bluuebunny/crossref_metadata_2025 huggingface dataset:
Numerical Analysis and Experimental Investigation of a Common Rail-Type Diesel Injector
Authors: Coppo Marco , Dongiovanni Claudio , Negri Claudio
Published: November 2004

A production common rail-type injector has been investigated via numerical simulation and experimentation. The functioning principle of the injector has been carefully analyzed so as to obtain a mathematical model of the device. A zero-dimensional approach has been used for modeling the injector, thus considering the variables as function of time only. The analysis of the hydraulic part of the injector resulted in the definition of an equivalent hydraulic scheme, on which basis both the equations of continuity in chambers and flow through nozzles were written. The connecting pipe between common rail and injector, as well as the injector internal line, were modeled according to a one-dimensional approach. The moving mechanical components of the injector, such as needle, pressure rod, and control valve have been modeled using the mass-spring-damper scheme, thus obtaining the equation governing their motion. An electromagnetic model of the control valve solenoid has also been realized, in order to work out the attraction force on the anchor, generated by the electric current when flowing into its coil. The model obtained has been implemented using the MATLAB® toolbox SIMULINK®; the ordinary differential equations were solved by means of an implicit scheme of the second-order accuracy, suitable for problems with high level of stiffness, while the partial differential equations were integrated using the finite-difference Lax-Friedrichs method. The experimental investigation on the common-rail injection system was performed on a test bench at some standard test conditions. Electric current flowing through the injector coil, oil pressure in the common rail and at the injector inlet, injection rate, needle lift, and control valve lift were gauged and recorded during several injection phases. The mean reflux-flow rate and the mean quantity of fuel injected per stroke were also measured. Temperature and pressure of the feeding oil as well as pressure in the rail were continuously controlled during the experimental test. The numerical and experimental results were compared. Afterwards, the model was used to investigate the effect of control volume feeding and discharge holes and of their inlet fillet, as well as the effect of the control volume capacity, on the injector performance.
TEM-121, a Novel Complex Mutant of TEM-Type β-Lactamase from
Enterobacter aerogenes
Authors: Poirel Laurent , Mammeri Hedi , Nordmann Patrice
Published: November 2004

Enterobacter aerogenes
clinical isolate LOR was resistant to penicillins and ceftazidime but susceptible to cefuroxime, cephalothin, cefoxitin, cefotaxime, ceftriaxone, and cefepime. PCR and cloning experiments from this strain identified a novel TEM-type β-lactamase (TEM-121) differing by five amino acid substitutions from β-lactamase TEM-2 (Glu104Lys, Arg164Ser, Ala237Thr, Glu240Lys, and Arg244Ser) and by only one amino acid change from the extended-spectrum β-lactamase (ESBL) TEM-24 (Arg244Ser), with the last substitution also being identified in the inhibitor-resistant β-lactamase IRT-2. Kinetic parameters indicated that TEM-121 hydrolyzed ceftazidime and aztreonam (like TEM-24) and was inhibited weakly by clavulanic acid and strongly by tazobactam. Thus, TEM-121 is a novel complex mutant TEM β-lactamase (CMT-4) combining the kinetic properties of an ESBL and an inhibitor-resistant TEM enzyme.
In Vitro and In Vivo Activities of Novel 6-Methylidene Penems as β-Lactamase Inhibitors
Authors: Weiss William J. , Petersen Peter J. , Murphy Timothy M. , Tardio LuAnna , Yang Youjun , Bradford Patricia A. , Venkatesan Aranapakam M. , Abe Takao , Isoda Takeshi , Mihira Ado , Ushirogochi Hideki , Takasake Tsuyoshi , Projan Steve , O'Connell John , Mansour Tarek S.
Published: November 2004

Novel penem molecules with heterocycle substitutions at the 6 position via a methylidene linkage were investigated for their activities and efficacy as β-lactamase inhibitors. The concentrations of these molecules that resulted in 50% inhibition of enzyme activity were 0.4 to 3.1 nM for the TEM-1 enzyme, 7.8 to 72 nM for Imi-1, 1.5 to 4.8 nM for AmpC, and 14 to 260 nM for a CcrA metalloenzyme. All the inhibitors were more stable than imipenem against hydrolysis by hog and human dehydropeptidases. Piperacillin was combined with a constant 4-μg/ml concentration of each inhibitor for MIC determinations. The combinations reduced piperacillin MICs by 2- to 32-fold for extended-spectrum β-lactamase (ESBL)-producingEscherichia coliandKlebsiella pneumoniaestrains. The MICs for piperacillin-resistant (MIC of piperacillin, >64 μg/ml) strains ofEnterobacterspp.,Citrobacterspp., andSerratiaspp. were reduced to the level of susceptibility (MIC of piperacillin, ≤16 μg/ml) when the drug was combined with 4, 2, or 1 μg of these penem inhibitors/ml. Protection against acute lethal bacterial infections with class A and C β-lactamase- and ESBL-producing organisms in mice was also demonstrated with piperacillin plus inhibitor. Median effective doses were reduced by approximately two- to eightfold compared to those of piperacillin alone when the drug was combined with the various inhibitors at a 4:1 ratio. Pharmacokinetic analysis after intravenous administration of the various inhibitors showed mean residence times of 0.1 to 0.5 h, clearance rates of 15 to 81 ml/min/kg, and volumes of distribution between 0.4 and 2.5 liters/kg. The novel methylidene penem molecules inhibit both class A and class C enzymes and warrant further investigation for potential as therapeutic agents when used in combination with a β-lactam antibiotic.
Pharmacokinetics and Pharmacodynamics of Intravenous Levofloxacin at 750 Milligrams and Various Doses of Metronidazole in Healthy Adult Subjects
Authors: Sprandel Kelly A. , Schriever Christopher A. , Pendland Susan L. , Quinn John P. , Gotfried Mark H. , Hackett Suzanne , Graham Mary Beth , Danziger Larry H. , Rodvold Keith A.
Published: November 2004

The purpose of this investigation was to evaluate the steady-state pharmacokinetics, pharmacodynamics, and safety of intravenous levofloxacin at 750 mg administered once daily combined with three different dosages of intravenous metronidazole (500 mg every 8 h [q8h], 1,000 mg q24h, and 1,500 mg q24h). Eighteen healthy adult subjects received all three combinations in a randomized, crossover fashion. Serial blood and urine samples were collected on the third day of each study period. The 24-h areas under the inhibitory (AUIC
0-24
) and bactericidal (AUBC
0-24
) curves of these three combination regimens were determined against clinical isolates of
Bacteroides fragilis
,
Bacteroides thetaiotaomicron
,
Peptostreptococcus asaccharolyticus
, and
Escherichia coli
. The mean concentrations of levofloxacin were not different between study periods and were similar to those previously published. The mean (± standard deviation) areas under the metronidazole plasma concentration-time curve (AUC
0-24
) for 1,500-mg q24h (338 ± 105 mg · h/liter) and 500-mg q8h (356 ± 68 mg · h/liter) regimens were not different (
P
> 0.05), but both were significantly higher than the 1,000-mg q24h AUC
0-24
(
P
< 0.05, 227 ± 57 mg · h/liter). Mean (± standard deviation) total body clearance and renal clearance values were similar among the 500-mg q8h, 1,000-mg q24, and 1,500-mg q24h regimens (62 ± 7, 67 ± 13, and 67 ± 14 and 11 ± 3, 12 ± 2, and 12 ± 5 ml/min/1.73 m
2
, respectively). Levofloxacin at 750 mg q24h plus metronidazole at 500 mg q8h or 1,500 mg q24h resulted in similar AUIC
0-24
and AUBC
0-24
values with one exception: the AUIC
0-24
for the 1,500-mg q24h regimen against
B. thetaiotamicron
was significantly higher (
P
< 0.05) than those of the other regimens. Overall, the combination of levofloxacin at 750 mg once daily and metronidazole at 500 mg q8h or 1,500 mg q24h appeared to have greater AUIC
0-24
and AUBC
0-24
values than did the 1,000-mg q24h regimen. All combination regimens of levofloxacin and metronidazole were well tolerated, and no serious drug-related adverse effects were reported. The pharmacokinetic, safety, and pharmacodynamic data from our study suggest that a once-daily regimen of intravenous levofloxacin at 750 mg and metronidazole at 1,500 mg warrants further clinical investigation.
National Surveillance of Antimicrobial Resistance in
Pseudomonas aeruginosa
Isolates Obtained from Intensive Care Unit Patients from 1993 to 2002
Authors: Obritsch Marilee D. , Fish Douglas N. , MacLaren Robert , Jung Rose
Published: November 2004

Nosocomial infections caused by
Pseudomonas aeruginosa
in critically ill patients are often difficult to treat due to resistance to multiple antimicrobials. The purpose of this study was to evaluate antimicrobial resistance among
P. aeruginosa
isolates from intensive care unit patients in the United States from 1993 to 2002 by using the Intensive Care Unit Surveillance Study database. Over the 10-year period, susceptibility of 13,999 nonduplicate isolates of
P. aeruginosa
was analyzed. From 1993 to 2002, nationwide increases in antimicrobial resistance were greatest for ciprofloxacin, imipenem, tobramycin, and aztreonam. Rates of multidrug resistance (resistance to ≥3 of the following drugs: ceftazidime, ciprofloxacin, tobramycin, and imipenem) increased from 4% in 1993 to 14% in 2002. The lowest dual resistance rates were observed between aminoglycosides or fluoroquinolones with piperacillin-tazobactam while the highest were for those that included β-lactams and ciprofloxacin. Ongoing surveillance studies are crucial in monitoring antimicrobial susceptibility patterns and selecting empirical treatment regimens.
Metabolism of the Anti-Hepatitis C Virus Nucleoside β-
d
-
N
4
-Hydroxycytidine in Different Liver Cells
Authors: Hernandez-Santiago Brenda I. , Beltran Thierry , Stuyver Lieven , Chu Chung K. , Schinazi Raymond F.
Published: November 2004

β-
d
-
N
4
-Hydroxycytidine (NHC) was found to have selective anti-hepatitis C virus (HCV) activity in the HCV replicon system (clone A). The intracellular metabolism of tritiated NHC was investigated in the HCV replicon system, Huh-7 cells, HepG2 cells, and primary human hepatocytes. Incubation of cells with 10 μM radiolabeled NHC demonstrated extensive and rapid phosphorylation in all liver cells. Besides the 5′-mono, -di-, and -triphosphate metabolites of NHC, other metabolites were characterized. These included cytidine and uridine mono-, di-, and triphosphates. UTP was the predominant early metabolite in Huh-7 cells and primary human hepatocytes, suggesting deamination of NHC as the primary catabolic pathway. The intracellular half-lives of radiolabeled NHC-triphosphate and of CTP and UTP derived from NHC incubation in Huh-7 cells were calculated to be 3.0 ± 1.3, 10.4 ± 3.3, and 13.2 ± 3.5 h (means ± standard deviations), respectively. Studies using monkey and human whole blood demonstrated more-rapid deamination and oxidation in monkey cells than in human cells, suggesting that NHC may not persist long enough in plasma to be delivered to liver cells.
A Radio Frequency Electric Current Enhances Antibiotic Efficacy against Bacterial Biofilms
Authors: Caubet R. , Pedarros-Caubet F. , Chu M. , Freye E. , de Belém Rodrigues M. , Moreau J. M. , Ellison W. J.
Published: November 2004

Bacterial biofilms are notably resistant to antibiotic prophylaxis. The concentration of antibiotic necessary to significantly reduce the number of bacteria in the biofilm matrix can be several hundred times the MIC for the same bacteria in a planktonic phase. It has been observed that the addition of a weak continuous direct electric current to the liquid surrounding the biofilm can dramatically increase the efficacy of the antibiotic. This phenomenon, known as the bioelectric effect, has only been partially elucidated, and it is not certain that the electrical parameters are optimal. We confirm here the bioelectric effect for
Escherichia coli
biofilms treated with gentamicin and with oxytetracycline, and we report a new bioelectric effect with a radio frequency alternating electric current (10 MHz) instead of the usual direct current. None of the proposed explanations (transport of ions within the biofilm, production of additional biocides by electrolysis, etc.) of the direct current bioelectric effect are applicable to the radio frequency bioelectric effect. We suggest that this new phenomenon may be due to a specific action of the radio frequency electromagnetic field upon the polar parts of the molecules forming the biofilm matrix.
Impact of High-InoculumStaphylococcus aureuson the Activities of Nafcillin, Vancomycin, Linezolid, and Daptomycin, Alone and in Combination with Gentamicin, in an In Vitro Pharmacodynamic Model
Authors: LaPlante Kerry L. , Rybak Michael J.
Published: November 2004

We evaluated the impact of high (9.5 log10CFU/g) and moderate (5.5 log10CFU/g) inocula of methicillin-susceptible and -resistant Staphylococcus aureus(MSSA and MRSA, respectively) on the activities of nafcillin, linezolid, vancomycin, and daptomycin, alone and in combination with gentamicin in an in vitro pharmacodynamic model with simulated endocardial vegetations over 72 h. Human therapeutic dosing regimens for nafcillin, daptomycin, vancomycin, linezolid, and gentamicin were simulated. At a moderate inoculum, nafcillin (MSSA only), vancomycin, and daptomycin demonstrated equivalent and significant (P< 0.01) bactericidal (99.9% kill) activities (decreases of 3.34 ± 1.1, 3.28 ± 0.4, and 3.34 ± 0.8 log10CFU/g, respectively). Bactericidal activity was demonstrated at 4 h for nafcillin and daptomycin and at 32 h for vancomycin. Linezolid demonstrated bacteriostatic activity over the course of the study period. At a high inoculum, daptomycin exhibited bactericidal activity against both MSSA and MRSA by 24 h (decrease of 5.51 to 6.31 ± 0.10 log10CFU/g). Nafcillin (versus MSSA), vancomycin, and linezolid (MSSA and MRSA) did not achieve bactericidal activity throughout the 72-h experiment. The addition of gentamicin increased the rate of 99.9% kill to 8 h for daptomycin (P< 0.01) and 48 h for nafcillin (MSSA only) (P= 0.01). The addition of gentamicin did not improve the activity of vancomycin or linezolid for either isolate for the 72-h period. Overall, high-inoculumStaphylococcus aureushad a significant impact on the activities of nafcillin and vancomycin. In contrast, daptomycin was affected minimally and linezolid was not affected by inoculum.
n-6 Polyunsaturated Fatty Acids Enhance the Activities of Ceftazidime and Amikacin in Experimental Sepsis Caused by Multidrug-Resistant
Pseudomonas aeruginosa
Authors: Giamarellos-Bourboulis Evangelos J. , Mouktaroudi Maria , Adamis Theodoros , Koussoulas Vassilios , Baziaka Fotini , Perrea Despina , Karayannacos Panayotis E. , Giamarellou Helen
Published: November 2004

Recent in vitro and ex vivo studies disclosed an enhancement of the activity of antimicrobials on multidrug-resistant
Pseudomonas aeruginosa
by n-6 polyunsaturated fatty acids (PUFAS); therefore their effect was evaluated in experimental sepsis in 60 rabbits. Solutions of gamma-linolenic acid (GLA) and arachidonic acid (AA) were administered intravenously with ceftazidime and amikacin in rabbits with sepsis caused by one multidrug-resistant isolate. Therapy was started after bacterial challenge in five groups comprising 12 animals in each group: A, normal saline; B, antimicrobials; C, 99% ethanol and antimicrobials; D, GLA and antimicrobials; and E, AA and antimicrobials. Blood was sampled for the estimation of levels of endotoxins in serum (lipopolysaccharide), leukocytes, tumor necrosis factor alpha (TNF-α) and antimicrobials. Animals were sacrificed 210 min after bacterial challenge for tissue cultures. All animals had considerable endotoxemia and evolved leukopenia. The number of viable cells in blood, lung, and mesenteric lymph nodes was significantly reduced in groups D and E compared to that in other groups. Levels of antimicrobials in serum were inadequate to achieve bacterial killing due to the level of resistance. n-6 PUFAs did not influence TNF-α. It is concluded that intravenous coadministration of n-6 PUFAs and antimicrobials enhanced antimicrobial bacterial killing in experimental sepsis caused by multidrug-resistant
P. aeruginosa
.
Evolution of Ciprofloxacin-Resistant
Staphylococcus aureus
in In Vitro Pharmacokinetic Environments
Authors: Campion Jeffrey J. , McNamara Patrick J. , Evans Martin E.
Published: November 2004

The development of novel antibacterial agents is decreasing despite increasing resistance to presently available agents among common pathogens. Insights into relationships between pharmacodynamics and resistance may provide ways to optimize the use of existing agents. The evolution of resistance was examined in two ciprofloxacin-susceptible
Staphylococcus aureus
strains exposed to in vitro-simulated clinical and experimental ciprofloxacin pharmacokinetic profiles for 96 h. As the average steady-state concentration (
C
avg ss
) increased, the rate of killing approached a maximum, and the rate of regrowth decreased. The enrichment of subpopulations with mutations in
grlA
and low-level ciprofloxacin resistance also varied depending on the pharmacokinetic environment. A regimen producing values for
C
avg ss
slightly above the MIC selected resistant variants with
grlA
mutations that did not evolve to higher levels of resistance. Clinical regimens which provided values for
C
avg ss
intermediate to the MIC and mutant prevention concentration (MPC) resulted in the emergence of subpopulations with
gyrA
mutations and higher levels of resistance. A regimen producing values for
C
avg ss
close to the MPC selected
grlA
mutants, but the appearance of subpopulations with higher levels of resistance was diminished. A regimen designed to maintain ciprofloxacin concentrations entirely above the MPC appeared to eradicate low-level resistant variants in the inoculum and prevent the emergence of higher levels of resistance. There was no relationship between the time that ciprofloxacin concentrations remained between the MIC and the MPC and the degree of resistance or the presence or type of ciprofloxacin-resistance mutations that appeared in
grlA
or
gyrA
. Regimens designed to eradicate low-level resistant variants in
S. aureus
populations may prevent the emergence of higher levels of fluoroquinolone resistance.
Reduced Content of Lysyl-Phosphatidylglycerol in the Cytoplasmic Membrane Affects Susceptibility to Moenomycin, as Well as Vancomycin, Gentamicin, and Antimicrobial Peptides, in
Staphylococcus aureus
Authors: Nishi Hiromi , Komatsuzawa Hitoshi , Fujiwara Tamaki , McCallum Nadine , Sugai Motoyuki
Published: November 2004

An association between moenomycin resistance and vancomycin intermediate resistance in
Staphylococcus aureus
was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn
551
insertional mutagenesis of methicillin-resistant
S. aureus
strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn
551
insertions in the
fmtC
(
mprF
) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn
551
insertion was located in the
lysC
gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as β-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of
S. aureus
appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.
Evaluation of Antibiotic Susceptibilities of
Ehrlichia canis
,
Ehrlichia chaffeensis
, and
Anaplasma phagocytophilum
by Real-Time PCR
Authors: Branger S. , Rolain J. M. , Raoult D.
Published: November 2004

We determined MICs of antibiotics against
Anaplasma phagocytophilum
,
Ehrlichia chaffeensis
, and
Ehrlichia canis
by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for
E. chaffeensis
, 26 h for
A. phagocytophilum
, and 28 h for
E. canis.
In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones;
A. phagocytophilum
was susceptible, but
E. canis
and
E. chaffeensis
were only partly susceptible. β-Lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (
Escherichia coli
numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.
Atovaquone Maintenance Therapy Prevents Reactivation of Toxoplasmic Encephalitis in a Murine Model of Reactivated Toxoplasmosis
Authors: Dunay Ildiko R. , Heimesaat Markus M. , Bushrab Faris Nadiem , Müller Rainer H. , Stocker Hartmut , Arasteh Keikawus , Kurowski Michael , Fitzner Rudolf , Borner Klaus , Liesenfeld Oliver
Published: November 2004

Acute therapy with pyrimethamine plus sulfadiazine is the treatment of choice for reactivated toxoplasmic encephalitis (TE). Acute therapy is followed by lifelong maintenance therapy (secondary prophylaxis) with the same drugs at lower dosages. The use of pyrimethamine plus sulfadiazine is hampered by severe side effects including allergic reactions and hematotoxicity. Alternative treatment regimens with pyrimethamine plus clindamycin or other antiparasitic drugs are less efficacious. Atovaquone nanosuspensions show excellent therapeutic effects for “acute” intravenous (i.v.) treatment of reactivated TE in a murine model. In the present study, the therapeutic efficacy of atovaquone for oral “maintenance” therapy was investigated. Mice with a targeted mutation in the interferon regulatory factor 8 gene were latently infected with
Toxoplasma gondii
, developed reactivated TE, and received acute i.v. therapy with atovaquone nanosuspensions. Mice were then treated orally with atovaquone suspension or other antiparasitic drugs to prevent relapse of TE. Maintenance therapy with atovaquone at daily doses of 50 or 100 mg/kg (body weight) protected mice against reactivated TE and death. This maintenance treatment was superior to standard therapy with pyrimethamine plus sulfadiazine. The latter combination was superior to the combination of pyrimethamine plus clindamycin. Inflammatory changes in the brain parenchyma and meninges, as well as parasite numbers, in the brains of mice confirmed the therapeutic efficacy of atovaquone for maintenance therapy. Atovaquone was detectable in sera, brains, livers, and lungs of infected mice by high-performance liquid chromatography and/or mass spectrometry. In conclusion, atovaquone appears to be superior to the standard maintenance therapy regimens in a murine model of reactivated TE. The therapeutic efficacy of atovaquone for maintenance therapy against TE should be further investigated in clinical trials.
β-Lactamase Gene Expression in a Penicillin-Resistant
Bacillus anthracis
Strain
Authors: Chen Yahua , Tenover Fred C. , Koehler Theresa M.
Published: November 2004

Expression of the
bla1
and
bla2
genes in an archetypal
Bacillus anthracis
strain is insufficient for penicillin resistance. In a penicillin-resistant clinical isolate, both genes are highly transcribed, but
bla1
is the major contributor to high-level resistance to ampicillin. Differential expression of the
bla
genes is dependent upon strain background.
VanE-Type Vancomycin-Resistant
Enterococcus faecalis
Clinical Isolates from Australia
Authors: Abadía-Patiño Lorena , Christiansen Keryn , Bell Jan , Courvalin Patrice , Périchon Bruno
Published: November 2004

Three distinct
Enterococcus faecalis
VanE-type isolates—BM4574, BM4575, and BM4576-obtained in Australia were studied. Expression of the resistance genes was constitutive in BM4575, probably due to a 2-bp deletion into the

vanS
E

gene, and inducible in BM4574 and BM4576. Transcription analysis of the
vanE
operons suggested that the five genes were cotranscribed from an initiation site located 25 bp upstream from the ATG start codon of
vanE
.
Need for Early Antifungal Treatment Confirmed in Experimental Disseminated
Candida albicans
Infection
Authors: MacCallum Donna M. , Odds Frank C.
Published: November 2004

Groups of mice infected intravenously with
Candida albicans
were treated intraperitoneally with amphotericin B, caspofungin, or fluconazole, starting at intervals before and after challenge. Survival was longest and tissue burdens were most reduced with early treatment, and survival times fell proportionately as treatment was delayed, reinforcing clinical recommendations for the earliest possible initiation of antifungal therapy.
Identification of Severe Acute Respiratory Syndrome Coronavirus Replicase Products and Characterization of Papain-Like Protease Activity
Authors: Harcourt Brian H. , Jukneliene Dalia , Kanjanahaluethai Amornrat , Bechill John , Severson Kari M. , Smith Catherine M. , Rota Paul A. , Baker Susan C.
Published: November 2004

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe
trans
-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione
S
-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a ≈300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro
trans
-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in
trans
at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.
Dissection of the Kaposi's Sarcoma-Associated Herpesvirus Gene Expression Program by Using the Viral DNA Replication Inhibitor Cidofovir
Authors: Lu Michael , Suen Jacqueline , Frias Carolina , Pfeiffer Ruth , Tsai Mong-Hsun , Chuang Eric , Zeichner Steven L.
Published: November 2004

Treatment of primary effusion lymphoma cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8 [HHV-8]) with agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a lytic viral replication cycle, with an ordered gene expression program. Initial studies of the KSHV expression program following TPA induction using viral microarrays yielded useful information concerning the viral expression program, but precise kinetic assignments for some genes remained unclear. Classically, late herpesvirus genes require viral DNA replication for maximal expression. We used cidofovir (CDV), a nucleotide-analogue KSHV DNA polymerase inhibitor, to dissect KSHV expression into two components: genes expressed without viral DNA replication and those requiring it. The expression of known immediate-early or early genes (e.g., open reading frames [ORFs] 50, K8 bZIP, and 57) serving lytic regulatory roles was relatively unaffected by the presence of CDV, while known late capsid and tegument structural genes (e.g., ORFs 25, 26, 64, and 67) were CDV sensitive. Latency-associated transcript ORF 73 was unaffected by the presence of TPA or CDV, suggesting that it was constitutively expressed. Expression of several viral cellular gene homologs, including K2 (vIL-6), ORF 72 (vCyclin), ORF 74 (vGPCR), and K9 (vIRF-1), was unaffected by the presence of CDV, while that of others, such as K4.1 (vMIP-III), K11.1 (vIRF-2), and K10.5 (LANA2, vIRF-3), was inhibited. The results distinguish KSHV genes whose full expression required viral DNA replication from those that did not require it, providing additional insights into KSHV replication and pathogenesis strategies and helping to show which viral cell homologs are expressed at particular times during the lytic process.
Accelerated Prion Disease in the Absence of Interleukin-10
Authors: Thackray Alana M. , McKenzie Andrew N. , Klein Michael A. , Lauder Angus , Bujdoso Raymond
Published: November 2004

The identity of pro- and anti-inflammatory cytokines in the neuropathogenesis of prion diseases remains undefined. Here we have investigated the role of anti-inflammatory cytokines on the progression of prion disease through the use of mice that lack interleukin-4 (IL-4), IL-10, IL-13, or both IL-4 and IL-13. Collectively our data show that among these anti-inflammatory cytokines, IL-10 plays a prominent role in the regulation of prion disease. Mice deficient in IL-10 are highly susceptible to the development of prion disease and show a markedly shortened incubation time. In addition, we have correlated cytokine gene expression in prion-inoculated IL-10−/−mice to wild-type-inoculated animals. Our experiments show that in the absence of IL-10 there is an early expression of tumor necrosis factor alpha (TNF-α). In wild-type prion-inoculated mice, the expression of TNF-α mRNA occurs at a later time point that correlates with the extended incubation time for terminal disease development in these animals compared to those that lack IL-10. Elevated levels of IL-13 mRNA are found at early time points in the central nervous system of prion-inoculated IL-10−/−mice. At terminal disease, the brains of wild-type mice inoculated with RML or ME7 are characterized by elevated levels of mRNA for the proinflammatory cytokines TNF-α and IL-1β, together with the anti-inflammatory cytokines IL-10, IL-13, and transforming growth factor beta. Our data are consistent with a role for proinflammatory cytokines in the initiation of pathology during prion disease and an attempt by anti-inflammatory cytokines to regulate the ensuing, invariably fatal pathology.
Genome Assembly and Particle Maturation of the Birnavirus Infectious Pancreatic Necrosis Virus
Authors: Villanueva Rodrigo A. , Galaz José L. , Valdés Juan A. , Jashés Matilde M. , Sandino Ana María
Published: November 2004

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.
Overlapping CRE and E Box Motifs in the Enhancer Sequences of the Bovine Leukemia Virus 5′ Long Terminal Repeat Are Critical for Basal and Acetylation-Dependent Transcriptional Activity of the Viral Promoter: Implications for Viral Latency
Authors: Calomme Claire , Dekoninck Ann , Nizet Séverine , Adam Emmanuelle , Nguyên Thi Liên-Anh , Van Den Broeke Anne , Willems Luc , Kettmann Richard , Burny Arsène , Van Lint Carine
Published: November 2004

Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5′ long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known
cis
-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response.
Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells
Authors: Vana Marcy L. , Tang Yi , Chen Aiping , Medina Gisselle , Carter Carol , Leis Jonathan
Published: November 2004

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA
98:
11199-11204, 2001). We have now identified the complete 3′ end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.
Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase
Authors: Deng Wentao , Lin Biing Yuan , Jin Ge , Wheeler Crystal G. , Ma Tianlin , Harper J. Wade , Broker Thomas R. , Chow Louise T.
Published: November 2004

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.
Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes
Authors: Crill Wayne D. , Chang Gwong-Jen J.
Published: November 2004

The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.
Human Metapneumovirus Persists in BALB/c Mice despite the Presence of Neutralizing Antibodies
Authors: Alvarez Rene , Harrod Kevin S. , Shieh Wun-Ju , Zaki Sherif , Tripp Ralph A.
Published: November 2004

Human metapneumovirus (HMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. Recent epidemiological evidence indicates that HMPV may cocirculate with respiratory syncytial virus, and HMPV infection has been associated with other respiratory diseases. In this study, we show that BALB/c mice are susceptible to HMPV infection, the virus replicates in the lungs with biphasic growth kinetics in which peak titers occur at days 7 and 14 postinfection (p.i.), and infectious HMPV can be recovered from lungs up to day 60 p.i. In addition, we show that genomic HMPV RNA can be detected in the lungs for ≥180 days p.i. by reverse transcription-PCR; however, neither HMPV RNA nor infectious virus can be detected in serum, spleen, kidneys, heart, trachea, and brain tissue. Lung histopathology revealed prevalent mononuclear cell infiltration in the interstitium beginning at day 2 p.i. and peaking at day 4 p.i. which decreased by day 14 p.i. and was associated with airway remodeling. Increased mucus production evident at day 2 p.i. was concordant with increased bronchial and bronchiolar inflammation. HMPV-specific antibodies were detected by day 14 p.i., neutralizing antibody titers reached ≥6.46 log2end-point titers by day 28 p.i., and depletion of T cells or NK cells resulted in increased HMPV titers in the lungs, suggesting some immune control of viral persistence. This study shows that BALB/c mice are amenable for HMPV studies and indicates that HMPV persists as infectious virus in the lungs of normal mice for several weeks postinfection.
Definition of Sequence Requirements for Latency-Associated Nuclear Antigen 1 Binding to Kaposi's Sarcoma-Associated Herpesvirus DNA
Authors: Srinivasan Viswanathan , Komatsu Takashi , Ballestas Mary E. , Kaye Kenneth M.
Published: November 2004

In latent infection, Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA1)-specific binding to KSHV terminal repeat DNA mediates multicopy episome persistence. We now use electrophoretic mobility shift assays to investigate LANA1 binding to its 20-bp cognate sequence. Mutations at positions 6, 7, and 8 (
6
CCC
8
) severely reduced LANA1 binding, whereas mutations at other positions only modestly reduced binding. Since
6
CCC
8
is in the 5′ half of an inverted repeat sequence, these results are consistent with an asymmetric role for the inverted repeat in LANA1 binding.
Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway
Authors: Tan Yee-Joo , Fielding Burtram C. , Goh Phuay-Yee , Shen Shuo , Tan Timothy H. P. , Lim Seng Gee , Hong Wanjin
Published: November 2004

Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these “accessory” viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.
A Period of Transient Viremia and Occult Infection Precedes Persistent Viremia and Antiviral Immune Responses during Multiple Low-Dose Intravaginal Simian Immunodeficiency Virus Inoculations
Authors: Ma Zhong-Min , Abel Kristina , Rourke Tracy , Wang Yichuan , Miller Christopher J.
Published: November 2004

In rhesus macaques, classic systemic infection, characterized by persistent viremia and seroconversion, occurred after multiple low-dose (10
3
50% tissue culture infective doses) intravaginal (IVAG) inoculations with simian immunodeficiency virus (SIV) strain SIVmac251. Monkeys developed classic SIV infections after a variable number of low-dose IVAG exposures to SIVmac251. Once established, the systemic infection was identical to SIV infection following high-dose IVAG SIV inoculation. However, occult systemic infection characterized by transient cell-associated or cell-free viremia consistently occurred early in the series of multiple vaginal SIV exposures. Further, antiviral cellular immune responses were present prior to the establishment of a classic systemic infection in the low-dose vaginal SIV transmission model.
Reductions in pre-season training loads reduce training injury rates in rugby league players
Authors: Gabbett T J
Published: November 2004

Objectives:To investigate if reductions in pre-season training loads reduced the incidence of training injuries in rugby league players, and to determine if the reductions in training loads compromised the improvements in physical fitness obtained during the pre-season preparation period.

Methods:A total of 220 sub-elite rugby league players participated in this 3 year prospective study. Players underwent measurements of speed, muscular power, and maximal aerobic power before and after three 4 month (December to March) pre-season preparation periods (2001–2003). A periodised skills and conditioning program was implemented, with training loads progressively increased in the general preparatory phase of the season (December to February) and reduced slightly in March in preparation for the competitive phase of the season. Training loads were calculated by multiplying the training session intensity by the duration of the training session. Following the initial season (2001), training loads were reduced through reductions in training duration (2002) and training intensity (2003). The incidence of injury was prospectively recorded over the three pre-season periods.

Results:The training loads for the 2002 and 2003 pre-season periods were significantly lower (p<0.001) than those in 2001. The incidence of injury was significantly higher in the 2001 pre-season than the 2002 and 2003 pre-season periods. The increases in maximal aerobic power progressively improved across the three seasons with a 62–88% probability that the 2002 and 2003 pre-season improvements in maximal aerobic power were of greater physiological significance than the 2001 pre-season improvements in maximal aerobic power.

Conclusions:These findings demonstrate that reductions in pre-season training loads reduce training injury rates in rugby league players and result in greater improvements in maximal aerobic power.