bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the Linearization of the Kinematic Equations in Railroad Vehicle System Dynamics
Authors: Shabana Ahmed A. , Tobaa Mahmoud , Marquis Brian , El-Sibaie Magdy
Published: March 2007

The sensitivity of the wheel/rail contact problem to the approximations made in some of the creepage expressions is examined in this investigation. It is known that railroad vehicle models that employ kinematic linearization can predict, particularly at high speeds, significantly different dynamic response as compared to models that are based on fully nonlinear kinematic and dynamic equations. In order to analytically examine this problem and numerically quantify the effect of the approximations used in the linearized railroad vehicle models, the fully nonlinear kinematic and dynamic equations of a wheel set are presented. The linearized kinematic and dynamic equations used in some railroad vehicle models are obtained from the fully nonlinear model in order to shed light on the assumptions and approximations used in the linearized models. The assumptions of small angles that are often made in developing railroad vehicle models and their effect on the angular velocity, angular acceleration, and the inertia forces are investigated. The velocity creepage expressions that result from the use of the assumptions of small angles are obtained and compared with the fully nonlinear expressions. Newton-Euler equations for the wheel set are presented and their dependence on Euler angles and their time derivatives is discussed. The effect of the linearization assumptions on the form of Newton-Euler equations is examined. A suspended wheel set model is used as an example to obtain the numerical results required to quantify the effect of the linearization. The results obtained in this investigation show that linearization of the creepages can lead to significant errors in the values predicted for the longitudinal and tangential forces as well as the spin moment. There are also significant differences between the two models in the prediction of the lateral and vertical forces used to evaluate the L∕V ratios as demonstrated by the results presented in this investigation.
Dynamics and Stability of a Two Degree of Freedom Oscillator With an Elastic Stop
Authors: Pascal Madeleine
Published: March 2007

A two degree of freedom oscillator with a colliding component is considered. The aim of the study is to investigate the dynamic behavior of the system when the stiffness obstacle changes to a finite value to an infinite one. Several cases are considered. First, in the case of rigid impact and without external excitation, a family of periodic solutions are found in analytical form. In the case of soft impact, with a finite time duration of the shock, and no external excitation, the existence of periodic solutions, with an arbitrary value of the period, is proved. Periodic motions are also obtained when the system is submitted to harmonic excitation, in both cases of rigid or soft impact. The stability of these periodic motions is investigated for these four cases.
The Dynamic Response of Tuned Impact Absorbers for Rotating Flexible Structures
Authors: Shaw Steven W. , Pierre Christophe
Published: March 2007

This paper describes an analytical investigation of the dynamic response and performance of impact vibration absorbers fitted to flexible structures that are attached to a rotating hub. This work was motivated by experimental studies at NASA, which demonstrated the effectiveness of these types of absorbers for reducing resonant transverse vibrations in periodically excited rotating plates. Here we show how an idealized model can be used to describe the essential dynamics of these systems, and used to predict absorber performance. The absorbers use centrifugally induced restoring forces so that their nonimpacting dynamics are tuned to a given order of rotation, whereas their large amplitude dynamics involve impacts with the primary flexible system. The linearized, nonimpacting dynamics are first explored in detail, and it is shown that the response of the system has some rather unique features as the hub rotor speed is varied. A class of symmetric impacting motions is also analyzed and used to predict the effectiveness of the absorber when operating in its impacting mode. It is observed that two different types of grazing bifurcations take place as the rotor speed is varied through resonance, and their influence on absorber performance is described. The analytical results for the symmetric impacting motions are also used to generate curves that show how important absorber design parameters—including mass, coefficient of restitution, and tuning—affect the system response. These results provide a method for quickly evaluating and comparing proposed absorber designs.
Global Dynamics of an Autoparametric System With Multiple Pendulums
Authors: Vyas Ashwin , Bajaj Anil K.
Published: March 2007

The Hamiltonian dynamics of a resonantly excited linear spring-mass-damper system coupled to an array of pendulums is investigated in this study under 1:1:1:…:2 internal resonance between the pendulums and the linear oscillator. To study the small-amplitude global dynamics, a Hamiltonian formulation is introduced using generalized coordinates and momenta, and action-angle coordinates. The Hamilton’s equations are averaged to obtain equations for the first-order approximations to free and forced response of the system. Equilibrium solutions of the averaged Hamilton’s equations in action-angle or comoving variables are determined and studied for their stability characteristics. The system with one pendulum is known to be integrable in the absence of damping and external excitation. Exciting the system with even a small harmonic forcing near a saddle point leads to stochastic response, as clearly demonstrated by the Poincaré sections of motion. Poincaré sections are also computed for motions started with initial conditions near center-center, center-saddle and saddle-saddle-type equilibria for systems with two, three and four pendulums. In case of the system with more than one pendulum, even the free undamped dynamics exhibits irregular exchange of energy between the pendulums and the block. The increase in complexity is also demonstrated as the number of pendulums is increased, and when external excitation is present.
Verification of Absolute Nodal Coordinate Formulation in Flexible Multibody Dynamics via Physical Experiments of Large Deformation Problems
Authors: Yoo Wan-Suk , Park Su-Jin , Dmitrochenko Oleg N. , Pogorelov Dmitry Yu.
Published: March 2007

A review of the current state of the absolute nodal coordinate formulation (ANCF) is proposed for large-displacement and large-deformation problems in flexible multibody dynamics. The review covers most of the known implementations of different kinds of finite elements including thin and thick planar and spatial beams and plates, their geometrical description inherited from FEM, and formulations of the most important elements of equations of motion. Much attention is also paid to simulation examples that show reasonableness and accuracy of the formulations applied to real physical problems and that are compared with experiments having significant geometrical nonlinearity. Current and further development directions of the ANCF are also briefly outlined.
Kinetic and affinity analyses of hybridization reactions between peptide nucleic acid probes and DNA targets using surface plasmon field-enhanced fluorescence spectroscopy
Authors: Park Hyeyoung , Germini Andrea , Sforza Stefano , Corradini Roberto , Marchelli Rosangela , Knoll Wolfgang
Published: March 2007

Peptide nucleic acid (PNA), a polyamide DNA mimic, has inspired the development of a variety of hybridization-based methods for the detection, quantification, purification, and characterization of nucleic acids owing to the stability of the PNA/DNA duplex. In this work, PNA probes complementary to a specific sequence of Roundup Ready® soybean were immobilized onto a sensor surface via a self-assembled matrix employing streptavidin/biotin binding. The specific hybridization of PNA and DNA has been monitored by applying the chromophore-labeled DNA target oligonucleotides to the PNA modified Au sensor surface in real time using surface plasmon field-enhanced fluorescence spectroscopy. The authors demonstrate three kinds of experiments called global, titration, and kinetic analyses for the determination of rate constants for the association (kon) and dissociation processes (koff, and the affinity constant (KA) of the PNA/DNA duplex formation by fitting the data to a simple Langmuir model. Discrimination of a single base mismatched DNA (15mer) target on a 15mer PNA probe was documented, with a difference of the affinity constant of two orders of magnitude. Finally, the affinity constant for the hybridization of a long polymerase chain reaction product (169mer) obtained by amplification of DNA extracted from genetically modified soybean reference material has been determined by a kinetic-titration analysis. The results show the influence of a Coulomb barrier at high target surface coverage even for the hybridization to PNA at low ionic strength.
Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications
Authors: Morgenthaler Sara , Zink Christian , Städler Brigitte , Vörös Janos , Lee Seunghwan , Spencer Nicholas D. , Tosatti Samuele G. P.
Published: March 2007

A simple dipping process has been used to prepare PEGylated surface gradients from the polycationic polymer poly(l-lysine), grafted with poly(ethylene glycol) (PLL-g-PEG), on metal oxide substrates, such as TiO2 and Nb2O5. PLL-g-PEG coverage gradients were prepared during an initial, controlled immersion and characterized with variable angle spectroscopic ellipsometry and x-ray photoelectron spectroscopy. Gradients with a linear change in thickness and coverage were generated by the use of an immersion program based on an exponential function. These single-component gradients were used to study the adsorption of proteins of different sizes and shapes, namely, albumin, immunoglobulin G, and fibrinogen. The authors have shown that the density and size of defects in the PLL-g-PEG adlayer determine the amount of protein that is adsorbed at a certain adlayer thickness. In a second step, single-component gradients of functionalized PLL-g-PEG were backfilled with nonfunctionalized PLL-g-PEG to generate two-component gradients containing functional groups, such as biotin, in a protein-resistant background. Such gradients were combined with a patterning technique to generate individually addressable spots on a gradient surface. The surfaces generated in this way show promise as a useful and versatile biochemical screening tool and could readily be incorporated into a method for studying the behavior of cells on functionalized surfaces.
Creation of a functional heterogeneous vesicle array via DNA controlled surface sorting onto a spotted microarray
Authors: Städler Brigitte , Bally Marta , Grieshaber Dorothee , Vörös Janos , Brisson Alain , Grandin H. Michelle
Published: March 2007

Membrane protein microarrays are expected to play a key role in the future of drug screening and discovery. The authors present a method for the creation of functional heterogeneous vesicle arrays via DNA controlled surface sorting. Complexes of streptavidin and biotinylated DNA are spotted onto a biomolecule- and cell-resistant surface of biotinylated poly(l-lysine)-grafted-poly(ethylene glycol). Two kinds of vesicles functionalized with either the membrane-binding protein annexin A5 or loaded with bovine serum albumin, are tagged with DNA, mixed together, and guided to predefined spots on the surface. The authors show that the spotted complexes remain active and selective and that the background is resistant towards nonspecific adsorption of the vesicles and the proteins.
Longitudinally Tracking Fluoroquinolone Resistance and Its Determinants in Penicillin-Susceptible and -Nonsusceptible
Streptococcus pneumoniae
Isolates in Hong Kong, 2000 to 2005
Authors: Ip Margaret , Chau Shirley S. L. , Chi Fang , Cheuk Edmund S. C. , Ma Helen , Lai Raymond W. M. , Chan Paul K.
Published: March 2007

Of 1,388
Streptococcus pneumoniae
isolates collected from 2000 to 2005, 10.5% had a ciprofloxacin MIC of ≥4.0 μg/ml and 1.6% (range, 0.8% to 4.3% per year) had a levofloxacin MIC of ≥4.0 μg/ml. Molecular characterization indicated that fluoroquinolone resistance occurred independently in our prevalent Spain
23F
-1 clone, expressing serotypes 23F, 19F, and 14. Rates of resistance to levofloxacin in
S. pneumoniae
have remained stable at a Hong Kong hospital.
Mutations in 16S rRNA and Ribosomal Protein S5 Associated with High-Level Spectinomycin Resistance in
Pasteurella multocida
Authors: Kehrenberg Corinna , Schwarz Stefan
Published: March 2007

Pasteurella multocida
isolates with high-level spectinomycin resistance in which no adenyltransferase genes could be demonstrated exhibited a C1192G transversion in the 16S rRNA of all six or five of the six rRNA operons and/or two different types of 3-bp deletions in the
rpsE
gene that codes for the ribosomal protein S5.
High-Level Gentamicin Resistance Mediated by a Tn
4001
-Like Transposon in Seven Nonclonal Hospital Isolates of
Streptococcus pasteurianus
Authors: Chow Viola C. Y. , Hawkey Peter M. , Chan Edward W. C. , Chin Miu L. , Au T. K. , Fung Danny K. C. , Chan Raphael C. Y.
Published: March 2007

We report on the first occurrence of high-level gentamicin resistance (MICs ≥ 512 μg/ml) in seven clinical isolates of
Streptococcus pasteurianus
from Hong Kong. These seven isolates were confirmed to be the species
S. pasteurianus
on the basis of nucleotide sequencing of the superoxide dismutase (
sodA
) gene. Epidemiological data as well as the results of pulse-field gel electrophoresis analysis suggested that the seven
S. pasteurianus
isolates did not belong to the same clone. Molecular characterization showed that they carried a chromosomal, transposon-borne resistance gene [
aac(6′)Ie-aph(2″)Ia
] which was known to encode a bifunctional aminoglycoside-modifying enzyme. The genetic arrangement of this transposon was similar to that of Tn
4001
, a transposon previously recovered from
Staphylococcus aureus
and other gram-positive isolates. Genetic linkage with other resistance elements, such as the
ermB
gene for erythromycin resistance, was not evident. On the basis of these findings, we suggest that routine screening for high-level gentamicin resistance should be recommended for all clinically significant blood culture isolates. This is to avoid the inadvertent use of short-course combination therapy with penicillin and gentamicin, which may lead to the failure of treatment for endocarditis, the selection of drug-resistant
Streptococcus pasteurianus
and other gram-positive organisms, as well as the unnecessary usage of gentamicin, a drug with potential toxicity.
Daptomycin Produces an Enhanced Bactericidal Activity Compared to Ceftriaxone, Measured by [
3
H]Choline Release in the Cerebrospinal Fluid, in Experimental Meningitis Due to a Penicillin-Resistant Pneumococcal Strain without Lysing Its Cell Wall
Authors: Stucki A. , Cottagnoud M. , Winkelmann V. , Schaffner T. , Cottagnoud P.
Published: March 2007

Daptomycin monotherapy was superior to ceftriaxone monotherapy and was highly efficacious in experimental pneumococcal meningitis, sterilizing the cerebrospinal fluid (CSF) of three of three rabbits after 4 to 6 h. With daptomycin therapy only a negligible release of [
3
H]choline as marker of cell wall lysis was detectable in the CSF, peaking around 250 cpm/min after 4 h, compared to a peak of around 2,400 cpm/min after 4 to 6 h for the ceftriaxone-treated rabbits.
Prevention of Brain Injury by the Nonbacteriolytic Antibiotic Daptomycin in Experimental Pneumococcal Meningitis
Authors: Grandgirard Denis , Schürch Christian , Cottagnoud Philippe , Leib Stephen L.
Published: March 2007

Bacteriolytic antibiotics cause the release of bacterial components that augment the host inflammatory response, which in turn contributes to the pathophysiology of brain injury in bacterial meningitis. In the present study, antibiotic therapy with nonbacteriolytic daptomycin was compared with that of bacteriolytic ceftriaxone in experimental pneumococcal meningitis, and the treatments were evaluated for their effects on inflammation and brain injury. Eleven-day-old rats were injected intracisternally with 1.3 × 10
4
± 0.5 × 10
4
CFU of
Streptococcus pneumoniae
serotype 3 and randomized to therapy with ceftriaxone (100 mg/kg of body weight subcutaneously [s.c.];
n
= 55) or daptomycin (50 mg/kg s.c.;
n
= 56) starting at 18 h after infection. The cerebrospinal fluid (CSF) was assessed for bacterial counts, matrix metalloproteinase-9 levels, and tumor necrosis factor alpha levels at different time intervals after infection. Cortical brain damage was evaluated at 40 h after infection. Daptomycin cleared the bacteria more efficiently from the CSF than ceftriaxone within 2 h after the initiation of therapy (log
10
3.6 ± 1.0 and log
10
6.3 ± 1.4 CFU/ml, respectively;
P
< 0.02); reduced the inflammatory host reaction, as assessed by the matrix metalloproteinase-9 concentration in CSF 40 h after infection (
P
< 0.005); and prevented the development of cortical injury (cortical injury present in 0/30 and 7/28 animals, respectively;
P
< 0.004). Compared to ceftriaxone, daptomycin cleared the bacteria from the CSF more rapidly and caused less CSF inflammation. This combined effect provides an explanation for the observation that daptomycin prevented the development of cortical brain injury in experimental pneumococcal meningitis. Further research is needed to investigate whether nonbacteriolytic antibiotic therapy with daptomycin represents an advantageous alternative over current bacteriolytic antibiotic therapies for the treatment of pneumococcal meningitis.
A New
Aspergillus fumigatus
Resistance Mechanism Conferring In Vitro Cross-Resistance to Azole Antifungals Involves a Combination of
cyp51A
Alterations
Authors: Mellado E. , Garcia-Effron G. , Alcázar-Fuoli L. , Melchers W. J. G. , Verweij P. E. , Cuenca-Estrella M. , Rodríguez-Tudela J. L.
Published: March 2007

Fourteen
Aspergillus fumigatus
clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the
cyp51A
gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the
cyp51A
gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the
cyp51A
gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of
cyp51
A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the
cyp51A
gene of an azole drug-susceptible
A. fumigatus
wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the
cyp51A
gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of
cyp51A
expression, confirming that the combination of both alterations was responsible for the azole-resistant phenotype.
Treatment of
Staphylococcus aureus
Biofilm Infection by the Quorum-Sensing Inhibitor RIP
Authors: Balaban Naomi , Cirioni Oscar , Giacometti Andrea , Ghiselli Roberto , Braunstein Joel B. , Silvestri Carmela , Mocchegiani Federico , Saba Vittorio , Scalise Giorgio
Published: March 2007

The quorum-sensing inhibitor RIP inhibits staphylococcal TRAP/
agr
systems and both TRAP- and
agr
-negative strains are deficient in biofilm formation in vivo, indicating the importance of quorum sensing to biofilms in the host. RIP injected systemically into rats has been found to have strong activity in preventing methicillin-resistant
Staphylococcus aureus
graft infections, suggesting that RIP can be used as a therapeutic agent.
Clinical and Pharmacokinetic Data Support Once-Daily Low-Dose Boosted Saquinavir (1,200 Milligrams Saquinavir with 100 Milligrams Ritonavir) in Treatment-Naive or Limited Protease Inhibitor-Experienced Human Immunodeficiency Virus-Infected Patients
Authors: Marin-Niebla Ana , Lopez-Cortes Luis Fernando , Ruiz-Valderas Rosa , Viciana Pompeyo , Mata Rosario , Gutierrez Alicia , Pascual Rosario , Rodriguez Magdalena
Published: March 2007

We evaluated the plasma and intracellular pharmacokinetics, clinical efficacy, and safety of once-daily low-dose boosted saquinavir (SQVr; 1,200 of saquinavir [SQV] with 100 mg of ritonavir) plus two nucleotide reverse transcriptase inhibitors in treatment-naive or limited protease inhibitor (PI)-experienced human immunodeficiency virus (HIV)-infected patients. A prospective study without entry restrictions on the plasma HIV-RNA (VL) or CD4 cell count was carried out. Plasma and intracellular SQV levels were measured by high-performance liquid chromatography. Efficacy was evaluated by an intention-to-treat analysis; treatment failure was defined as virological failure (a VL of >50 copies/ml after 24 weeks or a confirmed rebound to >50 copies/ml) or interruption for any reason. A total of 151 patients were included in the study (106 of them either had never received PI or had no previous virological failure on PIs) and could be characterized as follows: previous C3 stage, 28.9%; injection-drug users, 69.1%; subjects with chronic viral hepatitis, 53%; and subjects with cirrhosis, 10%. The median baseline CD4 level was 184/μl, and the median VL was 4.8 log10copies/ml. MedianCmax, area under the concentration-time curve from 0 to 24 h, andCminplasma and intracellular SQV levels were 3,672 and 10,105 ng/ml, 34,283 and 99,535 ng·h/ml, and 359 and 1,062 ng/ml, respectively. The efficacy as determined by intention to treat at 52 weeks was 69.7% (96% in the on-treatment analysis), with similar results regardless of the baseline VL and CD4 counts. Only five patients had virological failure despite adequateCminlevels, but with a poor adherence (the only variable related to virological failure). Adverse events caused the withdrawal of the treatment in four patients (2.6%). In conclusion, given the pharmacokinetic profile, efficacy, and tolerability of this regimen, once-daily low-dose SQVr may be considered a treatment option in treatment-naive or limited PI-experienced HIV-infected patients, with the additional benefit of being currently the least-expensive PI-based regimen available.
Spread of Extended-Spectrum β-Lactamase CTX-M-Producing
Escherichia coli
Clinical Isolates in Community and Nosocomial Environments in Portugal
Authors: Mendonça Nuno , Leitão Joana , Manageiro Vera , Ferreira Eugénia , Caniça Manuela
Published: March 2007

Of the 181 unduplicated
Escherichia coli
strains isolated in nine different hospitals in three Portuguese regions, 119 were extended-spectrum β-lactamase (ESBL)-CTX-M producers and were selected for phenotype and genotype characterization. CTX-M producer strains were prevalent among community-acquired infections (56%), urinary tract infections (76%), and patients ≥60 years old (76%). In MIC tests, all strains were resistant to cefotaxime, 92% were resistant to ceftazidime, 93% were resistant to quinolones, 89% were resistant to aminoglycoside, and 26% were resistant to trimethoprim-sulfamethoxazole; all strains were sensitive to carbapenems, and 92% of the strains had a multidrug resistance phenotype. Molecular methods identified 109 isolates harboring a
bla
CTX-M-15
gene, 1 harboring the
bla
CTX-M-32
gene (first identification in the country), and 9 harboring the
bla
CTX-M-14
gene. All isolates presented the IS
Ecp1
element upstream from the
bla
CTX-M
genes; one presented the IS
903
element (downstream of
bla
CTX-M-14
gene), and none had the IS
26
element; 85% carried
bla
TEM-1B
, and 84% also carried a
bla
OXA-30
. Genetic relatedness analysis based on pulsed-field gel electrophoresis defined five clusters and indicated that 76% of all isolates (from cluster IV) corresponded to a single epidemic strain. Of the 47 strains from one hospital, 41 belonged to cluster IV and were disseminated in three main wards. CTX-M-producing
E. coli
strains are currently a problem in Portugal, with CTX-M-15 particularly common. This study suggests that the horizontal transfer of
bla
CTX-M
genes, mediated by plasmids and/or mobile elements, contributes to the dissemination of CTX-M enzymes to community and hospital environments. The use of extended-spectrum cephalosporins, quinolones, and aminoglycosides is compromised, leaving carbapenems as the therapeutic option for severe infections caused by ESBL producers.
A Novel Bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI), GRL-98065, Is Potent against Multiple-PI-Resistant Human Immunodeficiency Virus In Vitro
Authors: Amano Masayuki , Koh Yasuhiro , Das Debananda , Li Jianfeng , Leschenko Sofiya , Wang Yuan-Fang , Boross Peter I. , Weber Irene T. , Ghosh Arun K. , Mitsuya Hiroaki
Published: March 2007

We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(
R
),3a(
S
),6a(
R
)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC
50
], 0.0002 to 0.0005 μM) with minimal cytotoxicity (50% cytotoxicity, 35.7 μM in CD4
+
MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1
NL4-3
variants exposed to and selected by up to a 5 μM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 μM concentration of lopinavir or atazanavir (EC
50
, 0.0015 to 0.0075 μM), although it was less active against HIV-1
NL4-3
selected by amprenavir (EC
50
, 0.032 μM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.
Bis-Acridines as Lead Antiparasitic Agents: Structure-Activity Analysis of a Discrete Compound Library In Vitro
Authors: Caffrey Conor R. , Steverding Dietmar , Swenerton Ryan K. , Kelly Ben , Walshe Deirdre , Debnath Anjan , Zhou Yuan-Min , Doyle Patricia S. , Fafarman Aaron T. , Zorn Julie A. , Land Kirkwood M. , Beauchene Jessica , Schreiber Kimberly , Moll Heidrun , Ponte-Sucre Alicia , Schirmeister Tanja , Saravanamuthu Ahilan , Fairlamb Alan H. , Cohen Fred E. , McKerrow James H. , Weisman Jennifer L. , May Barnaby C. H.
Published: March 2007

Parasitic diseases are of enormous public health significance in developing countries—a situation compounded by the toxicity of and resistance to many current chemotherapeutics. We investigated a focused library of 18 structurally diverse bis-acridine compounds for in vitro bioactivity against seven protozoan and one helminth parasite species and compared the bioactivities and the cytotoxicities of these compounds toward various mammalian cell lines. Structure-activity relationships demonstrated the influence of both the bis-acridine linker structure and the terminal acridine heterocycle on potency and cytotoxicity. The bioactivity of polyamine-linked acridines required a minimum linker length of approximately 10 Å. Increasing linker length resulted in bioactivity against most parasites but also cytotoxicity toward mammalian cells. N alkylation, but less so N acylation, of the polyamine linker ameliorated cytotoxicity while retaining bioactivity with 50% effective concentration (EC50) values similar to or better than those measured for standard drugs. Substitution of the polyamine for either an alkyl or a polyether linker maintained bioactivity and further alleviated cytotoxicity. Polyamine-linked compounds in which the terminal acridine heterocycle had been replaced with an aza-acridine also maintained acceptable therapeutic indices. The most potent compounds recorded low- to mid-nanomolar EC50values againstPlasmodium falciparumandTrypanosoma brucei; otherwise, low-micromolar potencies were measured. Importantly, the bioactivity of the library was independent ofP. falciparumresistance to chloroquine. Compound bioactivity was a function of neither the potential to bis-intercalate DNA nor the inhibition of trypanothione reductase, an important drug target in trypanosomatid parasites. Our approach illustrates the usefulness of screening focused compound libraries against multiple parasite targets. Some of the bis-acridines identified here may represent useful starting points for further lead optimization.
In Vitro Activity of 2,4-Diamino-6-[2-(Phosphonomethoxy)Ethoxy]-Pyrimidine against Multidrug-Resistant Hepatitis B Virus Mutants
Authors: Brunelle M. N. , Lucifora J. , Neyts J. , Villet S. , Holy A. , Trepo C. , Zoulim F.
Published: March 2007

The susceptibilities of drug-resistant hepatitis B virus (HBV) mutants to lamivudine, adefovir, tenofovir, entecavir, and 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine (PMEO-DAPym), a novel acyclic pyrimidine analogue, were assessed in vitro. Most drug-resistant mutants, including multidrug-resistant strains, remained sensitive to tenofovir and PMEO-DAPym. Therefore, the latter molecule deserves further evaluation for the treatment of HBV infection.
Staphylococcus aureus
Strains That are Hypersusceptible to Resistance Gene Transfer from Enterococci
Authors: Sung Julia M.-L. , Lindsay Jodi A.
Published: March 2007

We identified naturally occurring
Staphylococcus aureus
mutants of the restriction modification pathway SauI, including bovine lineage ST151. In a model of vancomycin resistance transfer from
Enterococcus faecalis
, ST151 isolates are 500 times more susceptible than human
S. aureus
isolates. The eradication of “hyperrecipient” strains may reduce the evolution of vancomycin-resistant
S. aureus
.
Kinetic Characterization of Squalene Synthase fromTrypanosoma cruzi: Selective Inhibition by Quinuclidine Derivatives
Authors: Sealey-Cardona Marco , Cammerer Simon , Jones Simon , Ruiz-Pérez Luis M. , Brun Reto , Gilbert Ian H. , Urbina Julio A. , González-Pacanowska Dolores
Published: March 2007

The biosynthesis of sterols is a major route for the development of antitrypanosomals. Squalene synthase (SQS) catalyzes the first step committed to the biosynthesis of sterols within the isoprenoid pathway, and several inhibitors of the enzyme have selective antitrypanosomal activity both in vivo and in vitro. The enzyme fromTrypanosoma cruziis a 404-amino-acid protein with a clearly identifiable membrane-spanning region. In an effort to generate soluble recombinant enzyme, we have expressed inEscherichia coliseveral truncated versions ofT. cruziSQS with a His tag attached to the amino terminus. Deletions of both the amino- and carboxyl-terminal regions generated active and soluble forms of the enzyme. The highest levels of soluble protein were achieved when 24 and 36 amino acids were eliminated from the amino and carboxyl regions, respectively, yielding a protein of 41.67 kDa. The Michaelis-Menten constants of the purified enzyme for farnesyl diphosphate and NAD (NADPH) were 5.25 and 23.34 μM, respectively, whereas theVmaxwas 1,428.56 nmol min−1mg−1. Several quinuclidine derivatives with antiprotozoal activity in vitro were found to be selective inhibitors of recombinantT. cruzi SQS in comparative assays with the human enzyme, with 50% inhibitory concentration values in the nanomolar range. These data suggest that selective inhibition ofT. cruziSQS may be an efficient strategy for the development of new antitrypanosomal agents.
Pharmacokinetics of Penicillin G in Very-Low-Birth-Weight Neonates
Authors: Metsvaht Tuuli , Oselin Kersti , Ilmoja Mari-Liis , Anier Kaili , Lutsar Irja
Published: March 2007

Data on the pharmacokinetics (PKs) of penicillin G (PEN) in neonates date back to the 1970s and do not include data for very-low-birth-weight (VLBW) neonates. The aim of this study was to describe the steady-state PKs and to establish an optimal regimen for the dosing of PEN in neonates with gestational ages of less than 28 weeks and birth weights of less than 1,200 g. Two PEN dosing regimens were studied: 50,000 IU (30 mg)/kg of body weight every 12 h (q12h) (group 1;
n
= 9) and 25,000 IU (15 mg)/kg q12h (group 2;
n
= 9). Samples for PK analysis were drawn before the injection of PEN and at 2 and 30 min and 1.5, 4, 8, and 12 h after intravenous injection of the third to eighth PEN doses. The PEN concentration was measured by a high-performance liquid chromatography with UV detection technique. The median peak and trough concentrations of PEN were 147 μg/ml (lower and upper quartiles, 109 and 157 μg/ml, respectively) and 7 μg/ml (lower and upper quartiles, 5 and 13 μg/ml, respectively) after administration of the dose of 50,000 IU and 59 μg/ml (lower and upper quartiles, 53 and 78 μg/ml, respectively) and 3 μg/ml (lower and upper quartiles, 3 and 4 μg/ml, respectively) after administration of the dose of 25,000 IU. The PEN half-life (median and lower and upper quartiles for group 1, 3.9 h and 3.3 and 7.0 h, respectively; median and lower and upper quartiles for group 2, 4.6 h and 3.8 and 5.6 h, respectively) was longer in VLBW neonates than in adults and term infants. PEN renal clearance correlated with creatinine clearance (
R
2
= 0.309596;
P
= 0.038). Only a median of 34% (lower and upper quartiles, 28 and 37%, respectively) of the administered dose was excreted in urine within the following 12 h. We conclude that in VLBW infants a PEN dose of 25,000 IU (15 mg)/kg q12h is safe and sufficient to achieve serum concentrations above the MIC
90
for group B streptococci for the entire dosing interval.
Determination of the Genetic Support for
tet
(W) in Oral Bacteria
Authors: Villedieu A. , Roberts A. P. , Allan E. , Hussain H. , McNab R. , Spratt D. A. , Wilson M. , Mullany P.
Published: March 2007

The DNA sequence flanking a
tet
(W) gene in an oral
Rothia
sp. was determined. The gene was linked to two different transposases, and these were flanked by two almost identical
mef
(macrolide efflux) genes. This structure was found in 4 out of 20
tet
(W)-containing oral bacteria investigated.
Cold-Active Chemoorganotrophic Bacteria from Permanently Ice-Covered Lake Hoare, McMurdo Dry Valleys, Antarctica
Authors: Clocksin Kate M. , Jung Deborah O. , Madigan Michael T.
Published: March 2007

Eight strains of chemoorganotrophic bacteria were isolated from the water column of Lake Hoare, McMurdo Dry Valleys, Antarctica, using cold enrichment temperatures. The isolates wereAlpha-,Beta-, andGammaproteobacteriaandActinobacteriaspp. All isolates grew at 0°C, and all but one grew at subzero temperatures characteristic of the water column of Lake Hoare. Growth temperature optima varied among isolates, but the majority showed optima near 15°C, indicative of cold-active phenotypes. One isolate was truly psychrophilic, growing optimally around 10°C and not above 20°C. Half of the isolates grew at 2% salt while the other half did not, and all but one isolate grew at 2 atm of O2. Our isolates are the first prokaryotes from the water column of Lake Hoare to be characterized phylogenetically and physiologically and show that cold-active species of at least two major phyla ofBacteriainhabit Lake Hoare.
Phylogenetic Diversity and Cosymbiosis in the Bioluminescent Symbioses of “Photobacterium mandapamensis”
Authors: Kaeding Allison J. , Ast Jennifer C. , Pearce Meghan M. , Urbanczyk Henryk , Kimura Seishi , Endo Hiromitsu , Nakamura Masaru , Dunlap Paul V.
Published: March 2007

“Photobacterium mandapamensis” (proposed name) andPhotobacterium leiognathiare closely related, phenotypically similar marine bacteria that form bioluminescent symbioses with marine animals. Despite their similarity, however, these bacteria can be distinguished phylogenetically by sequence divergence of their luminescence genes,luxCDAB(F)E, by the presence (P. mandapamensis) or the absence (P. leiognathi) ofluxFand, as shown here, by the sequence divergence of genes involved in the synthesis of riboflavin,ribBHA. To gain insight into the possibility thatP. mandapamensisandP. leiognathiare ecologically distinct, we used these phylogenetic criteria to determine the incidence ofP. mandapamensisas a bioluminescent symbiont of marine animals. Five fish species,Acropoma japonicum(Perciformes, Acropomatidae),Photopectoralis panayensisandPhotopectoralis bindus(Perciformes, Leiognathidae),Siphamia versicolor(Perciformes, Apogonidae), andGadella jordani(Gadiformes, Moridae), were found to harborP. mandapamensisin their light organs. Specimens ofA. japonicus,P. panayensis, andP. bindusharboredP. mandapamensisandP. leiognathitogether as cosymbionts of the same light organ. Regardless of cosymbiosis,P. mandapamensiswas the predominant symbiont ofA. japonicum, and it was the apparently exclusive symbiont ofS. versicolorandG. jordani. In contrast,P. leiognathiwas found to be the predominant symbiont ofP. panayensisandP. bindus, and it appears to be the exclusive symbiont of other leiognathid fishes and a loliginid squid. A phylogenetic test for cospeciation revealed no evidence of codivergence betweenP. mandapamensisand its host fishes, indicating that coevolution apparently is not the basis for this bacterium's host preferences. These results, which are the first report of bacterial cosymbiosis in fish light organs and the first demonstration thatP. leiognathiis not the exclusive light organ symbiont of leiognathid fishes, demonstrate that the host species ranges ofP. mandapamensisandP. leiognathiare substantially distinct. The host range difference underscores possible differences in the environmental distributions and physiologies of these two bacterial species.
Effect of Bovine Manure on Fecal Coliform Attachment to Soil and Soil Particles of Different Sizes
Authors: Guber Andrey K. , Pachepsky Yakov A. , Shelton Daniel R. , Yu Olivia
Published: March 2007

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.
Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in
pyr4
Mutants of
Ustilago maydis
Allows Target Validation of DHODH Inhibitors In Vivo
Authors: Zameitat Elke , Freymark Gerald , Dietz Cornelia D. , Löffler Monika , Bölker Michael
Published: March 2007

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the
pyr4
gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen
Ustilago maydis
. We were able to show that
pyr4
contains a functional mitochondrial targeting signal. The deletion of
pyr4
resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified
U. maydis
DHODH overproduced in
Escherichia coli
revealed that the
U. maydis
enzyme uses quinone electron acceptor Q
6
and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the
U. maydis
mitochondrial targeting signal is able to complement the auxotrophic phenotype of
pyr4
mutants. While
U. maydis
wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered
U. maydis
strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.
Global Transcriptional Response of
Nitrosomonas europaea
to Chloroform and Chloromethane
Authors: Gvakharia Barbara O. , Permina Elizabeth A. , Gelfand Mikhail S. , Bottomley Peter J. , Sayavedra-Soto Luis A. , Arp Daniel J.
Published: March 2007

Upon exposure of
Nitrosomonas europaea
to chloroform (7 μM, 1 h), transcripts for 175 of 2,460 genes were found at higher levels in treated cells than in untreated cells and transcripts for 501 genes were found at lower levels. With chloromethane (3.2 mM, 1 h), transcripts for 67 genes were at higher levels and transcripts for 148 genes were at lower levels. Transcripts for 37 genes were at higher levels following both treatments and included genes for heat shock proteins, σ-factors of the extracytoplasmic function subfamily, and toxin-antitoxin loci.
N. europaea
has higher levels of transcripts for a variety of defense genes when exposed to chloroform or chloromethane.
Elucidation of theipso-Substitution Mechanism for Side-Chain Cleavage of α-Quaternary 4-Nonylphenols and 4-t-Butoxyphenol inSphingobium xenophagumBayram
Authors: Gabriel Frédéric L. P. , Cyris Maike , Jonkers Niels , Giger Walter , Guenther Klaus , Kohler Hans-Peter E.
Published: March 2007

Recently we showed that degradation of several nonylphenol isomers with α-quaternary carbon atoms is initiated byipso-hydroxylation inSphingobium xenophagumBayram (F. L. P. Gabriel, A. Heidlberger, D. Rentsch, W. Giger, K. Guenther, and H.-P. E. Kohler, J. Biol. Chem. 280:15526-15533, 2005). Here, we demonstrate with18O-labeling experiments that theipso-hydroxy group was derived from molecular oxygen and that, in the major pathway for cleavage of the alkyl moiety, the resulting nonanol metabolite contained an oxygen atom originating from water and not from theipso-hydroxy group, as was previously assumed. Our results clearly show that the alkyl cation derived from the α-quaternary nonylphenol 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol throughipso-hydroxylation and subsequent dissociation of the 4-alkyl-4-hydroxy-cyclohexadienone intermediate preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the metabolism of certain α,α-dimethyl-substituted nonylphenols appears to also involve a reaction of the cation with theipso-hydroxy group to form the corresponding 4-alkoxyphenols. Growth, oxygen uptake, and18O-labeling experiments clearly indicate that strain Bayram metabolized 4-t-butoxyphenol byipso-hydroxylation to a hemiketal followed by spontaneous dissociation to the corresponding alcohol andp-quinone. Hydroquinone effected high oxygen uptake in assays with induced resting cells as well as in assays with cell extracts. This further corroborates the role of hydroquinone as the ring cleavage intermediate during degradation of 4-nonylphenols and 4-alkoxyphenols.
Temporal Expression Analysis of the
Borrelia burgdorferi
Paralogous Gene Family 54 Genes BBA64, BBA65, and BBA66 during Persistent Infection in Mice
Authors: Gilmore Robert D. , Howison Rebekah R. , Schmit Virginia L. , Nowalk Andrew J. , Clifton Dawn R. , Nolder Christi , Hughes Jessica L. , Carroll James A.
Published: March 2007

Members of the
Borrelia burgdorferi
paralogous gene family 54 (pgf 54) are regulated by conditions simulating mammalian infection and are thought to be instrumental in borrelial host survival and pathogenesis. To explore the activities of these genes in vivo, a comprehensive analysis of pgf 54 genes BBA64, BBA65, and BBA66 was performed to assess the genetic stability, host antibody responses, and kinetics of gene expression in the murine model of persistent infection. DNA sequencing of pgf 54 genes obtained from reisolates at 1 year postinfection demonstrated that all genes of this family are stable and do not undergo recombination to generate variant antigens during persistent infection. Antibodies against BBA64 and BBA66 appeared soon after infection and were detectable throughout the infection, suggesting that there was gene expression during infection. However, quantitative reverse transcription-PCR revealed that BBA64 gene expression was considerably decreased in
Borrelia
residing in the mouse ear tissue compared to the expression in cultured spirochetes by 20 days postinfection and that the levels of expression remained low throughout the infection. Conversely, transcription of the BBA65 and BBA66 genes was increased, and both of these genes were continuously expressed until 100 days postinfection; this was followed by periods of differential expression late in infection. The expression profile of the BBA64 gene suggests that this gene has an important role during tick-to-host transmission and early infection, whereas the expression profile of the BBA65 and BBA66 genes suggests that these genes have a role in persistent infection. The differential regulation of pgf 54 genes observed during infection may help confer a survival advantage during persistent infection, influencing mechanisms for
B. burgdorferi
dissemination, tissue tropism, or evasion of the adaptive immune response.