bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Tilting Effects and Geometric Nonuniformities in Micro-hydrostatic Gas Thrust Bearings
Authors: Teo C. J. , Spakovszky Z. S.
Published: September 2006

The Massachusetts Institute of Technology (MIT) microengine rotors are supported by hydrostatic gas journal and hydrostatic gas thrust bearings. Due to the low length-to-diameter ratio of the devices, the thrust bearings play an important role in providing sufficient tilting stiffness to resist any tilting motion about the spinning axis of the rotor. The performance of the thrust bearings can be influenced by geometric nonuniformities such as thrust-bearing clearances and orifice diameters, and profiles which arise in the process of micro-fabrication. To enable stable high speed operation of the micro-devices, it is important to quantify these effects. Furthermore, a thrust-bearing analysis tool needs to be developed that is able to explore different thrust-bearing arrangements and configurations. In this work, an analytical model is established for analyzing the effects of rotor tilt and geometric nonuniformities in micro-hydrostatic gas thrust bearings for application to micro-turbomachinery. A previously developed model (Teo and Spakovszky, 2006, “Modeling and Experimental Investigation of Micro-hydrostatic Gas Thrust Bearings for Micro-turbomachines,” ASME J. Turbomach., 128, pp. 597–605) is generalized and extended for application to thrust bearings with orifices arranged in nonaxisymmetric configurations. As a consequence of rotor tilt or geometric nonuniformities, the flow through individual orifices of the thrust bearing becomes nonuniform. The orifice flows are in turn coupled to the hydrostatic pressure field in the thrust-bearing pad, and a Green’s function approach is adopted to solve the coupled system. The hydrodynamic thrust-bearing forces induced by the pumping action of the rotor rotation are determined by solving the Reynolds equation. The model is able to predict thrust-bearing tilting stiffness and variations in the thrust-bearing mass flow rates as a function of rotor tilting angle for a variety of orifice arrangements. The model can be applied to analyze the effects of nonuniformities in orifice diameter and the presence of clogged orifices on tilting and the concomitant reduction in tilting stiffness. In addition, the effects of orifice taper are analyzed using an influence-coefficient method for one-dimensional compressible, viscous flows. Results obtained for various taper ratios are presented and discussed. The model serves as a useful tool for specifying design tolerances during the fabrication of micro-hydrostatic gas thrust bearings and is used in the experiments to estimate the tilting angle of the rotor during operation.
Analysis of Airfoil
Trailing Edge Heat Transfer and Its Significance in Thermal-Mechanical Design and
Durability
Authors: Cunha F. J. , Dahmer M. T. , Chyu M.
K.
Published: September 2006

The trailing edge section of modern high-pressure turbine airfoils is an area that requires a high degree of attention from turbine performance and durability standpoints. Aerodynamic loss near the trailing edge includes expansion waves, normal shocks, and wake shedding. Thermal issues associated with trailing edge are also very complex and challenging. To maintain effective cooling ensuring metal temperature below design limit is particularly difficult, as it needs to be implemented in a relatively small area of the airfoil. To date, little effort has been devoted to advancing the fundamental understanding of the thermal characteristics in airfoil trailing edge regions. Described in this paper are the procedures leading to closed-form, analytical solutions for temperature profile for four most representative trailing edge configurations. The configurations studied are: (1) solid wedge shape without discharge, (2) wedge with slot discharge, (3) wedge with discrete-hole discharge, and (4) wedge with pressure-side cutback slot discharge. Comparison among these four cases is made primarily in the context of airfoil metal temperature and resulting cooling effectiveness. Further discussed in the paper are the overall and detail design parameters for preferred trailing edge cooling configurations as they affect turbine airfoil performance and durability.
Influence of Blade Leading Edge Geometry on Turbine Endwall Heat (Mass) Transfer
Authors: Han S. , Goldstein R. J.
Published: September 2006

The secondary flows, including passage and other vortices in a turbine cascade, cause significant aerodynamic losses and thermal gradients. Leading edge modification of the blade has drawn considerable attention as it has been shown to reduce the secondary flows. However, the heat transfer performance of a leading edge modified blade has not been investigated thoroughly. Since a fillet at the leading edge blade is reported to reduce the aerodynamic loss significantly, the naphthalene sublimation technique with a fillet geometry is used to study local heat (mass) transfer performance in a simulated turbine cascade. The present paper compares Sherwood number distributions on an endwall with a simple blade and a similar blade having a modified leading edge by adding a fillet. With the modified blades, a horseshoe vortex is not observed and the passage vortex is delayed or not observed for different turbulence intensities. However, near the blade trailing edge the passage vortex has gained as much strength as with the simple blade for low turbulence intensity. Near the leading edge on the pressure and the suction surface, higher mass transfer regions are observed with the fillets. Apparently the corner vortices are intensified with the leading edge modified blade.
Effect of PEX, a Noncatalytic Metalloproteinase Fragment with Integrin-Binding Activity, on Experimental
Chlamydophila pneumoniae
Infection
Authors: Caronzolo Dario , Lucini Valeria , Pannacci Marilou , Grosso Silvia , Kieffer Nelly , Bello Lorenzo , Bikfalvi Andreas , Scaglione Francesco
Published: September 2006

Chlamydophila pneumoniae
is a pathogen that is involved in acute and chronic respiratory infections and that is associated with asthma and coronary artery diseases. In this study, we evaluated the effects of PEX, a noncatalytic metalloproteinase fragment with integrin-binding activity, against experimental infections caused by
C. pneumoniae
. Moreover, we investigated the relationships between
C. pneumoniae
and α
v
β
3
integrin functions in order to explain the possible mechanism of action of PEX both in vitro and in vivo. For the in vitro experiments, HeLa cells were infected with
C. pneumoniae
and treated with either PEX or azithromycin. The results obtained with PEX were not significantly different (
P
> 0.05) from those achieved with azithromycin. Similar results were also obtained in a lung infection model. Male C57BL/J6 mice inoculated intranasally with 10
6
inclusion-forming units of
C. pneumoniae
were treated with either PEX or azithromycin plus rifampin. Infected mice treated with PEX showed a marked decrease in
C. pneumoniae
counts versus those for the controls; this finding did not differ significantly (
P
> 0.05) from the results observed for the antibiotic-treated group. Integrin α
v
β
3
plays an important role in
C. pneumoniae
infection. Blockage of integrin activation led to a significant inhibition of
C. pneumoniae
infection in HeLa cells. Moreover, CHO
DHFR
α
v
β
3
-expressing cells were significantly (
P
< 0.001) more susceptible to
C. pneumoniae
infection than CHO
DHFR
cells. These results offer new perspectives on the treatment of
C. pneumoniae
infection and indicate that α
v
β
3
could be a promising target for new agents developed for activity against this pathogen.
Antimicrobial Susceptibility among Pathogens Collected from Hospitalized Patients in the United States and In Vitro Activity of Tigecycline, a New Glycylcycline Antimicrobial
Authors: Waites Ken B. , Duffy Lynn B. , Dowzicky Michael J.
Published: September 2006

The activities of tigecycline and comparators against isolates collected from 76 U.S. centers between January 2004 and September 2005 were assessed. Tigecycline MIC
90
s were ≤2 μg/ml for
Klebsiella pneumoniae
,
Klebsiella oxytoca
,
Escherichia coli
,
Enterobacter aerogenes
,
Enterobacter cloacae
,
Serratia marcescens
,
Acinetobacter baumannii
,
Staphylococcus aureus
,
Enterococcus faecalis
,
Enterococcus faecium
, and
Streptococcus agalactiae
.
Compartmentalized Intrapulmonary Pharmacokinetics of Amphotericin B and Its Lipid Formulations
Authors: Groll Andreas H. , Lyman Caron A. , Petraitis Vidmantas , Petraitiene Ruta , Armstrong Derek , Mickiene Diana , Alfaro Raul M. , Schaufele Robert L. , Sein Tin , Bacher John , Walsh Thomas J.
Published: September 2006

We investigated the compartmentalized intrapulmonary pharmacokinetics of amphotericin B and its lipid formulations in healthy rabbits. Cohorts of three to seven noninfected, catheterized rabbits received 1 mg of amphotericin B deoxycholate (DAMB) per kg of body weight or 5 mg of either amphotericin B colloidal dispersion (ABCD), amphotericin B lipid complex (ABLC), or liposomal amphotericin B (LAMB) per kg once daily for a total of 8 days. Following sparse serial plasma sampling, rabbits were sacrificed 24 h after the last dose, and epithelial lining fluid (ELF), pulmonary alveolar macrophages (PAM), and lung tissue were obtained. Pharmacokinetic parameters in plasma were derived by model-independent techniques, and concentrations in ELF and PAM were calculated based on the urea dilution method and macrophage cell volume, respectively. Mean amphotericin B concentrations ± standard deviations (SD) in lung tissue and PAM were highest in ABLC-treated animals, exceeding concurrent plasma levels by 70- and 375-fold, respectively (in lung tissue, 16.24 ± 1.62 versus 2.71 ± 1.22, 6.29 ± 1.17, and 6.32 ± 0.57 μg/g for DAMB-, ABCD-, and LAMB-treated animals, respectively [P= 0.0029]; in PAM, 89.1 ± 37.0 versus 8.92 ± 2.89, 5.43 ± 1.75, and 7.52 ± 2.50 μg/ml for DAMB-, ABCD-, and LAMB-treated animals, respectively [P= 0.0246]). By comparison, drug concentrations in ELF were much lower than those achieved in lung tissue and PAM. Among the different cohorts, the highest ELF concentrations were found in LAMB-treated animals (2.28 ± 1.43 versus 0.44 ± 0.13, 0.68 ± 0.27, and 0.90 ± 0.28 μg/ml in DAMB-, ABCD-, and ABLC-treated animals, respectively [P= 0.0070]). In conclusion, amphotericin B and its lipid formulations displayed strikingly different patterns of disposition in lungs 24 h after dosing. Whereas the disposition of ABCD was overall not fundamentally different from that of DAMB, ABLC showed prominent accumulation in lung tissue and PAM, while LAMB achieved the highest concentrations in ELF.
Pharmacokinetics and Tolerability of Daptomycin at Doses up to 12 Milligrams per Kilogram of Body Weight Once Daily in Healthy Volunteers
Authors: Benvenuto Mark , Benziger David P. , Yankelev Sara , Vigliani Gloria
Published: September 2006

Daptomycin, a novel lipopeptide, is bactericidal against a broad range of gram-positive strains, including methicillin- (MRSA) and vancomycin-resistant
Staphylococcus aureus
. Daptomycin is approved at 4 mg/kg of body weight given intravenously once daily for the treatment of complicated skin and skin structure infections and at 6 mg/kg for the treatment of
S. aureus
bloodstream infections (bacteremia), including right-sided endocarditis caused by methicillin-susceptible
S. aureus
and MRSA. The present study was designed to evaluate the multiple-dose pharmacokinetics and safety of daptomycin at doses of 6 to 12 mg/kg in healthy volunteers. Three cohorts of 12 subjects each were given daptomycin (10 mg/kg) or placebo once daily for 14 days, daptomycin (12 mg/kg) or placebo once daily for 14 days, or daptomycin (6 or 8 mg/kg) once daily for 4 days. Daptomycin produced dose-proportional increases in the area under the plasma concentration-time curve and in trough daptomycin levels and nearly dose-proportional increases in peak daptomycin concentrations. Other pharmacokinetic parameters measured on day 1 and at steady state were independent of the dose, including the half-life (approximately 8 h), weight-normalized plasma clearance (9 to 10 ml/h/kg), and volume of distribution (approximately 100 ml/kg). Plasma protein binding was 90% to 93% and was independent of the daptomycin concentration. Daptomycin did not produce electrocardiographic abnormalities or electrophysiological evidence of muscle or nerve toxicity. Daptomycin was well tolerated in subjects dosed with up to 12 mg/kg intravenously for 14 days. Doses of daptomycin higher than 6 mg/kg once daily may be considered in further studies to evaluate the safety and efficacy of daptomycin in difficult-to-treat infections.
The
mef
(E)-Carrying Genetic Element (mega) of
Streptococcus pneumoniae
: Insertion Sites and Association with Other Genetic Elements
Authors: Del Grosso Maria , Camilli Romina , Iannelli Francesco , Pozzi Gianni , Pantosti Annalisa
Published: September 2006

The structure of the macrolide efflux genetic assembly (mega) element, its genomic locations, and its association with other resistance determinants and genetic elements were investigated in 16
Streptococcus pneumoniae
isolates carrying
mef
(E), of which 1 isolate also carried
tet
(M) and 4 isolates also carried
tet
(M) and
erm
(B). All isolates carried a mega element of similar size and structure that included the operon
mef
(E)-
msr
(D) encoding the efflux transport system. Among tetracycline-susceptible isolates, six different integration sites were identified, five of which were recognized inside open reading frames present in the R6 genome. In the five isolates also carrying
tet
(M), mega was inserted in different genetic contexts. In one isolate, it was part of previously described Tn
916
-like element Tn
2009
. In another isolate, mega was inserted in a transposon similar to Tn
2009
that also included an
erm
(B) element. This new composite transposon was designated Tn
2010
. Neither Tn
2009
nor Tn
2010
could be transferred by conjugation to pneumococcal or enterococcal recipients. In the three isolates in which mega was not physically linked with
tet
(M), this gene was associated with
erm
(B) in transposon Tn
3872
, a Tn
916
-like element. Homologies between the chromosomal insertions of these composite transposons and sequences of multidrug-resistant pneumococcal genomes in the databases indicate the presence of preferential sites for the integration of composite Tn
916
-like elements carrying multiple resistance determinants in
S. pneumoniae
.
Total Variation in the
penA
Gene of
Neisseria meningitidis
: Correlation between Susceptibility to β-Lactam Antibiotics and
penA
Gene Heterogeneity
Authors: Thulin Sara , Olcén Per , Fredlund Hans , Unemo Magnus
Published: September 2006

In recent decades, the prevalence of
Neisseria meningitidis
isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen
i
) for most strains is due mainly to mosaic structures in the
penA
gene, encoding penicillin-binding protein 2. In this study, susceptibility to β-lactam antibiotics was determined for 60 Swedish clinical
N. meningitidis
isolates and 19 reference strains. The
penA
gene was sequenced and compared to 237
penA
sequences from GenBank in order to explore the total identified variation of
penA
. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type
penA
gene. By studying the final 1,143 to 1,149 bp of
penA
in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of
penA
mosaic structures was found especially for penicillin G and ampicillin. The Pen
i
isolates comprised an MIC of >0.094 μg/ml for penicillin G and an MIC of >0.064 μg/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen
s
or Pen
i
. In comparison with the wild-type
penA
sequence, two specific Pen
i
sites were altered in all except two mosaic
penA
sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between
penA
sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the
penA
gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.
Assessment of the Drug Susceptibility of
Plasmodium falciparum
Clinical Isolates from Africa by Using a
Plasmodium
Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration
Authors: Kaddouri Halima , Nakache Serge , Houzé Sandrine , Mentré France , Le Bras Jacques
Published: September 2006

The extension of drug resistance among malaria-causing
Plasmodium falciparum
parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the
Plasmodium
lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five
Plasmodium falciparum
clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8-
3
H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC
50
) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation (
r
= 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC
50
s. Recently collected
Plasmodium falciparum
African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.
Structure-Activity Relationships of
Bacillus cereus
and
Bacillus anthracis
Dihydrofolate Reductase: toward the Identification of New Potent Drug Leads
Authors: Joska Tammy M. , Anderson Amy C.
Published: September 2006

New and improved therapeutics are needed for
Bacillus anthracis
, the etiological agent of anthrax. To date, antimicrobial agents have not been developed against the well-validated target dihydrofolate reductase (DHFR). In order to address whether DHFR inhibitors could have potential use as clinical agents against
Bacillus
, 27 compounds were screened against this enzyme from
Bacillus cereus
, which is identical to the enzyme from
B. anthracis
at the active site. Several 2,4-diamino-5-deazapteridine compounds exhibit submicromolar 50% inhibitory concentrations (IC
50
s). Four of the inhibitors displaying potency in vitro were tested in vivo and showed a marked growth inhibition of
B. cereus
; the most potent of these has MIC
50
and minimum bactericidal concentrations at which 50% are killed of 1.6 μg/ml and 0.09 μg/ml, respectively. In order to illustrate structure-activity relationships for the classes of inhibitors tested, each of the 27 inhibitors was docked into homology models of the
B. cereus
and
B. anthracis
DHFR proteins, allowing the development of a rationale for the inhibition profiles. A combination of favorable interactions with the diaminopyrimidine and substituted phenyl rings explains the low IC
50
values of potent inhibitors; steric interactions explain higher IC
50
values. These experiments show that DHFR is a reasonable antimicrobial target for
Bacillus anthracis
and that there is a class of inhibitors that possess sufficient potency and antibacterial activity to suggest further development.
Deletions of the Endocytic Components
VPS28
and
VPS32
in
Candida albicans
Lead to Echinocandin and Azole Hypersensitivity
Authors: Cornet Muriel , Gaillardin Claude , Richard Mathias L.
Published: September 2006

Vps28p and Vps32p act in both the endocytic and the pH signaling pathways in yeasts and are required for
Candida albicans
virulence. Here, we show that deletions of
VPS28
and
VPS32
increase the susceptibility of
C. albicans
to cell wall disruption agents, echinocandin and azole antifungal agents.
Phase I Dose Escalation Study To Evaluate the Safety and Pharmacokinetic Profile of Tefibazumab in Subjects with End-Stage Renal Disease Requiring Hemodialysis
Authors: Hetherington Seth , Texter Michele , Wenzel Eric , Patti Joseph M. , Reynolds Laurie , Shamp Trish , Swan Suzanne
Published: September 2006

Two cohorts of four subjects requiring hemodialysis received tefibazumab (10 or 20 mg/kg). The mean elimination half-life was between 17 and 18 days, the average volume of distribution was 7.3 liters, and the average clearance was 12 ml/h for both dose groups. At a dose of 20 mg/kg of body weight, plasma levels were 88 μg/ml at 21 days.
Potent Antihematozoan Activity of Novel Bisthiazolium Drug T16: Evidence for Inhibition of Phosphatidylcholine Metabolism in Erythrocytes Infected with
Babesia
and
Plasmodium
spp
Authors: Richier Eric , Biagini Giancarlo A. , Wein Sharon , Boudou Frederic , Bray Patrick G. , Ward Steve A. , Precigout Eric , Calas Michèle , Dubremetz Jean-François , Vial Henri J.
Published: September 2006

A leading bisthiazolium drug, T16, designed to mimic choline, was shown to exert potent antibabesial activity, with 50% inhibitory concentrations of 28 and 7 nM against
Babesia divergens
and
B. canis
, respectively. T16 accumulated inside
Babesia
-infected erythrocytes (cellular accumulation ratio, >60) by a saturable process with an apparent

K
m

of 0.65 μM. Subcellular fractionation of
Babesia
parasites revealed the accumulation of T16 into a low-density fraction, while in malaria-infected erythrocytes a significant fraction of the drug was associated with heme malaria pigment. T16 exerts an early and specific inhibition of the de novo biosynthesis of phosphatidylcholine both in
B. divergens-
and
Plasmodium falciparum
-infected erythrocytes. Choline accumulation into isolated
Babesia
parasites was highly sensitive to inhibition by T16. These data are consistent with the hypothesis that bisthiazolium drugs target the de novo phosphatidylcholine biosynthesis of intraerythrocytic hematozoan parasites. In malaria parasites, which generate ferriprotoporphyrin IX during hemoglobin digestion, T16 binding to heme may enhance the accumulation and activity of the drug. The selectivity of accumulation and potent activity of this class of drug into parasite-infected erythrocytes offers unique advantages over more traditional antihematozoan drugs.
High-Throughput and Facile Assay of Antimicrobial Peptides Using pH-Controlled Fluorescence Resonance Energy Transfer
Authors: Kim Young Soo , Cha Hyung Joon
Published: September 2006

Amphipathic antimicrobial peptides can destroy bacteria cells by inducing membrane permeabilization, forming one strategy for innate defense by various organisms. However, although the antimicrobial peptides are considered a promising alternative for use against multidrug-resistant bacteria, large-scale screening of potential candidate antimicrobial peptides will require a simple, rapid assay for antimicrobial activity. Here, we describe a novel fluorescence resonance energy transfer (FRET)-based assay system for antimicrobial peptides which takes advantage of pH-related changes in FRET efficiency due to the instability of enhanced yellow fluorescent protein versus the stability of enhanced cyan fluorescent protein in a reduced-pH environment. We successfully showed that quantification of antimicrobial activity is possible through a difference of FRET efficiency between ECFP-EYFP fusion molecules released from disrupted
Escherichia coli
in an extracellular environment (pH 6) and those retained in an intracellular environment (pH ∼7). Thus, we herein suggest a new simple, effective, and efficient pH-controlled FRET-based antimicrobial peptide screening method applicable to high-throughput screening of candidate peptide libraries.
In Vitro Susceptibility of
Clostridium difficile
Clinical Isolates from a Multi-Institutional Outbreak in Southern Québec, Canada
Authors: Bourgault Anne-Marie , Lamothe François , Loo Vivian G. , Poirier Louise ,   the CDAD-CSI Study Group
Published: September 2006

Clostridium difficile
isolates from a 2004 outbreak in Québec, Canada, were all found to be susceptible to metronidazole, vancomycin, rifampin, and meropenem but resistant to bacitracin, cefotaxime, ciprofloxacin, and levofloxacin, and most (>80%) were resistant to ceftriaxone, clarithromycin, gatifloxacin, and moxifloxacin. The predominant NAP1 isolates were susceptible to clindamycin, while the NAP2 isolates were resistant.
Enhanced Membrane Disruption and Antibiotic Action against Pathogenic Bacteria by Designed Histidine-Rich Peptides at Acidic pH
Authors: Mason A. James , Gasnier Claire , Kichler Antoine , Prévost Gilles , Aunis Dominique , Metz-Boutigue Marie-Hélène , Bechinger Burkhard
Published: September 2006

The
histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA
delivery capabilities. Here, we explore the interaction of peptides
from this family with model membranes as monitored by solid-state
2
H nuclear magnetic resonance and their antibiotic
activities against a range of bacteria. At neutral pH, the membrane
disruption is weak, but at acidic pH, the peptides strongly disturb the
anionic lipid component of bacterial membranes and cause bacterial
lysis. The peptides are effective antibiotics at both pH 7.2 and pH
5.5, although the antibacterial activity is strongly affected by the
change in pH. At neutral pH, the LAH peptides were active against both
methicillin-resistant and -sensitive
Staphylococcus aureus
strains but ineffective against
Pseudomonas aeruginosa
. In
contrast, the LAH peptides were highly active against
P.
aeruginosa
in an acidic environment, as is found in the
epithelial-lining fluid of cystic fibrosis patients. Our results show
that modest antibiotic activity of histidine-rich peptides can be
dramatically enhanced by inducing membrane disruption, in this case by
lowering the pH, and that histidine-rich peptides have potential as
future antibiotic
agents.
Comparative In Vitro Susceptibilities of 396 Unusual Anaerobic Strains to Tigecycline and Eight Other Antimicrobial Agents
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , Warren Yumi A. , Tyrrell Kerin L. , Fernandez Helen T.
Published: September 2006

Tigecycline was tested against 396 strains of lesser-known anaerobic species encountered in human infections. It was active against all gram-positive strains and 228 of 232 gram-negative anaerobes at ≤1 μg/ml. One strain of
Prevotella oralis
was nonsusceptible at 8 μg/ml.
Development of a Human Immunodeficiency Virus Vector-Based, Single-Cycle Assay for Evaluation of Anti-Integrase Compounds
Authors: Bona Roberta , Andreotti Mauro , Buffa Viviana , Leone Pasqualina , Galluzzo Clementina Maria , Amici Roberta , Palmisano Lucia , Mancini Maria Grazia , Michelini Zuleika , Di Santo Roberto , Costi Roberta , Roux Alessandra , Pommier Yves , Marchand Christophe , Vella Stefano , Cara Andrea
Published: September 2006

Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.
Origins and Evolution of Methicillin-ResistantStaphylococcus aureusClonal Lineages
Authors: Gomes A. R. , Westh H. , de Lencastre H.
Published: September 2006

Most methicillin-resistantStaphylococcus aureus(MRSA) isolates identified among blood isolates collected in Denmark between 1957 and 1970 belonged to either phage group III or the closely related 83A complex and had a PSTM antibiotype (resistance to penicillin [P], streptomycin [S], tetracycline [T], and methicillin [M]). Recently, some of these isolates were shown to have the same genetic backgrounds as contemporary epidemic MRSA isolates, and Danish methicillin-susceptibleS. aureus(MSSA) isolates from the 1960s with a PST antibiotype were proposed to have been the recipients of themecAgene in those lineages. In this study, we investigated the genetic backgrounds of isolates from the 83A complex that were fully susceptible or resistant to penicillin only in order to try to trace the evolutionary trajectory of contemporary MRSA lineages. We also studied MSSA and MRSA isolates from other phage groups in order to investigate if they had the potential to develop into contemporary MRSA clones. Most susceptible or penicillin-resistant isolates from phage group III or the 83A complex belonged to sequence type 8 (ST8) or ST5, while four isolates were ST254. STs 30, 45 and 25 were represented by MSSA isolates from other phage groups, which also included several singletons. Representatives of most of the current major epidemic MRSA lineages were identified among fully susceptible isolates collected in the 1960s, suggesting that these were MSSA lineages which carried genetic traits important for superior epidemicity before the acquisition of methicillin resistance.
Biofilm Formation by the Emerging Fungal Pathogen
Trichosporon asahii
: Development, Architecture, and Antifungal Resistance
Authors: Di Bonaventura Giovanni , Pompilio Arianna , Picciani Carla , Iezzi Manuela , D'Antonio Domenico , Piccolomini Raffaele
Published: September 2006

Trichosporon asahii
is the most common cause of fatal disseminated trichosporonosis, frequently associated with indwelling medical devices. Despite the use of antifungal drugs to treat trichosporonosis, infection is often persistent and is associated with high mortality. This drove our interest in evaluating the capability of
T. asahii
to form a biofilm on biomaterial-representative polystyrene surfaces through the development and optimization of a reproducible
T. asahii
-associated biofilm model. Time course analyses of viable counts and a formazan salt reduction assay, as well as microscopy studies, revealed that biofilm formation by
T. asahii
occurred in an organized fashion through four distinct developmental phases: initial adherence of yeast cells (0 to 2 h), germination and microcolony formation (2 to 4 h), filamentation (4 to 6 h), and proliferation and maturation (24 to 72 h). Scanning electron microscopy and confocal scanning laser microscopy revealed that mature
T. asahii
biofilms (72-h) displayed a complex, heterogeneous three-dimensional structure, consisting of a dense network of metabolically active yeast cells and hyphal elements completely embedded within exopolymeric material. Antifungal susceptibility testing demonstrated a remarkable rise in the MICs of sessile
T. asahii
cells against clinically used amphotericin B, caspofungin, voriconazole, and fluconazole compared to their planktonic counterparts. In particular,
T. asahii
biofilms were up to 16,000 times more resistant to voriconazole, the most active agent against planktonic cells (MIC, 0.06 μg/ml). Our results suggest that the ability of
T. asahii
to form a biofilm may be a major factor in determining persistence of the infection in spite of in vitro susceptibility of clinical isolates.
Maribavir Antagonizes the Antiviral Action of Ganciclovir on Human Cytomegalovirus
Authors: Chou Sunwen , Marousek Gail I.
Published: September 2006

The cytomegalovirus (CMV) UL97 kinase inhibitor maribavir antagonized the anti-CMV effect of ganciclovir, increasing the ganciclovir 50% inhibitory concentration against a sensitive strain by up to 13-fold. Antiviral activities of foscarnet and cidofovir were unaffected by maribavir.
Bile Salt-Stimulated Lipase from Human Milk Binds DC-SIGN and Inhibits Human Immunodeficiency Virus Type 1 Transfer to CD4
+
T Cells
Authors: Naarding Marloes A. , Dirac Annette M. , Ludwig Irene S. , Speijer Dave , Lindquist Susanne , Vestman Eva-Lotta , Stax Martijn J. , Geijtenbeek Teunis B. H. , Pollakis Georgios , Hernell Olle , Paxton William A.
Published: September 2006

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus,
Mycobacterium
,
Leishmania
, and
Helicobacter pylori
, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4
+
T lymphocytes in
trans
and enhance infection. We have previously demonstrated that a multimeric Le
X
component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (Le
X
)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4
+
T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in Le
X
expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.
Inhibition of Filamentation Can Be Used To Treat Disseminated Candidiasis
Authors: Saville Stephen P. , Lazzell Anna L. , Bryant Alexander P. , Fretzen Angelika , Monreal Alex , Solberg Erik O. , Monteagudo Carlos , Lopez-Ribot Jose L. , Milne G. Todd
Published: September 2006

Candida albicans
remains the leading causative agent of invasive fungal infection. Although the importance of filamentation in
C. albicans
pathogenesis has been extensively investigated, in vivo studies to date have been unable to dissect the role of this developmental process in the establishment of infection versus the development of active disease as characterized by damage to the host leading to mortality. To address this issue, we genetically engineered a
C. albicans tet-NRG1
strain in which filamentation and virulence can be modulated both in vitro and in vivo simply by the presence or absence of doxycycline (DOX): this strain enabled us, in a prior study, to demonstrate that yeast-form cells were able to infect the deep organs but caused no disease unless filamentation (induced by the addition of DOX) was allowed to occur. In the present study, we examined whether inhibiting filamentation (by withdrawing the DOX) at 24 or 48 h postinfection could serve as an effective therapeutic intervention against candidiasis. The results obtained indicate that DOX removal led to an alteration in the morphology of the infecting fungal cells and a dramatic increase in survival, but as with conventional antifungal drug therapy regimens, mortality rates increased markedly the longer this intervention was delayed. These observations reinforce the importance of invasive filamentous growth in causing the damage to the host and the lethality associated with active disease and suggest this process could be fruitfully targeted for the development of new antifungal agents.
Reactivity of (1→3)-β-
d
-Glucan Assay with Commonly Used Intravenous Antimicrobials
Authors: Marty Francisco M. , Lowry Colleen M. , Lempitski Steven J. , Kubiak David W. , Finkelman Malcolm A. , Baden Lindsey R.
Published: September 2006

Forty-four intravenous antimicrobials were tested for the presence of (1→3)-β-
d
-glucan (BG). Colistin, ertapenem, cefazolin, trimethoprim-sulfamethoxazole, cefotaxime, cefepime, and ampicillin-sulbactam tested positive for BG at reconstituted-vial concentrations but not when diluted to usual maximum plasma concentrations. False-positive BG assays may occur when some antimicrobials are administered; however, this needs to be confirmed.
Enhanced Antiscrapie Effect Using Combination Drug Treatment
Authors: Kocisko David A. , Caughey Byron , Morrey John D. , Race Richard E.
Published: September 2006

Combination treatment with pentosan polysulfate and Fe(III)
meso
-tetra(4-sulfonatophenyl)porphine in mice beginning 14 or 28 days after scrapie inoculation significantly increased survival times. This increase may be synergistic, implying that the compounds act cooperatively in vivo. Combination therapy may therefore be more effective for treatment of transmissible spongiform encephalopathies and other protein-misfolding diseases.
Weak Mutators Can Drive the Evolution of Fluoroquinolone Resistance in
Escherichia coli
Authors: Örlén Hanna , Hughes Diarmaid
Published: September 2006

Weak mutators are common among clinical isolates of
Escherichia coli
. We show that the relative mutation rate and the “evolvability of fluoroquinolone resistance” are related by a power law slope of 1.2 over 3 orders of magnitude. Thus, even weak mutators can drive the evolution of fluoroquinolone resistance under selection pressure.
Standardization of an Experimental Murine Model of Invasive Pulmonary Aspergillosis
Authors: Sheppard Donald C. , Graybill John R. , Najvar Laura K. , Chiang Lisa Y. , Doedt Thomas , Kirkpatrick William R. , Bocanegra Rosie , Vallor Ana C. , Patterson Thomas F. , Filler Scott G.
Published: September 2006

Evaluating new therapeutic agents for invasive aspergillosis requires animal models that are reproducible among different laboratories. We therefore evaluated a murine model of aerosol infection in two independent laboratories and found a high level of both intra- and interlaboratory reproducibility of survival, fungal burden over time, and the efficacy of liposomal amphotericin B.
Vaccinia Virus Infection Attenuates Innate Immune Responses and Antigen Presentation by Epidermal Dendritic Cells
Authors: Deng Liang , Dai Peihong , Ding Wanhong , Granstein Richard D. , Shuman Stewart
Published: September 2006

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-α), and IFN-γ, it actively inhibited the production of proinflammatory cytokines TNF-α and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-α secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the ΔE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IκBα. These results suggest a novel role for the E3L protein as an antagonist of the NF-κB signaling pathway. ΔE3L-infected XS52 cells secreted higher levels of TNF-α and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. ΔK1L-infected cells secreted higher levels of TNF-α and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.
Phosphorylation of MCM4 at Sites Inactivating DNA Helicase Activity of the MCM4-MCM6-MCM7 Complex during Epstein-Barr Virus Productive Replication
Authors: Kudoh Ayumi , Daikoku Tohru , Ishimi Yukio , Kawaguchi Yasushi , Shirata Noriko , Iwahori Satoko , Isomura Hiroki , Tsurumi Tatsuya
Published: September 2006

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.
The Amino Terminus of the Herpes Simplex Virus 1 Protein Vhs Mediates Membrane Association and Tegument Incorporation
Authors: Mukhopadhyay Aparna , Lee Grace E. , Wilson Duncan W.
Published: September 2006

Assembly of herpes simplex viruses (HSV) is a poorly understood process involving multiple redundant interactions between large number of tegument and envelope proteins. We have previously shown (G. E. Lee, G. A. Church, and D. W. Wilson, J. Virol.
77:
2038-2045, 2003) that the virion host shutoff (Vhs) tegument protein is largely insoluble in HSV-infected cells and is also stably associated with membranes. Here we demonstrate that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore, we have found that the amino-terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a green fluorescent protein (GFP) reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. We conclude that the amino terminus of Vhs mediates targeting to sites of HSV assembly and to the viral tegument.
EBNA-3B- and EBNA-3C-Regulated Cellular Genes in Epstein-Barr Virus-Immortalized Lymphoblastoid Cell Lines
Authors: Chen Adrienne , Zhao Bo , Kieff Elliott , Aster Jon C. , Wang Fred
Published: September 2006

The cellular pathways that Epstein-Barr virus (EBV) manipulates in order to effect its lifelong persistence within hosts and facilitate its transmission between hosts are not well understood. The EBV nuclear antigen 3 (EBNA-3) family of latent infection proteins consists of transcriptional regulators that influence viral and cellular gene expression in EBV-infected cells. To identify EBNA-3B- and EBNA-3C-regulated cellular genes potentially important for virus infection in vivo, we studied a lymphoblastoid cell line (LCL) infected with an unusual EBV mutant, where a genetic manipulation to delete EBNA-3B also resulted in a significant decrease in EBNA-3C expression and slower than normal growth (3B−/3Clow). Transcriptional profiling was performed on the 3B−/3ClowLCLs, and comparison of mutant and wild-type LCL profiles resulted in a group of 21 probe sets representing 16 individual genes showing statistically significant differences in expression. Further quantitative reverse transcription-PCR analyses comparing 3B−/3ClowLCLs to a previously described EBNA-3B mutant (3B−) where EBNA-3C expression was normal revealed three potential EBNA-3B-repressed genes, three potential EBNA-3C-repressed genes, and two potential EBNA-3C-activated genes. The most highly EBNA-3C-repressed gene was Jagged1, a cell surface ligand and inducer of the Notch receptor signaling pathway that is usurped by EBV genes essential for B-cell immortalization. 3B−/3ClowLCLs expressed increased levels of Jagged1 protein and were able to more efficiently induce functional Notch signaling, and this signaling was dependent on Notch cleavage by γ-secretase. However, inhibiting γ-secretase-mediated Notch cleavage did not rescue 3B−/3ClowLCL growth, suggesting that EBNA-3C-mediated repression of this signaling pathway did not contribute to LCL growth in tissue culture. Similarly, expression of the chemokine receptor CXCR4 was reproducibly upregulated in EBNA-3B-null LCLs. Since deletion of EBNA-3B has no significant impact on B-cell immortalization in tissue culture, this finding suggested that EBNA-3B-mediated regulation of CXCR4 may be an important viral strategy for alteration of B-cell homing in the infected host. These studies identify two cellular genes that do not contribute to EBV-induced B-cell growth but whose expression levels are strongly EBNA-3 regulated in EBV-infected primary B cells. These EBV-manipulated cellular pathways may be important for virus survival or transmission in humans, and their independence from EBV-induced B-cell growth makes them potential targets for testing in vivo with the rhesus lymphocryptovirus animal model for EBV infection.