bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic cough: Stepwise application in primary care practice of the ACCP guidelines for diagnosis and management of cough
Authors: Gahbauer Mary , Keane Patricia
Published: July 2009

Purpose: To illustrate decision points encountered when using evidence‐based guidelines for diagnosis and management of chronic cough by means of a composite case study based on primary care practice.

Data source: The American College of Chest Physicians (ACCP) Evidence‐Based Clinical Practice Guidelines for Diagnosis and Management of Cough, and review of the scientific literature on cough and related topics.

Conclusions: The ACCP guidelines offer a systematic approach that uses trials of empirical therapies to diagnose and resolve the very common, often perplexing complaint of chronic cough. The major diagnoses reached are upper airway cough syndrome, cough‐variant asthma, gastroesophageal reflux disease, and nonasthmatic eosinophilic bronchitis.

Implications for practice: Cough is the most common reason that patients seek an office visit. Many coughs resolve spontaneously, but coughs that persist significantly impair the quality of life. Use of the ACCP guidelines allows a stepwise empirical approach to the problem of unexplained chronic cough. This approach greatly increases the percentage of chronic coughs that are accurately diagnosed and effectively treated, and avoids unnecessary diagnostic testing.
Influence of metyrapone treatment during pregnancy on the development and maturation of brain monoaminergic systems in the rat
Authors: Leret M. L. , Rua C. , Garcia‐Montojo M. , Lecumberri M. , González J. C.
Published: July 2009

Aim:  This study examines the effect of reducing the corticosterone levels of gestating rat dams on the postnatal development and maturation of monoaminergic systems in their offspring’s brains.

Methods:  Metyrapone, an inhibitor of CORT synthesis, was administered to pregnant rats from E0 to E17 of gestation. Monoamine concentrations were determined in male and female offspring at postnatal days (PN) 23 and 90 in the hippocampus, hypothalamus and striatum.

Results:  Reducing maternal corticosterone (mCORT) during gestation led to alterations in dopamine and serotonin levels in all three brain areas studied at PN 23. Alterations persisted until at least PN 90 in the serotonergic systems; the dopamine content of the hippocampus also remained modified. Reduced mCORT during gestation also led to alterations in the development and maturation of the hypothalamic noradrenergic systems. Sexually dimorphic responses were observed in all these monoaminergic systems at different times.

Conclusion:  These results suggest that while they are still developing, brain monoaminergic systems are particularly sensitive to epigenetic influences. An adequate foetal level of CORT is required for the normal ontogeny of brain monoaminergic systems. The present data also provide that during the critical period of brain development, maternal CORT plays an important role in the sexual differentiation of monoaminergic systems, with particular influence on brain serotonergic neurones.
Electrophysiological determinants of hypokalaemia‐induced arrhythmogenicity in the guinea‐pig heart
Authors: Osadchii O. E. , Olesen S. P.
Published: July 2009

Aim: Hypokalaemia is an independent risk factor contributing to arrhythmic death in cardiac patients. In the present study, we explored the mechanisms of hypokalaemia‐induced tachyarrhythmias by measuring ventricular refractoriness, spatial repolarization gradients, and ventricular conduction time in isolated, perfused guinea‐pig heart preparations.

Methods: Epicardial and endocardial monophasic action potentials from distinct left ventricular (LV) and right ventricular (RV) recording sites were monitored simultaneously with volume‐conducted electrocardiogram (ECG) during steady‐state pacing and following a premature extrastimulus application at progressively reducing coupling stimulation intervals in normokalaemic and hypokalaemic conditions.

Results: Hypokalaemic perfusion (2.5 mmK+for 30 min) markedly increased the inducibility of tachyarrhythmias by programmed ventricular stimulation and rapid pacing, prolonged ventricular repolarization and shortened LV epicardial and endocardial effective refractory periods, thereby increasing the critical interval for LV re‐excitation. Hypokalaemia increased the RV‐to‐LV transepicardial repolarization gradients but had no effect on transmural dispersion of APD90and refractoriness across the LV wall. As determined by local activation time recordings, the LV‐to‐RV transepicardial conduction and the LV transmural (epicardial‐to‐endocardial) conduction were slowed in hypokalaemic heart preparations. This change was attributed to depressed diastolic excitability as evidenced by increased ventricular pacing thresholds.

Conclusion: These findings suggest that hypokalaemia‐induced arrhythmogenicity is attributed to shortened LV refractoriness, increased critical intervals for LV re‐excitation, amplified RV‐to‐LV transepicardial repolarization gradients and slowed ventricular conduction in the guinea‐pig heart.
Burst firing induces postsynaptic LTD at developing mossy fibre–CA3 pyramid synapses
Authors: Ho T. M. , Pelkey K. A. , Pelletier J. G. , Huganir R. L. , Lacaille J.‐C. , McBain C. J.
Published: July 2009

Synaptic development is an activity‐dependent process utilizing coordinated network activity to drive synaptogenesis and subsequent refinement of immature connections. Hippocampal CA3 pyramidal neurons (PYRs) exhibit intense burst firing (BF) early in development, concomitant with the period of mossy fibre (MF) development. However, whether developing MF–PYR synapses utilize PYR BF to promote MF synapse maturation remains unknown. Recently, we demonstrated that transient tonic depolarization of postsynaptic PYRs induces a persistent postsynaptic form of long‐term depression (depolarization‐induced long‐term depression, DiLTD) at immature MF–PYR synapses. DiLTD induction is NMDAR independent but does require postsynaptic Ca2+ influx through L‐type voltage gated Ca2+ channels (L‐VGCCs), and is expressed as a reduction in AMPAR function through the loss of GluR2‐lacking AMPARs present at immature MF–PYR synapses. Here we examined whether more physiologically relevant phasic L‐VGCC activation by PYR action potential (AP) BF activity patterns can trigger DiLTD. Using combined electrophysiological and Ca2+ imaging approaches we demonstrate that PYR BF effectively drives L‐VGCC activation and that brief periods of repetitive PYR BF, produced by direct current injection or intrinsic network activity induces NMDAR‐independent LTD by promoting Ca2+ influx through the activated L‐VGCCs. This BF induced LTD, just like DiLTD, is specific for developing MF–PYR synapses, is PICK1 dependent, and is expressed postsynaptically. Our results demonstrate that DiLTD can be induced by phasic L‐VGCC activation driven by PYR BF, suggesting the engagement of natural PYR network activity patterns for MF synapse maturation.
Structure and Composition of Aggregates in Two Large European Rivers, Based on Confocal Laser Scanning Microscopy and Image and Statistical Analyses
Authors: Luef Birgit , Neu Thomas R. , Zweimüller Irene , Peduzzi Peter
Published: July 2009

Floating riverine aggregates are composed of a complex mixture of inorganic and organic components from their respective aquatic habitats. Their architecture and integrity are supplemented by the presence of extracellular polymeric substances of microbial origin. They are also a habitat for virus-like particles, bacteria, archaea, fungi, algae, and protozoa. In this study we present different confocal laser scanning microscopy strategies to examine aggregates collected from the Danube and Elbe Rivers. In order to collect multiple types of information, various approaches were necessary. Small aggregates were examined directly. To analyze large and dense aggregates, limitations of the technique were overcome by cryo-sectioning and poststaining of the samples. The staining procedure included positive staining (specific glycoconjugates and cellular nucleic acid signals) as well as negative staining (aggregate volume) and multichannel recording. Data sets of cellular nucleic acid signals (CNAS) and the structure of aggregates were visualized and quantified using digital image analysis. The Danube and Elbe Rivers differed in their aggregate composition and in the relative contribution of specific glycoconjugate and CNAS volume to the aggregate volume; these contributions also changed over time. We report different spatial patterns of CNAS inside riverine aggregates, depending on aggregate size and season. The spatial structure of CNAS inside riverine aggregates was more complex in the Elbe River than in the Danube River. Based on our samples, we discuss the strengths and challenges involved in scanning and quantifying riverine aggregates.
Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification
Authors: Yankson Kweku K. , Steck Todd R.
Published: July 2009

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.
Increased Malonyl Coenzyme A Biosynthesis by Tuning the
Escherichia coli
Metabolic Network and Its Application to Flavanone Production
Authors: Fowler Zachary L. , Gikandi William W. , Koffas Mattheos A. G.
Published: July 2009

Identification of genetic targets able to bring about changes to the metabolite profiles of microorganisms continues to be a challenging task. We have independently developed a cipher of evolutionary design (CiED) to identify genetic perturbations, such as gene deletions and other network modifications, that result in optimal phenotypes for the production of end products, such as recombinant natural products. Coupled to an evolutionary search, our method demonstrates the utility of a purely stoichiometric network to predict improved
Escherichia coli
genotypes that more effectively channel carbon flux toward malonyl coenzyme A (CoA) and other cofactors in an effort to generate recombinant strains with enhanced flavonoid production capacity. The engineered
E. coli
strains were constructed first by the targeted deletion of native genes predicted by CiED and then second by incorporating selected overexpressions, including those of genes required for the coexpression of the plant-derived flavanones, acetate assimilation, acetyl-CoA carboxylase, and the biosynthesis of coenzyme A. As a result, the specific flavanone production from our optimally engineered strains was increased by over 660% for naringenin (15 to 100 mg/liter/optical density unit [OD]) and by over 420% for eriodictyol (13 to 55 mg/liter/OD).
Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “
Bacteroidales
” Populations
Authors: Lamendella Regina , Santo Domingo Jorge W. , Yannarell Anthony C. , Ghosh Shreya , Di Giovanni George , Mackie Roderick I. , Oerther Daniel B.
Published: July 2009

In this study, we evaluated the specificity, distribution, and sensitivity of
Prevotella
strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “
Bacteroidales
” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific
Prevotella-
based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the
Prevotella
markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the
Prevotella
assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal
Bacteroidales
species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.
Microbial Diversity and Host-Specific Sequences of Canada Goose Feces
Authors: Lu Jingrang , Santo Domingo Jorge W. , Hill Stephen , Edge Thomas A.
Published: July 2009

Methods to assess the impact of goose fecal contamination are needed as the result of the increasing number of Canada geese (Branta canadensis) near North American inland waters. However, there is little information on goose fecal microbial communities, and such data are important for the development of host-specific source-tracking methods. To address this issue, 16S rRNA gene clone libraries for Canada goose fecal samples from Ontario, Canada, and Ohio were analyzed. Analyses of fecal clones from Ontario (447) and Ohio (302) showed that goose fecal communities are dominated by the classes “Clostridia” (represented by 33.7% of clones) and “Bacilli” (38.1% of clones) and the phylum “Bacteroidetes” (10.1% of clones). Sequences not previously found in other avian fecal communities were used to develop host-specific assays. Fecal DNA extracts from sewage plants (10 samples) and different species of birds (11 samples) and mammals (18 samples) were used to test for host specificity. Of all the assays tested, one assay showed specificity for Canada goose fecal DNA. The PCR assay was positive for Canada goose fecal DNA extracts collected from three locations in North America (Ohio, Oregon, and Ontario, Canada). Additionally, of 48 DNA extracts from Lake Ontario waters presumed to be impacted by waterfowl feces, 19 tested positive by the assay, although 10 were positive only after a nested PCR approach was used. Due to the level of host specificity and the presence of signals in environmental waters, the assay is proposed as a part of the toolbox to detect Canada goose contamination in waterfowl-contaminated waters.
Role of Transporter Proteins in Bile Tolerance ofLactobacillus acidophilus
Authors: Pfeiler Erika A. , Klaenhammer Todd R.
Published: July 2009

Lactobacillus acidophilusNCFM derivatives containing deletion mutations in the transporter genes LBA0552, LBA1429, LBA1446, and LBA1679 exhibited increased sensitivity to bile. These strains showed unique patterns of sensitivity to a variety of inhibitory compounds, as well as differential accumulations of ciprofloxacin and taurocholate.
Contributions of O Island 48 to Adherence of Enterohemorrhagic
Escherichia coli
O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops
Authors: Yin Xianhua , Wheatcroft Roger , Chambers James R. , Liu Bianfang , Zhu Jing , Gyles Carlton L.
Published: July 2009

O island 48 (OI-48) of
Escherichia coli
consists of three functional gene clusters that encode urease, tellurite resistance (Te
r
), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic
E. coli
(EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Te
r
gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type
ter
genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Te
r
-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%;
P
= 0.168). The OI-48
aidA
gene deletion had no effect on adherence in vitro or in vivo. Deletion of the
iha
and
ureC
genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Te
r
, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium.
Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in
Thiocapsa roseopersicina
Authors: Maróti Gergely , Tong Yingkai , Yooseph Shibu , Baden-Tillson Holly , Smith Hamilton O. , Kovács Kornél L. , Frazier Marvin , Venter J. Craig , Xu Qing
Published: July 2009

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a
Thiocapsa roseopersicina
O
2
-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the
hyaA
and
hyaB
genes of an
Alteromonas macleodii
hydrogenase, indicating that it belongs to the
Alteromonas
clade. We were able to express our new
Alteromonas
hydrogenase in
T. roseopersicina
. Expression was accomplished by coexpressing only two accessory genes,
hyaD
and
hupH
, without the need to express any of the
hyp
accessory genes (
hypABCDEF
). These results suggest that the native accessory proteins in
T. roseopersicina
could substitute for the
Alteromonas
counterparts that are absent in the host to facilitate the assembly of a functional
Alteromonas
hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new
Alteromonas hyaD
gene into the
T. roseopersicina hynD
mutant. Interestingly,
Alteromonas
endopeptidase HyaD could complement
T. roseopersicina
HynD to cleave endoproteolytically the C-terminal end of the
T. roseopersicina
HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.
Cloning, Expression, and Characterization of a Peculiar Choline-Binding β-Galactosidase from
Streptococcus mitis
Authors: Campuzano Susana , Serra Beatriz , Llull Daniel , García José L. , García Pedro
Published: July 2009

A
Streptococcus mitis
genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in
Escherichia coli.
This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In
S. mitis
, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first β-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.
U(VI) Sequestration in Hydroxyapatite Produced by Microbial Glycerol 3-Phosphate Metabolism
Authors: Shelobolina Evgenya S. , Konishi Hiromi , Xu Huifang , Roden Eric E.
Published: July 2009

Previous studies have demonstrated the potential for removal of U(VI) from solution via precipitation of U(VI)-bearing calcium-phosphate (Ca-P) minerals coupled to microbial hydrolysis of glycerol phosphate compounds. We evaluated this process in circumneutral-pH groundwater from Area 2 of the U.S. Department of Energy Field Research Center at Oak Ridge National Laboratory. Area 2 groundwater contains high concentrations of dissolved calcium (ca. 4 mM), and thus, release of phosphate during glycerol phosphate metabolism has the potential to create conditions favorable for U(VI) sequestration in Ca-P minerals. Microbial enumeration and isolation studies verified the presence of aerobic and nitrate-reducing glycerol 3-phosphate (G3P)-metabolizing microorganisms in Area 2 sediments. Coprecipitation of U(VI) with Ca-P minerals coupled to microbial G3P hydrolysis was demonstrated in artificial groundwater under aerobic and nitrate-reducing conditions. Transmission electron microscopy analysis and mineral-washing experiments demonstrated that U(VI) was incorporated into the structure of the insoluble Ca-P mineral hydroxyapatite [Ca5(PO4)3OH]. Our results support the idea that U(VI) can be effectively removed from solution in contaminated aquifers through stimulation of microbial organophosphate metabolism.
Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-InfectiousCryptosporidiumspp
Authors: Alagappan A. , Bergquist P. L. , Ferrari B. C.
Published: July 2009

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification ofCryptosporidium parvumandC. hominiswas developed. The assay is a simple, rapid, and cost-effective tool for the detection of the majorCryptosporidiumspecies of concern to public health.
Fungal Diversity in Deep-Sea Hydrothermal Ecosystems
Authors: Le Calvez Thomas , Burgaud Gaëtan , Mahé Stéphane , Barbier Georges , Vandenkoornhuyse Philippe
Published: July 2009

Deep-sea hydrothermal ecosystems are considered oases of life in oceans. Since the discovery of these ecosystems in the late 1970s, many endemic species of
Bacteria
,
Archaea
, and other organisms, such as annelids and crabs, have been described. Considerable knowledge has been acquired about the diversity of (micro)organisms in these ecosystems, but the diversity of fungi has not been studied to date. These organisms are considered key organisms in terrestrial ecosystems because of their ecological functions and especially their ability to degrade organic matter. The lack of knowledge about them in the sea reflects the widely held belief that fungi are terrestrial organisms. The first inventory of such organisms in deep-sea hydrothermal environments was obtained in this study. Fungal diversity was investigated by analyzing the small-subunit rRNA gene sequences amplified by culture-independent PCR using DNA extracts from hydrothermal samples and from a culture collection that was established. Our work revealed an unsuspected diversity of species in three of the five fungal phyla. We found a new branch of
Chytridiomycota
forming an ancient evolutionary lineage. Many of the species identified are unknown, even at higher taxonomic levels in the
Chytridiomycota
,
Ascomycota
, and
Basidiomycota
. This work opens the way to new studies of the diversity, ecology, and physiology of fungi in oceans and might stimulate new prospecting for biomolecules. From an evolutionary point of view, the diversification of fungi in the oceans can no longer be ignored.
Genetic Subdivision of Chemosynthetic Endosymbionts of
Solemya velum
along the Southern New England Coast
Authors: Stewart Frank J. , Baik Alan Hyun Y. , Cavanaugh Colleen M.
Published: July 2009

Population-level genetic diversity in the obligate symbiosis between the bivalve
Solemya velum
and its thioautotrophic bacterial endosymbiont was examined. Distinct populations along the New England coast shared a single mitochondrial genotype but were fixed for unique symbiont genotypes, indicating high levels of symbiont genetic structuring and potential symbiont-host decoupling.
Use of Chimeric DNA-RNA Primers in Quantitative PCR for Detection of
Ehrlichia canis
and
Babesia canis
Authors: Peleg Ofer , Baneth Gad , Eyal Osnat , Inbar Jacob , Harrus Shimon
Published: July 2009

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the
Ehrlichia canis
16S rRNA,
Babesia canis
Hsp70, and canine β-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.
Isolation and Characterization of “
Dehalococcoides
” sp. Strain MB, Which Dechlorinates Tetrachloroethene to
trans
-1,2-Dichloroethene
Authors: Cheng Dan , He Jianzhong
Published: July 2009

In an attempt to understand the microorganisms involved in the generation of
trans
-1,2-dichloroethene (
trans
-DCE), pure-culture “
Dehalococcoides
” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate
cis
-DCE as the predominant product,
Dehalococcoides
sp. strain MB reductively dechlorinates PCE to
trans
-DCE and
cis
-DCE at a ratio of 7.3 (±0.4):1. It utilizes H
2
as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with
Dehalococcoides ethenogenes
strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of
trans
-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group,
Dehalococcoides
species.
Overexpression of the
groESL
Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium
Anabaena
sp. Strain PCC7120
Authors: Chaurasia Akhilesh Kumar , Apte Shree Kumar
Published: July 2009

The bicistronic
groESL
operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the
Anabaena
sp. strain PCC7120 genome. In the recombinant
Anabaena
strain, the additional
groESL
operon was expressed from a strong cyanobacterial P

psbA1

promoter without hampering the stress-responsive expression of the native
groESL
operon. The net expression of the two
groESL
operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant
Anabaena
strain to heat and salinity stresses.
Genome-Wide Identification of
S
accharomyces cerevisiae
Genes Required for Maximal Tolerance to Ethanol
Authors: Teixeira Miguel C. , Raposo Luís R. , Mira Nuno P. , Lourenço Artur B. , Sá-Correia Isabel
Published: July 2009

The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H
+
-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme β-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the
FPS1
gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated.
FPS1
expression contributes to decreased [
3
H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of
FPS1
confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.
rhs
Genes Are Potential Markers for Multilocus Sequence Typing of
Escherichia coli
O157:H7 Strains
Authors: Liu K. , Knabel S. J. , Dudley E. G.
Published: July 2009

DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping
Escherichia coli
O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that
rhs
genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of
E. coli
O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these
rhs
genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using
rhs
genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in
rhsA
and
rhsJ
that might be used to define clade 8 and clade 6. Therefore,
rhs
genes may be useful markers for phylogenetic analysis of
E. coli
O157:H7.
In Vitro Kinetics of Prebiotic Inulin-Type Fructan Fermentation by Butyrate-Producing Colon Bacteria: Implementation of Online Gas Chromatography for Quantitative Analysis of Carbon Dioxide and Hydrogen Gas Production
Authors: Falony Gwen , Verschaeren An , De Bruycker Feije , De Preter Vicky , Verbeke Kristin , Leroy Frédéric , De Vuyst Luc
Published: July 2009

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of five butyrate-producing clostridial cluster XIVa colon bacteria grown on acetate plus fructose, oligofructose, inulin, or lactate were performed. A gas chromatography method was set up to assess H2and CO2production online and to ensure complete coverage of all metabolites produced. Method accuracy was confirmed through the calculation of electron and carbon recoveries. Fermentations withAnaerostipes caccaeDSM 14662T,Roseburia faecisDSM 16840T,Roseburia hominisDSM 16839T, andRoseburia intestinalisDSM 14610Trevealed similar patterns of metabolite production with butyrate, CO2, and H2as the main metabolites.R. faecisDSM 16840TandR. intestinalisDSM 14610Twere able to degrade oligofructose, displaying a nonpreferential breakdown mechanism. Lactate consumption was only observed withA. caccaeDSM 14662T.Roseburia inulinivoransDSM 16841Twas the only strain included in the present study that was able to grow on fructose, oligofructose, and inulin. The metabolites produced were lactate, butyrate, and CO2, without H2production, indicating an energy metabolism distinct from that of otherRoseburiaspecies. Oligofructose degradation was nonpreferential. In a coculture ofR. inulinivoransDSM 16841Twith the highly competitive strainBifidobacterium longumsubsp.longumLMG 11047 on inulin, hardly any production of butyrate and CO2was detected, indicating a lack of competitiveness of the butyrate producer. Complete recovery of metabolites during fermentations of clostridial cluster XIVa butyrate-producing colon bacteria allowed stoichiometric balancing of the metabolic pathway for butyrate production, including H2formation.
Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in
Aspergillus oryzae
by Systematically Deleting Large Chromosomal Segments
Authors: Jin Feng Jie , Takahashi Tadashi , Machida Masayuki , Koyama Yasuji
Published: July 2009

We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in
Aspergillus oryzae
. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest
A. oryzae
chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Δ5 and Δ7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Δ5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Δ5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.
Macrolide Resistance in Microorganisms at Antimicrobial-Free Swine Farms
Authors: Zhou Zhi , Raskin Lutgarde , Zilles Julie L.
Published: July 2009

To investigate the relationship between agricultural antimicrobial use and resistance, a variety of methods for quantification of macrolide-lincosamide-streptogramin B (MLSB) resistance were applied to organic swine farm manure samples. Fluorescence in situ hybridization was used to indirectly quantify the specific rRNA methylation resulting in MLSBresistance. Using this method, an unexpectedly high prevalence of ribosomal methylation and, hence, predicted MLSBresistance was observed in manure samples from two swine finisher farms that reported no antimicrobial use (37.6% ± 6.3% and 40.5% ± 5.4%, respectively). A culture-based method targeting relatively abundant clostridia showed a lower but still unexpectedly high prevalence of resistance at both farms (27.7% ± 11.3% and 11.7% ± 8.6%, respectively), while the prevalence of resistance in cultured fecal streptococci was low at both farms (4.0%). These differences in the prevalence of resistance across microorganisms suggest the need for caution when extrapolating from data obtained with indicator organisms. A third antimicrobial-free swine farm, a breeder-to-finisher operation, had low levels of MLSBresistance in manure samples with all methods used (<9%). Tetracycline antimicrobials were detected in manure samples from one of the finisher farms and may provide a partial explanation for the high level of MLSBresistance. Taken together, these findings highlight the need for a more fundamental understanding of the relationship between antimicrobial use and the prevalence of antimicrobial resistance.
Interactions of Typical and Atypical EnteropathogenicEscherichia coliStrains with the Calf Intestinal Mucosa Ex Vivo
Authors: Girard Francis , Dziva Francis , Stevens Mark P. , Frankel Gad
Published: July 2009

EnteropathogenicEscherichia coli(EPEC) can be found in healthy and diarrheic cattle; however, little is known about the role of attaching and effacing (A/E) lesion formation in colonization of bovine intestinal mucosa by such strains. We show that typical and atypical EPEC induce A/E lesions on calf intestinal explants independently of Tir tyrosine phosphorylation and TccP. Our data support the existence of conserved Tir- and TccP-independent mechanisms of A/E lesion formation in a range of hosts and reinforce the zoonotic potential of EPEC in cattle.
Distribution of Sulfonamide Resistance Genes inEscherichia coliandSalmonellaIsolates from Swine and Chickens at Abattoirs in Ontario and Québec, Canada
Authors: Kozak Gosia K. , Pearl David L. , Parkman Julia , Reid-Smith Richard J. , Deckert Anne , Boerlin Patrick
Published: July 2009

Sulfonamide-resistantEscherichia coliandSalmonellaisolates from pigs and chickens in Ontario and Québec were screened forsul1,sul2, andsul3by PCR. Eachsulgene was distributed differently across populations, with a significant difference between distribution in commensalE. coliandSalmonellaisolates andsul3restricted mainly to porcineE. coliisolates.
Influence of Vegetation on the In Situ Bacterial Community and Polycyclic Aromatic Hydrocarbon (PAH) Degraders in Aged PAH-Contaminated or Thermal-Desorption-Treated Soil
Authors: Cébron Aurélie , Beguiristain Thierry , Faure Pierre , Norini Marie-Paule , Masfaraud Jean-François , Leyval Corinne
Published: July 2009

The polycyclic aromatic hydrocarbon (PAH) contamination, bacterial community, and PAH-degrading bacteria were monitored in aged PAH-contaminated soil (Neuves-Maisons [NM] soil; with a mean of 1,915 mg of 16 PAHs·kg
−1
of soil dry weight) and in the same soil previously treated by thermal desorption (TD soil; with a mean of 106 mg of 16 PAHs·kg
−1
of soil dry weight). This study was conducted in situ for 2 years using experimental plots of the two soils. NM soil was colonized by spontaneous vegetation (NM-SV), planted with
Medicago sativa
(NM-Ms), or left as bare soil (NM-BS), and the TD soil was planted with
Medicago sativa
(TD-Ms). The bacterial community density, structure, and diversity were estimated by real-time PCR quantification of the 16S rRNA gene copy number, temporal thermal gradient gel electrophoresis fingerprinting, and band sequencing, respectively. The density of the bacterial community increased the first year during stabilization of the system and stayed constant in the NM soil, while it continued to increase in the TD soil during the second year. The bacterial community structure diverged among all the plot types after 2 years on site. In the NM-BS plots, the bacterial community was represented mainly by
Betaproteobacteria
and
G
ammaproteobacteria
. The presence of vegetation (NM-SV and NM-Ms) in the NM soil favored the development of a wider range of bacterial phyla (
Alphaproteobacteria
,
Betaproteobacteria
,
G
ammaproteobacteria
,
Verrucomicrobia
,
Actinobacteria
,
Firmicutes
, and
Chlorof
l
exi
) that, for the most part, were not closely related to known bacterial representatives. Moreover, under the influence of the same plant, the bacterial community that developed in the TD-Ms was represented by different bacterial species (
Alphaproteobacteria
,
Betaproteobacteria
,
Gammaproteobacteria
, and
Actinobacteria
) than that in the NM-Ms. During the 2 years of monitoring, the PAH concentration did not evolve significantly. The abundance of gram-negative (GN) and gram-positive (GP) PAH-degrading bacteria was estimated by real-time PCR quantification of specific functional genes encoding the α subunit of PAH-ring hydroxylating dioxygenase (PAH-RHD
α
). The percentage of the PAH-RHD
α
GN bacterial genes relative to 16S rRNA gene density decreased with time in all the plots. The GP PAH-RHD
α
bacterial gene proportion decreased in the NM-BS plots but stayed constant or increased under vegetation influence (NM-SV, NM-Ms, and TD-Ms).
The tRNA Modification Complex Elongator Regulates the Cdc42-Dependent Mitogen-Activated Protein Kinase Pathway That Controls Filamentous Growth in Yeast
Authors: Abdullah Ummi , Cullen Paul J.
Published: July 2009

Signal transduction pathways control multiple aspects of cellular behavior, including global changes to the cell cycle, cell polarity, and gene expression, which can result in the formation of a new cell type. In the budding yeast
Saccharomyces cerevisiae
, the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth induces a dimorphic foraging response under nutrient-limiting conditions. How nutritional cues feed into MAPK activation remains an open question. Here we report a functional connection between the elongator tRNA modification complex (
ELP
genes) and activity of the filamentous growth pathway. Elongator was required for filamentous growth pathway signaling, and
elp
mutants were defective for invasive growth, cell polarization, and MAPK-dependent mat formation. Genetic suppression analysis showed that elongator functions at the level of Msb2p, the signaling mucin that operates at the head of the pathway, which led to the finding that elongator regulates the starvation-dependent expression of the
MSB2
gene. The Elp complex was not required for activation of related pathways (pheromone response or high osmolarity glycerol response) that share components with the filamentous growth pathway. Because protein translation provides a rough metric of cellular nutritional status, elongator may convey nutritional information to the filamentous growth pathway at the level of
MSB2
expression.
How a Cell Crawls and the Role of Cortical Myosin II
Authors: Soll David R. , Wessels Deborah , Kuhl Spencer , Lusche Daniel F.
Published: July 2009

The movements of
Dictyostelium discoideum
amoebae translocating on a glass surface in the absence of chemoattractant have been reconstructed at 5-second intervals and motion analyzed by employing 3D-DIAS software. A morphometric analysis of pseudopods, the main cell body, and the uropod provides a comprehensive description of the basic motile behavior of a cell in four dimensions (4D), resulting in a list of 18 characteristics. A similar analysis of the myosin II phosphorylation mutant 3XASP reveals a role for the cortical localization of myosin II in the suppression of lateral pseudopods, formation of the uropod, cytoplasmic distribution of cytoplasm in the main cell body, and efficient motility. The results of the morphometric analysis suggest that pseudopods, the main cell body, and the uropod represent three motility compartments that are coordinated for efficient translocation. It provides a contextual framework for interpreting the effects of mutations, inhibitors, and chemoattractants on the basic motile behavior of
D. discoideum
. The generality of the characteristics of the basic motile behavior of
D. discoideum
must now be tested by similar 4D analyses of the motility of amoeboid cells of higher eukaryotic cells, in particular human polymorphonuclear leukocytes.
Slt2 and Rim101 Contribute Independently to the Correct Assembly of the Chitin Ring at the Budding Yeast Neck in
Saccharomyces cerevisiae
Authors: Gomez Alberto , Perez Jaqueline , Reyes Abigail , Duran Angel , Roncero Cesar
Published: July 2009

In
Saccharomyces cerevisiae
, the simultaneous absence of Slt2 and Rim101 prevents growth in nonosmotically stabilized media (F. Castrejon et al., Eukaryot. Cell 5:507-517, 2006). The double mutant
slt2
Δ
rim101
Δ displays altered chitin rings, together with a significant reduction in the overall levels of chitin. Cultures of this mutant lyse upon transfer to nonosmotically stabilized media, mostly through the bud, and such lysis is partially prevented by deletion of the chitinase gene (
CTS1
). Growth of the
slt2
Δ
rim101
Δ double mutant was restored by the overexpression of the
GFA1
or
CCT7
genes, which code for two biologically unrelated proteins. Further characterization of the mutant and its suppressors indicated that both Slt2 and Rim101 were independently required for the correct assembly of the septum machinery and that their concomitant absence reduced Chs3 accumulation at the neck, leading to lower levels of chitin.
GFA1
overexpression, as well as the addition of glucosamine to the growth medium, specifically suppressed the growth defects by activating chitin synthesis at the neck and restoring the normal assembly of the chitin ring. In contrast, overexpression of
CCT7
, a Cct chaperonin subunit, alleviated the defect in the septum machinery without affecting chitin synthesis. Both suppressors thus act by reducing neck fragility through different mechanisms and allow growth in nonstabilized media. This work reports new roles for Slt2 and Rim101 in septum formation in budding yeast and confirms the homeostatic role of the chitin ring in the maintenance of neck integrity during cell division.
Functional Dissection of the Catalytic Carboxyl-Terminal Domain of Origin Recognition Complex Subunit 1 (PfORC1) of the Human Malaria Parasite
Plasmodium falciparum
Authors: Gupta Ashish , Mehra Parul , Deshmukh Abhijeet , Dar Ashraf , Mitra Pallabi , Roy Nilanjan , Dhar Suman Kumar
Published: July 2009

Origin recognition complex subunit 1 (ORC1) is essential for DNA replication in eukaryotes. The deadly human malaria parasite
Plasmodium falciparum
contains an ORC1/CDC6 homolog with several interesting domains at the catalytic carboxyl-terminal region that include a putative nucleoside triphosphate-binding and hydrolysis domain, a putative PCNA-interacting-protein (PIP) motif, and an extreme C-terminal region that shows poor homology with other ORC1 homologs. Due to the unavailability of a dependable inducible gene expression system, it is difficult to study the structure and function of essential genes in
Plasmodium
. Using a genetic yeast complementation system and biochemical experiments, here we show that the putative PIP domain in ORC1 that facilitates in vitro physical interaction with PCNA is functional in both yeast (
Saccharomyces cerevisiae
) and
Plasmodium
in vivo, confirming its essential biological role in eukaryotes. Furthermore, despite having less sequence homology, the extreme C-terminal region can be swapped between
S. cerevisiae
and
P. falciparum
and it binds to DNA directly, suggesting a conserved role of this region in DNA replication. These results not only provide us a useful system to study the function of the essential genes in
Plasmodium
, they help us to identify the previously undiscovered unique features of replication proteins in general.