bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of
Enterococcus
spp. and
Escherichia coli
in Recreational Waters
Authors: Noble Rachel T. , Blackwood A. Denene , Griffith John F. , McGee Charles D. , Weisberg Stephen B.
Published: September 2010

Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for
Enterococcus
spp. and
Escherichia coli
using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (
Enterococcus
spp.) and
uidA
(
E. coli
) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (
r
2
) was 0.69 for
Enterococcus
spp., which was less than that observed between the culture-based methods (
r
2
, 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by
Enterococcus
QPCR and culture-based methods during both studies but were higher for
E. coli
QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of
Enterococcus
QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between
E. coli
QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.
Inactivation of
Escherichia coli
by Nanoparticulate Zerovalent Iron and Ferrous Ion
Authors: Kim Jee Yeon , Park Hee-Jin , Lee Changha , Nelson Kara L. , Sedlak David L. , Yoon Jeyong
Published: September 2010

The mechanism of
Escherichia coli
inactivation by nanoparticulate zerovalent iron (nZVI) and Fe(II) was investigated using reactive oxygen species (ROS) quenchers and probes, an oxidative stress assay, and microscopic observations. Disruption of cell membrane integrity and respiratory activity was observed under deaerated conditions [more disruption by nZVI than Fe(II)], and OH or Fe(IV) appears to play a role.
Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria
Authors: Park Byoung-Joon , Park Soo-Je , Yoon Dae-No , Schouten Stefan , Sinninghe Damsté Jaap S. , Rhee Sung-Keun
Published: September 2010

The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and “
Candidatus
Nitrosopumilus maritimus” (99.1 and 94.9% 16S rRNA and
amoA
gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence
in situ
hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal
amoA
gene was observed during ammonia oxidation. Bacterial
amoA
genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [
13
C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by
Nitrospina
-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.
Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses
Authors: Pan Yao , Bodrossy Levente , Frenzel Peter , Hestnes Anne-Grethe , Krause Sascha , Lüke Claudia , Meima-Franke Marion , Siljanen Henri , Svenning Mette M. , Bodelier Paul L. E.
Published: September 2010

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a “quantifiable” bias.
Ralstonia solanacearum
Dps Contributes to Oxidative Stress Tolerance and to Colonization of and Virulence on Tomato Plants
Authors: Colburn-Clifford Jennifer M. , Scherf Jacob M. , Allen Caitilyn
Published: September 2010

Ralstonia solanacearum
, an economically important soilborne plant pathogen, infects host roots to cause bacterial wilt disease. However, little is known about this pathogen's behavior in the rhizosphere and early in pathogenesis. In response to root exudates from tomato,
R. solanacearum
strain UW551 upregulated a gene resembling Dps, a nonspecific
D
NA binding
p
rotein from
s
tarved cells that is critical for stress survival in other bacteria. An
R. solanacearum dps
mutant had increased hydrogen peroxide sensitivity and mutation rate under starvation. Furthermore,
dps
expression was positively regulated by the oxidative stress response regulator OxyR. These functional results are consistent with a Dps annotation. The
dps
mutant caused slightly delayed bacterial wilt disease in tomato after a naturalistic soil soak inoculation. However, the
dps
mutant had a more pronounced reduction in virulence when bacteria were inoculated directly into host stems, suggesting that Dps helps
R. solanacearum
adapt to conditions inside plants. Passage through a tomato plant conferred transient increased hydrogen peroxide tolerance on both wild-type and
dps
mutant strains, demonstrating that
R. solanacearum
acquires Dps-independent oxidative stress tolerance during adaptation to the host environment. The
dps
mutant strain was also reduced in adhesion to tomato roots and tomato stem colonization. These results indicate that Dps is important when cells are starved or in stationary phase and that Dps contributes quantitatively to host plant colonization and bacterial wilt virulence. They further suggest that
R. solanacearum
must overcome oxidative stress during the bacterial wilt disease cycle.
Endosomal and AP-3-Dependent Vacuolar Trafficking Routes Make Additive Contributions to Candida albicans Hyphal Growth and Pathogenesis
Authors: Palmer Glen E.
Published: September 2010

Candida albicans
mutants deficient in vacuolar biogenesis are defective in polarized hyphal growth and virulence. However, the specific vacuolar trafficking routes required for hyphal growth and virulence are unknown. In
Saccharomyces cerevisiae
, two trafficking routes deliver material from the Golgi apparatus to the vacuole. One occurs via the late endosome and is dependent upon Vps21p, while the second bypasses the endosome and requires the AP-3 complex, including Aps3p. To determine the significance of these pathways in
C. albicans
hyphal growth and virulence,
aps3
Δ/Δ,
vps21
Δ/Δ, and
aps3
Δ/
Δ vps21
Δ/Δ mutant strains were constructed. Analysis of vacuolar morphology and localization of the vacuolar protein Mlt1p suggests that
C. albicans
Aps3p and Vps21p mediate two distinct transport pathways. The
vps21
Δ/Δ mutant has a minor reduction in hyphal elongation, while the
aps3
Δ/Δ mutant has no defect in hyphal growth. Interestingly, the
aps3
Δ/
Δ vps21
Δ/Δ double mutant has dramatically reduced hyphal growth. Overexpression of the Ume6p transcriptional activator resulted in constitutive hyphal growth of wild-type,
aps3
Δ/Δ, and
vps21
Δ/Δ strains and formation of highly vacuolated subapical compartments. Thus, Ume6p-dependent transcriptional responses are sufficient to induce subapical vacuolation. However, the
aps3
Δ/
Δ vps21
Δ/Δ mutant formed mainly pseudohyphae that lacked vacuolated compartments. The
aps3
Δ/Δ strain was virulent in a mouse model of disseminated infection; the
vps21
Δ/Δ mutant failed to kill mice but persisted within kidney tissue, while the double mutant was avirulent and cleared from the kidneys. These results suggest that while the AP-3 pathway alone has little impact on hyphal growth or virulence, it is much more significant when endosomal trafficking is disrupted.
Identification of a Cell Death Pathway in Candida albicans during the Response to Pheromone
Authors: Alby Kevin , Schaefer Dana , Sherwood Racquel Kim , Jones Stephen K. , Bennett Richard J.
Published: September 2010

Mating in hemiascomycete yeasts involves the secretion of pheromones that induce sexual differentiation in cells of the opposite mating type. Studies in
Saccharomyces cerevisiae
have revealed that a subpopulation of cells experiences cell death during exposure to pheromone. In this work, we tested whether the phenomenon of pheromone-induced death (PID) also occurs in the opportunistic pathogen
Candida albicans
. Mating in
C. albicans
is uniquely regulated by white-opaque phenotypic switching; both cell types respond to pheromone, but only opaque cells undergo the morphological transition and cell conjugation. We show that approximately 20% of opaque cells, but not white cells, of laboratory strain SC5314 experience pheromone-induced death. Furthermore, analysis of mutant strains revealed that PID was significantly reduced in strains lacking Fig1 or Fus1 transmembrane proteins that are induced during the mating process and, we now show, are necessary for efficient mating in
C. albicans
. The level of PID was also Ca
2+
dependent, as chelation of Ca
2+
ions increased cell death to almost 50% of the population. However, in contrast to
S. cerevisiae
PID, pheromone-induced killing of
C. albicans
cells was largely independent of signaling via the Ca
2+
-dependent protein phosphatase calcineurin, even when combined with the loss of Cmk1 and Cmk2 proteins. Finally, we demonstrate that levels of PID vary widely between clinical isolates of
C. albicans
, with some strains experiencing close to 70% cell death. We discuss these findings in light of the role of prodeath and prosurvival pathways operating in yeast cells undergoing the morphological response to pheromone.
Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from
Escherichia coli
O157:H4
Authors: Marolda Cristina L. , Li Bo , Lung Michael , Yang Mei , Hanuszkiewicz Anna , Rosales Amanda Roa , Valvano Miguel A.
Published: September 2010

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the
Escherichia coli
O157 Wzx (Wzx
EcO157
) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx
EcO157
consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.
Control of the
Staphylococcus aureus
Toxic Shock
tst
Promoter by the Global Regulator SarA
Authors: Andrey Diego O. , Renzoni Adriana , Monod Antoinette , Lew Daniel P. , Cheung Ambrose L. , Kelley William L.
Published: September 2010

The
Staphylococcus aureus
SarA global regulator controls the expression of numerous virulence genes, often in conjunction with the
agr
quorum-sensing system and its effector RNA, RNAIII. In the present study, we have examined the role of both SarA and RNAIII on the regulation of the promoter of
tst
, encoding staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1).
In vitro
DNA-protein interaction studies with purified SarA using gel shift and DNase I protection assays revealed one strong SarA binding site and evidence for a weaker site nearby within the minimal 400-bp promoter region upstream of
tst. In vivo
analysis of
tst
promoter activation using a

p
tst

-
luxAB
reporter inserted in the chromosome revealed partial but not complete loss of
tst
expression in a Δ
hld
-
RNAIII
strain. In contrast, disruption of
sarA
abrogated
tst
expression. No significant
tst
expression was found for the double Δ
hld-RNAIII-
Δ
sarA
mutant. Introduction of a plasmid containing cloned
hld-RNAIII
driven by a non-
agr
-dependent promoter,

p
HU

, into isogenic parental wild-type or Δ
sarA
strains showed comparable levels of RNAIII detected by quantitative reverse transcription-PCR (qRT-PCR) but a two-log
10
reduction in

p
tst
-luxAB

reporter expression in the Δ
sarA
strain, arguing that RNAIII levels alone are not strictly determinant for
tst
expression. Collectively, our results indicate that SarA binds directly to the
tst
promoter and that SarA plays a significant and direct role in the expression of
tst
.
BB0250 of
Borrelia burgdorferi
Is a Conserved and Essential Inner Membrane Protein Required for Cell Division
Authors: Liang Fang Ting , Xu Qilong , Sikdar Rakesh , Xiao Ying , Cox James S. , Doerrler William T.
Published: September 2010

The gene
bb0250
of
Borrelia burgdorferi
is a homolog of the
dedA
family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any
dedA
gene. Unlike many bacterial species, such as
Escherichia coli
, which has eight
dedA
genes,
B. burgdorferi
possesses only one, annotated
bb0250
, providing a unique opportunity to investigate the functions of the
dedA
family. Here, we show that
bb0250
is able to restore normal growth and cell division to a temperature-sensitive
E. coli
mutant with simultaneous deletions of two
dedA
genes,
yqjA
and
yghB
, and encodes a protein that localizes to the inner membrane of
E. coli
. The
bb0250
gene could be deleted from
B. burgdorferi
only after introduction of a promoterless
bb0250
under the control of an inducible
lac
promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-β-
d
-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that
bb0250
is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the
dedA
gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.
Stability and Instability in the Lysogenic State of Phage Lambda
Authors: Little John W. , Michalowski Christine B.
Published: September 2010

Complex gene regulatory circuits exhibit emergent properties that are difficult to predict from the behavior of the components. One such property is the stability of regulatory states. Here we analyze the stability of the lysogenic state of phage λ. In this state, the virus maintains a stable association with the host, and the lytic functions of the virus are repressed by the viral CI repressor. This state readily switches to the lytic pathway when the host SOS system is induced. A low level of SOS-dependent switching occurs without an overt stimulus. We found that the intrinsic rate of switching to the lytic pathway, measured in a host lacking the SOS response, was almost undetectably low, probably less than 10
−8
/generation. We surmise that this low rate has not been selected directly during evolution but results from optimizing the rate of switching in a wild-type host over the natural range of SOS-inducing conditions. We also analyzed a mutant, λ
prm240
, in which the promoter controlling CI expression was weakened, rendering lysogens unstable. Strikingly, the intrinsic stability of λ
prm240
lysogens depended markedly on the growth conditions; lysogens grown in minimal medium were nearly stable but switched at high rates when grown in rich medium. These effects on stability likely reflect corresponding effects on the strength of the
prm240
promoter, measured in an uncoupled assay system. Several derivatives of λ
prm240
with altered stabilities were characterized. This mutant and its derivatives afford a model system for further analysis of stability.
An Extracellular Loop of the Mannose Phosphotransferase System Component IIC Is Responsible for Specific Targeting by Class IIa Bacteriocins
Authors: Kjos Morten , Salehian Zhian , Nes Ingolf F. , Diep Dzung B.
Published: September 2010

Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive
Listeria monocytogenes
(
mpt
) and the nonsensitive
Lactococcus lactis
(
ptn
) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins.
Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B
Authors: Rowland Susan L. , Wadsworth Kimberly D. , Robson Scott A. , Robichon Carine , Beckwith Jon , King Glenn F.
Published: September 2010

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The
Escherichia coli
divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied
in vivo
without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between
Bacillus subtilis
DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.
The Carboxysome Shell Is Permeable to Protons
Authors: Menon Balaraj B. , Heinhorst Sabine , Shively Jessup M. , Cannon Gordon C.
Published: September 2010

Bacterial microcompartments (BMCs) are polyhedral organelles found in an increasingly wide variety of bacterial species. These structures, typified by carboxysomes of cyanobacteria and many chemoautotrophs, function to compartmentalize important reaction sequences of metabolic pathways. Unlike their eukaryotic counterparts, which are surrounded by lipid bilayer membranes, these microbial organelles are bounded by a thin protein shell that is assembled from multiple copies of a few different polypeptides. The main shell proteins form hexamers whose edges interact to create the thin sheets that form the facets of the polyhedral BMCs. Each hexamer contains a central pore hypothesized to mediate flux of metabolites into and out of the organelle. Because several distinctly different metabolic processes are found in the various BMCs studied to date, it has been proposed that a common advantage to packaging these pathways within shell-bound compartments is to optimize the concentration of volatile metabolites in the BMC by maintaining an interior pH that is lower than that of the cytoplasm. We have tested this idea by recombinantly fusing a pH-sensitive green fluorescent protein (GFP) to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the major enzyme component inside the carboxysome. Our results suggest that the carboxysomal pH is similar to that of its external environment and that the protein shell does not constitute a proton barrier. The explanation for the sundry BMC functions must therefore be sought in the characteristics of the pores that traverse their shells.
Resequencing the
Mycobacterium avium
subsp.
paratuberculosis
K10 Genome: Improved Annotation and Revised Genome Sequence
Authors: Wynne James W. , Seemann Torsten , Bulach Dieter M. , Coutts Scott A. , Talaat Adel M. , Michalski Wojtek P.
Published: September 2010

We report the resequencing and revised annotation of the
Mycobacterium avium
subsp.
paratuberculosis
K10 genome. A total of 90 single-nucleotide errors and a 51-bp indel in the original K10 genome were corrected, and the whole genome annotation was revised. Correction of these sequencing errors resulted in 28 frameshift alterations. The amended genome sequence is accessible via the supplemental section of study SRR060191 in the NCBI Sequence Read Archive and will serve as a valuable reference genome for future studies.
High-Redundancy Draft Sequencing of 15 Clinical and EnvironmentalBurkholderiaStrains
Authors: Mukhopadhyay Sanghamitra , Thomason Maureen K. , Lentz Shannon , Nolan Nichole , Willner Kristin , Gee Jay E. , Glass Mindy B. , Inglis Timothy J. J. , Merritt Adam , Levy Avram , Sozhamannan Shanmuga , Mateczun Al , Read Timothy D.
Published: September 2010

The Gram-negativeBurkholderiagenus includes several species of intracellular bacterial pathogens that pose substantial risk to humans. In this study, we have generated draft genome sequences of 15 strains ofB. oklahomensis,B. pseudomallei,B. thailandensis, andB. ubonensisto an average sequence read coverage of 25- to 40-fold.
Molecular Evolution of the
Helicobacter pylori
Vacuolating Toxin Gene
vacA
Authors: Gangwer Kelly A. , Shaffer Carrie L. , Suerbaum Sebastian , Lacy D. Borden , Cover Timothy L. , Bordenstein Seth R.
Published: September 2010

Helicobacter pylori
is a genetically diverse organism that is adapted for colonization of the human stomach. All strains contain a gene encoding a secreted, pore-forming toxin known as VacA. Genetic variation at this locus could be under strong selection as
H. pylori
adapts to the host immune response, colonizes new human hosts, or inhabits different host environments. Here, we analyze the molecular evolution of VacA. Phylogenetic reconstructions indicate the subdivision of VacA sequences into three main groups with distinct geographic distributions. Divergence of the three groups is principally due to positively selected sequence changes in the p55 domain, a central region required for binding of the toxin to host cells. Divergent amino acids map to surface-exposed sites in the p55 crystal structure. Comparative phylogenetic analyses of
vacA
sequences and housekeeping gene sequences indicate that
vacA
does not share the same evolutionary history as the core genome. Further, rooting the VacA tree with outgroup sequences from the close relative
Helicobacter acinonychis
reveals that the ancestry of VacA is different from the African origin that typifies the core genome. Finally, sequence analyses of the virulence determinant CagA reveal three main groups strikingly similar to the three groups of VacA sequences. Taken together, these results indicate that positive selection has shaped the phylogenetic structure of VacA and CagA, and each of these virulence determinants has evolved separately from the core genome.
Correlation between quantitative serum HBsAg and HBV DNA test in Korean patients who showed high level of HBsAg
Authors: Lee Jong-Han , Kim Sue Jung , Ahn Sang Hoon , Lee Jinhwa , Park Yongjung , Kim Hyon-Suk
Published: September 2010

Aims

The authors aimed to identify the correlation between quantitative hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA in Korean patients with a high level of HBsAg (>250 IU/ml) and characteristics of patients for whom quantitative HBsAg can be more suitably used to monitor HBV infection.

Methods

Quantitative HBsAg and HBV DNA were measured in collected sera from 565 patients with a high level of HBsAg (>250 IU/ml). The correlation between HBsAg and HBV DNA was analysed by Spearman rank test. We also retrospectively analysed the correlation according to the HBV infection phase in which quantitative HBsAg is more appropriate to be used in clinical practice.

Results

The overall correlation between quantitative HBsAg and HBV DNA was significant but very weak (Spearman ρ=0.121, p=0.004). Weak correlations were also noted in chronic hepatitis B patients (ρ=0.123, p=0.019) and in patients with hepatocellular carcinoma (ρ=0.328, p=0.002). No correlation was noted in liver cirrhosis patients (ρ=0.156, p=0.095). Relatively higher correlation was noted in hepatitis B e antigen positive patients without antiviral treatment and patients with a high HBV DNA to HBsAg ratio (>57.93).

Conclusions

There was a weak correlation between quantitative HBsAg and HBV DNA. However, quantitative HBsAg appears to be more useful to reflect HBV DNA in the early replicative phase of chronic hepatitis B patients than those in the late non-replicative phase.
Radiological diagnosis and follow-up of pulmonary tuberculosis
Authors: Roy Molly , Ellis Stephen
Published: September 2010

Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), is one of the oldest and still one of the deadliest pathogens known to man. Approximately 1 in 10 people with primary pulmonary tuberculosis (PTB) present clinically; of untreated cases, approximately 1 in 10 reactivate usually at a time of relative immunodeficiency. The spectrum of radiologic manifestations of PTB can pose a variety of diagnostic and management challenges. PTB infection often leaves long term sequelae of infection, particularly granulomatous nodules, cavitation, and fibrosis; distinguishing dormant disease from reactivation is not always clear-cut. The radiologic presentation of primary PTB infection tends to differ from that of post-primary PTB, but there is significant overlap in the appearances. Primary PTB typically presents with consolidation and regional lymphadenopathy, whereas post-primary PTB more often results in cavitation. The pathology and therefore the radiology of TB infection will be altered based on the efficacy of the immune response and will therefore vary depending on the immune competency. Clinically, in the presence of infection, the main questions are whether M tuberculosis is the infecting organism and, if treated, does the radiology indicate response to treatment. In order to interpret the radiology of TB one needs to be aware of the spectrum of presentation, the expected reaction to treatment, and the myriad of non-pulmonary sites of infection that may prove to be more clinically significant than the pulmonary infection.
Self-reported price of cigarettes, consumption and compensatory behaviours in a cohort of Mexican smokers before and after a cigarette tax increase
Authors: Saenz-de-Miera Belen , Thrasher James F , Chaloupka Frank J , Waters Hugh R , Hernandez-Avila Mauricio , Fong Geoffrey T
Published: September 2010

Objective

To assess the impact of a 2007 cigarette tax increase from 110% to 140% of the price to the retailer on cigarette price and consumption among Mexican smokers, including efforts to offset price increases.

Methods

Data were analysed from the 2006 and 2007 administrations of the International Tobacco Control (ITC) Policy Evaluation Survey in Mexico, which is a population-based cohort of adult smokers. Self-reported price of last cigarette purchase, place of last purchase, preferred brand, daily consumption and quit behaviour were assessed at baseline and follow-up.

Results

Self-reported cigarette prices increased by 12.7% after the tax increase, with prices for international brands increasing more than for national brands (13.5% vs 8.7%, respectively). Although the tax increases were not fully passed onto consumers particularly on national brands, no evidence was found for smokers changing behaviour to offset price increases. Consistent declines in consumption across groups defined by sociodemographic and smoking-related psychosocial variables suggest a relatively uniform impact of the tax increase across subpopulations. However, decreased consumption appeared limited to people who smoked relatively more cigarettes a day (>5 cigarettes/day). Average daily consumption among lighter smokers did not significantly decline. A total of 13% (n=98) of the sample reported being quit for a month or more at follow-up. In multivariate models, lighter smokers were more likely than heavier smokers to be quit.

Conclusions

Results suggest that the 2007 tax increase was passed on to consumers, whose consumption generally declined. Since no other tobacco control policies or programmes were implemented during the period analysed, the tax increase appears likely to have decreased consumption.
Using policy to increase prescribing of smoking cessation medications in the VA healthcare system
Authors: Smith Mark W , Chen Shuo , Siroka Andrew M , Hamlett-Berry Kim
Published: September 2010

Background

Since 2002 the US Veterans Affairs (VA) healthcare system has initiated national policies and programmes to reduce smoking among its patients and to increase evidence-based treatment for smoking.

Objective

To document changes in dispensing rates of cessation-related medications in VA from 2004 to 2008.

Design

Retrospective analysis of VA administrative data.

Results

Prescription fills for nicotine replacement therapy (NRT), and for bupropion among NRT users, each grew more than 60% in four years. The increase stemmed primarily from treating more people rather than from filling more prescriptions per person.

Conclusion

The results provide strong support for the efficacy of these policies and illustrate how healthcare systems can successfully employ multiple strategies to increase evidence-based smoking-cessation treatment.
Economic effects of Ohio's smoke-free law on Kentucky and Ohio border counties: Table 1
Authors: Pyles Mark K , Hahn Ellen J
Published: September 2010

Objective

To determine if the Ohio statewide smoke-free law is associated with economic activity in Ohio or Kentucky counties that lie on the border between the two states. In November 2006, Ohio implemented a comprehensive statewide smoke-free law for all indoor workplaces.

Design

A feasible generalised least squares (FLGS) time series design to estimate the impact of the Ohio smoke-free law on Kentucky and Ohio border counties.

Setting

Six Kentucky and six Ohio counties that lie on the border between the two states.

Subjects

All reporting hospitality and accommodation establishments in all Kentucky and Ohio counties including but not limited to food and drinking establishments, hotels and casinos.

Main outcome measures

Total number of employees, total wages paid and number of reported establishments in all hospitality and accommodation services, 6 years before Ohio's law and 1 year after.

Results

There is no evidence of a disproportionate change in economic activity in Ohio or Kentucky border counties relative to their non-border counterparts. There was no evidence of a relation between Ohio's smoke-free law and economic activity in Kentucky border counties. The law generated a positive influence on wages and number of establishments in Ohio border counties. The null result cannot be explained by low test power, as minimum changes necessary in the dependent variables to detect a significant influence are very reasonable in size.

Conclusions

Our data add to the large body of evidence that smoke-free laws are neutral with respect to the hospitality business across jurisdictions with and without laws.
PERISTALTIC FLOW CHARACTERSTICS OF MAXWELL AND MAGNETOHYDRODYNAMIC FLUIDS IN FINITE CHANNELS: MODELS FOR OESOPHAGEAL SWALLOWING
Authors: PANDEY S. K. , TRIPATHI DHARMENDRA
Published: September 2010

This is an attempt to investigate swallowing of a food bolus through oesophagus by modelling it as an unsteady peristaltic transport of Maxwell and magnetohydrodynamic (MHD) fluids in channels of finite length. The walls of the channels are subjected to progressive transverse contraction waves so that the natural oesophageal wall contractions are matched. The analysis is carried out in non-dimensional form by using long wavelength approximations. The expressions for axial and transverse velocities are derived and pressure across a wavelength is estimated. The reflux limit is determined for both the fluids. Mechanical efficiency for MHD fluids is also obtained. Physical interpretations reveal the behaviour of the flows of masticated viscoelastic food materials such as bread, white eggs etc. and saline water that is represented by MHD model in the oesophagus. It is found that fluids represented by Maxwell fluid are more swallow-friendly than Newtonian fluids while normal water is easier to swallow than saline water which is an MHD fluid. It is also revealed that relaxation time, a parameter of Maxwell model, has no effects on local wall shear stress and reflux limit whereas magneto-hydrodynamic parameters make the fluid more prone to flow reversal. It is found that if the transverse magnetic field and the electric conductivity increase, the pumping machinery requires more pressure for pushing the fluid forward. In other words, pumping has to work more efficiently. Finally, it is revealed that the peaks of pressure are identical, in case, an integral number of waves propagate along the channel while the peaks are of unequal size in the non-integral case.
Mechanosensitivity of STREX-lacking BKCa channels in the colonic smooth muscle of the mouse
Authors: Wang Wei , Huang Haixia , Hou Dongyan , Liu Ping , Wei Hua , Fu Xiaosuo , Niu Weizhen
Published: September 2010

Stretch sensitivity of Ca2+-activated large-conductance K+ channels (BKCa) has been observed in a variety of cell types and considered to be a potential mechanism in mechanoelectric transduction (MET). Mechanical stress is a major stimulator for the smooth muscle in the gastrointestinal (GI) tract. However, much about the role and mechanism of MET in GI smooth muscles remains unknown. The BKCa shows a functional diversity due to intensive Slo I alternative splicing and different α/β-subunit assembly in various cells. The stress-regulated exon (STREX) insert is suggested to be an indispensable domain for the mechanosensitivity of BKCa. The purpose of this study was to determine whether the BKCa in colonic myocytes of the adult mouse is sensitive to mechanical stimulation and whether the STREX insert is a crucial segment for the BKCa mechanosensitivity. The α- and β1-subunit mRNAs and the α-subunit protein of the BKCa channels were detected in the colonic muscularis. We found that the BKCa STREX-lacking variant was abundantly expressed in the smooth muscle, whereas the STREX variant was not detectable. We demonstrated that the STREX-lacking BKCa channels were also sensitive to membrane stretch. We suggest that in addition to the STREX domain, there are other additional structures in the channel responsible for mechanically coupling with the cell membrane.
Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survival
Authors: Li Hongxia , Zuo Shi , He Zhisong , Yang Yueting , Pasha Zeeshan , Wang Yigang , Xu Meifeng
Published: September 2010

Transplanted mesenchymal stem cells (MSC) release soluble factors that contribute to cardiac repair and vascular regeneration. We hypothesized that overexpression of GATA-4 enhances the MSC secretome, thereby increasing cell survival and promoting postinfarction cardiac angiogenesis. MSCs harvested from male rat bone marrow were transduced with GATA-4 (MSCGATA-4) using the murine stem cell virus retroviral expression system; control cells were either nontransduced (MSCbas) or transduced with empty vector (MSCNull). Compared with these control cells, MSCGATA-4 were shown by immunofluorescence, real-time PCR, and Western blotting to have higher expression of GATA-4. An increased expression of angiogenic factors in MSCGATA-4 and higher MSC resistance against hypoxia were observed. Human umbilical vein endothelial cells (HUVEC) treated with MSCGATA-4 conditioned medium exhibited increased formation of capillary-like structures and promoted migration, compared with HUVECs treated with MSCNull conditioned medium. MSCGATA-4 were injected into the peri-infarct region in an acute myocardial infarction model in Sprague-Dawley rats developed by ligation of the left anterior descending coronary artery. Survival of MSCGATA-4, determined by Sry expression, was increased at 4 days postengraftment. MSCGATA-4-treated animals showed significantly improved cardiac function as assessed by echocardiography. Furthermore, fluorescent microsphere and histological studies revealed increased blood flow and blood vessel density and reduced infarction size in MSCGATA-4-treated animals. We conclude that GATA-4 overexpression in MSCs increased both MSC survival and angiogenic potential in ischemic myocardium and may therefore represent a novel and efficient therapeutic approach for postinfarct remodeling.
Effects of aging on muscle mechanical function and muscle fiber morphology during short-term immobilization and subsequent retraining
Authors: Hvid Lars , Aagaard Per , Justesen Lene , Bayer Monika L. , Andersen Jesper L. , Ørtenblad Niels , Kjaer Michael , Suetta Charlotte
Published: September 2010

Very little attention has been given to the combined effects of aging and disuse as separate factors causing deterioration in muscle mechanical function. Thus the purpose of this study was to investigate the effects of 2 wk of immobilization followed by 4 wk of retraining on knee extensor muscle mechanical function (e.g., maximal strength and rapid force capacity) and muscle fiber morphology in 9 old (OM: 67.3 ± 1.3 yr) and 11 young healthy men (YM: 24.4 ± 0.5 yr) with comparable levels of physical activity. Following immobilization, OM demonstrated markedly larger decreases in rapid force capacity (i.e., rate of force development, impulse) than YM (∼20–37 vs. ∼13–16%; P < 0.05). In contrast, muscle fiber area decreased in YM for type I, IIA, and IIx fibers (∼15–30%; P < 0.05), whereas only type IIa area decreased in OM (13.2%; P < 0.05). Subsequent retraining fully restored muscle mechanical function and muscle fiber area in YM, whereas OM showed an attenuated recovery in muscle fiber area and rapid force capacity (tendency). Changes in maximal isometric and dynamic muscle strength were similar between OM and YM. In conclusion, the present data reveal that OM may be more susceptible to the deleterious effects of short-term muscle disuse on muscle fiber size and rapid force capacity than YM. Furthermore, OM seems to require longer time to recover and regain rapid muscle force capacity, which may lead to a larger risk of falling in aged individuals after periods of short-term disuse.
WNT11 Expression Is Induced by Estrogen-Related Receptor α and β-Catenin and Acts in an Autocrine Manner to Increase Cancer Cell Migration
Authors: Dwyer Mary A. , Joseph James D. , Wade Hilary E. , Eaton Matthew L. , Kunder Rebecca S. , Kazmin Dmitri , Chang Ching-yi , McDonnell Donald P.
Published: September 2010

Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, β-catenin (β-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by β-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or β-cat. Furthermore, silencing of ERRα and β-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/β-cat–dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/β-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and β-cat that influences the migratory capacity of cancer cells. Cancer Res; 70(22); 9298–308. ©2010 AACR.
Multiple Roles of Protein Kinase A in Arachidonic Acid–Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration
Authors: Fiorio Pla Alessandra , Genova Tullio , Pupo Emanuela , Tomatis Cristiana , Genazzani Armando , Zaninetti Roberta , Munaron Luca
Published: September 2010

We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca2+ entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca2+ signaling in the same cells. B-TEC stimulation with 5 μmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser1177, and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI14-22 completely prevented both AA- and NO-induced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor NG-nitro-l-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs. Mol Cancer Res; 8(11); 1466–76. ©2010 AACR.