bluuebunny/crossref_metadata_2025 huggingface dataset:
Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria
Authors: Kaczmarczyk Andreas , Vorholt Julia A. , Francez-Charlot Anne
Published: August 2013

Tunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of the
Pseudomonas putida
F1
cym
/
cmt
system. The resulting promoter, termed P
Q5
, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in other
Alphaproteobacteria
, such as the model organisms
Caulobacter crescentus
,
Paracoccus denitrificans
, and
Methylobacterium extorquens
. In the noninduced state, expression from P
Q5
is low enough to allow gene depletion analysis, as demonstrated with the essential gene
phyP
of
Sphingomonas
sp. strain Fr1. A set of P
Q5
-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.
Prevalences of Shiga Toxin Subtypes and Selected Other Virulence Factors among Shiga-Toxigenic Escherichia coli Strains Isolated from Fresh Produce
Authors: Feng Peter C. H. , Reddy Shanker
Published: August 2013

Shiga-toxigenic
Escherichia coli
(STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have the
eae
gene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among the
eae
-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. The
ehxA
gene, which encodes enterohemolysin, was found in ∼60% of the isolates, and the
saa
and
subAB
genes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. The
stx
1a
and
stx
2a
subtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. The
stx
2d
subtype was the second most common subtype (28% overall), followed by
stx
2c
(7.5%), and only 2 to 3% of the produce STEC strains had the
stx
2e
and
stx
2g
subtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.
Modeling of Fungal and Bacterial Spore Germination under Static and Dynamic Conditions
Authors: Peleg Micha , Normand Mark D.
Published: August 2013

Isothermal germination curves, sigmoid and nonsigmoid, can be described by a variety of models reminiscent of growth models. Two of these, which are consistent with the percent of germinated spores being initially zero, were selected: one, Weibullian (or “stretched exponential”), for more or less symmetric curves, and the other, introduced by Dantigny's group, for asymmetric curves (P. Dantigny, S. P.-M. Nanguy, D. Judet-Correia, and M. Bensoussan, Int. J. Food Microbiol. 146:176–181, 2011). These static models were converted into differential rate models to simulate dynamic germination patterns, which passed a test for consistency. In principle, these and similar models, if validated experimentally, could be used to predict dynamic germination from isothermal data. The procedures to generate both isothermal and dynamic germination curves have been automated and posted as freeware on the Internet in the form of interactive Wolfram demonstrations. A fully stochastic model of individual and small groups of spores, developed in parallel, shows that when the germination probability is constant from the start, the germination curve is nonsigmoid. It becomes sigmoid if the probability monotonically rises from zero. If the probability rate function rises and then falls, the germination reaches an asymptotic level determined by the peak's location and height. As the number of individual spores rises, the germination curve of their assemblies becomes smoother. It also becomes more deterministic and can be described by the empirical phenomenological models.
A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System
Authors: Alperi Anabel , Larrea Delfina , Fernández-González Esther , Dehio Christoph , Zechner Ellen L. , Llosa Matxalen
Published: August 2013

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of
Bartonella henselae
, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of
B. henselae
. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a
Bartonella
intracellular delivery (BID) domain, the translocation signal for the
Bartonella
VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.
Regulation of Host Hemoglobin Binding by the Staphylococcus aureus Clp Proteolytic System
Authors: Farrand Allison J. , Reniere Michelle L. , Ingmer Hanne , Frees Dorte , Skaar Eric P.
Published: August 2013

Protein turnover is a key process for bacterial survival mediated by intracellular proteases. Proteolytic degradation reduces the levels of unfolded and misfolded peptides that accumulate in the cell during stress conditions. Three intracellular proteases, ClpP, HslV, and FtsH, have been identified in the Gram-positive bacteriumStaphylococcus aureus, a pathogen responsible for significant morbidity and mortality worldwide. Consistent with their crucial role in protein turnover, ClpP, HslV, and FtsH affect a number of cellular processes, including metabolism, stress responses, and virulence. The ClpP protease is believed to be the principal degradation machinery inS. aureus. This study sought to identify the effect of the Clp protease on theiron-regulatedsurfacedeterminant (Isd) system, which extracts heme-iron from host hemoglobin during infection and is critical toS. aureuspathogenesis. Inactivation of components of the Clp protease alters abundance of several Isd proteins, including the hemoglobin receptor IsdB. Furthermore, the observed changes in IsdB abundance are the result of transcriptional regulation, since transcription ofisdBis decreased byclpPorclpXinactivation. In contrast, inactivation ofclpCenhancesisdBtranscription and protein abundance. Loss ofclpPorclpXimpairs host hemoglobin binding and utilization and results in severe virulence defects in a systemic mouse model of infection. These findings suggest that the Clp proteolytic system is important for regulating nutrient iron acquisition inS. aureus. The Clp protease and Isd complex are widely conserved in bacteria; therefore, these data reveal a novel Clp-dependent regulation pathway that may be present in other bacterial pathogens.
ppGpp-Dependent Negative Control of DNA Replication of Shiga Toxin-Converting Bacteriophages in Escherichia coli
Authors: Nowicki Dariusz , Kobiela Wioletta , Węgrzyn Alicja , Węgrzyn Grzegorz , Szalewska-Pałasz Agnieszka
Published: August 2013

The pathogenicity of enterohemorrhagic
Escherichia coli
(EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acid-starved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an
E. coli
wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp
0
). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp
0
bacteria than on those formed by wild-type
E. coli
. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.
Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp
Authors: Smith Margaret C. M. , Hendrix Roger W. , Dedrick Rebekah , Mitchell Kaitlin , Ko Ching-Chung , Russell Daniel , Bell Emma , Gregory Matthew , Bibb Maureen J. , Pethick Florence , Jacobs-Sera Deborah , Herron Paul , Buttner Mark J. , Hatfull Graham F.
Published: August 2013

The genome sequences of eight
Streptomyces
phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating
Streptomyces
spp. We also extracted five apparently intact prophages from recent
Streptomyces
spp. genome projects and, together with six phage genomes in the database, we analyzed all 19
Streptomyces
phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the
Streptomyces
phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from
Streptomyces
sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The
Streptomyces venezuelae
generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different
Streptomyces
species.
Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent
Authors: Westbye A. B. , Leung M. M. , Florizone S. M. , Taylor T. A. , Johnson J. A. , Fogg P. C. , Beatty J. T.
Published: August 2013

The gene transfer agent of
Rhodobacter capsulatus
(RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about
cis
-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes
rcc00555
and
rcc00556
, which were expressed and studied in
Escherichia coli
to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of
rcc00555
and for maturation of RcGTA to yield gene transduction-functional particles.
Depletion of Glycolytic Intermediates Plays a Key Role in Glucose-Phosphate Stress in Escherichia coli
Authors: Richards Gregory R. , Patel Maulik V. , Lloyd Chelsea R. , Vanderpool Carin K.
Published: August 2013

In bacteria like
Escherichia coli
, the accumulation of glucose-6-phosphate (G6P) or its analogs such as α-methyl glucoside-6-phosphate (αMG6P) results in stress that appears in the form of growth inhibition. The small RNA SgrS is an essential part of the response that helps
E. coli
combat glucose-phosphate stress; the growth of
sgrS
mutants during stress caused by αMG is significantly impaired. The cause of this stress is not currently known but may be due to either toxicity of accumulated sugar-phosphates or to depletion of metabolic intermediates. Here, we present evidence that glucose-phosphate stress results from depletion of glycolytic intermediates. Addition of glycolytic compounds like G6P and fructose-6-phosphate rescues the αMG growth defect of an
sgrS
mutant. These intermediates also markedly decrease induction of the stress response in both wild-type and
sgrS
strains grown with αMG, implying that cells grown with these intermediates experience less stress. Moreover, αMG transport assays confirm that G6P relieves stress even when αMG is taken up by the cell, strongly suggesting that accumulated αMG6P
per se
does not cause stress. We also report that addition of pyruvate during stress has a novel lethal effect on the
sgrS
mutant, resulting in cell lysis. The phosphoenolpyruvate (PEP) synthetase PpsA, which converts pyruvate to PEP, can confer resistance to pyruvate-induced lysis when
ppsA
is ectopically expressed in the
sgrS
mutant. Taken as a whole, these results provide the strongest evidence thus far that depletion of glycolytic intermediates is at the metabolic root of glucose-phosphate stress.
Cyclic AMP Receptor Protein Regulates Pheromone-Mediated Bioluminescence at Multiple Levels in Vibrio fischeri ES114
Authors: Lyell Noreen L. , Colton Deanna M. , Bose Jeffrey L. , Tumen-Velasquez Melissa P. , Kimbrough John H. , Stabb Eric V.
Published: August 2013

Bioluminescence in
Vibrio fischeri
ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45–50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040–4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199–1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87–91, 1983). Consistent with this model, we found that bioluminescence in
V. fischeri
ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δ
crp
mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking
crp
were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only
luxR
but also the alternative pheromone synthase gene
ainS
. We found that His-tagged
V. fischeri
CRP could bind sequences upstream of both
luxR
and
ainS
, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the
ainS
or
luxR
system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic
Escherichia coli
, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the
luxI
promoter was enhanced by CRP. Second, the effect of CRP on the
ainS
promoter depended on whether the
V. fischeri
regulatory gene
litR
was also introduced. These results suggest an integral role for CRP in pheromone signaling that goes beyond sensing cell density.
Amino Acid Racemization in Pseudomonas putida KT2440
Authors: Radkov Atanas D. , Moe Luke A.
Published: August 2013

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organismPseudomonas putidaKT2440 to elaborate on the genomics and enzymology ofd-amino acid metabolism.P. putidaKT2440 catabolized thed-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized byP. putidaKT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterizedin vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and thekcat/Kmvalues withl- andd-lysine were 3 orders of magnitude greater than thekcat/Kmvalues withl- andd-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higherkcat/Kmvalues than Alr withl- andd-alanine, respectively. The annotated proline racemase ProR ofP. putidaKT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.
Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-σ
K
Authors: Zhang Yang , Luethy Paul M. , Zhou Ruanbao , Kroos Lee
Published: August 2013

Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates
Bacillus subtilis
endospore formation by cleaving Pro-σ
K
and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σ
K
(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in
Escherichia coli
, and proximity was tested by disulfide cross-linking
in vivo
. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity.
The Genome of Pseudomonas fluorescens Strain R124 Demonstrates Phenotypic Adaptation to the Mineral Environment
Authors: Barton Michael D. , Petronio Michael , Giarrizzo Juan G. , Bowling Bethany V. , Barton Hazel A.
Published: August 2013

Microbial adaptation to environmental conditions is a complex process, including acquisition of positive traits through horizontal gene transfer or the modification of existing genes through duplication and/or mutation. In this study, we examined the adaptation of aPseudomonas fluorescensisolate (R124) from the nutrient-limited mineral environment of a silica cave in comparison withP. fluorescensisolates from surface soil and the rhizosphere. Examination of metal homeostasis gene pathways demonstrated a high degree of conservation, suggesting that such systems remain functionally similar across chemical environments. The examination of genomic islands unique to our strain revealed the presence of genes involved in carbohydrate metabolism, aromatic carbon metabolism, and carbon turnover, confirmed through phenotypic assays, suggesting the acquisition of potentially novel mechanisms for energy metabolism in this strain. We also identified a twitching motility phenotype active at low-nutrient concentrations that may allow alternative exploratory mechanisms for this organism in a geochemical environment. Two sets of candidate twitching motility genes are present within the genome, one on the chromosome and one on a plasmid; however, a plasmid knockout identified the functional gene as being present on the chromosome. This work highlights the plasticity of thePseudomonasgenome, allowing the acquisition of novel nutrient-scavenging pathways across diverse geochemical environments while maintaining a core of functional stress response genes.
Streptomyces coelicolor Encodes a Urate-Responsive Transcriptional Regulator with Homology to PecS from Plant Pathogens
Authors: Huang Hao , Mackel Brian J. , Grove Anne
Published: August 2013

Many transcriptional regulators control gene activity by responding to specific ligands. Members of the multiple-antibiotic resistance regulator (MarR) family of transcriptional regulators feature prominently in this regard, and they frequently function as repressors in the absence of their cognate ligands. Plant pathogens such as
Dickeya dadantii
encode a MarR homolog named PecS that controls expression of a gene encoding the efflux pump PecM in addition to other virulence genes. We report here that the soil bacterium
Streptomyces coelicolor
also encodes a PecS homolog (
SCO2647
) that regulates a
pecM
gene (
SCO2646
).
S. coelicolor
PecS, which exists as a homodimer, binds the intergenic region between
pecS
and
pecM
genes with high affinity. Several potential PecS binding sites were found in this intergenic region. The binding of PecS to its target DNA can be efficiently attenuated by the ligand urate, which also quenches the intrinsic fluorescence of PecS, indicating a direct interaction between urate and PecS.
In vivo
measurement of gene expression showed that activity of
pecS
and
pecM
genes is significantly elevated after exposure of
S. coelicolor
cultures to urate. These results indicate that
S. coelicolor
PecS responds to the ligand urate by attenuated DNA binding
in vitro
and upregulation of gene activity
in vivo
. Since production of urate is associated with generation of reactive oxygen species by xanthine dehydrogenase, we propose that PecS functions under conditions of oxidative stress.
Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1
Authors: Bendall Matthew L. , Luong Khai , Wetmore Kelly M. , Blow Matthew , Korlach Jonas , Deutschbauer Adam , Malmstrom Rex R.
Published: August 2013

We performed whole-genome analyses of DNA methylation in
Shewanella oneidensis
MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that
S. oneidensis
MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in
S. oneidensis
MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in
Escherichia coli
. Furthermore, comparative analyses suggest that many
Gammaproteobacteria
, including all members of the
Shewanellaceae
family, may also utilize DNA methylation to regulate genome replication.
EutR Is a Direct Regulator of Genes That Contribute to Metabolism and Virulence in Enterohemorrhagic Escherichia coli O157:H7
Authors: Luzader Deborah H. , Clark David E. , Gonyar Laura A. , Kendall Melissa M.
Published: August 2013

Ethanolamine (EA) metabolism is a trait associated with enteric pathogens, including enterohemorrhagic
Escherichia coli
O157:H7 (EHEC). EHEC causes severe bloody diarrhea and hemolytic uremic syndrome. EHEC encodes the ethanolamine utilization (
eut
) operon that allows EHEC to metabolize EA and gain a competitive advantage when colonizing the gastrointestinal tract. The
eut
operon encodes the transcriptional regulator EutR. Genetic studies indicated that EutR expression is induced by EA and vitamin B
12
and that EutR promotes expression of the
eut
operon; however, biochemical evidence for these interactions has been lacking. We performed EA-binding assays and electrophoretic mobility shift assays (EMSAs) to elucidate a mechanism for EutR gene regulation. These studies confirmed EutR interaction with EA, as well as direct binding to the
eutS
promoter. EutR also contributes to expression of the locus of enterocyte effacement (LEE) in an EA-dependent manner. We performed EMSAs to examine EutR activation of the LEE. The results demonstrated that EutR directly binds the regulatory region of the
ler
promoter. These results present the first mechanistic description of EutR gene regulation and reveal a novel role for EutR in EHEC pathogenesis.
Substance P reduces TNF-α-induced apoptosis in human tenocytes through NK-1 receptor stimulation
Authors: Backman Ludvig J , Eriksson Daniella E , Danielson Patrik
Published: August 2013

Background

It has been hypothesised that an upregulation of the neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), is a causative factor in inducing tenocyte hypercellularity, a characteristic of tendinosis, through both proliferative and antiapoptotic stimuli. We have demonstrated earlier that SP stimulates proliferation of human tenocytes in culture.

Aim

The aim of this study was to investigate whether SP can mediate an antiapoptotic effect in tumour necrosis factor-α (TNF-α)-induced apoptosis of human tenocytes in vitro.

Results

A majority (approximately 75%) of tenocytes in culture were immunopositive for TNF Receptor-1 and TNF Receptor-2. Exposure of the cells to TNF-α significantly decreased cell viability, as shown with crystal violet staining. TNF-α furthermore significantly increased the amount of caspase-10 and caspase-3 mRNA, as well as both BID and cleaved-poly ADP ribosome polymerase (c-PARP) protein. Incubation of SP together with TNF-α resulted in a decreased amount of BID and c-PARP, and in a reduced lactate dehydrogenase release, as compared to incubation with TNF-α alone. The SP effect was blocked with a NK-1 R inhibitor.

Discussion

This study shows that SP, through stimulation of the NK-1 R, has the ability to reduce TNF-α-induced apoptosis of human tenocytes. Considering that SP has previously been shown to stimulate tenocyte proliferation, the study confirms SP as a potent regulator of cell-turnover in tendon tissue, capable of stimulating hypercellularity through different mechanisms. This gives further support for the theory that the upregulated amount of SP seen in tendinosis could contribute to hypercellularity.
Cognitive debiasing 2: impediments to and strategies for change
Authors: Croskerry Pat , Singhal Geeta , Mamede Sílvia
Published: August 2013

In a companion paper, we proposed that cognitive debiasing is a skill essential in developing sound clinical reasoning to mitigate the incidence of diagnostic failure. We reviewed the origins of cognitive biases and some proposed mechanisms for how debiasing processes might work. In this paper, we first outline a general schema of how cognitive change occurs and the constraints that may apply. We review a variety of individual factors, many of them biases themselves, which may be impediments to change. We then examine the major strategies that have been developed in the social sciences and in medicine to achieve cognitive and affective debiasing, including the important concept of forcing functions. The abundance and rich variety of approaches that exist in the literature and in individual clinical domains illustrate the difficulties inherent in achieving cognitive change, and also the need for such interventions. Ongoing cognitive debiasing is arguably the most important feature of the critical thinker and the well-calibrated mind. We outline three groups of suggested interventions going forward: educational strategies, workplace strategies and forcing functions. We stress the importance of ambient and contextual influences on the quality of individual decision making and the need to address factors known to impair calibration of the decision maker. We also emphasise the importance of introducing these concepts and corollary development of training in critical thinking in the undergraduate level in medical education.
Sign-out snapshot: cross-sectional evaluation of written sign-outs among specialties
Authors: Schoenfeld Amy R , Salim Al-Damluji Mohammed , Horwitz Leora I
Published: August 2013

Background

Sign-out is the process (written, verbal or both) by which one clinical team transmits information about patients to another team. Poor quality sign-outs are associated with adverse events and delayed treatment. How different specialties approach written sign-outs is unknown.

Objective

To compare written sign-out practices across specialties and to determine consistency of content, format and timeliness.

Methods

The authors evaluated all non-Intensive Care Unit written sign-outs from five inpatient specialties on 18 January 2012, at Yale-New Haven Hospital, focusing on content elements, format style and whether the sign-outs had been updated within 24 h. In our institution, all specialties used a single standardised sign-out template, which was built into the electronic medical record.

Results

The final cohort included 457 sign-outs: 313 medicine, 64 general surgery, 36 paediatrics, 30 obstetrics, and 14 gynaecology. Though nearly all sign-outs (96%) had been updated within 24 h, they frequently lacked key information. Hospital course prevalence ranged from 57% (gynaecology) to 100% (paediatrics) (p<0.001). Clinical condition prevalence ranged from 34% (surgery) to 72% (paediatrics) (p=0.005).

Conclusions

Specialties have varied sign-out practices, and thus structured templates alone do not guarantee inclusion of critical content. Sign-outs across specialties often lacked complex clinical information such as clinical condition, anticipatory guidance and overnight tasks.
Intraprocedural parenchymal blood volume as a marker of reperfusion status in acute ischemic stroke intervention
Authors: Elijovich Lucas , Doss Vinodh T , Theessen Heike , Khan Maheen , Arthur Adam S
Published: August 2013

Parenchymal blood volume (PBV) mapping with flat panel detectors may provide real-time estimates of tissue perfusion during endovascular ischemic stroke procedures. We present two cases of acute middle cerebral artery (MCA) occlusion to demonstrate how PBV may: (1) be used in acute stroke; (2) influence intraprocedural decision-making; and (3) potentially serve as a predicator of clinical outcome. Both cases were successfully recanalized with endovascular embolectomy. Intraprocedural PBV maps were obtained immediately before and after recanalization. Pre-intervention reductions in PBV were seen throughout the MCA territory in both cases, with significant improvement in PBV in one case with good radiographic and clinical outcome and a lack of improvement in PBV in the second case with a large infarct volume. PBV deficit normalization may occur with recanalization of the parent artery and probably represents successful reperfusion. Baseline PBV maps should therefore be interpreted with caution and not interpreted to represent irreversible core infarct.
Target delineation for radiosurgery of a small brain arteriovenous malformation using high-resolution contrast-enhanced cone beam CT
Authors: van der Bom Imramsjah M J , Gounis Matthew J , Ding Linda , Kühn Anna Luisa , Goff David , Puri Ajit S , Wakhloo Ajay K
Published: August 2013

Three years following endovascular embolization of a 3 mm ruptured arteriovenous malformation (AVM) of the left superior colliculus in a 42-year-old man, digital subtraction angiography showed continuous regrowth of the lesion. Thin-slice MRI acquired for treatment planning did not show the AVM nidus. The patient was brought back to the angiography suite for high-resolution contrast-enhanced cone beam CT (VasoCT) acquired using an angiographic c-arm system. The lesion and nidus were visualized with VasoCT. MRI, CT and VasoCT data were transferred to radiation planning software and mutually co-registered. The nidus was annotated for radiation on VasoCT data by an experienced neurointerventional radiologist and a dose/treatment plan was completed. Due to image registration, the treatment area could be directly adopted into the MRI and CT data. The AVM was completely obliterated 10 months following completion of the radiosurgery treatment.
Integrated bio-behavioural HIV surveillance surveys among female sex workers in Sudan, 2011–2012
Authors: Elhadi Magda , Elbadawi Abdulateef , Abdelrahman Samira , Mohammed Ibtisam , Bozicevic Ivana , Hassan Ehab A , Elmukhtar Mohammed , Ahmed Sally , Abdelraheem Mohammed Sidahmed , Mubarak Nazik , Elsanousi Salwa , Setayesh Hamidreza
Published: August 2013

Objectives

To assess HIV and syphilis prevalence, HIV-related behaviours and testing for HIV in female sex workers (FSW) in Sudan.

Design

Bio-behavioural surveys using respondent-driven sampling were carried out among FSW in the capital cities of 14 states in Sudan in 2011–2012. HIV and syphilis testing was done by rapid tests.

Results

4220 FSW aged 15–49 years were recruited. The median age of recruited women varied from 21 to 28 years per site. The highest HIV prevalence was measured at two sites in the eastern zone (5.0% and 7.7%), while in the other zones it ranged from 0% to 1.5%. Syphilis prevalence ranged from 1.5% in the northern zone to 8.9% in the eastern zone. Ever having been tested for HIV was reported by 4.4%–23.9% of FSW across all sites. Condom use at last sex with a client varied from 4.7% to 55.1%, while consistent condom use with clients in the month preceding the surveys was reported by 0.7%–24.5% of FSW. The highest reporting of ever injecting drugs was measured at a site in the western zone (5.0%).

Conclusions

The surveys’ findings indicate that the highest burden of HIV in FSW is in the eastern states of the country. Condom use and HIV testing data demonstrate the need for HIV interventions that should focus on HIV testing and risk reduction strategies that include stronger condom promotion programmes in FSW and their clients.
Exploring the mechanics of thunniform propulsion: a model study
Authors: Ben-Zvi Micha , Shadwick Robert E.
Published: August 2013

Thunniform propulsion is considered a case study in convergent evolution. Independently derived at least four times, it is characterized by uniquely high lift-based thrust and efficient performance. As such, it has been the focus of studies from biologists, engineers, and physicists. Unfortunately, direct physical measurements of this phenomenon are difficult to obtain. Therefore, the majority of research so far has consisted of theoretical modeling or experimental testing with models of low biofidelity. We created a test apparatus that would more accurately mimic thunniform propulsion as seen in the skipjack tuna (Katsuwonus pelamis (L., 1758)). Motion parameters and swimming speeds, as well as caudal fin size, shape, and material properties, were all taken into account and closely matched with in vivo measurements. Instantaneous lateral and in-flow forces were measured in tests over a range of motion regimes. Overall, general motion parameter requirements for thrust generation were determined and quantified. Thrust production, of up to 0.42 N (per whole caudal fin) with a coefficient of thrust of approximately 0.2, were in line with estimates of whole-body drag. Propulsive efficiency estimates were low (≤35%) compared with estimates in the literature of up to 90%. Quasi-static analysis was also conducted and shown to underpredict measured thrust values by up to 50%.
How competitive trade-offs limit elevation ranges for temperate-breeding hummingbirds
Authors: Feldman R.E. , McGill B.J.
Published: August 2013

One intriguing hypothesis about range limits of species along environmental gradients is that interspecific interference competition limits the activity of the better exploitation competitor. The hypothesis works if the costs of interference and (or) exploitation vary along the gradient. However, in some systems, species turnover happens over gradients that may be too short to induce changes in costs associated with competition. An example is breeding Black-chinned Hummingbirds (Archilochus alexandri (Bourcier and Mulsant, 1846)) and Broad-tailed Hummingbirds (Selasphorus platycercus (Swainson, 1827)) in western Colorado, USA, where turnover happens over ∼400 m. We recorded foraging and chasing activity of the two species at feeders and found that their foraging activity changed with elevation but interspecific competition did not. Because the foraging activity of the two species changed inversely with each other, it may be the presence of Black-chinned Hummingbirds rather than active interference that limits the activity of Broad-tailed Hummingbirds. Importantly, the relationship between foraging activity and elevation depended on the distance between the two feeders, which shows that relationships with elevation are contingent on other factors such as spatial distribution of resources. Our results suggest that interspecific differences in flight performance are not manifested over the short elevation gradient and do not account for changes in activity. Flight performance may indirectly affect patterns in breeding-season activity by influencing how species interact with other competitors during the nonbreeding season.
The impact of a moderate chronic temperature increase on spleen immune-relevant gene transcription depends on whether Atlantic cod (Gadus morhua) are stimulated with bacterial versus viral antigens
Authors: Hori Tiago S. , Gamperl A. Kurt , Nash Gord , Booman Marije , Barat Ashoktaru , Rise Matthew L.
Published: August 2013

Exposure to elevated temperature is an inherent feature of Atlantic cod (Gadus morhua) sea-cage culture in some regions (e.g., Newfoundland) and may also become an increasingly prevalent challenge for wild fish populations because of accelerated climate change. Therefore, understanding how elevated temperatures impacts the immune response of this commercially important species may help to reduce the potential negative impacts of such challenges. Previously, we investigated the impacts of moderately elevated temperature on the antiviral responses of Atlantic cod (Hori et al. 2012) and reported that elevated temperature modulated the spleen transcriptome response to polyriboinosinic polyribocytidylic acid (pIC, a viral mimic). Herein, we report a complementary microarray study that investigated the impact of the same elevated temperature regime on the Atlantic cod spleen transcriptome response to intraperitoneal (IP) injection of formalin-killed Aeromonas salmonicida (ASAL). Fish were held at two different temperatures (10 °C and 16 °C) prior to immune stimulation and sampled 6 and 24 h post-injection (HPI). In this experiment, we identified 711 and 666 nonredundant ASAL-responsive genes at 6HPI and 24HPI, respectively. These included several known antibacterial genes, including hepcidin, cathelicidin, ferritin heavy subunit, and interleukin 8. However, we only identified 15 differentially expressed genes at 6HPI and 2 at 24HPI (FDR 1%) when comparing ASAL-injected fish held at 10 °C versus 16 °C. In contrast, the same comparisons with pIC-injected fish yielded 290 and 339 differentially expressed genes (FDR 1%) at 6HPI and 24HPI, respectively. These results suggest that moderately elevated temperature has a lesser effect on the Atlantic cod spleen transcriptome response to ASAL (i.e., the antibacterial response) than to pIC (i.e., antiviral response). Thus, the impacts of high temperatures on the cod’s immune response may be pathogen dependent.
The role of mitochondrial bioenergetics and reactive oxygen species in coronary collateral growth
Authors: Pung Yuh Fen , Sam Wai Johnn , Hardwick James P. , Yin Liya , Ohanyan Vahagn , Logan Suzanna , Di Vincenzo Lola , Chilian William M.
Published: August 2013

Coronary collateral growth is a process involving coordination between growth factors expressed in response to ischemia and mechanical forces. Underlying this response is proliferation of vascular smooth muscle and endothelial cells, resulting in an enlargement in the caliber of arterial-arterial anastomoses, i.e., a collateral vessel, sometimes as much as an order of magnitude. An integral element of this cell proliferation is the process known as phenotypic switching in which cells of a particular phenotype, e.g., contractile vascular smooth muscle, must change their phenotype to proliferate. Phenotypic switching requires that protein synthesis occurs and different kinase signaling pathways become activated, necessitating energy to make the switch. Moreover, kinases, using ATP to phosphorylate their targets, have an energy requirement themselves. Mitochondria play a key role in the energy production that enables phenotypic switching, but under conditions where mitochondrial energy production is constrained, e.g., mitochondrial oxidative stress, this switch is impaired. In addition, we discuss the potential importance of uncoupling proteins as modulators of mitochondrial reactive oxygen species production and bioenergetics, as well as the role of AMP kinase as an energy sensor upstream of mammalian target of rapamycin, the master regulator of protein synthesis.
Tafazzin knockdown interrupts cell cycle progression in cultured neonatal ventricular fibroblasts
Authors: He Quan , Wang Miao , Harris Nicole , Han Xianlin
Published: August 2013

Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. Previous studies have shown that tafazzin knockdown promotes hypertrophy of neonatal cardiac myocytes. The current investigation was designed to show whether tafazzin knockdown affects cardiac fibroblast proliferation and collagen secretion, which contribute to fibrosis in dilated cardiomyopathy. In primary cultures of neonatal ventricular fibroblasts (NVFs) transduced with a tafazzin short hairpin RNA adenovirus, tafazzin knockdown increased production of reactive oxygen species and activation of mitogen-activated protein kinases and induced protein and DNA synthesis via cell cycle regulators. It also reduced intracellular ATP, activated AMPK, and caused multinucleation, hypertrophy, and enhanced collagen secretion. We concluded that tafazzin knockdown interrupts the NVF cell cycle and this in turn may contribute to fibrosis and dilated cardiomyopathy in Barth syndrome.
Oxidative stress in cardiomyocytes contributes to decreased SERCA2a activity in rats with metabolic syndrome
Authors: Balderas-Villalobos Jaime , Molina-Muñoz Tzindilu , Mailloux-Salinas Patrick , Bravo Guadalupe , Carvajal Karla , Gómez-Viquez Norma L.
Published: August 2013

Ca+mishandling due to impaired activity of cardiac sarco(endo)plasmic reticulum Ca2+ATPase (SERCA2a) has been associated with the development of left ventricular diastolic dysfunction in insulin-resistant cardiomyopathy. However, the molecular causes underlying SERCA2a alterations induced by insulin resistance and related metabolic disorders, such as metabolic syndrome (MetS), are not completely understood. In this study, we used a sucrose-fed rat model of MetS to test the hypothesis that decreased SERCA2a activity is mediated by elevated oxidative stress produced in the MetS heart. Production of ROS and cytosolic Ca2+concentration were recorded in left ventricular myocytes using confocal imaging. The level of SERCA2a oxidation was determined in left ventricular homogenates by biotinylated iodoacetamide labeling. Compared with control rats, sucrose-fed rats exhibited several characteristics of MetS, including central obesity, insulin resistance, hyperinsulinemia, and hypertriglyceridemia. Moreover, relative to myocytes from control rats, myocytes from MetS rats exhibited elevated basal production of ROS accompanied by slowed cytosolic Ca2+removal, reflected by prolonged Ca2+transients. The slowed cytosolic Ca2+removal was associated with a significant decrease in SERCA2a-mediated Ca2+reuptake and increased SERCA2a oxidation. Importantly, myocytes from MetS rats treated with the antioxidant N-acetylcysteine showed normal ROS levels and SERCA2a-mediated Ca2+reuptake as well as accelerated cytosolic Ca2+removal. These data suggest that elevated oxidative stress may induce oxidative modifications on SERCA2a leading to abnormal function of this protein in the MetS heart.