bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Coxiella burnetii DNA in Inhalable Airborne Dust Samples from Goat Farms after Mandatory Culling
Authors: Hogerwerf Lenny , Borlée Floor , Still Kelly , Heederik Dick , van Rotterdam Bart , de Bruin Arnout , Nielen Mirjam , Wouters Inge M.
Published: May 2012

Coxiella burnetii
is thought to infect humans primarily via airborne transmission. However, air measurements of
C. burnetii
are sparse. We detected
C. burnetii
DNA in inhalable and PM10 (particulate matter with an aerodynamic size of 10 μm or less) dust samples collected at three affected goat farms, demonstrating that low levels of
C. burnetii
DNA are present in inhalable size fractions.
Syntrophic Oxidation of Propionate in Rice Field Soil at 15 and 30°C under Methanogenic Conditions
Authors: Gan Yanlu , Qiu Qiongfen , Liu Pengfei , Rui Junpeng , Lu Yahai
Published: May 2012

Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H2-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-13C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating13C were traced by DNA-based stable isotope probing.Syntrophobacterspp.,Pelotomaculumspp., andSmithellaspp. were found significantly incorporating13C into their nucleic acids after [13C]propionate incubation at 30°C. The activity ofSmithellaspp. increased in the later stage, and concurrently that ofSyntrophomonasspp. increased. AceticlasticMethanosaetaceaeand hydrogenotrophicMethanomicrobialesandMethanocellalesacted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C.Syntrophobacterspp. were significantly labeled with13C, whereasPelotomaculumspp. were less active at this temperature. In addition,Methanomicrobiales,Methanocellales, andMethanosarcinaceaedominated the methanogenic community, whileMethanosaetaceaedecreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly,Geobacterspp. and some other anaerobic organisms likeRhodocyclaceae,Acidobacteria,Actinobacteria, andThermomicrobiaprobably also assimilated propionate-derived13C. The mechanisms for the involvement of these organisms remain unclear.
Novel Phytases from Bifidobacterium pseudocatenulatum ATCC 27919 and Bifidobacterium longum subsp.
infantis
ATCC 15697
Authors: Tamayo-Ramos Juan Antonio , Sanz-Penella Juan Mario , Yebra María J. , Monedero Vicente , Haros Monika
Published: May 2012

Two novel phytases have been characterized from
Bifidobacterium pseudocatenulatum
and
Bifidobacterium longum
subsp.
infantis
. The enzymes belong to a new subclass within the histidine acid phytases, are highly specific for the hydrolysis of phytate, and render
myo
-inositol triphosphate as the final hydrolysis product. They represent the first phytases characterized from this group of probiotic microorganisms, opening the possibilities for their use in the processing of high-phytate-content foods.
Associations of Yeasts with Spotted-Wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in Cherries and Raspberries
Authors: Hamby Kelly A. , Hernández Alejandro , Boundy-Mills Kyria , Zalom Frank G.
Published: May 2012

A rich history of investigation documents variousDrosophila-yeast mutualisms, suggesting thatDrosophila suzukiisimilarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts ofD. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested withD. suzukiior not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts ofD. suzukii, representing 28 species of yeasts, withHanseniaspora uvarumpredominating. This suggests an association betweenD. suzukiiandH. uvarumthat could be utilized for pest management of the highly pestiferousD. suzukii.
Phylomark, a Tool To Identify Conserved Phylogenetic Markers from Whole-Genome Alignments
Authors: Sahl Jason W. , Matalka Malcolm N. , Rasko David A.
Published: May 2012

The sequencing and analysis of multiple housekeeping genes has been routinely used to phylogenetically compare closely related bacterial isolates. Recent studies using whole-genome alignment (WGA) and phylogenetics from >100
Escherichia coli
genomes has demonstrated that tree topologies from WGA and multilocus sequence typing (MLST) markers differ significantly. A nonrepresentative phylogeny can lead to incorrect conclusions regarding important evolutionary relationships. In this study, the Phylomark algorithm was developed to identify a minimal number of useful phylogenetic markers that recapitulate the WGA phylogeny. To test the algorithm, we used a set of diverse draft and complete
E. coli
genomes. The algorithm identified more than 100,000 potential markers of different fragment lengths (500 to 900 nucleotides). Three molecular markers were ultimately chosen to determine the phylogeny based on a low Robinson-Foulds (RF) distance compared to the WGA phylogeny. A phylogenetic analysis demonstrated that a more representative phylogeny was inferred for a concatenation of these markers compared to all other MLST schemes for
E. coli
. As a functional test of the algorithm, the three markers (genomic guided
E. coli
markers, or GIG-EM) were amplified and sequenced from a set of environmental
E. coli
strains (ECOR collection) and informatically extracted from a set of 78 diarrheagenic
E. coli
strains (DECA collection). In the instances of the 40-genome test set and the DECA collection, the GIG-EM system outperformed other
E. coli
MLST systems in terms of recapitulating the WGA phylogeny. This algorithm can be employed to determine the minimal marker set for any organism that has sufficient genome sequencing.
A Straightforward DOPE (Double Labeling of Oligonucleotide Probes)-FISH (FluorescenceIn SituHybridization) Method for Simultaneous Multicolor Detection of Six Microbial Populations
Authors: Behnam Faris , Vilcinskas Andreas , Wagner Michael , Stoecker Kilian
Published: May 2012

Fluorescencein situhybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.
Sensitive Quantification of Clostridium difficile Cells by Reverse Transcription-Quantitative PCR Targeting rRNA Molecules
Authors: Matsuda Kazunori , Tsuji Hirokazu , Asahara Takashi , Takahashi Takuya , Kubota Hiroyuki , Nagata Satoru , Yamashiro Yuichiro , Nomoto Koji
Published: May 2012

We established a sensitive and accurate quantification system for
Clostridium difficile
in human intestines, based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). We newly developed a species-specific primer set for
C. difficile
targeting 23S rRNA gene sequences. Both the vegetative cells and the spores of
C. difficile
in human feces were quantified by RT-qPCR, with a lower detection limit of 10
2.4
cells/g of feces. In an analysis of the feces of residents (
n
= 83; age, 85 ± 8 years) and staff (
n
= 19; age, 36 ± 10 years) at a care facility for the elderly,
C. difficile
was detected by RT-qPCR in 43% of the residents (average count, log
10
4.0 ± 2.0 cells/g of feces) and 16% of the staff (average count, log
10
2.2 ± 0.1 cells/g of feces); these rates were far higher than those detected by qPCR (residents, 19%; staff, 0%) or selective cultivation (residents, 18%; staff, 5%). Another analysis of healthy adults (
n
= 63; age, 41 ± 11 years) also revealed the significant carriage rate of
C. difficile
in the intestines (detection rate, 13%; average count, log
10
4.9 ± 1.2 cells/g of feces). From these results, it was suggested that rRNA-targeted RT-qPCR should be an effective tool for analyzing population levels of
C. difficile
in the human intestine.
Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1
Authors: Johnson Michael E. , Edlind Thomas D.
Published: May 2012

Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated
Saccharomyces cerevisiae
Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted
in silico
for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2.
ENVOY Is a Major Determinant in Regulation of Sexual Development in Hypocrea jecorina (
Trichoderma reesei
)
Authors: Seibel Christian , Tisch Doris , Kubicek Christian P. , Schmoll Monika
Published: May 2012

Light is one crucial environmental signal which can determine whether a fungus reproduces asexually or initiates sexual development. Mating in the ascomycete
Hypocrea jecorina
(anamorph
Trichoderma reesei
) occurs preferentially in light. We therefore investigated the relevance of the light response machinery for sexual development in
H. jecorina
. We found that the photoreceptors BLR1 and BLR2 and the light-regulatory protein ENV1 have no effect on male fertility, while ENV1 is essential for female fertility. BLR1 and BLR2 were found to impact fruiting body formation although they are not essential for mating. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that BLR1, BLR2, and ENV1 negatively regulate transcript levels of both pheromone receptors as well as peptide pheromone precursors in light but not in darkness and in a mating type-dependent manner. The effect of BLR1 and BLR2 on regulation of pheromone precursor and receptor genes is less severe than that of ENV1 as strains lacking
env1
show 100-fold (for
ppg1
) to more than 100,000-fold (for
hpp1
) increased transcript levels of pheromone precursor genes as well as more than 20-fold increased levels of
hpr1
, the pheromone receptor receiving the HPP1 signal in a MAT1-1 strain. ENV1 likely integrates additional signals besides light, and our results indicate that its function is partially mediated via regulation of
mat1-2-1
. We conclude that ENV1 is essential for balancing the levels of genes regulated in a mating-type-dependent manner, which contributes to determination of sexual identity and fruiting body formation.
Salmonella Biofilm Development Depends on the Phosphorylation Status of RcsB
Authors: Latasa Cristina , García Begoña , Echeverz Maite , Toledo-Arana Alejandro , Valle Jaione , Campoy Susana , García-del Portillo Francisco , Solano Cristina , Lasa Iñigo
Published: May 2012

The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that
Salmonella
biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in
Salmonella enterica
serovar Typhimurium.
Electron Bifurcation Involved in the Energy Metabolism of the Acetogenic Bacterium Moorella thermoacetica Growing on Glucose or H
2
plus CO
2
Authors: Huang Haiyan , Wang Shuning , Moll Johanna , Thauer Rudolf K.
Published: May 2012

Moorella thermoacetica
ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO
2
molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fd
red
2−
) molecules. In the reductive part, 2 CO
2
molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO
2
are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO
2
reduction to acetic acid are generated by the reduction of 2 NADP
+
molecules with 1 NADH and 1 Fd
red
2−
catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP
+
oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in
Escherichia coli
, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD.
M. thermoacetica
contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD
+
and Fd with 2 H
2
molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H
2
-CO
2
-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H
2
-CO
2
-grown cells additionally contain high specific activities of an NADP
+
-dependent hydrogenase that catalyzes the reduction of NADP
+
with H
2
. This activity is hardly detectable in glucose-grown cells.
Contributions of Individual σ
B
-Dependent General Stress Genes to Oxidative Stress Resistance of Bacillus subtilis
Authors: Reder Alexander , Höper Dirk , Gerth Ulf , Hecker Michael
Published: May 2012

The general stress regulon of
Bacillus subtilis
comprises approximately 200 genes and is under the control of the alternative sigma factor σ
B
. The activation of σ
B
occurs in response to multiple physical stress stimuli as well as energy starvation conditions. The expression of the general stress proteins provides growing and stationary nonsporulating vegetative cells with nonspecific and broad stress resistance. A previous comprehensive phenotype screening analysis of 94 general stress gene mutants in response to severe growth-inhibiting stress stimuli, including ethanol, NaCl, heat, and cold, indicated that secondary oxidative stress may be a common component of severe physical stress. Here we tested the individual contributions of the same set of 94 mutants to the development of resistance against exposure to the superoxide-generating agent paraquat and hydrogen peroxide (H
2
O
2
). In fact, 62 mutants displayed significantly decreased survival rates in response to paraquat and/or H
2
O
2
stress compared to the wild type at a confidence level of an α value of ≤0.01. Thus, we were able to assign 47 general stress genes to survival against superoxide, 6 genes to protection from H
2
O
2
stress, and 9 genes to the survival against both. Furthermore, we show that a considerable overlap exists between the phenotype clusters previously assumed to be involved in oxidative stress management and the actual group of oxidative-stress-sensitive mutants. Our data provide information that many general stress proteins with still unknown functions are implicated in oxidative stress resistance and further support the notion that different severe physical stress stimuli elicit a common secondary oxidative stress.
Flavobacterium johnsoniae RemA Is a Mobile Cell Surface Lectin Involved in Gliding
Authors: Shrivastava Abhishek , Rhodes Ryan G. , Pochiraju Soumya , Nakane Daisuke , McBride Mark J.
Published: May 2012

Cells of
Flavobacterium johnsoniae
move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the motor that propels the cell surface adhesin SprB. Cells with mutations in
sprB
are partially defective in motility and are also resistant to some bacteriophages. Transposon mutagenesis of a strain carrying a deletion spanning
sprB
identified eight mutants that were resistant to additional phages and exhibited reduced motility. Four of the mutants had transposon insertions in
remA
, which encodes a cell surface protein that has a lectin domain and appears to interact with polysaccharides. Three other genes identified in this screen (
remC
,
wza
, and
wzc
) encode proteins predicted to be involved in polysaccharide synthesis and secretion. Myc-tagged versions of RemA localized to the cell surface and were propelled rapidly along the cell at speeds of 1 to 2 μm/s. Deletion of
gldN
and
gldO
, which encode components of a bacteroidete protein secretion system, blocked the transport of RemA to the cell surface. Overexpression of RemA resulted in the formation of cell aggregates that were dispersed by the addition of galactose or rhamnose. Cells lacking RemC, Wza, and Wzc failed to aggregate. Cells of a
remC
mutant and cells of a
remA
mutant, neither of which formed aggregates in isolation, aggregated when they were mixed together, suggesting that polysaccharides secreted by one cell may interact with RemA on another cell. Fluorescently labeled lectin
Ricinus communis
agglutinin I detected polysaccharides secreted by
F. johnsoniae
. The polysaccharides bound to cells expressing RemA and were rapidly propelled on the cell surface. RemA appears to be a mobile cell surface adhesin, and secreted polysaccharides may interact with the lectin domain of RemA and enhance motility.
Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii
Authors: Ruiz Diego M. , Paggi Roberto A. , Giménez Maria I. , De Castro Rosana E.
Published: May 2012

Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon
Natrialba magadii
, and the recombinant active enzyme has been synthesized in
Haloferax volcanii
. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (G
RR
SVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in
H. volcanii
, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase.
Strategies to increase influenza vaccination rates: outcomes of a nationwide cross-sectional survey of UK general practice
Authors: Dexter Laura J , Teare M Dawn , Dexter Matthew , Siriwardena A Niroshan , Read Robert C
Published: May 2012

Objective

To identify practice strategies associated with higher flu vaccination rates in primary care.

Design

Logistic regression analysis of data from a cross-sectional online questionnaire.

Setting

795 general practices across England.

Participants

569 practice managers, 335 nursing staff and 107 general practitioners.

Primary outcome measures

Flu vaccination rates achieved by each practice in different groups of at-risk patients.

Results

7 independent factors associated with higher vaccine uptake were identified. Having a lead staff member for planning the flu campaign and producing a written report of practice performance predicted an 8% higher vaccination rate for at-risk patients aged <65 years (OR 1.37, 95% CI 1.10 to 1.71). These strategies, plus sending a personal invitation to all eligible patients and only stopping vaccination when Quality and Outcomes Framework targets are reached, predicted a 7% higher vaccination rate (OR 1.45, 95% CI 1.10 to 1.92) in patients aged ≥65 years. Using a lead member of staff for identifying eligible patients, with either a modified manufacturer's or in-house search programme for interrogating the practice IT system, independently predicted a 4% higher vaccination rate in patients aged ≥65 years (OR 1.22, 95% CI 1.06 to 1.41/OR 1.20, 95% CI 1.03 to 1.40). The provision of flu vaccine by midwives was associated with a 4% higher vaccination rate in pregnant women (OR 1.19, 95% CI 1.02 to 1.40).

Conclusions

Clear leadership, effective communication about performance and methods used to identify and contact eligible patients were independently associated with significantly higher rates of flu vaccination. Financial targets appear to incentivise practices to work harder to maximise seasonal influenza vaccine uptake. The strategies identified here could help primary care providers to substantially increase their seasonal flu vaccination rates towards or even above the Chief Medical Officer's targets.
Public health concerns for anti-obesity medicines imported for personal use through the internet: a cross-sectional study
Authors: Khan Mohiuddin Hussain , Tanimoto Tsuyoshi , Nakanishi Yoko , Yoshida Naoko , Tsuboi Hirohito , Kimura Kazuko
Published: May 2012

Objective

To explore the circulation of anti-obesity medicines via the internet and their quality.

Design

Cross-sectional study.

Setting

Internet pharmacies and pharmaceutical suppliers accessible from Japan.

Participants

Anti-obesity medicines were purchased using relevant keywords on Japanese Google search engine. Blogs and advertisement-only sites were excluded.

Primary and secondary outcome measures

The authenticity of the samples was investigated in collaboration with the manufacturers of the samples and medicine regulatory authorities. Quality of the samples was assessed by pharmacopoeial analyses using high-performance liquid chromatography.

Results

82 samples were purchased from 36 internet sites. Approximately half of the sites did not mention a physical address, and 45% of the samples did not contain a package insert. A variety of custom declarations were made for the shipments of the samples: personal health items, supplement, medicines, general merchandise, tea and others. Among 82 samples, 52 samples were analysed to check their pharmacopoeial quality. Authenticity responses were received from only five of 20 manufacturing companies. According to the pharmacopoeial analyses and authenticity investigation, three of the samples were identified as counterfeits and did not contain any active ingredients. Two of these samples were confirmed as counterfeits by the manufacturer of the authentic products. The manufacturer of the other sample did not respond to our request for an authenticity check even after several communication attempts. These counterfeit cases have been reported at the rapid alert system of Western Pacific Region of the WHO.

Conclusions

Many counterfeit and unapproved anti-obesity medicines may be easily bypassing regulatory checks during shipping and are widely circulated through the internet. Regulatory authorities should take measures to prevent these medicines from entering countries to safeguard their citizens.
Antidopaminergic drugs and acute pancreatitis: a population-based study
Authors: Bodén Robert , Bexelius Tomas S , Mattsson Fredrik , Lagergren Jesper , Lindblad Mats , Ljung Rickard
Published: May 2012

Objectives

To evaluate the suggested association between antidopaminergic drugs and acute pancreatitis.

Design

A large population-based nested case–control study.

Setting

Swedish nationwide study from 2006 to 2008.

Participants

The Patient Register was used to identify 6161 cases of acute pancreatitis. The 61 637 control subjects were randomly selected from the Register of the Total Population by frequency-based density sampling, matched for age, sex and calendar year.

Exposure

Exposure data were extracted from the Prescribed Drug Register. Antidopaminergic drugs were grouped into antiemetic/anxiolytic and other antipsychotics. Current use of antidopaminergic drugs was defined as filling a prescription 1–114 days before index date, while previous use was 115 days to 3.5 years before index date.

Main outcome measures

Cases were defined as being diagnosed as having acute pancreatitis. ORs and 95% CIs were calculated using unconditional logistic regression.

Results

The unadjusted OR indicated an increased risk of acute pancreatitis among current users of antiemetic/anxiolytics (OR 1.9, 95% CI 1.4 to 2.6), but not in the multivariable model adjusting for alcohol-related comorbidity, chronic obstructive lung disease, ischaemic heart disease, obesity, diabetes, opioid use, gallstone disease, educational level, marital status and number of concomitant medications (OR 0.9, 95% CI 0.6 to 1.2). Similarly, among current users of other antipsychotics, the unadjusted OR was 1.4 (95% CI 1.1 to 1.6), while the adjusted OR was 0.8 (95% CI 0.6 to 0.9). Results regarding previous use of antidopaminergic drugs followed a similar risk pattern as for current use.

Conclusions

The lack of association between antidopaminergic drugs and acute pancreatitis after adjustment for confounding factors in this study suggests that the previously reported positive associations might be explained by confounding.
Impact of obesity, overweight and underweight on life expectancy and lifetime medical expenditures: the Ohsaki Cohort Study
Authors: Nagai Masato , Kuriyama Shinichi , Kakizaki Masako , Ohmori-Matsuda Kaori , Sone Toshimasa , Hozawa Atsushi , Kawado Miyuki , Hashimoto Shuji , Tsuji Ichiro
Published: May 2012

Objectives

People who are obese have higher demands for medical care than those of the normal weight people. However, in view of their shorter life expectancy, it is unclear whether obese people have higher lifetime medical expenditure. We examined the association between body mass index, life expectancy and lifetime medical expenditure.

Design

Prospective cohort study using individual data from the Ohsaki Cohort Study.

Setting

Miyagi Prefecture, northeastern Japan.

Participants

The 41 965 participants aged 40–79 years.

Primary and secondary outcome measures

The life expectancy and lifetime medical expenditure aged from 40 years.

Results

In spite of their shorter life expectancy, obese participants might require higher medical expenditure than normal weight participants. In men aged 40 years, multiadjusted life expectancy for those who were obese participants was 41.4 years (95% CI 38.28 to 44.70), which was 1.7 years non-significantly shorter than that for normal weight participants (p=0.3184). Multiadjusted lifetime medical expenditure for obese participants was £112 858.9 (94 954.1–131 840.9), being 14.7% non-significantly higher than that for normal weight participants (p=0.1141). In women aged 40 years, multiadjusted life expectancy for those who were obese participants was 49.2 years (46.14–52.59), which was 3.1 years non-significantly shorter than for normal weight participants (p=0.0724), and multiadjusted lifetime medical expenditure was £137 765.9 (123 672.9–152 970.2), being 21.6% significantly higher (p=0.0005).

Conclusions

According to the point estimate, lifetime medical expenditure might appear to be higher for obese participants, despite their short life expectancy. With weight control, more people would enjoy their longevity with lower demands for medical care.
Physiological aspects of cardiac tissue engineering
Authors: Eschenhagen Thomas , Eder Alexandra , Vollert Ingra , Hansen Arne
Published: May 2012

Cardiac tissue engineering aims at repairing the diseased heart and developing cardiac tissues for basic research and predictive toxicology applications. Since the first description of engineered heart tissue 15 years ago, major development steps were directed toward these three goals. Technical innovations led to improved three-dimensional cardiac tissue structure and near physiological contractile force development. Automation and standardization allow medium throughput screening. Larger constructs composed of many small engineered heart tissues or stacked cell sheet tissues were tested for cardiac repair and were associated with functional improvements in rats. Whether these approaches can be simply transferred to larger animals or the human patients remains to be tested. The availability of an unrestricted human cardiac myocyte cell source from human embryonic stem cells or human-induced pluripotent stem cells is a major breakthrough. This review summarizes current tissue engineering techniques with their strengths and limitations and possible future applications.
Bridging experiments, models and simulations: an integrative approach to validation in computational cardiac electrophysiology
Authors: Carusi Annamaria , Burrage Kevin , Rodríguez Blanca
Published: May 2012

Computational models in physiology often integrate functional and structural information from a large range of spatiotemporal scales from the ionic to the whole organ level. Their sophistication raises both expectations and skepticism concerning how computational methods can improve our understanding of living organisms and also how they can reduce, replace, and refine animal experiments. A fundamental requirement to fulfill these expectations and achieve the full potential of computational physiology is a clear understanding of what models represent and how they can be validated. The present study aims at informing strategies for validation by elucidating the complex interrelations among experiments, models, and simulations in cardiac electrophysiology. We describe the processes, data, and knowledge involved in the construction of whole ventricular multiscale models of cardiac electrophysiology. Our analysis reveals that models, simulations, and experiments are intertwined, in an assemblage that is a system itself, namely the model-simulation-experiment (MSE) system. We argue that validation is part of the whole MSE system and is contingent upon 1) understanding and coping with sources of biovariability; 2) testing and developing robust techniques and tools as a prerequisite to conducting physiological investigations; 3) defining and adopting standards to facilitate the interoperability of experiments, models, and simulations; 4) and understanding physiological validation as an iterative process that contributes to defining the specific aspects of cardiac electrophysiology the MSE system targets, rather than being only an external test, and that this is driven by advances in experimental and computational methods and the combination of both.
Glc-6-PD and PKG contribute to hypoxia-induced decrease in smooth muscle cell contractile phenotype proteins in pulmonary artery
Authors: Chettimada Sukrutha , Rawat Dhwajbahadur K. , Dey Nupur , Kobelja Robert , Simms Zachary , Wolin Michael S. , Lincoln Thomas M. , Gupte Sachin A.
Published: May 2012

Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20–30 Torr) for 12 h in vitro significantly ( P < 0.05) reduces (by 30–50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80–90% ( P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.
Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells
Authors: Nimishakavi Shilpa , Besprozvannaya Marina , Raymond Wilfred W. , Craik Charles S. , Gruenert Dieter C. , Caughey George H.
Published: May 2012

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.
Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum
Authors: Wang Jian , Shimoda Larissa A. , Sylvester J. T.
Published: May 2012

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca2+ concentration ([Ca2+]i) by inducing Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx through store- and voltage-operated Ca2+ channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca2+ release, we measured [Ca2+]i with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP3R), respectively, or 4% O2 caused rapid transient increases in [Ca2+]i, indicating intracellular Ca2+ release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca2+-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca2+ release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca2+ release to caffeine and hypoxia but not norepinephrine. The IP3R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca2+ release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca2+]i that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca2+]i induced by hypoxia, as well as the subsequent Ca2+ influx that sustained this increase, required release of Ca2+ from both RyR and IP3R, and 2) the SR Ca2+ stores accessed by RyR, IP3R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca2+-ATPase.
IL-10 Directly Activates and Expands Tumor-Resident CD8+ T Cells withoutDe NovoInfiltration from Secondary Lymphoid Organs
Authors: Emmerich Jan , Mumm John B. , Chan Ivan H. , LaFace Drake , Truong Hoa , McClanahan Terrill , Gorman Daniel M. , Oft Martin
Published: May 2012

The presence of activated intratumoral T cells correlates clinically with better prognosis in patients with cancer. Although tumor vaccines can increase the number of tumor-specific CD8+ T cells in systemic circulation, they frequently fail to increase the number of active and tumor reactive T cells within the tumor. Here we show that treatment with the pleiotropic cytokine interleukin-10 (IL-10) induces specific activation of tumor-resident CD8+ T cells as well as their intratumoral expansion in several mouse tumor models. We found that inhibition of T-cell trafficking from lymphoid organs did not impair IL-10–induced tumor rejection or the activation of tumor-resident CD8+ T cells. Tumor-resident CD8+ T cells expressed elevated levels of the IL-10 receptor and were directly activated by IL-10, resulting in prominent phosphorylation of STAT3 and STAT1. Although CD4+ T cells, regulatory T cells, NK cells, and dendritic cells have been reported as prominent targets of IL-10 in the tumor microenvironment, we found that expression of the IL-10R was required only on CD8+ T cells to facilitate IL-10–induced tumor rejection as well as in situ expansion and proliferation of tumor-resident CD8 T cells. Together, our findings indicate that IL-10 activates CD8+ T-cell–mediated tumor control and suggest that IL-10 may represent a potential tumor immunotherapy in human patients with cancer. Cancer Res; 72(14); 3570–81. ©2012 AACR.
Assessing Individual Breast Cancer Risk within the U.K. National Health Service Breast Screening Program: A New Paradigm for Cancer Prevention
Authors: Evans D. Gareth R. , Warwick Jane , Astley Susan M. , Stavrinos Paula , Sahin Sarah , Ingham Sarah , McBurney Helen , Eckersley Barbara , Harvie Michelle , Wilson Mary , Beetles Ursula , Warren Ruth , Hufton Alan , Sergeant Jamie C. , Newman William G. , Buchan Iain , Cuzick Jack , Howell Anthony
Published: May 2012

The aim of this study is to determine breast cancer risk at mammographic screening episodes and integrate standard risk factors with mammographic density and genetic data to assess changing the screening interval based on risk and offer women at high risk preventive strategies. We report our experience of assessing breast cancer risk within the U.K. National Health Service Breast Screening Program using results from the first 10,000 women entered into the "Predicting Risk Of breast Cancer At Screening" study. Of the first 28,849 women attending for screening at fifteen sites in Manchester 10,000 (35%) consented to study entry and completed the questionnaire. The median 10-year Tyrer–Cuzick breast cancer risk was 2.65% (interquartile range, 2.10–3.45). A total of 107 women (1.07%) had 10-year risks 8% or higher (high breast cancer risk), with a further 8.20% having moderately increased risk (5%–8%). Mammographic density (percent dense area) was 60% or more in 8.3% of women. We collected saliva samples from 478 women for genetic analysis and will extend this to 18% of participants. At time of consent to the study, 95.0% of women indicated they wished to know their risk. Women with a 10-year risk of 8% or more or 5% to 8% and mammographic density of 60% or higher were invited to attend or be telephoned to receive risk counseling; 81.9% of those wishing to know their risk have received risk counseling and 85.7% of these were found to be eligible for a risk-reducing intervention. These results confirm the feasibility of determining breast cancer risk and acting on the information in the context of population-based mammographic screening. Cancer Prev Res; 5(7); 943–51. ©2012 AACR.
Liver X Receptor Agonist Modulation of Cholesterol Efflux in Mice With Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein
Authors: Xie Yan , Kennedy Susan , Sidhu Rohini , Luo Jianyang , Ory Daniel S. , Davidson Nicholas O.
Published: May 2012

Objective—

Previous work demonstrated that intestinal cholesterol absorption and regulated expression of intestinal Niemann-Pick C1-like 1 and ATP-binding cassette protein A1 are required for liver X receptor (LXR) agonist–mediated increases in high-density lipoprotein biogenesis. We re-examined those conclusions in mice with intestine-specific deletion of the microsomal triglyceride transfer protein (
MTTP-IKO
), where chylomicron formation is eliminated.

Methods and Results—

MTTP-IKO
mice demonstrated sustained ≈90% reduction in cholesterol absorption and >80% reduction in Niemann-Pick C1-like 1 expression, yet LXR agonist treatment increased serum high-density lipoprotein and upregulated intestinal ATP-binding cassette protein A1 expression. Hepatic lipogenesis and triglyceride content increased with LXR agonist treatment in both genotypes. Biliary cholesterol secretion was increased in
MTTP-IKO
mice without further increase upon LXR agonist administration. LXR agonist treatment caused a paradoxical increase in cholesterol absorption in
MTTP-IKO
mice and decreased fecal neutral sterol excretion, but to levels that still exceeded fecal neutral sterol excretion in LXR agonist–treated control mice. Finally,
MTTP-IKO
mice demonstrated indistinguishable patterns of increased cholesterol turnover and efflux after intravenous radiolabeled cholesterol administration, with or without LXR agonist treatment.

Conclusion—

Both intestinal and hepatic cholesterol efflux pathways are basally upregulated in
MTTP-IKO
mice. Moreover, LXR-dependent pathways modulate intestinal cholesterol absorption, transport, efflux, and high-density lipoprotein production independent of chylomicron assembly and secretion.
Proprotein Convertase Subtilisin/Kexin Type 9 Interacts With Apolipoprotein B and Prevents Its Intracellular Degradation, Irrespective of the Low-Density Lipoprotein Receptor
Authors: Sun Hua , Samarghandi Amin , Zhang Ningyan , Yao Zemin , Xiong Momiao , Teng Ba-Bie
Published: May 2012

Objective—

proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified.

Methods and Results—

We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (
Ldlr
−/−
and
Ldlr
−/−
Apobec1
−/−
). Irrespective of LDLR expression, mice transduced with the
PCSK9
gene invariably exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB. Consistent with these findings, the levels of very-low-density lipoprotein and LDL were also increased whereas high-density lipoprotein levels were unchanged. Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in hepatocytes. The PCSK9/apoB interaction resulted in increased production of apoB, possibly through the inhibition of intracellular apoB degradation via the autophagosome/lysosome pathway.

Conclusion—

We propose a new role for PCSK9 that involves shuttling between apoB and LDLR. The present study thus provides new insights into the action of PCSK9 in regulating apoB metabolism. Furthermore, our results indicate that targeting PCSK9 expression represents a new paradigm in therapeutic intervention against hyperlipidemia.