bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing the Molecular Determinant for the Catalytic Efficiency of
l
-Arabinose Isomerase from
Bacillus licheniformis
Authors: Prabhu Ponnandy , Jeya Marimuthu , Lee Jung-Kul
Published: January 2010

Bacillus licheniformis
l
-arabinose isomerase (
l
-AI) is distinguished from other
l
-AIs by its high degree of substrate specificity for
l
-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of
B. licheniformis
l
-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type
B. licheniformis
l
-AI was identified as an important residue affecting the catalytic efficiency of
B. licheniformis
l
-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward
l
-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with
l
-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of
B. licheniformis
l
-AI.
Immunogenicity of a Heptavalent Conjugate Pneumococcal Vaccine Administered Concurrently with a Combination Diphtheria, Tetanus, Five-Component Acellular Pertussis, Inactivated Polio, and
Haemophilus influenzae
Type b Vaccine and a Meningococcal Group C Conjugate Vaccine at 2, 3, and 4 Months of Age
Authors: Moss S. J. , Fenton A. C. , Toomey J. , Grainger A. , Borrow R. , Balmer P. , Smith J. , Gennery A. R.
Published: December 2009

The immunogenicities of conjugate pneumococcal vaccines have been demonstrated when they are administered at 2, 3, and 4 months of age. There is a paucity of data on the immunogenicity of this vaccine when it is administered concurrently with other vaccines in the primary immunization schedule of the United Kingdom. We immunized 55 term infants at 2, 3, and 4 months of age with the seven-valent pneumococcal conjugate vaccine (PCV7), the meningococcal group C conjugate (MCC) vaccine, and the diphtheria, tetanus, five-component acellular pertussis, inactivated polio, and
Haemophilus influenzae
type b (DTaP
5
/IPV/Hib-TT) vaccine. The immune responses to the
H. influenzae
type b (Hib), MCC, and tetanus vaccines were measured at 2, 5, and 12 months of age; and the immune responses to PCV7 were measured at 2 and 5 months and then either at 12 months or following a 4th dose of PCV7. There were increases in the geometric mean concentrations (GMCs) of all antigens postimmunization. Greater than or equal to 90% of the infants achieved putatively protective levels postimmunization for all vaccine antigens except pneumococcal serotype 6B and Hib. The GMCs of the PCV7 serotypes increased following a 4th dose, although one infant had not reached putative levels of protection against serotype 6B. In conclusion, when infants were vaccinated according to the schedule described above, they had lower postprimary immunization responses to Hib, meningococcus group C capsular polysaccharide, and pneumococcal serotype 6B than the responses demonstrated by use of the other schedules. Despite this finding, there was a good response following a 4th dose of PCV7.
Discrepancy in Dengue Virus Neutralizing Antibody Titers between Plaque Reduction Neutralizing Tests with Fcγ Receptor (FcγR)-Negative and FcγR-Expressing BHK-21 Cells
Authors: Moi Meng Ling , Lim Chang-Kweng , Kotaki Akira , Takasaki Tomohiko , Kurane Ichiro
Published: December 2009

Protective immunity against dengue virus (DENV) is best reflected by the presence of neutralizing antibodies. The conventional plaque reduction neutralizing test (PRNT) is performed using Fcγ receptor (FcγR)-negative cells. Because FcγR plays a key role in antibody-dependent enhancement, we examined neutralizing antibody titers of mouse monoclonal antibodies and human serum samples in PRNTs using FcγRIIA-negative and FcγRIIA-expressing BHK cells. There was a discrepancy in dengue virus neutralizing antibody titers between PRNTs using FcγRIIA-negative versus FcγRIIA-expressing BHK cells. Neutralizing antibody titers to DENV-1 and DENV-2 tested with monoclonal antibodies, and with most of the human serum samples, were higher in assays using BHK cells than those using FcγRIIA-expressing BHK cells. The results suggest that neutralizing antibody titers determined using FcγRIIA-expressing cells may better reflect the protective capacity of anti-DENV antibodies, as the major target cells of DENV infection are FcγR-positive cells.
High Seroprevalence of Neutralizing Capacity against Human Metapneumovirus in All Age Groups Studied in Bonn, Germany
Authors: Lüsebrink Jessica , Wiese Christoph , Thiel Anne , Tillmann Ramona-Liza , Ditt Vanessa , Müller Andreas , Schildgen Oliver , Schildgen Verena
Published: December 2009

Human metapneumovirus (hMPV) infections occur frequently despite high rates of perpetual seroprevalence for all age groups. Analyses of ∼2,000 archived, randomly selected serum samples demonstrated that neutralizing capacities remain high, with a minor decrease for individuals over 69 years of age, leading to the hypothesis that reinfections occur because humoral immune responses play minor roles in the clearance of hMPV infections.
Evaluation of a Rapid Serological Test for the Determination of
Mycobacterium bovis
Infection in Badgers (
Meles meles
) Found Dead
Authors: Chambers Mark A. , Lyashchenko Konstantin P. , Greenwald Rena , Esfandiari Javan , James Eurig , Barker Leslie , Jones Jeff , Watkins Gavin , Rolfe Simon
Published: December 2009

Between October 2005 and May 2006, a total of 727 badgers found dead in Wales were reported, and 550 were delivered to the Regional Laboratories of the Veterinary Laboratories Agency (VLA). Of the 459 carcasses suitable for examination, 55 were deemed to be infected with
Mycobacterium bovis
on the basis of culture, spoligotyping, and variable-number tandem repeat typing. Acid-fast bacteria were observed histologically in a further six badgers, but these bacteria were not confirmed as
M. bovis
by culture. A rapid serological test (BrockTB Stat-Pak) performed on thoracic blood showed a sensitivity of 35% and a specificity of 99%. Presence of
M. bovis
infection was 45 times more likely to be confirmed postmortem by culture in BrockTB Stat-Pak-reactive animals than in seronegative ones. Using visible carcass lesions as a marker of bovine tuberculosis (bTB) infection had a similar sensitivity (38%) but was significantly less specific (84%) than serology. The overall accuracy of the antibody detection was 93% (346 correct results from 374 tests), whereas the accuracy of regarding visible lesions as a marker for bTB infection was 78% (354 correct from 453 carcasses examined). Culture remains the gold standard method for detecting
M. bovis
infection in badgers. However, where resources are limited and/or an instant result is preferred, the BrockTB Stat-Pak could be used in field surveillance efforts to identify animals which should be examined further by only submitting test-negative animals to more detailed postmortem examination and culture.
Specific IgG Responses to Recombinant Antigen B and Em18 in Cystic and Alveolar Echinococcosis in China
Authors: Li Tiaoying , Ito Akira , Chen Xingwang , Sako Yasuhito , Qiu Jiamin , Xiao Ning , Qiu Dongchuan , Nakao Minoru , Yanagida Tetsuya , Craig Philip S.
Published: December 2009

An understanding of the correlation of the specific antibody responses and the disease phase is essential in evaluating diagnostic values of immunological tests in human echinococcosis. In this study, 422 echinococcosis patients diagnosed by ultrasonography, including 246 with cystic echinococcosis (CE), 173 with alveolar echinococcosis (AE), and 3 with dual infection, were tested for specific IgG in sera against recombinant AgB (rAgB) and recombinant Em18 (rEm18) in an enzyme-linked immunosorbent assay. As a result, rAgB-specific antibody was detected in 77.6% of CE and 86.1% of AE patients, while rEm18-specific antibody was present in 28.9% of CE and 87.3% of AE patients. Additionally, all three patients with dual infection exhibited specific antibodies responding to rAgB and rEm18. Further analysis revealed that rAgB-specific antibody was elevated in a significantly greater proportion (87.3%) of CE patients with cysts at active or transitional stages (CE1, CE2, or CE3), compared to 54.8% of other patients with cysts at an early or an inactive stage (CL or CE4 or CE5). Furthermore, rAgB-specific antibody was detected in 95.6% of CE2 cases, which was statistically greater than that (73.7%) in CE1 patients. Although rEm18-specific antibody was elevated in 28.9% of CE patients, the positive reaction was much weaker in CE than in AE cases. Serum levels and concentrations of rEm18-specific antibody were further indicated to be strongly disease phase correlated in AE patients, with positive rates of 97.4% in cases with alveolar lesions containing central necrosis and 66.7% in patients with early alveolar lesions that measured ≤5 cm.
Evaluation of a Newly Developed GenoArray Human Papillomavirus (HPV) Genotyping Assay and Comparison with the Roche Linear Array HPV Genotyping Assay
Authors: Liu Stephanie S. , Leung Rebecca C. Y. , Chan Karen K. L. , Cheung Annie N. Y. , Ngan Hextan Y. S.
Published: December 2009

Persistent infection with high-risk types of human papillomavirus (HPV) is a necessary step in the development of cervical cancer. The incorporation of HPV detection into cervical screening programs may improve the ability to identify women at risk of cervical cancer. We recently evaluated the performance characteristics of a newly developed HPV detection assay, the GenoArray (GA) genotyping assay, for the detection of HPV infections by comparing it with the commercial Roche Linear Array (LA) HPV genotyping assay. The GA assay has an analytical sensitivity for the detection of HPV types 16 (HPV-16) and HPV-18 of as few as 10 to 50 copies, and its reproducibility is adequate. The GA and LA assays showed no significant difference in the rates of detection of genotypes detected by both HPV genotyping assays and oncogenic genotypes, and the interassay agreement was excellent. The GA and LA assays revealed either concordant or compatible genotyping results for 97.5% of the samples and discordant results for only eight (2.5%) samples. Compatible results were also observed for the detection of single or multiple HPV infections and the detection of most of the genotypes. The GA assay also demonstrated good clinical performance characteristics when the comparisons were carried out with clinical subgroups of samples from patients with normal cytologies, low-grade or high-grade squamous intraepithelial lesions, and cancers. Therefore, the GA assay appears to be highly sensitive and specific for the genotyping of HPV. It has the advantage that it specifically detects HPV-52, which overcomes a limitation of the LA assay, and hence, it has potential value for use for genotyping, especially in regions where HPV-52 has a high prevalence.
Prevalence and Associations of
tcpC
, a Gene Encoding a Toll/Interleukin-1 Receptor Domain-Containing Protein, among
Escherichia coli
Urinary Tract Infection, Skin and Soft Tissue Infection, and Commensal Isolates
Authors: Starčič Erjavec Marjanca , Jesenko Blaž , Petkovšek Živa , Žgur-Bertok Darja
Published: December 2009

TcpC, a new Toll/interleukin-1 receptor domain-containing protein of uropathogenic
Escherichia coli
involved in the suppression of innate immunity, was found in 2008. The aim of the present study was to determine the prevalence of
tcpC
and its association with virulence factors and phylogenetic groups among strains from a collection of 212
E. coli
isolates from urinary tract and skin and soft tissue infections and 90 commensal
E. coli
strains.
Streptococcus pyogenes
Isolates Causing Severe Infections in Norway in 2006 to 2007:
emm
Types, Multilocus Sequence Types, and Superantigen Profiles
Authors: Meisal Roger , Andreasson Ida K. G. , Høiby E. Arne , Aaberge Ingeborg S. , Michaelsen Terje E. , Caugant Dominique A.
Published: December 2009

To investigate the epidemiological patterns and genetic characteristics of disease caused by group A
Streptococcus
(GAS), all available isolates from invasive cases in Norway during 2006 to 2007 (262 isolates) were subjected to antimicrobial susceptibility testing, T serotyping,
emm
typing, and multilocus sequence typing and screened for known streptococcal pyrogenic exotoxin (Spe) genes,
smeZ
, and
ssa
. The average incidence rate was 3.1 cases per 100,000 individuals. The most prevalent sequence types (STs) were STs 52, 28, and 334. In association with
emm
types 28, 77, and 87, the serotype T-28 comprised 24.8% of the strains.
emm
types 28, 1, and 82 were dominating. In 2007, a sharp increase in the number of
emm-
6 strains was noted. All strains were sensitive to penicillin and quinupristin-dalfopristin, while 3.4% and 6.1% of the strains were resistant to macrolides and tetracycline, respectively. Furthermore, the
emm-
6 strains had intermediate susceptibility to ofloxacin. Isolates displayed a wide variety of gene profiles, as shown by the presence or absence of the Spe genes,
smeZ
, and
ssa
, but 48% of the isolates fell into one of three profiles. In most cases, an
emm
type was restricted to one gene profile. Although the incidence decreased during this study, invasive GAS disease still has a high endemic rate, with involvement of both established and emerging
emm
types displaying variability in virulence gene profiles as well as differences in gender and age group preferences.
Prevalence of the
sodC
Gene in Nontypeable
Haemophilus influenzae
and
Haemophilus haemolyticus
by Microarray-Based Hybridization
Authors: McCrea Kirk W. , Wang Myron L. , Xie Jingping , Sandstedt Sara A. , Davis Gregg S. , Lee Joseph H. , Marrs Carl F. , Gilsdorf Janet R.
Published: December 2009

The
sodC
gene has been reported to be a useful marker for differentiating nontypeable (NT)
Haemophilus influenzae
from
Haemophilus haemolyticus
in respiratory-tract samples, but discrepancies exist as to the prevalence of
sodC
in NT
H. influenzae
. Therefore, we used a microarray-based, “library-on-a-slide” method to differentiate the species and found that 21 of 169 (12.4%) NT
H. influenzae
strains and all 110 (100%)
H. haemolyticus
strains possessed the
sodC
gene. Multilocus sequence analysis confirmed that the 21 NT
H. influenzae
strains were
H. influenzae
and not
H. haemolyticus
. An inactive
sodC
gene has been reported in encapsulated
H. influenzae
strains belonging to phylogenetic division II. Capsule-specific Southern hybridization and PCR and a lack of copper/zinc-cofactored superoxide dismutase (CuZnSOD) expression indicated that 6 of the 21
sodC
-containing NT
H. influenzae
strains in our study were likely capsule-deficient mutants belonging to phylogenetic division II. DNA sequence comparisons of the 21
H. influenzae sodC
genes with
sodC
from
H. haemolyticus
or encapsulated
H. influenzae
demonstrated that the
sodC
genes of the six
H. influenzae
capsule-deficient mutants were, on average, 99% identical to
sodC
from encapsulated
H. influenzae
but only 85% identical to
sodC
from
H. haemolyticus
. The
sodC
genes from 2/15 NT
H. influenzae
strains were similarly more closely related to
sodC
from encapsulated strains, while
sodC
genes from 13 NT
H. influenzae
strains were almost 95% identical to
sodC
genes from
H. haemolyticus
, suggesting the possibility of interspecies recombination in these strains. In summary, this study demonstrates that
sodC
is not completely absent (9.2%) in true NT
H. influenzae
strains.
Comparison of Whole Blood, Serum, and Plasma for Early Detection of Candidemia by Multiplex-Tandem PCR
Authors: Lau Anna , Halliday Catriona , Chen Sharon C.-A. , Playford E. Geoffrey , Stanley Keith , Sorrell Tania C.
Published: December 2009

We applied multiplex-tandem PCR (MT-PCR) to 255 EDTA whole-blood specimens, 29 serum specimens, and 24 plasma specimens from 109 patients with
Candida
bloodstream infection (candidemia) to determine whether a diagnosis could be expedited in comparison with the time to diagnosis by the use of standard blood culture. Overall, the MT-PCR performed better than blood culture with DNA extracted from whole blood from 52/74 (70%) patients, accelerating the time to detection (blood culture flagging) and determination of the pathogenic species (by use of the API 32C system [bioMérieux, Marcy l'Etoile, France]) by up to 4 days (mean, 2.2 days; range, 0.5 to 8 days).
Candida
DNA was detected more often in serum (71%) and plasma (75%) than in whole blood (54%), although relatively small numbers of serum and plasma specimens were tested. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay with whole blood were 75%, 97%, 95%, and 85%, respectively. Fungal DNA was not detected by MT-PCR in 6/24 (25%) whole-blood samples drawn simultaneously with the positive blood culture sample. MT-PCR performed better with whole-blood specimens stored at −20°C (75%) and when DNA was extracted within 1 week of sampling (66%). The molecular and culture identification results correlated for 61 of 66 patients (92%); one discrepant result was due to misidentification by culture. All but one sample from 53 patients who were at high risk of candidemia but did not have proven disease were negative by MT-PCR. The results demonstrate the good potential of MT-PCR to detect candidemia, to provide
Candida
species identification prior to blood culture positivity, and to provide improved sensitivity when applied to with serum and plasma specimens.
The Nonserotypeable Pneumococcus: Phenotypic Dynamics in the Era of Anticapsular Vaccines
Authors: Marsh R. , Smith-Vaughan H. , Hare K. M. , Binks M. , Kong F. , Warning J. , Gilbert G. L. , Morris P. , Leach A. J.
Published: December 2009

Nonserotypeable pneumococci (NSP) are commonly carried by Australian Indigenous children in remote communities. The purpose of this study was to characterize carriage isolates of NSP from Indigenous children vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV7) and to use these data to guide decisions on reporting of NSP. A total of 182 NSP were characterized by BOX typing, antibiogram analysis, and multilocus sequence typing (MLST) of common BOX types. NSP positive for the
wzg
capsule gene were analyzed by a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB-H) targeting capsule genes to determine the serotype. Among 182 NSP, 49 BOX types were identified. MLST of 10 representative isolates found 7 STs, including ST448 (which accounted for 11% of NSP). Non-penicillin susceptibility was evident in 51% of the isolates. Pneumococcal
wzg
sequences were detected in only 23 (13%) NSP, including 10 that contained an ∼1.2-kb insert in the region. mPCR/RLB-H identified serotype 14
wzy
sequences in all 10 NSP, and 1 also contained a serotype 3-specific
wze
sequence. Among the remaining 13
wzg-
positive NSP, few belonged to the serotypes represented in PCV7. It appears that most NSP identified in Australian Indigenous children are from a true nonencapsulated lineage. Few NSP represented serotypes in PCV7 that suppress capsular expression. High rates of carriage and penicillin resistance and the occasional presence of capsule genes suggest a role for NSP in the maintenance and survival of capsulated pneumococci. To avoid the inflation of pneumococcal carriage and antibiotic resistance rates, in clinical trials, we recommend separate reporting of rates of capsular strains and NSP and the exclusion of data for NSP from primary analyses.
Comparison of the Clinical Performance of PapilloCheck Human Papillomavirus Detection with That of the GP5+/6+-PCR-Enzyme Immunoassay in Population-Based Cervical Screening
Authors: Hesselink A. T. , Heideman D. A. M. , Berkhof J. , Topal F. , Pol R. P. , Meijer C. J. L. M. , Snijders P. J. F.
Published: December 2009

We compared the clinical performance of the PapilloCheck human papillomavirus (HPV) assay with that of the GP5+/6+-PCR method with an enzyme immunoassay readout (GP5+/6+-PCR-EIA) for the detection of high-risk HPV (hrHPV) types by the use of cervical samples originating from women in a population-based by the use of cervical screening cohort tested by combined cytology and GP5+/6+-PCR-EIA (POBASCAM trial). Specimens from a random sample of 1,437 controls (women ages 40 to 60 years with normal cytological findings and without evidence of cervical intraepithelial neoplasia grade 2 or higher [≥CIN2] within up to 8 years of follow-up) and 192 cases (women ages 30 to 60 years in whom ≥CIN3 was detected within up to 3 years of follow-up) were subjected to analysis by the PapilloCheck method. When all 17 (probably) hrHPV types were taken into account, the PapilloCheck assay had a clinical sensitivity for the detection of ≥CIN3 of 96.4% (185/192 samples; 95% confidence interval [CI], 93.7 to 99.7) and a clinical specificity for the detection of ≥CIN2 of 96.3% (95% CI, 95.3 to 97.3). After restriction of the analysis by the PapilloCheck assay to the 14 hrHPV types targeted by GP5+/6+-PCR-EIA, the clinical sensitivity and clinical specificity values were 95.8% (95% CI, 92.8 to 98.8) and 96.7% (95% CI, 95.7 to 97.7), respectively. By comparison, these values were 96.4% (95% CI, 93.9 to 98.9) and 97.7% (95% CI, 96.9 to 98.5), respectively, for the GP5+/6+-PCR-EIA. When all 17 (probably) hrHPV types were included in the analysis, noninferiority score testing revealed that the clinical sensitivity of the PapilloCheck assay for the detection of ≥CIN3 was noninferior to that of the GP5+/6+-PCR-EIA (
P
< 0.0001), but the clinical specificity of the PapilloCheck assay for the detection of ≥CIN2 was inferior to that of the GP5+/6+-PCR-EIA (
P
= 0.08) when lower bounds of 90% for sensitivity and 98% for specificity were used. When the analysis was restricted to the 14 hrHPV types targeted by the GP5+/6+-PCR-EIA, both the clinical sensitivity and the clinical specificity of the PapilloCheck assay were noninferior to those of the GP5+/6+-PCR-EIA (noninferiority score test;
P
< 0.0001 and
P
= 0.007, respectively). Thus, when the findings obtained for the 14 hrHPV types detectable by the GP5+/6+-PCR-EIA are considered, the PapilloCheck assay is clinically compatible with the GP5+/6+-PCR-EIA.
Sodium Polyanethole Sulfonate as an Inhibitor of Activation of Complement Function in Blood Culture Systems
Authors: Palarasah Yaseelan , Skjoedt Mikkel-Ole , Vitved Lars , Andersen Thomas Emil , Skjoedt Karsten , Koch Claus
Published: December 2009

Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three complement activation pathways: the classical, alternative, and lectin pathways, respectively. Inhibition of complement activity by SPS is caused by a blocking of complement activation and is not a result of complement consumption. The classical pathway is inhibited at SPS concentrations greater than 0.1 mg/ml, and complete inhibition is seen at 0.4 mg/ml. An SPS concentration of 0.5 mg/ml completely inhibits the binding of C1q and subsequent incorporation of C3, C4, and C9. The same was observed for the alternative pathway with an inhibition at SPS concentrations from 0.1 mg/ml and a complete inhibition from 0.4 mg/ml. Here, properdin binding was completely absent, and no incorporation of C3 and C9 was observed. In contrast, the lectin complement pathway remains unaffected at these SPS concentrations, and inhibition is first observed from 0.7 mg/ml. A complete inhibition required concentrations greater than 1 mg/ml. SPS is used in growth media (e.g., BACTEC and BacT/Alert) at concentrations from 0.3 to 0.5 mg/ml. The well-known finding that certain bacteria are growth inhibited by blood factors could therefore be a consequence of the lectin pathway, which is not inhibited at these concentrations. In addition, our findings also open up the possibility of a new assay for the assessment of the functional capacity of the lectin complement pathway.
GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product, Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein
Authors: Kalt Inna , Borodianskiy-Shteinberg Tatyana , Schachor Adi , Sarid Ronit
Published: December 2009

KS-Bcl-2, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins.
Inability of Plasmacytoid Dendritic Cells To Directly Lyse HIV-Infected Autologous CD4
+
T Cells despite Induction of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
Authors: Chehimi Jihed , Papasavvas Emmanouil , Tomescu Costin , Gekonge Bethsebah , Abdulhaqq Shaheed , Raymond Andrea , Hancock Aidan , Vinekar Kavita , Carty Craig , Reynolds Griffin , Pistilli Maxwell , Mounzer Karam , Kostman Jay , Montaner Luis J.
Published: December 2009

The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-α) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4
+
T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-α/β were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-α upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV
+
subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4
+
T cells from viremic HIV
+
subjects compared to controls or following
in vitro
infection/exposure to infectious and noninfectious virus or exogenous IFN-α, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV
+
CD4
+
T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4
+
T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-α in chronic HIV-1 infection
in vivo
and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4
+
T cells.
Measles Virus Infection of Alveolar Macrophages and Dendritic Cells Precedes Spread to Lymphatic Organs in Transgenic Mice Expressing Human Signaling Lymphocytic Activation Molecule (SLAM, CD150)
Authors: Ferreira Claudia S. Antunes , Frenzke Marie , Leonard Vincent H. J. , Welstead G. Grant , Richardson Christopher D. , Cattaneo Roberto
Published: December 2009

Recent studies of primate models suggest that wild-type measles virus (MV) infects immune cells located in the airways before spreading systemically, but the identity of these cells is unknown. To identify cells supporting primary MV infection, we took advantage of mice expressing the MV receptor human signaling lymphocyte activation molecule (SLAM, CD150) with human-like tissue specificity. We infected these mice intranasally (IN) with a wild-type MV expressing green fluorescent protein. One, two, or three days after inoculation, nasal-associated lymphoid tissue (NALT), the lungs, several lymph nodes (LNs), the spleen, and the thymus were collected and analyzed by microscopy and flow cytometry, and virus isolation was attempted. One day after inoculation, MV replication was documented only in the airways, in about 2.5% of alveolar macrophages (AM) and 0.5% of dendritic cells (DC). These cells expressed human SLAM, and it was observed that MV infection temporarily enhanced SLAM expression. Later, MV infected other immune cell types, including B and T lymphocytes. Virus was isolated from lymphatic tissue as early as 2 days post-IN inoculation; the mediastinal lymph node was an early site of replication and supported high levels of infection. Three days after intraperitoneal inoculation, 1 to 8% of the mediastinal LN cells were infected. Thus, MV infection of alveolar macrophages and subepithelial dendritic cells in the airways precedes infection of lymphocytes in lymphatic organs of mice expressing human SLAM with human-like tissue specificity.
Epstein-Barr Virus-Encoded Bcl-2 Homologue Functions as a Survival Factor in Wp-Restricted Burkitt Lymphoma Cell Line P3HR-1
Authors: Watanabe Ami , Maruo Seiji , Ito Taku , Ito Miho , Katsumura Koichi Ricardo , Takada Kenzo
Published: December 2009

Burkitt lymphoma (BL) is etiologically associated with Epstein-Barr virus (EBV). EBV-positive BL tumors display two latent forms of infection. One is referred to as latency I infection, in which EBV expresses the virus genome maintenance protein EBNA1 as the only viral protein. The other is referred to as Wp-restricted latency and was recently identified in a subset of BL tumors. In these tumors, EBV expresses EBNA1, EBNA3A, EBNA3B, EBNA3C, a truncated form of EBNA-LP, and the viral Bcl-2 homologue BHRF1, all of which are driven by the BamHI W promoter (Wp). To investigate the role of EBV in Wp-restricted BL, we conditionally expressed a dominant-negative EBNA1 (dnEBNA1) mutant which interrupts the virus genome maintenance function of EBNA1 in the P3HR-1 BL cell line. Induction of dnEBNA1 expression caused loss of the EBV genome and resulted in apoptosis of P3HR-1 cells in the absence of exogenous apoptosis inducers, indicating that P3HR-1 cells cannot survive without EBV. Stable transfection of the BHRF1 gene into P3HR-1 cells rescued the cells from the apoptosis induced by dnEBNA1 expression, whereas stable transfection of truncated EBNA-LP, EBNA3A, or EBNA3C did not. Moreover, knockdown of BHRF1 expression in P3HR-1 cells resulted in increased cell death. These results indicate that EBV is essential for the survival of P3HR-1 cells and that BHRF1 functions as a survival factor. Our finding implies a critical contribution of BHRF1 to the pathogenesis of Wp-restricted BLs.
Effect of Mutations in the Human Immunodeficiency Virus Type 1 Protease on Cleavage of the gp41 Cytoplasmic Tail
Authors: Waheed Abdul A. , Ablan Sherimay D. , Sowder Raymond C. , Roser James D. , Schaffner Carl P. , Chertova Elena , Freed Eric O.
Published: December 2009

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.
Identification of an ICP27-Responsive Element in the Coding Region of a Herpes Simplex Virus Type 1 Late Gene
Authors: Sedlackova Lenka , Perkins Keith D. , Meyer Julia , Strain Anna K. , Goldman Oksana , Rice Stephen A.
Published: December 2009

During productive herpes simplex virus type 1 (HSV-1) infection, a subset of viral delayed-early (DE) and late (L) genes require the immediate-early (IE) protein ICP27 for their expression. However, the
cis
-acting regulatory sequences in DE and L genes that mediate their specific induction by ICP27 are unknown. One viral L gene that is highly dependent on ICP27 is that encoding glycoprotein C (gC). We previously demonstrated that this gene is posttranscriptionally transactivated by ICP27 in a plasmid cotransfection assay. Based on our past results, we hypothesized that the gC gene possesses a
cis
-acting inhibitory sequence and that ICP27 overcomes the effects of this sequence to enable efficient gC expression. To test this model, we systematically deleted sequences from the body of the gC gene and tested the resulting constructs for expression. In so doing, we identified a 258-bp “silencing element” (SE) in the 5′ portion of the gC coding region. When present, the SE inhibits gC mRNA accumulation from a transiently transfected gC gene, unless ICP27 is present. Moreover, the SE can be transferred to another HSV-1 gene, where it inhibits mRNA accumulation in the absence of ICP27 and confers high-level expression in the presence of ICP27. Thus, for the first time, an ICP27-responsive sequence has been identified in a physiologically relevant ICP27 target gene. To see if the SE functions during viral infection, we engineered HSV-1 recombinants that lack the SE, either in a wild-type (WT) or ICP27-null genetic background. In an ICP27-null background, deletion of the SE led to ICP27-independent expression of the gC gene, demonstrating that the SE functions during viral infection. Surprisingly, the ICP27-independent gC expression seen with the mutant occurred even in the absence of viral DNA synthesis, indicating that the SE helps to regulate the tight DNA replication-dependent expression of gC.
Interaction Domains of the UL16 and UL21 Tegument Proteins of Herpes Simplex Virus
Authors: Harper Amy L. , Meckes David G. , Marsh Jacob A. , Ward Michael D. , Yeh Pei-Chun , Baird Nicholas L. , Wilson Carol B. , Semmes O. John , Wills John W.
Published: December 2009

The UL16 protein of herpes simplex virus is capsid associated and was previously identified as a binding partner of the membrane-associated UL11 tegument protein (J. S. Loomis, R. J. Courtney, and J. W. Wills, J. Virol. 77:11417-11424, 2003). In those studies, a less-prominent, ∼65-kDa binding partner of unknown identity was also observed. Mass spectrometry studies have now revealed this species to be UL21, a tegument protein that has been implicated in the transport of capsids in the cytoplasm. The validity of the mass spectrometry results was tested in a variety of coimmunoprecipitation and glutathione
S
-transferase pull-down experiments. The data revealed that UL21 and UL16 can form a complex in the absence of other viral proteins, even when the assays used proteins purified from
Escherichia coli
. Moreover, UL11 was able to pull down UL21 only when UL16 was present, suggesting that all three proteins can form a complex. Deletion analyses revealed that the second half of UL21 (residues 268 to 535) is sufficient for the UL16 interaction and packaging into virions; however, attempts to map a subdomain of UL16 were largely unsuccessful, with only the first 40 (of 373) residues being found to be dispensable. Nevertheless, it is clear that UL16 must have two distinct binding sites, because covalent modification of its free cysteines with
N
-ethylmaleimide blocked binding to UL11 but not UL21. These findings should prove useful for elucidating the molecular machinery used to transmit a signal into a virion when it attaches to cells, a recently discovered mechanism in which UL16 is a central player.
Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription
Authors: Tran Karen , Mahr Jeffrey A. , Spector Deborah H.
Published: December 2009

We have continued studies to further understand the role of the ubiquitin-proteasome system (UPS) in human cytomegalovirus (HCMV) infection. With specific inhibitors of the proteasome, we show that ongoing proteasome activity is necessary for facilitating the various stages of the infection. Immediate-early protein 2 expression is modestly reduced with addition of proteasome inhibitors at the onset of infection; however, both early and late gene expression are significantly delayed, even if the inhibitor is removed at 12 h postinfection. Adding the inhibitor at later times during the infection blocks the further accumulation of viral early and late gene products, the severity of which is dependent on when the proteasome is inhibited. This can be attributed primarily to a block in viral RNA transcription, although DNA synthesis is also partially inhibited. Proteasome activity and expression increase as the infection progresses, and this coincides with the relocalization of active proteasomes to the periphery of the viral DNA replication center, where there is active RNA transcription. Interestingly, one 19S subunit, Rpn2, is specifically recruited into the viral DNA replication center. The relocalization of the subunits requires viral DNA replication, but their maintenance around or within the replication center is not dependent on continued viral DNA synthesis or the proteolytic activity of the proteasome. These studies highlight the importance of the UPS at all stages of the HCMV infection and support further studies into this pathway as a potential antiviral target.
Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region
Authors: Ofek Gilad , McKee Krisha , Yang Yongping , Yang Zhi-Yong , Skinner Jeff , Guenaga F. Javier , Wyatt Richard , Zwick Michael B. , Nabel Gary J. , Mascola John R. , Kwong Peter D.
Published: December 2009

The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (
P
< 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ∼10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.
Gamma Interferon Signaling in Oligodendrocytes Is Critical for Protection from Neurotropic Coronavirus Infection
Authors: Parra Gabriel I. , Bergmann Cornelia C. , Phares Timothy W. , Hinton David R. , Atkinson Roscoe , Stohlman Stephen A.
Published: December 2009

Neurotropic coronavirus induces acute encephalomyelitis and demyelination in mice. Infection of BALB/c (H-2d) mice expressing a dominant negative gamma interferon (IFN-γ) receptor specifically in oligodendrocytes was examined to determine the influence of IFN-γ signaling on pathogenesis. Inhibition of IFN-γ signaling in oligodendrocytes increased viral load, infection of oligodendrocytes, oligodendrocyte loss, demyelination, and axonal damage resulting in increased mortality. IFN-γ levels and the inflammatory response were not altered, although the level of tumor necrosis factor (TNF) mRNA was increased. These data indicate that IFN-γ signaling by oligodendroglia reduces viral replication but affects both demyelination and tissue destruction in a host-specific manner.
Seasonal variations in humic substances and their reduction through water treatment processes
Authors: Alarcon-Herrera M. T. , Bewtra J. K. , Biswas N.
Published: January 2010

The apparent molecular mass concentrations of dissolved humic substances from the Detroit River and treated water from the Windsor Water Treatment Plant were determined by gel permeation chromatography over a 1-year period. The results of this research show significant seasonal variations in humic substance concentrations and these values are influenced by the turnover of water bodies. The gel permeation chromatograms of the samples indicate that there is a gradual reduction in the concentration of humic substances during the treatment process, the greatest change occurring after coagulation-sedimentation process. In this process, 76%, 40%, and 30% of the high, intermediate, and low molecular mass compounds, respectively, were either transformed in chemical composition or removed. Other parameters such as turbidity, chlorine demand (during prechlorination), and trihalomethanes concentration in the treated water were also analyzed and there appears to be a relationship between them. When the humic substance concentrations are high, generally the turbidity, chlorine demand, and total trihalomethanes concentrations in the drinking water are also high. Key words: water treatment, humic substances, molecular mass distribution.
Sedimentation — Exclusion and removal of sediment from diverted water
Authors: Rajaratnam N.
Published: January 2010

not available
Prediction of wind effects on buildings using computational methods — review of the state of the art
Authors: Baskaran A. , Stathopoulos T.
Published: January 2010

Advancements in computer software and hardware technology provide a new direction for analyzing engineering problems. Recently the field of wind engineering has gained significant momentum in the computer modelling process. This paper reviews the state of the art in computational wind engineering, including the finite element method, finite difference method, and control volume technique. A portion of this paper summarizes the research in this area carried out by the authors. Computations have been made for a variety of building configurations, including normal wind flow conditions for a building with different aspect ratios, and modelling wind environmental conditions around groups of buildings. The computer modelling technique may eventually enhance the design of buildings and structures against wind loading and supplement the current design practice of using building codes and standards or performing experiments in wind tunnels. Key words: buildings, computer modelling, pressure, velocity, wind engineering, wind tunnels.
Issues in structural fatigue design and evaluation
Authors: Agarwal A. C. , Birkemoe P. C. , Goel A. P. , Sweeney R. A. P. , Thompson J. C. , Topper T. H.
Published: January 2010

Current practices in the design and evaluation of structural components for fatigue are generally semi-empirical. This is because either it is not practical or economical to realistically simulate field conditions in the laboratory testing or the actual conditions are not known precisely. Fatigue life curves based on the constant amplitude cyclic loading may result in unconservative design and evaluations where real load cycles are variable and the loads are of transient nature. There is a need to apply probability-based principles to the fatigue considerations, particularly in the areas of developing representative fatigue loading, and fatigue crack detection. In the area of power transmission lines, there is a need for better understanding of the failure mechanism and rationalize the fatigue design provisions. The state of stress within a stress concentration region depends only on the local boundary geometry, the magnitude of the resultant force, and the material type. This equivalence of stress concentration regions can be exploited to develop design aids for stress concentration factors independent of the global conditions. This paper identifies the above issues in detail and establishes some specific areas where there is a need for improvement in the current practice. Key words: fatigue, structural design, structural evaluation, bridges, power transmission lines, offshore structures.
Strong motion accelerograph records from the 1993 Napierville earthquake
Authors: Tinawi René , Filiatrault André , Léger Pierre
Published: January 2010

An earthquake of magnitude ML = 4.3 occurred near Napierville, Quebec, on November 16, 1993. An accelerograph at the liquefaction, storage, and regasification plant of Gaz Metropolitain in Montreal, about 55 km from the epicentre, recorded the ground motion. Although the maximum accelerations and velocities from this event are small, the acceleration time histories do confirm the high energy content in the very short period range. The recorded ground motion and corresponding absolute acceleration response spectra are presented and various attenuation relationships, proposed for eastern North America, are utilized to compare the measured and predicted ground motion parameters. Key words: Napierville earthquake, attenuation relationships, acceleration spectra, strong motion records.
Auscultation des structures hydrauliques en béton par tomographie sonique
Authors: Kharrat Yahya , Rhazi Jamal , Ballivy Gérard , Côte Philippe
Published: January 2010

Concrete aging is a problem that affects more and more service structures. Detailed knowledge of their state is necessary prior to rehabilitation. Sonic tomography, a relatively new, nondestructive global auscultation approach allows to reconstitute the internal image of the structure. Transmitted sonic wave velocity in the material is the parameter considered. The relative variation in wave propagation velocity in the material provides information about changes in the structure, and therefore the state of degradation. Two hydraulic concrete structures were studied. The principle and the experimental procedure of this method are explained. Key words: structure, concrete, degradation, auscultation, nondestructive test, sonic tomography