bluuebunny/crossref_metadata_2025 huggingface dataset:
Is it possible to detect the presence of adenovirus in conjunctiva before the onset of conjunctivitis?
Authors: Kimura Ryoji , Migita Hironori , Kadonosono Kazuaki , Uchio Eiichi
Published: March 2008

Purpose:  It is widely accepted that patients with adenoviral conjunctivitis are contagious approximately as long as 14 days after the onset. However, there is controversy about whether patients with adenoviral conjunctivitis are contagious during the incubation period. We investigated the potential of the eye as a source for spreading infection during the incubation period by analysing the presence of adenovirus (Ad) in contralateral eyes before onset in patients with bilateral adenoviral conjunctivitis using the immunochromatography (IC) test and polymerase chain reaction (PCR).

Methods:  Patients with adenoviral conjunctivitis were directed to visit an outpatient clinic at approximately 3‐day intervals until onset in the contralateral eye or 2 weeks after onset in the first eye. Conjunctival scrapings obtained from the first infected eye and the contralateral eye before and after onset were tested to detect Ad antigen using the IC test and PCR.

Results:  Out of the 32 cases, 23 (72%) developed conjunctivitis in the contralateral eye. Among these bilateral cases, 18 (78%) were found positive for Ad by PCR after onset in the contralateral eye. However, no specimen obtained before onset showed a positive result for Ad in both tests.

Conclusion:  Although there is a possibility that eyes are contagious during the incubation period of adenoviral conjunctivitis in some situations, these results suggest that the possibility of the eye acting as a source for spreading infection during the incubation period is very small.
Acacia ochracea (Leguminosae‐Mimosoideae), a new species from Somalia
Authors: Thulin Mats , Hassan Abdisalam Sheikh
Published: March 2008

Acacia ochracea, a new species in the A. Senegal complex, is described and illustrated. It is a common and conspicuous tree over large areas in the western Bay Region and in the Gedo Region in SW Somalia.
Multilevel Regulation of Gene Expression by MicroRNAs
Authors: Makeyev Eugene V. , Maniatis Tom
Published: March 2008

MicroRNAs (miRNAs) are ∼22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.
Complex Riboswitches
Authors: Breaker Ronald R.
Published: March 2008

Using simple biochemical tricks, metabolite-binding riboswitches take on gene control functions that have long been thought to be the work of protein factors. Although modern riboswitches might be the last holdouts of primitive genetic elements, some are capable of sensory and regulatory feats that are competitive with their protein counterparts.
Aversive Learning Enhances Perceptual and Cortical Discrimination of Indiscriminable Odor Cues
Authors: Li Wen , Howard James D. , Parrish Todd B. , Gottfried Jay A.
Published: March 2008

Learning to associate sensory cues with threats is critical for minimizing aversive experience. The ecological benefit of associative learning relies on accurate perception of predictive cues, but how aversive learning enhances perceptual acuity of sensory signals, particularly in humans, is unclear. We combined multivariate functional magnetic resonance imaging with olfactory psychophysics to show that initially indistinguishable odor enantiomers (mirror-image molecules) become discriminable after aversive conditioning, paralleling the spatial divergence of ensemble activity patterns in primary olfactory (piriform) cortex. Our findings indicate that aversive learning induces piriform plasticity with corresponding gains in odor enantiomer discrimination, underscoring the capacity of fear conditioning to update perceptual representation of predictive cues, over and above its well-recognized role in the acquisition of conditioned responses. That completely indiscriminable sensations can be transformed into discriminable percepts further accentuates the potency of associative learning to enhance sensory cue perception and support adaptive behavior.
Structure of the Immature Dengue Virus at Low pH Primes Proteolytic Maturation
Authors: Yu I-Mei , Zhang Wei , Holdaway Heather A. , Li Long , Kostyuchenko Victor A. , Chipman Paul R. , Kuhn Richard J. , Rossmann Michael G. , Chen Jue
Published: March 2008

Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo–electron microscopy we show that the immature dengue particles undergo a reversible conformational change at low pH that renders them accessible to furin cleavage. At a pH of 6.0, the E proteins are arranged in a herringbone pattern with the pr peptides docked onto the fusion loops, a configuration similar to that of the mature virion. After cleavage, the dissociation of pr is pH-dependent, suggesting that in the acidic environment of the trans-Golgi network pr is retained on the virion to prevent membrane fusion. These results suggest a mechanism by which flaviviruses are processed and stabilized in the host cell secretory pathway.
Dynamics of Saturn's South Polar Vortex
Authors: Dyudina Ulyana A. , Ingersoll Andrew P. , Ewald Shawn P. , Vasavada Ashwin R. , West Robert A. , Del Genio Anthony D. , Barbara John M. , Porco Carolyn C. , Achterberg Richard K. , Flasar F. Michael , Simon-Miller Amy A. , Fletcher Leigh N.
Published: March 2008

The camera onboard the Cassini spacecraft has allowed us to observe many of Saturn's cloud features. We present observations of Saturn's south polar vortex (SPV) showing that it shares some properties with terrestrial hurricanes: cyclonic circulation, warm central region (the eye) surrounded by a ring of high clouds (the eye wall), and convective clouds outside the eye. The polar location and the absence of an ocean are major differences. It also shares properties with the polar vortices on Venus, such as polar location, cyclonic circulation, warm center, and long lifetime, but the Venus vortices have cold collars and are not associated with convective clouds. The SPV's combination of properties is unique among vortices in the solar system
Surface Trapping of Atoms and Molecules with Dipole Rings
Authors: Dil Hugo , Lobo-Checa Jorge , Laskowski Robert , Blaha Peter , Berner Simon , Osterwalder Jürg , Greber Thomas
Published: March 2008

The trapping of single molecules on surfaces without the formation of strong covalent bonds is a prerequisite for molecular recognition and the exploitation of molecular function. On nanopatterned surfaces, molecules may be selectively trapped and addressed. In a boron nitride nanomesh formed on Rh(111), the pattern consisted of holes 2 nanometers in diameter on a hexagonal superlattice, separated by about 3 nanometers. The trapping was further investigated with density functional theory and the photoemission of adsorbed xenon, where the holes were identified as regions of low work function. The analysis showed that the trapping potential was localized at the rims of the holes.
Self-Assembly of Large and Small Molecules into Hierarchically Ordered Sacs and Membranes
Authors: Capito Ramille M. , Azevedo Helena S. , Velichko Yuri S. , Mata Alvaro , Stupp Samuel I.
Published: March 2008

We report here the self-assembly of macroscopic sacs and membranes at the interface between two aqueous solutions, one containing a megadalton polymer and the other, small self-assembling molecules bearing opposite charge. The resulting structures have a highly ordered architecture in which nanofiber bundles align and reorient by nearly 90° as the membrane grows. The formation of a diffusion barrier upon contact between the two liquids prevents their chaotic mixing. We hypothesize that growth of the membrane is then driven by a dynamic synergy between osmotic pressure of ions and static self-assembly. These robust, self-sealing macroscopic structures offer opportunities in many areas, including the formation of privileged environments for cells, immune barriers, new biological assays, and self-assembly of ordered thick membranes for diverse applications.
Silica-on-Silicon Waveguide Quantum Circuits
Authors: Politi Alberto , Cryan Martin J. , Rarity John G. , Yu Siyuan , O'Brien Jeremy L.
Published: March 2008

Quantum technologies based on photons will likely require an integrated optics architecture for improved performance, miniaturization, and scalability. We demonstrate high-fidelity silica-on-silicon integrated optical realizations of key quantum photonic circuits, including two-photon quantum interference with a visibility of 94.8 ± 0.5%; a controlled-NOT gate with an average logical basis fidelity of 94.3 ± 0.2%; and a path-entangled state of two photons with fidelity of >92%. These results show that it is possible to directly “write” sophisticated photonic quantum circuits onto a silicon chip, which will be of benefit to future quantum technologies based on photons, including information processing, communication, metrology, and lithography, as well as the fundamental science of quantum optics.
Explaining the Cosmos to the People
Authors: Pauly Philip J.
Published: March 2008

Victorian Popularizers of Science
. Designing Nature for New Audiences.
By Bernard Lightman
. University of Chicago Press, Chicago, 2007. 563 pp. $45, £23.50. ISBN 9780226481180.

The author provides a scholarly, detailed consideration of the bloom of books by writers and journalists who presented science to the British public through the second half of the 19th century
Survival of Influenza Virus on Banknotes
Authors: Thomas Yves , Vogel Guido , Wunderli Werner , Suter Patricia , Witschi Mark , Koch Daniel , Tapparel Caroline , Kaiser Laurent
Published: March 2008

Successful control of a viral disease requires knowledge of the different vectors that could promote its transmission among hosts. We assessed the survival of human influenza viruses on banknotes given that billions of these notes are exchanged daily worldwide. Banknotes were experimentally contaminated with representative influenza virus subtypes at various concentrations, and survival was tested after different time periods. Influenza A viruses tested by cell culture survived up to 3 days when they were inoculated at high concentrations. The same inoculum in the presence of respiratory mucus showed a striking increase in survival time (up to 17 days). Similarly, B/Hong Kong/335/2001 virus was still infectious after 1 day when it was mixed with respiratory mucus. When nasopharyngeal secretions of naturally infected children were used, influenza virus survived for at least 48 h in one-third of the cases. The unexpected stability of influenza virus in this nonbiological environment suggests that unusual environmental contamination should be considered in the setting of pandemic preparedness.
Collateral Effects of Antibiotics: Carbadox and Metronidazole Induce VSH-1 and Facilitate Gene Transfer among
Brachyspira hyodysenteriae
Strains
Authors: Stanton Thaddeus B. , Humphrey Samuel B. , Sharma Vijay K. , Zuerner Richard L.
Published: March 2008

Brachyspira hyodysenteriae
is an anaerobic spirochete and the etiologic agent of swine dysentery. The genome of this spirochete contains a mitomycin C-inducible, prophage-like gene transfer agent designated VSH-1. VSH-1 particles package random 7.5-kb fragments of the
B. hyodysenteriae
genome and transfer genes between
B. hyodysenteriae
cells. The chemicals and conditions inducing VSH-1 production are largely unknown. Antibiotics used in swine management and stressors inducing traditional prophages might induce VSH-1 and thereby stimulate lateral gene transfer between
B. hyodysenteriae
cells. In these studies, VSH-1 induction was initially detected by a quantitative real-time reverse transcriptase PCR assay evaluating increased transcription of
hvp38
(VSH-1 head protein gene). VSH-1 induction was confirmed by detecting VSH-1-associated 7.5-kb DNA and VSH-1 particles in
B. hyodysenteriae
cultures. Nine antibiotics (chlortetracycline, lincomycin, tylosin, tiamulin, virginiamycin, ampicillin, ceftriaxone, vancomycin, and florfenicol) at concentrations affecting
B. hyodysenteriae
growth did not induce VSH-1 production. By contrast, VSH-1 was detected in
B. hyodysenteriae
cultures treated with mitomycin C (10 μg/ml), carbadox (0.5 μg/ml), metronidazole (0.5 μg/ml), and H
2
O
2
(300 μM). Carbadox- and metronidazole-induced VSH-1 particles transmitted tylosin and chloramphenicol resistance determinants between
B. hyodysenteriae
strains. The results of these studies suggest that certain antibiotics may induce the production of prophage or prophage-like elements by intestinal bacteria and thereby impact intestinal microbial ecology.
Unexpected Importance of Potential Parasites in the Composition of the Freshwater Small-Eukaryote Community
Authors: Lepère Cécile , Domaizon Isabelle , Debroas Didier
Published: March 2008

The diversity of small eukaryotes (0.2 to 5 μm) in a mesotrophic lake (Lake Bourget) was investigated using 18S rRNA gene library construction and fluorescent in situ hybridization coupled with tyramide signal amplification (TSA-FISH). Samples collected from the epilimnion on two dates were used to extend a data set previously obtained using similar approaches for lakes with a range of trophic types. A high level of diversity was recorded for this system with intermediate trophic status, and the main sequences from Lake Bourget were affiliated with ciliates (maximum, 19% of the operational taxonomic units [OTUs]), cryptophytes (33%), stramenopiles (13.2%), and cercozoa (9%). Although the comparison of TSA-FISH results and clone libraries suggested that the level of Chlorophyceae may have been underestimated using PCR with 18S rRNA primers, heterotrophic organisms dominated the small-eukaryote assemblage. We found that a large fraction of the sequences belonged to potential parasites of freshwater phytoplankton, including sequences affiliated with fungi and Perkinsozoa. On average, these sequences represented 30% of the OTUs (40% of the clones) obtained for each of two dates for Lake Bourget. Our results provide information on lacustrine small-eukaryote diversity and structure, adding to the phylogenetic data available for lakes with various trophic types.
Comparative Genomics of “
Dehalococcoides ethenogenes
” 195 and an Enrichment Culture Containing Unsequenced “
Dehalococcoides
” Strains
Authors: West Kimberlee A. , Johnson David R. , Hu Ping , DeSantis Todd Z. , Brodie Eoin L. , Lee Patrick K. H. , Feil Helene , Andersen Gary L. , Zinder Stephen H. , Alvarez-Cohen Lisa
Published: March 2008

Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by some “
Dehalococcoides
” organisms. A
Dehalococcoides
-organism-containing microbial consortium (referred to as ANAS) with the ability to degrade TCE to ethene, an innocuous end product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of “
Dehalococcoides ethenogenes
” 195. This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray results revealed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, cobalamin-salvaging system, nitrogen fixation pathway, and five hydrogenase complexes, are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase,
tceA
, was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of
Dehalococcoides
genomes will improve our understanding of the physiology and evolution of these bacteria, which is essential in developing effective strategies for the bioremediation of PCE and TCE in the environment.
Comparison of Methods for Processing Drinking Water Samples for the Isolation of
Mycobacterium avium
and
Mycobacterium intracellulare
Authors: Thomson Rachel , Carter Robyn , Gilpin Chris , Coulter Chris , Hargreaves Megan
Published: March 2008

Several protocols for isolation of mycobacteria from water exist, but there is no established standard method. This study compared methods of processing potable water samples for the isolation of
Mycobacterium avium
and
Mycobacterium intracellulare
using spiked sterilized water and tap water decontaminated using 0.005% cetylpyridinium chloride (CPC). Samples were concentrated by centrifugation or filtration and inoculated onto Middlebrook 7H10 and 7H11 plates and Lowenstein-Jensen slants and into mycobacterial growth indicator tubes with or without polymyxin, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. The solid media were incubated at 32°C, at 35°C, and at 35°C with CO
2
and read weekly. The results suggest that filtration of water for the isolation of mycobacteria is a more sensitive method for concentration than centrifugation. The addition of sodium thiosulfate may not be necessary and may reduce the yield. Middlebrook M7H10 and 7H11 were equally sensitive culture media. CPC decontamination, while effective for reducing growth of contaminants, also significantly reduces mycobacterial numbers. There was no difference at 3 weeks between the different incubation temperatures.
Occurrence and Expression of Gene Transfer Agent Genes in Marine Bacterioplankton
Authors: Biers Erin J. , Wang Kui , Pennington Catherine , Belas Robert , Chen Feng , Moran Mary Ann
Published: March 2008

Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine
Roseobacter
clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured
Alphaproteobacteria
, and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles (∼50 to 70 nm) was demonstrated experimentally for the
Roseobacter
clade member
Silicibacter pomeroyi
DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear.
Diverse Responses of the Bivalve-Killing Dinoflagellate
Heterocapsa circularisquama
to Infection by a Single-Stranded RNA Virus
Authors: Mizumoto Hiroyuki , Tomaru Yuji , Takao Yoshitake , Shirai Yoko , Nagasaki Keizo
Published: March 2008

Viruses are believed to be significant pathogens for phytoplankton. Usually, they infect a single algal species, and often their infection is highly strain specific. However, the detailed molecular background of the strain specificity and its ecological significance have not been sufficiently understood. Here, we investigated the temporal changes in viral RNA accumulation and virus-induced cell lysis using a bloom-forming dinoflagellate
Heterocapsa circularisquama
and its single-stranded RNA virus, HcRNAV. We observed at least three host response patterns to virus inoculation: sensitive, resistant, and delayed lysis. In the sensitive response, the host cell culture was permissive for viral RNA replication and apparent cell lysis was observed; in contrast, resistant cell culture was nonpermissive for viral RNA replication and not lysed. In the delayed-lysis response, although viral RNA replication occurred, virus-induced cell lysis was faint and remarkably delayed. In addition, the number of infectious virus particles released to the culture supernatant at 12 days postinoculation was comparable to that of the sensitive strain. By further analysis, a few strains were characterized as variants of the delayed-lysis strain. These observations indicate that the response of
H. circularisquama
to HcRNAV infection is highly diverse.
Bactericidal Effect of Solar Water Disinfection under Real Sunlight Conditions
Authors: Boyle M. , Sichel C. , Fernández-Ibáñez P. , Arias-Quiroz G. B. , Iriarte-Puña M. , Mercado A. , Ubomba-Jaswa E. , McGuigan K. G.
Published: March 2008

Batch solar disinfection (SODIS) inactivation kinetics are reported for suspensions in water of
Campylobacter jejuni
,
Yersinia enterocolitica
, enteropathogenic
Escherichia coli
,
Staphylococcus epidermidis
, and endospores of
Bacillus subtilis
, exposed to strong natural sunlight in Spain and Bolivia. The exposure time required for complete inactivation (at least 4-log-unit reduction and below the limit of detection, 17 CFU/ml) under conditions of strong natural sunlight (maximum global irradiance, ∼1,050 W m
−2
± 10 W m
−2
) was as follows:
C. jejuni
, 20 min;
S. epidermidis
, 45 min; enteropathogenic
E. coli
, 90 min;
Y. enterocolitica
, 150 min. Following incomplete inactivation of
B. subtilis
endospores after the first day, reexposure of these samples on the following day found that 4% (standard error, 3%) of the endospores remained viable after a cumulative exposure time of 16 h of strong natural sunlight. SODIS is shown to be effective against the vegetative cells of a number of emerging waterborne pathogens; however, bacterial species which are spore forming may survive this intervention process.
Phylogenetic Analysis of Members of the
Phycodnaviridae
Virus Family, Using Amplified Fragments of the Major Capsid Protein Gene
Authors: Larsen J. B. , Larsen A. , Bratbak G. , Sandaa R.-A.
Published: March 2008

Algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. Members of the family
Phycodnaviridae
are also interesting due to their extraordinary genome size. Few algal viruses in the
Phycodnaviridae
family have been sequenced, and those that have been have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study, we screened the nine type I core genes of the nucleocytoplasmic large DNA viruses for sequences suitable for designing a general set of primers. Sequence comparison between members of the
Phycodnaviridae
family, including three partly sequenced viruses infecting the prymnesiophyte
Pyramimonas orientalis
and the haptophytes
Phaeocystis pouchetii
and
Chrysochromulina ericina
(
Pyramimonas orientalis
virus 01B [PoV-01B],
Phaeocystis pouchetii
virus 01 [PpV-01], and
Chrysochromulina ericina
virus 01B [CeV-01B], respectively), revealed eight conserved regions in the major capsid protein (MCP). Two of these regions also showed conservation at the nucleotide level, and this allowed us to design degenerate PCR primers. The primers produced 347- to 518-bp amplicons when applied to lysates from algal viruses kept in culture and from natural viral communities. The aim of this work was to use the MCP as a proxy to infer phylogenetic relationships and genetic diversity among members of the
Phycodnaviridae
family and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the
Phycodnaviridae
based on alga host species. However, while placing the mimivirus in close proximity to the type species, PBCV-1, of
Phycodnaviridae
along with the three new viruses assigned to the family (PoV-01B, PpV-01, and CeV-01B), the results also indicate that the coccolithoviruses and phaeoviruses are more diverged from this group. Phylogenetic analysis of amplicons from virus assemblages from Norwegian coastal waters as well as from isolated algal viruses revealed a cluster of viruses infecting members of the prymnesiophyte and prasinophyte alga divisions. Other distinct clusters were also identified, containing amplicons from this study as well as sequences retrieved from the Sargasso Sea metagenome. This shows that closely related sequences of this family are present at geographically distant locations within the marine environment.
Correlation between Detection of a Plasmid and High-Level Virulence of
Vibrio nigripulchritudo
, a Pathogen of the Shrimp
Litopenaeus stylirostris
Authors: Reynaud Yann , Saulnier Denis , Mazel Didier , Goarant Cyrille , Le Roux Frédérique
Published: March 2008

Vibrio nigripulchritudo
, the etiological agent of
Litopenaeus stylirostris
summer syndrome, is responsible for mass mortalities of shrimp in New Caledonia. Epidemiological studies led to the suggestion that this disease is caused by an emergent group of pathogenic strains. Genomic subtractive hybridization was carried out between two isolates exhibiting low and high virulence. Our subtraction library was constituted of 521 specific fragments; 55 of these were detected in all virulent isolates from our collection (
n
= 32), and 13 were detected only in the isolates demonstrating the highest pathogenicity (
n
= 19), suggesting that they could be used as genetic markers for high virulence capacity. Interestingly, 10 of these markers are carried by a replicon of 11.2 kbp that contains sequences highly similar to those of a plasmid detected in
Vibrio shilonii
, a coral pathogen. The detection of this plasmid was correlated with the highest pathogenicity status of the isolates from our collection. The origin and consequence of this plasmid acquisition are discussed.
Monitoring of Vaccine-Specific Gamma Interferon Induction in Genital Mucosa of Mice by Real-Time Reverse Transcription-PCR
Authors: Revaz Véronique , Debonneville Anne , Bobst Martine , Nardelli-Haefliger Denise
Published: March 2008

Monitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-γ) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1165-173H-2Db-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN-γ enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN-γ mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN-γ induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.
Reciprocal Expression of
ospA
and
ospC
in Single Cells of
Borrelia burgdorferi
Authors: Srivastava Siddharth Y. , de Silva Aravinda M.
Published: March 2008

Outer surface proteins (Osp) A and C of the Lyme disease spirochete (
Borrelia burgdorferi
) are selectively produced and of functional significance in the tick vector and mammalian host, respectively. Some studies indicate a simple, reciprocal relationship where the signals and pathways that turn on
ospC
also turn off
ospA
. Other studies indicate a more complex regulation where many spirochetes produce both proteins and others produce one of the proteins or neither protein. Here, we have used flow cytometry to characterize
ospA
and
ospC
transcript and protein levels in individual bacterial cells grown in culture. The results support a simple, reciprocal model where, at the level of single cells, the transcription of
ospC
is linked to the repression of
ospA
. We also demonstrate that under conditions conducive for OspC production, spirochetes display an “all or none” response, with some cells displaying high levels of
ospC
transcription and others demonstrating little or no transcription. Despite the reciprocal regulation of
ospA
and
ospC
at the single-cell level, we propose that spirochetes display an array of phenotypes due to stochasticity in the pathways that regulate
osp
expression and the slow turnover of outer surface proteins.
An Ortholog of OxyR in
Legionella pneumophila
Is Expressed Postexponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase (
ahpC2D
) Operon
Authors: LeBlanc Jason J. , Brassinga Ann Karen C. , Ewann Fanny , Davidson Ross J. , Hoffman Paul S.
Published: March 2008

Legionella pneumophila
expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the
ahpC2D
operon. The major protein captured was an ortholog of OxyR (OxyR
Lp
). Genetic studies indicated that

oxyR
Lp

was an essential gene expressed postexponentially and only partially complemented an
Escherichia coli oxyR
mutant (GS077). Gel shift assays confirmed specific binding of OxyR
Lp
to
ahpC2D
promoter sequences, but not to promoters of
ahpC1
or

oxyR
Lp

; however, OxyR
Lp
weakly bound to
E. coli
OxyR-regulated promoters (
katG
,
oxyR
, and
ahpCF
). DNase I protection studies showed that the OxyR
Lp
binding motif spanned the promoter and transcriptional start sequences of
ahpC2
and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H
2
O
2
). Moreover, the OxyR
Lp
(pBADLp
oxyR
)-mediated repression of an
ahpC2
-
gfp
reporter construct in
E. coli
GS077 (the
oxyR
mutant) was not reversed by H
2
O
2
challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR
Lp
in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.
RNase E Regulates the
Yersinia
Type 3 Secretion System
Authors: Yang Jing , Jain Chaitanya , Schesser Kurt
Published: March 2008

Yersinia
spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in
Yersinia pseudotuberculosis
and
Yersinia pestis
. Here we report that
Y. pseudotuberculosis
cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble Δ
pnp
cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from
Y. pseudotuberculosis
cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity.
Differential Regulation by Magnesium of the Two MsbB Paralogs ofShigella flexneri
Authors: Goldman Seth R. , Tu Yupeng , Goldberg Marcia B.
Published: March 2008

Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream ofmsbB2were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion oflacZto this promoter. Thus, the virulence plasmid-encoded paralog ofmsbBis induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of manyEnterobacteriaceaeto environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins ofS. flexnerilikely contributes to pathogenesis.
Differential Mechanisms of Binding of Anti-Sigma Factors
Escherichia coli
Rsd and Bacteriophage T4 AsiA to
E. coli
RNA Polymerase Lead to Diverse Physiological Consequences
Authors: Sharma Umender K. , Chatterji Dipankar
Published: March 2008

Anti-sigma factors
Escherichia coli
Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ
70
, of
E. coli
. Though both factors are known to interact with the C-terminal region of σ
70
, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which
E. coli
Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of
E. coli
RNA polymerase, which leads to the inhibition of
E. coli
cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of
E. coli
cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ
70
with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ
70
studied by using the yeast two-hybrid system revealed that region 4 of σ
70
is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ
70
as well. Taken together, these results suggest that the higher inhibition of
E. coli
growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ
70
.
The Gene Encoding RNase III in
Streptomyces coelicolor
Is Transcribed during Exponential Phase and Is Required for Antibiotic Production and for Proper Sporulation
Authors: Sello Jason K. , Buttner Mark J.
Published: March 2008

Phenotypic analysis of a constructed RNase III null mutant of
Streptomyces coelicolor
revealed that RNase III is required for both antibiotic production and proper formation of sporulation septa. Transcriptional analysis of the gene encoding RNase III indicated that it is transcribed exclusively during exponential phase as part of a tricistronic message.
Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression
Authors: Ravin Nikolai V. , Rech Jérôme , Lane David
Published: March 2008

The mitotic stability of the linear plasmid-prophage N15 of
Escherichia coli
depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (
sopC
) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and
lacZ
reporter activity confirmed the identity of the N15 late promoter (
p52
), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between
p52
and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene
Q
) or IR3 (upstream of
p52
) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of
ir3
−
-
p52
::
lacZ
during N15 wild-type lytic growth was strongly reduced relative to the equivalent
ir3
+
fusion. The provision of Q protein and the IR2 and SopAB proteins in
trans
to
ir3
+
-
p52
::
lacZ
increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.
Evaluation of a Commercial Direct Fluorescent-Antibody Assay for Human Metapneumovirus in Respiratory Specimens
Authors: Vinh Donald C. , Newby Diane , Charest Hugues , McDonald Jane
Published: March 2008

The performance of a commercially manufactured direct fluorescent-antibody assay kit for human metapneumovirus was compared to that of reverse transcriptase PCR, the current “gold standard.” The kit demonstrated a sensitivity of 95.2%, a specificity of 100%, and an accuracy of 98.9%.
Diagnostic and Clinical Implications of a Nested PCR Specific for Ribosomal DNA of the Feline Lungworm
Aelurostrongylus abstrusus
(Nematoda, Strongylida)
Authors: Traversa Donato , Iorio Raffaella , Otranto Domenico
Published: March 2008

Aelurostrongylus abstrusus
(Nematoda, Strongylida, Metastrongyloidea) is a cosmopolitan parasite of cats and causes severe respiratory distress. Information on the biology and epidemiology of feline aelurostrongylosis is fragmentary, mainly due to the limits inherent in the classical diagnosis. In the present work, a two-step nested PCR based on the use of genetic markers in the second internal transcribed spacer (ITS2) of ribosomal DNA was established for
A. abstrusus
in different biological samples. Characterization of the ITS2 (321 bp of length) revealed a G+C content of 39.5%. To exploit the sequence difference between the ITS2 of
A. abstrusus
and those of other common feline endoparasites, specific primers were designed and tested by PCR for their specificities and sensitivities. The PCR assay was validated on a panel of fecal (i.e., feces, flotation supernatant, and Baermann sediment) and pharyngeal swab samples from cats with known histories of lungworm infections, and it showed a specificity of 100% and a sensitivity of up to 96.6%. Also, the nested PCR was able to identify cats that were actually infected but that tested negative by the classical diagnostic methods. This PCR method was shown to be a powerful tool for the molecular diagnosis of feline aelurostrongylosis, overcoming the constraints of the classical diagnosis. The implications of such a molecular tool for further bioepidemiological studies of both intermediate and definitive hosts have been discussed.
Assessment of
Echinococcus granulosus
Somatic Protoscolex Antigens for Serological Follow-Up of Young Patients Surgically Treated for Cystic Echinococcosis
Authors: Nouir Nadia Ben , Nuñez Sandra , Gianinazzi Christian , Gorcii Mohamed , Müller Norbert , Nouri Abdellatif , Babba Hamouda , Gottstein Bruno
Published: March 2008

Echinococcus granulosus
protoscolex soluble somatic antigens (PSSAs) were assessed for their prognostic value in the serological follow-up of young patients treated for cystic echinococcosis (CE), compared to conventional hydatid fluid (HF) antigen. Based on different clinical courses and outcome of infection, as well as imaging findings, patients were retrospectively classified into two different groups including either cured CE (CCE; i.e., absence of active cysts or presence of inactive cysts, respectively) and noncured CE (NCCE) patients still presenting active cysts at the end of an up to 5-year follow-up period. An immunoglobulin G (IgG)-PSSA enzyme-linked immunosorbent assay (ELISA) showed a gradual decrease in antibody levels in CCE cases, reaching seronegativity in 20% of the cases at least within 5 years postsurgery. In comparison, the conventional IgG-HF ELISA showed a significantly lower progressive decrease in antibody levels, serology becoming negative in only 15% of CCE patients at the endpoint of the follow-up period. Serological analysis of PSSA by immunoblotting yielded an interesting immunoreactive double band of 27 and 28 kDa that, in 15 (75%) of 20 CCE cases, exhibited a rapid decrease and subsequent disappearance of respective antibody reactivities within 3 years postsurgery. Conversely, anti-27- and -28-kDa antibody reactivity strongly persisted until the endpoint of the follow-up period in all of the five NCCE patients. Further analysis of the 27- and 28-kDa doublet by using affinity-purified antibodies showed that the double band was not detectable in HF. Furthermore, a predominantly IgG4 subclass-restricted humoral immune response against the 27- and 28-kDa antigens was demonstrated in seroreactive CE patients. Overall, an anti-27- and -28-kDa response appeared to correlate with cyst activity. In conclusion, PSSA represents a useful candidate to carry out a serologic follow-up of CE subsequent to treatment and deserves further respective evaluation for other age groups of CE patients.
Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA
Authors: Ginocchio C. C. , Barth D. , Zhang F.
Published: March 2008

This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay).
Characterization of “
Mycobacterium paraffinicum
” Associated with a Pseudo-Outbreak
Authors: Wang Shu-Hua , Mangino Julie E. , Stevenson Kurt , Yakrus Mitchell A. , Cooksey Robert , Butler W. Ray , Healy Mimi , Wise Mark G. , Schlesinger Larry S. , Pancholi Preeti
Published: March 2008

We describe the first “
Mycobacterium paraffinicum
” (unofficial taxon) pseudo-outbreak in a tertiary-care medical center. Fifteen clinical nontuberculous mycobacterium isolates from 10 patients were initially identified by biochemical tests and high-performance liquid chromatography as
Mycobacterium scrofulaceum
. However, further testing by molecular analysis revealed “
M. paraffinicum
.” Epidemiological and environmental investigation determined that the ice machine was the source of the pseudo-outbreak.
Comparison of Serological and Real-Time PCR Assays To Diagnose
Bordetella pertussis
Infection in 2007
Authors: André Philippe , Caro Valerie , Njamkepo Elisabeth , Wendelboe Aaron M. , Van Rie Annelies , Guiso Nicole
Published: March 2008

Bacterial culture for diagnosing pertussis infection has high specificity but poor sensitivity and is slow. Highly sensitive real-time PCR assays and single-serum pertussis serology have been developed to overcome these limitations, but there are few data available on the relative sensitivities and specificities of such assays for pertussis diagnosis. Using data on 195 participants (≥7 years old) from an epidemiological study, we assessed the sensitivity, specificity, and performance (Youden index) for pertussis diagnosis of the pertussis toxin enzyme-linked immunosorbent assay (using single and paired serology) and of real-time PCR assays (using the IS
481
and
ptxA
-Pr targets). All available diagnostic information (clinical and laboratory) was pooled to serve as the gold standard. Single serology was the most efficient diagnostic test (Youden index, 0.57 to 0.58), with relatively high sensitivity (>64%) and high specificity (>90%), independent of the cutoff level. IS
481
PCR performance was superior to that of
ptxA
-Pr PCR, and it was the second-most-efficient tool (Youden index, 0.30). Performing both
ptxA
-Pr and IS
481
PCRs did not improve diagnostic performance. The greatest test efficiency (Youden index, 0.69 to 0.74) was achieved when single-serum serology was used in combination with IS
481
or
ptxA
-Pr PCR or paired serology. Combining single serology with one PCR or paired serology increased the sensitivity with an associated limited decrease in specificity. The most specific tests for diagnosis of pertussis were single serology and
ptxA
-Pr PCR, and the most sensitive diagnostic tool was the combination of IS
481
PCR with single serology.