bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotypes and Virulence Profiles of Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Bovine Farms
Authors: Monaghan Áine , Byrne Brian , Fanning Séamus , Sweeney Torres , McDowell David , Bolton Declan J.
Published: October 2011

Non-O157 Shiga toxin-producing
Escherichia coli
(STEC) strains are clinically significant food-borne pathogens. However, there is a dearth of information on serotype prevalence and virulence gene distribution, data essential for the development of public health protection monitoring and control activities for the meat and dairy industries. Thus, the objective of this study was to examine the prevalence of non-O157 STEC on beef and dairy farms and to characterize the isolates in terms of serotype and virulence markers. Bovine fecal samples (
n
= 1,200) and farm soil samples (
n
= 600) were collected from 20 farms throughout Ireland over a 12-month period. Shiga toxin-positive samples were cultured and colonies examined for the presence of
stx
1
and/or
stx
2
genes by PCR. Positive isolates were serotyped and examined for a range of virulence factors, including
eaeA
,
hlyA
,
tir
,
espA
,
espB
,
katP
,
espP
,
etpD
,
saa
,
sab
,
toxB
,
iha
,
lpfA
O157/OI-141
,
lpfA
O113
, and
lpfA
O157/OI-154
. Shiga toxin and intimin genes were further examined for known variants. Significant numbers of fecal (40%) and soil (27%) samples were
stx
positive, with a surge observed in late summer-early autumn. One hundred seven STEC isolates were recovered, representing 17 serotypes. O26:H11 and O145:H28 were the most clinically significant, with O113:H4 being the most frequently isolated. However, O2:H27, O13/O15:H2, and ONT:H27 also carried
stx
1
and/or
stx
2
and
eaeA
and may be emerging pathogens.
Enhanced Growth of Acidovorax sp. Strain 2AN during Nitrate-Dependent Fe(II) Oxidation in Batch and Continuous-Flow Systems
Authors: Chakraborty Anirban , Roden Eric E. , Schieber Jürgen , Picardal Flynn
Published: October 2011

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations.
Acidovorax
sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO
3
−
, 8 to 9 mM Fe
2+
, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO
3
−
, 20 μM acetate, and 50 to 250 μM Fe
2+
, showed that the growth yield of
Acidovorax
sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.
Nitrate-Dependent N
2
O Emission from Intact Soybean Nodules via Denitrification by Bradyrhizobium japonicum Bacteroids
Authors: Hirayama Junta , Eda Shima , Mitsui Hisayuki , Minamisawa Kiwamu
Published: October 2011

In the presence of nitrate, N
2
O emission increased markedly from soybean roots inoculated with
nosZ
mutant of
Bradyrhizobium japonicum
, but not from soybean roots inoculated with a
napA nosZ
double mutant, indicating that
B. japonicum
bacteroids in soybean nodules are able to convert the exogenously supplied nitrate into N
2
O via a denitrification pathway.
Global Genome Transcription Profiling of Bifidobacterium bifidum PRL2010 under
In Vitro
Conditions and Identification of Reference Genes for Quantitative Real-Time PCR
Authors: Turroni Francesca , Foroni Elena , Montanini Barbara , Viappiani Alice , Strati Francesco , Duranti Sabrina , Ferrarini Alberto , Delledonne Massimo , van Sinderen Douwe , Ventura Marco
Published: October 2011

Bifidobacteria have attracted significant scientific attention due to their perceived role as health-promoting microorganisms, although the genetics of the bacterial group is still underexplored. In this study, we investigated the transcriptome of
Bifidobacterium bifidum
PRL2010 during
in vitro
growth by microarray technology. When
B. bifidum
PRL2010 was grown in liquid broth, 425 of the 1,644 PRL2010 genes represented on the array were expressed in at least one of the three investigated growth phases, i.e., the lag, exponential, and stationary phases. These transcriptional analyses identified a core
in vitro
transcriptome encompassing 150 genes that are expressed in all phases. A proportion of these genes were further investigated as potential reference genes by quantitative real-time reverse transcription-PCR (qRT-PCR) assays. Their expression stability was evaluated under different growth conditions, which included cultivation on different carbon sources, exposure to environmental stresses (thermal, acidic, and osmotic), and growth phases. Our analyses validated six reference genes suitable for normalizing mRNA expression levels in qRT-PCR experiments applied to bifidobacteria.
Connection between Multimetal(loid) Methylation in Methanoarchaea and Central Intermediates of Methanogenesis
Authors: Thomas Frank , Diaz-Bone Roland A. , Wuerfel Oliver , Huber Britta , Weidenbach Katrin , Schmitz Ruth A. , Hensel Reinhard
Published: October 2011

In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by
Methanosarcina mazei in vivo
are also methylated by
in vitro
assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH
3
Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis. Detailed studies revealed that cob(I)alamin [Cob(I)], formed by MtaA-catalyzed demethylation of CH
3
Cob(III), is the causative agent for the multimetal(loid) methylation observed. Moreover, Cob(I) is also capable of metal(loid) hydride generation. Global transcriptome profiling of
M. mazei
cultures exposed to bismuth did not reveal induced methyltransferase systems but upregulated regeneration of methanogenic cofactors in the presence of bismuth. Thus, we conclude that the multimetal(loid) methylation
in vivo
is attributed to side reactions of CH
3
Cob(III) with reduced cofactors formed in methanogenesis. The close connection between metal(loid) methylation and methanogenesis explains the general capability of methanoarchaea to methylate metal(loid)s.
Lactate Oxidation Coupled to Iron or Electrode Reduction by Geobacter sulfurreducens PCA
Authors: Call Douglas F. , Logan Bruce E.
Published: October 2011

Geobacter sulfurreducens
PCA completely oxidized lactate and reduced iron or an electrode, producing pyruvate and acetate intermediates. Compared to the current produced by
Shewanella oneidensis
MR-1,
G. sulfurreducens
PCA produced 10-times-higher current levels in lactate-fed microbial electrolysis cells. The kinetic and comparative analyses reported here suggest a prominent role of
G. sulfurreducens
strains in metal- and electrode-reducing communities supplied with lactate.
Genetic Background and Mobility of Variants of the Gene
nleA
in Attaching and Effacing Escherichia coli
Authors: Creuzburg Kristina , Heeren Sabine , Lis Claudia M. , Kranz Markus , Hensel Michael , Schmidt Herbert
Published: October 2011

In this study, we characterized the genetic background of various
nleA
variants in 106 Shiga toxin-producing
Escherichia coli
(STEC) and enteropathogenic
Escherichia coli
(EPEC) strains. The flanking regions of eight
nleA
variants were analyzed by DNA sequencing and compared with the corresponding regions of five previously described NleA-encoding prophages. The analyzed
nleA
variants were all located downstream of the DNA region responsible for phage morphogenesis. In particular, the type III effector genes
avrA
,
ospB
,
nleH
, and
nleG
and IS elements were detected in the neighborhood of
nleA
. The structure of the eight analyzed regions flanking
nleA
primarily resembled the corresponding region of the NleA
4795
-encoding prophage BP-4795. Using PCR, the gene order flanking 13
nleA
variants in strains of different serogroups was compared to the respective regions in reference strains. The analyses showed that strains which harbor prophages with conserved flanking regions of a particular
nleA
variant predominantly occurred, and IS elements were additionally detected in these regions. We were able to mobilize
nleA
by transduction in 20% of strains determined, which comprised in particular EPEC strains harboring an
nleA
variant, the gene encoding the protein known as “EspI-like.” Plaque hybridization was used to identify phages that harbor the genes
stx
and
nleA
. However, only two strains harbored variant
nleA
4795
in the genome of an Stx1 prophage.
A Whole-Genome Single Nucleotide Polymorphism-Based Approach To Trace and Identify Outbreaks Linked to a Common Salmonella enterica subsp. enterica Serovar Montevideo Pulsed-Field Gel Electrophoresis Type
Authors: den Bakker Henk C. , Moreno Switt Andrea I. , Cummings Craig A. , Hoelzer Karin , Degoricija Lovorka , Rodriguez-Rivera Lorraine D. , Wright Emily M. , Fang Rixun , Davis Margaret , Root Tim , Schoonmaker-Bopp Dianna , Musser Kimberlee A. , Villamil Elizabeth , Waechter HaeNa , Kornstein Laura , Furtado Manohar R. , Wiedmann Martin
Published: October 2011

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of
Salmonella enterica
subsp.
enterica
serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.
Novel Shuttle Markers for Nuclear Transformation of the Green Alga Chlamydomonas reinhardtii
Authors: Meslet-Cladière Laurence , Vallon Olivier
Published: October 2011

The green alga
Chlamydomonas reinhardtii
today is a premier model organism for the study of green algae and plants. Yet the efficient engineering of its nuclear genome requires development of new antibiotic resistance markers. We have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used previously for chloroplast transformation. The recoded AadA gene, placed under the control of the
HSP70A-RBCS2
hybrid promoter and preceded by the RbcS2 chloroplast-targeting peptide, can be integrated into the nuclear genome by electroporation, conferring resistance to spectinomycin and streptomycin. Transformation efficiency is markedly increased when vector sequences are completely eliminated from the transforming DNA. Antibiotic resistance is stable for several months in the absence of selection pressure. Shuttle markers allowing selection in both
Chlamydomonas
and
Escherichia coli
would also be a useful asset. By placing an artificial bacterial promoter and Shine-Dalgarno sequence in frame within the AadA coding sequence, we generated such a shuttle marker. To our surprise, we found that the classical AphVIII construct already functions as a shuttle marker. Finally, we developed a method to introduce the AadA and AphVIII markers into the vector part of the bacterial artificial chromosomes (BACs) of the
Chlamydomonas
genomic DNA library. Our aim was to facilitate complementation studies whenever the test gene cannot be selected for directly. After transformation of a
petC
mutant with a modified BAC carrying the AphVIII marker along with the
PETC
gene in the insert, almost half of the paromomycin-resistant transformants obtained showed restoration of phototrophy, indicating successful integration of the unselected test gene. With AadA, cotransformation was also observed, but with a lower efficiency.
The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling Circuit during High-Temperature Stress Responses in Cryptococcus neoformans
Authors: Aboobakar Eanas F. , Wang Xuying , Heitman Joseph , Kozubowski Lukasz
Published: October 2011

Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogenCryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation inC. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. Thects1Δ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506.In vitrotreatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.
Reg1 Protein Regulates Phosphorylation of All Three Snf1 Isoforms but Preferentially Associates with the Gal83 Isoform
Authors: Zhang Yuxun , McCartney Rhonda R. , Chandrashekarappa Dakshayini G. , Mangat Simmanjeet , Schmidt Martin C.
Published: October 2011

The phosphorylation status of the Snf1 activation loop threonine is determined by changes in the rate of its dephosphorylation, catalyzed by the yeast PP1 phosphatase Glc7 in complex with the Reg1 protein. Previous studies have shown that Reg1 can associate with both Snf1 and Glc7, suggesting substrate binding as a mechanism for Reg1-mediated targeting of Glc7. In this study, the association of Reg1 with the three Snf1 isoforms was measured by two-hybrid analysis and coimmunoprecipitation. We found that Reg1 association with Snf1 occurred almost exclusively with the Gal83 isoform of the Snf1 complex. Nonetheless, Reg1 plays an important role in determining the phosphorylation status of all three Snf1 isoforms. We found that the rate of dephosphorylation for isoforms of Snf1 did not correlate with the amount of associated Reg1 protein. Functional chimeric β subunits containing residues from Gal83 and Sip2 were used to map the residues needed to promote Reg1 association with the N-terminal 150 residues of Gal83. The Gal83 isoform of Snf1 is the only isoform capable of nuclear localization. A Gal83-Sip2 chimera containing the first 150 residues of Gal83 was able to associate with the Reg1 protein but did not localize to the nucleus. Therefore, nuclear localization is not required for Reg1 association. Taken together, these data indicate that the ability of Reg1 to promote the dephosphorylation of Snf1 is not directly related to the strength of its association with the Snf1 complex.
Disulfide Bonding within Components of the Chlamydia Type III Secretion Apparatus Correlates with Development
Authors: Betts-Hampikian H. J. , Fields K. A.
Published: October 2011

Chlamydia
spp. exhibit a unique biphasic developmental cycle whereby infectious elementary bodies (EBs) invade host epithelial cells and differentiate into noninfectious, metabolically active reticulate bodies (RBs). EBs posses a unique outer envelope where rigidity is achieved by disulfide bonding among cysteine-rich envelope-associated proteins. Conversely, these disulfide bonds become reduced in RBs to accommodate vegetative growth, thereby linking the redox status of cysteine-rich envelope proteins with progression of the developmental cycle. We investigated the potential role of disulfide bonding within the chlamydial type III secretion system (T3SS), since activity of this system is also closely linked to development. We focused on structural components of the T3S apparatus that contain an unusually high number of cysteine residues compared to orthologs in other secretion systems. Nonreducing SDS-PAGE revealed that EB-localized apparatus proteins such as CdsF, CdsD, and CdsC form higher-order complexes mediated by disulfide bonding. The most dramatic alterations were detected for the needle protein CdsF. Significantly, disulfide bonding patterns shifted during differentiation of developmental forms and were completely reduced in RBs. Furthermore, at later time points during infection following RB to EB conversion, we found that CdsF is reoxidized into higher-order complexes. Overall, we conclude that the redox status of specific T3SS apparatus proteins is intimately linked to the developmental cycle and constitutes a newly appreciated aspect of functionally significant alterations within proteins of the chlamydial envelope.
Translocators YopB and YopD from Yersinia enterocolitica Form a Multimeric Integral Membrane Complex in Eukaryotic Cell Membranes
Authors: Montagner Caroline , Arquint Christian , Cornelis Guy R.
Published: October 2011

The type III secretion systems are contact-activated secretion systems that allow bacteria to inject effector proteins across eukaryotic cell membranes. The secretion apparatus, called injectisome or needle complex, includes a needle that terminates with a tip structure. The injectisome exports its own distal components, like the needle subunit and the needle tip. Upon contact, it exports two hydrophobic proteins called translocators (YopB and YopD in
Yersinia enterocolitica
) and the effectors. The translocators, assisted by the needle tip, form a pore in the target cell membrane, but the structure of this pore remains elusive. Here, we purified the membranes from infected sheep erythrocytes, and we show that they contain integrated and not simply adherent YopB and YopD. In blue native PAGE, these proteins appeared as a multimeric 500- to 700-kDa complex. This heteropolymeric YopBD complex could be copurified after solubilization in 0.5% dodecyl maltoside but not visualized in the electron microscope. We speculate that this complex may not be stable and rigid but only transient.
The Extended Signal Peptide of the Trimeric Autotransporter EmaA of Aggregatibacter actinomycetemcomitans Modulates Secretion
Authors: Jiang X. , Ruiz T. , Mintz K. P.
Published: October 2011

The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium
Aggregatibacter actinomycetemcomitans
is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in
Escherichia coli
and
A. actinomycetemcomitans
using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in
E. coli
.
A. actinomycetemcomitans
strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.
Global Transcription Profiles of the Nitrogen Stress Response Resulting in Heterocyst or Hormogonium Development in Nostoc punctiforme
Authors: Christman Harry D. , Campbell Elsie L. , Meeks John C.
Published: October 2011

The filamentous cyanobacterium
Nostoc punctiforme
differentiates from vegetative cells into three distinct cell types, heterocysts, hormogonia, and akinetes, in response to different stimuli. Cultures growing with ammonium can be induced to form hormogonia or heterocysts upon removal of the combined nitrogen. A DNA microarray consisting of 94% of the open reading frames predicted from the 9.059-Mb
N. punctiforme
genome was used to generate a global transcription data set consisting of seven time points over a 24-h period of nitrogen deprivation, which results in heterocyst formation. This data set was compared to a similarly generated data set of nitrogen-starved
N. punctiforme
resulting in hormogonium formation that had previously been published (E. L. Campbell, H. Christman, and J. C. Meeks, J. Bacteriol. 190:7382-7391, 2008). The transition from vegetative cells to either heterocysts or hormogonia resulted in rapid and sustained expression of genes required for utilization of alternate nitrogen sources. Overall, 1,036 and 1,762 genes were found to be differentially transcribed during the heterocyst and hormogonium time courses, respectively, as analyzed with the Bayesian user-friendly software for analyzing time series microarray experiments (BATS). Successive transcription of heterocyst regulatory, structural, and functional genes occurred over the 24 h required to form a functional heterocyst. During hormogonium differentiation, some heterocyst structural and functional genes were upregulated, while the heterocyst master regulator
hetR
was downregulated. There are commonalities in differential expression between cells bound for differentiation into heterocysts or hormogonia, yet the two paths are distinguished by their developmentally specific transcription profiles.
Selective hydration of ferrocenylethyne mediated by a palladium complex with a camphorhydrazone ligand
Authors: Tauchman Jiří , Carvalho M. Fernanda N. N. , Štěpnička Petr
Published: October 2011

Complex [PdCl2L2], where L is a camphor hydrazone ligand, (1R,4S)-1,7,7-trimethyl-3-(2,2-dimethylhydrazone)-bicyclo[2.2.1]heptane-2,3-dione, efficiently promotes Markovnikov hydration of ethynylferrocene to acetylferrocene in aqueous methanol at room temperature. 1-Ferrocenylprop-1-yne and simple organic alkynes such as 1-octyne or ethynylbenzenes are not affected or polymerize under the reaction conditions.
Neuroanatomical Characteristics of Acupuncture Points: Relationship between Their Anatomical Locations and Traditional Clinical Indications
Authors: Cheng Kwokming James
Published: October 2011

This study examines the relationship between the anatomical location of traditional acupuncture points and their clinical indications as stated in two textbooks of traditional Chinese medicine (TCM). The following relationships are noted: (1) The acupuncture points in the trunk and their stated effects on the internal organs in the trunk have a segmental relationship—that is, acupuncture points within certain spinal segments in the trunk affect the functioning of the organs that receive autonomic innervation from the same spinal segments. This is consistent with the concept of segmental acupuncture and the idea that acupuncture may act via the somatic sympathetic reflex with a spinal pathway to affect the trunk organs. (2) The acupuncture points in the trunk and extremities have a musculoskeletal effect that is local or regional, but not distal. This is consistent with some of the models of acupuncture mechanisms on musculoskeletal effects in the Western medical acupuncture approach. (3) The acupuncture points on the head and neck preferentially affect the nearest organ. This presumably reflects the belief in TCM that acupuncture can somehow regulate the functioning of the proximal organ. No clear relationship can be identified between the myotome level of the acupuncture points in the extremities and their non-musculoskeletal clinical indications.
Adolescent and parental utilities for the health states associated with pelvic inflammatory disease
Authors: Trent Maria , Lehmann Harold P , Qian Qiang , Thompson Carol B , Ellen Jonathan M , Frick Kevin D
Published: October 2011

Purpose

There is limited information about how the consumers of adolescent pelvic inflammatory disease (PID) care value health states associated with the disorder. The aim of this study is to determine and compare adolescent and parent PID-related health utilities.

Methods

Adolescent girls (N=134) and parents (N=121) completed a web-based utility elicitation survey. Participants reviewed five scenarios describing the health states associated with PID (outpatient treatment (mild–moderate disease), inpatient treatment (severe disease), ectopic pregnancy, infertility and chronic abdominal pain). After each scenario, participants were asked to rate health-related quality of life (HRQL) using a Visual Analogue Scale (VAS) and to complete a time trade-off (TTO) assessment. Data were evaluated using multiple linear (VAS) and quantile (TTO) regression analyses.

Results

Adolescents had significantly lower mean valuations (p<0.01) than the parents on the VAS for HRQL in each health state (outpatient (62 vs 76), inpatient (57 vs 74), ectopic (55 vs 73), infertility (59 vs 68) and chronic abdominal pain (48 vs 61)). Using quantile regression analysis, adolescents were also willing to give up more time for health gains indicated by lower median TTO scores (p<0.01) for outpatient treatment (0.98 vs 1.0), inpatient treatment (0.96 vs 1.0) and ectopic pregnancy (0.98 vs 1.0).

Conclusions

The authors demonstrate that adolescents assign more disutility (lower valuations) than parents for HRQL and three of five of the TTO assessments for PID-related health states. Future economic evaluations using patient-specific preferences to determine resource allocation for PID management in adolescents should include adolescent health outcomes and utilities.
Loss of the preconditioning effect of rosuvastatin during sustained therapy: a human in vivo study
Authors: Liuni Andrew , Luca Mary Clare , Gori Tommaso , Parker John D.
Published: October 2011

Studies have demonstrated that the acute administration of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has protective effects in the setting of ischemia-reperfusion (IR). Previously, we demonstrated that a single dose of rosuvastatin prevented IR-induced endothelial dysfunction in humans through a cyclooxygenase-2-dependent mechanism. Whether the chronic administration of HMG-CoA reductase inhibitors provides similar protection remains controversial and is unknown in humans. Eighteen male volunteers were randomized to receive a single dose of rosuvastatin (20 mg) or placebo. Twenty-four hours later, endothelium-dependent, radial artery flow-mediated dilation (FMD) was measured before and after IR (15 min of upper arm ischemia followed by 15 min of reperfusion). In a separate protocol, 30 healthy volunteers were randomized in a double-blind fashion to receive oral rosuvastatin (20 mg/day) and placebo, rosuvastatin, and celecoxib (100 mg bid) or placebo alone, all for 21 days. Twenty-four hours after the final administration of study medication, FMD was measured before and after IR. Pre-IR FMD was similar between groups in both protocols. In the acute administration protocol, rosuvastatin significantly prevented the blunting of FMD associated with IR (FMD pre-IR: 8.4 ± 1.3%; post-IR: 6.2 ± 1.3%; P = 0.01 ANOVA, treatment group interaction). In the daily administration protocol, IR significantly blunted FMD in the placebo group (FMD pre-IR: 7.5 ± 0.9%; post-IR: 3.3 ± 0.7%; P < 0.001). Chronic treatment with rosuvastatin did not modify this ischemic injury (FMD pre-IR: 6.9 ± 0.4%; post-IR: 1.6 ± 1.0%; P < 0.001; P = NS ANOVA, treatment group interaction). Similarly, FMD responses post-IR in volunteers receiving rosuvastatin and celecoxib did not significantly differ from placebo (FMD pre-IR: 8.3 ± 0.9%; post-IR: 2.1 ± 0.8%; P < 0.001; P = NS ANOVA, treatment group interaction). In contrast to acute administration, chronic rosuvastatin does not prevent the development of IR-induced endothelial dysfunction in normal humans.
Use of the Frank-Starling mechanism during exercise is linked to exercise-induced changes in arterial load
Authors: Chantler Paul D. , Melenovsky Vojtech , Schulman Steven P. , Gerstenblith Gary , Becker Lewis C. , Ferrucci Luigi , Fleg Jerome L. , Lakatta Edward G. , Najjar Samer S.
Published: October 2011

Effective arterial elastance(EA) is a measure of the net arterial load imposed on the heart that integrates the effects of heart rate(HR), peripheral vascular resistance(PVR), and total arterial compliance(TAC) and is a modulator of cardiac performance. To what extent the change in EA during exercise impacts on cardiac performance and aerobic capacity is unknown. We examined EA and its relationship with cardiovascular performance in 352 healthy subjects. Subjects underwent rest and exercise gated scans to measure cardiac volumes and to derive EA[end-systolic pressure/stroke volume index(SV)], PVR[MAP/(SV*HR)], and TAC(SV/pulse pressure). EA varied with exercise intensity: the ΔEA between rest and peak exercise along with its determinants, differed among individuals and ranged from −44% to +149%, and was independent of age and sex. Individuals were separated into 3 groups based on their ΔEAI. Individuals with the largest increase in ΔEA(group 3;ΔEA≥0.98 mmHg.m2/ml) had the smallest reduction in PVR, the greatest reduction in TAC and a similar increase in HR vs. group 1(ΔEA<0.22 mmHg.m2/ml). Furthermore, group 3 had a reduction in end-diastolic volume, and a blunted increase in SV(80%), and cardiac output(27%), during exercise vs. group 1. Despite limitations in the Frank-Starling mechanism and cardiac function, peak aerobic capacity did not differ by group because arterial-venous oxygen difference was greater in group 3 vs. 1. Thus the change in arterial load during exercise has important effects on the Frank-Starling mechanism and cardiac performance but not on exercise capacity. These findings provide interesting insights into the dynamic cardiovascular alterations during exercise.
Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS
Authors: Chatterjee Shampa , Browning Elizabeth A. , Hong NanKang , DeBolt Kris , Sorokina Elena M. , Liu Weidong , Birnbaum Morris J. , Fisher Aron B.
Published: October 2011

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K+(KATP) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with KATPchannel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a KATPchannel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.
Early postnatal exposure of mice to side-steam tobacco smoke increases neuropeptide Y in lung
Authors: Wu Z.-X. , Benders K. B. , Hunter D. D. , Dey R. D.
Published: October 2011

Our recent study showed that prenatal and early postnatal exposure of mice to side-steam tobacco smoke (SS), a surrogate to environmental tobacco smoke (ETS), leads to increased airway responsiveness and sensory innervation later in life. However, the underlying mechanism initiated in early life that affects airway responses later in life remains undefined. The concomitant increase in nerve growth factor (NGF) after exposures suggests that NGF may be involved the regulation of airway innervation. Since NGF regulates sympathetic nerve responses, as well as sensory nerves, we extended previous studies by examining neuropeptide Y (NPY), a neuropeptide associated with sympathetic nerves. Different age groups of mice, postnatal day (PD) 2 and PD21, were exposed to either SS or filtered air (FA) for 10 consecutive days. The level of NPY protein in lung and the density of NPY nerve fibers in tracheal smooth muscle were significantly increased in the PD2–11SS exposure group compared with PD2–11FA exposure. At the same time, the level of NGF in lung tissue was significantly elevated in the PD2–11SS exposure groups. However, neither NPY (protein or nerves) nor NGF levels were significantly altered in PD21–30SS exposure group compared with the PD21–30FA exposure group. Furthermore, pretreatment with NGF antibody or K252a, which inhibits a key enzyme (tyrosine kinase) in the transduction pathway for NGF receptor binding, significantly diminished SS-enhanced NPY tracheal smooth muscle innervation and the increase in methacholine-induced airway resistance. These findings show that SS exposure in early life increases NPY tracheal innervation and alters pulmonary function and that these changes are mediated through the NGF.
NF-κB activation and polyubiquitin conjugation are required for pulmonary inflammation-induced diaphragm atrophy
Authors: Haegens Astrid , Schols Annemie M. , Gorissen Stefan H. , van Essen Anon L. , Snepvangers Frank , Gray Douglas A. , Shoelson Steven E. , Langen Ramon C.
Published: October 2011

Loss of diaphragm muscle strength in inflammatory lung disease contributes to mortality and is associated with diaphragm fiber atrophy. Ubiquitin (Ub) 26S-proteasome system (UPS)-dependent protein breakdown, which mediates muscle atrophy in a number of physiological and pathological conditions, is elevated in diaphragm muscle of patients with chronic obstructive pulmonary disease. Nuclear factor kappa B (NF-κB), an essential regulator of many inflammatory processes, has been implicated in the regulation of poly-Ub conjugation of muscle proteins targeted for proteolysis by the UPS. Here, we test if NF-κB activation in diaphragm muscle and subsequent protein degradation by the UPS are required for pulmonary inflammation-induced diaphragm atrophy. Acute pulmonary inflammation was induced in mice by intratracheal lipopolysaccharide instillation. Fiber cross-sectional area, ex vivo tyrosine release, protein poly-Ub conjugation, and inflammatory signaling were determined in diaphragm muscle. The contribution of NF-κB or the UPS to diaphragm atrophy was assessed in mice with intact or genetically repressed NF-κB signaling or attenuated poly-Ub conjugation, respectively. Acute pulmonary inflammation resulted in diaphragm atrophy measured by reduced muscle fiber cross-sectional area. This was accompanied by diaphragm NF-κB activation, and proteolysis, measured by tyrosine release from the diaphragm. Poly-Ub conjugation was increased in diaphragm, as was the expression of muscle-specific E3 Ub ligases. Genetic suppression of poly-Ub conjugation prevented inflammation-induced diaphragm muscle atrophy, as did muscle-specific inhibition of NF-κB signaling. In conclusion, the present study is the first to demonstrate that diaphragm muscle atrophy, resulting from acute pulmonary inflammation, requires NF-κB activation and UPS-mediated protein degradation.
Airway epithelial cells suppress T cell proliferation by an IFNγ/STAT1/TGFβ-dependent mechanism
Authors: Deppong Christine M. , Xu Jian , Brody Steven L. , Green Jonathan M.
Published: October 2011

Organ-specific regulation of immune responses relies on the exchange of information between nonimmune and immune cells. In a primary culture model of the lung airway, we demonstrate that T cell proliferation is potently inhibited by airway epithelial cells (ECs). This is mediated by activation of the IFNγ/STAT1 pathway in the EC and transforming growth factor-β (TGFβ)-dependent suppression of T cell proliferation. In this way, the EC can restrict the expansion of T cells. Given the constant exposure of the airway to inhaled antigen, this may be important in setting a threshold for the initiation of T cell-dependent immune responses and preventing unwanted, chronic inflammation.
Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury
Authors: Vohra Pawan K. , Hoeppner Luke H. , Sagar Gunisha , Dutta Shamit K. , Misra Sanjay , Hubmayr Rolf D. , Mukhopadhyay Debabrata
Published: October 2011

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0–75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR−/− mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.
Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia
Authors: Tang Jen-Ruey , Karumanchi S. Ananth , Seedorf Gregory , Markham Neil , Abman Steven H.
Published: October 2011

Epidemiological studies have shown that maternal preeclampsia (PE) increases the risk of bronchopulmonary dysplasia (BPD), but the underlying mechanism is unknown. Soluble vascular endothelial growth factor receptor-1 (soluble VEGFR1, known as soluble fms-like tyrosine kinase 1, or sFlt-1), an endogenous antagonist of vascular endothelial growth factor (VEGF), is markedly elevated in amniotic fluid and maternal blood in PE. Therefore, we hypothesized that antenatal exposure to excess sFlt-1 disrupts lung development through impaired VEGF signaling in utero, providing a mechanistic link between PE and BPD. To determine whether increased sFlt-1 in amniotic fluid is sufficient to cause sustained abnormalities of lung structure during infancy, sFlt-1 or saline was injected into amniotic sacs of pregnant Sprague-Dawley rats at 20 days of gestation (term, 22 days). After birth, pups were observed through 14 days of age for study. We found that intra-amniotic sFlt-1 treatment decreased alveolar number, reduced pulmonary vessel density, and caused right and left ventricular hypertrophy in 14-day-old rats. In addition, intra-amniotic sFlt-1 treatment suppressed activation of lung VEGF receptor-2 and increased apoptosis in endothelial and mesenchymal cells in the newborn lung. We conclude that exposure to excess sFlt-1 in amniotic fluid during late gestation causes sustained reductions in alveolarization and pulmonary vascular growth during infancy, accompanied by biventricular hypertrophy suggesting pulmonary and systemic hypertension. We speculate that impaired VEGF signaling in utero due to exposure of high amniotic fluid levels of sFlt-1 in PE disrupts lung growth and contributes to the increased risk of BPD in infants born to mothers with PE.
Leukocyte-derived extracellular superoxide dismutase does not contribute to airspace EC-SOD after interstitial pulmonary injury
Authors: Manni Michelle L. , Epperly Michael W. , Han Wei , Blackwell Timothy S. , Duncan Steven R. , Piganelli Jon D. , Oury Tim D.
Published: October 2011

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is known to limit inflammation and fibrosis following numerous pulmonary insults. Previous studies have reported a loss of full-length EC-SOD from the pulmonary parenchyma with accumulation of proteolyzed EC-SOD in the airspace after an interstitial lung injury. However, following airspace only inflammation, EC-SOD accumulates in the airspace without a loss from the interstitium, suggesting this antioxidant may be released from an extrapulmonary source. Because leukocytes are known to express EC-SOD and are prevalent in the bronchoalveolar lavage fluid (BALF) after injury, it was hypothesized that these cells may transport and release EC-SOD into airspaces. To test this hypothesis, C57BL/6 wild-type and EC-SOD knockout mice were irradiated and transplanted with bone marrow from either wild-type mice or EC-SOD knockout mice. Bone marrow chimeric mice were then intratracheally treated with asbestos and killed 3 and 7 days later. At both 3 and 7 days following asbestos injury, mice without pulmonary EC-SOD expression but with EC-SOD in infiltrating and resident leukocytes did not have detectable levels of EC-SOD in the airspaces. In addition, leukocyte-derived EC-SOD did not significantly lessen inflammation or early stage fibrosis that resulted from asbestos injury in the lungs. Although it is not influential in the asbestos-induced interstitial lung injury model, EC-SOD is still known to be present in leukocytes and may play an influential role in attenuating pneumonias and other inflammatory diseases.
TGF-β enhances deposition of perlecan from COPD airway smooth muscle
Authors: Ichimaru Yukikazu , Krimmer David I. , Burgess Janette K. , Black Judith L. , Oliver Brian G. G.
Published: October 2011

Chronic obstructive pulmonary disease (COPD) and asthma are characterized by irreversible remodeling of the airway walls, including thickening of the airway smooth muscle layer. Perlecan is a large, multidomain, proteoglycan that is expressed in the lungs, and in other organ systems, and has been described to have a role in cell adhesion, angiogenesis, and proliferation. This study aimed to investigate functional properties of the different perlecan domains in relation to airway smooth muscle cells (ASMC). Primary human ASMC obtained from donors with asthma ( n = 13), COPD ( n = 12), or other lung disease ( n = 20) were stimulated in vitro with 1 ng/ml transforming growth factor-β1 (TGF-β1) before perlecan deposition and cytokine release were analyzed. In some experiments, inhibitors of signaling molecules were added. Perlecan domains I–V were seeded on tissue culture plates at 10 μg/ml with 1 μg/ml collagen I as a control. ASM was incubated on top of the peptides before being analyzed for attachment, proliferation, and wound healing. TGF-β1 upregulated deposition of perlecan by ASMC from COPD subjects only. TGF-β1 upregulated release of IL-6 into the supernatant of ASMC from all subjects. Inhibitors of SMAD and JNK signaling molecules decreased TGF-β1-induced perlecan deposition by COPD ASMC. Attachment of COPD ASMC was upregulated by collagen I and perlecan domains IV and V, while perlecan domain II upregulated attachment only of asthmatic ASMC. Seeding on perlecan domains did not increase proliferation of any ASMC type. TGF-β1-induced perlecan deposition may enhance attachment of migrating ASMC in vivo and thus may be a mechanism for ASMC layer hypertrophy in COPD.