bluuebunny/crossref_metadata_2025 huggingface dataset:
Extended-Spectrum Beta-Lactamase Gene Sequences in Gram-Negative Saprophytes on Retail Organic and Nonorganic Spinach
Authors: Raphael Eva , Wong Lisa K. , Riley Lee W.
Published: January 2011

A substantial proportion of infections caused by drug-resistant Gram-negative bacteria (GNB) in community and health care settings are recognized to be caused by evolutionarily related GNB strains. Their global spread has been suggested to occur due to human activities, such as food trade and travel. These multidrug-resistant GNB pathogens often harbor mobile drug resistance genes that are highly conserved in their sequences. Because they appear across different GNB species, these genes may have origins other than human pathogens. We hypothesized that saprophytes in common human food products may serve as a reservoir for such genes. Between July 2007 and April 2008, we examined 25 batches of prepackaged retail spinach for cultivatable GNB population structure by 16S rRNA gene sequencing and for antimicrobial drug susceptibility testing and the presence of extended-spectrum beta-lactamase (ESBL) genes. We found 20 recognized GNB species among 165 (71%) of 231 randomly selected colonies cultured from spinach. Twelve strains suspected to express ESBLs based on resistance to cefotaxime and ceftazidime were further examined for
bla
CTX-M
and
bla
TEM
genes. We found a 712-bp sequence in
Pseudomonas teessidea
that was 100% identical to positions 10 to 722 of an 876-bp
bla
CTX-M-15
gene of an
E. coli
strain. Additionally, we identified newly recognized ESBL
bla
RAHN-2
sequences from
Rahnella aquatilis
. These observations demonstrate that saprophytes in common fresh produce can harbor drug resistance genes that are also found in internationally circulating strains of GNB pathogens; such a source may thus serve as a reservoir for drug resistance genes that ultimately enter pathogens to affect human health.
Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from
Jeotgalicoccus
Species
Authors: Rude Mathew A. , Baron Tarah S. , Brubaker Shane , Alibhai Murtaza , Del Cardayre Stephen B. , Schirmer Andreas
Published: January 2011

Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria,
Jeotgalicoccus
spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in
Jeotgalicoccus
sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme,
Jeotgalicoccus
sp. OleT (OleT
JE
), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of
Jeotgalicoccus
sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to
Escherichia coli.
OleT
JE
is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.
Subpopulation-Specific Metabolic Pathway Usage in Mixed Cultures as Revealed by Reporter Protein-Based
13
C Analysis
Authors: Rühl Martin , Hardt Wolf-Dietrich , Sauer Uwe
Published: January 2011

Most large-scale biological processes, like global element cycling or decomposition of organic matter, are mediated by microbial consortia. Commonly, the different species in such consortia exhibit mutual metabolic dependencies that include the exchange of nutrients. Despite the global importance, surprisingly little is known about the metabolic interplay between species in particular subpopulations. To gain insight into the intracellular fluxes of subpopulations and their interplay within such mixed cultures, we developed here a
13
C flux analysis approach based on affinity purification of the recombinant fusion glutathione
S
-transferase (GST) and green fluorescent protein (GFP) as a reporter protein. Instead of detecting the
13
C labeling patterns in the typically used amino acids from the total cellular protein, our method detects these
13
C patterns in amino acids from the reporter protein that has been expressed in only one species of the consortium. As a proof of principle, we validated our approach by mixed-culture experiments of an
Escherichia coli
wild type with two metabolic mutants. The reporter method quantitatively resolved the expected mutant-specific metabolic phenotypes down to subpopulation fractions of about 1%.
The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes
Authors: Vallejo Milene C. , Matsuo Alisson L. , Ganiko Luciane , Medeiros Lia C. Soares , Miranda Kildare , Silva Luiz S. , Freymüller-Haapalainen Edna , Sinigaglia-Coimbra Rita , Almeida Igor C. , Puccia Rosana
Published: January 2011

Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.
Nutrient Transport and Pathogenesis in Selected Parasitic Protozoa
Authors: Landfear Scott M.
Published: January 2011

Parasitic protozoa, such as malaria parasites, trypanosomes, andLeishmania, acquire a plethora of nutrients from their hosts, employing transport proteins located in the plasma membrane of the parasite. Application of molecular genetic approaches and the completion of genome projects have allowed the identification and functional characterization of a cohort of transporters and their genes in these parasites. This review focuses on a subset of these permeases that have been studied in some detail, that import critical nutrients, and that provide examples of approaches being undertaken broadly with these and other parasite transporters. Permeases reviewed include those for hexoses, purines, iron, polyamines, carboxylates, and amino acids. Topics of special emphasis include structure-function approaches, critical roles for transporters in parasite viability and physiology, regulation of transporter expression, and subcellular targeting. Investigations of parasite transporters impact a broad spectrum of basic biological problems in these protozoa.
Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1
Authors: Liu Yang , Xu Xinjing , Carlson Marian
Published: January 2011

The
Saccharomyces cerevisiae
SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.
ThetibAdherence Locus of EnterotoxigenicEscherichia coliIs Regulated by Cyclic AMP Receptor Protein
Authors: Espert Shirley M. , Elsinghorst Eric A. , Munson George P.
Published: January 2011

EnterotoxigenicEscherichia coli(ETEC) is a Gram-negative enteric pathogen that causes profuse watery diarrhea through the elaboration of heat-labile and/or heat-stable toxins. Virulence is also dependent upon the expression of adhesive pili and afimbrial adhesins that allow the pathogen to adhere to the intestinal epithelium or mucosa. Both types of enterotoxins are regulated at the level of transcription by cyclic AMP (cAMP) receptor protein (CRP). To further our understanding of virulence gene regulation, anin silicoapproach was used to identify putative CRP binding sites in the genome ofH10407(O78:H11), an ETEC strain that was originally isolated from the stool of a Bangledeshi patient with cholera-like symptoms circa 1971. One of the predicted binding sites was located within an intergenic region upstream oftibDBCA. TibA is an autotransporter and afimbrial adhesin that is glycosylated by TibC. Expression of the TibA glycoprotein was abolished in anH10407crpmutant and restored whencrpwas provided intrans. TibA-dependent aggregation was also abolished in acyaA::kanstrain and restored by addition of exogenous cAMP to the growth medium. DNase I footprinting confirmed that the predicted site upstream oftibDBCAis bound by CRP. Point mutations within the CRP binding site were found to abolish or significantly impair CRP-dependent activation of thetibDBpromoter. Thus, these studies demonstrate that CRP positively regulates the expression of the glycosylated afimbrial adhesin TibA through occupancy of a binding site withintibDBp.
Purification and Characterization of Dimethylsulfide Monooxygenase fromHyphomicrobium sulfonivorans
Authors: Boden Rich , Borodina Elena , Wood Ann P. , Kelly Donovan P. , Murrell J. Colin , Schäfer Hendrik
Published: January 2011

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase fromHyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH2-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had aKmof 17.2 (± 0.48) μM for DMS (kcat= 5.45 s−1) and aVmaxof 1.25 (± 0.01) μmol NADH oxidized min−1(mg protein−1). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg2+, Cd2+, and Pb2+ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH2-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.
Control of Heme Homeostasis inCorynebacterium glutamicumby the Two-Component System HrrSA
Authors: Frunzke Julia , Gätgens Cornelia , Brocker Melanie , Bott Michael
Published: January 2011

The response regulator HrrA of the HrrSA two-component system (previously named CgtSR11) was recently found to be repressed by the global iron-dependent regulator DtxR inCorynebacterium glutamicum. Here, we provide evidence that HrrA mediates heme-dependent gene regulation in this nonpathogenic soil bacterium. Growth experiments and DNA microarray analysis revealed thatC. glutamicumis able to use hemin as an alternative iron source and emphasize the involvement of the putative hemin ABC transporter HmuTUV and heme oxygenase (HmuO) in heme utilization. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of anhrrAdeletion mutant andC. glutamicumwild-type strain in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase. Based on our results, it can be furthermore deduced that HrrA activates the expression of heme-containing components of the respiratory chain, namely,ctaDand thectaE-qcrCABoperon encoding subunits I and III of cytochromeaa3oxidase and three subunits of the cytochromebc1complex. In addition, HrrA was found to repress almost all genes involved in heme biosynthesis, including those for glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Growth experiments with anhrrAdeletion mutant showed that this strain is significantly impaired in heme utilization. In summary, our results provide evidence for a central role of the HrrSA system in the control of heme homeostasis inC. glutamicum.
In VivoRecognition of thefecA3Target Promoter byHelicobacter pyloriNikR
Authors: Romagnoli Simona , Agriesti Francesca , Scarlato Vincenzo
Published: January 2011

InHelicobacter pylori, the transcriptional regulatorHpNikR represses transcription of thefecA3gene by binding to two adjacent operators spanning a region of almost 80 nucleotides along thefecA3promoter in a nickel-dependent manner. By employing hydroxyl radical footprinting, we mapped the protected nucleotides within each operator. Three short sequences rich in A and T nucleotides were identified within each operator, comprising just 24 bases for both operators, with 4 or 5 protected bases interspaced by 4 to 7 free nucleotides, with no center of symmetry. Base substitutions at any site strongly reduced the affinity ofHpNikR for the operators and also affected the stability of the DNA-protein complex, when the promoter-regulator interaction was analyzedin vitro. The effect of these substitutions was remarkably different when transcription of the mutant promoters was analyzedin vivo. Base changes introduced at the farthest subsites impaired theHpNikR-dependent repression, with the mutations closer to +1 completely abolishing the repression, the more distal one still allowing almost 50% of transcription, and the mutations in the middle being ineffective. The data presented here show thatHpNikR may first select its targets by identifying sequences within the previously defined consensus and subsequently establish base-specific contacts to firmly bind DNA. In particular,HpNikR seems to interact in an asymmetric mode with thefecA3target to repress its transcription.
Activators of the Glutamate-Dependent Acid Resistance System Alleviate Deleterious Effects of YidC Depletion inEscherichia coli
Authors: Yu Zhong , Bekker Martijn , Tramonti Angela , Cook Gregory M. , van Ulsen Peter , Scheffers Dirk-Jan , de Mattos Joost Teixeira , De Biase Daniela , Luirink Joen
Published: January 2011

The function of the essential inner membrane protein (IMP) YidC inEscherichia colihas been studied for a limited number of model IMPs and primarily using targeted approaches. These studies suggested that YidC acts at the level of insertion, folding, and quality control of IMPs, both in the context of the Sec translocon and as a separate entity. To further our understanding of YidC's role in IMP biogenesis, we screened a random overexpression library for factors that rescued the growth of cells upon YidC depletion. We found that the overexpression of the GadX and GadY regulators of the glutamate-dependent acid resistance system complemented the growth defect of YidC-depleted cells. Evidence is presented that GadXY overexpression counteracts the deleterious effects of YidC depletion on at least two fronts. First, GadXY prepares the cells for the decrease in respiratory capacity upon the depletion of YidC. Most likely, GadXY-regulated processes reduce the drop in proton-motive force that impairs the fitness of YidC-depleted cells. Second, in GadXY-overproducing cells increased levels of the general chaperone GroEL cofractionate with the inner membranes, which may help to keep newly synthesized inner membrane proteins in an insertion-competent state when YidC levels are limiting.
The Integrity of the Periplasmic Domain of the VirA Sensor Kinase Is Critical for Optimal Coordination of the Virulence Signal Response inAgrobacterium tumefaciens
Authors: Nair Gauri R. , Lai Xiaoqin , Wise Arlene A. , Rhee Benjamin Wonjae , Jacobs Mark , Binns Andrew N.
Published: January 2011

The plant pathogenAgrobacterium tumefaciensresponds to three main signals at the plant-bacterium interface: phenolics, such as acetosyringone (AS), monosaccharides, and acidic pH (∼5.5). These signals are transduced via the chromosomally encoded sugar binding protein ChvE and the Ti plasmid-encoded VirA/VirG two-component regulatory system, resulting in the transcriptional activation of the Ti plasmid virulence genes. Here, we present genetic and physical evidence that the periplasmic domain of VirA dimerizes independently of other parts of the protein, and we examine the effects of several engineered mutations in the periplasmic and transmembrane regions of VirA onvir-inducing capacity as indicated by AS sensitivity and maximal level ofvir-inducing activity at saturating AS levels. The data indicate that helix-breaking mutations throughout the periplasmic domain of VirA or mutations that reposition the second transmembrane domain (TM2) of VirA relieve the periplasmic domain's repressive effects on the maximal activity of this kinase in response to phenolics, effects normally relieved only when ChvE, sugars, and low pH are also present. Such relief, however, does not sensitize VirA to low concentrations of phenolics, the other major effect of the ChvE-sugar and low pH signals. We further demonstrate that amino acid residues in a small Trg-like motif in the periplasmic domain of VirA are crucial for transmission of the ChvE-sugar signal to the cytoplasmic domain. These experiments provide evidence that small perturbations in the periplasmic domain of VirA can uncouple sugar-mediated changes in AS sensitivity from the sugar-mediated effects on maximal activity.
Identification ofEscherichia coliZapC (YcbW) as a Component of the Division Apparatus That Binds and Bundles FtsZ Polymers
Authors: Hale Cynthia A. , Shiomi Daisuke , Liu Bing , Bernhardt Thomas G. , Margolin William , Niki Hironori , de Boer Piet A. J.
Published: January 2011

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified inEscherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission processper sebut contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring inE. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.
TheCoxiella burnetiiCryptic Plasmid Is Enriched in Genes Encoding Type IV Secretion System Substrates
Authors: Voth Daniel E. , Beare Paul A. , Howe Dale , Sharma Uma M. , Samoilis Georgios , Cockrell Diane C. , Omsland Anders , Heinzen Robert A.
Published: January 2011

The intracellular bacterial pathogenCoxiella burnetiidirects biogenesis of a phagolysosome-like parasitophorous vacuole (PV), in which it replicates. The organism encodes a Dot/Icm type IV secretion system (T4SS) predicted to deliver to the host cytosol effector proteins that mediate PV formation and other cellular events. AllC. burnetiiisolates carry a large, autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences (IPS), suggesting that plasmid and IPS genes are critical for infection. Bioinformatic analyses revealed two candidate Dot/Icm substrates with eukaryotic-like motifs uniquely encoded by the QpH1 plasmid from the Nine Mile reference isolate. CpeC, containing an F-box domain, and CpeD, possessing kinesin-related and coiled-coil regions, were secreted by the closely relatedLegionella pneumophilaDot/Icm T4SS. An additional QpH1-specific gene,cpeE, situated in a predicted operon withcpeD, also encoded a secreted effector. Further screening revealed that three hypothetical proteins (CpeA, CpeB, and CpeF) encoded by allC. burnetiiplasmids and IPS are Dot/Icm substrates. By use of new genetic tools, secretion of plasmid effectors byC. burnetiiduring host cell infection was confirmed using β-lactamase and adenylate cyclase translocation assays, and a C-terminal secretion signal was identified. When ectopically expressed in HeLa cells, plasmid effectors trafficked to different subcellular sites, including autophagosomes (CpeB), ubiquitin-rich compartments (CpeC), and the endoplasmic reticulum (CpeD). Collectively, these results suggest thatC. burnetiiplasmid-encoded T4SS substrates play important roles in subversion of host cell functions, providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen.
Inactivation of σ
E
and σ
G
in
Clostridium acetobutylicum
Illuminates Their Roles in Clostridial-Cell-Form Biogenesis, Granulose Synthesis, Solventogenesis, and Spore Morphogenesis
Authors: Tracy Bryan P. , Jones Shawn W. , Papoutsakis Eleftherios T.
Published: January 2011

Central to all clostridia is the orchestration of endospore formation (i.e., sporulation) and, specifically, the roles of differentiation-associated sigma factors. Moreover, there is considerable applied interest in understanding the roles of these sigma factors in other stationary-phase phenomena, such as solvent production (i.e., solventogenesis). Here we separately inactivated by gene disruption the major sporulation-specific sigma factors, σ
E
and σ
G
, and performed an initial analysis to elucidate their roles in sporulation-related morphogenesis and solventogenesis in
Clostridium acetobutylicum
. The terminal differentiation phenotype for the
sigE
inactivation mutant stalled in sporulation prior to asymmetric septum formation, appeared vegetative-like often with an accumulation of DNA at both poles, frequently exhibited two longitudinal internal membranes, and did not synthesize granulose. The
sigE
inactivation mutant did produce the characteristic solvents (i.e., butanol and acetone), but the extent of solventogenesis was dependent on the physiological state of the inoculum. The
sigG
inactivation mutant stalled in sporulation during endospore maturation, exhibiting engulfment and partial cortex and spore coat formation. Lastly, the
sigG
inactivation mutant did produce granulose and exhibited wild-type-like solventogenesis.
Resolution of Holliday Junction Recombination Intermediates by Wild-Type and Mutant IntDOT Proteins
Authors: Kim Seyeun , Gardner Jeffrey F.
Published: January 2011

CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the
attDOT
site in CTnDOT and an
attB
site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs
in vitro
. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.
Complete Genome Sequence of
Campylobacter jejuni
Strain S3
Authors: Cooper Kerry K. , Cooper Margarethe A. , Zuccolo Andrea , Law Bibiana , Joens Lynn A.
Published: January 2011

Campylobacter jejuni
is one of the leading causes of bacterial gastroenteritis in the world; however, there is only one complete genome sequence of a poultry strain to date. Here we report the complete genome sequence and annotation of the second poultry strain,
C. jejuni
strain S3. This strain has been shown to be nonmotile, to be a poor invader
in vitro
, and to be a poor colonizer of poultry after minimal
in vitro
passage.
Complete Genome Sequence of the Hyperthermophilic, Piezophilic, Heterotrophic, and Carboxydotrophic Archaeon
Thermococcus barophilus
MP
Authors: Vannier Pauline , Marteinsson Viggo Thor , Fridjonsson Olafur Hedinn , Oger Philippe , Jebbar Mohamed
Published: January 2011

Thermococcus barophilus
is a hyperthermophilic, anaerobic, mixed heterotrophic, and carboxydotrophic euryarchaeon isolated from the deep sea hydrothermal vent Snakepit site on the mid-Atlantic ridge at a depth of 3,550 m.
T. barophilus
is the first true piezophilic, hyperthermophilic archaeon isolated, having an optimal growth at 40 MPa. Here we report the complete genome sequence of strain MP, the type strain of
T. barophilus
. The genome data reveal a close proximity with
T
hermococcus
sibiricus
, another
Thermococcus
isolated from the deep biosphere and a possible connection to life in the depths.
Glycosylation Is Required for Outer Membrane Localization of the Lectin LecB inPseudomonas aeruginosa
Authors: Bartels Kai-Malte , Funken Horst , Knapp Andreas , Brocker Melanie , Bott Michael , Wilhelm Susanne , Jaeger Karl-Erich , Rosenau Frank
Published: January 2011

The fucose-/mannose-specific lectin LecB fromPseudomonas aeruginosais transported to the outer membrane; however, the mechanism used is not known so far. Here, we report that LecB is present in the periplasm ofP. aeruginosain two variants of different sizes. Both were functional and could be purified by their affinity to mannose. The difference in size was shown by a specific enzyme assay to be a result of N glycosylation, and inactivation of the glycosylation sites was shown by site-directed mutagenesis. Furthermore, we demonstrate that this glycosylation is required for the transport of LecB.
Whole Genome Sequence of an UnusualBorrelia burgdorferiSensu Lato Isolate
Authors: Casjens Sherwood R. , Fraser-Liggett Claire M. , Mongodin Emmanuel F. , Qiu Wei-Gang , Dunn John J. , Luft Benjamin J. , Schutzer Steven E.
Published: January 2011

Human Lyme disease is caused by a number of relatedBorrelia burgdorferisensu lato species. We report here the complete genome sequence ofBorreliasp. isolate SV1 from Finland. This isolate is to date the closest known relative ofB. burgdorferisensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named “Borrelia finlandensis.”
Detection of Klebsiella pneumoniae Carbapenemase (KPC) Production in Non-Klebsiella pneumoniae Enterobacteriaceae Isolates by Use of the Phoenix, Vitek 2, and Disk Diffusion Methods
Authors: Doern Christopher D. , Dunne W. Michael , Burnham Carey-Ann D.
Published: January 2011

In this study, we tested the abilities of the Vitek 2, BD Phoenix, and Kirby Bauer disk diffusion tests to detect carbapenemase production in a collection of 14
Klebsiella pneumoniae
carbapenemase (KPC)-producing non-
Klebsiella pneumoniae
isolates. In addition, we evaluated 13 KPC-positive
K. pneumoniae
isolates by using each of these methods and applied both 2009 and 2010 CLSI carbapenem interpretive guidelines.
Considerations for uniform and accurate biospecimen labelling in a biorepository and research environment
Authors: Kay Andrew B , Estrada Daniel K , Mareninov Sergey , Silver Steven S , Magyar Clara E , Dry Sarah M , Cloughesy Timothy F , Yong William H
Published: January 2011

Correct labelling of specimens in a biorepository or research laboratory is vital, especially for translational or clinical studies linking clinical data with biospecimens. While patient privacy must be carefully protected, confusing or inadequate labelling can potentially result in the study of the wrong biospecimens with detrimental effects to the accuracy of published findings or a requirement for invaluable biospecimens to be discarded. Labelling guidelines are described in the biorepository of the University of California–Los Angeles Brain Tumour Translational Resource, and in recipient neuro-oncology laboratories to which biospecimens and derivatives are provided. This approach includes specifying identifier types, types of dates and institutions on the biospecimen labels; using multiple identifiers on each specimen when feasible; and developing a three to four-letter alphanumeric code to aid in label recognition. In addition, steps are being taken to educate recipient laboratories on best practices in labelling.
Mixed mould species in laboratory cultures of respiratory specimens: how should they be reported, and what are the indications for susceptibility testing?: Table 1
Authors: Purcell J , McKenna J , Critten P , Denning D W , Hassan I A
Published: January 2011

Aims

To investigate how clinical microbiology laboratories should report and interpret mixed mould isolates includingAspergillusspecies from clinical samples and the criteria for susceptibility testing of the isolates.

Methods

Retrospectively collected data from our laboratory information system of moulds isolated between January 2005 and December 2007. Patient case notes were also reviewed.

Results

A total of 502 isolates (from 273 patients) were found. 20 patients with clinical diagnosis of a probable fungal infection had mixedAspergillusspecies.

Conclusions

In most instances, the isolation ofAspergillusspecies from non-sterile sites does not represent clinical disease, but only colonisation/contamination. However, for high-risk patients including transplant recipients, a positive culture is associated with invasive disease. Our tertiary centre routinely reports single fungal isolates and mixed cultures with appropriate comments, and those considered significant will also have susceptibility testing carried out. The correlation of culture results with clinical features can differentiate between invasive disease and contamination.
Haemostatic response to hypoxaemic/exercise stress: the dilemma of plasma volume correction: Figure 1
Authors: Fall Lewis , Evans Kevin A , Lewis Michael H , Bailey Damian M
Published: January 2011

Objective

Patients with arterial occlusive disease are typically hypoxaemic, and exercise is prescribed for rehabilitation. Both stressors independently contract plasma volume (PV), which may influence clinical interpretation of a patient's thrombogenicity. The aim of the study was to emphasise the conceptual significance of PV correction.

Methods and Results

Venous plasma samples were obtained from 18 healthy men at rest in normoxia for the measurement of fibrinogen, prothrombin (PT), thrombin (TT) and activated partial thromboplastin (aPTT) times. Additional samples were obtained in hypoxia (12% oxygen) after 6 h of rest and immediately after a maximal exercise challenge. Haemostatic parameters were expressed before and after volume-shift correction. Passive hypoxia reduced PV by 3±5% (p<0.05 vs normoxia), with a further decrease observed during exercise (14±5%, p<0.05). The latter increased the absolute concentration of fibrinogen and shortened aPTT (p<0.05), but these changes were no longer apparent after PV correction (p>0.05). Likewise, the lack of change in absolute values for PT and TT (p>0.05) translated into an elongation after correction (p<0.05).

Conclusions

These findings highlight the important, but previously ignored, interpretive implications of PV correction when haemostasis is assessed.
A practical application of analysing weighted kappa for panels of experts and EQA schemes in pathology
Authors: Wright Karen C , Harnden Patricia , Moss Sue , Berney Dan M , Melia Jane
Published: January 2011

Background

Kappa statistics are frequently used to analyse observer agreement for panels of experts and External Quality Assurance (EQA) schemes and generally treat all disagreements as total disagreement. However, the differences between ordered categories may not be of equal importance (eg, the difference between grades 1 vs 2 compared with 1 vs 3). Weighted kappa can be used to adjust for this when comparing a small number of readers, but this has not as yet been applied to the large number of readers typical of a national EQA scheme.

Aim

To develop and validate a method for applying weighted kappa to a large number of readers within the context of a real dataset: the UK National Urological Pathology EQA Scheme for prostatic biopsies.

Methods

Data on Gleason grade recorded by 19 expert readers were extracted from the fixed text responses of 20 cancer cases from four circulations of the EQA scheme. Composite kappa, currently used to compute an unweighted kappa for large numbers of readers, was compared with the mean kappa for all pairwise combinations of readers. Weighted kappa generalised for multiple readers was compared with the newly developed ‘pairwise-weighted’ kappa.

Results

For unweighted analyses, the median increase from composite to pairwise kappa was 0.006 (range −0.005 to +0.052). The difference between the pairwise-weighted kappa and generalised weighted kappa for multiple readers never exceeded ±0.01.

Conclusion

Pairwise-weighted kappa is a suitable and highly accurate approximation to weighted kappa for multiple readers.
Mature fat cells in the myocardium of patients with tuberous sclerosis complex: Figure 1
Authors: Adriaensen Miraude E A P M , van Oosterhout Matthijs F M , Feringa Harm H H , Schaefer-Prokop Cornelia M , Zonnenberg Bernard A , Prokop Mathias
Published: January 2011

Aim

Routine abdominal CT scans in patients with tuberous sclerosis complex (TSC) showed characteristic fatty foci in the depicted caudal portions of the myocardium. The purpose of this study was to investigate if areas of abnormal myocardium in patients with TSC could also be found in post-mortem specimens.

Methods

A retrospective search of our histopathology database was performed to identify specimens of the heart of patients with TSC. Institutional review board approval was obtained, and patient informed consent was waived. Four specimens were included (mean age, 44 years; range 32–68 years; 2 females).

Results

Two specimens (50%) of the heart showed areas of mature fat cells in the myocardium, without associated inflammation, without associated fibrosis, without entrapped myocardial cells and without a capsule.

Conclusion

Post-mortem specimens of the heart of patients with TSC showed areas of mature fat cells in the myocardium which seem to be unique for TSC.
Ethically complex decisions in the neonatal intensive care unit: impact of the new French legislation on attitudes and practices of physicians and nurses: Table 1
Authors: Garel Micheline , Caeymaex Laurence , Goffinet François , Cuttini Marina , Kaminski Monique
Published: January 2011

Objectives

A statute enacted in 2005 modified the legislative framework of the rights of terminally ill persons in France. Ten years after the EURONIC study, which described the self-reported practices of neonatal caregivers towards ethical decision-making, a new study was conducted to assess the impact of the new law in neonatal intensive care units (NICU) and compare the results reported by EURONIC with current practices.

Setting and design

The study was carried out in the same two NICU as in the EURONIC qualitative study. A third centre was added to increase the sample size. From February to October 2007, 19 physicians and 17 nurses participated in semistructured interviews very similar to those for EURONIC. Content analysis identified the recurring themes emerging from the interviews.

Results

Compared with the EURONIC results, the caregivers reported that they pay greater attention to the views of parents and provided respectful support to the neonates when life-sustaining treatment is withdrawn. Active termination of life has become exceptional. The possibility of withdrawal of treatment, the administration of sedatives to control pain even at the risk of hastening death, the emphasis on sparing parents the burden of decision, and the relative ignorance of the law were very similar to the EURONIC findings.

Conclusion

Both the medical and the legal regulation of practices has allowed more dialogue with the parents and more humane care for dying newborns. A new European study is necessary to investigate the possible changes in practices and attitudes also in other countries.
HUMAN-LIKE GAIT GENERATION FOR BIPED ANDROID ROBOT USING MOTION CAPTURE AND ZMP MEASUREMENT SYSTEM
Authors: KIM JUNG-YUP , KIM YOUNG-SEOG
Published: January 2011

This article proposes a novel strategy to generate both a human-like walking pattern and a human-like zero moment point (ZMP) trajectory for a biped android robot. In general, the motion-capture technique has been widely utilized to obtain a walking pattern that is kinematically similar to the walking of a human. However, in addition to kinematic considerations, a suitable ZMP shaping technique is necessary to apply the human gait derived by motion capturing to biped robots more effectively. In previous research by the authors, a walking pattern generation strategy was developed considering the kinematics using motion capturing and Fourier fitting. However, it was found that there were differences between the calculated ZMP trajectory of the earlier research and the measured ZMP trajectory directly derived from the sensor in this research. Therefore, the differences and their factors are analyzed and a new strategy is proposed that effectively reduces the differences between them. Finally, the proposed strategy is shown to be effective for generating human-like walking pattern and ZMP trajectory for biped android robots through stick figure simulations.
Estimates of yet-to-find impact crater population on Earth
Authors: Stewart S.A.
Published: January 2011

One hundred and seventy-six craters on Earth are currently accepted as resulting from bolide impact; this list is growing at a rate of approximately two craters per year because of new discoveries and revised interpretations. Here we take the subset of craters most relevant to sedimentary basin exploration and utilization (Phanerozoic kilometre-scale craters) and tackle the question of how many remain to be found in Phanerozoic strata on Earth. A surprising range in predictions of crater numbers is obtained from published crater population functions, reaching two orders of magnitude variation for craters in the range 1–5 km diameter. This range in predictions is largely due to variations in treatment of impactor breakup in the atmosphere. Also required is quantification of the total area and timespan of Phanerozoic sediment currently preserved on Earth; an estimate of 8.87 × 10
15
km
2
a is derived here. Together with a relatively conservative crater population function this yields a prediction of 845 craters; subtracting the 131 known Phanerozoic craters larger than 1 km leaves a predicted population yet to find of 714, of which 228 are larger than 2.5 km. Uncertainties, subsets of this population, and spatial distribution are also considered.
Phase equilibrium constraints on conditions of granulite-facies metamorphism at Scourie, NW Scotland
Authors: Johnson T.E. , White R.W.
Published: January 2011

The metamorphic evolution of a metapyroxenite and metagabbro from Scourie, NW Scotland, is investigated using phase equilibria modelled in the NCFMASHTO (Na
2
O–CaO–FeO–MgO–Al
2
O
3
–SiO
2
–H
2
O–TiO
2
–O) system. The calculated stability fields for the observed assemblages in each rock overlap and imply conditions of 8.5–11.5 kbar and 875–975 °C for the peak of granulite-facies (Badcallian) metamorphism. The lack of any evidence for the former presence of garnet in the metapyroxenite suggests that the rocks cannot have reached pressures much in excess of those recorded at the metamorphic peak. The growth of coronas of plagioclase, orthopyroxene and magnetite replacing garnet in the metagabbro is consistent with near-isothermal retrograde decompression from the metamorphic peak to pressures of 7–9 kbar. The constraints on the pressure–temperature evolution imposed by the modelled phase equilibria are more consistent with a process of crustal growth dominated by magmatic accretion rather than by significant tectonic thickening during the early Scourian.

Supplementary material:

Mineral chemical data are available at
http://www.geolsoc.org.uk/SUP18436
.
"Towards Collaboratively Learning and Populating Ontologies for the Social-Semantic Web" by Mathias Niepert, with Jessica Rubart as coordinator
Authors: Niepert Mathias
Published: January 2011

In this article we present the Indiana Philosophy Ontology (InPhO) project as one of the first social-semantic web endeavors which aims to harness feedback from users who are unfamiliar with designing and populating ontologies to build a precise conceptual representation of a particular domain. This representation is initially a dynamically maintained taxonomy with several non-taxonomic relations. More complex components like class axioms and additional non-taxonomic relations will be iteratively added once more intricate reasoning techniques and a finer granularity of the representation is needed. Our approach combines statistical text processing methods, feed-back gathered through user interfaces, and logic programming approaches to create a dynamically changing, semantic representation of the discipline of philosophy. The long-term goal of the project is to build novel search and exploration applications on top of this populated representation. We describe the basic ideas and methodology of the project.
Up and coming SIGWEB supported conferences
Authors: Deshpande Yogesh
Published: January 2011

SIGWEB supports several specialized conferences, short courses, and workshops, as well as the Annual Hypertext Conference. SIGWEB sponsored conferences focus on timely topics in applied and computational hypertext and Web disciplines and provide a place for members and the entire applied Hypermedia and Web community to exchange ideas and to meet with and expand their network of colleagues. In this article, we provide a brief overview of SIGWEB sponsored conferences, in addition to events that are in cooperation with SIGWEB.
Design as learning---or "knowledge creation"---the SECI model
Authors: Dubberly Hugh , Evenson Shelley
Published: January 2011

Models help bridge the gap between observing and making---especially when systems are involved (as in designing for interaction, service, and evolution). This forum introduces new models, links them to existing models, and describes their history and why they matter.

        

Hugh Dubberly, Editor | hugh@dubberly.com
Capacity and delay in mobile ad hoc networks under Gaussian channel model
Authors: Wang Cheng , Li Xiang-Yang , Tang Shaojie , Jiang Changjun , Liu Yunhao
Published: January 2011

We study the asymptotic delay and throughput in mobile ad hoc networks where η ad hoc nodes are distributed uniformly on a 2-D square (torus) region of area η. The communications between nodes are characterized by Gaussian Channel model, instead of the simplified protocol model or physical model. The mobility of nodes is characterized by two general broad classes of practical mobility models, i.e., hybrid random walk models and discrete random direction models, which generalize many mobility models used in the literature. Our results either fill the gap in this area or generalize a stream of milestone results on asymptotic capacity, delay, and the tradeoffs developed recently.
CMAC-ORS
Authors: Kim Soungwoo , Roh Heejun , Lee Wonjun
Published: January 2011

In recent years, several studies have attempted to find and explore MAC protocols for supporting cooperative communication to improve reliability and throughput in wireless networks. The performance of these MAC protocols depends on how to select relay nodes. We propose a novel cooperative MAC protocol with reactive relay selection for wireless LANs that reduces collisions and overheads in relay selection. Since our MAC protocol is a modification of IEEE 802.11 distributed coordination function, it can be applied in the existing network interfaces with software MAC. Simulation results demonstrate that the proposed MAC protocol increases network throughput substantially compared to the legacy IEEE 802.11b system.