bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Stapling Method and Device for Nasal Surgery
Authors: Buesseler Ryan K. , Hom David B. , Erdman Arthur G.
Published: December 2009

This paper presents the design and prototyping of a novel stapling device and method intended for—though not limited to—the nasal septal mucosa. The pistol shaped device has two long projections: one for each nostril. The internal mechanism pushes the male portion of the rivet, nearly 10 cm down one tube, where it pierces the mucosa and mates with the female portion. This is all accomplished with one grip of the handle, rotating the input handle only 30 deg. The internal mechanism was designed by solving loop equations for a five-bar, dual-slider single degree-of-freedom mechanism. Components were then rapid prototyped before the eventual functional prototype was completed.
A Preliminary Study of the Effect of Surface Texture on Algae Cell Attachment for a Mechanical-Biological Energy Manufacturing System
Authors: Cao Jian , Yuan Wenqiao , Pei Z. J. , Davis Tiffany , Cui Yan , Beltran Michael
Published: December 2009

A grand vision of an algal biofuel energy manufacturing system is presented here. The proposed system, from manufacturing engineering and system points of view, aims to provide technical solutions to two major challenges that the algal biofuel industry faces, i.e., low productivity and energy intensive harvesting and drying, which result in prohibitively high costs. The proposed idea is to have an integrated “conveyor belt” system floating on the water surface powered by windmills or a hybrid energy source. The conveyor belt is made of corrosion-resistant steel sheets that have microdimple surface features to significantly enhance the attachment of algae cells to the “belt” compared with a surface without microdimple features. The grown algae on the belt will then be mechanically scraped off, collected, dried, and squeezed for oil extraction. This paper addresses one of many fundamental problems in this vision, i.e., whether algae can grow effectively on textured stainless steel surfaces. Through both static and dynamic tests, it was found that the growth of algae on textured surfaces was several times more active than that on a flat sample.
Influence of Plasmid pO157 on
Escherichia coli
O157:H7 Sakai Biofilm Formation
Authors: Lim Ji Youn , La Hyun Joon , Sheng Haiqing , Forney Larry J. , Hovde Carolyn J.
Published: December 2009

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured
Escherichia coli
O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.
Effect of Chlorine on Incorporation of
Helicobacter pylori
into Drinking Water Biofilms
Authors: Gião M. S. , Azevedo N. F. , Wilks S. A. , Vieira M. J. , Keevil C. W.
Published: December 2009

The use of a specific peptide nucleic acid (PNA) probe demonstrated that
Helicobacter pylori
persisted inside biofilms exposed to low concentrations of chlorine (0.2 and 1.2 mg liter
−1
) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained using viability stains in pure culture, this result suggests that
H. pylori
can survive chlorination but remain undetectable by culture methods, which can be effectively replaced by PNA hybridization.
Role of
Saccharomyces cerevisiae
Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol
Authors: González Eva , Fernández M. Rosario , Marco Didac , Calam Eduard , Sumoy Lauro , Parés Xavier , Dequin Sylvie , Biosca Josep A.
Published: December 2009

NAD-dependent butanediol dehydrogenase (Bdh1p) from
Saccharomyces cerevisiae
reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of
BDH1
resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two
S. cerevisiae
strains under two different growth conditions. The concentrations of (2
R
,3
R
)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (
meso
)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be
d
-arabinose dehydrogenase (Ara1p), which converts (
R
/
S
)-acetoin to
meso
-2,3-butanediol and (2
S
,3
S
)-2,3-butanediol. Deletion of
BDH2
, a gene adjacent to
BDH1
, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that
BDH1
and
BDH2
are reciprocally regulated.
LccA, an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by
Haloferax volcanii
Authors: Uthandi Sivakumar , Saad Boutaiba , Humbard Matthew A. , Maupin-Furlow Julie A.
Published: December 2009

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon
Haloferax volcanii
. LccA was secreted at high levels into the culture supernatant of a recombinant
H. volcanii
strain, with peak activity (170 ± 10 mU·ml
−
1
) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U·mg
−
1
with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO
4
. Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent
K
m
values for SGZ, bilirubin, and ABTS were 35, 236, and 670 μM, with corresponding
k
cat
values of 22, 29, and 10 s
−
1
, respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h.
Use of
Hydrogenophaga pseudoflava
Penetration To Quantitatively Assess the Impact of Filtration Parameters for 0.2-Micrometer-Pore-Size Filters
Authors: Lee A. , McVey J. , Faustino P. , Lute S. , Sweeney N. , Pawar V. , Khan M. , Brorson K. , Hussong D.
Published: December 2009

Filters rated as having a 0.2-μm pore size (0.2-μm-rated filters) are used in laboratory and manufacturing settings for diverse applications of bacterial and particle removal from process fluids, analytical test articles, and gasses. Using
Hydrogenophaga pseudoflava
, a diminutive bacterium with an unusual geometry (i.e., it is very thin), we evaluated passage through 0.2-μm-rated filters and the impact of filtration process parameters and bacterial challenge density. We show that consistent
H. pseudoflava
passage occurs through 0.2-μm-rated filters. This is in contrast to an absence of significant passage of nutritionally challenged bacteria that are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.
Extracellular Production of an RNA Aptamer by Ribonuclease-Free Marine Bacteria Harboring Engineered Plasmids: a Proposal for Industrial RNA Drug Production
Authors: Suzuki Hiromichi , Ando Tomoaki , Umekage So , Tanaka Terumichi , Kikuchi Yo
Published: December 2009

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for
in vivo
production of artificial RNAs using the marine phototrophic bacterium
Rhodovulum sulfidophilum
. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA
in vivo
, which will pave the way for inexpensive production of RNA drugs.
Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach
Authors: Abdelzaher Amir M. , Wright Mary E. , Ortega Cristina , Solo-Gabriele Helena M. , Miller Gary , Elmir Samir , Newman Xihui , Shih Peter , Bonilla J. Alfredo , Bonilla Tonya D. , Palmer Carol J. , Scott Troy , Lukasik Jerzy , Harwood Valerie J. , McQuaig Shannon , Sinigalliano Chris , Gidley Maribeth , Plano Lisa R. W. , Zhu Xiaofang , Wang John D. , Fleming Lora E.
Published: December 2009

Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms,
Escherichia coli
, enterococci, and
Clostridium perfringens
), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and
Enterococcus faecium esp
gene), and pathogens (
Vibrio vulnificus
,
Staphylococcus aureus
, enterovirus, norovirus, hepatitis A virus,
Cryptosporidium
spp., and
Giardia
spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium
V. vulnificus
in sand and water, detection of the allochthonous protozoans
Giardia
spp. in water, and detection of
Cryptosporidium
spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.
Use of
rpsL
as a Counterselectable Marker in
Borrelia burgdorferi
Authors: Drecktrah Dan , Douglas J. Miles , Samuels D. Scott
Published: December 2009

We have demonstrated that
rpsL
, encoding the S12 protein of the small ribosomal subunit, can be used as a counterselectable marker in
Borrelia burgdorferi
, the causative agent of Lyme disease. Mutations in
rpsL
confer streptomycin resistance. Streptomycin susceptibility is dominant in an
rpsL
merodiploid, and streptomycin selects for the loss of wild-type
rpsL
carried in
trans
. This is the first description of a counterselectable marker in
B. burgdorferi
.
Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the
tdh
and
trh
Genes of
Vibrio parahaemolyticus
and Related
Vibrio
Species
Authors: Yamazaki Wataru , Kumeda Yuko , Misawa Naoaki , Nakaguchi Yoshitsugu , Nishibuchi Mitsuaki
Published: December 2009

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of
Vibrio parahaemolyticus
. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the
tdh
,
trh
1, and
trh
2 genes of
V. parahaemolyticus
. The LAMP assay was designed for both combined and individual detection of the
tdh
,
trh
1, and
trh
2 genes and combined detection of the
trh
1 and
trh
2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of
V. parahaemolyticus
, 3 strains of
Grimontia hollisae
, and 2 strains of
Vibrio mimicus
carrying the
tdh
,
trh
1, and
trh
2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the
tdh
,
trh
1, and
trh
2 genes. The sensitivities of the LAMP assay for detection of
tdh
-,
trh
1-, and
trh
2-carrying
V. parahaemolyticus
strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the
tdh
,
trh
1, and
trh
2 genes of
V. parahaemolyticus
and related
Vibrio
species.
New Architectures for Tet-On and Tet-Off Regulation in
Staphylococcus aureus
Authors: Stary Elena , Gaupp Rosmarie , Lechner Sabrina , Leibig Martina , Tichy Evelyn , Kolb Martina , Bertram Ralph
Published: December 2009

Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (
tet
) system for
Staphylococcus aureus
. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of
tetR
or a variant allele, rev
-tetR
r2
. The encoded regulators respond differently to the effector anhydrotetracycline (ATc), which causes target gene expression to be induced with TetR or repressed with rev
-
TetR. To quantify and compare regulation mediated by episomal or chromosomal (rev-)
tetR
constructs, expression from a chromosomal P
xyl/tet
-
gfpmut2
fusion was measured. Chromosomally encoded TetR showed tight repression and allowed high levels of dose-dependent gene expression in response to ATc. Regulatory abilities were further verified using a strain in which a native
S. aureus
gene (
zwf
) was put under
tet
control in its native chromosomal location. Tight repression was reflected by transcript amounts, which were barely detectable under repressed conditions and high in ATc-treated cells. In reporter gene assays, this type of control, termed Tet-on, was more efficient than Tet-off regulation, in which addition of ATc causes downregulation of a target gene. The latter was achieved and quantified by direct rev
-
TetR control of P

xyl/tet

-
gfpmut2
. Additionally, TetR was used in
trans
to control the expression of antisense RNA for posttranscriptional gene silencing. Induction of antisense RNA expression of the
fabI
gene caused pronounced growth retardation lasting several hours. These results demonstrate the efficiency of the new
tet
systems and their flexible use for different purposes.
Double Labeling of Oligonucleotide Probes for Fluorescence
In Situ
Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility
Authors: Stoecker Kilian , Dorninger Christiane , Daims Holger , Wagner Michael
Published: December 2009

Fluorescence
in situ
hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase
in situ
accessibility of rRNA target sites and thus provide more flexibility for probe design.
Multiple Genetic Mechanisms Lead to Loss of Functional TbAT1 Expression in Drug-Resistant Trypanosomes
Authors: Stewart Mhairi L. , Burchmore Richard J. S. , Clucas Caroline , Hertz-Fowler Christiane , Brooks Karen , Tait A. , MacLeod A. , Turner C. Michael R. , De Koning Harry P. , Wong Pui Ee , Barrett Michael P.
Published: December 2009

The P2 aminopurine transporter, encoded by Tb
AT1
in African trypanosomes in the
Trypanosoma brucei
group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of Tb
AT1
to be in the subtelomeric region of chromosome 5 and determined the status of the Tb
AT1
gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a
Trypanosoma equiperdum
clone selected for resistance to the diamidine, diminazene aceturate. In the
Trypanosoma brucei
gambiense
STIB 386 melarsamine hydrochloride-resistant line, Tb
AT1
is deleted, while in the
Trypanosoma brucei brucei
STIB 247 melarsamine hydrochloride-resistant and
T. equiperdum
diminazene-resistant lines, Tb
AT1
is present, but expression at the RNA level is no longer detectable. Further characterization of Tb
AT1
in
T. equiperdum
revealed that a loss of heterozygosity at the Tb
AT1
locus accompanied loss of expression and that P2-mediated uptake of [
3
H]diminazene is lost in drug-resistant
T. equiperdum
. Adenine-inhibitable adenosine uptake is still detectable in a ΔTb
at1
T. b. brucei
mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.
The NDR Kinase DBF-2 Is Involved in Regulation of Mitosis, Conidial Development, and Glycogen Metabolism in Neurospora crassa
Authors: Dvash Efrat , Kra-Oz Galia , Ziv Carmit , Carmeli Shmuel , Yarden Oded
Published: December 2009

Neurospora crassa
dbf-2
encodes an NDR (nuclear Dbf2-related) protein kinase, homologous to LATS1, a core component of the Hippo pathway. This pathway plays important roles in restraining cell proliferation and promoting apoptosis in differentiating cells. Here, we demonstrate that DBF-2 is involved in three fundamental processes in a filamentous fungus: cell cycle regulation, glycogen biosynthesis, and conidiation. DBF-2 is predominantly localized to the nucleus, and most (approximately 60%)
dbf-2
null mutant nuclei are delayed in mitosis, indicating that DBF-2 activity is required for properly completing the cell cycle. The
dbf-2
mutant exhibits reduced basal hyphal extension rates accompanied by a carbon/nitrogen ratio-dependent bursting of hyphal tips, vast glycogen leakage, defects in aerial hypha formation, and impairment of all three asexual conidiation pathways in
N. crassa
. Our findings also indicate that DBF-2 is essential for sexual reproduction in a filamentous fungus. Defects in other Hippo and glycogen metabolism pathway components (
mob-1
,
ccr-4
,
mst-1
, and
gsk-3
) share similar phenotypes such as mitotic delay and decreased CDC-2 (cell division cycle 2) protein levels, massive hyphal swellings, hyphal tip bursting, glycogen leakage, and impaired conidiation. We propose that DBF-2 functions as a link between Hippo and glycogen metabolism pathways.
The S Helix Mediates Signal Transmission as a HAMP Domain Coiled-Coil Extension in the NarX Nitrate Sensor fromEscherichia coliK-12
Authors: Stewart Valley , Chen Li-Ling
Published: December 2009

In the nitrate-responsive, homodimeric NarX sensor, two cytoplasmic membrane α-helices delimit the periplasmic ligand-binding domain. The HAMP domain, a four-helix parallel coiled-coil built from two α-helices (HD1 and HD2), immediately follows the second transmembrane helix. Previous computational studies identified a likely coiled-coil-forming α-helix, the signaling helix (S helix), in a range of signaling proteins, including eucaryal receptor guanylyl cyclases, but its function remains obscure. In NarX, the HAMP HD2 and S-helix regions overlap and apparently form a continuous coiled-coil marked by a heptad repeat stutter discontinuity at the distal boundary of HD2. Similar composite HD2-S-helix elements are present in other sensors, such as Sln1p fromSaccharomyces cerevisiae. We constructed deletions and missense substitutions in the NarX S helix. Most caused constitutive signaling phenotypes. However, strongly impaired induction phenotypes were conferred by heptad deletions within the S-helix conserved core and also by deletions that remove the heptad stutter. The latter observation illuminates a key element of the dynamic bundle hypothesis for signaling across the heptad stutter adjacent to the HAMP domain in methyl-accepting chemotaxis proteins (Q. Zhou, P. Ames, and J. S. Parkinson, Mol. Microbiol. 73:801-814, 2009). Sequence comparisons identified other examples of heptad stutters between a HAMP domain and a contiguous coiled-coil-like heptad repeat sequence in conventional sensors, such as CpxA, EnvZ, PhoQ, and QseC; other S-helix-containing sensors, such as BarA and TorS; and theNeurospora crassaNik-1 (Os-1) sensor that contains a tandem array of alternating HAMP and HAMP-like elements. Therefore, stutter elements may be broadly important for HAMP function.
Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn
1549
, a Member of a New Family of Conjugative Elements
Authors: Tsvetkova Krassimira , Marvaud Jean-Christophe , Lambert Thierry
Published: December 2009

Conjugative transfer from
Clostridium symbiosum
to enterococci of Tn
1549
, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (
oriT
) in the element. Transcription analysis of Tn
1549
indicated that
orf29
,
orf28
,
orfz
, and
orf27
were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to
orf29-orf30
of Tn
1549
was mobilized in
Escherichia coli recA
::RP4::Δ
nic
provided that
orf28
and
orf29
were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in
trans
, but a
cis
-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp
oriT
was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp
oriT
fragment. The minimal
oriT
contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB
p
superfamily. The nick site was identified within
oriT
by an oligonucleotide cleavage assay. Closely related
oriT
s linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn
1549
is a member of a MOB
p
clade. Interestingly, the Tn
1549
-derived constructs were mobilized by RP4 in
E. coli
, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn
1549
to be mobilized by a heterologous transfer system.
Glycerol Is Metabolized in a Complex and Strain-Dependent Manner in
Enterococcus faecalis
Authors: Bizzini Alain , Zhao Chen , Budin-Verneuil Aurélie , Sauvageot Nicolas , Giard Jean-Christophe , Auffray Yanick , Hartke Axel
Published: December 2009

Enterococcus faecalis
is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the
glpK
pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the
dhaK
pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the
glpK
pathway operates during aerobiosis and that the
dhaK
pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the
E. faecalis
JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the
dhaK
pathway under anaerobic conditions. Comparison of different
E. faecalis
isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the
glpK
pathway whereas others probably exclusively the
dhaK
pathway under aerobic conditions. Our results demonstrate that the species
E. faecalis
cannot be represented by only one model of aerobic glycerol catabolism.
Identification, Functional Studies, and Genomic Comparisons of New Members of the NnrR Regulon in
Rhodobacter sphaeroides
Authors: Hartsock Angela , Shapleigh James P.
Published: December 2009

Analysis of the
Rhodobacter sphaeroides
2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated
paz
,
norEF
, and
cdgA
, in the NnrR regulon, while the status of the fourth sequence, within the
tat
operon promoter, remains uncertain.
nnrV
, under control of a previously identified NnrR site, was also identified.
paz
encodes a pseudoazurin that is a donor of electrons to nitrite reductase.
paz
inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome

c
2

together reduced nitrite reduction. Inactivation of
norEF
reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of
norEF
increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of
R. sphaeroides
with
norEF
, even though all four of the strains whose genomes have been sequenced have the
norCBQD
operon and
nnrR
.
norEF
was shown to provide resistance to nitrite when it was mobilized into
R. sphaeroides
strain 2.4.1 containing
nirK
. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in
R. sphaeroides
revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions.
fpr
, a Deficient Xer Recombination Site from a
Salmonella
Plasmid, Fails To Confer Stability by Dimer Resolution: Comparative Studies with the pJHCMW1
mwr
Site
Authors: Tran Tung , Sherratt David J. , Tolmasky Marcelo E.
Published: December 2009

Salmonella
plasmid pFPTB1 includes a Tn
3
-like transposon and a Xer recombination site,
fpr
, which mediates site-specific recombination at efficiencies lower than those required for stabilizing a plasmid by dimer resolution. Mutagenesis and comparative studies with
mwr
, a site closely related to
fpr
, indicate that there is an interdependence of the sequences in the XerC binding region and the central region in Xer site-specific recombination sites.
Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the GenusCorynebacterium
Authors: Barth Enrico , Barceló Miriam Agulló , Kläckta Christian , Benz Roland
Published: December 2009

Two small polypeptides, PorA and PorH, are known to form cell wall channels inCorynebacteriumglutamicumand inCorynebacteriumefficiens. The genes coding for both polypeptides are localized in close proximity to one another between the genes coding for GroEl2 and a polyphosphate kinase (PKK2). In this study, we investigated the relationship of PorA and PorH to one another. The results suggested that the major cell wall channels ofCorynebacteriumglutamicum,Corynebacteriumefficiens, andCorynebacteriumdiphtheriaeneed the obligatory presence of two distinct polypeptides, one of class PorA and one of class PorH, to form an active cell wall channel. Identification of genes coding for homologous proteins in the chromosome ofCorynebacteriumcallunaesuggested a similar result for this strain. Contrary to our previous reports on channel-forming proteins in these strains, a heterooligomeric structure composed of PorA and PorH is needed in all of them to form the major cell wall channel. This was concluded from complementation experiments using aporH- andporA-deficientC.glutamicumstrain. The stringent necessity of proteins of either class to recover the wild-type channels was demonstrated by black lipid bilayer experiments using detergent or organic solvent extracts of the complementedporH- andporA-deficientC.glutamicumstrain. The channel-forming capability of recombinant expressed, affinity-purified PorA and PorH proteins ofC.glutamicumrevealed that the channels consisted solely of these two components. This agreed with results obtained from a transcript coding for both channel-forming components identified inC.glutamicumby Northern blot analysis and reverse transcription-PCR analysis. The transcription start point of the genes was determined by the rapid amplification of cDNA ends approach, allowing the prediction of the −35 and −10 regions of the promoter. The results demonstrate that the cell wall channels within the genusCorynebacteriummay be formed by two-component oligomers.
Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production inStreptomyces coelicolorA3(2)
Authors: D'Alia Davide , Nieselt Kay , Steigele Stephan , Müller Jonas , Verburg Ilse , Takano Eriko
Published: December 2009

Overexpression of antisense chromosomalcis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production inStreptomyces coelicolor. In addition, we predicted 3,597cis-encoded ncRNAs and validated 13 of them experimentally, including several ncRNAs that are differentially expressed in bacterial hormone-defective mutants.
Contributions of Four Cortex Lytic Enzymes to Germination of
Bacillus anthracis
Spores
Authors: Heffron Jared D. , Lambert Emily A. , Sherry Nora , Popham David L.
Published: December 2009

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs).
Bacillus anthracis
has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs
in vivo
was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca
2+
-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore's integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca
2+
-DPA-induced germination. SleL is an
N
-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of
B. anthracis
spores, greatly simplifying decontamination measures.
Coexistence of Two Different Photosynthetic Operons in
Citromicrobium bathyomarinum
JL354 As Revealed by Whole-Genome Sequencing
Authors: Jiao Nianzhi , Zhang Rui , Zheng Qiang
Published: December 2009

Citromicrobium bathyomarinum
is a bacteriochlorophyll
a
-containing aerobic anoxygenic phototrophic bacterium. Here, we report the draft genome sequence of
C. bathyomarinum
JL354, which was the first
C. bathyomarinum
strain isolated from a euphotic marine environment. The JL354 genome sequence reveals, for the first time, the coexistence of two different photosynthetic operons in a single strain.
Genome Sequences of
Mannheimia haemolytica
Serotype A2: Ovine and Bovine Isolates
Authors: Lawrence Paulraj K. , Kittichotirat Weerayuth , Bumgarner Roger E. , McDermott Jason E. , Herndon David R. , Knowles Donald P. , Srikumaran Subramaniam
Published: December 2009

This report describes the genome sequences of
Mannheimia haemolytica
serotype A2 isolated from pneumonic lungs of two different ruminant species, one from
Ovis aries
, designated ovine (O), and the other from
Bos taurus
, designated bovine (B).
Determination of 5-amino-6-nitroquinoline at a carbon paste electrode
Authors: Němcová Lenka , Zima Jiří , Barek Jiří
Published: December 2009

Differential pulse voltammetry, direct current voltammetry, adsorptive stripping voltammetry and HPLC with electrochemical detection were used for the determination of 5-amino-6-nitroquinoline at a carbon paste electrode. The methods are based either on anodic oxidation or cathodic reduction of this substance, whose electrochemical behavior at carbon paste electrode was further studied by cyclic voltammetry. Practical applicability of these methods was demonstrated on the determination of 5-amino-6-nitroquinoline in model samples of drinking and river water. The detection limit was 2.0 × 10–6 mol l–1 for anodic differential pulse voltammetry in a mixture of Britton–Robinson buffer (pH 11)–methanol 1:1 (v/v) and 1.6 × 10–7 mol l–1 for HPLC with electrochemical detection (E = +1.2 V) in a mobile phase Britton–Robinson buffer (pH 7)–methanol 1:9 (v/v).
Isopotential points in square-wave voltammetry of reversible electrode reactions
Authors: Zelić Marina , Lovrić Milivoj
Published: December 2009

Isopotential points in square-wave voltammetry are described for the first time. Model calculations and real measurements (performed with UO22+ and Eu3+ in perchlorate and bromide solutions, respectively) indicate that such an intersection could be observed when backward components of the net response, resulting from an increase in frequency or reactant concentration, are presented together. The electrode reaction should be fully reversible because quasireversible or slower electron transfer processes give the isopoints only at increasing reactant concentrations but not at increasing square-wave frequencies. The effect could be used as an additional diagnostic criterion for recognition of reversible electrode reactions where products remain dissolved in the electrolyte solution.
Verification of accuracy of standard thermodynamic data of inorganic electrolytes
Authors: Balej Jan
Published: December 2009

Accuracy of standard thermodynamic data for some inorganic electrolytes of the types 1-1, 1-2 and 2-1 at 25 °C, in the solid state or aqueous standard solutions, presented in NBS Tables (1982), has been checked. For the purpose, the condition for the pure solid–saturated solution equilibrium and reliable values of solubilities, mean activity coefficients and water activity in saturated solution at the given temperature were used. In most cases, a very good reliability and consistency of all relevant data have been found. On the other hand, some discrepancies have been observed for NaNO3, NaClO4/NaClO4 pair, Na2HPO4·12H2O/Na2HPO4 pair, K2CrO4, K2Cr2O7, (NH4)2SO4 and SrCl2·6H2O/SrCl2 pair the reason of which is not yet known. The verification method can be used for the determination of lacking thermodynamic data. The method has been applied to ΔGşf,cr of NaCNS·2H2O, NaH2PO4·2H2O, Na2CrO4·6H2O, Na2WO4·2H2O, MgI2·8H2O and CaCl2·6H2O at 25 °C. A suitable method has been used for the determination of the most probable solubility in the case when the values scatter. The used methods are applicable to all types of electrolytes and nonelectrolytes.
Comparison of Pathvysion and Poseidon HER2 FISH assays in measuring HER2 amplification in breast cancer: a validation study
Authors: Francz Monika , Egervari Kristof , Kardos Laszlo , Toth Judit , Nemes Zoltan , Szanto Janos , Szollosi Zoltan
Published: December 2009

Aims

The current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial Poseidon HER2 fluorescence in situ hybridisation (FISH) assay. However, it was felt that the experience of the authors of this study may be of interest to other laboratories when considering setting up a HER2 diagnostic facility.

Methods

122 patients who had been diagnosed with invasive breast cancer were selected. Immunolabelling with HercepTest, PathVysion and Poseidon FISH assays were carried out using tissue microarray blocks.

Results

Concordance correlation coefficients showed near perfect agreement in average HER2 and centromere specific signal counts per cell and in the HER2/CEP17 ratios between the PathVysion and the Poseidon FISH assays. In addition, the κ measure showed perfect agreement (κ 0.9441, p<0.0001), and if only 2+ cases were considered there was substantial agreement (κ 0.7671, p=0.0006), between the two assays. The sensitivity and the specificity of the Poseidon FISH kit were calculated to be 95.2% and 100%, respectively, whereas the positive predictive value (PPV) and negative predictive value (NPV) were 100% and 99%, respectively. With regard to the ability to presume HER2 polysomy, the Poseidon FISH kit had a sensitivity of 93.3% and a specificity of 99.1%, with PPV and NPV of 93.3% and 99.1%, respectively, as assessed with PathVysion classification as the reference.

Conclusions

Statistical analysis confirmed that the two FISH assays are comparable in terms of detection of HER2 gene amplification. Proceeding from these findings, the genetic diagnoses obtained with the Poseidon kit can be considered to be as valuable as the results from the Food and Drug Administration approved PathVysion assay, and its utilisation in routine HER2 diagnostics is proposed.
Do patients with basilar or vertebral artery stenosis have a higher stroke incidence poststenting?
Authors: Jiang W-J , Du B , Hon S F K , Jin M , Xu X-T , Ma N , Gao F , Dong K-H
Published: December 2009

Background and aim

Posterior circulation stenosis may be a risk factor associated with stroke after intracranial stenting as compared with anterior circulation stenosis. Our aim was to test our hypothesis that there was no difference in clinical outcome poststenting between patients with severe stenosis of the basilar artery (BA) and intracranial vertebral artery (VA).

Methods

Using the Cox proportional hazards regression model adjusted for prespecified factors (qualifying event, and timing of stenting after the qualifying event), we compared primary endpoint (ischemic stroke in the vertebrobasilar territory, including any stroke or death within 30 days of stenting) between patients with severe symptomatic atherosclerotic BA and VA stenosis who underwent elective stenting in our prospective database. Analysis was by intention-to-treat principle.

Results

Primary endpoint event occurred in 13 (18.8%) of 69 patients with BA stenosis during a mean 23.4 months (9 within 30 days and 4 afterward) and 3 (4.3%) of 70 patients with VA stenosis during a mean 26.4 months (2 within 30 days and 1 afterward). Patients with BA stenosis had a significantly higher risk of the primary endpoint (adjusted HR=4.87, 95% CI 1.37 to 17.29; p=0.014) or any stroke or death within 30 days of stenting (adjusted HR=5.13, 95% CI 1.10 to 23.96; p=0.038) than those with VA stenosis.

Conclusion

A significantly higher stroke risk poststenting exists in patients with severe BA stenosis than those with VA stenosis. The discrepancy in clinical outcome after stenting between patients with BA and VA stenosis should be considered in clinical practice and stenting trials.
Endovascular treatment of carotid embolic occlusions has a higher recanalization rate compared with cardioembolic occlusions
Authors: Hussain M S , Lin R , Cheng-Ching E , Jovin T G , Moskowitz S I , Bain M , Horowitz M , Gupta R
Published: December 2009

Background and purpose

Treatment of large artery cerebral occlusions is rapidly evolving. We hypothesized that patients with intracranial embolic occlusions secondary to an extracranial carotid artery stenosis or occlusion have a higher probability of successful endovascular recanalization compared with those with cardioembolic occlusions.

Methods

We retrospectively reviewed the databases of three institutions (University of Pittsburgh Medical Center (UPMC), Michigan State University (MSU) and Cleveland Clinic Foundation (CCF)) for acute anterior circulation ischemic strokes treated with endovascular therapies from January 2006 to July 2008. After collection of demographic, radiographic and angiographic variables, two groups were identified: artery to artery embolic occlusions and cardioembolic/cryptogenic intracranial occlusions. We defined recanalization as TIMI 2 or 3 flow. A binary logistic regression model was constructed to determine which characteristics were unique to patients with carotid embolic occlusions.

Results

A total of 207 patients were identified (UPMC=100, CCF=71, MSU=36) with a mean age of 69±11 years and mean NIHSS of 17±5. Of these, 157 (75%) were due to a cardiac or cryptogenic source and 50 (25%) were from a carotid embolic source. The use of multimodal therapy (OR 2.6 (1.2–5.6), p<0.009) and the presence of a carotid embolic intracranial occlusion (OR 3.6 (1.2–7.1), p<0.012) were associated with successful recanalization, while carotid terminus occlusions were associated with unsuccessful recanalization (OR 0.35 (0.18–0.68), p<0.002).

Conclusions

Patients with intracranial occlusions secondary to an extracranial carotid stenosis or total occlusion appear to have more successful recanalization rates when treated with endovascular therapy compared with those with cardioembolic occlusions.
Endovascular treatment of unruptured intracranial aneurysms in the elderly: analysis of procedure related complications
Authors: Stiefel M F , Park M S , McDougall C G , Albuquerque F C
Published: December 2009

Introduction

The management of unruptured intracranial aneurysms in the elderly remains controversial. Treatment risks are thought to be higher in this group. Large series assessing endovascular treatment of unruptured intracranial aneurysms in the elderly are lacking. Our single center endovascular experience in treating unruptured intracranial aneurysms in the elderly is presented.

Methods

77 patients, 70 years or older, were referred to the endovascular neurosurgery service for treatment of an unruptured intracranial aneurysm between February 2000 and May 2008. Hospital records, operative reports, angiograms and radiology reports were reviewed and analyzed retrospectively.

Results

99 aneurysms were treated in 77 patients in 102 procedures. Mean patient age was 75±4 years, and the average aneurysm size was 11±7 mm. Adjuvant techniques were used in 66% of cases. Endovascular procedures included coiling alone (32%), balloon assisted coiling (19%), stent assisted coiling (37%), balloon assisted stent and coiling (8%), stent only (1%) and glue (2%). The permanent morbidity and mortality rates were 1% and 3%, respectively. Four adverse events were attributed to the patient's age. Posterior circulation aneurysms were associated with more adverse events (41%) than anterior circulation aneurysms (14%). Endovascular treatments using adjuvant techniques were associated with a higher complication rate than coiling alone.

Conclusions

With only a 4% permanent rate of neurological morbidity and mortality, endovascular treatment of unruptured aneurysms can be performed safely in the elderly. Age should not be the limiting factor when considering endovascular therapy.
Acutely ruptured intracranial saccular aneurysms treated with stent assisted coiling: complications and outcomes in 42 consecutive patients
Authors: Taylor R A , Callison R C , Martin C O , Hayakawa M , Chaloupka J C
Published: December 2009

Background

Antiplatelet agents are required to prevent thromboembolic complications from recently deployed intracranial stents, yet they carry a risk of bleeding complications that may be serious in patients with recent subarachnoid hemorrhage.

Method

Consecutive patients at a single institution who had ruptured intracranial saccular aneurysms treated with stent assisted coiling were retrospectively reviewed. Our primary outcomes were ischemic stroke related to the stent and bleeding complications possibly related to antithrombotic therapy. Secondary outcomes included 3 month follow-up National Institute of Health Stroke Scale (NIHSS) scores and modified Rankin Scale (mRS) scores.

Results

44 aneurysms in 42 patients were treated. Seven patients experienced ischemic strokes during their hospitalization. Five ischemic strokes were secondary to vasospasm; one was definitely related to thrombus formation within the stent and one was possibly related to the stent. Two patients had asymptomatic intracranial hemorrhages and one patient had a symptomatic intracranial hemorrhage. Patients with Hunt and Hess grades I–II (n=25) experienced no stent associated ischemic strokes or symptomatic intracranial hemorrhages. The two stent associated ischemic strokes and one symptomatic intracranial hemorrhage occurred in patients with Hunt and Hess grades III–V (n=17) and patients with external ventricular drains (EVDs) (n=17). Only one patient had disability at the 3 month follow-up that was possibly related to the stent (mRS score of 3 and NIHSS score of 2).

Conclusion

These data suggest that higher grade hemorrhage patients, especially those with EVDs, are at greater risk for ischemic stroke and/or bleeding complications than lower grade patients. However, the complications had a small impact on mid-term disability outcomes in this cohort.