bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification ofYersinia enterocoliticaat the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy
Authors: Kuhm Andrea Elisabeth , Suter Daniel , Felleisen Richard , Rau Jörg
Published: July 2009

Yersinia enterocoliticaand otherYersiniaspecies, such asY. pseudotuberculosis,Y. bercovieri, andY. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well definedYersiniastrains from Switzerland and Germany was used to create a method for FT-IR-based differentiation ofYersiniaisolates at the species level. The isolates ofY. enterocoliticawere also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and “non-O:3, O:5, and O:9”). For external validation of the constructed methods, independently obtained isolates of differentYersiniaspecies were used. A total of 79.9% ofY. enterocoliticasensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of allY. bercovieri,Y. intermedia, andY. rohdeistrains fromY. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification ofY. enterocoliticaisolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of theailgene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning theailgene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case ofY. enterocoliticaalso subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.
Cell Surface Engineering of a β-Galactosidase for Galactooligosaccharide Synthesis
Authors: Li Yumei , Lu Lili , Wang Hongmei , Xu Xiaodong , Xiao Min
Published: July 2009

A novel gene encoding transglycosylating β-galactosidase (BGase) was cloned from
Penicillium expansum
F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of
Saccharomyces cerevisiae
EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25°C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.
Sporicidal Activity of Synthetic Antifungal Undecapeptides and Control of
Penicillium
Rot of Apples
Authors: Badosa Esther , Ferré Rafael , Francés Jesús , Bardají Eduard , Feliu Lidia , Planas Marta , Montesinos Emilio
Published: July 2009

The antifungal activity of cecropin A(2-8)-melittin(6-9) hybrid undecapeptides, previously reported as active against plant pathogenic bacteria, was studied. A set of 15 sequences was screened in vitro against
Fusarium oxysporum
,
Penicillium expansum
,
Aspergillus niger
, and
Rhizopus stolonifer
. Most compounds were highly active against
F. oxysporum
(MIC < 2.5 μM) but were less active against the other fungi. The best peptides were studied for their sporicidal activity and for Sytox green uptake in
F. oxysporum
microconidia. A significant inverse linear relationship was observed between survival and fluorescence, indicating membrane disruption. Next, we evaluated the in vitro activity against
P. expansum
of a 125-member peptide library with the general structure R-X
1
KLFKKILKX
10
L-NH
2
, where X
1
and X
10
corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. Fifteen sequences with MICs below 12.5 μM were identified. The most active compounds were BP21 {Ac,F,V} and BP34 {Ac,L,V} (MIC < 6.25 μM), where the braces denote R, X
1
, and X
10
positions and where Ac is an acetyl group. The peptides had sporicidal activity against
P. expansum
conidia. Seven of these peptides were tested in vivo by evaluating their preventative effect of inhibition of
P. expansum
infection in apple fruits. The peptide Ts-FKLFKKILKVL-NH
2
(BP22), where Ts is a tosyl group, was the most active with an average efficacy of 56% disease reduction, which was slightly lower than that of a commercial formulation of the fungicide imazalil.
Assessment of Microbial Communities by Graph Partitioning in a Study of Soil Fungi in Two Alpine Meadows
Authors: Zinger L. , Coissac E. , Choler P. , Geremia R. A.
Published: July 2009

Understanding how microbial community structure and diversity respond to environmental conditions is one of the main challenges in environmental microbiology. However, there is often confusion between determining the phylogenetic structure of microbial communities and assessing the distribution and diversity of molecular operational taxonomic units (MOTUs) in these communities. This has led to the use of sequence analysis tools such as multiple alignments and hierarchical clustering that are not adapted to the analysis of large and diverse data sets and not always justified for characterization of MOTUs. Here, we developed an approach combining a pairwise alignment algorithm and graph partitioning by using MCL (Markov clustering) in order to generate discrete groups for nuclear large-subunit rRNA gene and internal transcript spacer 1 sequence data sets obtained from a yearly monitoring study of two spatially close but ecologically contrasting alpine soils (namely, early and late snowmelt locations). We compared MCL with a classical single-linkage method (Ccomps) and showed that MCL reduced bias such as the chaining effect. Using MCL, we characterized fungal communities in early and late snowmelt locations. We found contrasting distributions of MOTUs in the two soils, suggesting that there is a high level of habitat filtering in the assembly of alpine soil fungal communities. However, few MOTUs were specific to one location.
Anaerobic Fermentation of Glycerol in
Paenibacillus macerans
: Metabolic Pathways and Environmental Determinants
Authors: Gupta Ashutosh , Murarka Abhishek , Campbell Paul , Gonzalez Ramon
Published: July 2009

Paenibacillus macerans
is one of the species with the broadest metabolic capabilities in the genus
Paenibacillus
, able to ferment hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose. However, little is known about glycerol metabolism in this organism, and some studies have reported that glycerol is not fermented. Despite these reports, we found that several
P. macerans
strains are capable of anaerobic fermentation of glycerol. One of these strains,
P. macerans
N234A, grew fermentatively on glycerol at a maximum specific growth rate of 0.40 h
−1
and was chosen for further characterization. The use of [U-
13
C]glycerol and further analysis of extracellular metabolites and proteinogenic amino acids via nuclear magnetic resonance (NMR) spectroscopy allowed identification of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) as fermentation products and demonstrated that glycerol is incorporated into cellular components. A medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO
2
-enriched atmosphere stimulated glycerol fermentation and are proposed to be environmental determinants of this process. The pathways involved in glycerol utilization and synthesis of fermentation products were identified using NMR spectroscopy in combination with enzyme assays. Based on these studies, the synthesis of ethanol and 1,2-PDO is proposed to be a metabolic determinant of glycerol fermentation in
P. macerans
N234A. Conversion of glycerol to ethanol fulfills energy requirements by generating one molecule of ATP per molecule of ethanol synthesized. Conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents, thus facilitating redox balance. Given the availability, low price, and high degree of reduction of glycerol, the high metabolic rates exhibited by
P. macerans
N234A are of paramount importance for the production of fuels and chemicals.
Carbonate Crystals Precipitated by Freshwater Bacteria and Their Use as a Limestone Consolidant
Authors: Zamarreño Dania V. , Inkpen Robert , May Eric
Published: July 2009

Bacterial carbonate precipitation is known to be a natural phenomenon associated with a wide range of bacterial species. Recently, the ability of bacteria to produce carbonates has been studied for its value in the conservation of limestone monuments and concrete. This paper describes investigations of carbonate crystals precipitated by freshwater bacteria by means of histological (Loeffler's methylene blue and alcian blue-periodic acid-Schiff stain) and fluorescence (CTC [5-cyano-2,3-ditolyl tetrazolium chloride]) stains, determination of cell viability inside carbonate crystals, and pore size reduction in limestone by image analysis. Carbonate crystals were found to be composed of bacteria embedded in a matrix of neutral and acid polysaccharides. Cell viability inside the carbonate crystals decreased with time. On stone, bacteria were found to form carbonate crystals, with only a few bacteria remaining as isolated cells or as cell aggregates. Pore size was reduced by about 50%, but no blockage was detected. Taken together, the results of this research provide some reassurance to conservators that biocalcification by bacteria could be a safe consolidation tool in a restoration strategy for building stone conservation.
Alteration of the Ileal Microbiota of Weanling Piglets by the Growth-Promoting Antibiotic Chlortetracycline
Authors: Rettedal Elizabeth , Vilain Sébastien , Lindblom Stacy , Lehnert Kelly , Scofield Clay , George Sajan , Clay Sharon , Kaushik Radhey S. , Rosa Artur J. M. , Francis David , Brözel Volker S.
Published: July 2009

Antibiotics such as chlortetracycline (CTC) have been used to promote growth of pigs for decades, but concerns over increased antibiotic-resistant infections in humans have prompted the development of alternative strategies. Developing alternatives to antibiotic growth promoters (AGPs) could be informed by information on the mechanisms of growth promotion, notably, how AGPs affect the microbial populations of the gastrointestinal tract. Pigs from three sows were aseptically delivered by cesarean section. Six piglets were distributed to each of two foster mothers until weaning, when piglets were fed a diet with or without 50 mg/kg CTC for 2 weeks. The ileal bacterial microbiota was characterized by using a cultivation-independent approach based on DNA extraction, PCR amplification, cloning, and sequencing of the 16S rRNA gene pool. The ileal and mucosal communities of these growing pigs were dominated byLactobacillusbacteria, various members of the familyClostridiaceae, and members of the poorly known genusTuricibacter. Overall, CTC treatment resulted in three shifts: a decrease inLactobacillusjohnsonii, an increase inL.amylovorus, and a decrease inTuricibacterphylotypes. The composition of the microbiota varied considerably between individual pigs, as revealed by shared operational taxonomic units (OTUs) and similarity (SONS) analysis (θYCvalues). While the observed variation between untreated pigs obscured the possible effect of CTC, ∫-LIBSHUFF and SONS analyses of pooled libraries indicated a significant shift due to CTC in both the lumen and the mucosa, with some OTUs unique to either treated or control ileum. DOTUR analysis revealed little overlap between control and treated communities at the 3% difference level, indicating unique ileal communities in the presence of CTC.
The
ARG9
Gene Encodes the Plastid-Resident
N
-Acetyl Ornithine Aminotransferase in the Green Alga
Chlamydomonas reinhardtii
Authors: Remacle Claire , Cline Sara , Boutaffala Layla , Gabilly Stéphane , Larosa Véronique , Barbieri M. Rosario , Coosemans Nadine , Hamel Patrice P.
Published: July 2009

Here we report the characterization of the
Chlamydomonas reinhardtii
gene
ARG9
, encoding the plastid resident
N
-acetyl ornithine aminotransferase, which is involved in arginine synthesis. Integration of an engineered
ARG9
cassette in the plastid chromosome of the nuclear
arg9
mutant restores arginine prototrophy. This suggests that
ARG9
could be used as a new selectable marker for plastid transformation.
Downregulation of Mitochondrial Porin Inhibits Cell Growth and Alters Respiratory Phenotype in
Trypanosoma brucei
Authors: Singha Ujjal K. , Sharma Shvetank , Chaudhuri Minu
Published: July 2009

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in
Trypanosoma brucei
, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a β-barrel structure similar to the bacterial OM porin OmpA.
T. brucei
porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in
T. brucei
according to mitochondrial energy demand.
Role for Chitin and Chitooligomers in the Capsular Architecture of
Cryptococcus neoformans
Authors: Fonseca Fernanda L. , Nimrichter Leonardo , Cordero Radames J. B. , Frases Susana , Rodrigues Jessica , Goldman David L. , Andruszkiewicz Ryszard , Milewski Sławomir , Travassos Luiz R. , Casadevall Arturo , Rodrigues Marcio L.
Published: July 2009

Molecules composed of β-1,4-linked
N
-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus
Cryptococcus neoformans
. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in
C. neoformans
varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to
C. neoformans
cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of
C. neoformans
in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.
Genetic Analysis of Repair and Damage Tolerance Mechanisms for DNA-Protein Cross-Links in
Escherichia coli
Authors: Salem Amir M. H. , Nakano Toshiaki , Takuwa Minako , Matoba Nagisa , Tsuboi Tomohiro , Terato Hiroaki , Yamamoto Kazuo , Yamada Masami , Nohmi Takehiko , Ide Hiroshi
Published: July 2009

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. We have recently shown that nucleotide excision repair (NER) and RecBCD-dependent homologous recombination (HR) collaboratively alleviate the lethal effect of DPCs in
Escherichia coli
. In this study, to gain further insight into the damage-processing mechanism for DPCs, we assessed the sensitivities of a panel of repair-deficient
E. coli
mutants to DPC-inducing agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies primarily on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Together with the genes responsible for HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair and transactions, such as
recQ
,
xth nfo
,
dksA
, and
topA
, rendered cells slightly but significantly sensitive to FA but not azaC, possibly reflecting the complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional role outside NER, since the
uvrD
mutation conferred a slight azaC sensitivity on cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the repair of DPCs, presumably by removing 5-azacytosine or its degradation product from the chromosome.
An Alternative Route for Recycling of
N
-Acetylglucosamine from Peptidoglycan Involves the
N
-Acetylglucosamine Phosphotransferase System in
Escherichia coli
Authors: Plumbridge Jacqueline
Published: July 2009

A set of enzymes dedicated to recycling of the amino sugar components of peptidoglycan has previously been identified in
Escherichia coli
. The complete pathway includes the
nagA
-encoded enzyme,
N
-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase, of the catabolic pathway for use of
N
-acetylglucosamine (GlcNAc). Mutations in
nagA
result in accumulation of millimolar concentrations of GlcNAc6P, presumably by preventing peptidoglycan recycling. Mutations in the genes encoding the key enzymes upstream of
nagA
in the dedicated recycling pathway (
ampG
,
nagZ
,
nagK
,
murQ
, and
anmK
), which were expected to interrupt the recycling process, reduced but did not eliminate accumulation of GlcNAc6P. A mutation in the
nagE
gene of the GlcNAc phosphotransferase system (PTS) was found to reduce by 50% the amount of GlcNAc6P which accumulated in a
nagA
strain and, together with mutations in the dedicated recycling pathway, eliminated all the GlcNAc6P accumulation. This shows that the
n
agE
-encoded PTS transporter makes an important contribution to the recycling of peptidoglycan. The
manXYZ
-encoded PTS transporter makes a minor contribution to the formation of cytoplasmic GlcNAc6P but appears to have a more important role in secretion of GlcNAc and/or GlcNAc6P from the cytoplasm.
Processive Endoglucanases Mediate Degradation of Cellulose by
Saccharophagus degradans
Authors: Watson Brian J. , Zhang Haitao , Longmire Atkinson G. , Moon Young Hwan , Hutcheson Steven W.
Published: July 2009

Bacteria and fungi are thought to degrade cellulose through the activity of either a complexed or a noncomplexed cellulolytic system composed of endoglucanases and cellobiohydrolases. The marine bacterium
Saccharophagus degradans
2-40 produces a multicomponent cellulolytic system that is unusual in its abundance of GH5-containing endoglucanases. Secreted enzymes of this bacterium release high levels of cellobiose from cellulosic materials. Through cloning and purification, the predicted biochemical activities of the one annotated cellobiohydrolase Cel6A and the GH5-containing endoglucanases were evaluated. Cel6A was shown to be a classic endoglucanase, but Cel5H showed significantly higher activity on several types of cellulose, was the highest expressed, and processively released cellobiose from cellulosic substrates. Cel5G, Cel5H, and Cel5J were found to be members of a separate phylogenetic clade and were all shown to be processive. The processive endoglucanases are functionally equivalent to the endoglucanases and cellobiohydrolases required for other cellulolytic systems, thus providing a cellobiohydrolase-independent mechanism for this bacterium to convert cellulose to glucose.
Genomic Insights into the Convergence and Pathogenicity Factors of
Campylobacter jejuni
and
Campylobacter coli
Species
Authors: Caro-Quintero Alejandro , Rodriguez-Castaño Gina P. , Konstantinidis Konstantinos T.
Published: July 2009

Whether or not bacteria form coherent evolutionary groups via means of genetic exchange and, hence, elicit distinct species boundaries remains an unsettled issue. A recent report implied that not only may the former be true but also, in fact, the clearly distinct
Campylobacter jejuni
and
Campylobacter coli
species may be converging as a consequence of increased interspecies gene flow fostered, presumably, by the recent invasion of an overlapping ecological niche (S. K. Sheppard, N. D. McCarthy, D. Falush, and M. C. Maiden, Science 320:237-239, 2008). We have reanalyzed the
Campylobacter
multilocus sequence typing database used in the previous study and found that the number of interspecies gene transfer events may actually be too infrequent to account, unequivocally, for species convergence. For instance, only 1 to 2% of the 4,507
Campylobacter
isolates examined appeared to have imported gene alleles from another
Campylobacter
species. Furthermore, by analyzing the available
Campylobacter
genomic sequences, we show that although there seems to be a slightly higher number of exchanged genes between
C. jejuni
and
C. coli
relative to other comparable species (∼10% versus 2 to 3% of the total genes in the genome, respectively), the function and spatial distribution in the genome of the exchanged genes are far from random, and hence, inconsistent with the species convergence hypothesis. In fact, the exchanged genes appear to be limited to a few environmentally selected cellular functions. Accordingly, these genes may represent important pathogenic determinants of pathogenic
Campylobacter
, and convergence of (any) two bacterial species remains to be seen.
Crystal Structures of
Streptococcus suis
Mannonate Dehydratase (ManD) and Its Complex with Substrate: Genetic and Biochemical Evidence for a Catalytic Mechanism
Authors: Zhang Qiangmin , Gao Feng , Peng Hao , Cheng Hao , Liu Yiwei , Tang Jiaqi , Thompson John , Wei Guohua , Zhang Jingren , Du Yuguo , Yan Jinghua , Gao George F.
Published: July 2009

Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of
d
-mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting is of therapeutic interest in the treatment of human
Streptococcus suis
infections. Here, we report the overexpression, purification, functional characterization, and crystallographic structure of ManD from
S. suis
. Importantly, by Fourier transform mass spectrometry, we show that 2-KDG is formed when the chemically synthesized substrate (
d
-mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry revealed the presence of Mn
2+
in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of two 41-kDa subunits. The crystal structures of
S. suis
ManD in native form and in complex with its substrate and Mn
2+
ion have been solved at a resolution of 2.9 Å. The core structure of
S. suis
ManD is a TIM barrel similar to that of other members of the xylose isomerase-like superfamily. Structural analyses and comparative amino acid sequence alignments provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction and a plausible mechanism for the ManD-catalyzed reaction is proposed.
Paenibacillus dendritiformis
Bacterial Colony Growth Depends on Surfactant but Not on Bacterial Motion
Authors: Be'er Avraham , Smith Rachel S. , Zhang H. P. , Florin E.-L. , Payne Shelley M. , Swinney Harry L.
Published: July 2009

Most research on growing bacterial colonies on agar plates has concerned the effect of genetic or morphotype variation. Some studies have indicated that there is a correlation between microscopic bacterial motion and macroscopic colonial expansion, especially for swarming strains, but no measurements have been obtained for a single strain to relate the microscopic scale to the macroscopic scale. We examined here a single strain (
Paenibacillus dendritiformis
type
T
; tip splitting) to determine both the macroscopic growth of colonies and the microscopic bacterial motion within the colonies. Our multiscale measurements for a variety of growth conditions revealed that motion on the microscopic scale and colonial growth are largely independent. Instead, the growth of the colony is strongly affected by the availability of a surfactant that reduces surface tension.
Region of Difference 1 in Nontuberculous
Mycobacterium
Species Adds a Phylogenetic and Taxonomical Character
Authors: van Ingen Jakko , de Zwaan Rina , Dekhuijzen Richard , Boeree Martin , van Soolingen Dick
Published: July 2009

The
esat-6
and
cfp-10
genes are essential for virulence in
Mycobacterium tuberculosis
. Among nontuberculous mycobacteria, we found these genes only in
M. kansasii
,
M. szulgai
,
M. marinum
, and
M. riyadhense
, with unique sequences. This adds a phylogenetic and taxonomical characteristic and may represent a virulence factor for nontuberculous mycobacteria.
DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of V
ibrio cholerae
Authors: Falero Alina , Caballero Andy , Ferrán Beatriz , Izquierdo Yovanny , Fando Rafael , Campos Javier
Published: July 2009

The native product of open reading frame 112 (
orf112
) and a recombinant variant of the RstB protein, encoded by
Vibrio cholerae
pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases.
Cellular Localization of Predicted Transmembrane and Soluble Chemoreceptors in
Sinorhizobium meliloti
Authors: Meier Veronika M. , Scharf Birgit E.
Published: July 2009

Bacterial chemoreceptors primarily locate in clusters at the cell pole, where they form large sensory complexes which recruit cytoplasmic components of the signaling pathway. The genome of the soil bacterium
Sinorhizobium meliloti
encodes seven transmembrane and two soluble chemoreceptors. We have investigated the localization of all nine chemoreceptors in vivo using genome-encoded fusions to a variant of the enhanced green fluorescent protein and to monomeric red fluorescent protein. Six of the transmembrane (McpT to McpX and McpZ) and both soluble (McpY and IcpA) receptors localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile
S. meliloti
cells form one major chemotaxis cluster that harbors all chemoreceptors except for McpS. Colocalization and deletion analysis demonstrated that formation of polar foci by the majority of receptors is dependent on other chemoreceptors and that receptor clusters are stabilized by the presence of the chemotaxis proteins CheA and CheW. The transmembrane McpV and the soluble IcpA localize to the pole independently of CheA and CheW. However, in mutant strains McpV formed delocalized polar caps that spread throughout the cell membrane while IcpA exhibited increased bipolarity. Immunoblotting of fractionated cells revealed that IcpA, which lacks any hydrophobic domains, nevertheless is associated to the cell membrane.
Identification and Characterization of Growth Suppressors ofEscherichia coliStrains Lacking Phosphorolytic Ribonucleases
Authors: Jain Chaitanya
Published: July 2009

RNases are involved in critical aspects of RNA metabolism in all organisms. Two classes of RNases that digest RNA from an end (exo-RNases) are known: RNases that use water as a nucleophile to catalyze RNA degradation (hydrolytic RNases) and RNases that use inorganic phosphate (phosphorolytic RNases). It has been shown previously that the absence of the two knownEscherichia coliphosphorolytic RNases, polynucleotide phosphorylase and RNase PH, leads to marked growth and ribosome assembly defects. To investigate the basis for these defects, a screen for growth suppressors was performed. The majority of suppressor mutations were found to lie withinnsrR, which encodes a nitric oxide (NO)-sensitive transcriptional repressor. Further analysis showed that the suppressors function not by inactivatingnsrRbut by causing overexpression of a downstream gene that encodes a hydrolytic RNase, RNase R. Additional studies revealed that overexpression of another hydrolytic RNase, RNase II, similarly suppressed the growth defects. These results suggest that the requirement for phosphorolytic RNases for robust cellular growth and efficient ribosome assembly can be bypassed by increased expression of hydrolytic RNases.
Effect of taping on the shoulders of Australian football players
Authors: Bradley T , Baldwick C , Fischer D , Murrell G A C
Published: July 2009

Background:

Taping of the shoulder is common in many sports, particularly Australian football, a contact sport that often involves marking (catching) the ball overhead and has a high incidence of shoulder instability.

Hypothesis:

Taping of the shoulder reduces glenohumeral joint laxity and improves proprioception without impairing function.

Study design:

Crossover study design.

Methods:

33 male players aged 18–31 years were recruited from a local Australian football club. The dominant shoulder of each player was tested with and without taping in a randomised fashion by an examiner blinded to the presence or absence of taping. The tests were (1) inferior glenohumeral joint laxity (the Orthopaedic Research Institute laxometer), (2) shoulder joint position sense accuracy using an optical tracking system, and (3) handballing accuracy.

Results:

The methods for testing laxity and joint position sense had good intraobserver reliability and sensitivity. All subjects tolerated the taping and testing. Glenohumeral joint laxity (p = 0.75), joint position sense (p = 0.56) and handballing accuracy (p = 0.6) were not changed by taping.

Conclusions:

Taping of the shoulder joint in uninjured and non-symptomatic Australian football players in a pattern that attempted not to restrict their range of overhead movement did not significantly affect the accuracy of joint position sense, inferior laxity or handball accuracy.

Clinical relevance:

These data suggest that taping of the shoulder is unlikely to decrease the incidence of injury—specifically dislocation—of the shoulder in Australian football players.
The effect of smoke-free policies on revenue in bars in Tasmania, Australia
Authors: Lal A , Siahpush M
Published: July 2009

Objective:

To examine the impact of smoke-free policies on revenue in Tasmanian bars.

Method:

Monthly sales turnover from January 2002 to March 2007, provided by the Australian Bureau of Statistics was analysed. There were two outcome measures: (1) the ratio of monthly bar sales turnover in Tasmania to monthly bar sales turnover in four other Australian states, and (2) the ratio of monthly bar turnover to monthly retail turnover in Tasmania. Linear regression was used to assess the impact of the smoke-free policy on expenditure.

Results:

The smoke-free policy had no effect on sales turnover.

Conclusion:

The smoke-free policy protects hospitality workers and patrons from exposure to secondhand smoke and has had no adverse effect on sales turnover.
The hazards and benefits associated with smoking and smoking cessation in Asia: a meta-analysis of prospective studies
Authors: Nakamura K , Huxley R , Ansary-Moghaddam A , Woodward M
Published: July 2009

Objective:

To provide the most reliable evidence as to the nature of the associations between smoking and cause-specific illness, as well as the expected benefits from quitting smoking, in studies conducted in Asia, where smoking remains popular among men.

Data sources:

Studies published between January 1966 and October 2008, identified in the Medline search strategy with medical subject headings, in addition to studies from the Asia Pacific Cohort Studies Collaboration.

Study selection:

Studies were considered to be relevant if they were prospective studies, in an Asian setting that reported on the association between smoking, quitting and cause-specific illness.

Data extraction:

Two reviewers independently screened all identified articles for possible inclusion and extracted data.

Data synthesis:

The pooled relative risks (RRs) for incidence or mortality, comparing current to never smokers were always significantly higher than unity; the highest was for lung cancer: 3.54 (95% confidence interval 3.00 to 4.17). The pooled RRs for former smokers (compared to never smokers) were also always significantly higher than unity, and were lower than in current smokers, for coronary heart disease, stroke, lung and upper aero-digestive tract cancer. Only for respiratory disease was the RR for former smokers higher than that for current smokers.

Conclusions:

This meta-analysis has shown that, despite the relative immaturity of the smoking epidemic in Asia, smoking is unquestionably a major contributor to ill health and death. However, the beneficial effects of quitting are not yet always apparent, most probably because quitting is a consequence of ill health and the relative unpopularity of smoking cessation in many Asian populations.
TRPV4-dependent dilation of peripheral resistance arteries influences arterial pressure
Authors: Earley Scott , Pauyo Thierry , Drapp Rebecca , Tavares Matthew J. , Liedtke Wolfgang , Brayden Joseph E.
Published: July 2009

Transient receptor potential vanilloid 4 (TRPV4) channels have been implicated as mediators of calcium influx in both endothelial and vascular smooth muscle cells and are potentially important modulators of vascular tone. However, very little is known about the functional roles of TRPV4 in the resistance vasculature or how these channels influence hemodynamic properties. In the present study, we examined arterial vasomotor activity in vitro and recorded blood pressure dynamics in vivo using TRPV4 knockout (KO) mice. Acetylcholine-induced hyperpolarization and vasodilation were reduced by ∼75% in mesenteric resistance arteries from TRPV4 KO versus wild-type (WT) mice. Furthermore, 11,12-epoxyeicosatrienoic acid (EET), a putative endothelium-derived hyperpolarizing factor, activated a TRPV4-like cation current and hyperpolarized the membrane of vascular smooth muscle cells, resulting in the dilation of mesenteric arteries from WT mice. In contrast, 11,12-EET had no effect on membrane potential, diameter, or ionic currents in the mesenteric arteries from TRPV4 KO mice. A disruption of the endothelium reduced 11,12-EET-induced hyperpolarization and vasodilatation by ∼50%. A similar inhibition of these responses was observed following the block of endothelial (small and intermediate conductance) or smooth muscle (large conductance) K+ channels, suggesting a link between 11,12-EET activity, TRPV4, and K+ channels in endothelial and smooth muscle cells. Finally, we found that hypertension induced by the inhibition of nitric oxide synthase was greater in TRPV4 KO compared with WT mice. These results support the conclusion that both endothelial and smooth muscle TRPV4 channels are critically involved in the vasodilation of mesenteric arteries in response to endothelial-derived factors and suggest that in vivo this mechanism opposes the effects of hypertensive stimuli.
Aging impairs flow-induced dilation in coronary arterioles: role of NO and H2O2
Authors: Kang Lori S. , Reyes Rafael A. , Muller-Delp Judy M.
Published: July 2009

Aging contributes significantly to the development of cardiovascular disease and is associated with elevated production of reactive oxygen species (ROS). The beneficial effects of nitric oxide (NO)-mediated vasodilation are quickly abolished in the presence of ROS, and this effect may be augmented with aging. We previously demonstrated an age-induced impairment of flow-induced dilation in rat coronary arterioles. Therefore, the purpose of this study was to determine the effects of O2− scavenging, as well as removal of H2O2, the byproduct of O2− scavenging, on flow-mediated dilation in coronary resistance arterioles of young (4 mo) and old (24 mo) male Fischer 344 rats. Flow increased NO and H2O2 production as evidenced by enhanced diaminofluorescein and dichlorodihydrofluorescein fluorescence, respectively, whereas aging reduced flow-induced NO and H2O2 production. Endothelium-dependent vasodilation was evaluated by increasing intraluminal flow (5–60 nl/s) before and after treatment with the superoxide dismutase mimetic Tempol (100 μM), the H2O2 scavenger catalase (100 U/ml), or Tempol plus catalase. Catalase reduced flow-induced dilation in both groups, whereas Tempol and Tempol plus catalase diminished vasodilation in young but not old rats. Tempol plus deferoxamine (100 μM), an inhibitor of hydroxyl radical formation, reversed Tempol-mediated impairment of flow-induced vasodilation in young rats and improved flow-induced vasodilation in old rats compared with control. Immunoblot analysis revealed increases in endogenous superoxide dismutase, catalase, and nitrotyrosine protein levels with aging. Collectively, these data indicate that NO- and H2O2-mediated flow-induced signaling decline with age in coronary arterioles and that elevated hydroxyl radical formation contributes to the age-related impairment of flow-induced vasodilation.
Ca2+ entry via α1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium
Authors: Wu Songwei , Jian Ming-Yuan , Xu Yan-Chun , Zhou Chun , Al-Mehdi Abu-Bakr , Liedtke Wolfgang , Shin Hee-Sup , Townsley Mary I.
Published: July 2009

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca2+ influx in response to diverse environmental stimuli. However, it is not clear whether Ca2+ influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the α1G T-type Ca2+ channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca2+, immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K+ perfusate induces Ca2+ entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca2+ entry in septal endothelium evoked by the selective TRPV4 activator 4α-phorbol-12,13-didecanoate (4α-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of α1G abolishes depolarization-induced Ca2+ entry and surface expression of P-selectin but does not prevent 4α-PDD-activated Ca2+ entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4α-PDD-activated Ca2+ entry and the increase in permeability, while not impacting depolarization-induced Ca2+ entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca2+ entry through α1G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.
l-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets
Authors: Ananthakrishnan Madhumita , Barr Frederick E. , Summar Marshall L. , Smith Heidi A. , Kaplowitz Mark , Cunningham Gary , Magarik Jordan , Zhang Yongmei , Fike Candice D.
Published: July 2009

Newborn piglets develop pulmonary hypertension and have diminished pulmonary vascular nitric oxide (NO) production when exposed to chronic hypoxia. NO is produced by endothelial NO synthase (eNOS) in the pulmonary vascular endothelium using l-arginine as a substrate and producing l-citrulline as a byproduct. l-Citrulline is metabolized to l-arginine by two enzymes that are colocated with eNOS in pulmonary vascular endothelial cells. The purpose of this study was to determine whether oral supplementation with l-citrulline during exposure of newborn piglets to 10 days of chronic hypoxia would prevent the development of pulmonary hypertension and increase pulmonary NO production. A total of 17 hypoxic and 17 normoxic control piglets were studied. Six of the 17 hypoxic piglets were supplemented with oral l-citrulline starting on the first day of hypoxia. l-Citrulline supplementation was provided orally twice a day. After 10 days of hypoxia or normoxia, the animals were anesthetized, hemodynamic measurements were performed, and the lungs were perfused in situ. Pulmonary arterial pressure and pulmonary vascular resistance were significantly lower in hypoxic animals treated with l-citrulline compared with untreated hypoxic animals ( P < 0.001). In vivo exhaled NO production ( P = 0.03) and nitrite/nitrate accumulation in the perfusate of isolated lungs ( P = 0.04) were significantly higher in l-citrulline-treated hypoxic animals compared with untreated hypoxic animals. l-Citrulline supplementation ameliorated the development of pulmonary hypertension and increased NO production in piglets exposed to chronic hypoxia. We speculate that l-citrulline may benefit neonates exposed to prolonged periods of hypoxia from cardiac or pulmonary causes.
Trust and Reliance on an Automated Combat Identification System
Authors: Wang Lu , Jamieson Greg A. , Hollands Justin G.
Published: July 2009

Objective: We examined the effects of aid reliability and reliability disclosure on human trust in and reliance on a combat identification (CID) aid. We tested whether trust acts as a mediating factor between belief in and reliance on a CID aid. Background: Individual CID systems have been developed to reduce friendly fire incidents. However, these systems cannot positively identify a target that does not have a working transponder. Therefore, when the feedback is “unknown”, the target could be hostile, neutral, or friendly. Soldiers have difficulty relying on this type of imperfect automation appropriately. Method: In manual and aided conditions, 24 participants completed a simulated CID task. The reliability of the aid varied within participants, half of whom were told the aid reliability level. We used the difference in response bias values across conditions to measure automation reliance. Results: Response bias varied more appropriately with the aid reliability level when it was disclosed than when not. Trust in aid feedback correlated with belief in aid reliability and reliance on aid feedback; however, belief was not correlated with reliance. Conclusion: To engender appropriate reliance on CID systems, users should be made aware of system reliability. Application: The findings can be applied to the design of information displays for individual CID systems and soldier training.
Coordination and Continuity of Intensive Care Unit Patient Care
Authors: Miller Anne , Scheinkestel Carlos , Joseph Michele
Published: July 2009

Objective: Using Klein’s model of team coordination, we explored the null hypothesis that intensive care unit (ICU) care coordination unfolds as a linear sequence. Our intent was to generate hypotheses for further research and to provide interim recommendations for improving care coordination. We also explored factors contributing to care coordination (e.g., role responsibilities, support tools). Background: Although the body of clinical communications research is considerable, few studies address broader team processes in real-world environments; hence, these processes are not well understood. Methods: All bedside communications for 5 ICU patients were recorded for 5 days per patient and were coded using Klein’s model. Markov analysis was used to describe the care coordination process. Multivariate contingency table analysis and standardized parameter estimates described important contributing factors, and support tools were described using descriptive statistics. Results: First-, second-, and third-order Markov analyses show that care coordination does not unfold as a linear sequence; however, Markov diagrams suggest some process structure. Standardized parameter estimates of factors contributing to care coordination were calculated from a statistically significant three-way model (χ 2[ df = 18] = 36.95, p < .005). Role-based differences depend on context, with important differences in contributions to care coordination occurring within rounds. Tools supported only 48% of conversations. Conclusion: Three alternative research hypotheses were defined with at least a minimal level of support. Testing these hypotheses present substantial theoretical, methodological, and data analysis challenges. Application: Within a research framework, recommendations for change could achieve significant gains for understanding and for reducing breakdowns in care coordination.