bluuebunny/crossref_metadata_2025 huggingface dataset:
Composite Materials with Viscoelastic Stiffness Greater Than Diamond
Authors: Jaglinski T. , Kochmann D. , Stone D. , Lakes R. S.
Published: February 2007

We show that composite materials can exhibit a viscoelastic modulus (Young's modulus) that is far greater than that of either constituent. The modulus, but not the strength, of the composite was observed to be substantially greater than that of diamond. These composites contain bariumtitanate inclusions, which undergo a volume-change phase transformation if they are not constrained. In the composite, the inclusions are partially constrained by the surrounding metal matrix. The constraint stabilizes the negative bulk modulus (inverse compressibility) of the inclusions. This negative modulus arises from stored elastic energy in the inclusions, in contrast to periodic composite metamaterials that exhibit negative refraction by inertial resonant effects. Conventional composites with positive-stiffness constituents have aggregate properties bounded by a weighted average of constituent properties; their modulus cannot exceed that of the stiffest constituent.
Yeast Rtt109 Promotes Genome Stability by Acetylating Histone H3 on Lysine 56
Authors: Driscoll Robert , Hudson Amanda , Jackson Stephen P.
Published: February 2007

Posttranslational modifications of the histone octamer play important roles in regulating responses to DNA damage. Here, we reveal that
Saccharomyces cerevisiae
Rtt109p promotes genome stability and resistance to DNA-damaging agents, and that it does this by functionally cooperating with the histone chaperone Asf1p to maintain normal chromatin structure. Furthermore, we show that, as for Asf1p, Rtt109p is required for histone H3 acetylation on lysine 56 (K56) in vivo. Moreover, we show that Rtt109p directly catalyzes this modification in vitro in a manner that is stimulated by Asf1p. These data establish Rtt109p as a member of a new class of histone acetyltransferases and show that its actions are critical for cell survival in the presence of DNA damage during S phase.
A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission
Authors: Tumpey Terrence M. , Maines Taronna R. , Van Hoeven Neal , Glaser Laurel , Solórzano Alicia , Pappas Claudia , Cox Nancy J. , Swayne David E. , Palese Peter , Katz Jacqueline M. , García-Sastre Adolfo
Published: February 2007

The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Here, we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch in receptor binding preference from the human α-2,6 to the avian α-2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual α-2,6 and α-2,3 specificity suggests that a predominant human α-2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals.
Recent Climate Observations Compared to Projections
Authors: Rahmstorf Stefan , Cazenave Anny , Church John A. , Hansen James E. , Keeling Ralph F. , Parker David E. , Somerville Richard C. J.
Published: February 2007

We present recent observed climate trends for carbon dioxide concentration, global mean air temperature, and global sea level, and we compare these trends to previous model projections as summarized in the 2001 assessment report of the Intergovernmental Panel on Climate Change (IPCC). The IPCC scenarios and projections start in the year 1990, which is also the base year of the Kyoto protocol, in which almost all industrialized nations accepted a binding commitment to reduce their greenhouse gas emissions. The data available for the period since 1990 raise concerns that the climate system, in particular sea level, may be responding more quickly to climate change than our current generation of models indicates.
Autophagy-Dependent Viral Recognition by Plasmacytoid Dendritic Cells
Authors: Lee Heung Kyu , Lund Jennifer M. , Ramanathan Balaji , Mizushima Noboru , Iwasaki Akiko
Published: February 2007

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes by means of Toll-like receptors (TLRs). Yet, pDC responses to certain single-stranded RNA (ssRNA) viruses occur only after live viral infection. We present evidence here that the recognition of such viruses by TLR7 requires transport of cytosolic viral replication intermediates into the lysosome by the process of autophagy. In addition, autophagy was found to be required for the production of interferon-α by pDCs. These results support a key role for autophagy in mediating ssRNA virus detection and interferon-α secretion by pDCs and suggest that cytosolic replication intermediates of viruses serve as pathogen signatures recognized by TLR7.
Application of the Fluorescence Polarization Assay for Detection of Caprine Antibodies to
Brucella melitensis
in Areas of High Prevalence and Widespread Vaccination
Authors: Ramírez-Pfeiffer C. , Nielsen K. , Smith P. , Marín-Ricalde F. , Rodríguez-Padilla C. , Gomez-Flores R.
Published: February 2007

The screening Rose Bengal test (RBT), the buffered plate agglutination test (BPAT), and the confirmatory complement fixation test (CFT) are currently approved by the World Organization for Animal Health (OIE) for diagnosis of goat brucellosis. However, RBT (at 3% or 8% cell concentration) is known to be affected by vaccinal antibodies. In the present study, Mexican and Canadian OIE tests were compared with the fluorescence polarization assay (FPA), alone or in combination, using indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from an area of high prevalence and widespread vaccination. The relative sensitivities and specificities were, respectively, 99.7% and 32.5% for RBT3, 92.8% and 68.8% for RBT8, 98.4% and 84.8% for Canadian CFT, 83.7% and 65.5% for Mexican CFT, and 78.1% and 89.3% for FPA. The use of FPA as the confirmatory test in combination with other tests significantly increased the final specificities of the screening tests alone; BPAT, RBT3, and RBT8 plus FPA resulted in final specificities of 90%, 91.2%, and 91.3%, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, specificities were 65.5%, 63.2%, and 91.7%, respectively. We suggest that FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis and as a confirmatory test for screening test series. Some advantages of FPA are that its cutoff can be adjusted to improve its sensitivity or specificity, it is a low-cost and easy-to-perform test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of misdiagnosed and killed goats.
Cellular Immunity in Adolescents and Adults following Acellular Pertussis Vaccine Administration
Authors: Meyer Claudius U. , Zepp Fred , Decker Michael , Lee Martin , Chang Swei-Ju , Ward Joel , Yoder Sandra , Bogaert Hugues , Edwards Kathryn M.
Published: February 2007

Cell-mediated immune (CMI) responses to an acellular pertussis vaccine administered to 49 subjects, a subset of participants in the National Institutes of Health-funded adult acellular pertussis vaccine efficacy trial, were evaluated and compared with antibody responses to vaccine antigens. Levels of proliferation of and cytokine secretion from lymphocytes cultured in the presence of pertussis toxin, filamentous hemagglutinin, or pertactin were measured before vaccination and 1 month and 1 year after vaccination. Statistically significant increases in lymphocyte stimulation indices and cytokine secretion were noted at both 1 month and 1 year after vaccination. Brisk pertussis antigen-specific immunoglobulin G responses were also noted at 1 month after vaccination, but these responses had declined by nearly 50% at 1 year after vaccination. These studies clearly demonstrate that both cellular and humoral immune responses occur after the administration of acellular pertussis vaccines to adolescents and adults but that the CMI responses are of greater magnitude and longer duration. CMI responses may be a better correlate of long-term protection.
Evaluation of the Partec Flow Cytometer against the BD FACSCalibur System for Monitoring Immune Responses of Human Immunodeficiency Virus-Infected Patients in Zimbabwe
Authors: Manasa Justen , Musabaike Hazvineyi , Masimirembwa Collen , Burke Eileen , Luthy Ruedi , Mudzori James
Published: February 2007

A single-platform volumetric flow cytometer, the Partec Cyflow SL_3, was evaluated against a BD FACSCalibur/Sysmex XT1800i dual platform for measuring CD4+lymphocytes, total lymphocytes, and the percentage of CD4 lymphocytes in whole-blood samples for monitoring the immune systems of human immunodeficiency virus (HIV)/AIDS patients. Statistical analyses for precision, correlation, and agreement were performed. Coefficients of variation (CV) of 5.8, 4.6, and 3.9% were obtained for low, medium, and high CD4+cell counts, respectively, using the SL_3, and CV of 3.7, 4.0, and 0.94 were obtained for the same categories, using the BD FACSCalibur. Significant correlations (P< 0.005) between the two assays for CD4 counts, total lymphocyte counts, and percentages of CD4 were obtained, with correlation coefficients of 0.99, 0.96, and 0.99, respectively (n= 229). Using the Bland-Altman plot, mean biases of −18 cell/μl (95% confidence interval (CI); −91 to 54 cells/μl), −0.8% (95% CI; −3.6 to 2%), and −36.8 cells/μl (95% CI; −477 to 404 cells/μl) were obtained for comparisons of CD4 counts, percentages of CD4 cells, and total lymphocyte counts, respectively. The effects of the age of the samples on the three parameters were also analyzed by comparing results from the same samples analyzed at 6, 24, and 48 h after collection. The correlation coefficients for comparisons among different time points for the same machine and among all the time points for the two different machines were greater than 0.90. These data showed that the Partec Cyflow SL_3 assay is comparable to the BD FACSCalibur/Sysmex XT1800i dual-platform method for measuring the amount of CD4+cells and total lymphocytes and the percentages of CD4 cells in blood samples for the purpose of monitoring HIV/AIDS patients.
Structure-Activity Analysis of Quorum-Sensing Signaling Peptides from
Streptococcus mutans
Authors: Syvitski Raymond T. , Tian Xiao-Lin , Sampara Kamal , Salman Alan , Lee Song F. , Jakeman David L. , Li Yung-Hua
Published: February 2007

Streptococcus mutans
secretes and utilizes a 21-amino-acid signaling peptide pheromone to initiate quorum sensing for genetic competence, biofilm formation, stress responses, and bacteriocin production. In this study, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions to investigate their structure-activity relationships based on the three-dimensional structures of
S. mutans
wild-type signaling peptide UA159sp and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence. By analyzing these peptides, we demonstrated that the signaling peptide of
S. mutans
has at least two functional domains. The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activation of the signal transduction pathway, while the core α-helical structure extending from residue 5 to the end of the peptide is required for receptor binding. Peptides in which three or more residues were deleted from the C terminus did not induce genetic competence but competitively inhibited quorum sensing activated by UA159sp. Disruption of the amphipathic α-helix by replacing the Phe-7, Phe-11, or Phe-15 residue with a hydrophilic residue resulted in a significant reduction in or complete loss of the activity of the peptide. In contrast to the C-terminally truncated peptides, these peptides with amino acid substitutions did not compete with UA159sp to activate quorum sensing, suggesting that disruption of the hydrophobic face of the α-helical structure results in a peptide that is not able to bind to the receptor. This study is the first study to recognize the importance of the signaling peptide C-terminal residues in streptococcal quorum sensing.
Siamycin Attenuates
fsr
Quorum Sensing Mediated by a Gelatinase Biosynthesis-Activating Pheromone in
Enterococcus faecalis
Authors: Nakayama Jiro , Tanaka Emi , Kariyama Reiko , Nagata Koji , Nishiguchi Kenzo , Mitsuhata Ritsuko , Uemura Yumi , Tanokura Masaru , Kumon Hiromi , Sonomoto Kenji
Published: February 2007

The expression of two
Enterococcus faecalis
virulence-related proteases, gelatinase (GelE) and serine protease (SprE), is positively regulated by a quorum-sensing system encoded by the
fsr
gene cluster. In this system,
E. faecalis
secretes an autoinducing peptide, gelatinase biosynthesis-activating pheromone (GBAP), which triggers the FsrC-FsrA two-component regulatory system controlling the expression of two transcripts,
fsrBDC
and
gelE-sprE
. In the present study, we screened actinomycete metabolites for inhibitors of
fsr
quorum sensing.
E. faecalis
was cultured with each actinomycete culture supernatant tested, and the production of gelatinase and the production of GBAP were examined using the first screening and the second screening, respectively. Culture supernatant of
Streptomyces
sp. strain Y33-1 had the most potent inhibitory effect on both gelatinase production and GBAP production without inhibiting
E. faecalis
cell growth. The inhibitor in the culture supernatant was identified as a known peptide antibiotic, siamycin I. Siamycin I inhibited both gelatinase production and GBAP production at submicromolar concentrations, and it inhibited
E. faecalis
cell growth at concentrations above micromolar concentrations. Quantitative analysis of
fsrBDC
and
gelE-sprE
transcripts revealed that siamycin I suppressed the expression of both transcripts at a sublethal concentration. Siamycin I attenuated gelatinase production even when an overdose of GBAP was exogenously added to the culture. These results suggested that siamycin I inhibited the GBAP signaling via the FsrC-FsrA two-component regulatory system in a noncompetitive manner. The sublethal concentrations of siamycin I also attenuated biofilm formation. Treatment with siamycin could be a novel means of treating enterococcal infections.
Control of EpsE, the Phosphoglycosyltransferase Initiating Exopolysaccharide Synthesis inStreptococcus thermophilus, by EpsD Tyrosine Kinase
Authors: Minic Zoran , Marie Corinne , Delorme Christine , Faurie Jean-Michel , Mercier Gérald , Ehrlich Dusko , Renault Pierre
Published: February 2007

We studied the roles ofStreptococcus thermophilusphosphogalactosyltransferase (EpsE) (the priming enzyme), tyrosine kinase (EpsD), phosphatase (EpsB), and a membrane-associated protein with no known biochemical function (EpsC) in exopolysaccharide (EPS) synthesis. These proteins are well-conserved among bacteria and are usually encoded by clustered genes. Exopolysaccharide synthesis took place in the wild-type strain and a mutant lacking EpsB but not in mutants lacking EpsC, EpsD, or EpsE. The three mutants unable to synthesize EPS lacked the EpsE phosphogalactosyltransferase activity, while the two EPS-synthesizing strains possessed this activity, showing that EpsC and EpsD are required for EpsE function. An EpsD phosphorylated form was found in all strains except theepsCmutant, indicating that EpsC is necessary for EpsD phosphorylation. Moreover, the phosphorylated form of EpsD, a supposedly cytoplasmic protein, was found to be associated with the plasma membrane, possibly due to interaction with EpsC. Finally, the EpsD and EpsE elution profiles in a gel filtration chromatography assay were similar, suggesting that these two proteins colocalize in the membrane. Mutation of Tyr200, predicted to be a phosphorylation site and present in a conserved motif in bacterial phosphoglycosyltransferases, led to EpsE inactivation. In contrast, mutation of Tyr162 or Tyr199 had no effect. Taken together, these data show that EpsD controls EpsE activity. Possible mechanisms for this control are discussed.
Regulation of Gene Expression inStreptococcus pneumoniaeby Response Regulator 09 Is Strain Dependent
Authors: Hendriksen Wouter T. , Silva Nuno , Bootsma Hester J. , Blue Clare E. , Paterson Gavin K. , Kerr Alison R. , de Jong Anne , Kuipers Oscar P. , Hermans Peter W. M. , Mitchell Tim J.
Published: February 2007

Recent murine studies have demonstrated that the role of response regulator 09 (RR09) ofStreptococcus pneumoniaein virulence is different in different strains. In the present study, we used a murine pneumonia model of infection to assess the virulence of a TIGR4 rr09 mutant, and we found that TIGR4Δrr09 was attenuated after intranasal infection. Furthermore, we investigated the in vitro transcriptional changes in pneumococcal rr09 mutants of two strains, D39 and TIGR4, by microarray analysis. The transcriptional profiles of the rr09 mutants of both strains had clear differences compared to the profiles of the parental wild-type strains. In D39Δrr09, but not in TIGR4Δrr09, genes involved in competence (e.g.,comAB) were upregulated. In TIGR4, genes located on therlrApathogenicity islet, which are not present in the D39 genome, appeared to be regulated by RR09. Furthermore, several phosphotransferase systems (PTSs) believed to be involved in sugar uptake (e.g., the PTS encoded by sp0060 to sp0066) were strongly downregulated in D39Δrr09, while they were not regulated by RR09 in TIGR4. To examine the role of one of these PTSs in virulence, D39Δsp0063 was constructed and tested in a murine infection model. No difference between the virulence of this strain and the virulence of the wild type was found, indicating that downregulation of the sp0063 gene alone is not the cause of the avirulent phenotype of D39Δrr09. Finally, expression of rr09 and expression of three of our identified RR09 targets during infection in mice were assessed. This in vivo experiment confirmed that there were differences between expression in wild-type strain TIGR4 and expression in the rr09 mutant, as well as differences between expression in wild-type strain D39 and expression in wild-type strain TIGR4. In conclusion, our results indicate that there is strain-specific regulation of pneumococcal gene expression by RR09.
Membrane Composition Changes and Physiological Adaptation byStreptococcus mutansSignal Recognition Particle Pathway Mutants
Authors: Hasona Adnan , Zuobi-Hasona Kheir , Crowley Paula J. , Abranches Jacqueline , Ruelf Michael A. , Bleiweis Arnold S. , Brady L. Jeannine
Published: February 2007

Previously, we presented evidence that the oral cariogenic speciesStreptococcus mutansremains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Δffh, ΔscRNA, and ΔftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the β subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression inS. mutansUA159 (parent strain) and AH333 (Δffhmutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.
The Two-Component Regulatory System TCS08 Is Involved in Cellobiose Metabolism ofStreptococcus pneumoniaeR6
Authors: McKessar Stuart J. , Hakenbeck Regine
Published: February 2007

The two-component system TCS08 is one of the regulatory systems that is important for virulence ofStreptococcus pneumoniae. In order to investigate the TCS08 regulon, we have analyzed transcription profiles of mutants derived fromS. pneumoniaeR6 by microarray analysis. Since deletion mutants are often without a significant phenotype, we constructed a mutation in the histidine kinase HK08, T133P, in analogy to the phosphatase mutation T230P in the H box of theS. pneumoniaeCiaH kinase described recently (D. Zähner, K. Kaminski, M. van der Linden, T. Mascher, M. Merai, and R. Hakenbeck, J. Mol. Microbiol. Biotechnol. 4:211-216, 2002). In addition, a deletion mutation was constructed inrr08, encoding the cognate response regulator. The most heavily suppressed genes in thehk08mutant were spr0276 to spr0282, encoding a putative cellobiose phosphoenolpyruvate sugar phosphotransferase system (PTS). Whereas the R6 Smrparent strain and the Δrr08mutant readily grew on cellobiose, thehk08mutant and selected mutants with deletions in the PTS cluster did not, strongly suggesting that TCS08 is involved in the catabolism of cellobiose. Homologues of the TCS08 system were found in closely related streptococci and other gram-positive cocci. However, the genes spr0276 to spr0282, encoding the putative cellobiose PTS, represent a genomic island inS. pneumoniaeand homologues were found inStreptococcus gordoniionly, suggesting that this system might contribute to the pathogenicity potential of the pneumococcus.
The Plasminogen-Binding Group A Streptococcal M Protein-Related Protein Prp Binds Plasminogen via Arginine and Histidine Residues
Authors: Sanderson-Smith Martina L. , Dowton Mark , Ranson Marie , Walker Mark J.
Published: February 2007

The migration of the human pathogen
Streptococcus pyogenes
(group A streptococcus) from localized to deep tissue sites may result in severe invasive disease, and sequestration of the host zymogen plasminogen appears crucial for virulence. Here, we describe a novel plasminogen-binding M protein, the
p
lasminogen-binding group A streptococcal M protein (PAM)-
r
elated
p
rotein (Prp). Prp is phylogenetically distinct from previously described plasminogen-binding M proteins of group A, C, and G streptococci. While competition experiments indicate that Prp binds plasminogen with a lower affinity than PAM (50% effective concentration = 0.34 μM), Prp nonetheless binds plasminogen with high affinity and at physiologically relevant concentrations of plasminogen (

K
d

= 7.8 nM). Site-directed mutagenesis of the putative plasminogen binding site indicates that unlike the majority of plasminogen receptors, Prp does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys
96
and Lys
101
reduced but did not abrogate plasminogen binding by Prp. Plasminogen binding was abolished only with the additional mutagenesis of Arg
107
and His
108
to alanine. Furthermore, mutagenesis of Arg
107
and His
108
abolished plasminogen binding by Prp despite the presence of Lys
96
and Lys
101
in the binding site. Thus, binding to plasminogen via arginine and histidine residues appears to be a conserved mechanism among plasminogen-binding M proteins.
Complete Genome of Acute Rheumatic Fever-Associated Serotype M5
Streptococcus pyogenes
Strain Manfredo
Authors: Holden Matthew T. G. , Scott Annabel , Cherevach Inna , Chillingworth Tracey , Churcher Carol , Cronin Ann , Dowd Linda , Feltwell Theresa , Hamlin Nancy , Holroyd Simon , Jagels Kay , Moule Sharon , Mungall Karen , Quail Michael A. , Price Claire , Rabbinowitsch Ester , Sharp Sarah , Skelton Jason , Whitehead Sally , Barrell Bart G. , Kehoe Michael , Parkhill Julian
Published: February 2007

Comparisons of the 1.84-Mb genome of serotype M5
Streptococcus pyogenes
strain Manfredo with previously sequenced genomes emphasized the role of prophages in diversification of
S. pyogenes
and the close relationship between strain Manfredo and MGAS8232, another acute rheumatic fever-associated strain.
Five-Hour Diagnosis of Dermatophyte Nail Infections with Specific Detection ofTrichophyton rubrum
Authors: Brillowska-Dąbrowska Anna , Saunte Ditte Marie , Arendrup Maiken Cavling
Published: February 2007

A rapid two-step DNA extraction method and a multiplex PCR for the detection of dermatophytes in general andTrichophyton rubrumspecifically were developed and evaluated with DNA extracted from pure cultures and from clinically diseased nails. DNA from the following dermatophytes was used:Epidermophyton floccosum,Microsporum audouinii,Microsporum canis,Microsporum gypseum,Microsporum nanum,Trichophyton mentagrophytes,Trichophyton rubrum,Trichophyton schoenleinii,Trichophyton soudanense,Trichophyton terrestre,Trichophyton tonsurans,Trichophyton verrucosum, andTrichophyton violaceum. Human DNA and DNA from the following nondermatophyte fungi were included as controls:Alternaria,Aspergillus niger,Candida albicans,Candida glabrata,Candida krusei,Malassezia furfur,Saccharomyces cerevisiae, andScopulariopsis brevicaulis.A total of 118 nail samples received for routine microscopy and culture for dermatophytes were subsequently tested by the two PCRs separately and in a multiplex format. Using DNA extracted from pure cultures and the pan-dermatophyte PCR, theT. rubrum-specific PCR sequentially and in a multiplex format correctly detected all dermatophytes and additionally correctly identifiedT. rubrum.Comparison of the traditional diagnostic evaluation (microscopy and culture) of nail samples with PCR on DNA directly extracted from the nails showed excellent agreement between PCR and microscopy, but the number of samples with dermatophyte species identification was increased considerably from 22.9% to 41.5%, mainly due to the identification ofT. rubrumby PCR in microscopy-positive but culture-negative samples. In conclusion, this 5-hour diagnostic test was shown to increase not only the speed but also the sensitivity of investigation for nail dermatophytosis.
Evaluation of Disk Diffusion and Etest Compared to Broth Microdilution for Antifungal Susceptibility Testing of Posaconazole against Clinical Isolates of Filamentous Fungi
Authors: Messer Shawn A. , Diekema Daniel J. , 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 posaconazole against 146 clinical isolates of filamentous fungi. By using provisional breakpoints for comparison purposes only, categorical agreement between the results of the agar-based methods and those of broth microdilution were 96 to 98%, with no very major errors. These agar-based methods hold promise as simple and reliable methods for determining the posaconazole susceptibilities of filamentous fungi.
MDA-5 Is Cleaved in Poliovirus-Infected Cells
Authors: Barral Paola M. , Morrison Juliet M. , Drahos Jennifer , Gupta Pankaj , Sarkar Devanand , Fisher Paul B. , Racaniello Vincent R.
Published: February 2007

Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2A
pro
or 3C
pro
. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is ∼90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.
Mutation in the Glycosylated Gag Protein of Murine Leukemia Virus Results in Reduced In Vivo Infectivity and a Novel Defect in Viral Budding or Release
Authors: Low Audrey , Datta Shoibal , Kuznetsov Yurii , Jahid Sohail , Kothari Nayantara , McPherson Alexander , Fan Hung
Published: February 2007

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80

gag

) in addition to the polyprotein precursor of the viral capsid proteins (Pr65

gag

). gPr80

gag

is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65

gag

. The role of glyco-Gag in MuLV replication has been unclear, since gPr80

gag

-negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80

gag

mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80

gag

negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80

gag

in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80

gag

produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80

gag

is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.
The Large and Small Isoforms of Human Papillomavirus Type 16 E6 Bind to and Differentially Affect Procaspase 8 Stability and Activity
Authors: Filippova Maria , Johnson Melyssa M. , Bautista Marnelli , Filippov Valery , Fodor Nadja , Tungteakkhun Sandy S. , Williams Kadia , Duerksen-Hughes Penelope J.
Published: February 2007

Human papillomavirus type 16 (HPV-16) has developed numerous ways to modulate host-initiated immune mechanisms. The HPV-16 E6 oncoprotein, for example, can modulate the cellular level, and consequently the activity, of procaspase 8, thus modifying the cellular response to cytokines of the tumor necrosis factor family. E6 from HPV-16, but not E6 from the low-risk types 6b and 11, alters the cellular level of procaspase 8 in a dose-dependent manner. Both the large and small (E6*) isoforms of E6, which originate by way of alternate splicing, can modulate procaspase 8 stability. Intriguingly, although both isoforms bind to procaspase 8, the large isoform accelerates the degradation of procaspase 8 while the small isoform stabilizes it. Binding leads to a change in the ability of procaspase 8 to bind either to itself or to FADD (Fas-associated death domain), with the large version of E6 able to inhibit this binding while the small isoform does not. Consistent with this model, knockdown of the large version of E6 by small interfering RNA leads to increases in the levels of procaspase 8 and its binding to both itself and FADD. Thus, these alternatively spliced isoforms can modulate both the level and the activity of procaspase 8 in opposite directions.
Emergence of Influenza A Virus Variants after Prolonged Shedding from Pheasants
Authors: Humberd Jennifer , Boyd Kelli , Webster Robert G.
Published: February 2007

We previously demonstrated the susceptibility of pheasants to infection with influenza A viruses of 15 hemagglutinin (HA) subtypes: 13/23 viruses tested were isolated for ≥14 days, all in the presence of serum-neutralizing antibodies; one virus (H10) was shed for 45 days postinfection. Here we confirmed that 20% of pheasants shed low-pathogenic influenza viruses for prolonged periods. We aimed to determine why the antibody response did not clear the virus in the usual 3 to 10 days, because pheasants serve as a long-term source of influenza viruses in poultry markets. We found evidence of virus replication and histological changes in the large intestine, bursa of Fabricius, and cecal tonsil. The virus isolated 41 days postinfection was antigenically distinct from the parental H10 virus, with corresponding changes in the HA and neuraminidase. Ten amino acid differences were found between the parental H10 and the pheasant H10 virus; four were in potential antigenic sites of the HA molecule. Prolonged shedding of virus by pheasants results from a complex interplay between the diversity of virus variants and the host response. It is often argued that vaccination pressure is a mechanism that contributes to the generation of antigenic-drift variants in poultry. This study provided evidence that drift variants can occur naturally in pheasants after prolonged shedding of virus, thus strengthening our argument for the removal of pheasants from live-bird retail markets.
Dimer Initiation Signal of Human Immunodeficiency Virus Type 1: Its Role in Partner Selection during RNA Copackaging and Its Effects on Recombination
Authors: Moore Michael D. , Fu William , Nikolaitchik Olga , Chen Jianbo , Ptak Roger G. , Hu Wei-Shau
Published: February 2007

Frequent human immunodeficiency virus type 1 (HIV-1) recombination occurs during DNA synthesis when portions of the two copackaged RNAs are used as templates to generate a hybrid DNA copy. Therefore, the frequency of copackaging of genomic RNAs from two different viruses (heterozygous virion formation) affects the generation of genotypically different recombinants. We hypothesized that the selection of copackaged RNA partners is largely determined by Watson-Crick pairing at the dimer initiation signal (DIS), a 6-nucleotide palindromic sequence at the terminal loop of stem-loop 1 (SL1). To test our hypothesis, we examined whether heterozygous virion formation could be encouraged by manipulation of the DIS. Three pairs of viruses were generated with compensatory DIS mutations, designed so that perfect DIS base pairing could only occur between RNAs derived from different viruses, not between RNAs from the same virus. We observed that vector pairs with compensatory DIS mutations had an almost twofold increase in recombination rates compared with wild-type viruses. These data suggest that heterozygous virion formation was enhanced in viruses with compensatory DIS mutations (from 50% to more than 90% in some viral pairings). The role of the SL1 stem in heterozygous virion formation was also tested; our results indicated that the intermolecular base pairing of the stem sequences does not affect RNA partner selection. In summary, our results demonstrate that the Watson-Crick pairing of the DIS is a major determinant in the selection of the copackaged RNA partner, and altering the base pairing of the DIS can change the proportion of heterozygous viruses in a viral population. These results also strongly support the hypothesis that HIV-1 RNA dimers are formed prior to encapsidation.
Susceptibility of CCR5-Deficient Mice to Genital Herpes Simplex Virus Type 2 Is Linked to NK Cell Mobilization
Authors: Thapa Manoj , Kuziel William A. , Carr Daniel J. J.
Published: February 2007

Following genital herpes simplex virus type 2 (HSV-2) exposure, NK cells and T cells are mobilized to sites of infection to control viral replication and spread. The present investigation sought to determine the role of the chemokine receptor CCR5 in this process. Mice deficient in CCR5 (CCR5−/−) displayed a significant reduction in cumulative survival following infection in comparison to wild-type, HSV-2-infected controls. Associated with decreased resistance to viral infection, CCR5−/−mice yielded significantly more virus and expressed higher levels of tumor necrosis factor alpha, CXCL1, CCL2, CCL3, and CCL5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. Whereas there was no difference in absolute number of leukocytes (CD45high), CD4 T cells, or CD8 T cells residing in the draining lymph nodes, spleen, spinal cord, or brain stem comparing HSV-2-infected wild-type to CCR5−/−mice prior to or after infection, there were significantly more NK cells (NK1.1+CD3−) residing in the brain stem and spleen of infected wild-type mice. Functionally, NK activity from cells isolated from the brain stem of HSV-2-infected wild-type mice was greater than that from HSV-2-infected CCR5−/−mice. In addition, antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vaginal, spinal cord, and brain stem tissue of wild-type but not CCR5−/−mice. Collectively, the absence of CCR5 expression significantly impacts the ability of the host to control genital HSV-2 infection, inflammation, and spread associated with a specific reduction in NK cell expansion, infiltration, and activity in the nervous system.
New Natural Intergenotypic (2/5) Recombinant of Hepatitis C Virus
Authors: Legrand-Abravanel Florence , Claudinon Julie , Nicot Florence , Dubois Martine , Chapuy-Regaud Sabine , Sandres-Saune Karine , Pasquier Christophe , Izopet Jacques
Published: February 2007

A 9.2-kb sequence from a hepatitis C virus (HCV) strain found in southwest France was compared to sequences from reference strains in HCV sequence databases. We found a recombinant virus with genotype 2 at the 5′ end and genotype 5 at the 3′ end. The crossover point was located between genes NS2 and NS3. Recombination between HCV genotypes must now be considered in studies on HCV epidemiology and evolution and in predictions of the virus response to antiviral therapy. Knowing the location of the recombination point may also be useful for constructing infectious chimeric viruses.
Canine Distemper Virus Infection Requires Cholesterol in the Viral Envelope
Authors: Imhoff Heidi , von Messling Veronika , Herrler Georg , Haas Ludwig
Published: February 2007

Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-β-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.
Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration
Authors: Murphy Mary , Gomos-Klein Janette , Stankic Marko , Falck-Pedersen Erik
Published: February 2007

The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.
Antigenic and Mutational Analyses of Herpes Simplex Virus Glycoprotein B Reveal Four Functional Regions
Authors: Bender Florent C. , Samanta Minu , Heldwein Ekaterina E. , de Leon Manuel Ponce , Bilman Elina , Lou Huan , Whitbeck J. Charles , Eisenberg Roselyn J. , Cohen Gary H.
Published: February 2007

Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by α-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.
Critical Role of Dendritic Cells in Mouse Mammary Tumor Virus In Vivo Infection
Authors: Courreges Maria Cecilia , Burzyn Dalia , Nepomnaschy Irene , Piazzon Isabel , Ross Susan R.
Published: February 2007

Mouse mammary tumor virus (MMTV) is a milk-transmitted betaretrovirus that causes mammary tumors in mice. Although mammary epithelial cells are the ultimate targets of MMTV, the virus utilizes components of the host immune system to establish infection. Previous studies indicated that dendritic cells play a role in MMTV infection. Here we show that dendritic cells are the first cells to be infected by MMTV in vivo and that they are capable of producing infectious virus that can be transmitted to other cell types. Moreover, upon contact with the virus, dendritic cells became more mature and migrated in response to the chemokine macrophage inflammatory protein 3β. Finally, we demonstrate that targeted ablation of dendritic cells in vivo dramatically attenuated MMTV infection. These data indicate that MMTV infection of dendritic cells is critical to initial propagation of the virus in vivo.
Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking
Authors: Patient Romuald , Hourioux Christophe , Sizaret Pierre-Yves , Trassard Sylvie , Sureau Camille , Roingeard Philippe
Published: February 2007

Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.