bluuebunny/crossref_metadata_2025 huggingface dataset:
Abundance of Novel and Diverse
tfdA
-Like Genes, Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases, in Soil
Authors: Zaprasis Adrienne , Liu Ya-Jun , Liu Shuang-Jiang , Drake Harold L. , Horn Marcus A.
Published: October 2009

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe
2+
-dependent dioxygenases encoded by
tfdA
-like genes (i.e.,
tfdA
and
tfdA
α). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of
tfdA
-like genes in soil. Five primer sets targeting
tfdA
-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified
tfdA
-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated
tfdA
-like genes indicated that soil
tfdA
-like genes were related to those of group 2 and 3
Bradyrhizobium
spp.,
Sphingomonas
spp., and uncultured soil bacteria. Soil-derived
tfdA
-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse
tfdA
-like genes. Correlation analysis of 16S rRNA and
tfdA
-like gene similarity indicated that any two bacteria with
D
> 20% of group 2
tfdA
-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2
tfdA
-like genes. Novel qPCR assays were established to quantify such new
tfdA
-like genes. Copy numbers of
tfdA
-like genes were 1.0 × 10
6
to 65 × 10
6
per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse
tfdA
-like genes are abundant in soils.
Aromatic Hydroxylation of Indan by
o
-Xylene-Degrading
Rhodococcus
sp. Strain DK17
Authors: Kim Dockyu , Lee Choong Hwan , Choi Jung Nam , Choi Ki Young , Zylstra Gerben J. , Kim Eungbin
Published: October 2009

The metabolically versatile
Rhodococcus
sp. strain DK17 utilizes indan as a growth substrate via the
o
-xylene pathway. Metabolite and reverse transcription-PCR analyses indicate that
o
-xylene dioxygenase hydroxylates indan at the 4,5 position of the aromatic moiety to form
cis
-indan-4,5-dihydrodiol, which is dehydrogenated to 4,5-indandiol by a dehydrogenase. 4,5-Indandiol undergoes ring cleavage by a
meta
-cleavage dioxygenase.
Crucial Role for Insertion Sequence Elements in
Lactobacillus helveticus
Evolution as Revealed by Interstrain Genomic Comparison
Authors: Kaleta Pawel , O'Callaghan John , Fitzgerald Gerald F. , Beresford Thomas P. , Ross R. Paul
Published: October 2009

Lactobacillus helveticus
is a versatile dairy bacterium found to possess heterogeneous genotypes depending on the ecosystem from which it was isolated. The recently published genome sequence showed the remarkable flexibility of its structure, demonstrated by a substantial level of insertion sequence (IS) element expansion in association with massive gene decay. To assess this diversity and examine the level of genome plasticity within the
L. helveticus
species, an array-based comparative genome hybridization (aCGH) experiment was designed in which 10 strains were analyzed. The aCGH experiment revealed 16 clusters of open reading frames (ORFs) flanked by IS elements. Four of these ORFs are associated with restriction/modification which may have played a role in accelerated evolution of strains in a commercially intensive ecosystem undoubtedly challenged through successive phage attack. Furthermore, analysis of the IS-flanked clusters demonstrated that the most frequently encountered ISs were also those most abundant in the genome (IS
1201
, ISL
2
, ISLhe
1
, ISLhe
2
, ISLhe
65
, and ISLhe
63
). These findings contribute to the overall viewpoint of the versatile character of IS elements and the role they may play in bacterial genome plasticity.
CLM1ofFusarium graminearumEncodes a Longiborneol Synthase Required for Culmorin Production
Authors: McCormick S. P. , Alexander N. J. , Harris L. J.
Published: October 2009

Fusarium graminearumis a fungal pathogen of cereal crops (e.g., wheat, barley, maize) and produces a number of mycotoxins, including 15-acetyldeoxynivalenol, butenolide, zearalenone, and culmorin. To identify a biosynthetic gene for the culmorin pathway, an expressed-sequence-tag database was examined for terpene cyclase genes. A gene designatedCLM1was expressed under trichothecene-inducing conditions. Expression ofCLM1in yeast (Saccharomyces cerevisiae) resulted in the production of a sesquiterpene alcohol, longiborneol, which has the same ring structure as culmorin. Gene disruption and add-back experiments inF. graminearumshowed thatCLM1was required for culmorin biosynthesis.CLM1gene disruptants were able to convert exogenously added longiborneol to culmorin. Longiborneol accumulated transiently in culmorin-producing strains. The results indicate thatCLM1encodes a longiborneol synthase and is required for culmorin biosynthesis inF. graminearum.
“CandidatusCurculioniphilus buchneri,” a Novel Clade of Bacterial Endocellular Symbionts from Weevils of the GenusCurculio
Authors: Toju Hirokazu , Hosokawa Takahiro , Koga Ryuichi , Nikoh Naruo , Meng Xian Ying , Kimura Nobutada , Fukatsu Takema
Published: October 2009

Here we investigated the bacterial endosymbionts of weevils of the genusCurculio. From all four species ofCurculioweevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in theGammaproteobacteria, which was not related to previously known groups of weevil endosymbionts such asNardonellaspp. andSodalis-allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont ofCurculioweevils and proposed the designation “CandidatusCurculioniphilus buchneri.” In addition to “Ca.Curculioniphilus,” we identifiedSodalis-allied gammaproteobacterial endosymbionts from the chestnut weevil,Curculio sikkimensis, which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that suchSodalis-allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor ofSitophilusgrain weevils.
Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in
Methylophilus methylotrophus
AS1 Cells Expressing the
aroP
-Encoded Transporter of
Escherichia coli
Authors: Yomantas Yurgis A. V. , Tokmakova Irina L. , Gorshkova Natalya V. , Abalakina Elena G. , Kazakova Svetlana M. , Gak Evgueni R. , Mashko Sergey V.
Published: October 2009

The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium
Methylophilus methylotrophus
AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the
Escherichia coli
gene
aroP
, which encodes the common aromatic amino acid transporter, into the genome of
M. methylotrophus
. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp.
M. methylotrophus
AS1 genes
trpE
,
tyrA
,
pheA
, and
aroG
were cloned in
E. coli
, sequenced, disrupted in vitro using a Km
r
marker, and electroporated into an
aroP
carrier recipient strain. This approach led to the construction of a set of marker-less
M. methylotrophus
AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.
7-Ketocholesterol Catabolism by
Rhodococcus jostii
RHA1
Authors: Mathieu Jacques M. , Mohn William W. , Eltis Lindsay D. , LeBlanc Justin C. , Stewart Gord R. , Dresen Carola , Okamoto Kenji , Alvarez Pedro J. J.
Published: October 2009

Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by
Rhodococcus jostii
RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.
Organization and Evolutionary Trajectory of the Mating Type (MAT) Locus in Dermatophyte and Dimorphic Fungal Pathogens
Authors: Li Wenjun , Metin Banu , White Theodore C. , Heitman Joseph
Published: October 2009

Sexual reproduction in fungi is governed by a specialized genomic region, the mating type (MAT) locus, whose gene identity, organization, and complexity are diverse. We identified theMATlocus of five dermatophyte fungal pathogens (Microsporum gypseum,Microsporum canis,Trichophyton equinum,Trichophyton rubrum, andTrichophyton tonsurans) and a dimorphic fungus,Paracoccidioides brasiliensis, and performed phylogenetic analyses. The identifiedMATlocus idiomorphs ofM. gypseumcontrol cell type identity in mating assays, and recombinant progeny were produced. Virulence tests inGalleria mellonellalarvae suggest the two mating types ofM. gypseummay have equivalent virulence. Synteny analysis revealed common features of theMATlocus shared among these five dermatophytes: namely, a small size (∼3 kb) and a novel gene arrangement. TheSLA2,COX13, andAPN2genes, which flank theMATlocus in otherAscomycotaare instead linked on one side of the dermatophyteMATlocus. In addition, the transcriptional orientations of theAPN2andCOX13genes are reversed compared to the dimorphic fungiHistoplasma capsulatum,Coccidioides immitis, andCoccidioides posadasii. A putative transposable element,pogo, was found to have inserted in theMAT1-2idiomorph of oneP. brasiliensisstrain but not others. In conclusion, the evolution of theMATlocus of the dermatophytes and dimorphic fungi from the last common ancestor has been punctuated by both gene acquisition and expansion, and asymmetric gene loss. These studies further support a foundation to develop molecular and genetic tools for dermatophyte and dimorphic human fungal pathogens.
Campylobacter jejuni
FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence
Authors: Konkel Michael E. , Larson Charles L. , Flanagan Rebecca C.
Published: October 2009

Campylobacter jejuni
is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of
C. jejuni
to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of
C. jejuni flpA
(Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of
C. jejuni
to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in
C. jejuni
binding to host cells by in vitro adherence assays with a
C. jejuni
wild-type strain and a
C. jejuni flpA
mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the
C. jejuni flpA
mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked
C. jejuni
adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates
C. jejuni
attachment to host epithelial cells via Fn binding.
Maturation of 23S rRNA inBacillus subtilisin the Absence of Mini-III
Authors: Redko Yulia , Condon Ciarán
Published: October 2009

23S rRNA maturation inBacillus subtilisis catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5′-to-3′ exoribonuclease RNase J1 and the 3′-to-5′ exoribonucleases, principally RNase PH and YhaM.
Isolation of Basal Bodies with C-Ring Components from the Na
+
-Driven Flagellar Motor of
Vibrio alginolyticus
Authors: Koike Masafumi , Terashima Hiroyuki , Kojima Seiji , Homma Michio
Published: October 2009

To investigate the Na
+
-driven flagellar motor of
Vibrio alginolyticus
, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.
HspR Mutations Are Naturally Selected in
Bifidobacterium longum
When Successive Heat Shock Treatments Are Applied
Authors: Berger B. , Moine D. , Mansourian R. , Arigoni F.
Published: October 2009

The development of molecular tools allowed light to be shed on several widespread genetic mechanisms aiming at limiting the effect of molecular damage on bacterial survival. For some bacterial taxa, there are limited tools in the genetic toolbox, which restricts the possibilities to investigate the molecular basis of their stress response. In that case, an alternative strategy is to study genetic variants of a strain under stress conditions. The comparative study of the genetic determinants responsible for their phenotypes, e.g., an improved tolerance to stress, offers precious clues on the molecular mechanisms effective in this bacterial taxon. We applied this approach and isolated two heat shock-tolerant strains derived from
Bifidobacterium longum
NCC2705. A global analysis of their transcriptomes revealed that the
dnaK
operon and the
clpB
gene were overexpressed in both heat shock-tolerant strains. We sequenced the
hspR
gene coding for the negative regulator of
dnaK
and
clpB
and found point mutations affecting protein domains likely responsible for the binding of the regulators to the promoter DNA. Complementation of the mutant strains by the wild-type regulator
hspR
restored its heat sensitivity and thus demonstrated that these mutations were responsible for the observed heat tolerance phenotype.
Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme, Involved in the Late Steps of Coenzyme B
12
Biosynthesis in
Salmonella enterica
Authors: Claas Kathy R. , Parrish J. R. , Maggio-Hall L. A. , Escalante-Semerena J. C.
Published: October 2009

In
Salmonella enterica
, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M
cobT
mutations were deleterious, we determined the nature of three class M
cobT
alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT
WT
) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT
WT
. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M
cobT
alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.
Linear Plasmid SLP2 Is Maintained by Partitioning, Intrahyphal Spread, and Conjugal Transfer inStreptomyces
Authors: Hsu Chin-Chen , Chen Carton W.
Published: October 2009

Low-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids inStreptomyces. SLP2 is a 50-kb low-copy-number linear plasmid inStreptomyces lividans, which contains a typicalparABpartitioning operon. InS. lividansandStreptomyces coelicolor, aparABdeletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-containing hyphae to plasmidless hyphae. Deletion of the transfer (traB) gene eliminated conjugal transfer, lessened the growth retardation of colonies, and increased plasmid loss through sporulation cycles. The additional deletion of an intrahyphal spread gene (spd1) caused almost complete plasmid loss in a sporulation cycle and eliminated all growth retardation. Moreover, deletion ofspd1alone severely reduced conjugal transfer and stability of SLP2 inS. coelicolorM145 but had no effect onS. lividansTK64. These results revealed the following three systems for SLP2 maintenance: partitioning and spread for moving the plasmid DNA along the hyphae and into spores and conjugal transfer for rescuing plasmidless hyphae. InS. lividans, both spread and partitioning appear to overlap functionally, but inS. coelicolor, spread appears to play the main role.
Discovery and Characterization of Three NewEscherichia coliSeptal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA
Authors: Arends S. J. Ryan , Williams Kyle , Scott Renada J. , Rolong Silvana , Popham David L. , Weiss David S.
Published: October 2009

SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins fromEscherichia coliandCaulobacter crescentushave been shown to bind peptidoglycan. Besides FtsN,E. colihas three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring inE. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, includingAquifex aeolicus, localized to septal rings when produced inE. coliand bound to purifiedE. colipeptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.
A Type VI Secretion System Effector Protein, VgrG1, from
Aeromonas hydrophila
That Induces Host Cell Toxicity by ADP Ribosylation of Actin
Authors: Suarez G. , Sierra J. C. , Erova T. E. , Sha J. , Horneman A. J. , Chopra A. K.
Published: October 2009

We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of
Aeromonas hydrophila
and showed that VasH, a σ
54
activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of
A. hydrophila
, namely, a Δ
act
mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Δ
act
Δ
vasH
mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the
vgrG1
gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and
Escherichia coli
, respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of
A. hydrophila
possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.
Competitive Inhibitions of the Chlorophyll Synthase of
Synechocystis
sp. Strain PCC 6803 by Bacteriochlorophyllide
a
and the Bacteriochlorophyll Synthase of
Rhodobacter sphaeroides
by Chlorophyllide
a
Authors: Kim Eui-Jin , Lee Jeong K.
Published: October 2009

The photosynthetic growth of
Synechocystis
sp. strain PCC 6803 is hampered by exogenously added bacteriochlorophyllide
a
(Bchlide
a
) in a dose-dependent manner. The growth inhibition caused by Bchlide
a
, however, is relieved by an increased level of exogenously added chlorophyllide
a
(Chlide
a
). The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide
a
, with inhibition constants (
K
I
s) of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of
Rhodobacter sphaeroides
is inhibited competitively by Chlide
a
, with
K
I
s of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, exogenously added Chlide
a
inhibits the metabolic conversion of exogenously added Bchlide
a
to bacteriochlorophyll
a
by an
R. sphaeroides bchFNB-bchZ
mutant that neither synthesizes nor metabolizes Chlide
a
. The metabolic inhibition by Chlide
a
, however, is relieved by the elevated level of Bchlide
a
. Thus, the chlorophyll synthase of
Synechocystis
sp. PCC 6803 and the bacteriochlorophyll synthase of
R. sphaeroides
, both of which perform ping-pong-type reactions, are inhibited by Bchlide
a
and Chlide
a
, respectively. Although neither inhibitor is catalyzed by the target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites.
Computational fluid dynamic simulation to assess flow characteristics of an in vitro aneurysm model
Authors: Lott D A , Siegel M , Chaudhry H R , Prestigiacomo C J
Published: October 2009

Background

Modifications of in vitro aneurysm modeling to study the effects of morphology on flow dynamics are time consuming, costly and analysis tends to be more qualitative than quantitative. This study develops a virtual two-dimensional flow model replicating an in vitro aneurysm model and analyzes how changes in morphology modify flow characteristics.

Methods

Using finite volume analysis, a two-dimensional saccular aneurysm model was created with a configuration matching a published, experimental, in vitro model. Qualitative comparisons were made determining whether a two-dimensional fluid dynamic model can replicate the results of an in vitro model. Quantitative changes in flow patterns, wall shear stress, dynamic pressure and maximum velocities were assessed by modifying the shape of the neck and proximal dome without modifying the overall size of the aneurysm.

Results

A two-dimensional computational fluid dynamic model reproducing the shape of a published aneurysm demonstrated excellent qualitative fidelity to an in vitro flow model. Additional information regarding dynamic pressure, shear stress and velocity along the aneurysm neck and within the aneurysm dome were determined. Although all dimensions were kept constant, slight modifications of the neck and proximal dome resulted in quantitative changes in studied parameters, such as wall shear stress and dynamic pressure.

Conclusions

Computer generated aneurysm flow models, when carefully developed, reproduce flow events within in vitro aneurysms providing objective data on biophysical parameters. Effective flow modeling of aneurysms depends on flow input, size of the parent vessel and aneurysm, and other factors. These data suggest that neck and proximal dome configuration, independent of size, are important characteristics of flow.
Balloon test occlusion and endosurgical parent artery sacrifice for the evaluation and management of complex intracranial aneurysmal disease
Authors: Lesley W S , Rangaswamy R
Published: October 2009

The lifetime risk of developing a cerebral aneurysm is about 5%. For some patients, aneurysms can be reasonably managed by conservative measures, including periodic clinical and imaging surveillance. However, the definitive treatment of cerebral aneurysm disease requires securing the aneurysm by surgically excluding it from the cerebrovascular circulation. Most commonly, this can be achieved by either open surgical clipping or embosurgery. Unfortunately, for a minority of patients, these interventions are inadequate because of many aneurysmal factors, such as complex anatomy, giant and wide neck aneurysmal morphology, peripheral small branch origin and skull base location. In situations like these, sacrifice of the parent artery may be preferable, especially when clinical tolerance or image based vascular reserve can be preoperatively demonstrated during temporary occlusion of the vessel. This preoperative procedure, which is known as the Balloon Test Occlusion (BTO), has several variations and technical nuances that can assist the surgeon in predicting which patients may best benefit from parent artery sacrifice (PAS). Together, BTO and PAS are invaluable tools in the management of complicated and atypical cerebral aneurysms. With regard to cerebrovascular aneurysm disease, this review will summarize the development of these procedures, condense the predictability of the numerous BTO variations and provide an overview of the currently available PAS techniques.
Balloon assisted treatment of intracranial aneurysms: the conglomerate coil mass technique
Authors: Fiorella D , Woo H H
Published: October 2009

Endovascular techniques for the treatment of intracranial aneurysms have rapidly evolved over the past 15 years since the introduction and subsequent US Food and Drug administration approval of the Gugleilmi detachable coil. During this period, a number of different coil designs and adjunctive devices have been developed to facilitate the treatment of more complex and challenging cerebral aneurysms. One such adjunctive device, the hypercompliant occlusion balloon, can be temporarily inflated during the delivery of embolization coils to prevent their prolapse into the parent vessel. This technique, known as balloon assisted treatment (BAT), remains somewhat controversial as many operators do not incorporate this approach into their practice, favoring stent supported techniques instead. Moreover, those operators who do practice BAT use a variety of different approaches. In this review, we discuss the theoretical concepts underlying BAT, the potential advantages and disadvantages of this approach and finally the technical evolution of BAT in our endovascular practice.
The impact of timing on outcomes of carotid artery stenting in recently symptomatic patients
Authors: Lin R , Mazighi M , Yadav J , Abou-Chebl A
Published: October 2009

Objectives

We sought to determine whether the timing of carotid artery stenting (CAS) affects peri-procedural outcomes in recently symptomatic patients.

Background

Early carotid endarterectomy following a stroke lowers the risk of recurrent ischemic events but has been associated with an increased risk of intracerebral hemorrhage. The optimal timing of CAS following a stroke is unknown.

Methods

Using a single-center prospective CAS registry, we retrospectively analyzed data on consecutive CAS procedures over an 8-year period. Patients were separated into early (≤4 weeks) and late (>4 weeks) treatment groups based on time to CAS from symptom onset. Post-procedural hypertension was treated. Thirty-day incidence of stroke, intracerebral hemorrhage, myocardial infarction and death were recorded.

Results

A total of 224 patients were treated for symptomatic lesions. The mean age was 71±10 years, and 63% were men. The median time for CAS was 8 days (0.2–30 days) and 90 days (31–180 days) respectively, for the early (n=122) and late (n=102) intervention groups. There were 4 (3.45%) periprocedural strokes in the early and 5 (5.95%) in the late CAS group (p=0.5). There was one hyperperfusion syndrome in each group. The 30-day stroke, myocardial infarction or death rates were similar between the early (6.03%) and late (8.33%) CAS groups, p=0.58. A multivariate analysis showed that age >80 years rather than timing of CAS was associated with adverse events.

Conclusions

CAS may be performed safely immediately following non-disabling strokes. Octogenarians may be at higher risk of complications.
The use of a covered stent graft for obliteration of high-flow carotid cavernous fistula presenting with life-threatening epistaxis
Authors: Kalia J S , Niu T , Zaidat O O
Published: October 2009

Background

We present a rare complication of trans-sphenoidal adenectomy (TSA) for pituitary macroadenoma: carotid cavernous fistula (CCF) that was treated with endovascular therapy. The incidence of internal carotid artery (ICA) injury following TSA is 1% and may spontaneously heal by packing and rarely manifest as symptomatic CCF/aneurysm. Treatment of post-TSA CCF may be challenging due to the breach of nasal floor and may be prone to recurrence.

Presentation/intervention

Uncontrolled intra-operative bleeding during a TSA led to an emergent angiogram to show slow-flow left CCF. Due to clinical deterioration with nasal bleeding, angiography was repeated after 4 h; the fistula had transformed into high flow with significant increase in size, and was therefore embolized using stent-assisted coiling. The fistula recanalized in a month with massive epistaxis and was re-treated using a covered stent graft.

Conclusion

This case represents several unique learning points: (1) CCF as a complication of TSA due to close anatomical proximity; (2) the role of endovascular management post-TSA complication; (3) stent-assisted coil embolization of high-flow fistula with moderate ICA laceration; (4) recanalization of CCF causing massive epistaxis; (5) rare use of covered stent graft stent in distal intracranial circulation maintaining integrity and patency of ICA; (6) long-term results after covered stent graft with no in-stent restenosis.
PKA, Rap1, ERK1/2, and p90RSK mediate PGE2and EP4 signaling in neonatal ventricular myocytes
Authors: He Quan , Harding Pamela , LaPointe Margot C.
Published: October 2009

We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE2production reduces hypertrophy after myocardial infarction in mice and 2) PGE2acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE2activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE2activation of ERK1/2. PGE2activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE2regulation of gene expression. PGE2stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE2stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE2regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos.
Nitric oxide is not obligatory for radial artery flow-mediated dilation following release of 5 or 10 min distal occlusion
Authors: Pyke Kyra , Green Daniel J. , Weisbrod Cara , Best Matthew , Dembo Lawrence , O'Driscoll Gerry , Tschakovsky Michael
Published: October 2009

This study investigated the nitric oxide (NO) dependence of radial artery (RA) flow-mediated dilation (FMD) in response to three different reactive hyperemia (RH) shear stimulus profiles. Ten healthy males underwent the following three RH trials: 1) 5 min occlusion (5 trial), 2) 10 min occlusion (10 trial), and 3) 10 min occlusion with cuff reinflation at 30 s (10–30 trial). Trials were performed during saline infusion and repeated during NG-monomethyl-l-arginine (l-NMMA) infusion in the brachial artery. RA blood flow velocity was measured with Doppler ultrasound, and B-mode RA images were analyzed using automated edge detection software. Shear rate estimation of shear stress was calculated as the blood flow velocity/vessel diameter. l-NMMA decreased baseline vascular conductance by 35%. l-NMMA infusion did not affect the peak shear rate stimulus ( P = 0.681) or the area under the curve (AUC) of shear rate to peak FMD ( P = 0.088). The AUC was significantly larger in the 10 trial vs. the 10–30 or 5 trial ( P < 0.001). Although percent FMD (%change in diameter) in the 10 trial was larger than that in the 5 trial ( P = 0.035), there was no significant difference in %FMD between the saline and l-NMMA conditions in any trial: 5 trial, 5.62 ± 1.48 vs. 5.63 ± 1.27%; 10 trial, 9.07 ± 1.16 vs. 11.22 ± 2.21%; 10–30 trial, 6.52 ± 1.43 vs. 7.98 ± 1.51% for saline and l-NMMA, respectively ( P = 0.158). We conclude the following: 1) RH following 10 min of occlusion results in an enhanced stimulus and %FMD compared with 5 min of occlusion. 2) When the occlusion cuff is reinflated 30 s postrelease of a 10 min occlusion, it does not result in an enhanced %FMD compared with that which results from RH following 5 min of occlusion. 3) The lack of effect of l-NMMA on FMD suggests that NO may not be obligatory for radial artery FMD in response to either 5 or 10 min of occlusion in healthy volunteers.
Arterial [H+] and the ventilatory response to hypoxia in humans: influence of acetazolamide-induced metabolic acidosis
Authors: Teppema Luc J. , van Dorp Eveline L. A. , Dahan Albert
Published: October 2009

In this study, we investigated possible separate effects of H+ions and CO2on hypoxic sensitivity in humans. We also examined whether hypoxic sensitivity, conventionally defined as the ratio of (hypoxic − normoxic) ventilation over (hypoxic − normoxic) Hb oxygen saturation can also be estimated by taking the ratio (hypoxic − normoxic) ventilation over (logPaO2hypoxia − logPaO2normoxia), enabling one to measure the hypoxic response independently from potential confounding influences of changes in position of the Hb oxygen saturation curve. We used acetazolamide to induce a metabolic acidosis. To determine the acute hypoxic response (AHR), we performed step decreases in end-tidal Po2to ∼50 Torr lasting 5 min each at three different constant end-tidal Pco2levels. Nine subjects ingested 250 mg of acetazolamide or placebo every 8 h for 3 days in a randomized double-blind crossover design. The metabolic acidosis was accompanied by a rise in ventilation, a substantial fall in PaCO2, and a parallel leftward shift of the ventilatory CO2response curve. In placebo, CO2induced equal relative increases in hypoxic sensitivity (O2-CO2interaction) regardless of the way it was defined. Acetazolamide shifted the response line representing the relationship between hypoxic sensitivity and arterial [H+] ([H+]a) to higher values of [H+]awithout altering its slope, indicating that it did not affect the O2-CO2interaction. So, in contrast to an earlier belief, CO2and H+have separate effects on hypoxic sensitivity. This was also supported by the finding that infusion of bicarbonate caused a leftward shift of the hypoxic sensitivity-[H+]aresponse lines in placebo and acetazolamide. A specific inhibitory effect of acetazolamide on hypoxic sensitivity was not demonstrated.
An α
2C
-Adrenergic Receptor Polymorphism Alters the Norepinephrine-Lowering Effects and Therapeutic Response of the β-Blocker Bucindolol in Chronic Heart Failure
Authors: Bristow Michael R. , Murphy Guinevere A. , Krause-Steinrauf Heidi , Anderson Jeffrey L. , Carlquist John F. , Thaneemit-Chen Surai , Krishnan Vaishali , Abraham William T. , Lowes Brian D. , Port J. David , Davis Gordon W. , Lazzeroni Laura C. , Robertson Alastair D. , Lavori Phillip W. , Liggett Stephen B.
Published: October 2009

Background—

Adrenergic activation is an important determinant of outcomes in chronic heart failure. Adrenergic activity is regulated in part by prejunctional α
2C
-adrenergic receptors (ARs), which exhibit genetic variation in humans. Bucindolol is a novel β-AR blocking agent that also lowers systemic norepinephrine and thus is also a sympatholytic agent. This study investigated whether α
2C
-AR polymorphisms affect sympatholytic effects of bucindolol in patients with heart failure.

Methods and Results—

In the β-Blocker Evaluation of Survival Trial, adrenergic activation was estimated by systemic venous norepinephrine measured at baseline, 3 months, and 12 months posttreatment in patients treated with placebo or bucindolol. In the β-Blocker Evaluation of Survival Trial AR polymorphism substudy, DNA was collected from 1040 of the 2708 randomized patients, and α
2C
-AR gene polymorphisms (α
2C
Del322-325 or the wild-type counterpart) were measured by polymerase chain reaction and gel electrophoresis. Patients who were α
2C
Del carriers (heterozygotes or homozygotes) exhibited a much greater sympatholytic response to bucindolol (decrease in norepinephrine at 3 months of 153±57 pg/mL,
P
=0.012 compared with placebo versus decrease of 50±13 pg/mL in α
2C
wild type,
P
=0.0005 versus placebo;
P
=0.010 by interaction test). α
2C
Del carriers had no evidence of a favorable survival benefit from bucindolol (mortality compared with placebo hazard ratio, 1.09; 95% CI, 0.57 to 2.08;
P
=0.80), whereas bucindolol-treated subjects who were wild type for the α
2C
-AR had a 30% reduction in mortality (hazard ratio, 0.70; 95% CI, 0.51 to 0.96;
P
=0.025).

Conclusions—

In the β-Blocker Evaluation of Survival Trial AR polymorphism substudy, the norepinephrine lowering and clinical therapeutic responses to bucindolol were strongly influenced by α
2C
receptor genotype.
The Cytoprotective Effects of Tumor Necrosis Factor Are Conveyed Through Tumor Necrosis Factor Receptor–Associated Factor 2 in the Heart
Authors: Burchfield Jana S. , Dong Jian-Wen , Sakata Yasushi , Gao Feng , Tzeng Huei-Ping , Topkara Veli K. , Entman Mark L. , Sivasubramanian Natarajan , Mann Douglas L.
Published: October 2009

Background—

Activation of both type 1 and type 2 tumor necrosis factor (TNF) receptors (TNFR1 and TNFR2) confers cytoprotection in cardiac myocytes. Noting that the scaffolding protein TNF receptor–associated factor 2 (TRAF2) is common to both TNF receptors, we hypothesized that the cytoprotective responses of TNF were mediated through TRAF2.

Methods and Results—

Mice with cardiac-restricted overexpression of low levels of TNF (MHCsTNF
3
) and TRAF2 (MHC-TRAF2
LC
) and mice lacking TNFR1, TNFR2, and TNFR1/TNFR2 were subjected to ischemia (30 minutes) reperfusion (30 minutes) injury ex vivo using a Langendorff apparatus. MHCsTNF
3
mice were protected against ischemia-reperfusion injury as shown by a significant ≈30% greater left ventricular developed pressure, ≈80% lower creatine kinase release, and Evans blue dye uptake compared with littermates. The extent of ischemia-reperfusion induced injury was similar in wild-type, TNFR1, and TNFR2 deficient mice; however, mice lacking TNFR1/TNFR2 had a significant ≈40% lower left ventricular developed pressure, a ≈65% greater creatine kinase release, and ≈40% greater Evans blue dye uptake compared with littermates. Interestingly, MHC-TRAF2
LC
mice had a significant ≈50% lower left ventricular developed pressure, a ≈70% lower creatine kinase release, and ≈80% lower Evans blue dye uptake compared with littermate controls after ischemia-reperfusion injury. Biochemical analysis of the MHC-TRAF2
LC
hearts showed that there was activation of nuclear factor-kappaB but not c-Jun N-terminal kinase activation.

Conclusion—

Taken together, these results suggest that TNF confers cytoprotection in the heart through TRAF2-mediated activation of nuclear factor-κB.
Application of Wool in High-velocity Ballistic Protective Fabrics
Authors: Sinnppoo Kanesalingam , Arnold Lyndon , Padhye Rajiv
Published: October 2009

The protective power of typical aramid-based ballistic fabrics, when assembled into multi-layered panels designed to defeat high-velocity ballistic impacts, can be improved if wool is incorporated into the weave structure. Although the synthetic is still the primary energy-absorbent material, the wool plays a complementary role by increasing resistive interactions between the yarns and filaments. Wool restricts the lateral separation of the synthetic yarns and ensures that more directly impacted yarns are held in place to dissipate the impact energy. Wool increases the energy-absorption mechanism of yarn pull-in by increasing the longitudinal friction along the yarns/filaments, in particular near the free edges of the fabric layers. The wool absorbs water that may otherwise lubricate synthetic filaments and so improves the wet performance. Ballistics tests have shown that synthetic fabrics blended with wool can at least match the dry or wet ballistic performance of an equivalent pure Kevlar fabric when tested under National Institute of Justice (NIJ) Ballistic Standard Level III A. The inclusion of the wool can significantly improve the tear strength of pure synthetic ballistic fabrics.
A Semi‐Mechanistic Model of CP‐690,550‐Induced Reduction in Neutrophil Counts in Patients With Rheumatoid Arthritis
Authors: Gupta Pankaj , Friberg Lena E. , Karlsson Mats O. , Krishnaswami Sriram , French Jonathan
Published: October 2009

CP‐690,550, a selective inhibitor of the Janus kinase family, is being developed as an oral disease‐modifying antirheumatic drug for the treatment of rheumatoid arthritis (RA). A semi‐mechanistic model was developed to characterize the time course of drug‐induced absolute neutrophil count (ANC) reduction in a phase 2a study. Data from 264 RA patients receiving 6‐week treatment (placebo, 5, 15, 30 mg bid) followed by a 6‐week off‐treatment period were analyzed. The model included a progenitor cell pool, a maturation chain comprising transit compartments, a circulation pool, and a feedback mechanism. The model was adequately described by system parameters (BASEh, ktrh, γ, and kcirc), disease effect parameters (DIS), and drug effect parameters (koff and kD). The disease manifested as an increase in baseline ANC and reduced maturation time due to increased demand from the inflammation site. The drug restored the perturbed system parameters to their normal values via an indirect mechanism. ANC reduction due to a direct myelosuppressive drug effect was not supported. The final model successfully described the dose‐ and time‐dependent changes in ANC and predicted the incidence of neutropenia at different doses reasonably well.
A Randomized, Placebo‐Controlled Study of the Effects of the p38 MAPK Inhibitor SB‐681323 on Blood Biomarkers of Inflammation in COPD Patients
Authors: Singh Dave , Smyth Lucy , Borrill Zoe , Sweeney Lisa , Tal‐Singer Ruth
Published: October 2009

The p38 mitogen‐activated protein kinase (MAPK) signaling upregulates inflammation and is known to be increased in chronic obstructive pulmonary disease (COPD). The authors assessed the pharmacology of the novel p38 MAPK inhibitor SB‐681323 using blood biomarkers in COPD. Seventeen COPD patients (forced expiratory volume in 1 second 50%–80% predicted) using short‐acting bronchodilators participated in a double‐blind, double‐dummy, randomized, crossover study. Patients received single oral doses of SB‐681323 7.5 mg and 25 mg, prednisolone 10 mg and 30 mg, and placebo. Blood was obtained predose and at 1, 2, 6, and 24 hours postdose. Whole‐blood sorbitol‐induced phosphorylated (p) heat shock protein (HSP) 27 levels as a marker of p38 pathway activation and lipopolysaccharide‐induced tumor necrosis factor (TNF)–α production were assessed. Both doses of SB‐681323, but not prednisolone, significantly (P < .0001) reduced weighted mean (WM) pHSP27 (0–6 hours) by 58% compared with placebo. WM TNF‐α production (0–24 hours) was significantly reduced compared with placebo by SB‐681323 25 mg (40%, P = .005) and 7.5 mg (33.4%, P = .02), while prednisolone 30 mg and 10 mg caused 81.5% and 58.2% suppression, respectively (both P < .0001). SB‐681323 inhibited the p38 MAPK pathway to a greater degree than prednisolone did. SB‐681323 inhibited TNF‐α production. SB‐681323 is a potent p38 MAPK inhibitor that potentially suppresses inflammation in COPD.