bluuebunny/crossref_metadata_2025 huggingface dataset:
Focused-ion beam fabrication of nanometer orifices for leak detection
Authors: Firpo G. , Repetto L. , Buatier de Mongeot F. , Valbusa U.
Published: October 2009

The leak devices most frequently used to calibrate leak-detection instruments are permeation leaks. These devices are very sensitive to temperature and can only be used with helium. The physical-leak types that could overcome this limits are prone to clog and their minimum size (about 1 μm) limits their applicability in the lowest flow range. Here, the authors propose a fabrication technique by means of focused-ion beam with which, in suitable materials, they are able to produce nanometer orifices. These devices [Università degli Studi di Genova, Italian Patent No., TO2008A000683 (18 September 2008)] work in the molecular-flow regime up to atmospheric pressure and do not clog. Other advantageous characteristics are the possibility of obtaining leak rates in the range equal to those of the permeation type and the linear dependence of the throughput on the inlet pressure.
Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A β-Lactamase from
Pseudomonas luteola
Authors: Doublet Benoît , Robin Frédéric , Casin Isabelle , Fabre Laëtitia , Le Fleche Anne , Bonnet Richard , Weill François-Xavier
Published: November 2009

Pseudomonas luteola
(formerly classified as CDC group Ve-1 and named
Chryseomonas luteola
) is an unusual pathogen implicated in rare but serious infections in humans. A novel β-lactamase gene,
bla
LUT-1
, was cloned from the whole-cell DNA of the
P. luteola
clinical isolate LAM, which had a weak narrow-spectrum β-lactam-resistant phenotype, and expressed in
Escherichia coli
. This gene encoded LUT-1, a 296-amino-acid Ambler class A β-lactamase with a pI of 6 and a theoretical molecular mass of 28.9 kDa. The catalytic efficiency of this enzyme was higher for cephalothin, cefuroxime, and cefotaxime than for penicillins. It was found to be 49% to 59% identical to other Ambler class A β-lactamases from
Burkholderia
sp. (PenA to PenL),
Ralstonia eutropha
(REUT),
Citrobacter sedlakii
(SED-1),
Serratia fonticola
(FONA and SFC-1),
Klebsiella
sp. (KPC and OXY), and CTX-M extended-spectrum β-lactamases. No gene homologous to the regulatory
ampR
genes of class A β-lactamases was found in the vicinity of the
bla
LUT-1
gene. The entire
bla
LUT-1
coding region was amplified by PCR and sequenced in five other genetically unrelated
P. luteola
strains (including the
P. luteola
type strain). A new variant of
bla
LUT-1
was found for each strain. These genes (named
bla
LUT-2
to
bla
LUT-6
) had nucleotide sequences 98.1 to 99.5% identical to that of
bla
LUT-1
and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The
bla
LUT
gene was located on a 700- to 800-kb chromosomal I-CeuI fragment, the precise size of this fragment depending on the
P. luteola
strain.
Isolation and Characterization of Two Members of the Siderophore-Microcin Family, Microcins M and H47
Authors: Vassiliadis Gaëlle , Destoumieux-Garzón Delphine , Lombard Carine , Rebuffat Sylvie , Peduzzi Jean
Published: November 2009

In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the
Enterobacteriaceae
family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several
Escherichia coli
strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the
Enterobacteriaceae
. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the
fepA
,
cir
, and
fiu
genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from
E. coli
Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.
Pharmacokinetics and Safety of S/GSK1349572, a Next-Generation HIV Integrase Inhibitor, in Healthy Volunteers
Authors: Min Sherene , Song Ivy , Borland Julie , Chen Shuguang , Lou Yu , Fujiwara Tamio , Piscitelli Stephen C.
Published: November 2009

S/GSK1349572 is a novel integrase inhibitor with potent
in vitro
anti-HIV activity, an
in vitro
resistance profile different from those of other integrase inhibitors, and favorable preclinical safety and pharmacokinetics (PK). Randomized, double-blind, placebo-controlled single-dose and multiple-dose, dose escalation studies evaluated the PK, safety, and tolerability of S/GSK1349572 for healthy subjects. In the single-dose study, two cohorts of 10 subjects each (8 active, 2 receiving placebo) received suspension doses of 2, 5, 10, 25, 50, and 100 mg in an alternating panel design. In the multiple-dose study, three cohorts of 10 subjects each (8 active, 2 receiving placebo) received suspension doses of 10, 25, and 50 mg once daily for 10 days. A cytochrome P450 3A (CYP3A) substudy with midazolam was conducted with the 25-mg dose. Laboratory testing, vital signs, electrocardiograms (ECGs), and PK sampling were performed at regular intervals. S/GSK1349572 was well tolerated. Most adverse events (AEs) were mild, with a few moderate AEs reported. Headache was the most common AE. No clinically significant laboratory trends or ECG changes were noted. PK was linear over the dosage range studied. The steady-state geometric mean area under the concentration-time curve over a dosing interval (AUC
0-τ
) and maximum concentration of the drug in plasma (
C
max
) ranged from 16.7 μg·h/ml (coefficient of variation [CV], 15%) and 1.5 μg/ml (CV, 24%) at a 10-mg dose to 76.8 μg·h/ml (CV, 19%) and 6.2 μg/ml (CV, 15%) at a 50-mg dose, respectively. The geometric mean steady-state concentration at the end of the dosing interval (
C
τ
) with a 50-mg dose was 1.6 μg/ml, approximately 25-fold higher than the protein-adjusted 90% inhibitory concentration (0.064 μg/ml). The half-life was approximately 15 h. S/GSK1349572 had no impact on midazolam exposure, indicating that it does not modulate CYP3A activity. The PK profile suggests that once-daily, low milligram doses will achieve therapeutic concentrations.
BEL-2, an Extended-Spectrum β-Lactamase with Increased Activity toward Expanded-Spectrum Cephalosporins in
Pseudomonas aeruginosa
Authors: Poirel Laurent , Docquier Jean-Denis , De Luca Filomena , Verlinde Annemie , Ide Louis , Rossolini Gian Maria , Nordmann Patrice
Published: November 2009

A
Pseudomonas aeruginosa
isolate recovered in Belgium produced a novel extended-spectrum ß-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The
bla
BEL-2
gene was identified from a
P. aeruginosa
isolate clonally related to another
bla
BEL-1
-positive isolate.
Antimicrobial Resistance in
Haemophilus influenzae
Respiratory Tract Isolates in Korea: Results of a Nationwide Acute Respiratory Infections Surveillance
Authors: Bae Songmee , Lee Jaehoon , Lee Jaehwa , Kim Eunah , Lee Sunhwa , Yu Jaeyon , Kang Yeonho
Published: November 2009

Antimicrobial susceptibility patterns and β-lactam resistance mechanisms of 544
Haemophilus influenzae
isolates through the nationwide Acute Respiratory Infections Surveillance (ARIS) network in Korea during 2005 and 2006 were determined. Resistance to ampicillin was 58.5%, followed by resistance to cefuroxime (23.3%), clarithromycin (18.7%), cefaclor (17.0%), amoxicillin-clavulanate (10.4%), and chloramphenicol (8.1%). Levofloxacin and cefotaxime were the most active agents tested in this study. β-Lactamase production (52.4%) was the main mechanism of ampicillin resistance, affecting 96.1% of TEM-1-type β-lactamase. According to their β-lactam resistance mechanisms, all isolates were classified into the following groups: β-lactamase-negative, ampicillin-sensitive (BLNAS) strains (
n
= 224; 41.5%); β-lactamase-positive, ampicillin-resistant (BLPAR) strains (
n
= 255; 47.2%); β-lactamase-negative, ampicillin-resistant (BLNAR) strains (
n
= 33; 6.1%); and β-lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (
n
= 28; 5.2%). Among the BLNAR and BLPACR strains, there were various patterns of multiple-amino-acid substitutions in penicillin-binding protein 3. Particularly, among BLNAR, group III isolates, which had three simultaneous substitutions (Met377Ile, Ser385Thr, and Leu389Phe), were identified for the first time in Korea. Three group III strains displayed the highest MIC of cefotaxime (1 to 2 μg/ml). The results indicate the importance of monitoring a changing situation pertaining to the increase and spread of BLNAR and BLPACR strains of
H. influenzae
for appropriate antibiotic therapy for patients with respiratory tract infections in Korea.
In Vitro and In Vivo Activities of 1-Hydroxy-2-Alkyl-4(1
H
)Quinolone Derivatives against
Toxoplasma gondii
Authors: Bajohr Lara Liv , Ma Ling , Platte Christian , Liesenfeld Oliver , Tietze Lutz F. , Groß Uwe , Bohne Wolfgang
Published: November 2009

1-Hydroxy-2-dodecyl-4(1
H
)quinolone (HDQ) was recently identified as a
Toxoplasma gondii
inhibitor. We describe here two novel 1-hydroxyquinolones, which displayed 50% inhibitory concentrations 10- and 5-fold lower than that of HDQ. In a mouse model of acute toxoplasmosis, these two compounds and HDQ reduced the percentages of infected peritoneal cells and decreased the parasite loads in lungs and livers. Compound B showed a tendency toward lowering parasite loads in brains in a mouse model of toxoplasmic encephalitis.
Screening and Quantification of the Expression of Antibiotic Resistance Genes in
Acinetobacter baumannii
with a Microarray
Authors: Coyne Sébastien , Guigon Ghislaine , Courvalin Patrice , Périchon Bruno
Published: November 2009

An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in
Acinetobacter baumannii
and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in
A. baumannii
and should allow detection of new efflux systems associated with antibiotic resistance.
An A643T Mutation in the Transcription Factor Upc2p Causes Constitutive
ERG11
Upregulation and Increased Fluconazole Resistance in
Candida albicans
Authors: Heilmann Clemens J. , Schneider Sabrina , Barker Katherine S. , Rogers P. David , Morschhäuser Joachim
Published: November 2009

The zinc cluster transcription factor Upc2p mediates upregulation of ergosterol biosynthesis genes in response to ergosterol depletion in the fungal pathogen
Candida albicans
. One mechanism of acquired resistance to the antifungal drug fluconazole, which inhibits ergosterol biosynthesis, is constitutively increased expression of the
ERG11
gene encoding the drug target enzyme. A G648D mutation in Upc2p has recently been shown to cause hyperactivity of the transcription factor, resulting in overexpression of ergosterol biosynthesis genes and increased fluconazole resistance. In order to investigate if gain-of-function mutations in Upc2p are a common mechanism of
ERG11
upregulation and fluconazole resistance, we sequenced the
UPC2
alleles of four
ERG11
-overexpressing, fluconazole-resistant
C. albicans
isolates and matched susceptible isolates from the same patients. In three of the isolate pairs, no differences in the
UPC2
alleles were found, suggesting that mechanisms other than Upc2p mutations can cause
ERG11
overexpression. One resistant isolate had become homozygous for a
UPC2
allele containing a G1927A substitution that caused an alanine-to-threonine exchange at amino acid position 643 of Upc2p. Replacement of one of the endogenous
UPC2
alleles in a fluconazole-susceptible strain by the
UPC2
A643T
allele resulted in
ERG11
overexpression and increased fluconazole resistance, which was further elevated when the A643T mutation was also introduced into the second
UPC2
allele. These results further establish gain-of-function mutations in
UPC2
, which can be followed by loss of heterozygosity for the mutated allele, as a mechanism of
ERG11
overexpression and increased fluconazole resistance in
C. albicans
, but other mechanisms of
ERG11
upregulation also exist.
Biologic Activities of the TolC-Like Protein of
Neisseria meningitidis
as Assessed by Functional Complementation in
Escherichia coli
Authors: Kamal Nazia , Shafer William M.
Published: November 2009

Neisseria meningitidis
can produce a TolC-like protein needed for secretion of FrpC but not efflux of antimicrobials. We now report that expression of the meningococcal
tolC
gene in a TolC-deficient strain of
Escherichia coli
can restore properties of α-hemolysis and antimicrobial resistance known to involve efflux pumps.
Increased Glucose Utilization in
Corynebacterium glutamicum
by Use of Maltose, and Its Application for the Improvement of
l
-Valine Productivity
Authors: Krause Felix S. , Henrich Alexander , Blombach Bastian , Krämer Reinhard , Eikmanns Bernhard J. , Seibold Gerd M.
Published: October 2009

Corynebacterium glutamicum
efficiently utilizes maltose as a substrate. We show here that the presence of maltose increases glucose utilization by raising the expression of
ptsG
, which encodes the glucose-specific EII permease of the phosphotransferase system. Consequently, the
l
-valine productivity of a pyruvate dehydrogenase complex-deficient
C. glutamicum
strain was improved by the presence of maltose.
Examination of
Xenorhabdus nematophila
Lipases in Pathogenic and Mutualistic Host Interactions Reveals a Role for
xlpA
in Nematode Progeny Production
Authors: Richards Gregory R. , Goodrich-Blair Heidi
Published: October 2009

Xenorhabdus nematophila
is a gammaproteobacterium and broad-host-range insect pathogen. It is also a symbiont of
Steinernema carpocapsae
, the nematode vector that transports the bacterium between insect hosts.
X. nematophila
produces several secreted enzymes, including hemolysins, lipases, and proteases, which are thought to contribute to virulence or nutrient acquisition for the bacterium and its nematode host in vivo.
X. nematophila
has two lipase activities with distinct in vitro specificities for Tween and lecithin. The gene encoding the Tween-specific lipase,
xlpA
, has been identified and is not required for
X. nematophila
virulence in one insect host, the tobacco hornworm
Manduca sexta
. However, the gene encoding the lecithin-specific lipase activity is not currently known. Here, we identify
X. nematophila estA
, a gene encoding a putative lecithinase, and show that an
estA
mutant lacks in vitro lipase activity against lecithin but has wild-type virulence in
Manduca sexta. X. nematophila
secondary-form phenotypic variants have higher in vitro lecithinase activity and
estA
transcript levels than do primary-form variants, and
estA
transcription is negatively regulated by NilR, a repressor of nematode colonization factors. We establish a role for
xlpA
, but not
estA
, in supporting production of nematode progeny during growth in
Galleria mellonella
insects. Future research is aimed at characterizing the biological roles of
estA
and
xlpA
in other insect hosts.
Diverse Genetic Markers Concordantly Identify Bovine OriginEscherichia coliO157 Genotypes Underrepresented in Human Disease
Authors: Whitworth Joshua , Zhang Yubei , Bono James , Pleydell Eve , French Nigel , Besser Thomas
Published: October 2009

Genetic markers previously reported to occur at significantly different frequencies in isolates ofEscherichia coliO157:H7 obtained from cattle and from clinically affected humans concordantly delineate at least five genetic groups. Isolates in three of these groups consistently carry one or more markers rarely found among clinical isolates.
Low-Density Macroarray Targeting Non-Locus of Enterocyte Effacement Effectors (
nle
Genes) and Major Virulence Factors of Shiga Toxin-Producing
Escherichia coli
(STEC): a New Approach for Molecular Risk Assessment of STEC Isolates
Authors: Bugarel Marie , Beutin Lothar , Fach Patrick
Published: October 2009

Rapid and specific detection of Shiga toxin-producing
Escherichia coli
(STEC) strains with a high level of virulence for humans has become a priority for public health authorities. This study reports on the development of a low-density macroarray for simultaneously testing the genes
stx
1
,
stx
2
,
eae
, and
ehxA
and six different
nle
genes issued from genomic islands OI-122 (
ent
,
nleB
, and
nleE
) and OI-71 (
nleF
,
nleH1
-
2
, and
nleA
). Various strains of
E. coli
isolated from the environment, food, animals, and healthy children have been compared with clinical isolates of various seropathotypes. The
eae
gene was detected in all enteropathogenic
E. coli
(EPEC) strains as well as in enterohemorrhagic
E. coli
(EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene
ehxA
was more prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally distributed. The
nle
genes were detected only in some EPEC and EHEC strains but with various distributions, showing that
nle
genes are strain and/or serotype specific, probably reflecting adaptation of the strains to different hosts or environmental niches. One characteristic
nle
gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H−, O55:H7, O123:H11, O172:H25, and O165:H25 was
ent/espL2
,
nleB
,
nleE
,
nleF
,
nleH1
-
2
,
nleA
. (Brackets indicate genotyping of the
flic
or
rfb
genes.) A second
nle
pattern (
ent/espL2
,
nleB
,
nleE
,
nleH1
-
2
) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and O15:H2. The presence of
eae
,
ent/espL2
,
nleB
,
nleE
, and
nleH1
-
2
genes is a clear signature of STEC strains with high virulence for humans.
Copper Stress Induces a Global Stress Response in
Staphylococcus aureus
and Represses
sae
and
agr
Expression and Biofilm Formation
Authors: Baker Jonathan , Sitthisak Sutthirat , Sengupta Mrittika , Johnson Miranda , Jayaswal R. K. , Morrissey Julie A.
Published: October 2009

Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify
Staphylococcus aureus
copper-responsive genes. Transcriptional profiling of
S. aureus
SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that
S. aureus
has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H
2
O
2
scavenging is an important
S. aureus
copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that
sae
,
agr
, and
eap
are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.
Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in
Schizosaccharomyces pombe
Authors: Berlanga Juan José , Rivero Damariz , Martín Ruth , Herrero Saturnino , Moreno Sergio , de Haro César
Published: October 2009

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in
Schizosaccharomyces pombe
. In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2α) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2α, and functionally replaced the endogenous
Saccharomyces cerevisiae
Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2α kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2α kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2α phosphorylation. Finally, the MAPK Sty1, but not the eIF2α kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2α kinase pathways for a particular range of stress responses.
Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and StarchlessChlamydomonas reinhardtii
Authors: Wang Zi Teng , Ullrich Nico , Joo Sunjoo , Waffenschmidt Sabine , Goodenough Ursula
Published: October 2009

When the unicellular green soil algaChlamydomonas reinhardtiiis deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in thesta6mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C16and C18) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C18:1). The FFA are ∼50% C16and ∼50% C18. The LB-derived TAG yield from a liter ofsta6cells at 107cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production inC. reinhardtiihas the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.
Virulence, Inflammatory Potential, and Adaptive Immunity Induced by
Shigella flexneri msbB
Mutants
Authors: Ranallo Ryan T. , Kaminski Robert W. , George Tonia , Kordis Alexis A. , Chen Qing , Szabo Kathleen , Venkatesan Malabi M.
Published: November 2009

The ability of genetically detoxified lipopolysaccharide (LPS) to stimulate adaptive immune responses is an ongoing area of investigation with significant consequences for the development of safe and effective bacterial vaccines and adjuvants. One approach to genetic detoxification is the deletion of genes whose products modify LPS. The
msbB1
and
msbB2
genes, which encode late acyltransferases, were deleted in the
Shigella flexneri
2a human challenge strain 2457T to evaluate the virulence, inflammatory potential, and acquired immunity induced by strains producing underacylated lipid A. Consistent with a reduced endotoxic potential,
S. flexneri
2a
msbB
mutants were attenuated in an acute mouse pulmonary challenge model. Attenuation correlated with decreases in the production of proinflammatory cytokines and in chemokine release without significant changes in lung histopathology. The levels of specific proinflammatory cytokines (interleukin-1β [IL-1β], macrophage inflammatory protein 1α [MIP-1α], and tumor necrosis factor alpha [TNF-α]) were also significantly reduced after infection of mouse macrophages with either single or double
msbB
mutants. Surprisingly, the
msbB
double mutant displayed defects in the ability to invade, replicate, and spread within epithelial cells. Complementation restored these phenotypes, but the exact nature of the defects was not determined. Acquired immunity and protective efficacy were also assayed in the mouse lung model, using a vaccination-challenge study. Both humoral and cellular responses were generally robust in
msbB
-immunized mice and afforded significant protection from lethal challenge. These data suggest that the loss of either
msbB
gene reduces the endotoxicity of
Shigella
LPS but does not coincide with a reduction in protective immune responses.
The Effects of PspC on Complement-Mediated Immunity to
Streptococcus pneumoniae
Vary with Strain Background and Capsular Serotype
Authors: Yuste Jose , Khandavilli Suneeta , Ansari Naadir , Muttardi Kairya , Ismail Laura , Hyams C. , Weiser Jeffrey , Mitchell Timothy , Brown Jeremy S.
Published: November 2009

Streptococcus pneumoniae
may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between
S. pneumoniae
strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several
S. pneumoniae
strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on
S. pneumoniae
, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia.
Salmonella enterica
Serovar Typhimurium
trxA
Mutants Are Protective against Virulent Challenge and Induce Less Inflammation than the Live-Attenuated Vaccine Strain SL3261
Authors: Peters S. E. , Paterson G. K. , Bandularatne E. S. D. , Northen H. C. , Pleasance S. , Willers C. , Wang J. , Foote A. K. , Constantino-Casas F. , Scase T. J. , Blacklaws B. A. , Bryant C. E. , Mastroeni P. , Charles I. G. , Maskell D. J.
Published: November 2009

In
Salmonella enterica
serovar Typhimurium,
trxA
encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of
trxA
in the mouse-virulent
S.
Typhimurium strain SL1344 (SL1344
trxA
), replacing the gene with a kanamycin resistance gene cassette. SL1344
trxA
was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344
trxA
induced less inflammation in murine spleens and livers than SL3261, the
aroA
mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344
trxA
was partially attributed to a reduction in the number of both CD4
+
and CD8
+
T cells and B lymphocytes in the spleen and reduced infiltration by CD11b
+
cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a
trxA
mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting
trxA
in SL3261. SL3261
trxA
was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344
trxA
.
A Novel Functional Variant in the Stem Cell Growth Factor Promoter Protects against Severe Malarial Anemia
Authors: Ouma Collins , Keller Christopher C. , Davenport Gregory C. , Were Tom , Konah Stephen , Otieno Michael F. , Hittner James B. , Vulule John M. , Martinson Jeremy , Ong'echa John M. , Ferrell Robert E. , Perkins Douglas J.
Published: November 2009

Plasmodium falciparummalaria is a leading global cause of infectious disease burden. In areas in whichP. falciparumtransmission is holoendemic, such as western Kenya, severe malarial anemia (SMA) results in high rates of pediatric morbidity and mortality. Although the pathophysiological basis of SMA is multifactorial, we recently discovered that suppression of unexplored hematopoietic growth factors that promote erythroid and myeloid colony development, such as stem cell growth factor (SCGF) (C-type lectin domain family member 11A [CLEC11A]), was associated with enhanced development of SMA and reduced erythropoietic responses. To extend these investigations, the relationships between a novel SCGF promoter variant (−539C/T, rs7246355), SMA (hemoglobin [Hb] < 6.0 g/dl), and reduced erythropoietic responses (reticulocyte production index [RPI], <2.0) were investigated with Kenyan children (n= 486) with falciparum malaria from western Kenya. Circulating SCGF was positively correlated with hemoglobin levels (r= 0.251;P= 0.022) and the reticulocyte production index (RPI) (r= 0.268;P= 0.025). Children with SMA also had lower SCGF levels than those in the non-SMA group (P= 0.005). Multivariate logistic regression analyses controlling for covariates demonstrated that individuals with the homologous T allele were protected against SMA (odds ratio, 0.57; 95% confidence interval [95% CI] 0.34 to 0.94;P= 0.027) relative to CC (wild-type) carriers. Carriers of the TT genotype also had higher SCGF levels in circulation (P= 0.018) and in peripheral blood mononuclear cell culture supernatants (P= 0.041), as well as an elevated RPI (P= 0.005) relative to individuals with the CC genotype. The results presented here demonstrate that homozygous T at −539 in the SCGF promoter is associated with elevated SCGF production, enhanced erythropoiesis, and protection against the development of SMA in children with falciparum malaria.
Burkholderia mallei
Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages
Authors: Burtnick Mary N. , DeShazer David , Nair Vinod , Gherardini Frank C. , Brett Paul J.
Published: November 2009

Burkholderia mallei
is a facultative intracellular pathogen that causes severe disease in animals and humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of
B. mallei tssE
mutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the
tssE
mutants were able to survive within RAW 264.7 cells, significant growth defects were observed in comparison to controls. In addition, analysis of infected monolayers by differential interference contrast and fluorescence microscopy demonstrated that the
tssE
mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy,
tssE
mutants were shown to undergo escape from lysosome-associated membrane protein 1-positive vacuoles. Curiously, however, following entry into the cytosol, the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in growth and actin-based motility following uptake of
B. mallei
by RAW 264.7 cells.
DesT Coordinates the Expression of Anaerobic and Aerobic Pathways for Unsaturated Fatty Acid Biosynthesis in
Pseudomonas aeruginosa
Authors: Subramanian Chitra , Rock Charles O. , Zhang Yong-Mei
Published: October 2009

The
fabA
and
fabB
genes are responsible for anaerobic unsaturated fatty acid formation in
Pseudomonas aeruginosa
. Expression of the
fabAB
operon was repressed by exogenous unsaturated fatty acids, and DNA sequences upstream of the translational start site were used to affinity purify DesT. The single protein interaction with the
fabAB
promoter detected in wild-type cell extracts was absent in the
desT
deletion strain, as was the repression of
fabAB
expression by unsaturated fatty acids. Thus, DesT senses the overall composition of the acyl-coenzyme A pool to coordinate the expression of the operons for the anaerobic (
fabAB
) and aerobic (
desCB
) pathways for unsaturated fatty acid synthesis.
Inefficient Tat-Dependent Export of Periplasmic Amidases in an
Escherichia coli
Strain with Mutations in Two DedA Family Genes
Authors: Sikdar Rakesh , Doerrler William T.
Published: October 2009

The DedA family genes are found in most bacterial genomes. Two of these proteins are
Escherichia coli
YqjA and YghB, predicted inner membrane proteins of unknown function sharing 61% amino acid identity. The
E. coli
single deletion mutants are largely without phenotype, but the double mutant (BC202; Δ
yqjA
::Tet
r
Δ
yghB
::Kan
r
) is characterized by incomplete cell division, temperature sensitivity, and altered phospholipid levels (K. Thompkins et al., J. Bacteriol. 190:4489-4500, 2008). In this report, we have better characterized the cell division chaining defect of BC202. Fluorescence recovery after photobleaching indicates that 58% of the cells in chains are compartmentalized by at least a cytoplasmic membrane. Green fluorescent protein fusions to the cell division proteins FtsZ, ZipA, FtsI, FtsL, and FtsQ are correctly localized to new septation sites in BC202. Periplasmic amidases AmiC and AmiA, secreted by the twin arginine transport (Tat) pathway, are localized to the cytoplasm in BC202. Overexpression of AmiA, AmiC, or AmiB, a periplasmic amidase secreted via the general secretory pathway, restores normal cell division but does not suppress the temperature sensitivity of BC202, indicating that YghB and YqjA may play additional roles in cellular physiology. Strikingly, overexpression of the Tat export machinery (TatABC) results in normal cell division and growth at elevated temperatures. These data collectively suggest that the twin arginine pathway functions inefficiently in BC202, likely due to the altered levels of membrane phospholipids in this mutant. These results underscore the importance of membrane composition in the proper function of the Tat protein export pathway.
Prediction and prevention of musculoskeletal injury: a paradigm shift in methodology
Authors: Quatman C E , Quatman C C , Hewett T E
Published: November 2009

Traditional methods employed to study musculoskeletal injury mechanisms and joint biomechanics utilise in vivo or in vitro techniques. The advent of new technology and improved methods has also given rise to in silico (computer modelling) techniques. Under the current research paradigm, in vivo, in vitro and in silico methods independently provide information regarding the mechanisms and prevention of musculoskeletal injury. However, individually, each of these methods has multiple, inherent limitations and is likely to provide incomplete answers about multifactorial, complex injury conditions. The purpose of this treatise is to review current methods used to study, understand, and prevent musculoskeletal injury and to develop new conceptual-methodological frameworks that may help create a paradigm shift in musculoskeletal injury prevention research. We term the fusion of these three techniques in simulacra amalgama, or simply in sim, meaning a “union of models done on the likeness of phenomena.” Anterior cruciate ligament (ACL) injury will be employed as a model example for the utility and applicability of the proposed, synthesised approach. Shifting the current experimental paradigm to incorporate a multifaceted, multidisciplinary, integration of in vivo, in vitro and in silico methods into the proposed in sim approaches may provide a platform for a more comprehensive understanding of the relationships between complex joint biomechanics and observed injury mechanisms.
Intra-arterial or intravenous thrombolysis for acute ischemic stroke? The SYNTHESIS pilot trial
Authors: Ciccone A , Valvassori L , Ponzio M , Ballabio E , Gasparotti R , Sessa M , Scomazzoni F , Tiraboschi P , Sterzi R ,   the SYNTHESIS Investigators
Published: October 2009

Objective

To assess the feasibility, safety and preliminary efficacy of intra-arterial thrombolysis (IAT) compared with standard intravenous thrombolysis (IVT) for acute ischemic stroke.

Methods

Eligible patients with ischemic stroke, who were devoid of contraindications, started IVT within 3 h or IAT as soon as possible within 6 h. Patients were randomized within 3 h of onset to receive either intravenous alteplase, in accordance with the current European labeling, or up to 0.9 mg/kg intra-arterial alteplase (maximum 90 mg), over 60 min into the thrombus, if necessary with mechanical clot disruption and/or retrieval. The purpose of the study was to determine the proportion of favorable outcome at 90 days. Safety endpoints included symptomatic intracranial hemorrhage (SICH), death and other serious adverse events.

Results

54 patients (25 IAT) were enrolled. Median time from stroke onset to start to treatment was 3 h 15 min for IAT and 2 h 35 min for IVT (p<0.001). Almost twice as many patients on IAT as those on IVT survived without residual disability (12/25 vs 8/29; OR 3.2; 95% CI 0.9 to 11.4; p=0.067). SICH occurred in 2/25 patients on IAT and in 4/29 on IVT (OR 0.5; CI 0.1 to 3.3; p=0.675). Mortality at day 7 was 5/25 (IAT) compared with 4/29 (IVT) (OR 1.6; CI 0.4 to 6.7; p=0.718). There was no significant difference in the rate of other serious adverse events.

Conclusions

Rapid initiation of IAT is a safe and feasible alternative to IVT in acute ischemic stroke.

Trial registration number NCT00540527.
PROTON STATIC PROPERTIES BY USING HYPERCENTRAL CONSTITUENT QUARK MODEL AND ISOSPIN
Authors: SALEHI NASRIN , RAJABI ALI AKBAR
Published: November 2009

The static properties of protons are useful for understanding the quark structure of the proton. In his work we have introduced the hypercentral constituent quark model and isospin dependent potentials. Here constituent quarks interact with each other via a potential in which we have taken into account the three-body force effect and the standard two-body potential contributions. According to our model the static properties of protons containing u and d quarks are better than the other models and closer to experimental results. The two key ingredients of this improvement are the effective quark–gluon hypercentral potentials, and hyperfine interaction and isospin dependence potential. Recently, Schrödinger equation has been solved by Giannini but we have solved the Dirac equation exact analytically and we have shown that a considerable improvement in the description of the static properties of proton is obtained with an isospin dependent potential and the complete interaction including spin and isospin terms reproduces the position of the quark.
CONSTRAINT STRUCTURE AND QUANTIZATION OF A NON-ABELIAN GAUGE THEORY BY MEANS OF DIRAC BRACKETS
Authors: BRACKEN PAUL
Published: November 2009

An SO(3) non-Abelian gauge theory is introduced. The Hamiltonian density is determined and the constraint structure of the model is derived. The first-class constraints are obtained and gauge-fixing constraints are introduced into the model. Finally, using the constraints, the Dirac brackets can be determined and a canonical quantization is found using Dirac's procedure.
ON THE "UNIVERSAL" QUANTUM AREA SPECTRUM
Authors: MEDVED A. J. M.
Published: November 2009

There has been much debate about the form of the quantum area spectrum for a black hole horizon, with the evenly spaced conception of Bekenstein having featured prominently in the discourse. Here, we refine a very recently proposed method for calibrating the Bekenstein form of the spectrum. Our refined treatment predicts, as did its predecessor, a uniform spacing between adjacent spectral levels of 8π in Planck units — notably, an outcome that already has a pedigree as a proposed "universal" value for this intrinsically quantum-gravitational measure. Although the two approaches are somewhat similar in logic and quite agreeable in outcome, we argue that our version is conceptually more elegant and formally simpler than its precursor. Moreover, our rendition is able to circumvent a previously unnoticed technical issue and, as an added bonus, translates to generic theories of gravity in a very direct manner.
THE TRAPPED-ION QUBIT: COHERENT CONTROL IN INFINITE-DIMENSIONAL QUANTUM SYSTEMS
Authors: RANGAN C.
Published: November 2009

Theories of quantum control have, until recently, made the assumption that the Hilbert space of a quantum system can be truncated to finite dimensions. Such truncations, which can be achieved for most quantum systems via bandwidth restrictions, have enabled the development of a rich variety of quantum control and optimal control schemes. Recent studies in quantum information processing have addressed the control of infinite-dimensional quantum systems such as the quantum states of a trapped-ion. Controllability in an infinite-dimensional quantum system is hard to prove with conventional methods, and infinite-dimensional systems provide unique challenges in designing control fields.

In this paper, we will discuss the control of a popular system for quantum computing the trapped-ion qubit. This system, modeled by a spin-half particle coupled to a quantized harmonic oscillator, is an example for a surprisingly rich variety of control problems. We will show how this infinite-dimensional quantum system can be examined via the lens of the Finite Controllability Theorem, two-color STIRAP, the generalized Heisenberg system, etc.

These results are important from the viewpoint of developing more efficient quantum control protocols, particularly in quantum computing systems. This work shows how one can expand the scope of quantum control research to beyond that of finite-dimensional quantum systems.
BIDIRECTIONAL QUANTUM SECURE DIRECT COMMUNICATION IN DRIVEN CAVITY QED
Authors: SHAN CHUAN-JIA , LIU JI-BING , CHENG WEI-WEN , LIU TANG-KUN , HUANG YAN-XIA , LI HONG
Published: November 2009

A theoretical scheme of bidirectional quantum secure direct communication is proposed in the context of driven cavity QED. We first present an entanglement swapping scheme in cavities where two atoms without previous interaction can be entangled with a success probability of unity. Then, based on a novel property of entanglement swapping, we propose a bidirectional quantum secure direct communication protocol, in which two legitimate users can exchange their different secret messages simultaneously in a direct way. The probability of success in our scheme is 1.0. This scheme does not involve apparent (or direct) Bell-state measurements and is insensitive to the cavity decay and the thermal field.
TWO SUPER-INTEGRABLE SYSTEMS AND ASSOCIATED SUPER-HAMILTONIAN STRUCTURES
Authors: ZHAO QIU-LAN , LI XIN-YUE , HE BAI-YING
Published: November 2009

The super extensions of g-cKdV and mKdV integrable systems are proposed. Two hierarchies of super-integrable nonlinear evolution equations are found. In addition, making use of the super-trace identity, we construct the super-Hamiltonian structures of zero-curvature equations associated with Lie superalgebras.
SUPERCONDUCTING RuSr2(Gd2-xCex)Cu2O8-δ PREPARED BY SOL–GEL ROUTE
Authors: YEOH L. M. , AHMAD M. , ABD-SHUKOR R.
Published: November 2009

A sol–gel route based on tatrate/acetate was used to synthesize RuSr 2( Gd 2-x Ce x) Cu 2 O 10-δ (x = 0.5 to 0.7) cuprate superconductors. A sample, with x = 0.6, was heated to 1050°C in oxygen flow for 24 h, followed by cooling at a rate of 1°C/min until it reached 100°C. The sample showed a single Ru -1222 phase with tetragonal symmetry. The lattice parameters for the sample were a = b = 3.863 Å and c = 28.57 Å. The onset transition temperature was 59 K, and the zero-resistance temperature was 40 K.