bluuebunny/crossref_metadata_2025 huggingface dataset:
Firm R&D, Absorptive Capacity and Learning by Exporting: Firm‐level Evidence from China
Authors: Dai Mi , Yu Miaojie
Published: January 2013

The absorptive capacity of firms developed through R&D promotes learning by exporting. In this paper, we estimate the instantaneous and long‐run productivity effects of exporting on the universe of Chinese manufacturing firms. We find that exporting has very different productivity effects for firms with different pre‐export R&D status. It has large and lasting productivity effects for firms with pre‐export R&D, while it has little effects for firms without pre‐export R&D. Furthermore, the effect of exporting increases with the number of years of pre‐export R&D investment.
The Trend and Mechanism of Intergenerational Income Mobility in China: An Analysis from the Perspective of Human Capital, Social Capital and Wealth
Authors: Zhigang Yuan , Lin Chen
Published: January 2013

Intergenerational income mobility is the extent to which children’s incomedepends on their parents. The increasing of intergenerational income mobilitycan relieve the social pressure caused by the income gap and provide the incentivefor hardworking and human capital investment. Using microdata and economicempirical technique, this study analyses the time trend and structuralmechanism of China’s intergenerational income mobility in 1988–2005. Theresults suggest the intergenerational income mobility improves in the early period,then becomes stable and even shows a deteriorating sign recently. Humancapital, social capital and wealth explain more than 60 per cent of the intergenerationalincome mobility in China, with wealth contributing the largest proportion.Promoting education equality, eliminating the labour market segmentationand deepening the market–oriented reform are suggested to help improve theintergenerational income mobility in China.
The Effect of Foreign Direct Investment on International Migration: Does Education Matter?
Authors: Wang Miao , Wong M. C. Sunny , Granato Jim
Published: January 2013

Using migration data in 1990 and 2000, we find that inward foreign direct investment (FDI) in non‐OECD countries affects the out‐migration of individuals with tertiary and secondary education to OECD countries originating the investments, but has no significant effect on the out‐migration of individuals with primary education. Distinguishing between linkage and home effects, our results show a dominant home effect of FDI for individuals with tertiary education, but a stronger linkage effect for those with secondary education. The existing stock of former migrants in foreign countries influences the out‐migration of individuals with primary education.
Prevalence of pain in a university veterinary intensive care unit
Authors: Moran Clara E. , Hofmeister Erik H.
Published: January 2013

Objective

The purposes of this study were to determine the prevalence of pain in veterinaryICUpatients, to determine if there are predictors for patients to be painful, and to relate 3 commonly used pain scoring systems with each other.

Design

Cross‐sectional observational study.

Setting

The study was conducted in the small animalICUat the University ofGeorgiaVeterinary teaching hospital.

Animals

All dogs admitted during June 1, 2010–August 5, 2010 were eligible for inclusion. Exclusion criteria included patients receiving supplemental oxygen, comatose animals, aggressive animals, and animals with a zoonotic disease.

Interventions

None.

Measurements and Main Results

Twice daily, at randomly generated times, an independent observer assessed every eligible dog for pain using three different methods: theGlasgow composite pain scale (Glasgow), a visual analogue scale (VAS), and a simple descriptive scale (SDS). Six hundred twenty‐nine observations were made on 230 individual dogs over a 63‐day time period. Dogs who were receiving hydromorphone or were on the orthopedic or neurosurgical service were more likely to be described as painful. Overall, 22% of dogs were described as painful, and this was not different depending on time of day or day of the week. There was no difference in slope betweenVASandGlasgow scores depending on order. The kappa statistic was 0.58 betweenGlasgow andSDS, 0.70 betweenVASandSDS, and 0.47 betweenGlasgow andVAS.

Conclusions

In this study population, dogs undergoing surgery needed a more intense analgesic approach to ensure that they were nonpainful. TheSDSidentified the highest number of painful observations of the scoring systems, at the given levels for defining pain.
Measuring the level of agreement between directly measured blood pressure and pressure readings obtained with a veterinary‐specific oscillometric unit in anesthetized dogs
Authors: Acierno Mark J. , Fauth Erika , Mitchell Mark A. , da Cunha Anderson
Published: January 2013

Objective

To determine if an oscillometric device optimized for use in dogs produces systolic, diastolic, and mean arterial pressures (MAPs) measurements that are in good agreement with directly obtained pressures

Design

Prospective study.

Setting

University teaching hospital.

Animals

Twenty‐one dogs under general anesthesia for surgical procedures.

Interventions

A 20‐Ga catheter was placed into the dorsal pedal artery and systolic, diastolic, and MAPs were directly measured using a validated blood pressure measurement system. Indirect blood pressure measurements were collected using a widely available veterinary oscillometric blood pressure unit. Results obtained by the 2 methods were then compared.

Measurements and Main Results

Agreement between the directly and indirectly measured pressure demonstrated a bias of 9.9 mm Hg and limits of agreement (LOA) 73.7 to −53.9, a bias of −8.9 mm Hg and LOA 23.3 to −41.2, and a bias of −6.3 mm Hg and LOA 28.2 to −40.8 for systolic, diastolic, and MAP, respectively.

Conclusions

There was poor agreement between the direct and indirect measured blood pressure measurement systems. Therefore, use of the oscillometric blood pressure unit evaluated in this study for monitoring patients under anesthesia cannot be recommended at this time.
A Parametric Study of Reversible Jet-Fan Blades Aerodynamic Performance
Authors: Daneshkhah Kasra , Sheard A. G.
Published: January 2013

This paper presents a fully reversible blade parametric design methodology. The blades are for application in jet fans that ventilate and provide emergency smoke control in road tunnels. The blade design variables are tip solidity, twist, and camber distribution. The authors base the design methodology on a sensitivity analysis which they derived from a response surface approximation. They construct the latter using a computational analysis of four experimental cases which they generated using an experimental design approach. The sensitivity analysis calculates a rank and a weight for each design variable that affects the jet-fan performance parameters thrust and efficiency, and thus facilitates insight into each variable's relative importance. Finally, the authors present a redesign of an existing reversible jet-fan blade by following the design guidelines which they obtained from the sensitivity analysis. The authors study the aerodynamic characteristics of the redesigned blade and compare it to that of the baseline design configuration. They then manufacture a redesigned blade prototype. Finally, the authors present thrust and power measurements for both the baseline jet fan, and the baseline jet fan when fitted with a redesigned blade.
The Effect of the Degree of Reaction on the Leakage Loss in Steam Turbines
Authors: Yoon Sungho
Published: January 2013

The degree of reaction selected in designing steam turbines is of paramount importance. There has been competition between 50% reaction and impulse turbines over a century. It is, therefore, important to understand the effect of the degree of reaction on aerodynamic performance. In particular, a change in the degree of reaction affects the leakage flow substantially in both the stationary and rotating blades due to a change in the blade loading. The effect of the degree of reaction on the efficiency loss due to leakage flows is systematically investigated in this paper using analytical models. It is shown that the appropriate way to understand the efficiency loss due to leakage flows is to estimate the kinetic energy dissipation rather than the leakage mass flow rate, as demonstrated by Yoon et al. (Yoon, S., Curtis, E., Denton, J., and Longley, J., 2010, “The Effect of Clearance on Shrouded and Unshrouded Turbine at Two Different Levels of Reaction,” ASME Paper No. GT2010-22541). In order to estimate the efficiency loss due to leakage flows, the well-known Denton model (Denton, J. D., 1993, “Loss Mechanisms in Turbomachinery,” ASME J. Turbomach., 115, pp. 621–656) is extended by considering the velocity triangles in a repeating turbine stage. The extended model is compared with experimental data, at different degrees of reaction, and shows good agreement with measurements. It is shown that a reduction in the degree of reaction, at a fixed flow coefficient and a fixed work coefficient, results in an increase in the efficiency loss across the stationary blade but a decrease in that across the rotating blade. However, the efficiency loss across the stationary blade hub is estimated to be smaller than the efficiency loss across the rotating blade tip. A stationary blade can be better sealed than a rotating blade by applying multiple seals and using a leakage path with a low radius. The efficiency loss due to the tip leakage flow is substantially influenced by the choice of the tip configuration. Shrouded blades show several aerodynamic advantages over unshrouded blades in reducing the tip leakage efficiency loss. Employing multiple seals over the shroud decreases the tip leakage mass flow rate significantly. Moreover, as the degree of reaction approaches zero, the tip leakage mass flow rate over the shroud becomes small since the axial pressure drop across the rotating blade becomes small. In unshrouded blades, a reduction in the degree of reaction is shown to increase the leakage mass flow rate over the tip because the circumferential pressure difference between the blade pressure side and blade suction side generally increases when the pitch-to-chord ratio remains unchanged.
An Experimental Investigation of Kerosene Droplet Breakup by Laser-Induced Blast Waves
Authors: Gebel Gregor C. , Mosbach Thomas , Meier Wolfgang , Aigner Manfred , Le Brun Stéphane
Published: January 2013

The work presented in this paper intends to deepen our understanding of the mechanisms involved in the spark ignition of liquid fuel sprays. An experimental study is presented regarding the ignition of monodisperse droplet chains of Jet A-1 aviation kerosene in a generic model combustor under well-defined boundary conditions. Breakdowns created by focused laser radiation were used as ignition sparks. They featured rapid spatial expansion, resulting in the formation of spherical blast waves in the surrounding air. The focus of this study lay on the effect of the blast waves on the fuel droplets. Blast wave trajectories were investigated by Schlieren imaging. Their interaction with kerosene droplets was observed with a high speed camera via a long distance microscope; the droplets were visualized by laser-induced Mie scattering. Droplets within a distance of 10 mm from the breakdown position were deformed and disintegrated by the aerodynamic forces of the postshock flow field. Different breakup modes were observed, depending on the distance from the breakdown position: Catastrophic breakup was observed at a 5 mm distance, resonant breakup was observed at a 10 mm distance. Breakup by blast waves from ignition sparks is expected to be a crucial mechanism for spray ignition because it supports evaporation. Weber number calculations revealed that the breakup modes observed under lab conditions will also appear in aviation gas turbines at high altitude relight conditions.
Effect of Engine Operating Conditions and Coolant Temperature on the Physical and Chemical Properties of Deposits From an Automotive Exhaust Gas Recirculation Cooler
Authors: Prabhakar Bhaskar , Boehman André L.
Published: January 2013

The effect of engine operating conditions on exhaust gas recirculation (EGR) cooler fouling was studied using a 6.4 L V-8 common rail turbodiesel engine. An experimental setup, which included a custom-made shell and tube heat exchanger (EGR cooler) with six surrogate tubes, was designed to control flow variables independently. The engine was operated at 2150 rpm, 203 Nm and 1400 rpm, 81 Nm, representing medium and low load conditions, respectively, and the coolant to the heat exchanger was circulated at 85 °C and 40 °C. Heat exchanger effectiveness and pressure drop was monitored throughout the tests. Deposits from the EGR cooler were collected every 1.5 h for a total of 9 h, and their microstructure was analyzed using a scanning electron microscope while their chemical composition was analyzed using a pyrolysis GC-MS apparatus, and the elemental weight percentages were obtained using a CHN analyzer. The results of these analyses showed that the effectiveness of the EGR cooler drops rapidly initially and asymptotes in a few hours. The medium load condition had a higher effectiveness loss due to a greater accumulation of deposits inside the EGR cooler, mostly due to increased thermophoresis, and produced smaller and coarse particles. The low load condition had lower effectiveness loss but produced bigger particles mostly due to excess hydrocarbons. Coolant temperature played a significant role in altering the deposit microstructure and in increasing the amount of condensed hydrocarbons. More deposits were produced for the cold coolant condition, indicating that lower coolant temperature promotes greater hydrocarbon condensation and thermophoresis. These results indicate the complex nature of fouling in automotive heat exchangers.
Superimposition of Elementary Thermodynamic Cycles and Separation of the Heat Transfer Section in Energy Systems Analysis
Authors: Morandin Matteo , Toffolo Andrea , Lazzaretto Andrea
Published: January 2013

In a wide variety of thermal energy systems, the high integration among components derives from the need to correctly exploit all the internal heat sources by a proper matching with the internal heat sinks. According to what has been suggested in previous works to address this problem in a general way, a “basic configuration” can be extracted from the system flowsheet including all components but the heat exchangers, in order to exploit the internal heat integration between hot and cold thermal streams through process integration techniques. It was also shown how the comprehension of the advanced thermodynamic cycles can be strongly facilitated by decomposing the system into elementary thermodynamic cycles which can be analyzed separately. The advantages of the combination of these approaches are summarized in this paper using the steam injected gas turbine (STIG) cycle and its evolution towards more complex system configurations as an example of application. The new concept of “baseline thermal efficiency” is introduced to combine the efficiencies of the elementary cycles making up the overall system, which demonstrates to be a useful reference to quantify the performance improvement deriving from heat integration between elementary cycles within the system.
Feature-Based Solid Model Reconstruction
Authors: Wang Jun , Gu Dongxiao , Gao Zhanheng , Yu Zeyun , Tan Changbai , Zhou Laishui
Published: January 2013

In this paper, we propose an effective solution to reconstruct solid models of existing objects. Specifically, we convert the model reconstruction problem into the issue of feature parameter extraction, and thereby design diverse methods to extract the parameters of basic design features from input surface meshes. After extracting the feature parameters, the corresponding features are constructed. By performing modeling operations on those features, the final solid model is constructed, and meanwhile the complete history of the model building operations is recorded. By introducing the concepts of “feature,” “constraint,” and “modeling history” into the reconstruction process, the design intent is captured and hence represented in the reconstructed model. As a result, the model is geometrically accurate and topologically consistent, and moreover it is flexibly editable, which makes it convenient to carry out model redesign and modification for the innovation applications. A variety of experimental results demonstrate the effectiveness and robustness of this solution.
Mechanical Characterization of Thin SOFC Electrolytes With Honeycomb Support
Authors: Berke Ryan B. , Walter Mark E.
Published: January 2013

Planar solid oxide fuel cells are made up of repeating sequences of electrolytes, electrodes, seals, and current collectors. The electrolyte should be as thin as possible for optimal electrochemical efficiency; however, for electrolyte-supported cells, the thin electrolytes are susceptible to damage during production, assembly, and operation. To produce cells that are sufficiently mechanically robust, electrolytes can be made having a cosintered honeycomb structure that supports thin, electrochemically efficient electrolyte membranes. Use of finite element analysis is desirable to mechanically characterize such electrolytes. To maintain reasonable numbers of elements and element aspect ratios, it is not possible to simultaneously model the small-scale details together with the overall membrane response. A two-scale approach is devised: the smaller mesoscale analyzes a representative area of the electrolyte, while the larger macroscale examines the electrolyte as a whole. Elastic properties for the mesoscale model are measured over a range of temperatures using a sonic resonance technique. Effective properties for the macroscale are obtained over a range of mesoscale geometries and can be obtained without needing to rerun the mesoscale simulations. The effective properties are experimentally validated using four-point bend experiments on representative samples. The bulk properties and the effective properties can then be used as material inputs for the macroscale model in order to design cells that are more sufficiently mechanically robust without sacrificing electrochemical performance.
Influence of the Vaginal Microbiota on Toxic Shock Syndrome Toxin 1 Production by Staphylococcus aureus
Authors: MacPhee Roderick A. , Miller Wayne L. , Gloor Gregory B. , McCormick John K. , Hammond Jo-Anne , Burton Jeremy P. , Reid Gregor
Published: January 2013

Menstrual toxic shock syndrome (TSS) is a serious illness that afflicts women of premenopausal age worldwide and arises from vaginal infection by
Staphylococcus aureus
and concurrent production of toxic shock syndrome toxin-1 (TSST-1). Studies have illustrated the capacity of lactobacilli to reduce
S. aureus
virulence, including the capacity to suppress TSST-1. We hypothesized that an aberrant microbiota characteristic of pathogenic bacteria would induce the increased production of TSST-1 and that this might represent a risk factor for the development of TSS. A
S. aureus
TSST-1 reporter strain was grown in the presence of vaginal swab contents collected from women with a clinically healthy vaginal status, women with an intermediate status, and those diagnosed with bacterial vaginosis (BV). Bacterial supernatant challenge assays were also performed to test the effects of aerobic vaginitis (AV)-associated pathogens toward TSST-1 production. While clinical samples from healthy and BV women suppressed toxin production,
in vitro
studies demonstrated that
Streptococcus agalactiae
and
Enterococcus
spp. significantly induced TSST-1 production, while some
Lactobacillus
spp. suppressed it. The findings suggest that women colonized by
S. aureus
and with AV, but not BV, may be more susceptible to menstrual TSS and would most benefit from prophylactic treatment.
Comparison of the Genome Sequences of “Candidatus Portiera aleyrodidarum” Primary Endosymbionts of the Whitefly Bemisia tabaci B and Q Biotypes
Authors: Jiang Zi-Feng , Xia Fangfang , Johnson Kipp W. , Brown Christopher D. , Bartom Elizabeth , Tuteja Jigyasa H. , Stevens Rick , Grossman Robert L. , Brumin Marina , White Kevin P. , Ghanim Murad
Published: January 2013

“
Candidatus
Portiera aleyrodidarum” is the primary endosymbiont of whiteflies. We report two complete genome sequences of this bacterium from the worldwide invasive B and Q biotypes of the whitefly
Bemisia tabaci
. Differences in the two genome sequences may add insights into the complex differences in the biology of both biotypes.
The Iron-Dependent Regulator Fur Controls Pheromone Signaling Systems and Luminescence in the Squid Symbiont Vibrio fischeri ES114
Authors: Septer Alecia N. , Lyell Noreen L. , Stabb Eric V.
Published: January 2013

Bacteria often use pheromones to coordinate group behaviors in specific environments. While high cell density is required for pheromones to achieve stimulatory levels, environmental cues can also influence pheromone accumulation and signaling. For the squid symbiont
Vibrio fischeri
ES114, bioluminescence requires pheromone-mediated regulation, and this signaling is induced in the host to a greater extent than in culture, even at an equivalent cell density. Our goal is to better understand this environment-specific control over pheromone signaling and bioluminescence. Previous work with
V. fischeri
MJ1 showed that iron limitation induces luminescence, and we recently found that ES114 encounters a low-iron environment in its host. Here we show that ES114 induces luminescence at lower cell density and achieves brighter luminescence in low-iron media. This iron-dependent effect on luminescence required ferric uptake regulator (Fur), which we propose influences two pheromone signaling master regulators, LitR and LuxR. Genetic and bioinformatic analyses suggested that under low-iron conditions, Fur-mediated repression of
litR
is relieved, enabling more LitR to perform its established role as an activator of
luxR
. Interestingly, Fur may similarly control the LitR homolog SmcR of
Vibrio vulnificus
. These results reveal an intriguing regulatory link between low-iron conditions, which are often encountered in host tissues, and pheromone-dependent master regulators.
Isolation of a Substantial Proportion of Forest Soil Bacterial Communities Detected via Pyrotag Sequencing
Authors: VanInsberghe David , Hartmann Martin , Stewart Gordon R. , Mohn William W.
Published: January 2013

We isolated 1,264 bacterial strains from forest soils previously surveyed via pyrosequencing of rRNA gene amplicons. Conventional culturing techniques recovered a substantial proportion of the community, with isolates representing 22% of 98,557 total pyrotags. Growth characteristics of isolates indicated that ecological traits were associated with relative abundances of corresponding pyrotag operational taxonomic units.
Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs
Authors: Jechalke Sven , Kopmann Christoph , Rosendahl Ingrid , Groeneweg Joost , Weichelt Viola , Krögerrecklenfort Ellen , Brandes Nikola , Nordwig Mathias , Ding Guo-Chun , Siemens Jan , Heuer Holger , Smalla Kornelia
Published: January 2013

Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes
sul1
and
sul2
in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both
sul
genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of
sul
genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the
sul
gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into
Escherichia coli
. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.
Hydrologic and Vegetative Removal of Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii Surrogate Microspheres in Coastal Wetlands
Authors: Hogan Jennifer N. , Daniels Miles E. , Watson Fred G. , Oates Stori C. , Miller Melissa A. , Conrad Patricia A. , Shapiro Karen , Hardin Dane , Dominik Clare , Melli Ann , Jessup David A. , Miller Woutrina A.
Published: January 2013

Constructed wetland systems are used to reduce pollutants and pathogens in wastewater effluent, but comparatively little is known about pathogen transport through natural wetland habitats. Fecal protozoans, including
Cryptosporidium parvum
,
Giardia lamblia
, and
Toxoplasma gondii
, are waterborne pathogens of humans and animals, which are carried by surface waters from land-based sources into coastal waters. This study evaluated key factors of coastal wetlands for the reduction of protozoal parasites in surface waters using settling column and recirculating mesocosm tank experiments. Settling column experiments evaluated the effects of salinity, temperature, and water type (“pure” versus “environmental”) on the vertical settling velocities of
C. parvum
,
G. lamblia
, and
T. gondii
surrogates, with salinity and water type found to significantly affect settling of the parasites. The mesocosm tank experiments evaluated the effects of salinity, flow rate, and vegetation parameters on parasite and surrogate counts, with increased salinity and the presence of vegetation found to be significant factors for removal of parasites in a unidirectional transport wetland system. Overall, this study highlights the importance of water type, salinity, and vegetation parameters for pathogen transport within wetland systems, with implications for wetland management, restoration efforts, and coastal water quality.
Isolation of a Bifidogenic Peptide from the Pepsin Hydrolysate of Bovine Lactoferrin
Authors: Oda Hirotsugu , Wakabayashi Hiroyuki , Yamauchi Koji , Sato Takumi , Xiao Jin-Zhong , Abe Fumiaki , Iwatsuki Keiji
Published: January 2013

Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals for which various biological functions have been reported, such as antimicrobial activity and bifidogenic activity. In this study, we compared the bifidogenic activity of bovine lactoferrin (bLF) and pepsin hydrolysate of bLF (bLFH), isolated bifidogenic peptide from bLFH, and investigated the bifidogenic spectra of bLF, bLFH, and its active peptide against 42 bifidobacterial strains comprising nine species. Against
Bifidobacterium breve
ATCC 15700
T
, minimal effective concentrations of bLF and bLFH were 300 and 10 μg/ml. Against
Bifidobacterium longum
subsp.
infantis
ATCC 15697
T
, the minimal effective concentration of bLFH was 30 μg/ml, and bLF did not show bifidogenic activity within 300 μg/ml. As an active peptide, a heterodimer of A
1
-W
16
and L
43
-A
48
linked by a disulfide bond was isolated. Previously, this peptide was identified as having antibacterial activity. An amino acid mixture with the same composition as this peptide showed no bifidogenic activity. The strains of each species whose growth was highly promoted (>150%) by this peptide at 3.75 μM were as follows:
B. breve
(7 out of 7 strains [7/7]),
B. longum
subsp.
infantis
(5/5),
Bifidobacterium bifidum
(2/5),
B. longum
subsp.
longum
(1/3),
Bifidobacterium adolescentis
(3/6),
Bifidobacterium catenulatum
(1/4),
Bifidobacterium pseudocatenulatum
(0/4),
Bifidobacterium dentium
(0/5), and
Bifidobacterium angulatum
(0/3). Growth of none of the strains was highly promoted by bLF at 3.75 μM. We demonstrated that bLFH showed stronger bifidogenic activity than natural bLF, especially against infant-representative species,
B. breve
and
B. longum
subsp.
infantis
; furthermore, we isolated its active peptide. This is the first report about a bifidogenic peptide derived from bLF.
Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR
Authors: Shi Xu , Gao Weimin , Chao Shih-hui , Zhang Weiwen , Meldrum Deirdre R.
Published: January 2013

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatomThalassiosira pseudonanaas a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels inT. pseudonanaand demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.
Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa
Authors: Kung Stephanie H. , Retchless Adam C. , Kwan Jessica Y. , Almeida Rodrigo P. P.
Published: January 2013

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in
Xylella fastidiosa
, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced
X. fastidiosa
subsp.
fastidiosa
genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of
X. fastidiosa
, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.
Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers
Authors: Drury Bradley , Rosi-Marshall Emma , Kelly John J.
Published: January 2013

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of
Deltaproteobacteria
,
Desulfococcus
,
Dechloromonas
, and
Chloroflexi
sequences and increases in abundances of
Nitrospirae
and
Sphingobacteriales
sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.
Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii: a Coenzyme A Loop Saves Energy for Caffeate Activation
Authors: Hess Verena , Gonzï¿½lez Josï¿½ M. , Parthasarathy Anutthaman , Buckel Wolfgang , Mï¿½ller Volker
Published: January 2013

The anaerobic acetogenic bacterium
Acetobacterium woodii
couples reduction of caffeate with electrons derived from molecular hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions. Caffeate is activated to caffeyl coenzyme A (caffeyl-CoA) prior to its reduction, and the caffeate reduction operon encodes an ATP-dependent caffeyl-CoA synthetase that is thought to catalyze the initial caffeate activation. The operon also encodes a potential CoA transferase, the product of
carA
, which was thought to be involved in subsequent ATP-independent caffeate activation. To prove the proposed function of
carA
, we overproduced its protein in
Escherichia coli
and then purified it. Purified CarA drives the formation of caffeyl-CoA from caffeate with hydrocaffeyl-CoA as the CoA donor. The dependence of the reaction on caffeate and hydrocaffeyl-CoA followed Michaelis-Menten kinetics, with apparent

K
m

values of 75 ï¿½ 5 μM for caffeate and 8 ï¿½ 2 μM for hydrocaffeyl-CoA. The enzyme activity had broad ranges of pH and temperature optima. In addition to being able to use caffeate, CarA could use
p
-coumarate and ferulate but not cinnamate, sinapate, or
p
-hydroxybenzoate as a CoA acceptor. Neither acetyl-CoA nor butyryl-CoA served as the CoA donor for CarA. The enzyme uses a ping-pong mechanism for CoA transfer and is the first classified member of a new subclass of family I CoA transferases that has two catalytic domains on one polypeptide chain. Apparently, CarA catalyzes an energy-saving CoA loop for caffeate activation in the steady state of caffeate respiration.
New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7
T
: Implications for DNA Content
Authors: P�rez-Cruz Carla , Carri�n Ornella , Delgado Lidia , Martinez Gemma , L�pez-Iglesias Carmen , Mercade Elena
Published: January 2013

Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium
Shewanella vesiculosa
M7
T
has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 � 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium
Shewanella vesiculosa
M7
T
that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).
Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai
Authors: Chen Chun , Lewis Carrie R. , Goswami Kakolie , Roberts Elisabeth L. , DebRoy Chitrita , Dudley Edward G.
Published: January 2013

Prophages make up 12% of the enterohemorrhagic
Escherichia coli
genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of
E. coli
O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by
E. coli
O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 ï¿½ 10
−4
. One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing
pchB
and
psrC
, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other
E. coli
O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of
E. coli
O157:H7.
Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria
Authors: Parikh Amit , Kumar Devanand , Chawla Yogesh , Kurthkoti Krishna , Khan Shazia , Varshney Umesh , Nandicoori Vinay K.
Published: January 2013

Escherichia coli
-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the
Mycobacterium tuberculosis pknK
gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5
attB
site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.
Characterization of an Acid-Inducible Sulfatase in Salmonella enterica Serovar Typhimurium
Authors: Das Seema , Singh Swati , McClelland Michael , Forst Steven , Gyaneshwar Prasad
Published: January 2013

Sulfatases of enteric bacteria can provide access to heavily sulfated mucosal glycans. In this study, we show that
aslA
(STM0084) of
Salmonella enterica
serovar Typhimurium LT2 encodes a sulfatase that requires mildly acidic pH for its expression and activity. AslA is not regulated by sulfur compounds or tyramine but requires the EnvZ-OmpR and PhoPQ regulatory systems, which play an important role in pathogenesis.
Antifungal Hydroxy Fatty Acids Produced during Sourdough Fermentation: Microbial and Enzymatic Pathways, and Antifungal Activity in Bread
Authors: Black Brenna A. , Zannini Emanuele , Curtis Jonathan M. , Gï¿½nzle Michael G.
Published: January 2013

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity.
Lactobacillus hammesii
exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of
L. hammesii
to a monohydroxy C
18:1
fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from
L. hammesii
and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter
−1
. Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter
−1
.
L. hammesii
accumulated the monohydroxy C
18:1
fatty acid in sourdough to a concentration of 0.73 ï¿½ 0.03 g liter
−1
(mean ï¿½ standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with
L. hammesii
or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion,
L. hammesii
converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.
Chemoreceptor VfcA Mediates Amino Acid Chemotaxis in Vibrio fischeri
Authors: Brennan Caitlin A. , DeLoney-Marino Cindy R. , Mandel Mark J.
Published: January 2013

Flagellar motility and chemotaxis by
Vibrio fischeri
are important behaviors mediating the colonization of its mutualistic host, the Hawaiian bobtail squid. However, none of the 43 putative methyl-accepting chemotaxis proteins (MCPs) encoded in the
V. fischeri
genome has been previously characterized. Using both an available transposon mutant collection and directed mutagenesis, we isolated mutants for 19 of these genes, and screened them for altered chemotaxis to six previously identified chemoattractants. Only one mutant was defective in responding to any of the tested compounds; the disrupted gene was thus named
vfcA
(

V
ibrio
f
ischeri

c
hemoreceptor
A
; locus tag VF_0777). In soft-agar plates, mutants disrupted in
vfcA
did not exhibit the serine-sensing chemotactic ring, and the pattern of migration in the mutant was not affected by the addition of exogenous serine. Using a capillary chemotaxis assay, we showed that, unlike wild-type
V. fischeri
, the
vfcA
mutant did not undergo chemotaxis toward serine and that expression of
vfcA
on a plasmid in the mutant was sufficient to restore the behavior. In addition to serine, we demonstrated that alanine, cysteine, and threonine are strong attractants for wild-type
V. fischeri
and that the attraction is also mediated by VfcA. This study thus provides the first insights into how
V. fischeri
integrates information from one of its 43 MCPs to respond to environmental stimuli.
Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms
Authors: Rajendran Ranjith , Williams Craig , Lappin David F. , Millington Owain , Martins Margarida , Ramage Gordon
Published: January 2013

Aspergillus fumigatushas been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized thatA. fumigatusbiofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component ofA. fumigatusECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.