bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehension of conversational implicature in L2 Chinese
Authors: Taguchi Naoko , Li Shuai , Liu Yan
Published: November 2013

This study examined the ability to comprehend conventional and non-conventional implicatures, and the effect of proficiency and learning context (foreign language learners vs. heritage learners) on comprehension of implicature in L2 Chinese. Participants were three groups of college students of Chinese: elementary-level foreign language learners (n=21), advanced-level foreign language learners (n=25), and advanced-level heritage learners (n=25). They completed a 36-item computer-delivered listening test measuring their ability to comprehend three types of implicature: conventional indirect refusals, conventional indirect opinions, and non-conventional indirect opinions. Comprehension was analyzed for accuracy (scores on a multiple-choice measure) and comprehension speed (average time taken to answer items correctly). There was a significant effect of implicature type on accuracy, but not on comprehension speed. A significant effect of participant group was observed on accuracy, but the effect was mixed on comprehension speed.
Rethinking communicative competence for typical speakers
Authors: Tsai Meng-Ju
Published: November 2013

The concept of communicative competence has been studied widely for over 40 years in several fields, including linguistics, psychology, and speech communication. Different definitions of communicative competence and measurement of communicative competence exist in these fields. A clear approach to communicative competence for typical speaking individuals and its measurement of communicative competence is unclear. This paper aims to: (1) review four main approaches to communicative competence and highlight strengths and weaknesses of each approach; (2) develop an integrated approach to communicative competence for typical speakers; and (3) address measurement of communicative competence. An integrated approach to communicative competence for typical speakers provides a fundamental access to communicative competence in different fields. For examples, scholars and speech, language pathologists have clear knowledge assessing communicative competence of individuals with communication disorders.
The coupling-constitution fallacy
Authors: Kagan Aaron , Lassiter Charles
Published: November 2013

The coupling-constitution fallacy claims that arguments for extended cognition involve the inference of “x and y constitute z” from “x is coupled to y” and that such inferences are fallacious. We argue that the coupling-constitution fallacy fails in its goal to undermine the hypothesis of extended cognition: appeal to the coupling-constitution fallacy to rule out possible empirical counterexamples to intracranialism is fallacious. We demonstrate that appeals to coupling-constitution worries are problematic by constructing the fallacious argument against the hypothesis of extended cognition. We consider several objections to our argument and ﬁnd them insufficient to rebut our conclusion.
Hedging and rounding in numerical expressions
Authors: Williams Sandra , Power Richard
Published: November 2013

Previous accounts of hedges assume that they cause language to become vague or fuzzy (Lakoff 1973); however, hedges can actually sharpen numerical concepts by giving explicit information about approximation, especially where bare numbers appear misleadingly round or precise. They can also tell hearers about the direction of approximation (greater or less than). This article provides a first empirical account of interactions between hedging and rounding in numerical expressions. We demonstrate that hedges occur more commonly with round numbers than with non-round ones. However, we also provide evidence from user studies that in the absence of hedges, readers interpret round numbers as approximations and non-round ones as precise; and that placing a hedge before a round number has no effect on its interpretation, whereas placing it before a non-round number shifts people’s interpretations from precise towards approximate. We attempt to explain this conundrum.
The communicative functions of five signing chimpanzees (Pan troglodytes)
Authors: Leeds Charles Austin , Jensvold Mary Lee A.
Published: November 2013

Speech act theory describes units of language as acts which function to change the behavior or beliefs of the partner. Therefore, with every utterance an individual seeks a communicative goal that is the underlying motive for the utterance’s production; this is the utterance’s function. Studies of deaf and hearing human children classify utterances into categories of communicative function. This study classified signing chimpanzees’ utterances into the categories used in human studies. The chimpanzees utilized all seven categories of communicative functions and used them in ways that resembled human children. The chimpanzees’ utterances functioned to answer questions, request objects and actions, describe objects and events, make statements about internal states, accomplish tasks such as initiating games, protest interlocutor behavior, and as conversational devices to maintain and initiate conversation.
Towards a New Jerusalem: The Committee for Regulating the Excise, 1649–1653
Authors: Coffman D’Maris
Published: November 2013

The development of the British excise establishment shows a high degree of path dependence. The Long Parliament and Commonwealth used the excise ordinances as collateral for a wide array of public debt instruments. Because of the extra-constitutionality and dubious legality of domestic commodity taxation and the necessity of satisfying creditors, the introduction of the excises in the 1640s were marked by an unprecedented degree of transparency, accountability, and a commitment to maintaining the ‘publike faith.’ The Long Parliament’s practice of ‘administration by legislation,’ coupled with aggressive oversight by the Commonwealth Committee for Regulating the Excise, created a body of procedures and precedents that the Restoration Treasury preserved and adapted in its attempts to settle the revenue. Analysis of Restoration redactions of the Committee’s minute and order books, coupled with examination of successive salary lists, offers insight into how this knowledge was preserved and transferred through successive regimes. To a surprising degree, the structure of the eighteenth-century excise establishment was in place by 1654. The resulting narrative complements and refines claims made by Braddick for the evolution of the Interregnum excise establishment. Although the restoration of Charles II marked a return to court borrowing, the domestic excise, the most important fiscal innovation of the Interregnum period, survived regime change—albeit in reduced form. North and Weingast associated legal protection of creditors and taxpayers, a commitment to servicing debt, and the idea of ‘public credit’ with the Revolution of 1688/89. In doing so, they neglect the specifically English origins of the Financial Revolution.
Hungatella effluvii gen. nov., sp. nov., an obligately anaerobic bacterium isolated from an effluent treatment plant, and reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov.
Authors: Kaur Sukhpreet , Yawar Mir , Kumar P. Anil , Suresh K.
Published: November 2013

A Gram-stain-positive, rod-shaped, spore-forming and strictly anaerobic bacterium, designated UB-B.2T, was isolated from an industrial effluent anaerobic digester sample. It grew optimally at 30 °C and pH 7.0. Comparative analysis of the 16S rRNA gene sequence confirmed that strain UB-B.2Twas closely related toClostridium hathewayiDSM 13479T(97.84 % similarity), a member of rRNA gene cluster XIVa of the genusClostridium, and formed a coherent cluster with other related members of theBlautia(Clostridium)coccoidesrRNA group in phylogenetic analyses. The end products of glucose fermentation by strain UB-B.2Twere acetate and propionate. The G+C content of the DNA was 51.4 mol%. Although strain UB-B.2Tshowed 97.8 % 16S rRNA gene sequence identity to the type strain ofC. hathewayi, it exhibited only 38.4 % relatedness at the whole-genome level. It also showed differences from its closest phylogenetic relative,C. hathewayiDSM 13479T, in phenotypic characteristics such as hydrolysis of aesculin, starch and urea and fermentation end products. Both strains showed phenotypic differences from the members of rRNA gene cluster XIVa of the genusClostridium. Based on these differences,C. hathewayiDSM 13479Tand strain UB-B.2Twere identified as representatives of a new genus of the familyClostridiaceae. Thus, we propose the reclassification ofClostridium hathewayiasHungatella hathewayigen. nov., comb. nov., the type species of the new genus (type strain DSM 13479T = CCUG 43506T = MTCC 10951T). Strain UB-B.2T( = MTCC 11101T = DSM 24995T) is assigned to the novel speciesHungatella effluviigen. nov., sp. nov as the type strain.
Killing rates exerted by caspofungin in 50 % serum and its correlation with in vivo efficacy in a neutropenic murine model against Candida krusei and Candida inconspicua
Authors: Kovács Renátó , Gesztelyi Rudolf , Berényi Réka , Domán Marianna , Kardos Gábor , Juhász Béla , Majoros László
Published: November 2013

Killing rates (K) of 1–32 µg ml−1 caspofungin were determined in RPMI-1640 and in 50 % serum using time–kill methodology against three Candida krusei (MICs of all three isolates 0.25 µg ml−1 in RPMI-1640 and 2 µg ml−1 in serum) and three Candida inconspicua clinical isolates (MIC ranges 0.06–0.12 µg ml−1 in RPMI-1640 and 0.25–0.5 µg ml−1 in serum), against C. krusei ATCC 6258 and against one C. krusei isolate that was resistant to echinocandins (MIC 8 µg ml−1 in RPMI-1640 and 32 µg ml−1 in serum). In RPMI-1640, the highest mean K values were observed at 4 (−1.05 h−1) and 16 (−0.27 h−1) μg ml−1 caspofungin for C. krusei and C. inconspicua clinical isolates, respectively. In 50 % serum, mean K value ranges at 1–32 and 4–32 µg ml−1 concentrations for C. inconspicua and C. krusei were −1.12 to −1.44 and −0.42 to −0.57 h−1, respectively. While K values against C. krusei in RPMI-1640 and 50 % serum were comparable, serum significantly increased the killing rate against C. inconspicua (P<0.0003 for all tested concentrations). In a neutropenic murine model, daily caspofungin at 1, 2, 3, 5 and 15 mg kg−1 significantly decreased the fungal tissue burden of C. inconspicua in the kidneys (P<0.05–0.001). Against C. krusei, doses of 3, 5 and 15 mg kg−1 caspofungin were effective (P<0.05–0.01). All effective doses were comparably efficacious for both species. Only the highest 15 mg kg−1 caspofungin dose was effective even against the echinocandin-resistant C. krusei isolate. In 50 % serum, killing was concentration independent at effective concentrations (≥4 and ≥1 µg ml−1 for C. krusei and C. inconspicua, respectively), suggesting that the efficacy of dose escalation is questionable. These in vitro results were also supported by the murine model.
Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus
Authors: Weinheimer Isabel , Boonrod Kajohn , Moser Mirko , Wassenegger Michael , Krczal Gabi , Butcher Sarah J. , Valkonen Jari P. T.
Published: November 2013

Sweet potato chlorotic stunt virus (SPCSV; genusCrinivirus, familyClosteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III–like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs.In vitroassays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2′-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg2+was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn2+. Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase IIIin vitroor make them unsuitable for processing by the enzyme.
Evolution in the influenza A H3 stalk – a challenge for broad-spectrum vaccines?
Authors: Lees William D. , Moss David S. , Shepherd Adrian J.
Published: November 2013

Recently, a number of broad-spectrum human antibodies binding to the stalk region of influenza A haemagglutinin (HA) have been isolated. As this region tends to develop substitutions at a slower rate than other regions of HA, a vaccine eliciting such antibodies could have a longer effective life. But this begs a question: is the stalk resistant to change even in the face of evolutionary pressure? In this paper, we analysed the known epitopes in the H3 stalk and, utilizing a collection of 3440 sequences, present a novel approach for detecting putative B-cell epitopes in regions such as this, in which mutations occur infrequently. We concluded that there have been periods of activity in the stalk that are consistent with the evolution of antigenic escape. This work casts light on the presence of stalk-binding antibodies in the population as a whole and, through the analysis of antigenically active regions in the stalk, may contribute to the identification of epitopes that are refractive to change and hence useful for vaccine development.
Ag as a surfactant for Co/MgO(111)-(3×3)R 30°
Authors: Johnson-Steigelman H. Trevor , Parihar Somendra S. , King Seth T. , Lyman Paul F.
Published: November 2013

Ag was examined as a potential surfactant to aid in the growth of smooth Co films deposited on MgO. Ag was deposited onto single-crystal MgO(111)-(3×3)R30° substrates. It was found that Ag formed islands upon annealing. Monolayer films of Co were subsequently deposited using an electrostatic electron-beam evaporator on single-crystal MgO(111)-(3×3)R30° substrates with 0.5–10 monolayers of Ag. The Ag/MgO substrates were held at room temperature or 450 °C during growth, with subsequent annealing of temperatures 400–800 °C. These films have been characterized using low-energy electron diffraction, x-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Despite the fact that Ag formed islands, it was found that the presence of Ag did have a surfactant effect upon the thin-film growth of Co on Ag/MgO(111)-(3×3)R30°. Co islands were still present, but the surface was much smoother than for films grown without the Ag surfactant. XPS peak intensity changes and AFM suggest strongly that Ag segregated to the top of these samples at temperatures above 400 °C. In the absence of Ag, AFM evidence suggests that the Co growth mode appears to be Volmer–Weber island growth, but the morphology is much smoother with Ag present.
Pseudomorphic growth of InAs on misoriented GaAs for extending quantum cascade laser wavelength
Authors: Meyer Charles , Cheng Emily , Grayer Justin , Mueller David , Triplett Gregory , Roberts Denzil , Graham Samuel
Published: November 2013

The authors have studied the impact of epilayer strain on the deposition of InAs/GaAs on (100) and (111)B with 2° offset toward⟨2-1-1⟩ surfaces. Consequences of a 7% lattice mismatch between these orientations in the form of three-dimensional growth are less apparent for (111)B with 2° offset toward⟨2-1-1⟩ surfaces compared to (100). By exploring a range of molecular beam epitaxy process parameters for InAs/GaAs growth and utilizing scanning electron microscopy, atomic force microscopy, and Raman spectroscopy to evaluate the quality of these strained layers, the authors develop empirical models that describe the influence of the process conditions in regards to surface roughness with >92% accuracy. The smoothest InAs/GaAs samples demonstrated average surface roughness of 0.08 nm for 10 μm2 areas, albeit at very low deposition rates. The authors have found the most important process conditions to be substrate temperature and deposition rate, leading us to believe that controlling diffusion length may be the key to reducing defects in severely strained structures. InGaAs/AlGaAs quantum cascade laser structures were also produced on (111)B with 2° offset toward⟨2-1-1⟩ to take advantage of the piezoelectric effect, and the modified laser transitions due to these effects were observed.
Atmospheric-pressure low-temperature plasma processes for thin film deposition
Authors: Kakiuchi Hiroaki , Ohmi Hiromasa , Yasutake Kiyoshi
Published: November 2013

Nonthermal plasmas generated under atmospheric pressure (AP) have been receiving increased attention in direct plasma technology applications for thin film deposition. This is because the atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD) is expected to realize low-cost and high-throughput processing with open air systems, which are of prime importance for various industrial applications. A large number of studies have been reported on the preparation of thin films using various types of AP plasma sources such as corona, dielectric barrier and AP glow discharges excited by pulsed or low-frequency power sources that can produce a nonequilibrium AP plasma. Most of the reported films using these common AP plasma sources have been related to polymers, oxides, and carbon materials. On the other hand, by virtue of the low ion energy due to the high collision frequency, AP-plasma process can have a nature of soft or gentle processing in addition to high-rate processing. Therefore, AP-PECVD also has a potential to form good-quality functional thin films, such as high-purity semiconductor or insulator thin films, which may be applicable for electronic devices. Although the development of AP-PECVD technology for such applications are attractive in the future advanced industry, the reports on these applications are limited. The reason may be related to the fact that the high collision frequency in AP plasma enhances secondary reactions in the gas phase to generate dust particles which will deteriorate the film quality, and also limits mass transport, which leads to poor uniformity of the resulting film. In the present article, the authors review the present status of AP low-temperature plasma processes, bearing in mind their application for high-purity functional thin films including silicon and related materials. The authors first summarize recent progress in the use of common AP plasma sources for direct PECVD processes. To grasp the present status of AP-PECVD technique, the authors have picked up popular materials for AP-PECVD, such as carbon, oxides, and other inorganic materials as well as silicon and related materials. Although there already exists a plenty of good review articles dealing with PECVD using common AP plasma sources, works on reviewing PECVD using radio-frequency (RF) and very-high-frequency (VHF) excitations of AP plasma seem to be insufficient. RF and VHF excitations of AP plasma are capable of generating continuous oscillating glow discharges without unstable streamers and filaments, which will be important to form uniform and dust-free films. So, secondly, the authors discuss the key distinguishing features of PECVD using RF and VHF excitations of AP plasma from the common AP plasma sources. Finally, they describe examples of the application of AP-VHF plasma to the preparation of silicon and related thin films.
Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types
Authors: Santamaría Rosa Isela , Bustos Patricia , Sepúlveda-Robles Omar , Lozano Luis , Rodríguez César , Fernández José Luis , Juárez Soledad , Kameyama Luis , Guarneros Gabriel , Dávila Guillermo , González Víctor
Published: November 2013

In this work, we isolated and characterized 14 bacteriophages that infect
Rhizobium etli
. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48
R. etli
strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and
in vivo
cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.
Genomic Investigation of Lysogen Formation and Host Lysis Systems of the Salmonella Temperate Bacteriophage SPN9CC
Authors: Shin Hakdong , Lee Ju-Hoon , Yoon Hyunjin , Kang Dong-Hyun , Ryu Sangryeol
Published: November 2013

To understand phage infection and host cell lysis mechanisms in pathogenic
Salmonella
, a novel
Salmonella enterica
serovar Typhimurium-targeting bacteriophage, SPN9CC, belonging to the
Podoviridae
family was isolated and characterized. The phage infects
S
. Typhimurium via the O antigen of lipopolysaccharide (LPS) and forms clear plaques with cloudy centers due to lysogen formation. Phylogenetic analysis of phage major capsid proteins revealed that this phage is a member of the lysogen-forming P22-like phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions and host cell lysis gene clusters show very low levels of identity, suggesting that lysogen formation and host cell lysis mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in
S
. Typhimurium and
Escherichia coli
hosts revealed that collaboration of these lysis proteins is important for the lysis of both hosts and that holin is a key protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a Δ
cI
mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge assays revealed that SPN9CCM has shorter eclipse/latency periods and a larger burst size, as well as higher host cell lysis activity, than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol agents similar to virulent phages.
Signature Protein of the PVC Superphylum
Authors: Lagkouvardos Ilias , Jehl Marc-André , Rattei Thomas , Horn Matthias
Published: November 2013

The phyla
Planctomycetes
,
Verrucomicrobia
,
Chlamydiae
,
Lentisphaerae
, and “
Candidatus
Omnitrophica (OP3)” comprise bacteria that share an ancestor but show highly diverse biological and ecological features. Together, they constitute the PVC superphylum. Using large-scale comparative genome sequence analysis, we identified a protein uniquely shared among all of the known members of the PVC superphylum. We provide evidence that this signature protein is expressed by representative members of the PVC superphylum. Its predicted structure, physicochemical characteristics, and overexpression in
Escherichia coli
and gel retardation assays with purified signature protein suggest a housekeeping function with unspecific DNA/RNA binding activity. Phylogenetic analysis demonstrated that the signature protein is a suitable phylogenetic marker for members of the PVC superphylum, and the screening of published metagenome data indicated the existence of additional PVC members. This study provides further evidence of a common evolutionary history of the PVC superphylum and presents a unique case in which a single protein serves as an evolutionary link among otherwise highly diverse members of major bacterial groups.
The Combined Effects of Bacterial Symbionts and Aging on Life History Traits in the Pea Aphid, Acyrthosiphon pisum
Authors: Laughton Alice M. , Fan Maretta H. , Gerardo Nicole M.
Published: November 2013

While many endosymbionts have beneficial effects on hosts under specific ecological conditions, there can also be associated costs. In order to maximize their own fitness, hosts must facilitate symbiont persistence while preventing symbiont exploitation of resources, which may require tight regulation of symbiont populations. As a host ages, the ability to invest in such mechanisms may lessen or be traded off with demands of other life history traits, such as survival and reproduction. Using the pea aphid,
Acyrthosiphon pisum
, we measured survival, lifetime fecundity, and immune cell counts (hemocytes, a measure of immune capacity) in the presence of facultative secondary symbionts. Additionally, we quantified the densities of the obligate primary bacterial symbiont,
Buchnera aphidicola
, and secondary symbionts across the host's lifetime. We found life history costs to harboring some secondary symbiont species. Secondary symbiont populations were found to increase with host age, while
Buchnera
populations exhibited a more complicated pattern. Immune cell counts peaked at the midreproductive stage before declining in the oldest aphids. The combined effects of immunosenescence and symbiont population growth may have important consequences for symbiont transmission and maintenance within a host population.
High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections
Authors: Palliyil Soumya , Downham Christina , Broadbent Ian , Charlton Keith , Porter Andrew J.
Published: November 2013

A number of bacteria, including pathogens like
Pseudomonas aeruginosa
, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in
P. aeruginosa
biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent
P. aeruginosa
infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in
P. aeruginosa
cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of
Pseudomonas
infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.
Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by
carB
-Based Oligonucleotide Microarray
Authors: Shin Hwa Hui , Hwang Byeong Hee , Seo Jeong Hyun , Cha Hyung Joon
Published: November 2013

It is important to rapidly and selectively detect and analyze pathogenic
Salmonella enterica
subsp.
enterica
in contaminated food to reduce the morbidity and mortality of
Salmonella
infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the
carB
gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three
S. enterica
subsp.
enterica
serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target
Salmonella
serotype from a bacterial mixture. Therefore, these results demonstrated that our novel
carB
-based oligonucleotide microarray can be used as an effective and specific detection system for
S. enterica
subsp.
enterica
serotypes.
Higher Water Temperature and Incubation under Aerobic and Microaerobic Conditions Increase the Recovery and Diversity of Arcobacter spp. from Shellfish
Authors: Levican Arturo , Collado Luis , Yustes Clara , Aguilar Carme , Figueras Maria José
Published: November 2013

Some
Arcobacter
species are considered emerging food-borne and waterborne pathogens, and shellfish have been suggested as one of their reservoirs. However, only a few studies have investigated the presence of
Arcobacter
in this kind of food. This study assesses the prevalence and diversity of
Arcobacter
spp. in shellfish by multiplex PCR (m-PCR) and culturing methods (under different atmospheric conditions) and evaluates the possible influence of environmental parameters (temperature, salinity, and harvesting bay).
Arcobacter
was detected by m-PCR and/or culturing in 61 (29.9%) of 204 shellfish samples. Of the positive samples by culturing, 41.1% were obtained under only aerobic incubation conditions, while 23.2% were obtained under only microaerobic conditions. Of 476 investigated isolates, 118 belonged to different enterobacterial repetitive intergenic consensus (ERIC)-PCR genotypes (strains) and to 11 different species. This study shows the highest diversity of
Arcobacter
species ever observed in samples from any origin. The most prevalent species was
Arcobacter butzleri
(60.2%), followed by
Arcobacter molluscorum
(21.2%). The prevalence of
Arcobacter
was significantly higher during the summer than in other seasons, being associated with an increase in water temperature. Results confirm that shellfish are a reservoir for a remarkable diversity of
Arcobacter
spp.
Community Dynamics and Activity of Ammonia-Oxidizing Prokaryotes in Intertidal Sediments of the Yangtze Estuary
Authors: Zheng Yanling , Hou Lijun , Newell Silvia , Liu Min , Zhou Junliang , Zhao Hui , You Lili , Cheng Xunliang
Published: November 2013

Diversity, abundance, and activity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using the ammonia monooxygenase α subunit (
amoA
) in the intertidal sediments of the Yangtze Estuary. Generally, AOB had a lower diversity of
amoA
genes than did AOA in this study. Clone library analysis revealed great spatial variations in both AOB and AOA communities along the estuary. The UniFrac distance matrix showed that all the AOB communities and 6 out of 7 AOA communities in the Yangtze Estuary were statistically indistinguishable between summer and winter. The studied AOB and AOA community structures were observed to correlate with environmental parameters, of which salinity, pH, ammonium, total phosphorus, and organic carbon had significant correlations with the composition and distribution of both communities. Also, the AOA communities were significantly correlated with sediment clay content. Quantitative PCR (qPCR) results indicated that the abundance of AOB
amoA
genes was greater than that of AOA
amoA
genes in 10 of the 14 samples analyzed in this study. Potential nitrification rates were significantly greater in summer than in winter and had a significant negative correlation with salinity. In addition, potential nitrification rates were correlated strongly only with archaeal
amoA
gene abundance and not with bacterial
amoA
gene abundance. However, no significant differences were observed between rates measured with and without ampicillin (AOB inhibitor). These results implied that archaea might play a more important role in mediating the oxidation of ammonia to nitrite in the Yangtze estuarine sediments.
The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures
Authors: Mascher Gerald , Derman Yağmur , Kirk David G. , Palonen Eveliina , Lindström Miia , Korkeala Hannu
Published: November 2013

In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II)
Clostridium botulinum
represents a major hazard in chilled foods, the cold adaption mechanisms of group II
C. botulinum
organisms are not known. Here, we show that the CLO3403/CLO3404 TCS of
C. botulinum
E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase (
clo3403
) and the response regulator (
clo3404
) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional
clo3403
and
clo3404
knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of
clo3403
had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of
C. botulinum
E1 Beluga.
Genetic and Functional Investigation of Zn2Cys6Transcription Factors RSE2 and RSE3 in Podospora anserina
Authors: Bovier Elodie , Sellem Carole H. , Humbert Adeline , Sainsard-Chanet Annie
Published: November 2013

InPodospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of theaoxgene. We identified 10 independent mutations in 9 positions inrse2and 11 mutations in 5 positions inrse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations inrse2orrse3. It showed that in addition toaox,fbp, andpck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both anrse2gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.
C-Terminal Truncations of the Saccharomyces cerevisiae PMA1 H
+
-ATPase Have Major Impacts on Protein Conformation, Trafficking, Quality Control, and Function
Authors: Mason A. Brett , Allen Kenneth E. , Slayman Carolyn W.
Published: November 2013

The C-terminal tail of yeast plasma membrane (PM) H
+
-ATPase extends approximately 38 amino acids beyond the final membrane-spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys
889
, removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala
881
(at the end of TM10) to Gly
888
is required for stable folding and PM targeting. (ii) The stretch between Lys
889
and Lys
916
, a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu
917
and Thr
918
) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H
+
-ATPase.
Global Expression Analysis of the Yeast Lachancea (Saccharomyces) kluyveri Reveals New
URC
Genes Involved in Pyrimidine Catabolism
Authors: Andersson Rasmussen Anna , Kandasamy Dineshkumar , Beck Halfdan , Crosby Seth D. , Björnberg Olof , Schnackerz Klaus D. , Piškur Jure
Published: November 2013

Pyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, the
URC
pathway, has been initially discovered in our laboratory in the yeast
Lachancea kluyveri
. Here, we present the global changes in gene expression in
L. kluyveri
in response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the known
URC
genes,
URC1-6
, helped to identify nine putative novel
URC
genes with a similar expression pattern. The microarray analysis provided evidence that both the
URC
and
PYD
genes are under nitrogen catabolite repression in
L. kluyveri
and are induced by uracil or dihydrouracil, respectively. We determined the function of
URC8
, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that the
L. kluyveri
Fui1p protein transported uridine, just like its homolog in
Saccharomyces cerevisiae
, but we demonstrated that is was not the only uridine transporter in
L. kluyveri
. We also showed that the
L. kluyveri
homologs of
DUR3
and
FUR4
do not have the same function that they have in
S. cerevisiae
, where they transport urea and uracil, respectively. In
L. kluyveri
, both of these deletion strains grew normally on uracil and urea.
WetA Is Required for Conidiogenesis and Conidium Maturation in the Ascomycete Fungus Fusarium graminearum
Authors: Son Hokyoung , Kim Myung-Gu , Min Kyunghun , Lim Jae Yun , Choi Gyung Ja , Kim Jin-Cheol , Chae Suhn-Kee , Lee Yin-Won
Published: November 2013

Fusarium graminearum
, a prominent fungal pathogen that infects major cereal crops, primarily utilizes asexual spores to spread disease. To understand the molecular mechanisms underlying conidiogenesis in
F. graminearum
, we functionally characterized the
F. graminearum
ortholog of
Aspergillus nidulans
wetA
, which has been shown to be involved in conidiogenesis and conidium maturation. Deletion of
F. graminearum
wetA
did not alter mycelial growth, sexual development, or virulence, but the
wetA
deletion mutants produced longer conidia with fewer septa, and the conidia were sensitive to acute stresses, such as oxidative stress and heat stress. Furthermore, the survival rate of aged conidia from the
F. graminearum
wetA
deletion mutants was reduced. The
wetA
deletion resulted in vigorous generation of single-celled conidia through autophagy-dependent microcycle conidiation, indicating that WetA functions to maintain conidial dormancy by suppressing microcycle conidiation in
F. graminearum
. Transcriptome analyses demonstrated that most of the putative conidiation-related genes are expressed constitutively and that only a few genes are specifically involved in
F. graminearum
conidiogenesis. The conserved and distinct roles identified for WetA in
F. graminearum
provide new insights into the genetics of conidiation in filamentous fungi.
The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis
Authors: Govindaraghavan Meera , Lad Alisha A. , Osmani Stephen A.
Published: November 2013

The G
2
-M transition in
Aspergillus nidulans
requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G
2
arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events.
N
-Acetylglucosamine (GlcNAc)-Inducible Gene
GIG2
Is a Novel Component of GlcNAc Metabolism in Candida albicans
Authors: Ghosh Swagata , Hanumantha Rao Kongara , Bhavesh Neel Sarovar , Das Gobardhan , Dwivedi Ved Prakash , Datta Asis
Published: November 2013

Candida albicans
is an opportunistic fungal pathogen that resides in the human body as a commensal and can turn pathogenic when the host is immunocompromised. Adaptation of
C. albicans
to host niche-specific conditions is important for the establishment of pathogenicity, where the ability of
C. albicans
to utilize multiple carbon sources provides additional flexibility. One alternative sugar is
N
-acetylglucosamine (GlcNAc), which is now established as an important carbon source for many pathogens and can also act as a signaling molecule. Although GlcNAc catabolism has been well studied in many pathogens, the importance of several enzymes involved in the formation of metabolic intermediates still remains elusive. In this context, microarray analysis was carried out to investigate the transcriptional responses induced by GlcNAc under different conditions. A novel gene that was highly upregulated immediately following the GlcNAc catabolic genes was identified and was named
GIG2
(GlcNAc-induced gene 2). This gene is regulated in a manner distinct from that of the GlcNAc-induced genes described previously in that GlcNAc metabolism is essential for its induction. Furthermore, this gene is involved in the metabolism of
N
-acetylneuraminate (sialic acid), a molecule equally important for initial host-pathogen recognition. Mutant cells showed a considerable decrease in fungal burden in mouse kidneys and were hypersensitive to oxidative stress conditions. Since
GIG2
is also present in many other fungal and enterobacterial genomes, targeted inhibition of its activity would offer insight into the treatment of candidiasis and other fungal or enterobacterial infections.
Bacterial Competition Reveals Differential Regulation of the
pks
Genes by Bacillus subtilis
Authors: Vargas-Bautista Carol , Rahlwes Kathryn , Straight Paul
Published: November 2013

Bacillus subtilis
is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The
pks
genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of
pks
genes appear to be organized as a giant operon (>74 kb from
pksC-pksR
). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the
pks
operon in
B. subtilis
was found to induce prodiginine production by
Streptomyces coelicolor
. Here, colonies of wild-type
B. subtilis
formed a spreading population that induced prodiginine production from
Streptomyces lividans
, suggesting differential regulation of
pks
genes and, as a result, bacillaene. While the parent colony showed widespread induction of
pks
expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the
pks
genes. To identify regulators that control
pks
genes, we first determined the pattern of
pks
gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of
pks
gene expression. We found that expression of the
pks
genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of
degU
,
comA
, and
scoC
had moderate effects, disrupting the timing and level of
pks
gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for
B. subtilis
.