bluuebunny/crossref_metadata_2025 huggingface dataset:
CagA and VacA Polymorphisms Are Associated with Distinct Pathological Features in
Helicobacter
pylori
-Infected Adults with Peptic Ulcer and Non-Peptic Ulcer Disease
Authors: Panayotopoulou Effrosini G. , Sgouras Dionyssios N. , Papadakos Konstantinos S. , Petraki Kalliopi , Breurec Sébastien , Michopoulos Spyros , Mantzaris Gerassimos , Papatheodoridis George , Mentis Andreas , Archimandritis Athanasios
Published: April 2010

Polymorphic variability in
Helicobacter
pylori
factors CagA and VacA contributes to bacterial virulence. The presence of one CagA EPIYA-C site is an independent risk factor for gastroduodenal ulceration (odds ratio [OR], 4.647; 95% confidence interval [CI], 2.037 to 10.602), while the presence of the
vacA
i1 allele is a risk factor for increased activity (OR, 5.310; 95% CI, 2.295 to 12.287) and severity of gastritis (OR, 3.862; 95% CI, 1.728 to 8.632).
Detection of Inducible Clindamycin Resistance in Beta-Hemolytic Streptococci by Using the CLSI Broth Microdilution Test and Erythromycin-Clindamycin Combinations
Authors: Bowling Jason E. , Owens Aaron E. , McElmeel M. Leticia , Fulcher Letitia C. , Herrera Monica L. , Wickes Brian L. , Jorgensen James H.
Published: April 2010

This study assessed an erythromycin-clindamycin (ERY-CC) broth test for inducible CC resistance in beta-hemolytic streptococci. One hundred one isolates of groups A, B, C, F, and G were tested by the CLSI broth microdilution method. Combinations of 1 and 0.25 μg/ml or 0.5 and 0.25 μg/ml of ERY and CC, respectively, detected all inducible isolates.
Alternative Use for Spectra MRSA Chromogenic Agar in Detection of Methicillin-Resistant
Staphylococcus aureus
from Positive Blood Cultures
Authors: Peterson Jess F. , Dionisio Alexander A. , Riebe Katherine M. , Hall Gerri S. , Wilson Deborah A. , Whittier Susan , DiPersio Joseph R. , Ledeboer Nathan A.
Published: April 2010

Spectra MRSA agar (Remel, Lenexa, KS), a novel chromogenic medium originally developed to detect methicillin-resistant
Staphylococcus aureus
(MRSA) from nasal swabs, was evaluated in this multicenter study for the detection of MRSA from positive blood cultures exhibiting Gram-positive cocci upon initial Gram staining.
Typhoid in Kenya Is Associated with a Dominant Multidrug-Resistant
Salmonella
enterica
Serovar Typhi Haplotype That Is Also Widespread in Southeast Asia
Authors: Kariuki Samuel , Revathi Gunturu , Kiiru John , Mengo Doris M. , Mwituria Joyce , Muyodi Jane , Munyalo Agnes , Teo Yik Y. , Holt Kathryn E. , Kingsley Robert A. , Dougan Gordon
Published: April 2010

In sub-Saharan Africa, the burden of typhoid fever, caused by
Salmonella enterica
serovar Typhi, remains largely unknown, in part because of a lack of blood or bone marrow culture facilities. We characterized a total of 323
S
. Typhi isolates from outbreaks in Kenya over the period 1988 to 2008 for antimicrobial susceptibilities and phylogenetic relationships using single-nucleotide polymorphism (SNP) analysis. There was a dramatic increase in the number and percentage of multidrug-resistant (MDR)
S
. Typhi isolates over the study period. Overall, only 54 (16.7%)
S
. Typhi isolates were fully sensitive, while the majority, 195 (60.4%), were multiply resistant to most commonly available drugs—ampicillin, chloramphenicol, tetracycline, and cotrimoxazole; 74 (22.9%) isolates were resistant to a single antimicrobial, usually ampicillin, cotrimoxazole, or tetracycline. Resistance to these antibiotics was encoded on self-transferrable IncHI1 plasmids of the ST6 sequence type. Of the 94 representative
S
. Typhi isolates selected for genome-wide haplotype analysis, sensitive isolates fell into several phylogenetically different groups, whereas MDR isolates all belonged to a single haplotype, H58, associated with MDR and decreased ciprofloxacin susceptibility, which is also dominant in many parts of Southeast Asia. Derivatives of the same
S
. Typhi lineage, H58, are responsible for multidrug resistance in Kenya and parts of Southeast Asia, suggesting intercontinental spread of a single MDR clone. Given the emergence of this aggressive MDR haplotype, careful selection and monitoring of antibiotic usage will be required in Kenya, and potentially other regions of sub-Saharan Africa.
Evaluation of Bio-Rad MRSA
Select
Agar for Detection of Methicillin-Resistant
Staphylococcus aureus
Directly from Blood Cultures
Authors: Riedel Stefan , Dam Lisa , Stamper Paul D. , Shah Syed A. R. , Carroll Karen C.
Published: April 2010

MRSA
Select
agar (Bio-Rad, Redmond, WA) was evaluated for its performance in detecting MRSA directly from positive blood cultures containing Gram-positive cocci in clusters. Agar plates were evaluated for the presence of pink colonies at 18 to 24 h. Results were compared to organism identification by using standard laboratory methods. Confirming coagulase on pink isolates, the sensitivity and specificity were both 99%.
Direct Identification of Urinary Tract Pathogens from Urine Samples by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Authors: Ferreira Laura , Sánchez-Juanes Fernando , González-Ávila Magdalena , Cembrero-Fuciños David , Herrero-Hernández Ana , González-Buitrago José Manuel , Muñoz-Bellido Juan Luis
Published: April 2010

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 ×
g
) to remove leukocytes and then at high revolutions (15,500 ×
g
) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >10
5
CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was
Escherichia coli
(173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%). Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved.
Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit
Authors: Garner Cherilyn D. , Starr Jennifer K. , McDonough Patrick L. , Altier Craig
Published: April 2010

Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods—traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit—showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common
Candida
spp., such as
C. albicans
,
C. glabrata
,
C. tropicalis
, and the
C. parapsilosis
group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.
Development and Evaluation of anompAQuantitative Real-Time PCR Assay forChlamydia trachomatisSerovar Determination
Authors: Stevens Matthew P. , Twin Jimmy , Fairley Christopher K. , Donovan Basil , Tan Sarah E. , Yu Jingxi , Garland Suzanne M. , Tabrizi Sepehr N.
Published: April 2010

Knowledge of circulatingChlamydia trachomatisserovars can be beneficial for sexual network surveillance, monitoring treatment success, and associating specific clinical manifestations. Typically,C. trachomatisserovars are predicted by nucleotide sequencing of four variable domains within theompAgene. However, sequencing procedures can be labor-intensive, are not readily available, and can lack the capacity to identify multiple serovars. This study describes the development and evaluation of a quantitative real-time PCR (qPCR) test algorithm for the rapid prediction ofC. trachomatisserovars, including ocular (A to C) and anogenital (D to L3) strains. This test comprises a primary qPCR to confirmC. trachomatispositivity and the phylogenetic group(s) present and a secondary set of qPCRs to determine specific serovars. Cell culture isolates from 15 prototypicC. trachomatisserovars were correctly identified using this assay, with no cross-reactivity observed among serovars or with other common pathogenic microorganisms. Five hundred clinical specimens (previously diagnosed as beingC. trachomatispositive) were evaluated by qPCR, with their results compared to results obtained by conventional sequencing. The qPCR identified 88.9% (423/476) complete matches (95% confidence interval [CI], 86 to 92%) of serovars compared to the results obtained using the sequence-based approach. Among the anogenital specimens, 2.4% (12/494) (95% CI, 1.3 to 4.2%) contained multiple serovars, categorized as single-serovar infections by conventional sequencing. Overall, this test exhibited high discriminatory success for predictingC. trachomatisserovars, particularly among anogenital infections. This is the first report of a qPCR typing assay offering differentiation ofC. trachomatisserovars associated with both anogenital and ocular diseases.
Rapid Antifungal Susceptibility Determination for Yeast Isolates by Use of Etest Performed Directly on Blood Samples from Patients with Fungemia
Authors: Guinea Jesús , Recio Sandra , Escribano Pilar , Torres-Narbona Marta , Peláez Teresa , Sánchez-Carrillo Carlos , Rodríguez-Créixems Marta , Bouza Emilio
Published: April 2010

We prospectively determined the antifungal susceptibility of yeast isolates causing fungemia using the Etest on direct blood samples (195 prospectively collected and 133 laboratory prepared). We compared the Etest direct (24 h of incubation) with CLSI M27-A3 and the standard Etest methodologies for fluconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B. Strains were classified as susceptible, resistant, or nonsusceptible using CLSI breakpoints (voriconazole breakpoints were used for posaconazole and isavuconazole). Categorical errors between Etest direct and CLSI M27-A3 for azoles were mostly minor. No errors were detected for caspofungin, and high percentages of major errors were detected for amphotericin B. For the azoles, false susceptibility (very major errors) was found in only two (0.6%) isolates (
Candida tropicalis
and
C. glabrata
). False resistance (major errors) was detected in 46 (14%) isolates for the three azoles (in 23 [7%] after excluding posaconazole). Etest direct of posaconazole yielded a higher number of major errors than the remaining azoles, especially for
C. glabrata
,
Candida
spp., and other yeasts. Excluding
C. glabrata
,
Candida
spp., and other yeasts, the remaining species did not yield major errors. Etest direct for fluconazole, voriconazole, isavuconazole, and caspofungin shows potential as an alternative to the CLSI M27-A3 procedure for performing rapid antifungal susceptibility tests on yeast isolates from patients with fungemia. Etest direct is a useful tool to screen for the presence of azole-resistant and caspofungin-nonsusceptible strains.
Evaluation of a New Selective Medium, BD BBL CHROMagar MRSA II, for Detection of Methicillin-Resistant
Staphylococcus aureus
in Different Specimens
Authors: Wendt C. , Havill N. L. , Chapin K. C. , Boyce J. M. , Dickenson R. , Eigner U. , Schütt S. , Fahr A. M.
Published: April 2010

The sensitivity of screening for methicillin-resistant
Staphylococcus aureus
(MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII.
S. aureus
was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as
S. aureus
(by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (
P
< 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.
Evaluation of a New Selective Medium, BD BBL CHROMagar MRSA II, for Detection of Methicillin-Resistant
Staphylococcus aureus
in Stool Specimens
Authors: Havill Nancy L. , Boyce John M.
Published: April 2010

We compared the recovery of methicillin-resistant
Staphylococcus aureus
(MRSA) on a new selective chromogenic agar, BD BBL CHROMagar MRSA II (CMRSAII), to that on traditional culture media with 293 stool specimens. The recovery of MRSA was greater on the CMRSAII agar. Screening of stool samples can identify patients who were previously unknown carriers of MRSA.
Multisite Study of Cryptosporidiosis in Children with Diarrhea in India
Authors: Ajjampur Sitara Swarna Rao , Liakath Farzana Begum , Kannan Arun , Rajendran Priya , Sarkar Rajiv , Moses Prabhakar Devarajan , Simon Anna , Agarwal Indira , Mathew Ann , O'Connor Roberta , Ward Honorine , Kang Gagandeep
Published: April 2010

Cryptosporidium
spp., a common cause of diarrhea in children, were investigated in the first multisite study in India. Diarrheal stools from hospitalized children aged <5 years from Delhi, Trichy, and Vellore were analyzed by microscopy, PCR-restriction fragment length polymorphism (RFLP), and/or sequencing at the small-subunit (SSU) rRNA and
Cpgp40/15
loci for species determination and subgenotyping, respectively. Seventy of 2,579 (2.7%) children, 75% of whom were <2 years old, had cryptosporidial diarrhea as determined by microscopy. Genotyping and subgenotyping showed that
C
ryptosporidium
hominis
was the most commonly identified species (59/67 children), and subgenotypes Ie, Ia, Ib, and Id were common in all centers. A novel
C. parvum
subgenotype, IIn, was identified in Vellore. Meteorological analysis revealed a higher rate of cryptosporidial positivity during hotter and drier weather in Delhi.
Selection of a Rare Neutralization-Resistant Variant following Passive Transfer of Convalescent Immune Plasma in Equine Infectious Anemia Virus-Challenged SCID Horses
Authors: Taylor Sandra D. , Leib Steven R. , Carpenter Susan , Mealey Robert H.
Published: April 2010

Vaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicatein vivounder relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.
An LYPSL Late Domain in the Gag Protein Contributes to the Efficient Release and Replication of Rous Sarcoma Virus
Authors: Dilley Kari A. , Gregory Devon , Johnson Marc C. , Vogt Volker M.
Published: April 2010

The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPX
n
L. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned 5 amino acids downstream of PPPY. LYPX
n
L motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. The overexpression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.
The RING Finger Domain of Varicella-Zoster Virus ORF61p Has E3 Ubiquitin Ligase Activity That Is Essential for Efficient Autoubiquitination and Dispersion of Sp100-Containing Nuclear Bodies
Authors: Walters Matthew S. , Kyratsous Christos A. , Silverstein Saul J.
Published: April 2010

Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate that disruption of the ORF61p RING finger domain by amino acid substitution (Cys19Gly) alters ORF61p intranuclear distribution and abolishes ORF61p-mediated dispersion of Sp100-containing nuclear bodies. In addition, we demonstrate that an intact ORF61p RING finger domain is necessary for E3 ubiquitin ligase activity and is required for autoubiquitination and regulation of protein stability.
Live-Cell Coimaging of the Genomic RNAs and Gag Proteins of Two Lentiviruses
Authors: Kemler Iris , Meehan Anne , Poeschla Eric M.
Published: April 2010

Human immunodeficiency virus type 1 (HIV-1) Gag and genomic RNA determinants required for encapsidation are well established, but where and when encapsidation occurs in the cell is unknown. We constructed MS2 phage coat protein labeling systems to track spatial dynamics of primate and nonprimate lentiviral genomic RNAs (HIV-1 and feline immunodeficiency virus [FIV]) vis-à-vis their Gag proteins in live cells. Genomic RNAs of both lentiviral genera were observed to traffic into the cytoplasm, and this was Rev dependent. In transit, FIV Gag and genomic RNA accumulated independently of each other at the nuclear envelope, and focal colocalizations of genomic RNA with an intact packaging signal (ψ) and Gag were observed to extend outward from the cytoplasmic face. In contrast, although HIV-1 genomic RNA was detected at the nuclear envelope, HIV-1 Gag was not. For both lentiviruses, genomic RNAs were seen at the plasma membrane if and only if Gag was present and ψ was intact. In addition, HIV-1 and FIV genomes accumulated with Gag in late endosomal foci, again, only ψ dependently. Thus, lentiviral genomic RNAs require specific Gag binding to accumulate at the plasma membrane, packaged genomes cointernalize with Gag into the endosomal pathway, and plasma membrane RNA incorporation by Gag does not trigger committed lentiviral particle egress from the cell. Based on the FIV results, we hypothesize that the Gag-genome association may initiate at the nuclear envelope.
The A20 Deubiquitinase Activity Negatively Regulates LMP1 Activation of IRF7
Authors: Ning Shunbin , Pagano Joseph S.
Published: April 2010

A20 possesses both deubiquitinase (DUB) and ubiquitin E3 ligase activities that are required for termination of Toll-like receptor (TLR) signaling leading to NF-κB activation and for blockage of tumor necrosis factor (TNF)-induced cytotoxicity and apoptosis. A20 is induced by the Epstein-Barr virus (EBV) oncoprotein LMP1. However, its dual ubiquitin-editing activities have not been investigated in the context of either EBV infection or IRF7 responses. Both A20 and IRF7 have oncogenic properties. We have recently shown that LMP1 activates IRF7 through K63-linked ubiquitination which requires RIP1 and TRAF6, but how this ubiquitination event is regulated has not been studied. Here, we show that A20 negatively regulates IRF7 transcriptional activity induced by LMP1. Deletion or mutation of A20 C-terminal zinc finger motifs had no effect on the inhibition of IRF7 activity, whereas DUB-deficient truncation or point mutation ablated the ability of A20 to inhibit IRF7. Correspondingly, the A20 N-terminal DUB domain, but not the C-terminal E3 ligase domain, interacts physically with IRF7. Transient expression of A20 reduced K63-linked ubiquitination of IRF7
in vivo
, but an
in vitro
deubiquitination assay with purified constituents shows that IRF7 did not act as a substrate for A20 DUB activity. Moreover, A20 interacts with IRF7 endogenously in latently EBV-infected type 3 Raji cells, in which expression of both A20 and IRF7 is constitutively induced by the considerable level of endogenous LMP1. Knockdown of endogenous A20 in Raji cells by expression of A20 short hairpin RNA (shRNA) vectors increases endogenous IRF7 activity and ubiquitination, as well as the protein level of LMP1, a target of IRF7. Thus, A20 negatively regulates LMP1-stimulated IRF7 ubiquitination and activity in EBV latency, and its DUB activity is indispensable for this function. Finally, we discussed the regulation and function of IRFs in EBV latency.
DDX6 (Rck/p54) Is Required for Efficient Hepatitis C Virus Replication but Not for Internal Ribosome Entry Site-Directed Translation
Authors: Jangra Rohit K. , Yi MinKyung , Lemon Stanley M.
Published: April 2010

DDX6 (Rck/p54) is an evolutionarily conserved member of the SF2 DEAD-box RNA helicase family that contributes to the regulation of translation and storage and the degradation of cellular mRNAs. It interacts with multiple proteins and is a component of the micro-RNA (miRNA)-induced silencing complex (miRISC). Since miRNA-122 (miR-122) is essential for efficient hepatitis C virus (HCV) replication, we investigated the requirement for DDX6 in HCV replication in cultured hepatoma cells. Small interfering RNA (siRNA)-mediated knockdown of DDX6 and rescue with an siRNA-resistant mutant demonstrated that DDX6 expression is indeed required for optimal HCV replication. However, DDX6 knockdown did not impair miR-122 biogenesis or alter HCV responsiveness to miR-122 supplementation. Overexpression of DDX6 fused to EYFP (EYFP-DDX6) enhanced replication, whereas a helicase-deficient mutant with a substitution in the conserved DEAD-box motif II (DQAD) had a dominant-negative effect, reducing HCV yields. Coimmunoprecipitation experiments revealed an intracellular complex containing DDX6, HCV core protein, and both viral and cellular RNAs, the formation of which was dependent upon the C-terminal domain of DDX6 but not DDX6 helicase activity. However, since DDX6 abundance influenced the replication of subgenomic HCV RNAs lacking core sequence, the relevance of this complex is uncertain. Importantly, DDX6 knockdown caused minimal reductions in cellular proliferation, generally stimulated cellular translation ([
35
S]Met incorporation), and did not impair translation directed by the HCV internal ribosome entry site. Thus, DDX6 helicase activity is essential for efficient HCV replication, reflecting essential roles for DDX6 in HCV genome amplification and/or maintenance of cellular homeostasis.
Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities
Authors: Tikhanovich Irina , Nasheuer Heinz Peter
Published: April 2010

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase α-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).
Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference
Authors: Pelletier Isabelle , Saulnier Aure , Brisac Cynthia , Jegouic Sophie , Vabret Nicolas , Tangy Frédéric , Blondel Bruno , Colbère-Garapin Florence
Published: April 2010

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.
RASCAL Is a New Human Cytomegalovirus-Encoded Protein That Localizes to the Nuclear Lamina and in Cytoplasmic Vesicles at Late Times Postinfection
Authors: Miller Matthew S. , Furlong Wendy E. , Pennell Leesa , Geadah Marc , Hertel Laura
Published: April 2010

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear

r

im-

as

sociated

c

ytomegalovir

al

protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.
Antibody-Mediated Protection against Mucosal Simian-Human Immunodeficiency Virus Challenge of Macaques Immunized with Alphavirus Replicon Particles and Boosted with Trimeric Envelope Glycoprotein in MF59 Adjuvant
Authors: Barnett Susan W. , Burke Brian , Sun Yide , Kan Elaine , Legg Harold , Lian Ying , Bost Kristen , Zhou Fengmin , Goodsell Amanda , zur Megede Jan , Polo John , Donnelly John , Ulmer Jeffrey , Otten Gillis R. , Miller Christopher J. , Vajdy Michael , Srivastava Indresh K.
Published: April 2010

We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIV
SF162P4
following sequential immunization with alpha
v
irus
r
eplicon
p
articles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1
SF162
gp140ΔV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIV
SF162P4
(intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against HIV infection at a relevant mucosal portal of entry.
2A Protease Is Not a Prerequisite for Poliovirus Replication
Authors: Igarashi Hiroko , Yoshino Yasuko , Miyazawa Miwako , Horie Hitoshi , Ohka Seii , Nomoto Akio
Published: April 2010

Poliovirus (PV) 2A
pro
has been considered important for PV replication and is known to be toxic to host cells. A 2A
pro
-deficient PV would potentially be less toxic and ideal as a vector. To examine whether 2A
pro
is needed to form progeny virus, a full-length cDNA of dicistronic (
dc
) PV with (pOME) or without (pOMEΔ2A) 2A
pro
was constructed in the strain PV1(M)OM. RNAs of both pOME and pOMEΔ2A were capable of forming progeny viruses, called OME and OMEΔ2A, respectively. In their ability to induce a cytopathic effect (CPE), the strains ranked as OMEΔ2A < OME ≒ PV1(M)OM. These results suggest that 2A
pro
is not essential for full-length
dc
PV to form progeny virus and that it contributes to the efficient viral replication and/or induction of a CPE. To clarify whether 2A
pro
is essential for P1-null (lacking the entire coding sequence for capsid proteins) PV, the RNA replication activity of P1-null PV (pOMΔP1) or P1-null PV without 2A
pro
(pOMΔP1Δ2A) or without both 2A
pro
and 2B (pOMΔP1Δ2AΔ2B) was examined. The RNAs of pOMΔP1 and pOMΔP1Δ2A could replicate and form progeny viruses under a
trans
supply of P1 protein, whereas the RNA of pOMΔP1Δ2AΔ2B could not. These results suggest that 2A
pro
is not needed for the replication of P1-null PV, although it is important for PV RNA replication and inducing a CPE. To know whether a 2A
pro
-deficient PV can be used as a vector, a P1-null PV containing the enhanced green fluorescent protein (EGFP) coding sequence with or without 2A
pro
was examined. It expressed fluorescent protein. This result suggests that 2A
pro
-deficient PV can express foreign genes.
In Vitro
Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity
Authors: Chutinimitkul Salin , van Riel Debby , Munster Vincent J. , van den Brand Judith M. A. , Rimmelzwaan Guus F. , Kuiken Thijs , Osterhaus Albert D. M. E. , Fouchier Ron A. M. , de Wit Emmie
Published: April 2010

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.
The effect of truncation and shift on virial coefficients of Lennard–Jones potentials
Authors: Shaul Katherine R. S. , Schultz Andrew J. , Kofke David A.
Published: April 2010

We present virial coefficients of up to fifth order computed by Mayer-sampling Monte Carlo for several truncated-and-shifted Lennard–Jones potentials. We employ these coefficients within the virial equation of state to compute vapor-branch spinodals and critical points for each potential considered. We find that truncation distances of 5.0σ and higher yield values in significantly better agreement with those of the unmodified potential than those resulting from the more commonly used truncation distances of 2.5 and 3.0σ. We also employ these virial coefficients to examine the perturbed virial expansion method of Nezbeda and Smith for estimating the critical point. We find that the first-order perturbation performs well in characterizing the effect of potential truncation on the critical point for the truncation distances considered, with errors in critical temperatures ranging from –3 to +2% and errors in critical densities about constant at –22%. Addition of higher-order terms to the perturbation treatment brings it closer to the behavior given by the virial equation of state, which at fifth order underestimates the critical temperatures by 2 to 4% and the critical densities by 20 to 30%.
Tool for group contribution methods – computational fragmentation
Authors: Kolská Zdeňka , Petrus Pavel
Published: April 2010

Group contribution methods are presently one of the universal and the most frequently used approach to estimate many physico-chemical properties of compounds. One of the important steps in development of group contribution method is a correct division of chemical structures of compounds into defined structural fragments. Computational program dividing automatically chemical structures of compounds (hydrocarbons and halogenated hydrocarbons) into structural fragments are now presented. For description of chemical structures of compounds and structural fragments we used SMILES format. New database of fragments and new record of fragments were created.
Solvation and ion pair association in aqueous metal sulfates: Interpretation of NDIS raw data by isobaric–isothermal molecular dynamics simulation
Authors: Chialvo Ariel A. , Simonson J. Michael
Published: April 2010

We analyzed the solvation behavior of aqueous lithium, nickel, and ytterbium sulfates at ambient conditions in terms of the relevant radial distributions functions and the corresponding first-order difference of the sulfur-site neutron-weighted distribution functions generated by isothermal-isobaric molecular dynamics simulation. We determined the partial contributions to the neutron-weighted distribution functions, to identify the main contributing peaks of the corresponding radial distribution functions, and the effect of the contact ion-pair configuration on the resulting water’s hydrogen coordination around the sulfate’s sulfur site. Finally, we assessed the extent of the ion-pair formation according to Poirier–DeLap formalism and highlighted the significant increase of the ion-pair association exhibited by these salts with cation charge.
Geology of the Gullbridge Copper Deposit, Newfoundland: Volcanogenic Sulfides In Cordierite–Anthophyllite Rocks
Authors: Upadhyay H. D. , Smitheringale W. G.
Published: April 2010

The Gullbridge copper deposit occurs in a folded and regionally metamorphosed sequence of Upper Ordovician mafic volcanic and tuffaceous rocks in the Central Mobile Belt of Newfoundland. The ore consists of chalcopyrite, pyrrhotite, and pyrite in a tabular zone of cordierite–andalusite–chlorite schist that is surrounded by cordierite–anthophyilite rock. Regional metamorphism to greenschist or amphibolite fades preceded and accompanied regional de formation that produced tight folds with a strong axial plane schistosity (S1) in the host and country rocks. Contact metamoprhism related to nearby plutons superimposed the cordierite, andalusite, and anthophyllite mineral assemblages upon S1. This was followed by a second deformation and by chloritization of the ore zone. Finally the ore body underwent some minor mineralogical and structural modifications. From various field and laboratory evidence the sulfides, and possibly the associated magnesium alteration, are interpreted as volcanogenic. The Gullbridge deposit is similar to a number of other copper sulfide deposits in cordierite anthophyilite host rocks in North America and Scandinavia.
Seismic Refraction Studies in Western Canada
Authors: Chandra N. N. , Cumming G. L.
Published: April 2010

A seismic refraction profile approximately 700 km long, from Greenbush Lake, British Columbia to Swift Current, Saskatchewan along latitude 50° 30′ is studied with the aid of delay time solutions. Additional refraction data in an area of large gravity anomalies southeast of Calgary, Alberta is included. The analysis indicates that upper crustal velocities vary laterally from 6.1 km/s to 6.5 km/s and that the boundaries between the velocity zones are most probably near vertical faults which involve the whole crustal section through to the Mohorovicic discontinuity.Upper mantle velocities vary along the profile from about 8.0 km/s west of the Rocky Mountains to 8.3 km/s in the region between Vulcan and Suffield where the profile crosses the Sweetgrass Arch. The data indicate a crustal root under the Rocky Mountains of about 5 km thickness.Comparisons between observed Bouguer gravity anomalies and those computed for the seismic model are used as a further constraint on the interpretation of the seismic data and a good fit is obtained when the initial seismic model is adjusted within the limits of the seismic control.
Observations on the Proterozoic Seton Formation, East Arm of Great Slave Lake, Northwest Territories
Authors: Olade M. A. D. , Morton R. D.
Published: April 2010

The Proterozoic (Aphebian) Seton Formation is shown to extend across almost the entire length of the East Arm structural subprovince of the Great Slave Lake region, Northwest Territories. Earlier described as greenstones or basalts and recently as an andesite–rhyolite suite, the volcanic rocks which characterize the Seton Formation are clearly of spilitic–keratophyric affinity. The formation is composed of a sequence of marine to subaerial, spilitic basalt flows, trachytic flows, quartz keratophyric–and spilitic–basic pyroclastics, volcanic sandstones, jasper, banded ironstones, and intercalated marine epiclastic sedimentary rocks. Small hypabyssal intrusions of albite granophyre, albite, and quartz porphyry represent minor subvolcanic phases. Petrographic descriptions of the lavas and pyroclastic rocks from Toopon Lake, the Fort Reliance area, and Seton Island are augmented by partial chemical analyses of 15 lavas from the latter locality. The volcanic–sedimentary Seton Formation, 1300 m thick in the SW of the East Arm, and 40 m thick in the Fort Reliance district, should be classified as a member of the Sosan Group, being in part laterally equivalent to the Akaitcho River Formation and the upper Kluziai Formation. The Aphebian Coronation Geosyncline during Seton times was thus characterized by effusive (partially submarine) island volcanism in the SW of the region, contemporaneous with shallow marine sedimentation towards the northeast part of the basin.
A Minimal Test of Significance of Nonuniform Density of Data Points in a Three Variable Closed Array
Authors: Smith F. G. , Watson D. F.
Published: April 2010

An algorithm and computer program are described for making a conservative test of significance of clustering of data points in a three-variable closed array. Normative compositions of granitic rocks, projected to points in the albite–orthoclase–quartz triangle, have a clustering that is significant at the 99% level, if the proportions of these three minerals are the only variables.
The Neruokpuk Formation of the Barn Mountains and Driftwood Hills, Northern Yukon; its Age and Graptolite Fauna
Authors: Lenz Alfred C. , Perry David G.
Published: April 2010

The pre-Carboniferous racks of the Bam Mountains consist of a very thick, highly deformed and dislocated sequence of mildly metamorphosed elastics and carbonates assignable to the Neruokpuk Formation. The Driftwood Hills, for the most part, are composed of dark shales possibly assignable to the Neruokpuk Formation. Graptolites collected from a number of thin shale beds in the two areas are correlative with the Arenigian, Llanvirnian, Ashgillian, ?Llandoverian, Ludlovian, and Siegenian or Emsian Stages. It is suggested that complete, or nearly complete Ordovician and Silurian sedimentary sequences are present, and that these sediments represent geosynclinal equivalents of cratonic sediments to the south and southwest.
Further Analysis of Vertical Crustal Movement Observations in the Lac St. Jean Area, Quebec
Authors: Vaníček Petr , Hamilton Angus C.
Published: April 2010

Further statistical tests have been made on levelling data in the Lac St. Jean area of Québec. These tests confirm that relative to Québec City there is downward movement focussed about Stoneham at the rate of 45.7 cm per century with a 95% confidence limit of ± 14.5 cm per century, and that there is uplift to the north and west with rates and 95% confidence limits of 58.8 ± 12.5 cm per century at St. Félicien and 83.5 ± 22.8 cm per century at Senneterre.A test for correlation between elevation differences and movement rates showed no significant effect; this is interpreted as evidence there was no appreciable systematic error in the levelling. From these analyses the possibility that the results might be explained in terms of errors, either random or systematic, can be rejected without any reservations.