bluuebunny/crossref_metadata_2025 huggingface dataset:
The outer membrane protein TolC is required for phytoalexin resistance and virulence of the fire blight pathogen Erwinia amylovora
Authors: Al‐Karablieh Nehaya , Weingart Helge , Ullrich Matthias S.
Published: February 2009

Erwinia amylovora causes fire blight on several plant species such as apple and pear, which produce diverse phytoalexins as defence mechanisms. An evolutionary successful pathogen thus must develop resistance mechanisms towards these toxic compounds. The E. amylovora outer membrane protein, TolC, might mediate phytoalexin resistance through its interaction with the multidrug efflux pump, AcrAB. To prove this, a tolC mutant and an acrB/tolC double mutant were constructed. The minimal inhibitory concentrations of diverse antimicrobials and phytoalexins were determined for these mutants and compared with that of a previously generated acrB mutant. The tolC and arcB/tolC mutants were considerably more susceptible than the wild type but showed similar levels as the acrB mutant. The results clearly indicated that neither TolC nor AcrAB significantly interacted with other transport systems during the efflux of the tested toxic compounds. Survival and virulence assays on inoculated apple plants showed that pathogenicity and the ability of E. amylovora to colonize plant tissue were equally impaired by mutations of tolC and acrB/tolC. Our results allowed the conclusion that TolC plays an important role as a virulence and fitness factor of E. amylovora by mediating resistance towards phytoalexins through its exclusive interaction with AcrAB.
Emphysema Mediated by Lung Overexpression of ADAM10
Authors: Saitoh Hiroki , Leopold Philip L. , Harvey Ben‐Gary , O'Connor Timothy P. , Worgall Stefan , Hackett Neil R. , Crystal Ronald G.
Published: February 2009

Cigarette smoking is the major risk factor for emphysema, a disorder of the lung parenchyma characterized by destruction of the alveolar walls. Current concepts of the pathogenesis of emphysema hold that the destruction of the lung parenchyma results, in part, from a local imbalance of proteases and antiproteases. Based on the knowledge that human alveolar macrophages express ADAM 10, a protease capable of destroying basement membrane collagen but not previously implicated in emphysema, we used adenovirus‐mediated lung expression of ADAM 10 in a mouse model to assess whether an increased burden of ADAM 10 was capable of inducing emphysema. To assess this, the human ADAM 10 cDNA under control of a constitutive promoter was inserted into an adenovirus gene transfer vector (AdhADAMlO), and the vector (1011 particle units) administered to the respiratory tract of wild type C57BI/6 mice. Lung levels of ADAM 10 mRNAand protein were upregulated following AdhADAMlO administration. After 8 weeks, quantitative morphometry of the lung parenchyma demonstrated that AdhADAMlO administration induced emphysema (mean linear intercept of 60.6 + 1.3 μm compared with 55.6 + 0.8 in mice treated with a control vector, p < 0.003). These results suggest a role of ADAM 10 in the pathogenesis of emphysema, adding to the list of proteases expressed in the lung that are capable of contributing to the development of lung destruction.
c‐Kit+ Bone Marrow Stem Cells Differentiate into Functional Cardiac Myocytes
Authors: Kubo Hajime , Berretta Remus M. , Jaleel Naser , Angert David , Houser Steven R.
Published: February 2009

The utility of bone marrow cells (BMCs) to regenerate cardiac myocytes is controversial. The present study examined the capacity of different types of BMCs to generate functional cardiac myocytes. Isolated c‐kit+ BMCs (BMSCs), c‐kit+ and crude BMCs from the adult feline femur were membrane stained with PKH26 dye or infected with a control enhanced green fluorescence protein transcript (EGFP)‐adenovirus prior to co‐culture upon neonatal rat ventricular myocytes (NRVM). Co‐cultured cells were immuno‐stained for c‐kit, α‐tropomyosin, α‐actinin, connexin 43 (C×43) and Ki67 and analyzed with confocal microscopy. Electrophysiology of BMSC derived myocytes were compared to NRVMs within the same culture dish. Gap junction function was analyzed by fluorescence recovery after photo‐bleaching (FRAP). BMCs proliferated and differentiated into cardiac myocytes during the first 48 hours of co‐culturing. These newly formed cardiac myocytes were able to contract spontaneously or synchronously with neighboring NRVMs. The myogenic rate of c‐kit+ BMSCs was significantly greater than c‐kit+ and crude BMCs (41.2 ± 2.1, 6.1 ± 1.2, and 17.1 ± 1.5%, respectively). The newly formed cardiac myocytes exhibited an immature electrophysiological phenotype until they became electrically coupled to NRVMs through functional gap junctions. BMSCs did not become functional myocytes in the absence of NRVMs. In conclusion, c‐kit+ BMSCs have the ability to transdifferentiate into functional cardiac myocytes.
A Paradise Within? Mary Astell, Sarah Scott and the Limits of Utopia
Authors: DEVEREAUX JOHANNA
Published: February 2009

Mary Astell and Sarah Scott's feminist texts A Serious Proposal and Milennium Hall, written sixty years apart from each other, both espouse theories of feminine retreat and education. However, the changing philosophical context in which these texts were written, from the 1690s Cambridge Platonists to the mid‐century theorists of sociable benevolence, makes them very different in their intellectual perspectives. The feminist possibilities identified by Astell and Scott arise from their responses to contemporary male philosophers. Their texts, far from straightforwardly utopian, identify how through the correct kind of philosophical education, women might learn to be of social and spiritual worth.
‘At This Period Peculiarly Necessary’: The Republication of Milton's Political Tracts in the 1790s
Authors: CRAWFORD JOSEPH
Published: February 2009

This article investigates the republication of Milton's political pamphlets in the 1790s, locating them in their context as part of the ‘pamphlet wars’ that followed the outbreak of the French Revolution. It discusses the reception history of Milton's political works between his death and the French Revolution, demonstrating their extreme rarity and low cultural profile before 1790. It then gives a detailed account of each of the reprinted Milton pamphlets of which any copies have survived, analysing their paratexts to explore how and why these 150‐year‐old pamphlets were deployed as contributions to the political and ideological debates of the 1790s.
Chromosome number, karyotype and pollen morphology of the rare endemic Centaurea goeksunensis
Authors: Gömürgen A. N. , Doğan C. , Özmen E. , Aytaç Z.
Published: February 2009

In this paper, chromosome number, karyotype and pollen morphology of Centaureagoeksunensis Aytaç & H. Duman, described in 2005, are studied for the first time. Centaurea goeksunensis is found to be tetraploid (2n=4x=60). This is the second record of polyploidy in Centaurea sect. Psephelloideae (Boiss.) Sons. The basic chromosome number of C. goeksunensis was found to be x=15 and the haploid karyotype formula is 18 m+10 sm+2 st. The pollen grains of C. goeksunensis were found to be radially symmetric, isopolar and tricolporate. They are intersemiangular in polar view and prolate–sphaeroidal in equatorial view. The exine is thickened at the poles.
Teucrium ramaswamii sp. nov. (Lamiaceae) from India
Authors: Viswanathan M. B. , Manikandan U.
Published: February 2009

A new species of Lamiaceae, Teucrium ramaswamii M. B. Viswan. & U. Manik. is described from the Kalakkad‐Mundanthurai Tiger Reserve (KMTR) in the Agastyamalai hills of the southwestern Ghats in peninsular India. It is allied to T. tomentosum Heyne ex Benth. but differs by stem, leaves and inflorescence being glandular strigose; leaves being deltoid–ovate, crenate–dentate or doubly crenate–dentate at margin, subcoriaceous, sparsely strigose above, densely strigose beneath; bracts being oblong–deltoid, ca 9.4×2.6 mm; calyx with uppermost teeth being lanceolate, ca 2.8×1.9 mm, lateral teeth being broadly triangular, lower teeth being oblong–lanceolate; corolla being glandular strigose outside below lateral lobes and ovary being glandular strigose. Using the IUCN criteria, conservation status of the species is assigned as Critically Endangered based on the field data (2000–2002). Life history studies, population ecology, genome resource banking and wild population management are recommended for conserving this species.
Modified quantum dimensions and re-normalized link invariants
Authors: Geer Nathan , Patureau-Mirand Bertrand , Turaev Vladimir
Published: February 2009

In this paper we give a re-normalization of the Reshetikhin–Turaev quantum invariants of links, using modified quantum dimensions. In the case of simple Lie algebras these modified quantum dimensions are proportional to the usual quantum dimensions. More interestingly, we give two examples where the usual quantum dimensions vanish but the modified quantum dimensions are non-zero and lead to non-trivial link invariants. The first of these examples is a class of invariants arising from Lie superalgebras previously defined by the first two authors. These link invariants are multivariable and generalize the multivariable Alexander polynomial. The second example is a hierarchy of link invariants arising from nilpotent representations of quantized$\mathfrak {sl}(2)$at a root of unity. These invariants contain Kashaev’s quantum dilogarithm invariants of knots.
The exceptional zero conjecture for Hilbert modular forms
Authors: Mok Chung Pang
Published: February 2009

Using ap-adic analogue of the convolution method of Rankin–Selberg and Shimura, we construct the two-variablep-adicL-function of a Hida family of Hilbert modular eigenforms of parallel weight. It is shown that the conditions of Greenberg–Stevens [R. Greenberg and G. Stevens,p-adic L-functions and p-adic periods of modular forms, Invent. Math.111(1993), 407–447] are satisfied, from which we deduce special cases of the Mazur–Tate–Teitelbaum conjecture in the Hilbert modular setting.
Tropical fans and the moduli spaces of tropical curves
Authors: Gathmann Andreas , Kerber Michael , Markwig Hannah
Published: February 2009

We give a rigorous definition of tropical fans (the ‘local building blocks for tropical varieties’) and their morphisms. For a morphism of tropical fans of the same dimension we show that the number of inverse images (counted with suitable tropical multiplicities) of a point in the target does not depend on the chosen point; a statement that can be viewed as one of the important first steps of tropical intersection theory. As an application we consider the moduli spaces of rational tropical curves (both abstract and in some ℝr) together with the evaluation and forgetful morphisms. Using our results this gives new, easy and unified proofs of various tropical independence statements, e.g. of the fact that the numbers of rational tropical curves (in any ℝr) through given points are independent of the points.
Genericity of supercuspidal representations of p-adic Sp4
Authors: Blondel Corinne , Stevens Shaun
Published: February 2009

We describe the supercuspidal representations of Sp4(F), for F a non-archimedean local field of residual characteristic different from two, and determine which are generic.
Metabolism of an Alkyl Polyamine Analog by a Polyamine Oxidase from the Microsporidian
Encephalitozoon cuniculi
Authors: Bacchi Cyrus J. , Yarlett Nigel , Faciane Evangeline , Bi Xiangdong , Rattendi Donna , Weiss Louis M. , Woster Patrick M.
Published: February 2009

Encephalitozoon cuniculi
is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{
N
-[
o
-(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an
E. cuniculi
amine oxidase activity. The primary natural substrate for this oxidase activity was
N
′-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and
K

m

of 2 μM. In the presence of
N
′-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of
13
C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of
13
C-labeled BW-1 to
E. cuniculi
preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with
E. cuniculi
. The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.
A Small-Molecule Dengue Virus Entry Inhibitor
Authors: Wang Qing-Yin , Patel Sejal J. , Vangrevelinghe Eric , Xu Hao Ying , Rao Ranga , Jaber Deana , Schul Wouter , Gu Feng , Heudi Olivier , Ma Ngai Ling , Poh Mee Kian , Phong Wai Yee , Keller Thomas H. , Jacoby Edgar , Vasudevan Subhash G.
Published: February 2009

The incidence of dengue fever epidemics has increased dramatically over the last few decades. However, no vaccine or antiviral therapies are available. Therefore, the need for safe and effective antiviral drugs has become imperative. The entry of dengue virus into a host cell is mediated by its major envelope (E) protein. The crystal structure of the E protein reveals a hydrophobic pocket that is presumably important for low-pH-mediated membrane fusion. High-throughput docking with this hydrophobic pocket was performed, and hits were evaluated in cell-based assays. Compound 6 was identified as one of the inhibitors and had an average 50% effective concentration of 119 nM against dengue virus serotype 2 in a human cell line. Mechanism-of-action studies demonstrated that compound 6 acts at an early stage during dengue virus infection. It arrests dengue virus in vesicles that colocalize with endocytosed dextran and inhibits NS3 expression. The inhibitors described in this report can serve as molecular probes for the study of the entry of flavivirus into host cells.
Prospectively Validated Dosing Nomograms for Maximizing the Pharmacodynamics of Vancomycin Administered by Continuous Infusion in Critically Ill Patients
Authors: Pea Federico , Furlanut Mario , Negri Camilla , Pavan Federica , Crapis Massimo , Cristini Francesco , Viale Pierluigi
Published: February 2009

The efficacy of vancomycin against methicillin-resistant
Staphylococcus aureus
(MRSA)-related infections has been called into question by recent findings of higher rates of failure of vancomycin treatment of infections caused by strains with high MICs. Continuous infusion may be the best way to maximize the time-dependent activity of vancomycin. The aim of this study was to create dosing nomograms in relation to different creatinine clearance (CL
Cr
) estimates for use in daily clinical practice to target the steady-state concentrations (
C
ss
s) of vancomycin during continuous infusion at 15 to 20 mg/liter (after the administration of an initial loading dose of 15 mg/kg of body weight over 2 h). The correlation between vancomycin clearance (CL
v
) and CL
Cr
was retrospectively assessed in a cohort of critically ill patients (group 1,
n
= 70) to create a formula for dosage calculation to target
C
ss
at 15 mg/liter. The performance of this formula was prospectively validated in a similar cohort (group 2,
n
= 63) by comparison of the observed and the predicted
C
ss
s. A significant relationship between CL
v
and CL
Cr
was observed in group 1 (
P
< 0.001). The application of the calculated formula to vancomycin dosing in group 2 {infusion rate (g/24 h) = [0.029 × CL
Cr
(ml/min) + 0.94] × target C
ss
× (24/1,000)} led to a significant correlation between the observed and the predicted
C
ss
s (
r
= 0.80,
P
< 0.001). Two dosing nomograms based on CL
Cr
were created to target the vancomycin
C
ss
at 15 and 20 mg/liter in critically ill patients. These nomograms could be helpful in improving the vancomycin treatment of MRSA infections, especially in the presence of borderline-susceptible pathogens and/or of pathophysiological conditions which may enhance the clearance of vancomycin, while potentially avoiding the increased risk of nephrotoxicity observed with the use of high intermittent doses of vancomycin.
Functional Characterization of the σ
B
-Dependent
yabJ
-
spoVG
Operon in
Staphylococcus aureus
: Role in Methicillin and Glycopeptide Resistance
Authors: Schulthess Bettina , Meier Stefan , Homerova Dagmar , Goerke Christiane , Wolz Christiane , Kormanec Jan , Berger-Bächi Brigitte , Bischoff Markus
Published: February 2009

The alternative sigma factor σ
B
of
Staphylococcus aureus
controls the expression of multiple genes, including virulence determinants and global regulators; promotes capsule production; and increases the resistance levels of methicillin-resistant
S. aureus
(MRSA) and glycopeptide-intermediate-resistant
S. aureus
(GISA) strains. We show here that deletion of the σ
B
-controlled
yabJ-spoVG
operon, which codes for potential downstream regulators of σ
B
, abolished capsule synthesis and reduced resistance in MRSA and GISA to the same extent that σ
B
inactivation did. Introduction of the
yabJ-spoVG
operon in
trans
restored the original phenotype. By genetic manipulations, we show that SpoVG but not YabJ is required for complementation. We therefore postulate that SpoVG is the major factor of the
yabJ-spoVG
operon required in
S. aureus
for capsule formation and antibiotic resistance.
Pharmacokinetics of EDP-420 after Ascending Single Oral Doses in Healthy Adult Volunteers
Authors: Jiang Li-Juan , Wang Michelle , Or Yat Sun
Published: February 2009

EDP-420 (EP-013420, S-013420) is a first-in-class bicyclolide (bridged bicyclic macrolide) currently in clinical development for the treatment of respiratory tract infections. It has good preclinical pharmacokinetic properties across multiple species and potent in vitro and in vivo activity against respiratory tract infection pathogens, including
Haemophilus influenzae
, atypical organisms (e.g.,
Chlamydia pneumoniae, Mycoplasma pneumoniae
, and
Legionella pneumophila
), and multidrug-resistant streptococci. The safety, tolerability, and pharmacokinetics of an orally administered EDP-420 suspension in 40 healthy adult subjects were assessed in a randomized, double-blind, placebo-controlled, ascending single-dose study. Eligible subjects were sequentially randomized into one of five study groups (i.e., 100-, 200-, 400-, 800-, or 1,200-mg dosing groups) consisting of eight subjects (six active and two placebo) each. EDP-420 was well tolerated. There were no serious adverse events reported, nor were there any dose-limiting clinical or laboratory adverse events reported. EDP-420 was rapidly absorbed after a single oral dose. The mean plasma terminal half-life ranged from 15.6 to 20.1 h with low clearance. At the 400-mg dose, the area under the curve was 14.4 μg·h/ml, which well exceeded the required area under the concentration-time curve to cover common respiratory tract infection pathogens based on preclinical pharmacokinetic/pharmacodynamic modeling. The long half-life and high systemic exposure of EDP-420 support once-daily dosing and may allow for shorter treatment durations compared to other macrolide antibiotics. Based on its human pharmacokinetic profiles, taken together with its in vitro/in vivo activity against common respiratory pathogens, EDP-420 warrants efficacy trials for the treatment of respiratory tract infections.
Emergence of Plasmid-Mediated Quinolone Resistance among Non-Typhi
Salmonella enterica
Isolates from Humans in the United States
Authors: Sjölund-Karlsson Maria , Folster Jason P. , Pecic Gary , Joyce Kevin , Medalla Felicita , Rickert Regan , Whichard Jean M.
Published: February 2009

Plasmid-mediated quinolone resistance determinants are emerging among gram-negative pathogens. Here we report results of a retrospective study investigating the prevalence of
aac(6′)-Ib-cr
,
qepA
, and
qnr
genes among 19,010 human isolates of non-Typhi
Salmonella enterica
collected in the United States from 1996 to 2006.
Surfactants, Aromatic and Isoprenoid Compounds, and Fatty Acid Biosynthesis Inhibitors Suppress
Staphylococcus aureus
Production of Toxic Shock Syndrome Toxin 1
Authors: McNamara Peter J. , Syverson Rae Ellen , Milligan-Myhre Kathy , Frolova Olga , Schroeder Sarah , Kidder Joshua , Hoang Thanh , Proctor Richard A.
Published: February 2009

Menstrual toxic shock syndrome is a rare but potentially life-threatening illness manifest through the actions of
Staphylococcus aureus
toxic shock syndrome toxin 1 (TSST-1). Previous studies have shown that tampon additives can influence staphylococcal TSST-1 production. We report here on the TSST-1-suppressing activity of 34 compounds that are commonly used additives in the pharmaceutical, food, and perfume industries. Many of the tested chemicals had a minimal impact on the growth of
S. aureus
and yet were potent inhibitors of TSST-1 production. The TSST-1-reducing compounds included surfactants with an ether, amide, or amine linkage to their fatty acid moiety (e.g., myreth-3-myristate, Laureth-3, disodium lauroamphodiacetate, disodium lauramido monoethanolamido, sodium lauriminodipropionic acid, and triethanolamine laureth sulfate); aromatic compounds (
e.g
. phenylethyl and benzyl alcohols); and several isoprenoids and related compounds (e.g., terpineol and menthol). The membrane-targeting and -altering effects of the TSST-1-suppressing compounds led us to assess the activity of molecules that are known to inhibit fatty acid biosynthesis (e.g., cerulenin, triclosan, and hexachlorophene). These compounds also reduced
S. aureus
TSST-1 production. This study suggests that more additives than previously recognized inhibit the production of TSST-1.
Serial Plasma Voriconazole Concentrations after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Trifilio Steven M. , Yarnold Paul R. , Scheetz Marc H. , Pi Judy , Pennick Gennethel , Mehta Jayesh
Published: February 2009

Plasma voriconazole concentrations vary considerably between patients receiving standard dosing, and trough voriconazole concentrations are known to affect efficacy and toxicity. Temporal variations in serial plasma voriconazole concentrations through the course of therapy in hematopoietic stem cell transplantation patients has not been carefully described. Paired voriconazole concentrations in 64 patients were studied to determine the predictability of the second concentration based on the first. The difference between the two values was ≤5% in six patients. In 25 patients, the second concentration was higher by a median of 40%. In 33 patients, the subsequent concentration was lower by a median of 59%. For patients with an initial concentration of <2 μg/ml, the correlation between the two values was poor (
r
= 0.24;
P
< 0.17). For those with an initial concentration of ≥2 μg/ml, the correlation was good (
r
= 0.72;
P
< 0.0001). There was no relationship between the magnitude of the change and the time elapsing between the two measurements. Among the 43 patients who had an initial concentration of ≥1 μg/ml, the two voriconazole measurements were strongly correlated (
r
= 0.66,
P
< 0.0001), but only 67% had a voriconazole serum concentration of ≥1 μg/ml on the second measurement. No studied variables were reliable predictors in identifying concentrations above or below 1 or 2 μg/ml. Our data suggest that variations in voriconazole concentrations are unpredictable despite standard dosing, and the acceptability of a concentration on one occasion cannot be extrapolated to future concentrations in the same patient. This suggests that ongoing therapeutic drug monitoring and dose adjustment may be beneficial in patients requiring prolonged voriconazole therapy.
Effect of Topical Liposomes Containing Paromomycin Sulfate in the Course of
Leishmania major
Infection in Susceptible BALB/c Mice
Authors: Jaafari Mahmoud R. , Bavarsad Neda , Fazly Bazzaz Bibi Sedigheh , Samiei Afshin , Soroush Dina , Ghorbani Serajodin , Lotfi Heravi Mohammad M. , Khamesipour Ali
Published: February 2009

The aim of this study was to evaluate the antileishmanial effects of topical liposomal paromomycin sulfate (PM) in
Leishmania major
-infected BALB/c mice. Liposomes containing 10 or 15% PM (Lip-PM-10 and Lip-PM-15, respectively) were prepared by the fusion method and were characterized for their size and encapsulation efficiency. The penetration of PM from the liposomal PM formulations (LPMFs) through and into skin was evaluated in vitro with Franz diffusion cells fitted with mouse skin at 37°C for 8 h. The in vitro permeation data showed that almost 15% of the LPMFs applied penetrated the mouse skin, and the amount retained in the skin was about 60% for both formulations. The 50% effective doses of Lip-PM-10 and Lip-PM-15 against
L. major
promastigotes in culture were 65.32 and 59.73 μg/ml, respectively, and those against
L. major
amastigotes in macrophages were 24.64 and 26.44 μg/ml, respectively. Lip-PM-10 or Lip-PM-15 was used topically twice a day for 4 weeks to treat
L. major
lesions on BALB/c mice, and the results showed a significantly (
P
< 0.001) smaller lesion size in the mice in the treated groups than in the mice in the control group, which received either empty liposomes or phosphate-buffered saline (PBS). Eight weeks after the beginning of the treatment, every mouse treated with LPMFs was completely cured. The spleen parasite burden was significantly (
P
< 0.001) lower in mice treated with Lip-PM-10 or Lip-PM-15 than in mice treated with PBS or control liposomes, but no significant difference was seen between the two groups treated with either Lip-PM-10 or Lip-PM-15. The results suggest that topical liposomal PM may be useful for the treatment of cutaneous leishmaniasis.
Fenticonazole Activity Measured by the Methods of the European Committee on Antimicrobial Susceptibility Testing and CLSI against 260
Candida
Vulvovaginitis Isolates from Two European Regions and Annotations on the Prevalent Genotypes
Authors: Antonopoulou Stavroula , Aoun Michel , Alexopoulos Evangelos C. , Baka Stavroula , Logothetis Emanuel , Kalambokas Theodoros , Zannos Andreas , Papadias Konstantine , Grigoriou Odysseas , Kouskouni Evangelia , Velegraki Aristea
Published: February 2009

The activity of fenticonazole was studied against 260 West and Southeast European vulvovaginal candidiasis isolates, and low MICs were displayed. Fenticonazole was assessed by European Committee on Antimicrobial Susceptibility Testing and CLSI microdilution methods for the first time, and the results showed excellent agreement (97%) and significant interclass correlation coefficient (
P
< 0.0001). Also, the levels of agreement for the results for itraconazole, fluconazole, and ketoconazole were 84%, 90%, and 98% (
P
< 0.0001), respectively. Multilocus typing by PCR fingerprinting and subsequent cluster analysis delineated geographically associated alignments for
Candida albicans
and fluconazole resistance-related clusters for
Candida glabrata
.
Human Immunodeficiency Virus Type 1 Isolates with the Reverse Transcriptase (RT) Mutation Q145M Retain Nucleoside and Nonnucleoside RT Inhibitor Susceptibility
Authors: Varghese Vici , Mitsuya Yumi , Shahriar Rajin , Bachmann Michael H. , Fessel W. Jeffrey , Kagan Ron M. , Shafer Robert W.
Published: February 2009

Q145M, a mutation in a conserved human immunodeficiency virus type 1 reverse transcriptase (RT) region, was reported to decrease susceptibility to multiple RT inhibitors. We report that Q145M and other Q145 mutations do not emerge with RT inhibitors nor decrease RT inhibitor susceptibility. Q145M should not, therefore, be considered an RT inhibitor resistance mutation.
ST-246 Antiviral Efficacy in a Nonhuman Primate Monkeypox Model: Determination of the Minimal Effective Dose and Human Dose Justification
Authors: Jordan Robert , Goff Arthur , Frimm Annie , Corrado Michael L. , Hensley Lisa E. , Byrd Chelsea M. , Mucker Eric , Shamblin Josh , Bolken Tove' C. , Wlazlowski Carly , Johnson Wendy , Chapman Jennifer , Twenhafel Nancy , Tyavanagimatt Shanthakumar , Amantana Adams , Chinsangaram Jarasvech , Hruby Dennis E. , Huggins John
Published: February 2009

Therapeutics for the treatment of pathogenic orthopoxvirus infections are being sought. In the absence of patients with disease, animal models of orthopoxvirus disease are essential for evaluation of the efficacies of antiviral drugs and establishment of the appropriate dose and duration of human therapy. Infection of nonhuman primates (NHP) by the intravenous injection of monkeypox virus has been used to evaluate a promising therapeutic drug candidate, ST-246. ST-246 administered at 3 days postinfection (which corresponds to the secondary viremia stage of disease) at four different doses (from 100 mg/kg of body weight down to 3 mg/kg) once a day for 14 days was able to offer NHP 100% protection from a lethal infection with monkeypox virus and reduce the viral load and lesion formation. In NHP, the administration of ST-246 at a dose of 10 mg/kg/day for 14 days resulted in levels of blood exposure comparable to the levels attained in humans administered 400 mg in the fed state. These results suggest that administration of an oral dosage of 400 mg once daily for 14 days will be effective for the prevention or treatment of smallpox or monkeypox infections in humans.
Intracellular Activity of Antibiotics against
Staphylococcus aureus
in a Mouse Peritonitis Model
Authors: Sandberg Anne , Hessler Jonas H. R. , Skov Robert L. , Blom Jens , Frimodt-Møller Niels
Published: February 2009

Antibiotic treatment of
Staphylococcus aureus
infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of
S. aureus
was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log
10
number of CFU/ml (Δlog
10
CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog
10
CFU/ml) > cefuroxime (3.56 Δlog
10
CFU/ml) > rifampin (1.86 Δlog
10
CFU/ml) > gentamicin (0.61 Δlog
10
CFU/ml) > azithromycin (0.21 Δlog
10
CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.
Development of Non-Antibiotic-Resistant, Chromosomally Based, Constitutive and Inducible Expression Systems for
aroA
-Attenuated
Salmonella enterica
Serovar Typhimurium
Authors: Matic Jake N. , Terry Tamsin D. , Van Bockel David , Maddocks Tracy , Tinworth David , Jennings Michael P. , Djordjevic Steven P. , Walker Mark J.
Published: February 2009

Live-vaccine delivery systems expressing two model antigens from
Mycoplasma hyopneumoniae
, F2
P97
(Adh) and NrdF, were constructed using
Salmonella enterica
serovar Typhimurium
aroA
(STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced
sspA
promoter and recombining the expression construct into
aroD
. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in
S. enterica
serovar Typhimurium
aroA
from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.
First
Streptococcus pyogenes
Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants
Authors: Kizy Anne E. , Neely Melody N.
Published: February 2009

The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify
Streptococcus pyogenes
virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as
mga
,
hasA
,
amrA
,
smeZ
, and two genes in the
sil
locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes,
silB
and
silC
of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis.
sil
locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of
S. pyogenes
factors required for successful invasive infection.
Concurrent
Helicobacter bilis
Infection in C57BL/6 Mice Attenuates Proinflammatory
H. pylori
-Induced Gastric Pathology
Authors: Lemke Laura B. , Ge Zhongming , Whary Mark T. , Feng Yan , Rogers Arlin B. , Muthupalani Sureshkumar , Fox James G.
Published: February 2009

Because coinfections can alter helicobacter gastritis, we investigated whether enterohepatic
Helicobacter bilis
modulates
Helicobacter pylori
gastritis in C57BL/6 mice. Thirty mice per group were sham dosed,
H. bilis
or
H. pylori
infected, or
H. bilis
infected followed in 2 weeks by
H. pylori
and then evaluated at 6 and 11 months postinfection (mpi) for gastritis and premalignant lesions. Compared to
H. pylori
-infected mice,
H. bilis/H. pylori
-infected mice at 6 and 11 mpi had less severe gastritis, atrophy, mucous metaplasia and hyperplasia (
P
< 0.01) and, additionally, at 11 mpi, less severe intestinal metaplasia and dysplasia (
P
< 0.05).
H. bilis/H. pylori
-infected mice at 11 mpi exhibited less Ki67 labeling of proliferating epithelial cells, reduced numbers of FoxP3
+
T-regulatory (T
REG
) cells, and lower FoxP3
+
mRNA levels than did
H. pylori
-infected mice (
P
< 0.05). Proinflammatory interleukin-1β (IL-1β), gamma interferon, and tumor necrosis factor alpha mRNA levels were attenuated in
H. bilis/H. pylori
-infected mice at 6 and 11 mpi (
P
< 0.01), although anti-inflammatory IL-10, IL-13, and transforming growth factor β1 mRNA levels were not consistently impacted by
H. bilis
coinfection. Decreased pathology in
H. bilis/H. pylori
-infected mice correlated with higher gastric
H. pylori
colonization at 6 mpi (
P
< 0.001) and lower Th1-associated immunoglobulin G2c responses to
H. pylori
at 6 and 10 mpi (
P
< 0.05). We hypothesized that reduced pathology in
H. bilis/H. pylori
-infected mice was due to
H. bilis
-primed T
REG
cells in the lower bowel that migrated to the gastric compartment and inhibited Th1 responses to subsequent
H. pylori
infection. Thus,
H. pylori
-induced gastric lesions may vary in mouse models of unknown enteric helicobacter infection status and, importantly, variable sequelae to human
H. pylori
infection, particularly in developing countries, may occur where coinfection with lower bowel helicobacters and
H. pylori
may be common.
Differential Expression and Glycosylation of
Anaplasma phagocytophilum
Major Surface Protein 2 Paralogs during Cultivation in Sialyl Lewis x-Deficient Host Cells
Authors: Troese Matthew J. , Sarkar Madhubanti , Galloway Nathan L. , Thomas Rachael J. , Kearns Sarah A. , Reneer Dexter V. , Yang Tian , Carlyon Jason A.
Published: February 2009

Many microbial pathogens alter expression and/or posttranslational modifications of their surface proteins in response to dynamics within their host microenvironments to retain optimal interactions with their host cells and/or to evade the humoral immune response.
Anaplasma phagocytophilum
is an intragranulocytic bacterium that utilizes sialyl Lewis x (sLe
x
)-modified P-selectin glycoprotein ligand 1 as a receptor for infecting myeloid cells. Bacterial populations that do not rely on this receptor can be obtained through cultivation in sLe
x
-defective cell lines.
A. phagocytophilum
major surface protein 2 [Msp2(P44)] is encoded by members of a paralogous gene family and is speculated to play roles in host adaptation. We assessed the complement of Msp2(P44) paralogs expressed by
A. phagocytophilum
during infection of sLe
x
-competent HL-60 cells and two HL-60 cell lines defective for sLe
x
expression. Multiple Msp2(P44) and N-terminally truncated 25- to 27-kDa isoforms having various isoelectric points and electrophoretic mobilities were expressed in each cell line. The complement of expressed
msp2
(
p44
) paralogs and the glycosyl residues modifying Msp2(P44) varied considerably among bacterial populations recovered from sLe
x
-competent and -deficient host cells. Thus, loss of host cell sLe
x
expression coincided with both differential expression and glycosylation of
A. phagocytophilum
Msp2(P44). This reinforces the hypothesis that this bacterium is able to generate a large variety of surface-exposed molecules that could provide great antigenic diversity and result in multiple binding properties.
ULTRASHORT PULSE MULTISPECTRAL NON-LINEAR OPTICAL MICROSCOPY
Authors: LARSON ADAM M. , LEE ANTHONY , LEE PO-FENG , BAYLESS KAYLA J. , YEH ALVIN T.
Published: February 2009

Ultrashort pulse, multispectral non-linear optical microscopy (NLOM) is developed and used to image, simultaneously, a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis. Broadband, sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen. A 16-channel multispectral detector is used to delineate the multiple non-linear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.
MULTIPHOTON MICROSCOPY: A NEW APPROACH, IN PHYSIOLOGICAL STUDIES AND PATHOLOGICAL DIAGNOSIS FOR OPHTHALMOLOGY
Authors: HSUEH CHIU-MEI , LO WEN , LIN SUNG-JAN , WANG TSUNG-JEN , HU FUNG-RUNG , TAN HSIN-YUAN , DONG CHEN-YUAN
Published: February 2009

Multiphoton microscopy (MPM), with the advantages of improved penetration depth, decreased photo-damage, and optical sectioning capability, has become an indispensable tool for biomedical imaging. The combination of multiphoton fluorescence (MF) and second-harmonic generation (SHG) microscopy is particularly effective in imaging tissue structures of the ocular surface. This work is intended to be a review of advances that MPM has made in ophthalmic imaging. The MPM not only can be used for the label-free imaging of ocular structures, it can also be applied for investigating the morphological alterations in corneal pathologies, such as keratoconus, infected keratitis, and corneal scar. Furthermore, the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty (CK) can also be studied with MPM. Finally, qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation. With additional development, multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.
INTRACELLULAR MANIPULATION BY FEMTOSECOND LASERS: REVIEW
Authors: WATANABE WATARU , MATSUNAGA SACHIHIIRO , FUKUI KIICHI , ITOH KAZUYOSHI
Published: February 2009

Multiphoton absorption of femtosecond laser pulses focused through an objective with high numerical aperture (NA) can be used to image and manipulate cellular and intracellular objects. This review highlights recent advances in intracellular manipulation, including nanosurgery and labeling in living cells with femtosecond lasers.
EFFICIENT FITTING RANGE FOR GLUCOSE MEASUREMENT WITH OPTICAL COHERENCE TOMOGRAPHY
Authors: FENG SHU , YUAN KEHONG , YE DATIAN
Published: February 2009

Studies of non-invasive glucose measurement with optical coherence tomography (OCT) in tissue-simulating phantoms and biological tissues show that glucose has an effect on the OCT signal slope. Choosing an efficient fitting range to calculate the OCT signal slope is important because it helps to improve the precision of glucose measurement. In this paper, we study the problem in two ways: (1) scattering-induced change of OCT signal slope versus depth in intralipid suspensions with different concentrations based on Monte Carlo (MC) simulations and experiments and (2) efficient fitting range for glucose measurement in 3% and 10% intralipid. The results show that the OCT signal slope expresses a contrary change with scattering coefficient below a certain depth in high intralipid concentrations, so that there is an effective fitting depth. With an efficient fitting range from 100 μm to the effective fitting depth, the precision of glucose measurement can be 4.4 mM for 10% intralipid and 2.2 mM for 3% intralipid.
OBSERVING NEURONAL ACTIVITIES WITH RANDOM ACCESS TWO-PHOTON MICROSCOPE
Authors: WU YUXIANG , LIU XIULI , ZHOU WEI , LV XIAOHUA , ZENG SHAOQUN
Published: February 2009

As a second messenger in signal transduction, calcium ion plays a very important role in neuronal information processing and integrating. Limited by the imaging technique, it is difficult to simultaneously perform deep tissue imaging and measure intracellular free calcium concentration ([ Ca 2+] i ) in different compartments of neurons in brain slice noncollinearly. By means of random access two-photon microscopy, which provides high optical penetration into tissues and low photo damage, we successfully measured [ Ca 2+] i of different parts of pyramidal neurons in neocortical layer V in rat brain slices with high spatial and temporal resolution. Combining the patch clamp technique, we stimulated the soma with depolarizing current and explored the dynamics of calcium in pyramidal neurons.
DEVELOPMENT OF HIGH-SPEED SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY SYSTEM AT 1320 nm
Authors: WU TONG , DING ZHIHUA , CHEN MINGHUI , XU LEI , SHI GUOHUA , ZHANG YUDONG
Published: February 2009

A swept-source optical coherence tomography (SSOCT) system based on a high-speed scanning laser source at center wavelength of 1320 nm and scanning rate of 20 kHz is developed. The axial resolution is enhanced to 8.3 μm by reshaping the spectrum in frequency domain using a window function and a wave number calibration method based on a Mach-Zender Interferometer (MZI) integrated in the SSOCT system. The imaging speed and depth range are 0.04 s per frame and 3.9 mm, respectively. The peak sensitivity of the SSOCT system is calibrated to be 112 dB. With the developed SSOCT system, optical coherence tomography (OCT) images of human finger tissue are obtained which enable us to view the sweat duct (SD), stratum corneum (SC) and epidermis (ED), demonstrating the feasibility of the SSOCT system for in vivo biomedical imaging.
GLUT2 mutations, translocation, and receptor function in diet sugar managing
Authors: Leturque Armelle , Brot-Laroche Edith , Le Gall Maude
Published: February 2009

Cloned 20 years ago, GLUT2 is a facilitative glucose transporter in the liver, pancreas, intestine, kidney, and brain. It ensures large bidirectional fluxes of glucose in and out the cell due to its low affinity and high capacity. It also transports other dietary sugars, such as fructose and galactose, within the range of physiological concentrations. Sugars and hormones regulate its gene expression. The contribution of GLUT2 to human metabolic diseases previously appeared modest. However, in the past decade, three major features of the GLUT2 protein have been revealed. First, GLUT2 mutations cause the severe but rare Fanconi-Bickel syndrome, mainly characterized by glycogenosis. Recently, a GLUT2 polymorphism has been associated with preferences for sugary food. Second, the GLUT2 location at the cell surface is regulated; this governs cellular activities dependent on glucose in the intestine and possibly those in the liver and pancreas. For instance, GLUT2 translocation from an intracellular pool to the apical membrane after a sugar meal transiently increases sugar uptake by enterocytes (reviewed in 32). Third, GLUT2 functions as a membrane receptor of sugar. Independently of glucose metabolism, GLUT2 detects the presence of extracellular sugar and transduces a signal to modulate cell functions, including β-cell insulin secretion, renal reabsorption, and intestinal absorption according to the sugar environment. These recent developments are examined here in heath and metabolic disease, highlighting various unanswered questions.