bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex PCR for the detection ofBrucella ovis,Actinobacillus seminisandHistophilus somniin ram semen
Authors: Saunders VF , Reddacliff LA , Berg T , Hornitzky M
Published: February 2007

Objective To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection ofBrucella ovis,Actinobacillus seminis,Histophilus somniin fresh ram semen samples.

Design The multiplex assay was based on the single PCR assays published for the detection ofA seminisandB ovis, and the forward primer published for the detection ofH somni; an alternative reverse primer forH somniwas designed in this study.

Procedure Culture and PCR of 295 fresh semen samples were carried out.

Results The multiplex PCR was far more successful in the detection ofH somni(45/295) than culture (23/295).A seminiswas also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) andB oviswas detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams.

Conclusion This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detectionB ovis,H somniandA seminis.
Applications of Modern Ferroelectrics
Authors: Scott J. F.
Published: February 2007

Long viewed as a topic in classical physics, ferroelectricity can be described by a quantum mechanical ab initio theory. Thin-film nanoscale device structures integrated onto Si chips have made inroads into the semiconductor industry. Recent prototype applications include ultrafast switching, cheap room-temperature magnetic-field detectors, piezoelectric nanotubes for microfluidic systems, electrocaloric coolers for computers, phased-array radar, and three-dimensional trenched capacitors for dynamic random access memories. Terabit-per-square-inch ferroelectric arrays of lead zirconate titanate have been reported on Pt nanowire interconnects and nanorings with 5-nanometer diameters. Finally, electron emission from ferroelectrics yields cheap, high-power microwave devices and miniature x-ray and neutron sources.
Maplike Representation of Celestial
E
-Vector Orientations in the Brain of an Insect
Authors: Heinze Stanley , Homberg Uwe
Published: February 2007

For many insects, the polarization pattern of the blue sky serves as a compass cue for spatial navigation.
E
-vector orientations are detected by photoreceptors in a dorsal rim area of the eye. Polarized-light signals from both eyes are finally integrated in the central complex, a brain area consisting of two subunits, the protocerebral bridge and the central body. Here we show that a topographic representation of zenithal
E
-vector orientations underlies the columnar organization of the protocerebral bridge in a locust. The maplike arrangement is highly suited to signal head orientation under the open sky.
Archaeal Type III RuBisCOs Function in a Pathway for AMP Metabolism
Authors: Sato Takaaki , Atomi Haruyuki , Imanaka Tadayuki
Published: February 2007

The type III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) present in the archaeon
Thermococcus kodakaraensis
was found to participate in adenosine 5′-monophosphate (AMP) metabolism, a role that is distinct from that of classical RuBisCOs of the Calvin-Benson-Bassham cycle. Genes annotated as thymidine phosphorylase (
deoA
) and eucaryal translation initiation factor 2B (
e2b2
) were found to encode AMP phosphorylase and ribose-1,5-bisphosphate isomerase, respectively. These enzymes supplied the RuBisCO substrate, ribulose-1,5-bisphosphate, from AMP and phosphate. Archaea with type III RuBisCOs all harbor both DeoA and the corresponding E2b2 homologs. In this pathway, adenine was released from AMP and the phosphoribose moiety entered central-carbon metabolism.
Multiple Energy Scales at a Quantum Critical Point
Authors: Gegenwart P. , Westerkamp T. , Krellner C. , Tokiwa Y. , Paschen S. , Geibel C. , Steglich F. , Abrahams E. , Si Q.
Published: February 2007

We report thermodynamic measurements in a magnetic-field-driven quantum critical point of a heavy fermion metal, YbRh
2
Si
2
. The data provide evidence for an energy scale in the equilibrium excitation spectrum that is in addition to the one expected from the slow fluctuations of the order parameter. Both energy scales approach zero as the quantum critical point is reached, thereby providing evidence for a new class of quantum criticality.
Magmatic and Crustal Differentiation History of Granitic Rocks from Hf-O Isotopes in Zircon
Authors: Kemp A. I. S. , Hawkesworth C. J. , Foster G. L. , Paterson B. A. , Woodhead J. D. , Hergt J. M. , Gray C. M. , Whitehouse M. J.
Published: February 2007

Granitic plutonism is the principal agent of crustal differentiation, but linking granite emplacement to crust formation requires knowledge of the magmatic evolution, which is notoriously difficult to reconstruct from bulk rock compositions. We unlocked the plutonic archive through hafnium (Hf) and oxygen (O) isotope analysis of zoned zircon crystals from the classic hornblende-bearing (I-type) granites of eastern Australia. This granite type forms by the reworking of sedimentary materials by mantle-like magmas instead of by remelting ancient metamorphosed igneous rocks as widely believed. I-type magmatism thus drives the coupled growth and differentiation of continental crust.
Experimental Realization of Wheeler's Delayed-Choice Gedanken Experiment
Authors: Jacques Vincent , Wu E , Grosshans Frédéric , Treussart François , Grangier Philippe , Aspect Alain , Roch Jean-François
Published: February 2007

Wave-particle duality is strikingly illustrated by Wheeler's delayed-choice gedanken experiment, where the configuration of a two-path interferometer is chosen after a single-photon pulse has entered it: Either the interferometer is closed (that is, the two paths are recombined) and the interference is observed, or the interferometer remains open and the path followed by the photon is measured. We report an almost ideal realization of that gedanken experiment with single photons allowing unambiguous which-way measurements. The choice between open and closed configurations, made by a quantum random number generator, is relativistically separated from the entry of the photon into the interferometer.
Fracture-Controlled Paleo-Fluid Flow in Candor Chasma, Mars
Authors: Okubo Chris H. , McEwen Alfred S.
Published: February 2007

Color observations from the High Resolution Imaging Science Experiment on board the Mars Reconnaissance Orbiter reveal zones of localized fluid alteration (cementation and bleaching) along joints within layered deposits in western Candor Chasma, Mars. This fluid alteration occurred within the subsurface in the geologic past and has been exposed at the surface through subsequent erosion. These findings demonstrate that fluid flow along fractures was a mechanism by which subsurface fluids migrated through these layered deposits. Fractured layered deposits are thus promising sites for investigating the geologic history of water on Mars.
Voices from Bird Bones
Authors: Diamond Jared
Published: February 2007

Extinct Birds of New Zealand
.
By Alan Tennyson and Paul Martinson
. Te Papa Press, Wellington, New Zealand, 2006. 190 pp. NZ$65. ISBN 9780909010218.

Extinction and Biogeography of Tropical Pacific Birds
.
By David W. Steadman
. University of Chicago Press, Chicago, 2006. 608 pp. $110, £69.50. ISBN 9780226771410. Paper, $45, £28.50. ISBN 9780226771427.

Steadman uses the paleontological and archaeological records to reconstruct the prehistoric avian communities of tropical Pacific islands, chart their postsettlement fates, and discuss the theories of island biogeography. Tennyson and Martinson describe and portray 58 endemic species of birds that disappeared within a few hundred years of the settlement of New Zealand.
Polymerizing Actin Fibers Position Integrins Primed to Probe for Adhesion Sites
Authors: Galbraith Catherine G. , Yamada Kenneth M. , Galbraith James A.
Published: February 2007

Migrating cells extend protrusions, probing the surrounding matrix in search of permissive sites to form adhesions. We found that actin fibers polymerizing along the leading edge directed local protrusions and drove synchronous sideways movement of β
1
integrin adhesion receptors. These movements lead to the clustering and positioning of conformationally activated, but unligated, β
1
integrins along the leading edge of fibroblast lamellae and growth cone filopodia. Thus, rapid actin-based movement of primed integrins along the leading edge suggests a “sticky fingers” mechanism to probe for new adhesion sites and to direct migration.
The
Calyptogena magnifica
Chemoautotrophic Symbiont Genome
Authors: Newton I. L. G. , Woyke T. , Auchtung T. A. , Dilly G. F. , Dutton R. J. , Fisher M. C. , Fontanez K. M. , Lau E. , Stewart F. J. , Richardson P. M. , Barry K. W. , Saunders E. , Detter J. C. , Wu D. , Eisen J. A. , Cavanaugh C. M.
Published: February 2007

Chemoautotrophic endosymbionts are the metabolic cornerstone of hydrothermal vent communities, providing invertebrate hosts with nearly all of their nutrition. The
Calyptogena magnifica
(Bivalvia: Vesicomyidae) symbiont,
Candidatus Ruthia magnifica
, is the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced, revealing a suite of metabolic capabilities. The genome encodes major chemoautotrophic pathways as well as pathways for biosynthesis of vitamins, cofactors, and all 20 amino acids required by the clam.
The Phosphothreonine Lyase Activity of a Bacterial Type III Effector Family
Authors: Li Hongtao , Xu Hao , Zhou Yan , Zhang Jie , Long Chengzu , Li Shuqin , Chen She , Zhou Jian-Min , Shao Feng
Published: February 2007

Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the
Shigella
type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal–regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the α proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.
Serum Antibody Responses in Ethiopian Meningitis Patients Infected withNeisseria meningitidisSerogroup A Sequence Type 7
Authors: Norheim Gunnstein , Aseffa Abraham , Yassin Mohammed Ahmed , Mengistu Getahun , Kassu Afework , Fikremariam Dereje , Tamire Wegene , Merid Yared , Høiby E. Arne , Caugant Dominique A. , Fritzsønn Elisabeth , Tangen Torill , Alebel Tsegaye , Berhanu Degu , Harboe Morten , Rosenqvist Einar
Published: February 2007

To elucidate critical components of protective immune responses induced during the natural course of serogroup A meningococcal disease, we studied acute-, early-convalescent-, and late-convalescent-phase sera from Ethiopian patients during outbreaks in 2002 to 2003. Sera were obtained from laboratory-confirmed patients positive for serogroup A sequence type 7 (ST-7) meningococci (A:4/21:P1.20,9) (n= 71) and from Ethiopian controls (n= 113). The sera were analyzed using an enzyme-linked immunosorbent assay to measure levels of immunoglobulin G (IgG) against serogroup A polysaccharide (APS) and outer membrane vesicles (OMVs) and for serum bactericidal activity (SBA) using both rabbit and human complement sources. Despite relatively high SBA titers and high levels of IgG against APS and OMVs in acute-phase patient sera, significant increases were seen in the early convalescent phase. Antibody concentrations returned to acute-phase levels in the late convalescent phase. Considering all patients' sera, a significant but low correlation (r= 0.46) was observed between SBA with rabbit complement (rSBA) using an ST-5 reference strain and SBA with human complement (hSBA) using an ST-7 strain from Ethiopia. While rSBA demonstrated a significant linear relation with IgG against APS, hSBA demonstrated significant linear relationships with IgG against both APS and OMV. This study indicates that antibodies against both outer membrane proteins and APS may be important in providing the protection induced during disease, as measured by hSBA. Therefore, outer membrane proteins could also have a role as components of future meningococcal vaccines for the African meningitis belt.
Antibodies against C-Reactive Protein Cross-React with 60-Kilodalton Heat Shock Proteins
Authors: Udvarnoki Katalin , Cervenak László , Uray Katalin , Hudecz Ferenc , Kacskovics Imre , Spallek Ralf , Singh Mahavir , Füst George , Prohászka Zoltán
Published: February 2007

C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinantMycobacterium tuberculosisHsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.
A Human Monoclonal Immunoglobulin M Reduces Bacteremia and Inflammation in a Mouse Model of Systemic Pneumococcal Infection
Authors: Fabrizio Kevin , Groner Abraham , Boes Marianne , Pirofski Liise-anne
Published: February 2007

Antibody-based approaches to pneumococcal disease may hold promise for immunocompromised patients in whom vaccines are less immunogenic and/or in the context of antimicrobial resistance. Antibody-mediated protection against experimental pneumococcal pneumonia has been shown to depend on immunoregulation, but the relationship between antibody and protection against pneumococcal sepsis and immunoregulation has not been examined. Similarly, the requirement for B and T cells for antibody efficacy is not known. In this study, we determined the efficacy of the human pneumococcal capsular polysaccharide serotype 3-specific antibody, A7 (immunoglobulin M [IgM]), in secretory IgM (sIgM)−/−, CD4−/−, CD8−/−, μMT−/−, and SCID mice and investigated its effect on cytokine and chemokine expression in sera and spleens from mice with intact cellular immunity. A7 is known to be protective against systemic infection with serotype 3 and to require complement for efficacy. Compared to that of an isotype control antibody, A7 administration prolonged the survival of mice of each immunodeficient strain and was associated with a significant reduction in CFU in blood, lung, and spleen samples and a significantly reduced level of keratinocyte-derived chemokine (KC), interleukin-6 (IL-6), and macrophage inflammatory protein-2 (MIP-2) expression in normal and sIgM−/−mice. Studies with mice treated with penicillin revealed similar reductions in CFU and similar levels of IL-6, KC, or MIP-2 expression in A7- and penicillin-treated mice. These findings demonstrate that natural IgM and B and T cells are dispensable for A7-mediated protection against experimental pneumococcal sepsis and suggest that the efficacy of antibody-mediated protection depends on immunomodulation. Taken together, our data extend the association between antibody-mediated protection and immunomodulation to protection against systemic pneumococcal infection and to a clinically important serotype often responsible for pneumococcal sepsis.
Limited Protection Conferred by a DNA Vaccine against a Lethal Pseudorabies Virus Infection at Day 5 Postvaccination
Authors: Dory Daniel , Torché Anne-Marie , Béven Véronique , Cariolet Roland , Jestin André
Published: February 2007

No pseudorabies virus (PRV)-specific neutralizing or immunoglobulin G1-type antibodies were detected in sera 5 days after injection of a DNA vaccine against PRV infection in pigs. PRV-stimulated peripheral blood mononuclear cells produced gamma interferon mRNA in vitro. Two out of five pigs recovered from lethal PRV infection without attenuation of nasal viral excretion.
In Vivo Expression of and Cell-Mediated Immune Responses to the Plasmid-Encoded Virulence-Associated Proteins ofRhodococcus equiin Foals
Authors: Jacks Stephanie , Giguère Steeve , Prescott John F.
Published: February 2007

Rhodococcus equiis a facultative intracellular pathogen that causes pneumonia in foals but does not induce disease in adult horses. Virulence ofR. equidepends on the presence of a large plasmid, which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH). Eradication ofR. equifrom the lungs depends on gamma interferon (IFN-γ) production by T lymphocytes. The objectives of the present study were to determine the relative in vivo expression of thevapgenes ofR. equiin the lungs of infected foals, to determine the recall response of bronchial lymph node (BLN) lymphocytes from foals and adult horses to each of the Vap proteins, and to compare the cytokine profiles of proliferating lymphocytes between foals and adult horses.vapA,vapD, andvapGwere preferentially expressed in the lungs of infected foals, and expression of these genes in the lungs was significantly (P< 0.05) higher than that achieved during in vitro growth. VapA and VapC induced the strongest lymphoproliferative responses for foals and adult horses. There was no significant difference in recall lymphoproliferative responses or IFN-γ mRNA expression by bronchial lymph node lymphocytes between foals and adults. In contrast, interleukin 4 (IL-4) expression was significantly higher for adults than for foals for each of the Vap proteins. The ratio of IFN-γ to IL-4 was significantly higher for foals than for adult horses for most Vap proteins. Therefore, foals are immunocompetent and are capable of mounting lymphoproliferative responses of the same magnitude and cytokine phenotype as those of adult horses.
Comparison of Mantoux and QuantiFERON TB Gold Tests for Diagnosis of Latent Tuberculosis Infection in Army Personnel
Authors: Franken Willeke P. J. , Timmermans Joost F. , Prins Corine , Slootman Evert-Jan H. J. , Dreverman Johan , Bruins Hans , van Dissel Jaap T. , Arend Sandra M.
Published: February 2007

The tuberculin skin test (TST) was compared with QuantiFERON-TB Gold in-tube (QFT-GIT) test for the diagnosis of tuberculosis in non-
Mycobacterium bovis
BCG-vaccinated military personnel. Among subjects positive by TST, 44.4% of recruits were positive by QFT-GIT compared with 11.5% subjects tested after missions abroad, suggesting that most TST conversions in the latter group were caused by nontuberculous mycobacteria.
Low Maternal Viral Loads and Reduced Granulocyte-Macrophage Colony-Stimulating Factor Levels Characterize Exposed, Uninfected Infants Who Develop Protective Human Immunodeficiency Virus Type 1-Specific Responses
Authors: Schramm Diana B. , Meddows-Taylor Stephen , Gray Glenda E. , Kuhn Louise , Tiemessen Caroline T.
Published: February 2007

Human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses are elicited in a proportion of infants born to HIV-1-infected mothers and are associated with protection against vertical transmission. To investigate correlates of these HIV-1-specific responses, we examined levels of the immune activation markers neopterin, β2-microglobulin (β2-m), and solublel-selectin (sl-selectin); the immunomodulatory and hematopoietic factors interleukin-7 (IL-7), stromal-cell-derived factor 1 alpha (CXCL12), and granulocyte-macrophage colony-stimulating factor (GM-CSF); and the immunoregulatory cytokine IL-10 among a group of newborns born to HIV-1-positive mothers who did not receive any antiretroviral drugs for prevention of perinatal HIV-1 transmission. Cellular immune responses to HIV-1 envelope (Env) peptides were also measured. We aimed to determine whether newborns who elicit HIV-1-specific cellular immune responses (Env+) and those who lack these responses (Env−) exhibit unique immune features. Our data confirmed that no Env+infants acquired HIV-1 infection. Among exposed, uninfected infants, Env+infants had reduced immune activation (as measured by β2-m and sl-selectin levels in cord blood plasma) compared to Env−infants as well as reduced GM-CSF levels in cord blood plasma. There was also a reduced ability of cord blood mononuclear cells to be induced to produce GM-CSF among Env+infants. Maternal viral load was lower in Env+infants, suggesting that exposure to low levels of antigen may be responsible for priming the protective responses. These findings suggest that infants who are able to develop apparently protective HIV-1-specific cellular immune responses have immunological features and viral exposure histories that distinguish them from their nonresponder counterparts, providing new insights into the development of HIV-1 protective immunity.
Molecular Analysis of Oral and Respiratory Bacterial Species Associated with Ventilator-Associated Pneumonia
Authors: Bahrani-Mougeot Farah K. , Paster Bruce J. , Coleman Shirley , Barbuto Sara , Brennan Michael T. , Noll Jenene , Kennedy Thomas , Fox Philip C. , Lockhart Peter B.
Published: February 2007

Trauma intensive care unit (TICU) patients requiring mechanical respiratory support frequently develop ventilator-associated pneumonia (VAP). Oral and oropharyngeal bacteria are believed to be responsible for many cases of VAP, but definitive evidence of this relationship is lacking. Earlier studies used conventional culture-based methods for identification of bacterial pathogens, but these methods are insufficient, as some bacteria may be uncultivable or difficult to grow. The purpose of this study was to use a culture-independent molecular approach to analyze and compare the bacterial species colonizing the oral cavity and the lungs of TICU patients who developed VAP. Bacterial samples were acquired from the dorsal tongue and bronchoalveolar lavage fluid of 16 patients. Bacterial DNA was extracted, and the 16S rRNA genes were PCR amplified, cloned into
Escherichia coli
, and sequenced. The sequencing data revealed the following: (i) a wide diversity of bacterial species in both the oral and pulmonary sites, some of them novel; (ii) known and putative respiratory pathogens colonizing both the oral cavity and lungs of 14 patients; and (iii) a number of bacterial pathogens (e.g.,
Dialister pneumosintes
,
Haemophilus segnis
,
Gemella morbillorum
, and
Pseudomonas fluorescens
) in lung samples that had not been reported previously at this site when culture-based methods were used. Our data indicate that the dorsal surface of the tongue serves as a potential reservoir for bacterial species involved in VAP. Furthermore, it is clear that the diversity of bacterial pathogens for VAP is far more complex than the current literature suggests.
Colorimetric Phage-Based Assay for Detection of Rifampin-Resistant
Mycobacterium tuberculosis
Authors: McNerney Ruth , Mallard Kim , Urassa Honorathy M. R. , Lemma Eshetu , Donoghue Helen D.
Published: February 2007

Tests based on bacteriophage replication enable rapid screening of
Mycobacterium tuberculosis
for drug resistance. We describe a novel broth-based colorimetric method for detecting phage replication. When clinical isolates were tested by this novel method, high concordance was observed with both the traditional phage assay and gene mutation analysis for detection of resistance to rifampin.
Evaluation of Etest and Disk Diffusion Methods Compared with Broth Microdilution Antifungal Susceptibility Testing of Clinical Isolates of
Candida
spp. against Posaconazole
Authors: Diekema Daniel J. , Messer Shawn A. , Hollis Richard J. , Boyken Linda B. , Tendolkar Shailesh , Kroeger Jennifer , Pfaller Michael A.
Published: February 2007

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of 2,171 clinical isolates of
Candida
spp. against posaconazole. By using provisional breakpoints for comparison purposes only, the categorical agreement between the agar-based methods and broth microdilution results ranged from 93 to 98%, with <1% very major errors. The essential agreement (within 2 well dilutions) between the Etest and broth microdilution methods was 94%. These agar-based methods hold promise as simple and reliable methods for determination of the posaconzole susceptibilities of
Candida
spp.
Determination of
bft
Gene Subtypes in
Bacteroides fragilis
Clinical Isolates
Authors: Avila-Campos Mario J. , Liu Chengxu , Song Yuli , Rowlinson Marie-Claire , Finegold Sydney M.
Published: February 2007

A rapid multiplex PCR approach was developed to detect the
bft
gene subtypes in
Bacteroides fragilis
clinical isolates. This technique could be used to look at the epidemiology of enterotoxigenic strains of
B. fragilis
in clinical infections and whether there is a correlation between disease and the presence of
B. fragilis
enterotoxin.
Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza A+B Test for Rapid Diagnosis of Influenza
Authors: Mehlmann Martin , Bonner Aleta B. , Williams John V. , Dankbar Daniela M. , Moore Chad L. , Kuchta Robert D. , Podsiad Amy B. , Tamerius John D. , Dawson Erica D. , Rowlen Kathy L.
Published: February 2007

The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A+B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses.
Detection of Extended-Spectrum β-Lactamases by Using Boronic Acid as an AmpC β-Lactamase Inhibitor in Clinical Isolates of
Klebsiella
spp. and
Escherichia coli
Authors: Song Wonkeun , Bae Il Kwon , Lee You-Nae , Lee Chae-Hoon , Lee Sang Hee , Jeong Seok Hoon
Published: February 2007

We evaluated highly sensitive methods using boronic acid (BA) to detect extended-spectrum β-lactamase (ESBL) production. A total of 182 clinical isolates of
Klebsiella
spp. (
n
= 118) and
Escherichia coli
(
n
= 64) were analyzed: 62 harbored only ESBLs, 80 harbored both ESBLs and plasmid-mediated AmpC β-lactamases (pAmpCs), and 40 harbored only pAmpCs. The CLSI confirmatory test detected all isolates that produce only ESBLs but detected 85% of isolates that produce both enzymes. When a ≥5-mm increase in the zone diameter of either the cefotaxime (CTX) or the ceftazidime (CAZ) disk in the presence of both clavulanic acid (CA) and BA was considered to be a positive result, the test detected all isolates that harbor ESBLs (± pAmpCs) but showed frequent false-positive results (50%) for isolates that produce only pAmpCs. Meanwhile, when a ≥3-mm increase in the zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result, the test also detected all isolates that harbor ESBLs (± pAmpCs) and showed less frequent false-positive results (5%) in isolates that produce only pAmpCs. The latter new interpretive guideline has enhanced detection of ESBLs in clinical isolates of
Klebsiella
spp. and
Escherichia coli
and allowed detection of an ESBL even when potentially masked by a pAmpC.
First Notification of Positive Blood Cultures and the High Accuracy of the Gram Stain Report
Authors: Søgaard Mette , Nørgaard Mette , Schønheyder Henrik C.
Published: February 2007

When blood cultures turn positive, the attending physicians are usually notified immediately about Gram stain findings. However, information on the accuracy of Gram staining is very limited. We examined the accuracy of preliminary blood culture reports provided by a regional laboratory in an observational study including the years 1996, 2000 to 2001, and 2003. We used data from computer files and technicians' laboratory notes. The study was restricted to cultures with one morphological type. Using cultural identification as a reference, we estimated the sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) for the following defined morphological groups: gram-positive cocci in clusters, gram-positive cocci in chains or diplococci, gram-positive rods, gram-negative cocci, gram-negative rods, and yeasts. We further evaluated the Gram stain and wet mount findings for the most frequent bacterial species/groups. We obtained 5,893 positive blood cultures and the following results for the defined groups: sensitivity, range of 91.3 to 99.7%; specificity, 98.9 to 100%; PPV, 94.6 to 100%; and NPV, 99.0 to 100%. The sensitivity for the most frequent species was in the range 91.3 to 100%, with nonhemolytic streptococci having the lowest value (sensitivity, 91.3%; 95% confidence interval, 86.2 to 94.9%). Wet mount reports were less accurate (sensitivity of 30 to 70% for species with peritrichous motility), and
Enterobacteriaceae
(notably
Salmonella
spp.) accounted for 25% of the reports stating polar motility. In conclusion, we demonstrated a high accuracy of Gram stain reports, whereas wet mount microscopy was generally less accurate.
Role of the Hydrophilic Channels of Simian Virus 40 T-Antigen Helicase in DNA Replication
Authors: Wang Weiping , Manna David , Simmons Daniel T.
Published: February 2007

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.
Participation of Rab5, an Early Endosome Protein, in Hepatitis C Virus RNA Replication Machinery
Authors: Stone Michelle , Jia Shuaizheng , Heo Won Do , Meyer Tobias , Konan Kouacou V.
Published: February 2007

Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce “membranous web” formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.
Reversal of Human Cytomegalovirus Major Immediate-Early Enhancer/Promoter Silencing in Quiescently Infected Cells via the Cyclic AMP Signaling Pathway
Authors: Keller Michael J. , Wu Allen W. , Andrews Janet I. , McGonagill Patrick W. , Tibesar Eric E. , Meier Jeffery L.
Published: February 2007

The human cytomegalovirus (HCMV) major immediate-early (MIE) enhancer contains five functional cyclic AMP (cAMP) response elements (CRE). Because the CRE in their native context do not contribute appreciably to MIE enhancer/promoter activity in lytically infected human fibroblasts and NTera2 (NT2)-derived neurons, we postulated that they might have a role in MIE enhancer/promoter reactivation in quiescently infected cells. Here, we show that stimulation of the cAMP signaling pathway by treatment with forskolin (FSK), an adenylyl cyclase activator, greatly alleviates MIE enhancer/promoter silencing in quiescently infected NT2 neuronal precursors. The effect is immediate, independent of de novo protein synthesis, associated with the phosphorylation of ATF-1 serine 63 and CREB serine 133, dependent on protein kinase A (PKA) and the enhancer's CRE, and linked to viral-lytic-cycle advancement. Coupling of FSK treatment with the inhibition of either histone deacetylases or protein synthesis synergistically activates MIE gene expression in a manner suggesting that MIE enhancer/promoter silencing is optimally relieved by an interplay of multiple regulatory mechanisms. In contrast, MIE enhancer/promoter silence is not overcome by stimulation of the gamma interferon (IFN-γ) signaling pathway, despite the enhancer having two IFN-γ-activated-site-like elements. We conclude that stimulation of the cAMP/PKA signaling pathway drives CRE-dependent MIE enhancer/promoter activation in quiescently infected cells, thus exposing a potential mode of regulation in HCMV reactivation.
Guanidine Hydrochloride Inhibits Mammalian Orthoreovirus Growth by Reversibly Blocking the Synthesis of Double-Stranded RNA
Authors: Murray Kenneth E. , Nibert Max L.
Published: February 2007

Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2C
ATPase
, resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on mammalian orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family
Reoviridae
. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.
Stability of a Receptor-Binding Active Human Immunodeficiency Virus Type 1 Recombinant gp140 Trimer Conferred by Intermonomer Disulfide Bonding of the V3 Loop: Differential Effects of Protein Disulfide Isomerase on CD4 and Coreceptor Binding
Authors: Billington J. , Hickling T. P. , Munro G. H. , Halai C. , Chung R. , Dodson G. G. , Daniels R. S.
Published: February 2007

Stable trimeric forms of human immunodeficiency virus recombinant gp140 (rgp140) are important templates for determining the structure of the glycoprotein to assist in our understanding of HIV infection and host immune response. Such information will aid the design of therapeutic drugs and vaccines. Here, we report the production of a highly stable and trimeric rgp140 derived from a HIV type 1 (HIV-1) subtype D isolate that may be suitable for structural studies. The rgp140 is functional in terms of binding to CD4 and three human monoclonal antibodies (17b, b12, and 2G12) that have broad neutralizing activities against a range of HIV-1 isolates from different subtypes. Treatment of rgp140 with protein disulfide isomerase (PDI) severely restricted 17b binding capabilities. The stable nature of the rgp140 was due to the lack of processing at the gp120/41 boundary and the presence of an intermonomer disulfide bond formed by the cysteines of the V3 loop. Further characterization showed the intermonomer disulfide bond to be a target for PDI processing. The relevance of these findings to the roles of the V3 domain and the timing of PDI action during the HIV infection process are discussed.
PrP
Sc
in Salivary Glands of Scrapie-Affected Sheep
Authors: Vascellari Marta , Nonno Romolo , Mutinelli Franco , Bigolaro Michela , Di Bari Michele Angelo , Melchiotti Erica , Marcon Stefano , D'Agostino Claudia , Vaccari Gabriele , Conte Michela , De Grossi Luigi , Rosone Francesca , Giordani Francesco , Agrimi Umberto
Published: February 2007

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP
Sc
). PrP
Sc
was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP
Sc
concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP
Sc
in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP
Sc
in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.
Persistence of Cyprinid Herpesvirus 3 in Infected Cultured Carp Cells
Authors: Dishon Arnon , Davidovich Maya , Ilouze Maya , Kotler Moshe
Published: February 2007

Cyprinid herpesvirus 3 (CyHV-3), previously designated carp interstitial nephritis and gill necrosis virus or koi herpesvirus, is the cause of a worldwide mortal disease of koi and carp. Morphologically, the virus resembles herpesviruses, yet it bears a genome of 277 to 295 kbp, which is divergent from most of the genomic sequences available in GenBank. The disease afflicts fish in the transient seasons, when the water temperature is 18 to 28°C, conditions which permit virus propagation in cultured cells. Here we report that infectious virus is preserved in cultured cells maintained for 30 days at 30°C. CyHV-3-infected vacuolated cells with deformed morphology converted to normal, and plaques disappeared following shifting up of the temperature and reappeared after transfer to the permissive temperature. Viral propagation and viral gene transcription were turned off by shifting cells to the nonpermissive temperature. Upon return of the cells to the permissive temperature, transcription of viral genes was reactivated in a sequence distinguished from that occurring in naïve cells following infection. Our results show that CyHV-3 persists in cultured cells maintained at the nonpermissive temperature and suggest that viruses could persist for long periods in the fish body, enabling a new burst of infection upon a shift to a permissive temperature.
Rab 5 Is Required for the Cellular Entry of Dengue and West Nile Viruses
Authors: Krishnan Manoj N. , Sukumaran Bindu , Pal Utpal , Agaisse Herve , Murray James L. , Hodge Thomas W. , Fikrig Erol
Published: February 2007

The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.
Xenopus
Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication
Authors: Alves-Rodrigues Isabel , Mas Antonio , Díez Juana
Published: February 2007

By using a Brome mosaic virus (BMV)-
Saccharomyces cerevisiae
system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the
dhh1Δ
and
lsm1Δ
strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.
Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution
Authors: Yap Thai Leong , Xu Ting , Chen Yen-Liang , Malet Helene , Egloff Marie-Pierre , Canard Bruno , Vasudevan Subhash G. , Lescar Julien
Published: February 2007

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the
Flavivirus
genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The
S
-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.
Bax Is Activated during Rotavirus-Induced Apoptosis through the Mitochondrial Pathway
Authors: Martin-Latil Sandra , Mousson Laurence , Autret Arnaud , Colbère-Garapin Florence , Blondel Bruno
Published: February 2007

Rotaviruses are the leading cause of infantile viral gastroenteritis worldwide. Mature enterocytes of the small intestine infected by rotavirus undergo apoptosis, and their replacement by less differentiated dividing cells probably leads to defective absorptive function of the intestinal epithelium, which, in turn, contributes to osmotic diarrhea and rotavirus pathogenesis. Here we show that infection of MA104 cells by the simian rhesus rotavirus strain RRV induced caspase-3 activation, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase; all three phenomena are features of apoptosis. RRV induced the release of cytochrome
c
from mitochondria to the cytosol, indicating that the mitochondrial apoptotic pathway was activated. RRV infection of MA104 cells activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change. Most importantly, Bax-specific small interfering RNAs partially inhibited cytochrome
c
release in RRV-infected cells. Thus, mitochondrial dysfunction induced by rotavirus is Bax dependent. Apoptosis presumably leads to impaired intestinal functions, so our findings contribute to improving our understanding of rotavirus pathogenesis at the cellular level.