bluuebunny/crossref_metadata_2025 huggingface dataset:
EuSplice: a unified resource for the analysis of splice signals and alternative splicing in eukaryotic genes
Authors: Bhasi Ashwini , Pandey Ram Vinay , Utharasamy Suriya Prabha , Senapathy Periannan
Published: March 2007

Motivation: Despite increased availability of genome annotation data, a comprehensive resource for in-depth analysis of splice signal distributions and alternative splicing (AS) patterns in eukaryote genomes is still lacking. To meet this need, we have developed EuSplice—a unique splice-centric database which provides reliable splice signal and AS information for 23 eukaryotes.

Results: The EuSplice database contains 95 822 AS events and 2.1 million splice signals associated with over 270 000 protein-coding genes. The intuitive, user-friendly EuSplice web interface has powerful data mining and graphics capabilities for inter-genomic comparative analysis of splice signals, putative cryptic splice sites and AS events. Moreover, the seamless integration of splicing data to extensive gene-specific annotations, such as homolog annotations, functional information, mutations and sequence details makes EuSplice a powerful one-stop information resource for investigating the molecular mechanisms of complex splicing events, disease associations and the evolution of splicing in eukaryotes.

Availability:  http://66.170.16.154/EuSplice

Contact:  ps@genome.com

Supplementary information: Supplementary tables and figures at Bioinfo online.
A new method to measure the semantic similarity of GO terms
Authors: Wang James Z. , Du Zhidian , Payattakool Rapeeporn , Yu Philip S. , Chen Chin-Fu
Published: March 2007

Motivation: Although controlled biochemical or biological vocabularies, such as Gene Ontology (GO) (http://www.geneontology.org), address the need for consistent descriptions of genes in different data sources, there is still no effective method to determine the functional similarities of genes based on gene annotation information from heterogeneous data sources.

Results: To address this critical need, we proposed a novel method to encode a GO term's semantics (biological meanings) into a numeric value by aggregating the semantic contributions of their ancestor terms (including this specific term) in the GO graph and, in turn, designed an algorithm to measure the semantic similarity of GO terms. Based on the semantic similarities of GO terms used for gene annotation, we designed a new algorithm to measure the functional similarity of genes. The results of using our algorithm to measure the functional similarities of genes in pathways retrieved from the saccharomyces genome database (SGD), and the outcomes of clustering these genes based on the similarity values obtained by our algorithm are shown to be consistent with human perspectives. Furthermore, we developed a set of online tools for gene similarity measurement and knowledge discovery.

Availability: The online tools are available at: http://bioinformatics.clemson.edu/G-SESAME

Contact:  jzwang@cs.clemson.edu

Supplementary information:  http://bioinformatics.clemson.edu/Publication/Supplement/gsp.htm
BioGuideSRS: querying multiple sources with a user-centric perspective
Authors: Cohen-Boulakia Sarah , Biton Olivier , Davidson Susan , Froidevaux Christine
Published: March 2007

Summary: Biologists are frequently faced with the problem of integrating information from multiple heterogeneous sources with their own experimental data. Given the large number of public sources, it is difficult to choose which sources to integrate without assistance. When doing this manually, biologists differ in their preferences concerning the sources to be queried as well as the strategies, i.e. the querying process they follow for navigating through the sources. In response to these findings, we have developed BioGuide to assist scientists search for relevant data within external sources while taking their preferences and strategies into account. In this article, we present BioGuideSRS, a user-friendly system which automatically retrieves instances of data by using BioGuide on top of the sequence retrieval system (SRS). BioGuideSRS is an Applet that can be run from its web page on any system with Java 5.0.

Availability:  http://www.bioguide-project.net

Contact:  sarahcb@seas.upenn.edu
Deforestation and apparent extinctions of endemic forest beetles in Madagascar
Authors: Hanski Ilkka , Koivulehto Helena , Cameron Alison , Rahagalala Pierre
Published: March 2007

Madagascar has lost about half of its forest cover since 1953 with much regional variation, for instance most of the coastal lowland forests have been cleared. We sampled the endemic forest-dwelling Helictopleurini dung beetles across Madagascar during 2002–2006. Our samples include 29 of the 51 previously known species for which locality information is available. The most significant factor explaining apparent extinctions (species not collected by us) is forest loss within the historical range of the focal species, suggesting that deforestation has already caused the extinction, or effective extinction, of a large number of insect species with small geographical ranges, typical for many endemic taxa in Madagascar. Currently, roughly 10% of the original forest cover remains. Species–area considerations suggest that this will allow roughly half of the species to persist. Our results are consistent with this prediction.
Biomimetic materials research: what can we really learn from nature's structural materials?
Authors: Fratzl Peter
Published: March 2007

Nature provides a wide range of materials with different functions and which may serve as a source of bio-inspiration for the materials scientist. The article takes the point of view that a successful translation of these ideas into the technical world requires more than the observation of nature. A thorough analysis of structure-function relations in natural tissues must precede the engineering of new bio-inspired materials. There are, indeed, many opportunities for lessons from the biological world: on growth and functional adaptation, about hierarchical structuring, on damage repair and self-healing. Biomimetic materials research is becoming a rapidly growing and enormously promising field. Serendipitous discovery from the observation of nature will be gradually replaced by a systematic approach involving the study of natural tissues in materials laboratories, the application of engineering principles to the further development of bio-inspired ideas and the generation of specific databases.
Experiments show importance of flow-induced pressure on endothelial cell shape and alignment
Authors: Voorhees Abram , Nackman Gary B , Wei Timothy
Published: March 2007

While the importance of haemodynamic forces on vascular disease is well recognized, the biologic response to these forces is not well understood. Indeed, as will be discussed, even the nature of the forces themselves is not understood; wall shear stress acting on arterial walls is commonly understood to be the primary, if not the sole, source of haemodynamic loading. The ultimate goal of this research is to experimentally quantify the complete force distribution acting on endothelial cells (ECs) and to directly examine the biochemical response of ECs to variations in haemodynamic loading. This report contains a description of the first spatially resolved microscopic particle image velocimetry (μPIV) flow measurements over individual human ECs. Using velocity data from multiple two-dimensional planes, it was possible to reconstruct the three-dimensional shear stress and pressure distributions over the cells. Data indicate that flow-induced pressure is of the same magnitude as shear with maximum magnitudes at the ‘toe’ and ‘heel’ of the ECs. Arguments are then made that EC response to pressure includes (i) reshaping to minimize drag, (ii) realigning to minimize shear-induced torque, and (iii) minimizing attachment forces required to maintain adhesion.
Explicit Poincaré equations of motion for general constrained systems. Part I. Analytical results
Authors: Udwadia Firdaus E , Phohomsiri Phailaung
Published: March 2007

This paper gives the general constrained Poincaré equations of motion for mechanical systems subjected to holonomic and/or nonholonomic constraints that may or may not satisfy d'Alembert's principle at each instant of time. It also extends Gauss's principle of least constraint to include quasi-accelerations when the constraints are ideal, thereby expanding the compass of this principle considerably. The new equations provide deeper insights into the dynamics of multi-body systems and point to new ways for controlling them.
On a ferroelastic mechanism governing the toughness of metastable tetragonal-prime (
t
′) yttria-stabilized zirconia
Authors: Mercer C , Williams J.R , Clarke D.R , Evans A.G
Published: March 2007

This article investigates the toughness of yttria-stabilized zirconia (YSZ) with the tetragonal-prime (
t
′) structure. Such materials are used as durable thermal barriers in gas turbines. Their durability has been attributed to high toughness, relative to materials in the cubic phase field. Based on prior literature, a ferroelastic toughening mechanism is hypothesized and this assertion is examined by characterizing the material in the wake of an indentation-induced crack. Assessment by transmission electron microscopy, Raman spectroscopy and optical interferometry has affirmed the existence of a process zone, approximately 3 μm in width, containing a high density of nano-scale domains, with equal proportions of all three crystallographic variants. Outside the zone, individual grains contain a single variant (no domains) implying that the toughening mechanism is controlled by domain
nucleation
(rather than the
motion
of pre-existing twin boundaries). The viability of the ferroelastic toughening mechanism is assessed using a process zone model that relates the observed toughening to the stress/strain hysteresis accompanying domain formation. Based on the measured process zone size, the known tetragonality of
t
′-7 wt% YSZ and the enhancement in toughness relative to cubic YSZ, consistency between the model and the observed toughening is demonstrated for a reasonable choice of the coercive stress.
Explicit Poincaré equations of motion for general constrained systems. Part II. Applications to multi-body dynamics and nonlinear control
Authors: Udwadia Firdaus E , Phohomsiri Phailaung
Published: March 2007

The power of the new equations of motion developed in part I of this paper is illustrated using three examples from multi-body dynamics. The first two examples deal with the problem of accurately controlling the orientation of a rigid body, while the third example deals with the synchronization of two rigid bodies so that their relative orientations are ‘locked’ through prescribed dynamical relationships. The ease, simplicity and accuracy with which control of such highly nonlinear systems is achieved are demonstrated.
Genetic studies of human diversity in East Asia
Authors: Zhang Feng , Su Bing , Zhang Ya-ping , Jin Li
Published: March 2007

East Asia is one of the most important regions for studying evolution and genetic diversity of human populations. Recognizing the relevance of characterizing the genetic diversity and structure of East Asian populations for understanding their genetic history and designing and interpreting genetic studies of human diseases, in recent years researchers in China have made substantial efforts to collect samples and generate data especially for markers on Y chromosomes and mtDNA. The hallmark of these efforts is the discovery and confirmation of consistent distinction between northern and southern East Asian populations at genetic markers across the genome. With the confirmation of an African origin for East Asian populations and the observation of a dominating impact of the gene flow entering East Asia from the south in early human settlement, interpretation of the north–south division in this context poses the challenge to the field. Other areas of interest that have been studied include the gene flow between East Asia and its neighbouring regions (i.e. Central Asia, the Sub-continent, America and the Pacific Islands), the origin of Sino-Tibetan populations and expansion of the Chinese.
Histone Replacement Marks the Boundaries of cis-Regulatory Domains
Authors: Mito Yoshiko , Henikoff Jorja G. , Henikoff Steven
Published: March 2007

Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. We have examined chromatin at
Drosophila
homeotic gene clusters by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. Our results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.
Inverse Relations Between Amounts of Air Pollution and Orographic Precipitation
Authors: Rosenfeld Daniel , Dai Jin , Yu Xing , Yao Zhanyu , Xu Xiaohong , Yang Xing , Du Chuanli
Published: March 2007

Particulate air pollution has been suggested as the cause of the recently observed decreasing trends of 10 to 25% in the ratio between hilly and upwind lowland precipitation, downwind of urban and industrial areas. We quantified the dependence of this ratio of the orographic-precipitation enhancement factor on the amounts of aerosols composed mostly of pollution in the free troposphere, based on measurements at Mt. Hua near Xi'an, in central China. The hilly precipitation can be decreased by 30 to 50% during hazy conditions, with visibility of less than 8 kilometers at the mountaintop. This trend shows the role of air pollution in the loss of significant water resources in hilly areas, which is a major problem in China and many other areas of the world.
Odor Cues During Slow-Wave Sleep Prompt Declarative Memory Consolidation
Authors: Rasch Björn , Büchel Christian , Gais Steffen , Born Jan
Published: March 2007

Sleep facilitates memory consolidation. A widely held model assumes that this is because newly encoded memories undergo covert reactivation during sleep. We cued new memories in humans during sleep by presenting an odor that had been presented as context during prior learning, and so showed that reactivation indeed causes memory consolidation during sleep. Re-exposure to the odor during slow-wave sleep (SWS) improved the retention of hippocampus-dependent declarative memories but not of hippocampus-independent procedural memories. Odor re-exposure was ineffective during rapid eye movement sleep or wakefulness or when the odor had been omitted during prior learning. Concurring with these findings, functional magnetic resonance imaging revealed significant hippocampal activation in response to odor re-exposure during SWS.
Development and Characterization of Monoclonal Antibodies and Aptamers against Major Antigens ofMycobacterium aviumsubsp.paratuberculosis
Authors: Bannantine John P. , Radosevich Thomas J. , Stabel Judith R. , Sreevatsan Srinand , Kapur Vivek , Paustian Michael L.
Published: March 2007

Specific antibodies, available in unlimited quantities, have not been produced againstMycobacterium aviumsubsp.paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) againstM. aviumsubsp.paratuberculosiswere produced from BALB/c mice immunized with a whole-cell extract ofM. aviumsubsp.paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening anM. aviumsubsp.paratuberculosislambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance inM. aviumsubsp.paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding toM. aviumsubsp.paratuberculosisas well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.
Immune Modulator Adamantylamide Dipeptide Stimulates Efficient Major Histocompatibility Complex Class I-Restricted Responses in Mice
Authors: Becker Pablo D. , Nörder Miriam , Guzmán Carlos A. , Grinstein Saul
Published: March 2007

Adamantylamide
l
-alanyl-
d
-isoglutamine (AdDP) is a synthetic adjuvant which belongs to the family of the desmuramyl peptides. AdDP exerts its adjuvant properties when it is administered either by the parenteral or by the mucosal route, leading to the elicitation of strong humoral responses at both the systemic and the mucosal levels. However, very little is known about the effect of AdDP on cellular immunity. Here we demonstrate that AdDP is able to stimulate cellular responses, which are characterized by the release of gamma interferon by CD8
+
T cells when they are restimulated with a major histocompatibility complex class I-restricted peptide and strong in vivo lymphocyte-mediated cytotoxic activity. The capacity of AdDP to stimulate the elicitation of both cellular and humoral adaptive responses makes this adjuvant a promising tool for the development of mucosal vaccine formulations.
Development of a Simple Latex Agglutination Assay for Detection of Shiga Toxin-Producing
Escherichia coli
(STEC) by Using Polyclonal Antibody against STEC
Authors: Hajra Tapas K. , Bag Prasanta K. , Das Suresh C. , Mukherjee Souryadeep , Khan Asis , Ramamurthy T.
Published: March 2007

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing
Escherichia coli
(STEC) strain VT3 (
stx
1
+
stx
2
+
eae
+
) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type γ. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of
E. coli
and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.
Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody
Authors: He Qigai , Velumani Sumathy , Du Qingyun , Lim Chee Wee , Ng Fook Kheong , Donis Ruben , Kwang Jimmy
Published: March 2007

The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.
Maternal Milk Contains Antimicrobial Factors That Protect Young Rabbits from Enteropathogenic
Escherichia coli
Infection
Authors: Gallois Mélanie , Gidenne Thierry , Tasca Christian , Caubet Cécile , Coudert Cécile , Milon Alain , Boullier Séverine
Published: March 2007

Enteropathogenic
Escherichia coli
(EPEC) colibacillosis represents a major cause of lethal diarrhea in young children in developing countries. EPEC strains also infect numerous mammal species and represent a major economical problem in rabbit industry. Protection against this pathogen is a challenging goal both in humans and in other mammal species. Despite a good knowledge of the pathogenicity mechanisms of EPEC, the intrinsic and environmental factors that control the expression of EPEC virulence in mammals remain unknown. For instance, the exacerbated sensitivity of young mammals to EPEC infection is still unexplained. Our goal was to investigate if age or other factors, like milk consumption, could be determinants that trigger the disease. We used rabbits as an animal model to study the role of milk in the sensitivity to an EPEC infection. Weaned and suckling rabbits were orally inoculated with EPEC strain E22 (O103:H2:K−) at 28 days of age, and the evolution of the disease was investigated in the two groups. In addition, in order to better characterize the interactions between milk and EPEC, we determined in vitro bacterial growth and the abilities of EPEC cells to adhere to epithelial cells in the presence of milk. Our results demonstrate a protective role of milk in vivo in association with in vitro antibacterial activity. These effects are independent of the presence of specific anti-EPEC antibodies.
Early Identification of Seronegative Human Immunodeficiency Virus Type 1 Infection with Severe Presentation
Authors: Chin Bum Sik , Lee Sun Hee , Kim Gab Jung , Kee Mee Kyung , Suh Soon Deok , Kim Sung Soon
Published: March 2007

Specific antibodies against human immunodeficiency virus (HIV), usually used for diagnosis, almost invariably become detectable within 3 months of exposure. We report on a patient whose HIV infection was identified early by a combined antigen/antibody test, but seroconversion did not occur for 7 months, until the implementation of antiretroviral therapy.
Variability of Properties of
Vibrio parahaemolyticus
Strains Isolated from Individual Patients
Authors: Bhoopong Phuangthip , Palittapongarnpim Prasit , Pomwised Rattanaruji , Kiatkittipong Arpapak , Kamruzzaman Muhammad , Nakaguchi Yoshitsugu , Nishibuchi Mitsuaki , Ishibashi Masanori , Vuddhakul Varaporn
Published: March 2007

Infections by strains belonging to the O3:K6 pandemic clone of
Vibrio parahaemolyticus
are prevalent in southern Thailand, and serovariants of these strains have also been detected.
V. parahaemolyticus
strains lacking important virulence genes (
tdh
and
trh
) were isolated from 6.5 to 10.9% of clinical specimens during the period from 2000 to 2003. In order to understand whether changes to the characteristics of
V. parahaemolyticus
occur during infection, 10 isolates collected from each of 63 patients who presented with diarrhea at the Hat Yai hospital from 2003 to 2004 were examined for the presence of the
tdh
and
trh
genes, the O:K serotype, and genetic markers for the pandemic clone. A total of 42 patients (66.7%) yielded identical isolates (homogeneous populations), and 21 of the patients (33.3%) yielded isolates that differed in at least one character from the other isolates (heterogeneous populations). The DNA fingerprints (examined by arbitrarily primed PCR and pulsed-field gel electrophoresis) of some, but not all, of the heterogeneous populations from single patients were indistinguishable. The results indicated that some patients were infected with a unique strain and that in vivo changes (
tdh
deletion or serotype conversion) might have occurred in certain individuals. It is therefore important to bear in mind that epidemiological studies based on the analysis of a single colony from a single patient might lead to misleading conclusions. Finally, the present study did not rule out the possibility that isolates lacking
tdh
and
trh
have unknown virulence mechanisms other than the
tdh
and
trh
genes.
Prevalence and Significance of a Negative Extended-Spectrum β-Lactamase (ESBL) Confirmation Test Result after a Positive ESBL Screening Test Result for Isolates of
Escherichia coli
and
Klebsiella pneumoniae
: Results from the SENTRY Asia-Pacific Surveillance Program
Authors: Bell Jan M. , Chitsaz Mohsen , Turnidge John D. , Barton Mary , Walters Luke J. , Jones Ronald N.
Published: March 2007

A negative extended-spectrum β-lactamase (ESBL) confirmation test result obtained after a positive ESBL screening test result using Clinical and Laboratory Standards Institute methods has been a common occurrence among isolates of
Escherichia coli
and
Klebsiella pneumoniae
in the SENTRY Antimicrobial Surveillance Program in the Asia-Pacific region. Among isolates collected between 1998 and 2004 this screen-positive, nonconfirmed profile (failed to show clavulanate synergy) was observed in 8.9% of 4,515
E. coli
isolates and 20.3% of 2,303
K. pneumoniae
isolates. We then selected 52
E. coli
isolates and 68
K. pneumoniae
isolates with a negative ESBL confirmation test, as well as comparable number of isolates with confirmed ESBL-positive tests, and examined them for the presence of TEM, SHV, plasmid-borne
ampC
, and CTX-M genes. We found that 62% of nonconfirming
E. coli
isolates and 75% of nonconfirming
K. pneumoniae
harbored a plasmid-borne AmpC enzyme of the CIT or DHA type. The majority of nonconfirming
E. coli
and
K. pneumoniae
from the Asia-Pacific region harbor important β-lactamases, and a positive screening test alone should be sufficient grounds to report resistance to extended-spectrum cephalosporins in this region.
Comparison of the SPF
10
-LiPA System to the Hybrid Capture 2 Assay for Detection of Carcinogenic Human Papillomavirus Genotypes among 5,683 Young Women in Guanacaste, Costa Rica
Authors: Safaeian Mahboobeh , Herrero Rolando , Hildesheim Allan , Quint Wim , Freer Enrique , Van Doorn Leen-Jan , Porras Carolina , Silva Sandra , González Paula , Bratti M. Concepcion , Rodriguez Ana Cecilia , Castle Philip
Published: March 2007

The objective of this analysis was to compare the performance characteristics of two human papillomavirus (HPV) DNA detections assays, the Hybrid Capture 2 assay (HC2) and the SPF
10
assay, for the detection of carcinogenic HPV. Data are from the enrollment visits of women who participated in the randomized, double-blind, placebo-controlled phase III HPV16/18 Vaccine Trial in Guanacaste, Costa Rica. We compared the results of HC2 and SPF
10
testing of cervical specimens. Since the line probe assay (LiPA) detection system does not distinguish between HPV type 68 (HPV68; which is targeted by HC2) and HPV73 (which is not targeted by HC2), for SPF
10
-LiPA, we defined the carcinogenic HPV types as the 12 HC2-targeted types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), HPV68/73, and the HC2-cross-reactive, carcinogenic type HPV66. The kappa values and the performance characteristics for the detection of cervical abnormalities were ascertained. Paired observations were available for 5,683 sexually active, young women (median age, 21 years). The prevalence of carcinogenic HPV types was 35% (
n
= 1,962) by HC2 and 35% (
n
= 2,003) by SPF
10
-LiPA. There were no differences in the prevalence of carcinogenic HPV types by HC2 and SPF
10
-LiPA among women with normal, atypical squamous cells of undetermined significance and high-grade squamous intraepithelial lesion cytology. Among women with low-grade squamous intraepithelial lesion cytology, HC2 was more likely to test positive than SPF
10
-LiPA for the carcinogenic HPV types (87% and 79%, respectively;
P
= 0.001) as a result of HC2 cross-reactivity with HPV types 40, 43, 44, 53, 54, 60, 70, and 74. The crude agreement between the two assays was 88%, with a kappa value of 0.75 (95% confidence limits, 0.73 to 0.76). We observed very good agreement between HC2 and SPF
10
-LiPA for carcinogenic HPV type detection.
N-Terminal Mutants of Herpes Simplex Virus Type 2 gH Are Transported without gL but Require gL for Function
Authors: Cairns Tina M. , Friedman Lisa S. , Lou Huan , Whitbeck J. Charles , Shaner Marie S. , Cohen Gary H. , Eisenberg Roselyn J.
Published: March 2007

Glycoprotein H (gH) is conserved among all herpesviruses and is essential for virus entry and cell fusion along with gL, gB, and, in most alphaherpesviruses, gD. Within the gH/gL heterodimer, it is thought that gH accounts for the fusion function and gL acts as a chaperone for the folding and transport of gH. Here, we found that the N terminus of gH2 contains important elements involved in both its folding and its transport. Our conclusions are based on the phenotypes of a series of gH deletion mutants in which the signal sequence (residues 1 to 18) was retained and N-terminal residues were removed up to the number indicated. The first mutant, gH2Δ29 (deletion of residues 19 to 28), like wild-type (WT) gH, required gL for both transport and function. To our surprise, two other mutants (gH2Δ64 and gH2Δ72) were transported to the cell surface independent of gL but were nonfunctional, even when complexed with gL. Importantly, a fourth mutant (gH2Δ48) was transported independent of gL but was functional only when complexed with gL. Using a panel of monoclonal antibodies against gH2, we found that when gH2Δ48 was expressed alone, its antigenic structure differed from that of gH2Δ48/gL or gH2-WT/gL. Mutation of gH2 residue R39, Y41, W42, or D44 allowed gL-independent transport of gH. Our results also show that gL is not merely required for gH transport but is also necessary for the folding and function of the complex. Since gH2Δ64/gL and gH2Δ72/gL were nonfunctional, we hypothesized that residues critical for gH/gL function lie within this deleted region. Additional mutagenesis identified L66 and L72 as important for function. Together, our results highlight several key gH residues: R39, Y41, W42, and D44 for gH transport and L66 and L72 for gH/gL structure and function.
Role of the Foamy Virus Pol Cleavage Site in Viral Replication
Authors: Roy Jacqueline , Linial Maxine L.
Published: March 2007

Foamy virus Pol precursor protein processing by the viral protease occurs at only one site, releasing a protease-reverse transcriptase and an integrase protein. To examine whether the cleavage of the Pol precursor protein is necessary for enzymatic activities and efficient viral replication, several mutations were generated around the cleavage site. All cleavage site mutants synthesize wild-type levels of Pol precursor protein. Mutants containing more than two amino acid substitutions around the cleavage site exhibit no detectable Pol processing. The Pol cleavage site is not required for the production of infectious particles in a single round of infection, but is important for subsequent rounds of viral infection. Mutations around the cleavage site affected the enzymatic activities of the protease and reverse transcriptase and prevented replication after two rounds of infection. Interestingly, Pol encapsidation is significantly reduced in some of the mutants.
Demonstration of Coinfection with and Recombination by Caprine Arthritis-Encephalitis Virus and Maedi-Visna Virus in Naturally Infected Goats
Authors: Pisoni Giuliano , Bertoni Giuseppe , Puricelli Maria , Maccalli Marina , Moroni Paolo
Published: March 2007

Recombination of different strains and subtypes is a hallmark of lentivirus infections, particularly for human immunodeficiency virus, and contributes significantly to viral diversity and evolution both within individual hosts and within populations. Recombinant viruses are generated in individuals coinfected or superinfected with more than one lentiviral strain or subtype. This, however, has never been described in vivo for the prototype lentivirus maedi-visna virus of sheep and its closely related caprine counterpart, the caprine arthritis-encephalitis virus. Cross-species infections occur in animals living under natural conditions, which suggests that dual infections with small-ruminant lentiviruses (SRLVs) are possible. In this paper we describe the first documented case of coinfection and viral recombination in two naturally infected goats. DNA fragments encompassing a variable region of the envelope glycoprotein were obtained from these two animals by end-limiting dilution PCR of peripheral blood mononuclear cells or infected cocultures. Genetic analyses, including nucleotide sequencing and heteroduplex mobility assays, showed that these goats harbored two distinct populations of SRLVs. Phylogenetic analysis permitted us to assign these sequences to the maedi-visna virus group (SRLV group A) or the caprine arthritis-encephalitis virus group (SRLV group B). SimPlot analysis showed clear evidence of A/B recombination within the
env
gene segment of a virus detected in one of the two goats. This case provides conclusive evidence that coinfection by different strains of SRLVs of groups A and B can indeed occur and that these viruses actually recombine in vivo.
Incorporation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase into Virus-Like Particles
Authors: Liao Wei-Hao , Huang Kuo-Jung , Chang Yu-Fen , Wang Shiu-Mei , Tseng Ying-Tzu , Chiang Chien-Cheng , Wang Jaang-Jiun , Wang Chin-Tien
Published: March 2007

We demonstrate that a genetically engineered human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) composed mainly of p66 or p51 subunits can be incorporated into virus-like particles (VLPs) when coexpressed with HIV-1 Pr55

gag

. VLP-associated RT exhibited a detergent-resistant association with immature cores during sucrose gradient equilibrium centrifugation, suggesting that RT is incorporated into VLPs. However, RT that retains downstream integrase (IN) is severely inhibited in terms of incorporation into VLPs. Results from immunofluorescence tests reveal that RT-IN is primarily localized at the perinuclear area and exhibits poor colocalization with Gag. IN removal leads to a redistribution of RT throughout the cytoplasm and improved RT incorporation into VLPs. Similar results were observed for RT-IN in which alanine was substituted for 186-Lys-Arg-Lys-188 residues of the IN putative nuclear localization signal, suggesting that IN karyophilic properties may partly account for the inhibitory effect of IN on RT incorporation. Although the membrane-binding capacity of RT was markedly reduced compared to that of wild-type Gag or Gag-Pol, the correlation of membrane-binding ability with particle incorporation efficiency was incomplete. Furthermore, we observed that membrane-binding-defective myristylation-minus RT can be packaged into VLPs at the same level as its normal myristylated counterpart. This suggests that the incorporation of RT into VLPs is independent of membrane affinity but very dependent on RT-Gag interaction. Results from a genetic analysis suggest that the Gag-interacting regions of RT mainly reside in the thumb subdomain and that the RT-binding domains of Gag are located in the matrix (MA) and p6 regions.
Severe Acute Respiratory Syndrome Coronavirus Accessory Protein 6 Is a Virion-Associated Protein and Is Released from 6 Protein-Expressing Cells
Authors: Huang Cheng , Peters C. J. , Makino Shinji
Published: March 2007

Analysis of severe acute respiratory syndrome coronavirus (SCoV) by either sucrose gradient equilibrium centrifugation or a virus capture assay using an anti-SCoV S protein antibody demonstrated that the SCoV 6 protein, which is one of the accessory proteins of SCoV, was incorporated into virus particles. Coexpression of the SCoV S, M, E, and 6 proteins was sufficient for incorporation of the 6 protein into virus-like particles. Cells transfected with plasmid expressing the 6 protein released SCoV 6 protein; however, infected cells released SCoV 6 protein only in association with SCoV particles.
Biologic, Antigenic, and Full-Length Genomic Characterization of a Bovine-Like Coronavirus Isolated from a Giraffe
Authors: Hasoksuz Mustafa , Alekseev Konstantin , Vlasova Anastasia , Zhang Xinsheng , Spiro David , Halpin Rebecca , Wang Shiliang , Ghedin Elodie , Saif Linda J.
Published: March 2007

Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.
The Human Cytomegalovirus Virion Possesses an Activated Casein Kinase II That Allows for the Rapid Phosphorylation of the Inhibitor of NF-κB, IκBα
Authors: Nogalski Maciej T. , Podduturi Jagat P. , DeMeritt Ian B. , Milford Liesl E. , Yurochko Andrew D.
Published: March 2007

We documented that the NF-κB signaling pathway was rapidly induced following human cytomegalovirus (HCMV) infection of human fibroblasts and that this induced NF-κB activity promoted efficient transactivation of the major immediate-early promoter (MIEP). Previously, we showed that the major HCMV envelope glycoproteins, gB and gH, initiated this NF-κB signaling event. However, we also hypothesized that there were additional mechanisms utilized by the virus to rapidly upregulate NF-κB. In this light, we specifically hypothesized that the HCMV virion contained IκBα kinase activity, allowing for direct phosphorylation of IκBα following virion entry into infected cells. In vitro kinase assays performed on purified HCMV virion extract identified bona fide IκBα kinase activity in the virion. The enzyme responsible for this kinase activity was identified as casein kinase II (CKII), a cellular serine-threonine protein kinase. CKII activity was necessary for efficient transactivation of the MIEP and IE gene expression. CKII is generally considered to be a constitutively active kinase. We suggest that this molecular characteristic of CKII represents the biologic rationale for the viral capture and utilization of this kinase early after infection. The packaging of CKII into the HCMV virion identifies that diverse molecular mechanisms are utilized by HCMV for rapid NF-κB activation. We propose that HCMV possesses multiple pathways to increase NF-κB activity to ensure that the correct temporal regulation of NF-κB occurs following infection and that sufficient threshold levels of NF-κB are reached in the diverse array of cells, including monocytes and endothelial cells, infected in vivo.
Mitogen-Activated Protein Kinases Activate the Nuclear Localization Sequence of Human Papillomavirus Type 11 E1 DNA Helicase To Promote Efficient Nuclear Import
Authors: Yu Jei-Hwa , Lin Biing Yuan , Deng Wentao , Broker Thomas R. , Chow Louise T.
Published: March 2007

Human and animal papillomavirus DNA replicates as multicopy nuclear plasmids. Replication requires two viral proteins, the origin-recognition protein E2 and the replicative DNA helicase E1. Using genetic, biochemical, and immunofluorescence assays, we demonstrated that efficient nuclear import of the human papillomavirus (HPV) type 11 E1 protein depends on a codominant bipartite nuclear localization sequence (NLS) and on phosphorylation of the serine residues S89 and S93 by the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase, and c-Jun N-terminal protein kinase. The NLS and the MAPK substrates are located within a 50-amino-acid-long peptide near the amino terminus, previously designated the localization regulatory region (LRR). The downstream NLS overlaps the cyclin-binding motif RRL, which is necessary for phosphorylation by the cyclin-dependent kinases to inactivate a dominant nuclear export sequence, also in the LRR. Alanine mutations of the MAPK substrates significantly impaired nuclear import, whereas phospho-mimetic mutations partially restored nuclear import. We further identified two MAPK docking motifs near the C terminus of E1 that are conserved among E1 proteins of many HPVs and bovine papillomavirus type 1. Mutations of these MAPK docking motifs or addition of specific MAPK inhibitors significantly reduced nuclear import. Interestingly, a fraction of the NLS-minus E1 protein was cotransported with the E2 protein into the nucleus and supported transient viral DNA replication. In contrast, E1 proteins mutated in the MAPK docking motifs were completely inactive in transient replication, an indication that additional properties were adversely affected by those changes.
Sphingomyelinase Restricts the Lateral Diffusion of CD4 and Inhibits Human Immunodeficiency Virus Fusion
Authors: Finnegan Catherine M. , Rawat Satinder S. , Cho Edward H. , Guiffre Danielle L. , Lockett Stephen , Merrill Alfred H. , Blumenthal Robert
Published: March 2007

Previously, we reported that treatment of cells with sphingomyelinase inhibits human immunodeficiency virus type 1 (HIV-1) entry. Here, we determined by measuring fluorescence recovery after photobleaching that the lateral diffusion of CD4 decreased 4-fold following sphingomyelinase treatment, while the effective diffusion rate of CCR5 remained unchanged. Notably, sphingomyelinase treatment of cells did not influence gp120 binding, HIV-1 attachment, or fluid-phase and receptor-mediated endocytosis. Furthermore, sphingomyelinase treatment did not affect the membrane disposition of the HIV receptor proteins CD4, CXCR4, and CCR5, as determined by Triton X-100 extraction. Restriction of CD4 diffusion by antibody cross-linking also inhibited HIV infection. We therefore interpret the decrease in CD4 lateral mobility following sphingomyelinase treatment in terms of clustering of CD4 molecules. Examination of fusion intermediates indicated that sphingomyelinase treatment inhibited HIV at a step in the fusion process after CD4 engagement. Maximal inhibition of fusion was observed following short coculture times and with target cells that express low levels of CD4. As HIV entry into cells requires the sequential engagement of viral envelope protein with CD4 and coreceptor, we propose that sphingomyelinase inhibits HIV infection by inducing CD4 clustering that prevents coreceptor engagement and HIV fusion.
Epigenetic Status of an Adenovirus Type 12 Transgenome upon Long-Term Cultivation in Hamster Cells
Authors: Hochstein Norbert , Muiznieks Indrikis , Mangel Laurence , Brondke Holger , Doerfler Walter
Published: March 2007

The epigenetic status of integrated adenovirus type 12 (Ad12) DNA in hamster cells cultivated for about 4 decades has been investigated. Cell line TR12, a fibroblastic revertant of the Ad12-transformed epitheloid hamster cell line T637 with 15 copies of integrated Ad12 DNA, carries one Ad12 DNA copy plus a 3.9-kbp fragment from a second copy. The cellular insertion site for the Ad12 integrate, identical in both cell lines, is a >5.2-kbp inverted DNA repeat. The Ad12 transgenome is packaged around nucleosomes. The cellular junction is more sensitive to micrococcal nuclease at Ad12-occupied sites than at unoccupied sites. Bisulfite sequencing reveals complete de novo methylation in most of the 1,634 CpGs of the integrated viral DNA, except for its termini. Isolated unmethylated CpGs extend over the entire Ad12 integrate. The fully methylated transgenome segments are characterized by promoter silencing and histone H3 and H4 hypoacetylation. Nevertheless, there is minimal transcriptional activity of the late viral genes controlled by the fully methylated major late promoter of Ad12 DNA.