bluuebunny/crossref_metadata_2025 huggingface dataset:
The Spread of Ras Activity Triggered by Activation of a Single Dendritic Spine
Authors: Harvey Christopher D. , Yasuda Ryohei , Zhong Haining , Svoboda Karel
Published: June 2008

In neurons, individual dendritic spines isolate
N
-methyl-
d
-aspartate (NMDA) receptor–mediated calcium ion (Ca
2+
) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca
2+
influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca
2+
-dependent Ras activation in single spines that decayed in ∼5 minutes. Ras activity spread over ∼10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca
2+
-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.
Environmental and Growth Phase Regulation of the
Streptococcus gordonii
Arginine Deiminase Genes
Authors: Liu Yaling , Dong Yiqian , Chen Yi-Ywan M. , Burne Robert A.
Published: June 2008

A 1,026-bp open reading frame sharing significant similarity with
queA
, which encodes a predicted
S
-adenosylmethionine:tRNA ribosyltransferase-isomerase responsible for queosine modification of tRNAs, was found immediately 5′ of the gene for the transcriptional activator (ArcR) of the arginine deiminase system (ADS) operon of
Streptococcus gordonii
. The role of QueA in bacterial physiology is enigmatic, but loss of QueA has been shown to compromise stationary-phase survival or virulence in certain enteric bacteria. Interestingly,
S. gordonii
appears to be unique among ADS-positive bacteria in the linkage of
queA
with the ADS genes. A putative σ
70
promoter (p

queA

; TTGCCA-N
21
-TATAAT) was mapped 5′ of
queA
by primer extension, and
queA
and
arcR
were shown to be cotranscribed. The expression from p

queA

was found to be constitutive under all conditions tested, but the expression of p

arcA

, which drives the expression of the
arc
structural genes, was enhanced in stationary phase and could be induced by low pH and arginine. QueA and CcpA acted repressively on
arc
transcription, but neither QueA-deficient strains nor CcpA-deficient strains showed significant differences in arginine deiminase enzyme activities compared with the wild-type strain. The growth rate of a QueA-deficient strain did not differ significantly from that of the parental strain, but the QueA-deficient strain did not compete well with the wild-type during serial passage. In addition to the finding that ADS expression can be regulated separately by growth phase and pH, a significant linkage between the ADS, translational efficiency modulated by QueA, and post-exponential-phase survival of
S. gordonii
was found.
Previously Unrecognized Vaccine Candidates ControlTrypanosoma cruziInfection and Immunopathology in Mice
Authors: Bhatia Vandanajay , Garg Nisha Jain
Published: June 2008

Trypanosoma cruziis the etiologic agent of Chagas' disease, a major health problem in Latin America and an emerging infectious disease in the United States. Previously, we screened aT. cruzisequence database by a computational-bioinformatic approach and identified antigens that exhibited the characteristics of good vaccine candidates. In this study, we tested the vaccine efficacy of three of the putative candidate antigens againstT. cruziinfection and disease in a mouse model. C57BL/6 mice vaccinated withT. cruzi G1(TcG1)-, TcG2-, or TcG4-encoding plasmids and cytokine (interleukin-12 and granulocyte-macrophage colony-stimulating factor) expression plasmids elicited a strong Th1-type antibody response dominated by immunoglobulin G2b (IgG2b)/IgG1 isotypes. The dominant IgG2b/IgG1 antibody response was maintained after a challenge infection and was associated with 50 to 90% control of the acute-phase tissue parasite burden and an almost undetectable level of tissue parasites during the chronic phase, as determined by a sensitiveT. cruzi18S rRNA gene-specific real-time PCR approach. Splenocytes from vaccinated-and-infected mice, compared to unvaccinated-and-infected mice, exhibited decreased (∼50% lower) proliferation and gamma interferon (IFN-γ) production when stimulated in vitro withT. cruziantigens, thus suggesting that protection from challenge infection was not provided by an active T-cell response. Subsequently, the serum and cardiac levels of IFN-γ and tumor necrosis factor alpha and infiltration of inflammatory infiltrate in the heart were decreased in vaccinated mice during the course of infection and chronic disease development. Taken together, these results demonstrate the identification of novel vaccine candidates that provided protection fromT. cruzi-induced immunopathology in experimental mice.
Development of a Fluorescent-Bead-Based Multiplex Immunoassay To Determine Immunoglobulin G Subclass Responses to
Neisseria meningitidis
Serogroup A and C Polysaccharides
Authors: de Voer Richarda M. , van der Klis Fiona R. M. , Engels Carla W. A. M. , Rijkers Ger T. , Sanders Elisabeth A. , Berbers Guy A. M.
Published: June 2008

A fluorescent-particle-based multiplex flow cytometric immunoassay (MIA) for the detection of serum immunoglobulin G (IgG) and two IgG subclasses, IgG1 and IgG2, specific for
Neisseria meningitidis
serogroup A (MenA) and C (MenC) polysaccharides (PS) was developed. The assay comprised three separate duplex assays, one for the detection of the IgG response to MenA and MenC PS, another for the detection of the IgG1 response to MenA and MenC PS, and a third for the detection of the IgG2 response to MenA and MenC PS. Next, the three separate duplex assays were combined and analyzed as a hexaplex assay. No interference between monoplex, duplex, and hexaplex assays was observed, and the assay was found to have low intra- and interassay variation (<9.0% and <27%, respectively). Comparison of the meningococcal subclass MIA to the in-house enzyme-linked inmmunosorbent assays showed a good correlation (
R
≥ 0.85) for each of the subclasses. We conclude that the hexaplex meningococcal subclass MIA is an easy and specific assay for the determination of anti-MenA and anti-MenC PS subclass IgG, requiring minimal amounts of serum to study IgG subclass responses to vaccines.
Mitogen-Activated Protein Kinase Hog1 Mediates Adaptation to G1Checkpoint Arrest during Arsenite and Hyperosmotic Stress
Authors: Migdal Iwona , Ilina Yulia , Tamás Markus J. , Wysocki Robert
Published: June 2008

Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G1and G2checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and thehog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx intohog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G1and G2delays in wild-type cells, whereas cells that lack theHOG1gene or are defective in Hog1 kinase activity displayed persistent G1cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treatedhog1Δ cells, prolonged the G1delay but did not cause a persistent G1arrest. In contrast, deletion of theSIC1gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G1exit inhog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treatedhog1Δ cells. Interestingly, Sic1-dependent persistent G1arrest was also observed inhog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G1cell cycle arrest.
Expression, Purification, and Structural Characterization of CfrA, a Putative Iron Transporter from
Campylobacter jejuni
Authors: Carswell Casey L. , Rigden Marc D. , Baenziger John E.
Published: June 2008

The gene for the
Campylobacter
ferric receptor (CfrA), a putative iron-siderophore transporter in the enteric food-borne pathogen
Campylobacter jejuni
, was cloned, and the membrane protein was expressed in
Escherichia coli
, affinity purified, and then reconstituted into model lipid membranes. Fourier transform infrared spectra recorded from the membrane-reconstituted CfrA are similar to spectra that have been recorded from other iron-siderophore transporters and are highly characteristic of a β-sheet protein (∼44% β-sheet and ∼10% α-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to
2
H
2
O and yet is resistant to thermal denaturation at temperatures up to 95°C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane β-barrel structure containing a plug domain. Sequence alignments indicate that CfrA contains many of the structural motifs conserved in other iron-siderophore transporters, including the Ton box, PGV, IRG, RP, and LIDG motifs of the plug domain. Surprisingly, a homology model reveals that regions of CfrA that are expected to play a role in enterobactin binding exhibit sequences that differ substantially from the sequences of the corresponding regions that play an essential role in binding/transport by the
E. coli
enterobactin transporter, FepA. The sequence variations suggest that there are differences in the mechanisms used by CfrA and FepA to interact with bacterial siderophores. It may be possible to exploit these structural differences to develop CfrA-specific therapeutics.
Genetic Analysis of Spirochete Flagellin Proteins and Their Involvement in Motility, Filament Assembly, and Flagellar Morphology
Authors: Li Chunhao , Wolgemuth Charles W. , Marko Michael , Morgan David G. , Charon Nyles W.
Published: June 2008

The filaments of spirochete periplasmic flagella (PFs) have a unique structure and protein composition. In most spirochetes, the PFs consist of a core of at least three related proteins (FlaB1, FlaB2, and FlaB3) and a sheath of FlaA protein. The functions of these filament proteins remain unknown. In this study, we used a multidisciplinary approach to examine the role of these proteins in determining the composition, shape, and stiffness of the PFs and how these proteins impact motility by using the spirochete
Brachyspira
(formerly
Treponema
,
Serpulina
)
hyodysenteriae
as a genetic model. A series of double mutants lacking combinations of these PF proteins was constructed and analyzed. The results show the following. First, the diameters of PFs are primarily determined by the sheath protein FlaA, and that FlaA can form a sheath in the absence of an intact PF core. Although the sheath is important to the PF structure and motility, it is not essential. Second, the three core proteins play unequal roles in determining PF structure and swimming speed. The functions of the core proteins FlaB1 and FlaB2 overlap such that either one of these proteins is essential for the spirochete to maintain the intact PF structure and for cell motility. Finally, linear elasticity theory indicates that flagellar stiffness directly affects the spirochete's swimming speed.
Duplication of Teichoic Acid Biosynthetic Genes in
Staphylococcus aureus
Leads to Functionally Redundant Poly(Ribitol Phosphate) Polymerases
Authors: Pereira Mark P. , D'Elia Michael A. , Troczynska Justyna , Brown Eric D.
Published: June 2008

Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of
Bacillus subtilis
and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen
Staphylococcus aureus
. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (
tarI
′
J
′ and
tarIJ
) as well as poly(ribitol phosphate) polymerization (
tarK
and
tarL
) in
S. aureus
. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by
tarK
and
tarL
) without affecting teichoic acid levels in the
S. aureus
cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (
tarI
′
J
′) could be obtained, while
tarIJ
remained essential. Suppression analysis of the
tarIJ
null mutant indicated that the mechanism of dysfunction in
tarI
′
J
′ is due to poor translation of the TarJ′ enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in
S. aureus
and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.
Evaluation of Linear Array Human Papillomavirus Genotyping Using Automatic Optical Imaging Software
Authors: Jeronimo J. , Wentzensen N. , Long R. , Schiffman M. , Dunn S. T. , Allen R. A. , Walker J. L. , Gold M. A. , Zuna R. E. , Sherman M. E. , Wacholder S. , Wang S. S.
Published: June 2008

Variations in biological behavior suggest that each carcinogenic human papillomavirus (HPV) type should be considered individually in etiologic studies. HPV genotyping assays might have clinical applications if they are approved for use by the FDA. A widely used genotyping assay is the Roche Linear Array HPV genotyping test (LA). We used LA to genotype the HPV isolates from cervical specimens from women with the full spectrum of cervical disease: cervical cancer, cervical intraepithelial neoplasia (CIN), and HPV infections. To explore the feasibility and value of the automated reading of the LA results, we custom-designed novel, optical imaging software that provides optical density measurements of LA bands. We compared unmagnified visual examination with the automated measurements. The two measurements were highly associated. By either method, the threshold between a negative and a positive result was fairly sharp, with a clear bimodal distribution. Visually, most positive results were judged to be strong or medium, with fewer equivocal results categorized as weak (9.5% of positive samples), very weak (6.5% of positive samples), or extremely weak (7.7% of positive samples). The automated measurements of the intensities were significantly associated with the strength of the visual categories (
P
< 0.001). At the extremes of the automated signal intensities (≤20 units or ≥120 units), the bands were almost always categorized visually as negative and positive, respectively. In the equivocal zone (20 to 119 units), specimens were more increasingly likely to be judged to be visually positive as the number of other, definite infections on the same strip increased (
P
for trend < 0.001). Multiple, concurrent infections comprise ≥25% of HPV infections; thus, any systematic visual tendency that influences their evaluation when the result is equivocal should be minimized. Therefore, automated reading is probably worth development if easy-to-calibrate hardware and software can be optimized.
Prevalence of Toxic Shock Syndrome Toxin 1 (TSST-1)-Producing Strains of
Staphylococcus aureus
and Antibody to TSST-1 among Healthy Japanese Women
Authors: Parsonnet Jeffrey , Goering Richard V. , Hansmann Melanie A. , Jones Michaelle B. , Ohtagaki Kumiko , Davis Catherine C. , Totsuka Kyoichi
Published: June 2008

Many cases of neonatal toxic shock syndrome (TSS)-like exanthematous disease but few cases of menstrual TSS (mTSS) have been reported in Japan. We determined the prevalence of mucosal colonization with
Staphylococcus aureus
and of positive antibodies to TSS toxin 1 (TSST-1) among 209 healthy Japanese women in Tokyo.
S. aureus
isolates from mucosal sites were characterized with respect to TSST-1 production and resistance genotype. Antibody titers were determined for test subjects and for 133 Japanese and 137 Caucasian control women living in the United States.
S. aureus
was isolated from at least one site in 108 of 209 women (52%) in Tokyo. Of the 159
S. aureus
isolates recovered, 14 (9%) were TSST-1 positive (12 unique strains). Twelve of 209 women (6%) were colonized with a TSST-1-producing strain; two (<1%) had vaginal colonization. Only 2 of 12 unique toxigenic strains (14%) were methicillin resistant. Of the 12 TSST-1-positive strains isolated, 6 (50%) were pulsed-field gel electrophoresis type USA200, multilocus sequence type clonal complex 30. Fewer Japanese women in Tokyo (47%) than Caucasian and Japanese women in the United States (89% and 75%, respectively) had TSST-1 antibodies. The prevalences of colonization with TSST-1-producing
S. aureus
were comparable in Japan and the United States, despite low seropositivity to TSST-1 in Japan. Environmental factors appear to be important in promoting the development of anti-TSST-1 antibodies, as there was a significant difference in titers between Japanese women living in Tokyo and those living in the United States. Most colonizing TSST-1-producing
S. aureus
strains in Japan were genotypically similar to mTSS strains found in the United States.
Evaluation of New Vitek 2 Card and Disk Diffusion Method for Determining Susceptibility of
Staphylococcus aureus
to Oxacillin
Authors: Roisin Sandrine , Nonhoff Claire , Denis Olivier , Struelens Marc J.
Published: June 2008

Detection of methicillin resistance in
Staphylococcus aureus
is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in
S. aureus
strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including
mecA
-negative
S. aureus
isolates (
n
= 56), hospital-acquired (
n
= 88) and community-acquired (
n
= 40)
S. aureus
isolates, and heterogeneous methicillin-resistant
S. aureus
isolates (
n
= 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 μg cefoxitin and 30 μg moxalactam. Oxacillin resistance was confirmed by PCR for the
mecA
gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-μg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-μg cefoxitin disks, respectively. Methicillin-susceptible
S. aureus
isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant
S. aureus
strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.
Evaluation and Strategy for Use of MIRU-VNTRplus, a Multifunctional Database for Online Analysis of Genotyping Data and Phylogenetic Identification ofMycobacterium tuberculosisComplex Isolates
Authors: Allix-Béguec Caroline , Harmsen Dag , Weniger Thomas , Supply Philip , Niemann Stefan
Published: June 2008

Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has become a major method for the epidemiological tracking ofMycobacterium tuberculosiscomplex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains has been available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build a database, which is freely accessible athttp://www.MIRU-VNTRplus.org. The geographical origin and the drug susceptibility profile of each strain were stored together with comprehensive genetic lineage information, including the 24-locus MIRU-VNTR profile, the spoligotyping pattern, the single-nucleotide- and large-sequence-polymorphism profiles, and the IS6110restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single or multiple user strains can be analyzed, e.g., for lineage identification with or without the use of reference strains, by best-match or tree-based analyses with single or combined marker data sets. The results can easily be exported. In the present study, we evaluated the database consistency and various analysis parameters both by testing the reference collection against itself and by using an external population-based data set comprising 629 different strains. Under the optimal conditions found, lineage predictions based on typing by 24-locus MIRU-VNTR analysis optionally combined with spoligotyping were verified in >99% of the cases. On the basis of this evaluation, a user strategy was defined, which consisted of best-match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplusdatabase is a powerful tool for high-resolution clonal identification and has little equivalent in terms of functionalities among the bacterial genotyping databases available so far.
Comparison of Two Immunoassays for Detection of
Entamoeba histolytica
Authors: Buss Sarah , Kabir Mamun , Petri William A. , Haque Rashidul
Published: June 2008

Effective diagnostic tools are essential in order to combat disease caused by the parasite
Entamoeba histolytica
. In this study, we compared the commercially available Ridascreen
Entamoeba
test (R-Biopharm) and the
E. histolytica
II test (Techlab), and we found that the
E. histolytica
II test detects
E. histolytica
infections more accurately.
Rifampin Resistance, Beijing-W Clade-Single Nucleotide Polymorphism Cluster Group 2 Phylogeny, and the Rv2629 191-C Allele inMycobacterium tuberculosisStrains
Authors: Chakravorty Soumitesh , Aladegbami Bola , Motiwala Alifiya S. , Dai Yang , Safi Hassan , Brimacombe Michael , Helb Danica , Alland David
Published: June 2008

Rifampin resistance is a key prognostic marker for treatment success in tuberculosis patients. Recently, Wang et al. demonstrated that Rv2629 A191C mutations were present in 99.1% of rifampin-resistant and 0% of rifampin-susceptible clinicalMycobacterium tuberculosisisolates and that overexpression of the Rv2629 191C allele inMycobacterium smegmatisproduced an eightfold increase in rifampin resistance. These results suggested that Rv2629 could be a cause of rifampin resistance and a valuable target for rifampin resistance detection assays. We developed a molecular-beacon assay to study the association between Rv2629 191 alleles and rifampin resistance in 246 geographically and phylogenetically diverse clinicalM. tuberculosisisolates. The 191C allele was present in 30/98 (30.6%) rifampin-resistant isolates and 25/148 (16.9%) rifampin-susceptible isolates and was more common in isolates from Asia. Phylogenetic analysis demonstrated complete overlap between the 191C allele and single nucleotide polymorphism cluster group 2 (SCG-2), a phylogenetic lineage that corresponds to the Beijing-W clade ofM. tuberculosis. All 55 (100%) 191C isolates were SCG-2, while none of the 191 191A isolates were SCG-2 (P< 0.001). No association was found between the 191C allele and rifampin resistance in an analysis that included the SCG type (P= 1.0). Also, in contrast to the findings of Wang et al., we found that overexpression of either Rv2629 191 allele inM. smegmatisdid not produce an increase in rifampin resistance. We conclude that the Rv2629 191C allele is not associated with rifampin resistance and that the allele cannot be used as a molecular target to detect rifampin resistance. The allele appears to be an excellent marker for the Beijing-W clade/SCG-2 phylogenetic group.
Fourier Transform Infrared Spectroscopy for Rapid Identification of Nonfermenting Gram-Negative Bacteria Isolated from Sputum Samples from Cystic Fibrosis Patients
Authors: Bosch Alejandra , Miñán Alejandro , Vescina Cecilia , Degrossi José , Gatti Blanca , Montanaro Patricia , Messina Matías , Franco Mirta , Vay Carlos , Schmitt Juergen , Naumann Dieter , Yantorno Osvaldo
Published: June 2008

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are
Pseudomonas aeruginosa, Staphylococcus aureus
, and
Haemophilus influenzae
, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as
Burkholderia cepacia
complex (BCC) bacteria,
Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter
spp., and
Achromobacter
spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by
recA
-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by
recA
species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of
P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter
spp.,
R. pickettii
, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC,
B. cepacia, B. multivorans, B. cenocepacia
, and
B. stabilis
(genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.
Lewis Antigen Expression by
Helicobacter pylori
Strains Colonizing Different Regions of the Stomach of Individual Patients
Authors: González-Valencia Gerardo , Muñoz-Perez Leopoldo , Morales-Espinosa Rosario , Camorlinga-Ponce Margarita , Muñoz Onofre , Torres Javier
Published: June 2008

The diversity in the expression of Lewis antigens (Le) of 226 single colonies of
Helicobacter pylori
isolated from four regions of the stomach of eight adults is shown. Le
y
was expressed more in strains colonizing antrum than in strains colonizing fundus, whereas Le
x
was more common in fundus strains.
cagA
+
strains were more associated with Le-negative strains.
Previously Infected Chimpanzees Are Not Consistently Protected against Reinfection or Persistent Infection after Reexposure to the Identical Hepatitis C Virus Strain
Authors: Bukh Jens , Thimme Robert , Meunier Jean-Christophe , Faulk Kristina , Spangenberg Hans Christian , Chang Kyong-Mi , Satterfield William , Chisari Francis V. , Purcell Robert H.
Published: June 2008

Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.
Replication Properties of Clade A/C Chimeric Feline Immunodeficiency Viruses and Evaluation of Infection Kinetics in the Domestic Cat
Authors: de Rozìeres Sohela , Thompson Jesse , Sundstrom Magnus , Gruber Julia , Stump Debora S. , de Parseval Aymeric P. , VandeWoude Sue , Elder John H.
Published: June 2008

Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats, with clinical course dependent on virus strain. For example, clade A FIV-PPR is predominantly neurotropic and causes a mild disease in the periphery, whereas clade C FIV-C36 causes fulminant disease with CD4
+
T-cell depletion and neutropenia but no significant pathology in the central nervous system. In order to map pathogenic determinants, chimeric viruses were prepared between FIV-C36 and FIV-PPR, with reciprocal exchanges involving (i) the 3′ halves of the viruses, including the Vif, OrfA, and Env genes; (ii) the 5′ end extending from the 5′ long terminal repeat (LTR) to the beginning of the capsid (CA)-coding region; and (iii) the 3′ LTR and Rev2-coding regions. Ex vivo replication rates and in vivo replication and pathologies were then assessed and compared to those of the parental viruses. The results show that FIV-C36 replicates ex vivo and in vivo to levels approximately 20-fold greater than those of FIV-PPR. None of the chimeric FIVs recapitulated the replication rate of FIV-C36, although most replicated to levels similar to those of FIV-PPR. The rates of chloramphenicol acetyltransferase gene transcription driven by the FIV-C36 and FIV-PPR LTRs were identical. Furthermore, the ratios of surface glycoprotein (SU) to capsid protein (CA) in the released particles were essentially the same in the wild-type and chimeric FIVs. Tests were performed in vivo on the wild-type FIVs and chimeras carrying the 3′ half of FIV-C36 or the 3′ LTR and Rev2 regions of FIV-C36 on the PPR background. Both chimeras were infectious in vivo, although replication levels were lower than for the parental viruses. The chimera carrying the 3′ half of FIV-C36 demonstrated an intermediate disease course with a delayed peak viral load but ultimately resulted in significant reductions in neutrophil and CD4
+
T cells, suggesting potential adaptation in vivo. Taken together, the findings suggest that the rapid-growth phenotype and pathogenicity of FIV-C36 are the result of evolutionary fine tuning throughout the viral genome, rather than being properties of any one constituent.
Structural Insights into Calicivirus Attachment and Uncoating
Authors: Bhella David , Gatherer Derek , Chaudhry Yasmin , Pink Rebecca , Goodfellow Ian G.
Published: June 2008

The
Caliciviridae
family comprises positive-sense RNA viruses of medical and veterinary significance. In humans, caliciviruses are a major cause of acute gastroenteritis, while in animals respiratory illness, conjunctivitis, stomatitis, and hemorrhagic disease are documented. Investigation of virus-host interactions is limited by a lack of culture systems for many viruses in this family. Feline calicivirus (FCV), a member of the
Vesivirus
genus, provides a tractable model, since it may be propagated in cell culture. Feline junctional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for FCV. We have analyzed the structure of this virus-receptor complex by cryo-electron microscopy and three-dimensional image reconstruction, combined with fitting of homology modeled high-resolution coordinates. We show that domain 1 of fJAM-1 binds to the outer face of the P2 domain of the FCV capsid protein VP1, inducing conformational changes in the viral capsid. This study provides the first structural view of a native calicivirus-protein receptor complex and insights into the mechanisms of virus attachment and uncoating.
A Simple Mathematical Model Helps To Explain the Immunodominance of CD8 T Cells in Influenza A Virus Infections
Authors: Handel Andreas , Antia Rustom
Published: June 2008

Understanding immunodominance, the phenomenon of epitope-specific T cells expanding in an often distinctly hierarchical fashion, is important for the design of T-cell-based intervention strategies. Several recent studies have investigated immunodominance of H-2D
b
-restricted CD8
+
T cells specific for the nucleoprotein NP366 and acid polymerase PA224 epitopes during influenza A virus infection of C57BL/6 mice. CD8
+
T cells specific for these two epitopes are codominant during primary infection; NP366 dominates during secondary infection. While a number of explanations for this observation have been proposed, none of them can fully account for all the observed data. In this article, we use a simple mathematical model to explain the seemingly inconsistent data. We show that the dynamic interactions between CD8
+
T cells and antigen presentation lead to a situation where CD8
+
T cells are limiting during the initial response whereas antigen is limiting in the secondary response. This “numbers game” between antigen and CD8
+
T cells can reproduce the observed immunodominance of the NP336- and PA224-specific CD8
+
T cells, thereby explaining the reported experimental data.
Small-Molecule Inhibitor Which Reactivates p53 in Human T-Cell Leukemia Virus Type 1-Transformed Cells
Authors: Jung Kyung-Jin , Dasgupta Arindam , Huang Keven , Jeong Soo-Jin , Pise-Masison Cynthia , Gurova Katerina V. , Brady John N.
Published: June 2008

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-κB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-κB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G
1
and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is “druggable” and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-κB suggests a promising strategy for the treatment of HTLV-1-transformed cells.
Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease
Authors: Robel Ivonne , Gebhardt Julia , Mesters Jeroen R. , Gorbalenya Alexander , Coutard Bruno , Canard Bruno , Hilgenfeld Rolf , Rohayem Jacques
Published: June 2008

Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific
trans
cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to
trans
cleavage by NS6 or NS6-7 proteins, whereas
cis
cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to
trans
cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.
The Secondary Structure of the Human Immunodeficiency Virus Type 1 Transcript Modulates Viral Splicing and Infectivity
Authors: Jablonski Joseph A. , Buratti Emanuele , Stuani Cristiana , Caputi Massimo
Published: June 2008

Splicing of human immunodeficiency virus type 1 (HIV-1) exon 6D is regulated by the presence of a complex splicing regulatory element (SRE) sequence that interacts with the splicing factors hnRNP H and SC35. In this work, we show that, in the context of the wild-type viral sequence, hnRNP H acts as a repressor of exon 6D inclusion independent of its binding to the SRE. However, hnRNP H binding to the SRE acts as an enhancer of exon 6D inclusion in the presence of a critical T-to-C mutation. These seemingly contrasting functional properties of hnRNP H appear to be caused by a change in the RNA secondary structure induced by the T-to-C mutation that affects the spatial location of bound hnRNP H with respect to the exon 6D splicing determinants. We propose a new regulatory mechanism mediated by RNA folding that may also explain the dual properties of hnRNP H in splicing regulation.
Human Immunodeficiency Virus (HIV) Type 1 Proviral Hypermutation Correlates with CD4 Count in HIV-Infected Women from Kenya
Authors: Land Allison M. , Ball T. Blake , Luo Ma , Pilon Richard , Sandstrom Paul , Embree Joanne E. , Wachihi Charles , Kimani Joshua , Plummer Francis A.
Published: June 2008

APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the
vpu
/
env
region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral
vif
. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.
Human Metapneumovirus Small Hydrophobic Protein Inhibits NF-κB Transcriptional Activity
Authors: Bao Xiaoyong , Kolli Deepthi , Liu Tianshuang , Shan Yichu , Garofalo Roberto P. , Casola Antonella
Published: June 2008

Human metapneumovirus, a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. In this study, we showed that infection of airway epithelial cells or mice with recombinant human metapneumovirus lacking SH expression (rhMPV-ΔSH) enhanced secretion of proinflammatory mediators, including interleukin 6 (IL-6) and IL-8, encoded by two NF-kB-dependent genes, compared to infection with wild-type rhMPV. RhMPV-ΔSH infection resulted in enhanced NF-kB-dependent gene transcription and in increased levels of phosphorylated and acetylated NF-kB without affecting its nuclear translocation, identifying a possible novel mechanism by which paramyxovirus SH proteins modulate NF-kB activation.
PHASE TRANSITIONS AND QUANTUM STABILIZATION IN QUANTUM ANHARMONIC CRYSTALS
Authors: KARGOL ALINA , KONDRATIEV YURI , KOZITSKY YURI
Published: June 2008

A unified theory of phase transitions and quantum effects in quantum anharmonic crystals is presented. In its framework, the relationship between these two phenomena is analyzed. The theory is based on the representation of the model Gibbs states in terms of path measures (Euclidean Gibbs measures). It covers the case of crystals without translation invariance, as well as the case of asymmetric anharmonic potentials. The results obtained are compared with those known in the literature.
Unital Positive Maps and Quantum States
Authors: Asorey M. , Kossakowski A. , Marmo G. , Sudarshan E. C. G.
Published: June 2008

We analyze the structure of the subset of states generated by unital completely positive quantum maps. A witness certifying that a state does not belong to the subset generated by a given map is constructed. We analyse the representations of positive maps and their relation to quantum Perron-Frobenius theory.
On Completely Positive Non-Markovian Evolution of a d-Level System
Authors: Kossakowski Andrzej , Rebolledo Rolando
Published: June 2008

A sufficient condition for non-Markovian master equation which ensures the complete positivity of the resulting time evolution is presented.
Noise Induced Current in a Double-Well Trap
Authors: Anderloni Sebastiano , Benatti Fabio , Floreanini Roberto
Published: June 2008

We study the behaviour of cold atoms trapped in optical double well potentials in the presence of noise, either generated by an external environment or by the trap itself. We show that quite in general the noise can induce a current between the two wells even when tunneling between the two wells is highly suppressed (Mott insulator phase). An engineered environment could provide a test ground for the behaviour of such a current.
Quantum Teleportation and Holomorphic Representation of CCR
Authors: Nagamachi S. , Brüning E.
Published: June 2008

For the treatment of teleportation of continuous quantum variables, often the Wigner functions are used. In this paper, we will show that by using the holomorphic representation of the canonical commutation relations (CCR), the teleportation of continuous quantum variables is treated in a parallel way as the teleportation of qubits.
Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart
Authors: Xie Yi , Ottolia Michela , John Scott A. , Chen Jau-Nian , Philipson Kenneth D.
Published: June 2008

The Na+/Ca2+ exchanger is the major Ca2+ extrusion mechanism in cardiac myocytes. The activity of the cardiac Na+/Ca2+ exchanger is dynamically regulated by intracellular Ca2+. Previous studies indicate that Ca2+ binding to a high-affinity Ca2+-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to yellow and cyan fluorescent protein. Ca2+ binding to CBD1 induces conformational changes, as detected by fluorescence resonance energy transfer. With this transgenic fish model, we were able to monitor conformational changes of the Ca2+ regulatory domain of Na+/Ca2+ exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca2+ transients and Ca2+-induced changes in fluorescence resonance energy transfer. The results indicate that Ca2+ regulation of the Na+/Ca2+ exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na+/Ca2+ exchanger in vivo.