bluuebunny/crossref_metadata_2025 huggingface dataset:
Microscale Mechanical Behavior of the Subsurface by Finishing Processes
Authors: Guo Y. B. , Warren A. W.
Published: April 2005

Hard turning, grinding, and honing are common finishing processes in today’s production. The machined subsurface undergoes severe deformation and possible microstructure changes in a small scale subsurface layer <20μm. Mechanical behavior of this shallow layer is critical for component performance such as fatigue and wear. Due to the small size of this region, mechanical behavior of this shallow layer is hard to measure using traditional material testing. With the nanoindentation method, mechanical behavior (nanohardness and modulus) at the microscale in subsurface was measured for AISI 52100 and AISI 1070 steel components machined by hard turning, grinding, and honing. The test results show that white layer increases nanohardness but decreases modulus of a turned surface. Nanohardness and modulus of the ground surface are slightly smaller than the honed one in the subsurface. However, grinding produces higher nanohardness and modulus in near-surface <10μm than honing, while honing produces more uniform hardness and modulus in the near-surface and subsurface, and would improve component performance. Nanohardness and modulus of the machined near-surface are strongly influenced by strain hardening, residual stress, size-effect, and microstructure changes.
Effect of Refrigerant Flow Control on the Heating Performance of a Variable-Speed Heat Pump Operating at Low Outdoor Temperature
Authors: Kim Bong H. , O’Neal Dennis L.
Published: April 2005

An experimental study was conducted to investigate the effect of electronic flow control on the performance of a variable-speed heat pump. A heat pump with two different expansion devices (capillary tube and electronic expansion valve) was tested in a psychrometric calorimeter over a range of outdoor temperatures from −15 to 7°C. Heat pump performance was first optimized with respect to charge for each expansion device through cycle-matching tests. Parametric tests also were conducted by changing compressor speed and opening angle for the electronic expansion valve at each outdoor temperature. The refrigeration cycle characteristics of the electronic valve were illustrated using pressure-enthalpy diagrams. Performance enhancement was also analyzed in terms of superheat, heating capacity, and energy efficiency ratio (EER). Comparison of the capillary tube and electronic valve indicated that the superheat significantly improved when using the electronic valve. Also, unit showed larger heating capacity and EER with the electronic valve than with the capillary tube except when the compressor speed was above 95 Hz. Enhancement of heating performance became larger as outdoor temperature decreased.
Binderless CBN Tools, a Breakthrough for Machining Titanium Alloys
Authors: Rahmath Zareena A. , Rahman M. , Wong Y. S.
Published: April 2005

In an attempt to find an appropriate cutting tool for machining titanium alloys, performance of binderless cubic boron nitride tools is evaluated. This new cutting tool material is expected to be a breakthrough in machining titanium alloys.
Optical Durability of Candidate Solar Reflectors
Authors: Kennedy C. E. , Terwilliger K.
Published: April 2005

Concentrating solar power (CSP) technologies use large mirrors to collect sunlight to convert thermal energy to electricity. The viability of CSP systems requires the development of advanced reflector materials that are low in cost and maintain high specular reflectance for extended lifetimes under severe outdoor environments. The long-standing goals for a solar reflector are specular reflectance above 90% into a 4 mrad half-cone angle for at least 10 years outdoors with a cost of less than $13.8/m2 (the 1992 $10.8/m2 goal corrected for inflation to 2002 dollars) when manufactured in large volumes. Durability testing of a variety of candidate solar reflector materials at outdoor test sites and in laboratory accelerated weathering chambers is the main activity within the Advanced Materials task of the CSP Program at the National Renewable Energy Laboratory (NREL) in Golden, Colorado. Test results to date for several candidate solar reflector materials will be presented. These include the optical durability of thin glass, thick glass, aluminized reflectors, front-surface mirrors, and silvered polymer mirrors. The development, performance, and durability of these materials will be discussed. Based on accelerated exposure testing the glass, silvered polymer, and front-surface mirrors may meet the 10 year lifetime goals, but at this time because of significant process changes none of the commercially available solar reflectors and advanced solar reflectors have demonstrated the 10 year or more aggressive 20 year lifetime goal.
On the Uncertainty of Wind Power Predictions—Analysis of the Forecast Accuracy and Statistical Distribution of Errors
Authors: Lange Matthias
Published: April 2005

In this work the uncertainty of wind power predictions is investigated with a special focus on the important role of the nonlinear power curve. Based on numerical predictions and measured data from six onshore wind farms the overall prediction accuracy is assessed and it is shown that due to the power curve the relative forecast error increases by a factor of 1.8–2.6 compared to the wind speed forecast. This factor can be considered as an effective nonlinearity factor. A decomposition of the commonly known root mean square error is beneficially used to distinguish different error sources related to either on-site conditions or global properties of the numerical weather prediction system. The statistical distribution of the wind speed prediction error is found to be Gaussian in contrast to the the one of power prediction error. Using the power curve and conditional probability density functions of the wind speed the unsymmetric distribution of the power prediction error can be explained and modeled such that it can be estimated even if no measurement data is available.
Development of a New Model of Single-Speed Air Conditioners at Part-Load Conditions for Hourly Simulations
Authors: Mara Thierry Alex , Fock Eric , Garde Franc¸ois , Lucas Frank
Published: April 2005

This paper deals with a new model of HVAC systems for hourly simulations. The former is derived from a short time step model that is priory described. The hourly model is appraised by comparing its predictions to those of the short time step simulations. Finally, a sensitivity analysis is carried out to understand the cause of the discrepancies between the two models when it occurs. The analysis also allows to identify the factors that mainly affect the system performance.
Wheel-Tooling Gap Measurement System for Conform™ Extrusion Machinery Based on a Capacitive Sensor
Authors: Khawaja K. , Seneviratne L. , Althoefer K.
Published: April 2005

Conform™ extrusion is a very versatile manufacturing process enabling the production of a wide range of extruded profiles. It is critical to maintain a precise predefined wheel-tooling gap for the efficient running of the Conform extrusion process and to maintain high product quality. However, this is a challenging task due to the hostile environment, high operating temperatures, and required accuracy. An accurate high-temperature gap measurement system for Conform extrusion machinery, using a capacitive sensing system, is developed in this study. The sensor is implemented in a copper Conform extrusion machine, and experimental results are presented, providing for the first time a detailed view of Conform Extrusion gap behavior during production. It is shown that the proposed gap-sensing and control system results in a number of advantages, including reduced machine setup times, reduced flash (waste) rates, and on-line monitoring and control of gap size. The research is carried out in collaboration with Holton Machinery Ltd., a leading manufacturer of Conform Extrusion machinery.
Robot Path Planning for Dimensional Measurement in Automotive Manufacturing
Authors: Sheng Weihua , Xi Ning , Song Mumin , Chen Yifan
Published: April 2005

This paper addresses the robot path planning problem in our effort to develop a fully automated dimensional measurement system using an eye-in-hand robotic manipulator. First, the CAD-based vision sensor planning system developed in our lab is briefly introduced; it uses both the CAD model and the camera model to plan camera viewpoints. The planning system employs a decomposition-based approach to generate camera viewpoints that satisfy given task constraints. Second, to improve the efficiency of the eye-in-hand robot inspection system, robot path planning is studied, which is the focus of this paper. This problem is rendered as a Traveling Salesman Problem (TSP). A new hierarchical approach is developed to solve the TSP into its suboptimality. Instead of solving a large size TSP, this approach utilizes the clustering nature of the viewpoints and converts the TSP into a clustered Traveling Salesman Problem (CTSP). A new algorithm, which favors the intergroup paths, is proposed to solve the CTSP quickly. Performance of the new algorithm is analyzed. It is shown that instead of a fixed performance ratio as reported in some existing work, a constant bound can be achieved which is related to the diameter of the clusters. Experimental results demonstrate the effectiveness of the robot motion planning system. The proposed path planning approach can obtain sub-optimal solutions quickly for many large scale TSPs, which are common problems in many robotic applications.
Antileishmanial Activity of the Terpene Nerolidol
Authors: Arruda Denise C. , D'Alexandri Fabio Luiz , Katzin Alejandro M. , Uliana Silvia R. B.
Published: April 2005

The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs, was evaluated againstLeishmaniaspecies. Nerolidol inhibited the growth ofLeishmania amazonensis,L. braziliensis, andL. chagasipromastigotes andL. amazonensisamastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 μM, respectively. The treatment ofL. amazonensis-infected macrophages with 100 μM nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis, as shown by reduced incorporation of [2-14C]mevalonic acid (MVA) or [1-14C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor [1(n)-3H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol.L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However, long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified inLeishmania, as described for other trypanosomatids and Apicomplexa.
Molecular Characterization of
vanB
Elements in Naturally Occurring Gut Anaerobes
Authors: Ballard S. A. , Pertile K. K. , Lim M. , Johnson P. D. R. , Grayson M. L.
Published: April 2005

Previously, we reported the isolation of 10 vancomycin-resistant gram-positive anaerobic bacilli carrying the
vanB
ligase gene from nine hemodialysis patients (S. A. Ballard et al., Antimicrob. Agents Chemother. 49:77-81, 2005; T. P. Stinear et al., Lancet 357:855-856, 2001). In the present study, the molecular and evolutionary relationship of the
vanB
resistance element within these 10 anaerobes and two vancomycin-resistant
Enterococcus faecium
strains were examined. PCR analysis and nucleotide sequencing demonstrated that all 12 isolates carried the
vanB
operon associated with an element identical to Tn
1549
and Tn
5382
of
Enterococcus
. Restriction fragment length polymorphism analysis of the
vanB
operon in these isolates revealed two distinct patterns, and sequencing showed that minor base differences existed. PCR amplification of the joint region of a circular intermediate was demonstrated in nine of these organisms, a finding indicative of an ability to excise and circularize, an intermediate step in transposition and conjugative transfer. Southern hybridization with a
vanB
-
vanX
B
probe suggests that there is one insert of the transposon in all isolates. Sequence analysis of the integration site revealed distinct sequences: the Tn
1549/5382
element within
E. faecium
was inserted within the host chromosome, whereas nucleotide sequences surrounding the Tn
1549/5382
element in the 10 anaerobes showed no significant homology to sequences in the GenBank database. We demonstrate considerable similarity between the Tn
1549
/
5382
element identified in 10 anaerobe isolates with that found in enterococci. The homology and potential to transpose suggest a recent horizontal transfer event may have occurred. However, the original direction of transposition and the mechanism involved remains unknown.
Mixed-Lipid Storage Disorder Induced in Macrophages and Fibroblasts by Oritavancin (LY333328), a New Glycopeptide Antibiotic with Exceptional Cellular Accumulation
Authors: Van Bambeke Françoise , Saffran Jennifer , Mingeot-Leclercq Marie-Paule , Tulkens Paul M.
Published: April 2005

Oritavancin, a semisynthetic derivative of vancomycin endowed with a cationic amphiphilic character, accumulates to large extent in the lysosomes of eukaryotic cells (F. Van Bambeke, S. Carryn, C. Seral, H. Chanteux, D. Tyteca, M. P. Mingeot-Leclercq, and P. M. Tulkens, Antimicrob. Agents Chemother.
48:
2853-2860, 2004). In the present study, we examined whether this accumulation could cause cell alterations in phagocytic (J774 mouse macrophages) and nonphagocytic (rat embryo fibroblasts) cells exposed to clinically meaningful (0- to 40-mg/liter) concentrations of oritavancin. Optical and electronic microscopy evidenced conspicuous alterations of the vacuolar apparatus in both cell types, characterized by the deposition of concentric lamellar structures, finely granular material, or other less-defined osmiophilic material, often deposed in giant vesicles. Biochemical studies showed an accumulation of phospholipids (1.5× control values) and free and esterified cholesterol (3 to 4× control values for total cholesterol). Accumulation of these lipids was in close relation to that of oritavancin (excess phospholipid/oritavancin and excess cholesterol/oritavancin molar ratios of 2 to 3 and 3 to 5, respectively). Cholesterol accumulation was rapid and reversible, and that of phospholipids was slower and poorly reversible. Vancomycin and teicoplanin, used as controls (50 and 100 mg/liter, respectively), did not cause any significant change in the lipid content of fibroblasts. The data therefore suggest that oritavancin has the potential to cause a mixed-lipid storage disorder in eukaryotic cells.
Antifungal Therapy of Experimental Cerebral Phaeohyphomycosis Due to
Cladophialophora bantiana
Authors: Al-Abdely Hail M. , Najvar Laura K. , Bocanegra Rosie , Graybill John R.
Published: April 2005

Cladophialophora bantiana
is associated with central nervous system infection and a poor outcome.
C. bantiana
tends to be resistant to amphotericin B. Accordingly, we evaluated amphotericin B and three triazoles—posaconazole, itraconazole, and fluconazole—for treatment of
C. bantiana
infection in mice. In immunosuppressed ICR mice infected intravenously, posaconazole, itraconazole, and amphotericin B prolonged survival. This improvement in survival corresponded with a reduction in brain fungal concentrations for mice which were given itraconazole and posaconazole, but not amphotericin B. In nonimmunosuppressed BALB/c mice infected intracerebrally, posaconazole showed dose-dependent responses in survival and reduction of brain tissue counts. These responses were observed for short, delayed, and prolonged therapy. Although posaconazole prolonged the survival of mice with reductions in brain fungal counts, it did not sterilize brain tissue with continuous therapy for 8 weeks. We concluded that posaconazole shows promise for the treatment of
C. bantiana
brain infections.
Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations
Authors: Smith Anthony J. , Meyer Peter R. , Asthana Deshratn , Ashman Margarita R. , Scott Walter A.
Published: April 2005

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3′-azido-3′-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [32P]ddAMP-terminated DNA primer/template gave rise to32P-labeled adenosine 2′,3′-dideoxyadenosine 5′,5′′′−P1,P4-tetraphosphate (Ap4ddA), ddATP, Gp4ddA, and Ap3ddA, corresponding to the transfer of [32P]ddAMP to ATP, PPi, GTP, and ADP, respectively. Incubation with [32P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous32P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PPilevels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4+or CD8+T cells, while PPiwas present at 7 to 15 μM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4+or CD8+T cells contained 1.4 to 2.7 mM ATP and 55 to 79 μM PPi. These cellular PPiconcentrations are lower than previously reported; nonetheless, the PPi-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.
Mutations in PA3574 (
nalD
) Lead to Increased MexAB-OprM Expression and Multidrug Resistance in Laboratory and Clinical Isolates of
Pseudomonas aeruginosa
Authors: Sobel Mara L. , Hocquet Didier , Cao Lily , Plesiat Patrick , Poole Keith
Published: April 2005

Mutations in genes
mexR
and
nalC
have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in
Pseudomonas aeruginosa
. A transposon insertion multidrug-resistant mutant of
P. aeruginosa
overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in
mexR
or
nalC
were also shown to carry mutations in
nalD
. Moreover, the cloned
nalD
gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the
nalD
mutations vis-à-vis MexAB-OprM overexpression in these isolates.
Integron Content of Extended-Spectrum-β-Lactamase-Producing
Escherichia coli
Strains over 12 Years in a Single Hospital in Madrid, Spain
Authors: Machado Elisabete , Cantón Rafael , Baquero Fernando , Galán Juan-Carlos , Rollán Azucena , Peixe Luísa , Coque Teresa M.
Published: April 2005

The contribution of integrons to the dissemination of extended-spectrum β-lactamases (ESBL) was analyzed on all ESBL-producing
Escherichia coli
isolates from 1988 to 2000 at Ramón y Cajal Hospital. We studied 133
E. coli
pulsed-field gel electrophoresis types: (i) 52 ESBL-producing clinical strains (C-ESBL) (16 TEM, 9 SHV, 21 CTX-M-9, 1 CTX-M-14, and 5 CTX-M-10); (ii) 43 non-ESBL blood clinical strains (C-nESBL); and (iii) 38 non-ESBL fecal isolates from healthy volunteers (V-nESBL). Class 1 integrons were more common among C-ESBL (67%) than among C-nESBL (40%) or V-nESBL (26%) (
P
< 0.001) due to the high number of strains with
bla

CTX
-
M
-
9

, which is linked to an In6-like class 1 integron. Without this bias, class 1 integron occurrence would be similar in C-ESBL and C-nESBL groups (47% versus 40%). Occurrence of class 2 integrons was similar among clinical and community isolates (13 to 18%). No isolates contained class 3 integrons. The relatively low rate of class 1 integrons within transferable elements carrying

bla
TEM

(23%) or

bla
SHV

(33%) and the absence of class 2 integrons in all ESBL transconjugants mirror the assembly of translocative pieces containing

bla
TEM

or

bla
SHV

on local available transferable elements lacking integrons. The low diversity of class 1 integrons (seven types recovered in all groups) might indicate a wide dissemination of specific genetic elements in which they are located. In our environment, the spread of genetic elements encoding ESBL has no major impact on the dispersion of integrons, nor do integrons have a major impact on the spread of ESBL, except when

bla
ESBL

genes are within an integron platform such as
bla

CTX
-
M
-
9

.
Combination of Candidate Microbicides Cellulose Acetate 1,2-Benzenedicarboxylate and UC781 Has Synergistic and Complementary Effects against Human Immunodeficiency Virus Type 1 Infection
Authors: Liu Shuwen , Lu Hong , Neurath A. Robert , Jiang Shibo
Published: April 2005

The combination of two candidate microbicides, cellulose acetate 1,2-benzenedicarboxylate (CAP), a polymer that blocks human immunodeficiency virus type 1 (HIV-1) entry by targeting gp120 and gp41, and UC781, a tight-binding HIV-1 reverse transcriptase inhibitor (RTI), resulted in effective synergy for inhibition of MT-2 cell infection by HIV-1
IIIB
, a laboratory-adapted virus strain. The 95% effective concentration values for the combination were reduced about 15- to 20-fold compared with those corresponding to the single compounds. The combination of CAP and UC781 is also synergistic in inhibiting infection of peripheral blood mononuclear cells by a primary HIV-1 isolate, 92US657. Combinations of CAP with other RTIs, such as efavirenz or zidovudine, also had significant synergistic effects on the inhibition of HIV-1 infection. In addition, CAP and UC781 had complementary effects against HIV-1 infection since (i) CAP inhibited infection by the UC781-resistant strain HIV-1
IIIB
A17 and (ii) pretreatment of MT-2 cells with UC781, but not CAP, abolished subsequent infection after removal of the compound. This suggests that the combination of CAP and UC781 represents a promising candidate microbicide for prevention of sexual transmission of HIV-1.
A Novel MATE Family Efflux Pump Contributes to the Reduced Susceptibility of Laboratory-Derived
Staphylococcus aureus
Mutants to Tigecycline
Authors: McAleese Fionnuala , Petersen Peter , Ruzin Alexey , Dunman Paul M. , Murphy Ellen , Projan Steven J. , Bradford Patricia A.
Published: April 2005

Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in
Staphylococcus aureus
. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant
S. aureus
strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster,
mepRAB
(multidrug export protein), encoding a MarR-like transcriptional regulator (
mepR
), a novel MATE family efflux pump (
mepA
), and a hypothetical protein of unknown function (
mepB
). Sequencing of the
mepR
gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of
mepA
. Overexpression of
mepA
in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the
mepR
defect by overexpressing a wild-type
mepR
gene reduced
mepA
transcription and lowered the tigecycline MIC in the mutants. Transcription of
tet
(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the
tet
(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of
mepA
but not
tet
(M) may contribute to decreased susceptibility of tigecycline in
S. aureus
.
Evolution of TEM-Type Extended-Spectrum β-Lactamases in Clinical
Enterobacteriaceae
Strains in Poland
Authors: Baraniak Anna , Fiett Janusz , Mrówka Agnieszka , Walory Jarosław , Hryniewicz Waleria , Gniadkowski Marek
Published: April 2005

Seventeen extended-spectrum β-lactamase (ESBL)-producing isolates of the family
Enterobacteriaceae
recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these β-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different
bla
TEM-1
gene alleles were precursors of the ESBL genes:
bla
TEM-1A
, which was the ancestor of
bla
TEM-93
, and
bla
TEM-1F
, from which all the remaining genes originated. The evolution of the
bla
TEM-1F
-related genes most probably consisted of three major separate lineages, one of which, including
bla
TEM-4
,
bla
TEM-47
,
bla
TEM-48
,
bla
TEM-49
,
bla
TEM-68
, and
bla
TEM-94
, was highly structured itself and could have been initiated by the
bla
TEM-25
gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related
bla
TEM
genes, which suggested either the intense diversification or transposition of
bla
TEM
genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.
Mechanism of Action of a Novel Series of Naphthyridine-Type Ribosome Inhibitors: Enhancement of tRNA Footprinting at the Decoding Site of 16S rRNA
Authors: Shen Linus L. , Black-Schaefer Candace , Cai Yingna , Dandliker Peter J. , Beutel Bruce A.
Published: April 2005

The novel ribosome inhibitors (NRIs) are a broad-spectrum naphthyridine class that selectively inhibits bacterial protein synthesis (P. J. Dandliker et al., Antimicrob. Agents Chemother. 47:3831-3839, 2003). Footprinting experiments, using a range of NRIs and chemical modification agents on
Escherichia coli
ribosomes, revealed no evidence for direct protection of rRNA. In the presence of tRNA, however, we found that NRIs enhanced the known ribosomal footprinting pattern of tRNA in a dose-dependent manner. The most prominent increase in protection, at A1492/3 and A1413 in helix-44 of 16S RNA, strictly required the presence of tRNA and poly(U), and the effect was correlated with the potency of the inhibitor. Radioligand binding studies with inhibitor [
3
H]A-424902 showed that the compound binds to tRNA, either in its charged or uncharged form. The dissociation constant for [
3
H]A-424902 binding to Phe-tRNA
Phe
was determined to be 1.8 μM, near its translation inhibition potency of 1.6 μM in a cell-free
S. pneumoniae
extract assay. The compound did not change the binding of radiolabeled tRNA to the 30S ribosomal subunit. Taken together, these results imply that the NRIs exert their effects on protein synthesis by structurally perturbing the tRNA/30S complex at the decoding site.
Selective Intracellular Activation of a Novel Prodrug of the Human Immunodeficiency Virus Reverse Transcriptase Inhibitor Tenofovir Leads to Preferential Distribution and Accumulation in Lymphatic Tissue
Authors: Lee William A. , He Gong-Xin , Eisenberg Eugene , Cihlar Tomas , Swaminathan Swami , Mulato Andrew , Cundy Kenneth C.
Published: April 2005

An isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir (GS 7340) was prepared, and its in vitro antiviral activity, metabolism, and pharmacokinetics in dogs were determined. The 50% effective concentration (EC
50
) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 μM compared to an EC
50
of 5 μM for the parent drug, tenofovir. The (
L
)-alaninyl analog (GS 7340) was >1,000-fold more active than the (
D
)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37°C and a half-life of 28.3 min in an MT-2 cell extract at 37°C. The antiviral activity (>10× the EC
50
) and the metabolic stability in MT-2 cell extracts (>35×) and plasma (>2.5×) were also sensitive to the stereochemistry at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an intravenous dose of tenofovir was 17%. The total intracellular concentration of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 μg-eq/ml compared to 0.2 μg-eq/ml in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the commercially available prodrug tenofovir DF (Viread).
Dual Targeting of Topoisomerase IV and Gyrase To Reduce Mutant Selection: Direct Testing of the Paradigm by Using WCK-1734, a New Fluoroquinolone, and Ciprofloxacin
Authors: Strahilevitz Jacob , Hooper David C.
Published: April 2005

Quinolones that act equally against DNA gyrase and topoisomerase IV are a desirable modality to decrease the selection of resistant strains. We first determined by genetic and biochemical studies in
Staphylococcus aureus
that the primary target enzyme of WCK-1734, a new quinolone, was DNA gyrase. A single mutation in gyrase, but not topoisomerase IV, caused a two- to fourfold increase in the MIC. Studies with purified topoisomerase IV and gyrase from
S. aureus
also showed that gyrase was more sensitive than topoisomerase IV to WCK-1734 (50% inhibitory concentration, 1.25 and 2.5 to 5.0 μg/ml, respectively; 50% stimulation of cleavage complex formation, 0.62 and 2.5 to 5.0 μg/ml, respectively). To test the effect of balanced activity of quinolones against the two target enzymes, we measured the frequency of selection of mutants with ciprofloxacin (which targets topoisomerase IV) and WCK-1734 alone and in combination. With the combination of ciprofloxacin and WCK-1734, each at its MIC, the ratio of frequency of mutants selected was significantly lower than that with each drug alone at two times their respective MICs. We further characterized resistant strains selected with the combination of ciprofloxacin and WCK-1734 and found evidence to suggest the existence of novel mutational mechanisms for low-level quinolone resistance. By use of a combination of differentially targeting quinolones, this study provides novel data in direct support of the paradigm for dual targeting of quinolone action and reduced development of resistance.
Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in
Leishmania infantum
Axenic and Intracellular Amastigotes
Authors: El Fadili Karima , Messier Nadine , Leprohon Philippe , Roy Gaétan , Guimond Chantal , Trudel Nathalie , Saravia Nancy G. , Papadopoulou Barbara , Légaré Danielle , Ouellette Marc
Published: April 2005

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite
Leishmania
. We present our studies on
Leishmania infantum
amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against
Leishmania
. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA),
S
-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the
MRPA
gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.
Altered PBP 2A and Its Role in the Development of Penicillin, Cefotaxime, and Ceftriaxone Resistance in a Clinical Isolate of
Streptococcus pneumoniae
Authors: Smith Anthony M. , Feldman Charles , Massidda Orietta , McCarthy Kerrigan , Ndiweni Dalu , Klugman Keith P.
Published: April 2005

We report the unusual involvement of altered PBP 2A in the development of β-lactam resistance in
Streptococcus pneumoniae
. This was investigated amid three identical serotype 14 isolates (designated isolates 1, 2, and 3, respectively) of pneumococci cultured successfully from the blood of a human immunodeficiency virus-seropositive child with recurrent pneumonia. The passage of this strain through its human host induced several changes in the bacterium, which is typical of the adaptive and evolving nature of the pneumococcus. An efflux resistance mechanism, which conferred increased ciprofloxacin resistance, was induced in isolates 2 and 3. In addition, faster growth rates and larger capsules were also observed for these isolates, with respect to isolate 1. Notably, compared to isolates 1 and 2, isolate 3 showed a decrease in penicillin, cefotaxime, and ceftriaxone resistance. This change was associated with the replacement of an altered PBP 2A for an unaltered PBP 2A. In all likelihood, these events produced a strain which evolved into a fitter and more virulent type, isolate 3, that resulted in an aggravated pneumococcal infection and ultimately in the patient's death.
Novel Human Immunodeficiency Virus Type 1 Protease Mutations Potentially Involved in Resistance to Protease Inhibitors
Authors: Svicher Valentina , Ceccherini-Silberstein Francesca , Erba Fulvio , Santoro Maria , Gori Caterina , Bellocchi Maria Concetta , Giannella Sara , Trotta Maria Paola , Monforte Antonella d'Arminio , Antinori Andrea , Perno Carlo Federico
Published: April 2005

Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naïve patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 Å of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naïve patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.
Mechanistic Study of the Photodynamic Inactivation of
Candida albicans
by a Cationic Porphyrin
Authors: Lambrechts S. A. G. , Aalders M. C. G. , Van Marle J.
Published: April 2005

The growing resistance against antifungal agents has renewed the search for alternative treatment modalities, and antimicrobial photodynamic inactivation (PDI) is a potential candidate. The cationic porphyrin 5-phenyl-10,15,20-Tris(
N
-methyl-4-pyridyl)porphyrin chloride (TriP[4]) is a photosensitizer that in combination with light can inactivate bacteria, fungi, and viruses. For future improvement of the efficacy of PDI of clinically relevant fungi such as
Candida albicans
, we sought to understand the working mechanism by following the response of
C. albicans
exposed to PDI using fluorescence confocal microscopy and freeze-fracture electron microscopy. The following events were observed under dark conditions: TriP[4] binds to the cell envelope of
C. albicans
, and none or very little TriP[4] enters the cell. Upon illumination the cell membrane is damaged and eventually becomes permeable for TriP[4]. After lethal membrane damage, a massive influx of TriP[4] into the cell occurs. Only the vacuole membrane is resistant to PDI-induced damage once TriP[4] passes the plasma membrane. Increasing the incubation time of
C. albicans
with TriP[4] prior to illumination did not increase the influx of TriP[4] into the cell or the efficacy of PDI. After the replacement of 100% phosphate-buffered saline (PBS) by 10% PBS as the medium,
C. albicans
became permeable for TriP[4] during dark incubation and the efficacy of PDI increased dramatically. In conclusion,
C. albicans
can be successfully inactivated by the cationic porphyrin TriP[4], and the cytoplasmic membrane is the target organelle. TriP[4] influx occurred only after cell death.
Targeting Tn
5
Transposase Identifies Human Immunodeficiency Virus Type 1 Inhibitors
Authors: Ason Brandon , Knauss Daniel J. , Balke Allison M. , Merkel George , Skalka Anna Marie , Reznikoff William S.
Published: April 2005

Human immunodeficiency virus (HIV) type 1 (HIV-1) integrase is an underutilized drug target for the treatment of HIV infection. One limiting factor is the lack of costructural data for use in the rational design or modification of integrase inhibitors. Tn
5
transposase is a structurally well characterized, related protein that may serve as a useful surrogate. However, little data exist on inhibitor cross-reactivity. Here we screened 16,000 compounds using Tn
5
transposase as the target and identified 20 compounds that appear to specifically inhibit complex assembly. Six were found to also inhibit HIV-1 integrase. These compounds likely interact with a highly conserved region presumably within the catalytic core. Most promising, several cinnamoyl derivatives were found to inhibit HIV transduction in cells. The identification of integrase inhibitors from a screen using Tn
5
transposase as the target illustrates the utility of Tn
5
as a surrogate for HIV-1 integration even though the relationship between the two systems is limited to the active site architecture and catalytic mechanism.
Replication Fitness and NS5B Drug Sensitivity of Diverse Hepatitis C Virus Isolates Characterized by Using a Transient Replication Assay
Authors: Ludmerer Steven W. , Graham Donald J. , Boots Evelyn , Murray Edward M. , Simcoe Amy , Markel Eric J. , Grobler Jay A. , Flores Osvaldo A. , Olsen David B. , Hazuda Daria J. , LaFemina Robert L.
Published: April 2005

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.
Hypermutable and Fluoroquinolone-Resistant Clinical Isolates of
Staphylococcus aureus
Authors: Trong Hiep N'Guyen , Prunier Anne-Laure , Leclercq Roland
Published: April 2005

Over 124 methicillin-susceptible
Staphylococcus aureus
0/74 fluoroquinolone-susceptible versus 5/50 fluoroquinolone-resistant isolates were hypermutable. Hypermutable isolates combined mutations in
gyrA
,
parC
, and/or
parE
genes. One strain had a large deletion of the mutator
mutS
and
mutL
genes. No relevant mutation in
mutS
and
mutL
genes was found in the other isolates.
Origin and Dissemination of Chloroquine-Resistant
Plasmodium falciparum
with Mutant
pfcrt
Alleles in the Philippines
Authors: Chen Nanhua , Wilson Danny W. , Pasay Cielo , Bell David , Martin Laura B. , Kyle Dennis , Cheng Qin
Published: April 2005

The
pfcrt
allelic type and adjacent microsatellite marker type were determined for 82
Plasmodium falciparum
isolates from the Philippines. Mutant
pfcrt
allelic types P1a and P2a/P2b were dominant in different locations. Microsatellite analysis revealed that P2a/P2b evolved independently in the Philippines, while P1a shared common ancestry with Papua New Guinea chloroquine-resistant parasites.
Florfenicol Resistance Gene
floR
Is Part of a Novel Transposon
Authors: Doublet Benoît , Schwarz Stefan , Kehrenberg Corinna , Cloeckaert Axel
Published: April 2005

The florfenicol/chloramphenicol resistance gene
floR
was found to be part of the novel 4,284-bp transposon Tn
floR
from
Escherichia coli
. Tn
floR
consists of the gene
floR
, a putative regulatory gene, and the transposase gene
tnpA
. A circular form of Tn
floR
was detected and suggested the potential mobility of this transposon.
Differences in Interactions between Azole Drugs Related to Modifications in the 14-α Sterol Demethylase Gene (
cyp51A
) of
Aspergillus fumigatus
Authors: Garcia-Effron G. , Mellado E. , Gomez-Lopez A. , Alcazar-Fuoli L. , Cuenca-Estrella M. , Rodriguez-Tudela Juan L.
Published: April 2005

The combined activity of different azole drugs was investigated. Thirty-one
Aspergillus fumigatus
strains were tested, including two
cyp51A
−
and one
cyp51B
−
gene-knockout strain and azole-susceptible and -resistant strains with different resistance mechanisms. The combination of itraconazole and voriconazole was synergistic for all strains except for those with gene knockouts.
STRUCTURE FORMATION WITH MIRROR DARK MATTER: CMB AND LSS
Authors: BEREZHIANI ZURAB , CIARCELLUTI PAOLO , COMELLI DENIS , VILLANTE FRANCESCO L.
Published: April 2005

In the mirror world hypothesis, the mirror baryonic component emerges as a possible dark matter candidate. An immediate question arises: how do the mirror baryons behave and what are their differences from the more familiar dark matter candidates such as cold dark matter? In this paper, we answer this question quantitatively. First, we discuss the dependence of the relevant scales for the structure formation (Jeans and Silk scales) on the two macroscopic parameters necessary to define the model: the temperature of the mirror plasma (limited by the Big Bang Nucleosynthesis) and the amount of mirror baryonic matter. Then we perform a complete quantitative calculation of the implications of mirror dark matter on the cosmic microwave background and large scale structure power spectrum. Finally, confronting with the present observational data, we obtain some bounds on the mirror parameter space.
ON THE PHYSICAL PROPERTIES OF SPHERICALLY SYMMETRIC SELF-SIMILAR SOLUTIONS
Authors: SHARIF M. , AZIZ SEHAR
Published: April 2005

In this paper, we are exploring some of the properties of the self-similar solutions of the first kind. In particular, we shall discuss the kinematic properties and also check the singularities of these solutions. We discuss these properties both in co-moving and also in non-co-moving (only in the radial direction) coordinates. Some interesting features of these solutions turn up.
SUPERNOVA EXPLOSIONS, 511 keV PHOTONS, GAMMA RAY BURSTS AND MIRROR MATTER
Authors: FOOT R. , SILAGADZE Z. K.
Published: April 2005

There are three astroparticle physics puzzles which fire the imagination: the origin of the "Great Positron Producer" in the galactic bulge, the nature of the gamma-ray bursts central engine and the mechanism of supernova explosions. We show that the mirror matter model has the potential to solve all three of these puzzles in one beautifully simple strike.
NONLINEARLY ELASTIC THIN PLATE MODELS FOR A CLASS OF OGDEN MATERIALS: I
Authors: TRABELSI KARIM
Published: April 2005

A new two-dimensional nonlinear membrane plate theory is derived via a formal asymptotic procedure for a family of hyperelastic nonlinear materials proposed by Ciarlet and Geymonat [11], whose stored energy function is polyconvex and becomes infinite, when the determinant of the deformation gradient tends to zero, and can be adjusted to arbitrary Lamé constants.