains is not as global as commonly claimed and is determined largely by human migration flows (rather than by pathobiological properties of these strains). Consequently, a host population factor appears to play a major role in shaping the
in situ
dissemination pattern of the imported strains in an autochthonous population.
Structure of Bacterial Transcription Factor SpoIIID and Evidence for a Novel Mode of DNA Binding
Authors: Chen Bin , Himes Paul , Liu Yu , Zhang Yang , Lu Zhenwei , Liu Aizhuo , Yan Honggao , Kroos Lee
Published: March 2014

SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of
Bacillus subtilis
. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding
in vitro
of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic
Bacilli
and
Clostridia
, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.
The association of the ‘additional height index’ with atopic diseases, non-atopic asthma, ischaemic heart disease and mortality: a population-based study
Authors: Fenger R V , Vidal C , Gonzalez-Quintela A , Husemoen L L N , Skaaby T , Aadahl M , Linneberg A
Published: March 2014

Objective

Intrauterine growth has been associated with atopic conditions. Growth and adult height have been associated with cardiovascular disease, cancers and mortality but are highly genetic traits. The objectives of the study were as follows: first, to define a height measure indicating an individual's height below or above that which could be expected based on parental height (genetic inheritance) and growth charts. It was named ‘the additional height index’ (AHI), defined as (attained—expected) height; second, to investigate possible associations of AHI with atopic versus non-atopic health outcomes and with ischaemic heart disease (IHD) and IHD mortality.

Design

General population-based study.

Setting

Research centre.

Participants

A random sample of 2656 men and women living in greater Copenhagen took part in the MONICA10 study (the Danish monitoring trends and determinants of cardiovascular disease). In total, 1900 participants with information of parental height were selected.

Outcome measures

Atopic sensitisation (serum IgE), questionnaire information of atopic dermatitis, rhinoconjunctivitis, asthma or wheezing, and registry-based diagnoses of IHD/IHD mortality from National Registries.

Results

Increasing levels of AHI were inversely associated with non-atopic asthma, non-atopic wheezing, IHD and IHD mortality (IHD-all). For one SD increase of AHI, the OR or HR with CI in adjusted analyses was non-atopic asthma OR=0.52 (0.36 to 0.74), non-atopic wheezing OR=0.67 (0.51 to 0.89), and IHD-all HR=0.89 (0.78 to 1.01). The level of AHI was higher among individuals with atopic dermatitis, allergic rhinoconjunctivitis and atopic sensitisation (all p values <0.001) compared with individuals without those conditions; however, the associations were not confirmed in adjusted analyses.

Conclusions

Individuals with childhood conditions that led them to attain tallness higher than expected from their parents’ height may be at lower risk of non-atopic asthma/wheeze and IHD/IHD mortality but possibly at higher risk of atopic conditions. The measure of tallness below or above the expected height could be a sensitive alternative to normal height in epidemiological analyses.
Changing community health service delivery in economically less-developed rural areas in China: impact on service use and satisfaction
Authors: Liu Yong , Yuan Zhaokang , Liu Yuxi , Jayasinghe Upali W , Harris Mark F
Published: March 2014

Objective

To evaluate the impact of a model of rural community health service (CHS) on the use and acceptability of primary healthcare services.

Design

Quasi-experimental.

Setting

Two adjacent rural counties in China.

Participants

5842 residents in 2009 and 3807 in 2010 from 980 households in 7 intervention townships and 49 villages; 2232 residents in 2009 and 2315 in 2010 from 628 households in 3 comparison townships and 9 villages. All residents were approached to participate, with no significant differences in age or sex between groups.

Intervention

Multilevel intervention in 2009 including training rural practitioners, encouraging clinic improvements, providing clinical guidelines, standards and subsidies.

Data collection

Surveys of community members from randomly sampled households in 2009 and 2010.

Primary outcome measures

Satisfaction with and utilisation of outpatient and public health services.

Analysis

Factor analysis confirmed two components of satisfaction. Univariate and multilevel analysis was used.

Results

Satisfaction scores for intervention county respondents increased from 21.4 (95% CI 21.1 to 21.7) to 22.1 (95% CI 21.7 to 22.4) with no change in comparison area. In multilevel analysis, satisfaction with patient-centred care was associated with chronic disease, shorter waiting times and county. Satisfaction with clinic environment and cost was associated with female gender, shorter waiting times but not county. The proportion of children receiving immunisation in intervention village clinics increased from 42.5% (95% CI 27.9% to 47.1%) to 59.2% (95% CI 53.8% to 64.6%) whereas this decreased in comparison villages (16.5%; 95% CI 10.3% to 22.7% to 6.0%; 95% CI 1.3% to 10.7%). Antenatal visits increased in intervention villages (from 69.0%, 95% CI 65.8% to 73.1% to 75.8%, 95% CI 72.2% to 79.4%) with no change in comparison villages.

Conclusions

Introduction of a CHS model adapted to economically less-developed rural areas was associated with some improvements in satisfaction with care and use of some village-based public health services. Further research is needed to determine its public health impact and application to other areas.
Chlamydia trachomatisIgG seroprevalence in the general population of the Netherlands in 1996 and in 2007: differential changes by gender and age
Authors: van Aar F , de Moraes M , Morré S A , van Bergen J E A M , van der Klis F R M , Land J A , van der Sande M A B , van den Broek I V F
Published: March 2014

Objectives

Chlamydia trachomatis(CT) reporting rates from sexually transmitted infection clinics and general practitioners have shown a rising trend in the Netherlands. It is unknown to what extent this reflects increased CT transmission or improved case finding. To achieve more insight into the CT epidemic, we explored the CT IgG seroprevalence (a marker of past CT infection) in the general population of the Netherlands in 1996 and in 2007.

Methods

From two population-based studies in 1996 and 2007, serum samples, demographic and sexual behaviour outcomes were examined, including 1246 men and 1930 women aged 15–39 years. Serum CT IgG antibodies were analysed using the Medac CT IgG ELISA test. Multivariate logistic regression analyses explored the seroprevalence and determinants over time.

Results

The CT IgG seroprevalence was higher in women than in men (10% vs 6%). Among women aged 25–39 years the seroprevalence was lower in 2007 (9%) than in 1996 (14%; adjusted OR (aOR) 0.6, 95% CI 0.4 to 0.8). There was no statistical evidence of a difference in seroprevalence within birth cohorts. Factors associated with seropositivity were male gender (aOR 0.4, 95% CI 0.3 to 0.7), a self-reported history of CT infection (aOR 5.1, 95% CI 2.6 to 10.0), age 25–39 years (aOR 1.7, 95% CI 1.1 to 2.7), non-Western ethnicity (aOR 2.2, 95% CI 1.4 to 3.3) and ≥2 recent sexual partners (aOR 2.2, 95% CI 1.3 to 3.5).

Conclusions

Between 1996 and 2007 the proportion of individuals in the general population with CT IgG antibodies was lower among women aged 25–39 years, but remained similar among younger women and men.
The release and persistence of radioactive anthropogenic nuclides
Authors: Hancock Gary J. , Tims Stephen G. , Fifield L. Keith , Webster Ian T.
Published: March 2014

Atmospheric testing of nuclear weapons during the period 1945–1980 ushered in the ‘atomic age’ and released large quantities of anthropogenic radiogenic nuclides into the atmosphere. These radionuclides were subsequently deposited as fallout to the entire surface of the planet. While many have decayed to negligible levels, long-lived radionuclides persist and will do so for thousands of years. Isotopes of plutonium,
239
Pu (half-life 24 100 years) and
240
Pu (half-life 6563 years), provide the best chronological markers for the onset of this anthropogenic event both now and into the future due to their long half-lives, particle-reactivity, and the fact that they were present in negligible quantities prior to anthropogenic production and release. Chronostratigraphic markers established by distinct Pu concentration profiles and Pu isotope changes in sediment sequences and ice and coral cores can provide high-resolution dating over the last 60 years. However, even though fallout has ceased, it is found that the Pu inventory currently held in surface soil layers and the oceans will continue to supply Pu to sediment deposition zones for millennia and centuries, respectively. The delivery of this Pu will depend on soil erosion and bioturbation rates, and the rate of removal of dissolved Pu from the ocean.