bluuebunny/crossref_metadata_2025 huggingface dataset:
Rational Be´zier Line-Symmetric Motions
Authors: Li Shutian , Ge Q. J.
Published: March 2005

This paper brings together line geometry, kinematic geometry of line-symmetric motions, and computer aided geometric design to develop a method for geometric design of rational Be´zier line-symmetric motions. By taking advantage of the kinematic geometry of a line-symmetric motion, the problem of synthesizing a rational Be´zier line-symmetric motion is reduced to that of designing a rational Be´zier ruled surface. In this way, a recently developed de Casteljau algorithm for line-geometric design of ruled surfaces can be applied. An example is presented in which the Bennet motion is represented as a rational Be´zier line-symmetric motion whose basic surface is a hyperboloid.
Topology Optimization of Multicomponent Beam Structure via Decomposition-Based Assembly Synthesis
Authors: Lyu Naesung , Saitou Kazuhiro
Published: March 2005

This paper presents a method for synthesizing multicomponent beam structural assemblies with maximum structural performance and manufacturability. The problem is posed as a relaxation of decomposition-based assembly synthesis, where both topology and decomposition of a structure are regarded as variables over a ground structure with nonoverlapping beams. A multiobjective genetic algorithm with graph-based crossover, coupled with FEM analyses, is used to obtain Pareto optimal solutions to this problem, exhibiting trade-offs among structural stiffness, total weight, component manufacturability (size and simplicity), and the number of joints. Case studies with a cantilever and a simplified automotive floor frame are presented, and representative designs in the Pareto front are examined for the trade-offs among the multiple criteria.
Numerical Analysis and Design for a Higher Jumping Rescue Robot Using a Pneumatic Cylinder
Authors: Tsukagoshi Hideyuki , Sasaki Masashi , Kitagawa Ato , Tanaka Takahiro
Published: March 2005

This paper describes the numerical analysis and design for a higher jumping rescue robot using a pneumatic cylinder. First, the basic equations for jumping are derived and the simulation is performed. Then, the relationship between the jumping height and the pressure-receiving area of the cylinder is considered when the volume or the stroke of the cylinder is kept constant. This allows calculation of the optimal cross sectional area. In addition, the jumping height is also affected by the weight ratio between the rod and the cylinder tube. Based on these results, a robot equipped with a cylinder of the appropriate dimensions controlled by a well-selected valve is reengineered and demonstrated. Experimental results show that the improved robot can jump considerably higher than the former design with the same energy efficiency, as shown in the video 〈http://www.cm.ctrl.titech.ac.jp/study/jump/home.html〉.
Shaking Force and Shaking Moment Balancing of Mechanisms: A Historical Review With New Examples
Authors: Arakelian Vigen H. , Smith M. R.
Published: March 2005

This paper deals with the solutions of the problem of the shaking force and shaking moment balancing of planar mechanisms by different methods based on the generation of the movements of counterweights. Some special cases are examined, such as balancing methods based on the copying properties of pantograph systems that carry the counterweights (formed by gears or by toothed-belt transmissions). The pantograph system executes a movement exactly opposite to the movement of the total center of the movable link masses. Such a solution provides the conditions for dynamic balancing with a relatively small increase of the total mass of the movable links. The methods are illustrated by many examples.
Numerical Analysis on the Performance of Control Valve in Variable Displacement Wobble Plate Compressor
Authors: Dou Chunpeng , Yang Xinjiang , Tian Changqing , Li Xianting
Published: March 2005

The performance of control valve is analyzed in this paper in order to study characteristics of the variable displacement wobble plate compressor. The forces acting on the control valve are analyzed and the mathematical model to analyze its performance is developed. A test bench is set up to get the steady and dynamic performance of control valve. The steady-state performances predicted by the mathematical model agree well with the experimental data while the predicted dynamic performances agree with the experimental results qualitatively. With the mathematical model, the steady-state performances of the control valve with four different preset control points and three different effective areas of the evacuated bellows have been analyzed, and the dynamic behavior of one type control valve has been studied. It is shown that: (1) the suction pressure at preset control point is inversely proportional to the discharge pressure; (2) the larger the starting force of spring in control valve or the smaller the effective area of evacuated bellows, the larger the suction pressure at preset control point; and the larger the starting force of spring in control valve and the smaller the maximum opening of ball valve, the more quickly the crankcase pressure changes and the smaller mass flow rate of control valve; the slope ratio of crankcase pressure to suction pressure cannot be influenced by the starting force of spring in control valve but can be influenced by the effective area of evacuated bellows; (3) the transition time of the crankcase pressure is about 1 s and can be neglected when working condition changes.
Fourier Methods for Kinematic Synthesis of Coupled Serial Chain Mechanisms
Authors: Nie Xichun , Krovi Venkat
Published: March 2005

Single degree-of-freedom coupled serial chain (SDCSC) mechanisms are a class of mechanisms that can be realized by coupling successive joint rotations of a serial chain linkage, by way of gears or cable-pulley drives. Such mechanisms combine the benefits of single degree-of-freedom design and control with the anthropomorphic workspace of serial chains. Our interest is in creating articulated manipulation-assistive aids based on the SDCSC configuration to work passively in cooperation with the human operator or to serve as a low-cost automation solution. However, as single-degree-of-freedom systems, such SDCSC-configuration manipulators need to be designed specific to a given task. In this paper, we investigate the development of a synthesis scheme, leveraging tools from Fourier analysis and optimization, to permit the end-effectors of such manipulators to closely approximate desired closed planar paths. In particular, we note that the forward kinematics equations take the form of a finite trigonometric series in terms of the input crank rotations. The proposed Fourier-based synthesis method exploits this special structure to achieve the combined number and dimensional synthesis of SDCSC-configuration manipulators for closed-loop planar path-following tasks. Representative examples illustrate the application of this method for tracing candidate square and rectangular paths. Emphasis is also placed on conversion of computational results into physically realizable mechanism designs.
Determination of the Dynamic Workspace of Cable-Driven Planar Parallel Mechanisms
Authors: Barrette Guillaume , Gosselin Cle´ment M.
Published: March 2005

In this paper, we present a general and systematic analysis of cable-driven planar parallel mechanisms. The equations for the velocities are derived, and the forces in the cables are obtained by the principle of virtual work. Then, a detailed analysis of the workspace is performed and an analytical method for the determination of the boundaries of an x-y two-dimensional subset is proposed. The new notion of dynamic workspace is defined, as its shape depends on the accelerations of the end-effector. We demonstrate that any subset of the workspace can be considered as a combination of three-cable subworkspaces, with boundaries being of two kinds: two-cable equilibrium loci and three-cable singularity loci. By using a parametric representation, we see that for the x-y workspace of a simple no-spring mechanism, the two-cable equilibrium loci represent a hyperbolic section, degenerating, in some particular cases, to one or two linear segments. Examples of such loci are presented. We use quadratic programming to choose which sections of the curves constitute the boundaries of the workspace for any particular dynamic state. A detailed example of workspace determination is included for a six-cable mechanism.
Multidimensional Clustering Interpretation and Its Application to Optimization of Coolant Passages of a Turbine Blade
Authors: Jeong Min Joong , Dennis Brian H. , Yoshimura Shinobu
Published: March 2005

A data-clustering method can be a useful tool for engineering design that is based on numerical optimization. The clustering method is an effective way of producing representative designs, or clusters, from a large set of potential designs. The results presented here focus on the application of clustering to single-objective optimization results. In the case of single-objective optimization, the method is used to determine the clusters in a set of quasi-optimal feasible solutions generated by an optimizer. A data-clustering procedure based on an evolutionary method is briefly described. The number of clusters is determined automatically and need not be known a priori. The method is demonstrated by application to the results of a turbine blade coolant passage shape-optimization problem. The solutions are transformed to a lower-dimensional space for better understanding of their variance and character. Engineering information, such as the shapes and locations of the internal passages, is supported by the visualization of clustered solutions. The clustering, transformation, and visualization methods presented in this study might be applicable to the increasing interpretation demands of design optimization.
Unsteady Heat Transfer Enhancement Around an Engine Cylinder in Order to Detect Knock
Authors: Loubar Khaled , Bellettre Je´ro^me , Tazerout Mohand
Published: March 2005

This paper deals with the transient thermal signal around an engine cylinder in order to propose a new and nonintrusive method of knock detection. Numerical simulations of unsteady heat transfer through the cylinder and inside the coolant flow are carried out to account for heat flux variations due to normal and knocking combustion. The effect of rib roughened surfaces on thermal signal amplification is investigated. The geometric parameters are fixed at Pi/h=10 and w/h=1 with a Reynolds number based on hydraulic diameter of 12,000. The results reveal that square ribs give better performance in term of thermal signal amplification within the fluid. An amplification of the temperature variation up to 20 times higher is found. Finally, flow analysis shows that amplification depends on the position where the thermal signal is collected.
A Thermodynamic Nonequilibrium Slug Flow Model
Authors: Barbosa, Jader R. , Hewitt Geoffrey F.
Published: March 2005

This paper presents a calculation methodology to predict the peaks in heat transfer coefficient at near zero equilibrium quality observed in forced convective boiling in vertical conduits. The occurrence of such peaks is typical of low latent heat, low thermal conductivity systems (such as refrigerants and hydrocarbons), and of systems in which the vapor volume formation rate for a given heat flux is large (low-pressure water). The methodology is based on a model that postulates that the mechanism behind the heat transfer coefficient enhancement is the existence of thermodynamic nonequilibrium slug flow, i.e., a type of slug flow in which rapid bubble growth in subcooled boiling leads to the formation of Taylor bubbles separated by slugs of subcooled liquid. Results are compared with experimental data for forced convective boiling of pure hydrocarbons and show considerable improvement over existing correlations.
Heat Transfer for Laminar Flow in Spiral Ducts of Rectangular Cross Section
Authors: Egner Michael W. , Burmeister Louis C.
Published: March 2005

Laminar flow and heat transfer in three-dimensional spiral ducts of rectangular cross section with aspect ratios of 1, 4, and 8 were determined by making use of the FLUENT computational fluid dynamics program. The peripherally averaged Nusselt number is presented as a function of distance from the inlet and of the Dean number. Fully developed values of the Nusselt number for a constant-radius-of-curvature duct, either toroidal or helical with small pitch, can be used to predict those quantities for the spiral duct in postentry regions. These results are applicable to spiral-plate heat exchangers.
Condensation of Zeotropic Mixtures in Horizontal Tubes: New Simplified Heat Transfer Model Based on Flow Regimes
Authors: Del Col D. , Cavallini A. , Thome J. R.
Published: March 2005

The need for optimal design of heat exchangers with in-tube condensation of zeotropic refrigerant mixtures has pushed the research of predictive methods in the last years. Some procedures have been developed, based on the Colburn and Drew (1937, Trans. AIChemE 33, pp. 197–215) analysis, that require significant numerical effort and the diffusivity properties of the mixture to calculate the mass transfer resistance in the process and, hence, are rarely used for heat exchanger design. Proposing a modified version of the well-known simplified approach of Silver (1947, Trans. Inst. Chem. Eng. 25, pp. 30–42) and Bell and Ghaly (1973, AIChE Symp. Ser. 69, pp. 72–79) to include the effects of interfacial roughness and nonequilibrium effects, the present study extends the flow-pattern-based model of Thome, El Hajal, and Cavallini (2003, Int. J. Heat Mass Transfer 46, pp. 3365–3387) for condensation of pure fluids and azeotropic mixtures to zeotropic mixtures. By implementing this within the above flow-pattern-based heat transfer model, it leads to an improved method for accurately predicting local mixture heat transfer coefficients, maintaining a clear relationship between flow regime and heat transfer, and achieving both the goals of higher prediction accuracy and low calculation effort. The method has been verified for refrigerant mixtures (both halogenated and hydrocarbon) having temperature glides of 3.5–22°C, that is temperature differences between the dew point and bubble point temperatures (at a fixed pressure and bulk composition).
On Population Balance Approach for Subcooled Boiling Flow Prediction
Authors: Tu J. Y. , Yeoh G. H. , Park G.-C. , Kim M.-O.
Published: March 2005

The capability of using the population balance approach combined with a three-dimensional two-fluid model for predicting subcooled boiling flow is investigated. Experiments were conducted to study the local flow characteristics of subcooled boiling flow and to provide measured local two-phase flow parameters. Calculations were performed using the newly developed population balance boiling model to study the effects of various factors on numerical predication of local two-phase flow parameters in the subcooled boiling regime. Comparison of model predictions against local measurements was made for the radial distribution of the bubble Sauter diameter and void fraction covering a range of different mass and heat fluxes and inlet subcooling temperatures. Additional comparison using recent active nucleation site density models and empirical relationships to determine the local bubble diameter adopted by other researchers was also investigated. Overall, good agreement was achieved between predictions and measurements using the newly formulated population balance approach based on the modified MUSIG (multiple-size-group) model for subcooled boiling and two-fluid model.
An Experimental and Theoretical Investigation of the Effect of Flow Maldistribution on the Thermal Performance of Plate Heat Exchangers
Authors: Prabhakara Rao B. , Sunden Bengt , Das Sarit K.
Published: March 2005

An experimental and theoretical study of the effect of flow maldistribution from port to channel on the thermal performance of single and multipass plate heat exchangers is presented. In general, flow maldistribution brings about an increase in pressure drop and decrease of the thermal performance in heat exchangers. This deterioration is found to depend on flow rate, number of channels, and port size. Experiments show that analytical predictions of pressure drop and thermal performance in presence of flow maldistribution are quite accurate for practical purposes. The results indicate that under identical conditions, maldistribution is more severe in Z-type plate heat exchanger compared to U type. Multipassing is found to reduce the maldistribution effect significantly. An insight to the physical aspects of maldistribution and its possible reduction through proper design strategy are also presented.
ATM Activation by DNA Double-Strand Breaks Through the Mre11-Rad50-Nbs1 Complex
Authors: Lee Ji-Hoon , Paull Tanya T.
Published: March 2005

The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.
Evidence for Multiple-Antibiotic Resistance in
Campylobacter jejuni
Not Mediated by CmeB or CmeF
Authors: Pumbwe Lilian , Randall Luke P. , Woodward Martin J. , Piddock Laura J. V.
Published: March 2005

An efflux system, CmeABC, in
Campylobacter jejuni
was previously described, and a second efflux system, CmeDEF, has now been identified. The substrates of CmeDEF include ampicillin, ethidium bromide, acridine, sodium dodecyl sulfate (SDS), deoxycholate, triclosan, and cetrimide, but not ciprofloxacin or erythromycin.
C. jejuni
NCTC11168 and two efflux pump knockout strains,
cmeB
::Kan
r
and
cmeF
::Kan
r
, were exposed to 0.5 to 1 μg of ciprofloxacin/ml in agar plates. All mutants arising from NCTC11168 were resistant to ciprofloxacin but not to other agents and contained a mutation resulting in the replacement of threonine 86 with isoleucine in the quinolone resistance-determining region of GyrA. Mutants with two distinct phenotypes were selected from the efflux pump knockout strains. Mutants with the first phenotype were resistant to ciprofloxacin only and had the same substitution within GyrA as the NCTC11168-derived mutants. Irrespective of the parent strain, mutants with the second phenotype were resistant to ciprofloxacin, chloramphenicol, tetracycline, ethidium bromide, acridine orange, and SDS and had no mutation in
gyrA
. These mutants expressed levels of the efflux pump genes
cmeB
and
cmeF
and the major outer membrane protein gene
porA
similar to those expressed by the respective parent strains. No mutations were detected in
cmeF
or
cmeB
. Accumulation assays revealed that the mutants accumulated lower concentrations of drug. These data suggest the involvement of a non-CmeB or -CmeF efflux pump or reduced uptake conferring multiple-antibiotic resistance, which can be selected after exposure to a fluoroquinolone.
Pseudomonas aeruginosaBloodstream Infection: Importance of Appropriate Initial Antimicrobial Treatment
Authors: Micek Scott T. , Lloyd Ann E. , Ritchie David J. , Reichley Richard M. , Fraser Victoria J. , Kollef Marin H.
Published: March 2005

Pseudomonas aeruginosabloodstream infection is a serious infection with significant patient mortality and health-care costs. Nevertheless, the relationship between initial appropriate antimicrobial treatment and clinical outcomes is not well established. This study was a retrospective cohort analysis employing automated patient medical records and the pharmacy database at Barnes-Jewish Hospital. Three hundred five patients withP. aeruginosabloodstream infection were identified over a 6-year period (January 1997 through December 2002). Sixty-four (21.0%) patients died during hospitalization. Hospital mortality was statistically greater for patients receiving inappropriate initial antimicrobial treatment (n= 75) compared to appropriate initial treatment (n= 230) (30.7% versus 17.8%;P= 0.018). Multiple logistic regression analysis identified inappropriate initial antimicrobial treatment (adjusted odds ratio [AOR], 2.04; 95% confidence interval [CI], 1.42 to 2.92;P= 0.048), respiratory failure (AOR, 5.18; 95% CI, 3.30 to 8.13;P< 0.001), and circulatory shock (AOR, 4.00; 95% CI, 2.71 to 5.91;P< 0.001) as independent determinants of hospital mortality. Appropriate initial antimicrobial treatment was administered statistically more often among patients receiving empirical combination antimicrobial treatment for gram-negative bacteria compared to empirical monotherapy (79.4% versus 65.5%;P= 0.011). Inappropriate initial empirical antimicrobial treatment is associated with greater hospital mortality among patients withP. aeruginosabloodstream infection. Inappropriate antimicrobial treatment ofP. aeruginosabloodstream infections may be minimized by increased use of combination antimicrobial treatment until susceptibility results become known.
Activity of Hoechst 33258 against
Pneumocystis carinii
f. sp.
muris
,
Candida albicans
, and
Candida dubliniensis
Authors: Disney Matthew D. , Stephenson Ruth , Wright Terry W. , Haidaris Constantine G. , Turner Douglas H. , Gigliotti Francis
Published: March 2005

Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against
Pneumocystis carinii
f. sp.
muris
in a mouse model for
P. carinii
pneumonia and against
Candida albicans
and
Candida dubliniensis
in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with
P. carinii
reduced by about 100-fold the number of
P. carinii
organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of
P. carinii
by about fourfold. In vitro inhibition of
P. carinii
group I intron splicing was observed with a 50% inhibitory concentration (IC
50
)of 30 μM in 2 or 4 mM Mg
2+
, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of
Candida
strains with and without group I introns. IC
50
s ranged from 1 to 9 μM for strains with group I introns and were 12 and 32 μM for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for
Pneumocystis
and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.
Pharmacokinetic and Maximum Tolerated Dose Study of Micafungin in Combination with Fluconazole versus Fluconazole Alone for Prophylaxis of Fungal Infections in Adult Patients Undergoing a Bone Marrow or Peripheral Stem Cell Transplant
Authors: Hiemenz J. , Cagnoni P. , Simpson D. , Devine S. , Chao N. , Keirns J. , Lau W. , Facklam D. , Buell D.
Published: March 2005

In this dose escalation study, 74 adult cancer patients undergoing bone marrow or peripheral blood stem cell transplantation received fluconazole (400 mg/day) and either normal saline (control) (12 subjects) or micafungin (12.5 to 200 mg/day) (62 subjects) for up to 4 weeks. The maximum tolerated dose (MTD) of micafungin was not reached, based on the development of Southwest Oncology Group criteria for grade 3 toxicity; drug-related toxicities were rare. Commonly occurring adverse events considered related to micafungin were headache (6.8%), arthralgia (6.8%), hypophosphatemia (4.1%), insomnia (4.1%), maculopapular rash (4.1%), and rash (4.1%). Pharmacokinetic profiles for micafungin on days 1 and 7 were similar. The mean half-life was approximately 13 h, with little variance after repeated or increasing doses. Mean maximum concentrations of the drug in serum and areas under the concentration-time curve from 0 to 24 h were approximately proportional to dose. There was no clinical or kinetic evidence of interaction between micafungin and fluconazole. Five of 12 patients (42%) in the control group and 14 of 62 (23%) in the micafungin-plus-fluconazole groups had a suspected fungal infection during treatment which resulted in empirical treatment with amphotericin B. The combination of micafungin and fluconazole was found to be safe in this high-risk patient population. The MTD of micafungin was not reached even at doses up to 200 mg/day for 4 weeks. The pharmacokinetic profile of micafungin in adult cancer patients with blood or marrow transplants is consistent with the profile in healthy volunteers, and the area under the curve is proportional to dose.
Comparison of the Pharmacodynamics of Meropenem in Patients with Ventilator-Associated Pneumonia following Administration by 3-Hour Infusion or Bolus Injection
Authors: Jaruratanasirikul Sutep , Sriwiriyajan Somchai , Punyo Jarurat
Published: March 2005

The time that concentrations in serum are above the MIC (
T
>MIC) is the pharmacokinetic/pharmacodynamic parameter correlating with the therapeutic efficacy of β-lactam antibiotics. The aim of this study was to demonstrate the
T
>MIC of meropenem when administered by a 3-h infusion compared with that when administered by bolus injection. The study was conducted with nine patients with ventilator-associated pneumonia. Each subject received meropenem in three regimens consecutively: (i) bolus injection of 1 g every 8 h for 24 h; (ii) 3-h infusion of 1 g every 8 h for 24 h; and (iii) 3-h infusion of 2 g every 8 h for 24 h. Following bolus injection, the percentages of the
T
>MICs of 16, 8, 4, and 1 μg/ml were 28.33% ± 11.67%, 45.89% ± 22.90%, 57.00% ± 24.82%, and 74.67% ± 17.94% of an 8-h interval, respectively. For the 3-h infusion of 1 g of meropenem, the percentages of the
T
>MICs of 16, 8, 4, and 1 μg/ml were 37.78% ± 20.57%, 58.11% ± 24.38%, 72.67% ± 21.97%, and 93.56% ± 6.84% of an 8-h interval, respectively. For the 3-h infusion of 2 g of meropenem, the percentages of the
T
>MICs of 16, 8, 4, and 1 μg/ml were 57.89% ± 24.26%, 72.89% ± 22.40%, 85.56% ± 16.42%, and 98.56% ± 3.28% of an 8-h interval, respectively. In conclusion, a 3-h infusion resulted in greater
T
>MICs than those after a bolus injection. For the treatment of infections caused by pathogens with intermediate resistance, a 3-h infusion of 2 g of meropenem every 8 h can provide concentrations in serum above the MIC of 16 μg/ml for almost 60% of an 8-h interval.
Bactericidal and Antiendotoxic Properties of Short Cationic Peptides Derived from a Snake Venom Lys49 Phospholipase A2
Authors: Santamaría Carlos , Larios Silda , Quirós Steve , Pizarro-Cerda Javier , Gorvel Jean-Pierre , Lomonte Bruno , Moreno Edgardo
Published: March 2005

The activities of short synthetic, nonhemolytic peptides derived from the C-terminal region of myotoxin II, a catalytically inactive phospholipase A2homologue present in the venom of the snakeBothrops asper, have been shown to reproduce the bactericidal activity of the parent protein. They combine cationic and hydrophobic-aromatic amino acids, thus functionally resembling the antimicrobial peptides of innate defenses. This study evaluated the antimicrobial and antiendotoxic properties of a 13-mer derivative peptide of the C-terminal sequence from positions 115 to 129 of myotoxin II, named pEM-2. This peptide (KKWRWWLKALAKK) showed bactericidal activity against both gram-positive and gram-negative bacteria. In comparison to previously described peptide variants derived from myotoxin II, the toxicity of pEM-2 toward eukaryotic cells in culture was significantly reduced, being similar to that of lactoferricin B but lower than that of polymyxin B. The all-denantiomer of pEM-2 [pEM-2 (d)] retained the same bactericidal potency of itsl-enantiomeric counterpart, but it showed an enhanced ability to counteract the lethal activity of an intraperitoneal lipopolysaccharide challenge in mice, which correlated with a significant reduction of the serum tumor necrosis factor alpha levels triggered by this endotoxin. Lethality induced by intraperitoneal infection of mice withEscherichia coliorSalmonella entericaserovar Typhimurium was reduced by the administration of pEM-2 (d). These results demonstrate that phospholipase A2-derived peptides may have the potential to counteract microbial infections and encourage further evaluations of their actions in vivo.
Successive Emergence of
Enterobacter aerogenes
Strains Resistant to Imipenem and Colistin in a Patient
Authors: Thiolas Aurélie , Bollet Claude , La Scola Bernard , Raoult Didier , Pagès Jean-Marie
Published: March 2005

Enterobacter aerogenes
is an agent of hospital-acquired infection that exhibits a remarkable resistance to β-lactam antibiotics during therapy. Five successive isolates of
E. aerogenes
infecting a patient and exhibiting a multiresistance phenotype to β-lactam antibiotics and fluoroquinolones were investigated. Among these clinical strains, four presented resistant phenotypes during successive imipenem and colistin treatments. The involved resistance mechanisms exhibited by the successive isolates were associated with alterations of the outer membrane that caused a porin decrease and lipopolysaccharide modifications.
Efficacy of Caspofungin against Central Nervous System
Aspergillus fumigatus
Infection in Mice Determined by TaqMan PCR and CFU Methods
Authors: Singh Gaurav , Imai Jackie , Clemons Karl V. , Stevens David A.
Published: March 2005

We have reported previously that prolonged caspofungin (CAS) dosing enhances survival in a murine model of central nervous system aspergillosis. In this study we determined by quantitative PCR (qPCR) and CFU enumeration whether CAS could reduce fungal burdens, prior to the deaths of untreated animals, and also assessed progressive infection in untreated mice. Mice were infected intracranially and treated for 4 days with CAS (1, 5, or 10 mg/kg of body weight/day) or amphotericin B (AMB) (3 mg/kg/day) starting 1 day postinfection. Fungal burdens in brains and kidneys of untreated controls were determined on days 1, 3, and 5 to assess progressive infection; burdens in treated animals were determined on day 5. qPCR showed higher burdens than CFU enumeration in all comparisons. In untreated animals, qPCR showed transiently increased burdens in brains, while CFU enumeration showed a decrease. qPCR showed increased burdens in kidneys, but CFU enumeration did not. Neither method indicated drug efficacy in the brain. Both methods showed AMB efficacy in the kidneys, and qPCR demonstrated CAS efficacy at all doses. Spearman correlations of qPCR and CFU determination results showed a significant correlation for most untreated groups; results correlated well for kidneys (
P
≤ 0.03) but not for brains in treated mice. Regression analyses of qPCR and CFU groups indicated different slopes for progressive infection in untreated animals but the same slopes for CAS dose-response efficacy. qPCR appeared to better reflect the progression of untreated infection. The lack of demonstration of efficacy in the brain suggests that longer dosing is necessary to cause burden reduction. These results also suggest that, when there is drug efficacy in a therapeutic study, either method appears to be useful for determining
Aspergillus fumigatus
burdens.
Susceptibility to Antibiotics and β-Lactamase Induction in Murein Hydrolase Mutants of
Escherichia coli
Authors: Korsak Dorota , Liebscher Sylvia , Vollmer Waldemar
Published: March 2005

The antibiotic susceptibilities and capabilities to induce β-lactamases were studied in multiple
Escherichia coli
murein (peptidoglycan) hydrolase mutants.
E. coli
mutants lacking either three amidases, three amidases and one lytic transglycosylase, or six lytic transglycosylases showed higher levels of susceptibility to bacitracin, erythromycin, gallidermin, and vancomycin than the wild type. Mutant cells without three amidases lost viability in the presence of vancomycin and gallidermin, whereas the wild type was resistant to both antibiotics. β-Lactamase induction was studied after introduction of a plasmid carrying the
ampC
and
ampR
genes. Upon addition of cefoxitin to the growth medium, the wild type as well as a mutant lacking all known amidases and
dd
-endopeptidases induced β-lactamase, whereas a mutant lacking all known lytic transglycosylases was unable to induce β-lactamase, showing that lytic transglycosylase activity is essential for β-lactamase induction. Consequently, cells lacking lytic transglycosylase activity lysed in the presence of penicillin, despite the presence of the inducible β-lactamase system. We discuss the potential of murein hydrolase inhibitors for antibiotic therapy.
Scintillation Proximity Assay for Inhibitors of
Escherichia coli
MurG and, Optionally, MraY
Authors: Ravishankar Sudha , Kumar Vidya Prasanna , Chandrakala B. , Jha Ramesh K. , Solapure Suresh M. , de Sousa Sunita M.
Published: March 2005

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of
Escherichia coli
membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[
3
H]
N
-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-
N
-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis—penicillin G, moenomycin, and bacitracin—had no effect. Surprisingly, however, the β-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.
Compensatory Adaptation to the Loss of Biological Fitness Associated with Acquisition of Fusidic Acid Resistance in
Staphylococcus aureus
Authors: Besier Silke , Ludwig Albrecht , Brade Volker , Wichelhaus Thomas A.
Published: March 2005

Recent studies have shown that individual amino acid exchanges within elongation factor G (EF-G) cause fusidic acid resistance in
Staphylococcus aureus
. The data from the present study illustrate that the fusidic acid resistance-mediating amino acid substitutions P406L and H457Y are associated with a marked impairment of the biological fitness of
S. aureus
. In particular, strains producing EF-G derivatives with these mutations showed reduced growth, decreased plasma coagulase activity, and an impaired capability to compete with the isogenic wild-type strain. Second-site mutations within EF-G, such as A67T and S416F, that have been encountered in clinical fusidic acid-resistant isolates containing the amino acid exchanges P406L and H457Y, respectively, were shown not to contribute to resistance. Furthermore, the substitution A67T had no impact on the biological fitness in vitro. The exchange S416F, however, was found to function as a fitness-compensating mutation in
S. aureus
carrying the substitution H457Y in EF-G. In conclusion, the data presented in this report provide evidence at the molecular level that the deleterious effects of fusidic acid resistance-mediating exchanges within EF-G of
S. aureus
can be reduced considerably by specific compensating mutations in this target protein. This compensatory adaptation most likely plays a significant role in the stabilization of resistant bacteria within a given population.
Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in
Leishmania donovani
Authors: Marquis Jean-François , Hardy Isabelle , Olivier Martin
Published: March 2005

The antitumor compound camptothecin (CPT) is also recognized for its specific activity against
Leishmania donovani
topoisomerase I (Topo-I). In consequence, defining CPT resistance mechanisms represents an important strategic tool in the acquisition of a better understanding of its mode of action. In the present study, we selected a single highly resistant
L
.
donovani
strain termed LdRCPT.160 by stepwise exposure to CPT. Gene sequencing revealed two single nucleotide mutations in the LdRCPT.160
LdTOP1A
gene, resulting in two amino acid substitutions (Gly185Arg and Asp325Glu) in the protein. Moreover, these two substitutions observed in the LdTOP1A protein were correlated with a decreased Topo-I DNA relaxation activity in these resistant parasites. Nevertheless, there was no change in the
LdTOP1A
gene expression level. Interestingly, transfection studies of the LdRCPT.160
LdTOP1A
gene in its wild-type counterpart showed that it induced CPT resistance. Site-directed mutagenesis studies demonstrated that, despite a substantial level of resistance conferred by the Gly185Arg and Asp325Glu substitutions separately, both were essential to reach a high-resistance phenotype. Of interest, the amino acid substitutions observed in LdRCPT.160 LdTOP1A protein occurred near the amino acids previously predicted to interact with CPT, providing new insight into the mechanism of CPT molecular action.
A
Helicobacter pylori
TolC Efflux Pump Confers Resistance to Metronidazole
Authors: van Amsterdam Karin , Bart Aldert , van der Ende Arie
Published: March 2005

In
Helicobacter pylori
, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of
H. pylori
26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in
H. pylori
and resembles the situation in other bacteria.
Phenotypic Tolerance: Antibiotic Enrichment of Noninherited Resistance in Bacterial Populations
Authors: Wiuff C. , Zappala R. M. , Regoes R. R. , Garner K. N. , Baquero F. , Levin B. R.
Published: March 2005

When growing bacteria are exposed to bactericidal concentrations of antibiotics, the sensitivity of the bacteria to the antibiotic commonly decreases with time, and substantial fractions of the bacteria survive. Using
Escherichia coli
CAB1 and antibiotics of five different classes (ampicillin, ciprofloxacin, rifampin, streptomycin, and tetracycline), we examine the details of this phenomenon and, with the aid of mathematical models, develop and explore the properties and predictions of three hypotheses that can account for this phenomenon: (i) antibiotic decay, (ii) inherited resistance, and (iii) phenotypic tolerance. Our experiments cause us to reject the first two hypotheses and provide evidence that this phenomenon can be accounted for by the antibiotic-mediated enrichment of subpopulations physiologically tolerant to but genetically susceptible to these antibiotics, phenotypic tolerance. We demonstrate that tolerant subpopulations generated by exposure to one concentration of an antibiotic are also tolerant to higher concentrations of the same antibiotic and can be tolerant to antibiotics of the other four types. Using a mathematical model, we explore the effects of phenotypic tolerance to the microbiological outcome of antibiotic treatment and demonstrate, a priori, that it can have a profound effect on the rate of clearance of the bacteria and under some conditions can prevent clearance that would be achieved in the absence of tolerance.
Efficacy of Ciprofloxacin-Releasing Bioabsorbable Osteoconductive Bone Defect Filler for Treatment of Experimental Osteomyelitis Due toStaphylococcus aureus
Authors: Koort Jyri K. , Mäkinen Tatu J. , Suokas Esa , Veiranto Minna , Jalava Jari , Knuuti Juhani , Törmälä Pertti , Aro Hannu T.
Published: March 2005

The concept of local antibiotic delivery via biodegradable bone defect fillers with multifunctional properties for the treatment of bone infections is highly appealing. Fillers can be used to obliterate surgical dead space and to provide targeted local bactericidal concentrations in tissue for extended periods. Eventually, the osteoconductive component of the filler could guide the healing of the bone defect. The present experimental study was carried out to test this concept in a localizedStaphylococcus aureusosteomyelitis model in the rabbit (n= 31). A metaphyseal defect of the tibia was filled with a block of bone cement, followed by insertion of a bacterial inoculum. After removal of the bone cement and surgical debridement at 2 weeks, the defect was filled with a ciprofloxacin-containing (7.6% ± 0.1%, by weight) composite (treated-infection group) or with a composite without antibiotic (sham-treated group). Both a positive control group (untreated-infection group) and a negative control group were also produced. The treatment response, monitored by positron emission tomography (PET) with fluorine-18-labeled fluorodeoxyglucose ([18F]FDG) at 3 and 6 weeks, showed rapidly decreasing amounts of [18F]FDG uptake in the treated-infection group (P= 0.001 compared with the results for the untreated-infection group at 6 weeks). The bacteriological analysis confirmed the eradication of the bone pathogen in the treated-infection group. However, three animals had culture-positive soft tissue infections. All animals in the sham-treated and untreated-infection groups had culture-positive bone infections with typical radiographic changes of osteomyelitis. Histomorphometry, peripheral quantitative computed tomography, and backscattered electron imaging of scanning electron microscopy images verified the osteoconductive properties of the bioactive glass microspheres within the composite. The median bone ciprofloxacin concentrations were 1.2 and 2.1 μg/g at two anatomic locations of the tibia. This is the first report to show the value of [18F]FDG PET for quantitative monitoring of the treatment response in bone infections. The collaborative results of bacteriologic and [18F-FDG] PET studies showed that use of the multifunctional composite was successful for eradication of theS. aureuspathogen from bone.
Expression ofccaR, Encoding the Positive Activator of Cephamycin C and Clavulanic Acid Production inStreptomyces clavuligerus, Is Dependent onbldG
Authors: Bignell Dawn R. D. , Tahlan Kapil , Colvin Kimberley R. , Jensen Susan E. , Leskiw Brenda K.
Published: March 2005

InStreptomyces coelicolor,bldGencodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment fromStreptomyces clavuligeruscontained an open reading frame that encoded a protein showing 92% identity to theS. coelicolorBldG protein and 91% identity to the BldG ortholog inStreptomyces avermitilis. Sequencing of the region downstream ofbldGinS. clavuligerusrevealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins inS. coelicolorandS. avermitilis, respectively. Northern analysis indicated that, as inS. coelicolor, theS. clavuligerus bldGgene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstreamorf3gene. High-resolution S1 nuclease mapping ofS. clavuligerus bldGtranscripts revealed the presence of threebldG-specific promoters, and analysis of expression of abldGp-egfpreporter indicated that thebldGpromoter is active at various stages of development and in both substrate and aerial hyphae. AbldGnull mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the5Sclavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makesbldGthe first regulatory protein identified inS. clavuligerusthat functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.