bluuebunny/crossref_metadata_2025 huggingface dataset:
Year-Round Prevalence of Norovirus in the Environment of Catering Companies without a Recently Reported Outbreak of Gastroenteritis
Authors: Boxman Ingeborg L. A. , Verhoef Linda , Dijkman Remco , Hägele Geke , te Loeke Nathalie A. J. M. , Koopmans Marion
Published: March 2011

Food handlers play an important role in the transmission of norovirus (NoV) in food-borne outbreaks of gastroenteritis (GE). In a year-round prevalence study, the prevalence of NoV in catering companies without recently reported outbreaks of GE was investigated and compared to the observed prevalence in catering companies with recently reported outbreaks. Swab samples were collected from surfaces in the kitchens and (staff) bathrooms in 832 randomly chosen companies and analyzed for the presence of NoV RNA. In total, 42 (1.7%) out of 2,496 environmental swabs from 35 (4.2%) catering companies tested positive. In contrast, NoV was detected in 147 (39.7%) of the 370 samples for 44 (61.1%) of the 72 establishments associated with outbreaks of gastroenteritis. NoV-positive swabs were more frequently found in winter, in specific types of companies (elderly homes and lunchrooms), and in establishments with separate bathrooms for staff. We found a borderline association with population density but no relation to the number of employees. Sequence analysis showed that environmental strains were interspersed with strains found in outbreaks of illness in humans. Thus, the presence of NoV in catering companies seemed to mirror the presence in the population but was strongly increased when associated with food-borne GE. Swabs may therefore serve as a valuable tool in outbreak investigations for the identification of the causative agent, although results should be interpreted with care, taking into account all other epidemiological data.
Isolation of Lactobacillus salivarius 1077 (NRRL B-50053) and Characterization of Its Bacteriocin, Including the Antimicrobial Activity Spectrum
Authors: Svetoch Edward A. , Eruslanov Boris V. , Levchuk Vladimir P. , Perelygin Vladimir V. , Mitsevich Evgeny V. , Mitsevich Irina P. , Stepanshin Juri , Dyatlov Ivan , Seal Bruce S. , Stern Norman J.
Published: March 2011

Lactobacillus salivarius
1077 (NRRL B-50053) was isolated from poultry intestinal materials, and
in vitro
anti-
Campylobacter jejuni
activity was demonstrated. The isolate was then used for bacteriocin production and its enrichment. The protein content of the cell-free supernatant from the spent medium was precipitated by ammonium sulfate and dialyzed to produce the crude antimicrobial preparation. A typical bacteriocin-like response of sensitivity to proteolytic enzymes and resistance to lysozyme, lipase, and 100°C was observed with this preparation. The polypeptide was further purified by gel filtration, ion-exchange, and hydrophobic-interaction chromatography. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), Edman degradation, and isoelectrofocusing were used to characterize its 3,454-Da molecular mass, the amino acid sequence of its 37 residue components, and the isoelectric point of pI 9.1 of the bacteriocin. Bacteriocin L-1077 contained the class IIa bacteriocin signature N-terminal sequence YGNGV. MICs of bacteriocin L-1077 against 33 bacterial isolates (both Gram negative and Gram positive) ranged from 0.09 to 1.5 μg/ml. Subsequently, the therapeutic benefit of bacteriocin L-1077 was demonstrated in market-age (40- to 43-day-old) broiler chickens colonized with both
C. jejuni
and
Salmonella enterica
serovar Enteritidis. Compared with untreated control birds, both
C. jejuni
and
S
. Enteritidis counts in colonized ceca were diminished by >4 log
10
and
S
. Enteritidis counts in both the liver and the spleen of treated birds were reduced by 6 to 8 log
10
/g compared with those in the nontreated control birds. Bacteriocin L-1077 appears to hold promise in controlling
C. jejuni
/
S
. Enteritidis among commercial broiler chickens.
Important Role of Class I Heat Shock Genes
hrcA
and
dnaK
in the Heat Shock Response and the Response to pH and NaCl Stress of Group I Clostridium botulinum Strain ATCC 3502
Authors: Selby Katja , Lindström Miia , Somervuo Panu , Heap John T. , Minton Nigel P. , Korkeala Hannu
Published: March 2011

Class I heat shock genes (HSGs) code for molecular chaperones which play a major role in the bacterial response to sudden increases of environmental temperature by assisting protein folding. Quantitative reverse transcriptase real-time PCR gene expression analysis of the food-borne pathogen
Clostridium botulinum
grown at 37°C showed that the class I HSGs
grpE
,
dnaK
,
dnaJ
,
groEL
, and
groES
and their repressor,
hrcA
, were expressed at constant levels in the exponential and transitional growth phases, whereas strong downregulation of all six genes was observed during stationary phase. After heat shock from 37 to 45°C, all HSGs were transiently upregulated. A mutant with insertionally inactivated
hrcA
expressed higher levels of class I HSGs during exponential growth than the wild type, followed by upregulation of only
groES
and
groES
after heat shock. Inactivation of
hrcA
or of
dnaK
encoding a major chaperone resulted in lower maximum growth temperatures than for the wild type and reduced growth rates under optimal conditions compared to the wild type. The
dnaK
mutant showed growth inhibition under all tested temperature, pH, and NaCl stress conditions. In contrast, the growth of an
hrcA
mutant was unaffected by mild temperature or acid stress compared to the wild-type strain, indicating that induced class I HSGs support growth under moderately nonoptimal conditions. We show that the expression of class I HSGs plays a major role for survival and growth of
C. botulinum
under the stressful environmental conditions that may be encountered during food processing or growth in food products, in the mammalian intestine, or in wounds.
Living Colors in the Gray Mold Pathogen Botrytis cinerea: Codon-Optimized Genes Encoding Green Fluorescent Protein and mCherry, Which Exhibit Bright Fluorescence
Authors: Leroch Michaela , Mernke Dennis , Koppenhoefer Dieter , Schneider Prisca , Mosbach Andreas , Doehlemann Gunther , Hahn Matthias
Published: March 2011

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus
Botrytis cinerea
because of low fluorescence intensity. The codon usage of
B. cinerea
genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the
B. cinerea
enhanced GFP (eGFP)-encoding gene (Bc
gfp
) for improved expression in
B. cinerea
. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bc
gfp
was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bc
gfp
fusions for quantitative evaluation of various toxic compounds as inducers of the
atrB
gene encoding an ABC-type drug efflux transporter of
B. cinerea
. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in
B. cinerea
.
Characterization of an Obligate Intracellular Bacterium in the Midgut Epithelium of the Bulrush Bug Chilacis typhae (Heteroptera, Lygaeidae, Artheneinae)
Authors: Kuechler Stefan Martin , Dettner Konrad , Kehl Siegfried
Published: March 2011

Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto).
Chilacis typhae
possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This “mycetocytic belt” might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the
groEL
genes showed that the bacterium belongs to the
Gammaproteobacteria
, and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of
Cimex lectularius
and free-living plant pathogens such as
Pectobacterium
and
Dickeya
. The distribution and ultrastructure of the rod-shaped
Chilacis
endosymbiont were studied in adults and nymph stages using fluorescence
in situ
hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, “
Candidatus
Rohrkolberia cinguli,” is proposed for this newly characterized clade of symbiotic bacteria.
The YaaA Protein of the Escherichia coli OxyR Regulon Lessens Hydrogen Peroxide Toxicity by Diminishing the Amount of Intracellular Unincorporated Iron
Authors: Liu Yuanyuan , Bauer Sarah C. , Imlay James A.
Published: March 2011

Hydrogen peroxide (H
2
O
2
) is commonly formed in microbial habitats by either chemical oxidation processes or host defense responses. H
2
O
2
can penetrate membranes and damage key intracellular biomolecules, including DNA and iron-dependent enzymes. Bacteria defend themselves against this H
2
O
2
by inducing a regulon that engages multiple defensive strategies. A previous microarray study suggested that
yaaA
, an uncharacterized gene found in many bacteria, was induced by H
2
O
2
in
Escherichia coli
as part of its OxyR regulon. Here we confirm that
yaaA
is a key element of the stress response to H
2
O
2
. In a catalase/peroxidase-deficient (Hpx
−
) background,
yaaA
deletion mutants grew poorly, filamented extensively, and lost substantial viability when they were cultured in aerobic LB medium. The results from a
thyA
forward mutagenesis assay and the growth defect of the
yaaA
deletion in a recombination-deficient (
recA56
) background indicated that
yaaA
mutants accumulated high levels of DNA damage. The growth defect of
yaaA
mutants could be suppressed by either the addition of iron chelators or mutations that slowed iron import, indicating that the DNA damage was caused by the Fenton reaction. Spin-trapping experiments confirmed that Hpx
−
yaaA
cells had a higher hydroxyl radical (HO
•
) level. Electron paramagnetic resonance spectroscopy analysis showed that the proximate cause was an unusually high level of intracellular unincorporated iron. These results demonstrate that during periods of H
2
O
2
stress the induction of YaaA is a critical device to suppress intracellular iron levels; it thereby attenuates the Fenton reaction and the DNA damage that would otherwise result. The molecular mechanism of YaaA action remains unknown.
Diversity of Expressed
vlhA
Adhesin Sequences and Intermediate Hemagglutination Phenotypes in Mycoplasma synoviae
Authors: May Meghan , Brown Daniel R.
Published: March 2011

A reservoir of pseudogene alleles encoding the primary adhesin VlhA occurs in the avian pathogen
Mycoplasma synoviae
. Recombination between this reservoir and its single expression site was predicted to result in lineages of
M. synoviae
that each express a different
vlhA
allele as a consequence of host immune responses to those antigens. Such interstrain diversity at the
vlhA
expression site, including major differences in the predicted secondary structures of their expressed adhesins, was confirmed in 14 specimens of
M. synoviae
. Corresponding functional differences in the extent to which they agglutinated erythrocytes, a quantitative proxy for VlhA-mediated cytadherence, were also evident. There was a >20-fold difference between the highest- and lowest-agglutinating strains and a rheostatic distribution of intermediate phenotypes among the others (Tukey-Kramer honestly significant difference [HSD],
P
< 0.001). Coincubation with the sialic acid analog 2-deoxy-2,3-didehydro-
N
-acetylneuraminate inhibited hemagglutination in a pattern correlated with endogenous sialidase activity (
r
= 0.91,
P
< 0.001), although not consistently to the same extent that erythrocyte pretreatment with sialidase purified from
Clostridium perfringens
did (
P
< 0.05). The striking correlation between the ranked hemagglutination and endogenous sialidase activities of these strains (Spearman's
r
= 0.874,
P
< 0.001) is evidence that host-induced
vlhA
allele switching indirectly drives sequence diversity in the passenger sialidase gene of
M. synoviae
.
Whole-Genome Sequencing of Staphylococcus aureus Strain RN4220, a Key Laboratory Strain Used in Virulence Research, Identifies Mutations That Affect Not Only Virulence Factors but Also the Fitness of the Strain
Authors: Nair Dhanalakshmi , Memmi Guido , Hernandez David , Bard Jonathan , Beaume Marie , Gill Steven , Francois Patrice , Cheung Ambrose L.
Published: March 2011

Staphylococcus aureus
RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.
Regulation and Characterization of the
dadRAX
Locus for
d
-Amino Acid Catabolism in Pseudomonas aeruginosa PAO1
Authors: He Weiqing , Li Congran , Lu Chung-Dar
Published: March 2011

d
-Amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In
Pseudomonas aeruginosa
, the
dadAX
operon, encoding the
d
-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for
d
- and
l
-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all
d
-amino acids tested as substrates except
d
-Glu and
d
-Gln. DadA also showed comparable
k
cat
and

K
m

values on
d
-Ala and several
d
-amino acids.
dadRAX
knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of
l
-Ala,
l
-Trp,
d
-Ala, and a specific set of
d
-amino acids as sole nitrogen sources was abolished in the
dadA
mutant and/or severely hampered in the
dadR
mutant while growth yield on
d
-amino acids was surprisingly improved in the
dadX
mutant. The
dadA
promoter was induced by several
l
-amino acids, most strongly by Ala, and only by
d
-Ala among all tested
d
-amino acids. Enhanced growth of the
dadX
mutant on
d
-amino acids is consistent with the finding that the
dadA
promoter was constitutively induced in the
dadX
mutant, where exogenous
d
-Ala but not
l
-Ala reduced the expression. Binding of DadR to the
dadA
regulatory region was demonstrated by electromobility shift assays, and the presence of
l
-Ala but not
d
-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the
dadA
regulatory region was demonstrated
in vitro
, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation
in vivo
. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed
d
-amino acid dehydrogenase activity of broad substrate specificity and the physiological functions of DadRAX in catabolism of several
d
-amino acids and support
l
-Ala as the signal molecule for induction of the
dadAX
genes through DadR binding to several putative operator sites.
Natural Transformation of Myxococcus xanthus
Authors: Wang Jing , Hu Wei , Lux Renate , He Xuesong , Li Yuezhong , Shi Wenyuan
Published: March 2011

Myxococcus xanthusbelongs to the delta class of the proteobacteria and is notable for its complex life-style with social behaviors and relatively large genome. Although previous observations have suggested the existence of horizontal gene transfer inM. xanthus, its ability to take up exogenous DNA via natural transformation has not been experimentally demonstrated. In this study, we achieved natural transformation inM. xanthususing the autonomously replicating myxobacterial plasmid pZJY41 as donor DNA.M. xanthusexopolysaccharide (EPS) was shown to be an extracellular barrier for transformation. Cells deficient in EPS production, e.g., mutant strains carrying ΔdifAor ΔepsA, became naturally transformable. Among the inner barriers to transformation were restriction-modification systems inM. xanthus, which could be partially overcome by methylating DNAin vitrousing cell extracts ofM. xanthusprior to transformation. In addition, the incubation time of DNA with cells and the presence of divalent magnesium ion affected transformation frequency ofM. xanthus. Furthermore, we also observed a potential involvement of the type IV pilus system in the DNA uptake machinery ofM. xanthus. The natural transformation was totally eliminated in the ΔpilQ/epsAand Δtgl/epsAmutants, and null mutation ofpilBorpilCin an ΔepsAbackground diminished the transformation rate. Our study, to the best of our knowledge, provides the first example of a naturally transformable species among deltaproteobacteria.
Identification of a Glycosyltransferase from Mycobacterium marinum Involved in Addition of a Caryophyllose Moiety in Lipooligosaccharides
Authors: Sarkar Debasmita , Sidhu Mandeep , Singh Albel , Chen Jiemin , Lammas David A. , van der Sar Astrid M. , Besra Gurdyal S. , Bhatt Apoorva
Published: March 2011

Deletion of
Mycobacterium marinum MMAR2333
resulted in the loss of three of four subclasses of lipooligosaccharides (LOSs). The mutant was unable to extend an intermediate (LOS-II*) by addition of caryophyllose. These data and the predicted domain structure suggest that MMAR2333 is a glycosyltransferase involved in the generation of a lipid-linked caryophyllose donor.
Genome Sequence of Neisseria meningitidis Serogroup B Strain H44/76
Authors: Piet J. R. , Huis in 't Veld R. A. G. , van Schaik B. D. C. , van Kampen A. H. C. , Baas F. , van de Beek D. , Pannekoek Y. , van der Ende A.
Published: March 2011

Neisseria meningitidis
is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.
YjbH-Enhanced Proteolysis of Spx by ClpXP in Bacillus subtilis Is Inhibited by the Small Protein YirB (YuzO)
Authors: Kommineni Sushma , Garg Saurabh K. , Chan Chio Mui , Zuber Peter
Published: March 2011

The Spx protein of
Bacillus subtilis
is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using
yjbH
as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity
in vivo
when overproduced from an isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible
yirB
construct. Pulldown experiments using extracts of
B. subtilis
cells producing a His-tagged YirB showed that native YjbH interacts with YirB in
B. subtilis
. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx.
In vitro
, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.
Effect of Amino Acid Substitutions in the GerAA Protein on the Function of the Alanine-Responsive Germinant Receptor of Bacillus subtilis Spores
Authors: Mongkolthanaruk Wiyada , Cooper Gareth R. , Mawer Julia S. P. , Allan Raymond N. , Moir Anne
Published: March 2011

Spores of
Bacillus subtilis
require the GerAA, GerAB, and GerAC receptor proteins for
l
-alanine-induced germination. Mutations in
gerAA
, both random and site directed, result in phenotypes that identify amino acid residues important for receptor function in broad terms. They highlight the functional importance of two regions in the central, integral membrane domain of GerAA. A P324S substitution in the first residue of a conserved PFPP motif results in a 10-fold increase in a spore's sensitivity to alanine; a P326S change results in the release of phase-dark spores, in which the receptor may be in an “activated” or “quasigerminated” state. Substitutions in residues 398 to 400, in a short loop between the last two likely membrane-spanning helices of this central domain, all affect the germination response, with the G398S substitution causing a temperature-sensitive defect. In others, there are wider effects on the receptor: if alanine is substituted for conserved residue N146, H304, or E330, a severe defect in
l
-alanine germination results. This correlates with the absence of GerAC, suggesting that the assembly or stability of the entire receptor complex has been compromised by the defect in GerAA. In contrast, severely germination-defective mutants such as E129K, L373F, S400F, and M409N mutants retain GerAC at normal levels, suggesting more local and specific effects on the function of GerAA itself. Further interpretation will depend on progress in structural analysis of the receptor proteins.
Complex Transcriptional Control of the Antibiotic Regulator
afsS
in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators
Authors: Santos-Beneit Fernando , Rodríguez-García Antonio , Martín Juan F.
Published: March 2011

The
afsS
gene of several
Streptomyces
species encodes a small sigma factor-like protein that acts as an activator of several pathway-specific regulatory genes (e.g.,
actII
-ORF4 and
redD
in
Streptomyces coelicolor
). The two pleiotropic regulators AfsR and PhoP bind to overlapping sequences in the −35 region of the
afsS
promoter and control its expression. Using mutated
afsS
promoters containing specific point mutations in the AfsR and PhoP binding sequences, we proved that the overlapping recognition sequences for AfsR and PhoP are displaced by 1 nucleotide. Different nucleotide positions are important for binding of AfsR or PhoP, as shown by electrophoretic mobility shift assays and by reporter studies using the
luxAB
gene coupled to the different promoters. Mutant promoter M5 (with a nucleotide change at position 5 of the consensus box) binds AfsR but not PhoP with high affinity (named “superAfsR”). Expression of the
afsS
gene from this promoter led to overproduction of actinorhodin. Mutant promoter M16 binds PhoP with extremely high affinity (“superPhoP”). Studies with Δ
afsR
and Δ
phoP
mutants (lacking AfsR and PhoP, respectively) showed that both global regulators are competitive transcriptional activators of
afsS
. AfsR has greater influence on expression of
afsS
than PhoP, as shown by reverse transcriptase PCR (RT-PCR) and promoter reporter (luciferase) studies. These two high-level regulators appear to integrate different nutritional signals (particularly phosphate limitation sensed by PhoR),
S
-adenosylmethionine, and other still unknown environmental signals (leading to AfsR phosphorylation) for the AfsS-mediated control of biosynthesis of secondary metabolites.
Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR
Authors: Hirakawa Hidetada , Oda Yasuhiro , Phattarasukol Somsak , Armour Christopher D. , Castle John C. , Raymond Christopher K. , Lappala Colin R. , Schaefer Amy L. , Harwood Caroline S. , Greenberg E. Peter
Published: March 2011

The
Rhodopseudomonas palustris
transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal
p
-coumaroyl-homoserine lactone (
p
C-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the
rpaI
promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by
p
C-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His
6
-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the
rpaI
transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although
p
C-HSL-RpaR bound to
rpaI
promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the
rpaI
operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is
p
C-HSL-RpaR-activated antisense transcription of
rpaR
. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.
DEAD-Box Proteins from Escherichia coli Exhibit Multiple ATP-Independent Activities
Authors: Zhao Xinliang , Jain Chaitanya
Published: March 2011

DEAD-box proteins (DBPs) are a widespread class of ATP-dependent RNA helicases that play a key role in unwinding RNA duplexes. In recent years, certain DBPs have also been found to exhibit activities that do not require ATP. To gain a better understanding of prokaryotic RNA metabolism, we investigated whether
Escherichia coli
DBPs harbor any ATP-independent activities. We show that each of the four
E. coli
DBPs tested in this study can accelerate the association of cRNA molecules, can stimulate strand displacement, and can function as an RNA chaperone without utilizing ATP. To the best of our knowledge, these prokaryotic DBPs constitute the first examples of proteins that harbor each of these three activities. The identification of these auxiliary functions indicates that the
E. coli
DBPs are versatile factors that possess significant RNA remodeling activity in addition to their canonical RNA helicase activity and might therefore participate in a greater variety of cellular processes than has been previously appreciated.
Characterization of Mutants Deficient in the
l,d
-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39
Authors: Barendt Skye M. , Sham Lok-To , Winkler Malcolm E.
Published: March 2011

Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as
Escherichia coli
and
Bacillus subtilis
, but remain relatively uncharacterized in ovococcus species, such as
Streptococcus pneumoniae
(pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of
S. pneumoniae
strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRK

Spn

two-component system regulon, which also contains the essential
pcsB
cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated
dacB
(
spd
_
0549
), which we showed encodes an
l,d
-carboxypeptidase involved in PG maturation. Notably,
dacB
mutants, similar to
dacA
(
d,d
-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in
d
-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRK

Spn

regulon gene
spd
_
0703
, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein.
Spontaneous calcium oscillations during diastole in the whole heart: the influence of ryanodine reception function and gap junction coupling
Authors: Plummer Bradley N. , Cutler Michael J. , Wan Xiaoping , Laurita Kenneth R.
Published: March 2011

Triggered arrhythmias due to spontaneous cytoplasmic calcium oscillations occur in a variety of disease conditions; however, their cellular mechanisms in tissue are not clear. We hypothesize that spontaneous calcium oscillations in the whole heart are due to calcium release from the sarcoplasmic reticulum and are facilitated by calcium diffusion through gap junctions. Optical mapping of cytoplasmic calcium from Langendorff perfused guinea pig hearts ( n = 10) was performed using oxygenated Tyrode's solution (in mM): 140 NaCl, 0.7 MgCl, 4.5 KCl, 5.5 dextrose, 5 HEPES, and 5.5 CaCl2 (pH 7.45, 34°C). Rapid pacing was used to induce diastolic calcium oscillations. In all preparations, pacing-induced multicellular diastolic calcium oscillations (m-SCR) occurred across most of the mapping field, at all pacing rates tested. Ryanodine (1 μM) eliminated all m-SCR activity. Low-dose caffeine (1 mM) increased m-SCR amplitude (+10.4 ± 4.4%, P < 0.05) and decreased m-SCR time-to-peak (−17.4 ± 6.7%, P < 0.05) and its temporal synchronization (i.e., range) across the mapping field (−26.9 ± 17.1%, P < 0.05). Surprisingly, carbenoxolone increased the amplitude of m-SCR activity (+14.8 ± 4.1%, P < 0.05) and decreased m-SCR time-to-peak (−11.3 ± 9.6%, P < 0.01) and its synchronization (−37.0 ± 19.1%, P < 0.05), similar to caffeine. In isolated myocytes, carbenoxolone (50 μM) had no effect on the frequency of aftercontractions, suggesting the effect of cell-to-cell uncoupling on m-SCR activity is tissue specific. Therefore, in the whole heart, overt m-SCR activity caused by calcium release from the SR can be induced over a broad range of pacing rates. Enhanced ryanodine receptor open probability and, surprisingly, decreased cell-to-cell coupling increased the amplitude and temporal synchronization of spontaneous calcium release in tissue.
Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia
Authors: Rastaldo Raffaella , Cappello Sandra , Folino Anna , Berta Giovanni N. , Sprio Andrea E. , Losano Gianni , Samaja Michele , Pagliaro Pasquale
Published: March 2011

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 μM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by NG-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 μM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 μM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area ( P < 0.001) and limited the postischemic myocardial contracture ( P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.
Tobacco-associated pulmonary vascular dysfunction in smokers: role of the ET-1 pathway
Authors: Henno Priscilla , Boitiaux Jean-François , Douvry Benoit , Cazes Aurélie , Lévy Marilyne , Devillier Philippe , Delclaux Christophe , Israël-Biet Dominique
Published: March 2011

Pulmonary vascular remodeling and dysfunction associated to tobacco smoking might pave the way for the subsequent development of pulmonary hypertension. Its prognosis is dreadful and its underlying mechanisms are so far largely unknown in humans. To assess the potential role of endothelin-1 and its receptors in smokers' pulmonary artery vasoactive properties. Endothelium-dependent vasodilation to ACh was assessed in pulmonary vascular rings from 34 smokers and compared with that of 10 nonsmokers. The effects of ET-A (BQ 123) or ET-B (BQ 788) blockers and that of an ET-B activator (sarafotoxin) were evaluated. Endothelin-1 was quantitated by ELISA. Expression of its receptors was quantitated by Western blotting. Smokers exhibited an impaired pulmonary endothelium-dependent vasodilation compared with nonsmokers ( P < 0.01). In the former group, 8 of 34 subjects exhibited a marked endothelial dysfunction (ED+) whereas 26 (ED−) ( P < 10−4) displayed a vasorelaxation to ACh that was comparable to that of nonsmokers. In ED+ subjects, ET-A was overexpressed ( P < 0.05) and inversely correlated ( P < 10−2) with the response to ACh. Sarafotoxin significantly improved vasodilation in all subjects ( P < 10−2). In conclusion, tobacco smoking is associated to an impaired pulmonary vasorelaxation at least partly mediated by an ET-1/ET-A-dependent dysfunction.
A role for decorin in a murine model of allergen-induced asthma
Authors: Marchica Cinzia L. , Pinelli Valentina , Borges Marcos , Zummer Jaryd , Narayanan Venkatesan , Iozzo Renato V. , Ludwig Mara S.
Published: March 2011

Decorin (Dcn) is an extracellular matrix proteoglycan, which affects airway mechanics, airway-parenchymal interdependence, airway smooth muscle proliferation and apoptosis, and transforming growth factor-β bioavailability. As Dcn deposition is differentially altered in asthma, we questioned whether Dcn deficiency would impact the development of allergen-induced asthma in a mouse model. Dcn−/−and Dcn+/+mice (C57Bl/6) were sensitized with ovalbumin (OA) and challenged intranasally 3 days/wk × 3 wk. After OA challenge, mice were anesthetized, and respiratory mechanics measured under baseline conditions and after delivery of increasing concentrations of methacholine aerosol. Complex impedance was partitioned into airway resistance and tissue elastance and damping. Bronchoalveolar lavage was performed. Lungs were excised, and tissue sections evaluated for inflammatory cell influx, α-smooth muscle actin, collagen, biglycan, and Dcn deposition. Changes in TH-2 cytokine mRNA and protein were also measured. Airway resistance was increased in OA-challenged Dcn+/+mice only ( P < 0.05), whereas tissue elastance and damping were increased in both OA-challenged Dcn+/+and Dcn−/−, but more so in Dcn+/+mice ( P < 0.001). Inflammation and collagen staining within the airway wall were increased with OA in Dcn+/+only ( P < 0.001 and P < 0.01, respectively, vs. saline). IL-5 and IL-13 mRNA were increased in lung tissue of OA-challenged Dcn+/+mice. Dcn deficiency resulted in more modest OA-induced hyperresponsiveness, evident at the level of the central airways and distal lung. Differences in physiology were accompanied by differences in inflammation and remodeling. These findings may be, in part, due to the well-described ability of Dcn to bind transforming growth factor-β and render it less bioavailable.
Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice
Authors: Bertok Szabolcs , Wilson Michael R. , Dorr Anthony D. , Dokpesi Justina O. , O'Dea Kieran P. , Marczin Nandor , Takata Masao
Published: March 2011

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.
Summary Verification Measures and Their Interpretation for Ensemble Forecasts
Authors: Bradley A. Allen , Schwartz Stuart S.
Published: March 2011

Ensemble prediction systems produce forecasts that represent the probability distribution of a continuous forecast variable. Most often, the verification problem is simplified by transforming the ensemble forecast into probability forecasts for discrete events, where the events are defined by one or more threshold values. Then, skill is evaluated using the mean-square error (MSE; i.e., Brier) skill score for binary events, or the ranked probability skill score (RPSS) for multicategory events. A framework is introduced that generalizes this approach, by describing the forecast quality of ensemble forecasts as a continuous function of the threshold value. Viewing ensemble forecast quality this way leads to the interpretation of the RPSS and the continuous ranked probability skill score (CRPSS) as measures of the weighted-average skill over the threshold values. It also motivates additional measures, derived to summarize other features of a continuous forecast quality function, which can be interpreted as descriptions of the function’s geometric shape. The measures can be computed not only for skill, but also for skill score decompositions, which characterize the resolution, reliability, discrimination, and other aspects of forecast quality. Collectively, they provide convenient metrics for comparing the performance of an ensemble prediction system at different locations, lead times, or issuance times, or for comparing alternative forecasting systems.
Glutamine Therapy Improves Outcome of In Vitro and In Vivo Experimental Colitis Models
Authors: Xue Hongyu , Sufit Alexandra J. D. , Wischmeyer Paul E.
Published: March 2011

Background: Pharmacologic doses of glutamine (GLN) can improve clinical outcome following acute illness and injury. Recent studies indicate enhanced heat shock protein (HSP) expression is a key mechanism underlying GLN's protection. However, such a link has not yet been tested in chronic inflammatory states, such as experimental inflammatory bowel disease (IBD). Methods: Experimental colitis was induced in Sprague‐Dawley rats via oral 5% dextran sulfate sodium (DSS) for 7 days. GLN (0.75 g/kg/d) or sham was administered to rats by oral gavage during 7‐day DSS treatment. In vitro inflammatory injury was studied using YAMC colonic epithelial cells treated with varying concentrations of GLN and cytokines (tumor necrosis factor–α/interferon‐γ). Results: Pharmacologic dose, bolus GLN attenuated DSS‐induced colitis in vivo with decreased area under curve for bleeding (8.06 ± 0.87 vs 10.38 ± 0.79, P < .05) and diarrhea (6.97 ± 0.46 vs 8.53 ± 0.39, P < .05). This was associated with enhanced HSP25 and HSP70 in colonic mucosa. In vitro, GLN enhanced cell survival and reduced proapoptotic caspase3 and poly(ADP‐ribose) polymerase cleavage postcytokine injury. Cytokine‐induced inducible nitric oxide synthase expression and nuclear translocation of nuclear factor‐κB p65 subunit were markedly attenuated at GLN concentrations above 0.5 mmol/L. GLN increased cellular HSP25 and HSP70 in a dose‐dependent manner. Conclusions: These data demonstrate the therapeutic potential of GLN as a “pharmacologically acting nutrient” in the setting of experimental IBD. GLN sufficiency is crucial for the colonic epithelium to mount a cell‐protective, antiapoptotic, and anti‐inflammatory response against inflammatory injury. The enhanced HSP expression observed following GLN treatment may be responsible for this protective effect.
Glutamine Restores Myocardial Cytochrome c Oxidase Activity and Improves Cardiac Function During Experimental Sepsis
Authors: Groening Portia , Huang Zhishan , La Gamma Edmund Frances , Levy Richard J.
Published: March 2011

Background: Cardiac dysfunction occurs commonly in sepsis. Impaired mitochondrial function is a potential cause of sepsis‐associated myocardial depression. Cytochrome oxidase (COX), the terminal oxidase of the electron transport chain, is inhibited in the septic heart. Glutamine (GLN) increases Krebs cycle intermediates and supports oxidative phosphorylation. Exogenous GLN has been shown to restore myocardial adenosine triphosphate levels and cardiac function following ischemia–reperfusion injury. The authors hypothesize that exogenous GLN will abrogate sepsis‐induced myocardial COX inhibition and improve sepsis‐associated myocardial depression. Methods: Under general anesthesia, male Sprague‐Dawley rats underwent cecal ligation and double puncture (CLP) or sham operation. At the time of operation, rats underwent intraperitoneal injection of either GLN (0.75 g/kg) or an equal volume of saline. Twenty‐four hours after the procedure, animals were killed, cardiac ventricles harvested, and mitochondria isolated. Steady‐state COX kinetic activity was measured and normalized to citrate synthase activity. Steady‐state levels of COX subunit I protein were determined with immunoblot analysis. Cardiac function was assessed using an isolated rat heart preparation. Five animals per group were evaluated. Significance was determined with analysis of variance and post hoc Tukey test. Results: CLP significantly decreased myocardial COX activity, oxygen consumption, left ventricular pressure (LVP), and pressure developed during isovolumic contraction (+dP/dt) and relaxation (−dP/dt). GLN restored COX activity to sham levels, significantly increased myocardial oxygen extraction and consumption, increased LVP toward sham values, and increased ±dP/dt by >30% following CLP. Conclusion: The beneficial effects of GLN therapy during sepsis may be in part due to restoration of oxidative phosphorylation and abrogation of sepsis‐associated myocardial depression.
Influence of Unclogging Agents on the Surface State of Enteral Feeding Tubes
Authors: Rucart Pierre‐Adelin , Boyer‐Grand Anne , Sautou‐Miranda Valérie , Bouteloup Corinne , Chopineau Jean
Published: March 2011

Background: Despite standardized prevention procedures, recalcitrant clogging of enteral feeding tubes is observed, which requires recourse to varied unclogging agents. Some of these agents have proved effective in routine use, but their impact on the surface state of the tube materials has never been studied. In this work, the authors tested the impact of different unclogging agents on the materials used for these tubes (polyurethane and silicone). Methods: Enteral feeding tubes were placed in contact with different agents in vitro, and the surface state of the material was analyzed using 2 different methods: infrared spectroscopy and scanning electron microscopy. To assess the surface state of the silicone and polyurethane tubes, negative controls (undamaged tubes) and positive controls (deliberately damaged tubes) were used for each type. Results: The infrared spectroscopy method did not reveal any damage to the surface of either the silicone or the polyurethane tubes with either treatment. The test results by scanning electron microscopy showed that orange juice, pineapple juice, and cola had no detrimental action on the tube biomaterials under current conditions of clinical practice. Conclusions: Although some studies have advocated using polyurethane tubes to administer medication, silicone appears to be less vulnerable to damage by the agents tested.
Vitamin B6 Deficiency
Authors: Gerlach Anthony T. , Thomas Sheela , Stawicki Stanislaw P. , Whitmill Melissa L. , Steinberg Steven M. , Cook Charles H.
Published: March 2011

Objective: In children, vitamin B6 (pyridoxine) deficiency has been described as a cause of seizures that are refractory to conventional antiepileptic medications. We describe the clinical presentation of 3 adults with refractory seizures (later diagnosed with vitamin B6 deficiency) that resolved after pyridoxine treatment. Design: Case series. Setting: Tertiary care surgical intensive care unit. Patients: In the first case, a 54‐year‐old male with history of alcoholic cirrhosis developed new‐onset seizures refractory to phenytoin and levetiracetam 8 days after liver transplantation. In the second case, a 59‐year‐old male with hepatitis C infection developed intracranial hemorrhage and new‐onset seizures refractory to phenytoin, levetiracetam, and pentobarbital. The third patient is a 78‐year‐old male with a history of alcohol dependence who was admitted for an intraventricular bleed and developed new onset of refractory seizures. Interventions: Intravenous pyridoxine followed by oral pyridoxine. Measurement and Main Results: In all 3 cases, seizures persisted despite escalation of conventional antiepileptic medications but resolved within 2 days of pyridoxine supplementation. In each case, low serum pyridoxal 5′‐phosphate concentrations normalized with pyroxidine administration. Conclusions: Although refractory seizures caused by vitamin B6 deficiency are rare in adults, it should be considered in critically ill adult patients with refractory seizures.