bluuebunny/crossref_metadata_2025 huggingface dataset:
A neural model for auditory scene analysis
Authors: Yang Dekun , Meyer Georg F. , Ainsworth William A.
Published: September 2008

The fundamental process of auditory scene analysis is the organization of elementary acoustic features in a complex auditory scene into grouped meaningful auditory streams. There are two important issues which need to be addressed for modeling auditory scene analysis. The first issue is concerned with the representation of elementary acoustic features, whilst the second issue is related to the binding mechanism. This paper presents a neural model for auditory scene analysis in which a two-dimensional amplitude modulation (AM) map is used to represent elementary acoustic features and the synchronization of neural oscillators is adopted as the binding mechanism. The AM map captures the modulation frequencies of sound signals filtered by an auditory filterbank. Since the modulation frequencies are the F0-related features for voiced speech signals, F0-based segregation can be utilized to group the auditory streams. The grouping of F0-related features is attained as the formation of the synchronization of nonlinear neural oscillators. Each oscillator is associated with a certain modulation frequency. A set of oscillators are synchronized only when their associated frequencies are harmonically related. The proposed model is tested on synthetic double-vowel identification and the results are in accordance with psychophysical data.
In-duct fan sound power measurement. Part II. Revision of ISO 5136
Authors: Neise Wolfgang , Arnold Frank
Published: September 2008

In recent investigations problems with the present version of the in-duct method ISO 5136 (1990) became apparent which indicate that a revision of the standard is necessary. The two major problem areas are: (1) Swirl flow existing in the test duct may interfere with the microphone probe to give too high sound power levels. Farzami and Guedel [1992 Proc. Fan Noise, CETIM Senlis, France, pp. 375–380] showed that this problem can be solved by placing a flow straightener between the fan outlet and measurement plane. (2) By comparison with other methods of sound power determination [Holste and Neise, JSV 152, 1–26 (1992)], the sound power levels obtained by using the in-duct method are too low by several decibels in the frequency region of higher-order mode sound propagation. It is shown that the discrepancy between the results of the in-duct method and other methods is caused by the so-called modal correction C4 which is to account for the directivity characteristic of the microphone fitted with a turbulence screen in view of the propagation angle of the higher-order duct modes. Experimental and theoretical results are presented for a new combined frequency correction C34 which includes the effects of the superimposed mean flow on the sensitivity and directivity of the microphone probe as well as on the modal sound propagation and characteristics in the duct.
An overview of hearing conservation practices in the U.S.A
Authors: Royster Larry H. , Royster Julia D.
Published: September 2008

Federal governmental regulations dealing with the prevention of occupational noise-induced hearing loss began in 1969 with the publication of the Walsh–Healey Act and was extended to general occupations when the U.S. Department of Labor published the OSHA (U.S. Occupational Safety and Health Administration) regulations in 1971. Finally, required procedures for developing an acceptable hearing conservation program were promulgated in 1983. This presentation will briefly review the history and implementation of the OSHA regulations in U.S. industry. In addition, the present activities by U.S. industry in implementing the OSHA regulations and by OSHA in enforcing them will be reviewed to answer the question: ‘‘Is the present implementation of the OSHA noise regulations effective in preventing noise-induced hearing loss in U.S. occupational environments?’’
Ocean monitoring by using underwater sound channel as a feedback loop
Authors: Furduev A. V.
Published: September 2008

A method is proposed to monitor variability of the medium parameters on an acoustic path. The method has been tested for temperature and current monitoring in the Black Sea. The technique is based on the regenerative scheme with a positive feedback. A specific feature of the method is that the medium to be studied forms a part of the measuring instrument, this approach leading to self-matching to a frequency of the interference maximum and thereby requiring lower acoustic power than that in other methods. The measurements have confirmed an accuracy of 1 sm/s in current monitoring. Two modes of monitoring have been tested: (1) a self-excitation mode when information on the variability is carried by the frequency, the spectrum of frequency deviations characterizing the physical nature of the variability; (2) a regeneration mode when the received ambient noise is reradiated from a distant point, this mode yielding higher contrast of the interference spectrum and allowing the delays between rays to be estimated, the interference variability characterizing processes on the path. The proposed method is promising for short paths, harbors, and straits to monitor heat- and mass-exchange processes, fish migrations, ship traffic, or ice drift.
Maximum a posteriori probability background estimation
Authors: Green Thomas J. , Payne William H. , Titus Vivian E. , Van Allen Eric J.
Published: September 2008

Signal detection in acoustic data is complicated by a structured, nonuniform background that obscures signal characteristics. Consequently, efforts to estimate and whiten the background can yield significant improvements in downstream performance. In fact, the structure of the Neyman–Pearson optimal signal detector includes an explicit background whitening step. Linear estimators suffer from the difficulty of distinguishing between signal and background in the region of interest. Consequently, signal contamination can cause substantial errors in background estimates that reduce signal detectibility via subsequent algorithms and analysis. A nonlinear estimator has been developed that exploits models of both the measured data and the presumed scene and maximum a posteriori probability (MAP) theory to produce a background estimate. The data model is guided by the measurement physics and the scene model is based on a Markov random field assumption. Both models are augmented to accommodate anomalies. In this seminar the MAP estimator is described, along with practical issues associated with algorithm initialization, simulated performance with both white and colored-noise inputs, and real data examples. [Work supported by SPAWAR, under Air Force Contract F19628-95-C-0002.]
Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses
Authors: Jones Siân , Zhang Xiaosong , Parsons D. Williams , Lin Jimmy Cheng-Ho , Leary Rebecca J. , Angenendt Philipp , Mankoo Parminder , Carter Hannah , Kamiyama Hirohiko , Jimeno Antonio , Hong Seung-Mo , Fu Baojin , Lin Ming-Tseh , Calhoun Eric S. , Kamiyama Mihoko , Walter Kimberly , Nikolskaya Tatiana , Nikolsky Yuri , Hartigan James , Smith Douglas R. , Hidalgo Manuel , Leach Steven D. , Klein Alison P. , Jaffee Elizabeth M. , Goggins Michael , Maitra Anirban , Iacobuzio-Donahue Christine , Eshleman James R. , Kern Scott E. , Hruban Ralph H. , Karchin Rachel , Papadopoulos Nickolas , Parmigiani Giovanni , Vogelstein Bert , Velculescu Victor E. , Kinzler Kenneth W.
Published: September 2008

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for ∼10
6
single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.
Effect of Spaceflight on Ability of Monocytes To Respond to Endotoxins of Gram-Negative Bacteria
Authors: Kaur Indreshpal , Simons Elizabeth R. , Kapadia Asha S. , Ott C. Mark , Pierson Duane L.
Published: September 2008

Astronauts live and work in relatively crowded, confined environments on the Space Shuttle and the International Space Station. They experience a unique set of stressors that contribute to a diminishment of many immune responses. This study investigated the ability of the shuttle crew members' monocytes to respond to gram-negative endotoxin that they could encounter during infections. Blood specimens were collected from 20 crew members and 15 control subjects 10 days before launch, 3 to 4 h after landing, and 15 days after landing and from crew members during their annual medical examination at 6 to 12 months after landing. When challenged with gram-negative endotoxin, the crew member's monocytes collected at all three time points produced lower levels of interleukin-6 (IL-6) and IL-1β and higher levels of IL-1ra and IL-8 compared to those of control subjects. Cytokines were assessed by measuring the number of cells positive for intracellular cytokines. These values returned to normal 6 to 12 months after landing, except for IL-1ra, which was still higher (five- to sixfold) than in controls. This phenomenon was accompanied by an increased expression of Toll-like receptor 4 and decreased expression of CD14 on the crew members' monocytes at all time points. There were also increased levels of the lipopolysaccharide binding protein in the plasma of the crew members 3 to 4 h and 15 days after landing. This study shows that spaceflight-associated factors (in-flight and preflight) modulate the response of monocytes to gram-negative endotoxins.
Evaluation of a Multianalyte Profiling Assay and an Enzyme-Linked Immunosorbent Assay for Serological Examination of Epstein-Barr Virus-Specific Antibody Responses in Diagnosis of Nasopharyngeal Carcinoma
Authors: Gu Ai-Di , Mo Hao-Yuan , Xie Yan-Bo , Peng Rou-Jun , Bei Jin-Xin , Peng Juan , Li Miao-Yan , Chen Li-Zhen , Feng Qi-Sheng , Jia Wei-Hua , Zeng Yi-Xin
Published: September 2008

Assessment of antibody responses to Epstein-Barr virus (EBV) antigens has been used to assist in nasopharyngeal carcinoma (NPC) diagnosis by several methods. In this study, we evaluated an in-house Luminex multianalyte profiling (xMAP) technology and commercial enzyme-linked immunosorbent assay (ELISA) kits for serological examination of EBV-specific antibody responses in 135 NPC patients and 130 healthy controls. Four EBV biomarkers were measured: immunoglobulin A (IgA) against viral capsid antigen (VCA), EBV nuclear antigen 1 (EBNA1), diffused early antigen (EA-D), and IgG against EA-D. The sensitivities and specificities of the four markers ranged between 71.5 and 90% for xMAP assays and 80 and 92% for ELISA. Logistic regression analysis revealed that the combined markers in the xMAP assay had overall sensitivity and specificity values of 82% and 92%, respectively. The correlation coefficient (
r
) values for the xMAP assay and ELISA were lowest for IgA-VCA (0.468) and highest for IgA-EBNA1 (0.846); for IgA-EA-D and IgG-EA-D, the
r
values were 0.719 and 0.798, respectively. The concordances of the two methods for NPC discrimination were good (79 to 88%). Our results suggest that both the xMAP assay and ELISA are satisfactory for EBV antibody evaluation when multiple antigens are included.
Hyperproliferation of B Cells Specific for a Weakly Immunogenic PorA in a Meningococcal Vaccine Model
Authors: Luijkx Thomas A. , van Gaans-van den Brink Jacqueline A. M. , van Dijken Harry H. , van den Dobbelsteen Germie P. J. M. , van Els Cécile A. C. M.
Published: September 2008

Highly homologous meningococcal porin A (PorA) proteins induce protective humoral immunity againstNeisseria meningitidisgroup B infection but with large and consistent differences in the levels of serum bactericidal activity achieved. We investigated whether a poor PorA-specific serological outcome is associated with a limited size of the specific B-cell subpopulation involved. The numbers of PorA-specific splenic plasma cells, bone marrow (BM) plasma cells, and splenic memory B cells were compared between mice that received priming and boosting with the weakly immunogenic PorA (P1.7-2,4) protein and those that received priming and boosting with the highly immunogenic PorA (P1.5-1,2-2) protein. Immunoglobulin G (IgG) titers (except at day 42), bactericidal activity, and the avidity of IgG produced against P1.7-2,4 were significantly lower at all time points after priming and boosting than against P1.5-1,2-2. These differences, however, were not associated with a lack of P1.7-2,4-specific plasma cells. Instead, priming with both of the PorAs resulted in the initial expansion of comparable numbers of splenic and BM plasma cells. Moreover, P1.7-2,4-specific BM plasma cells, but not P1.5-1,2-2-specific plasma cells, expanded significantly further after boosting. Likewise, after a relative delay during the priming phase, the splenic P1.7-2,4-specific memory B cells largely outnumbered those specific for P1.5-1,2-2, upon boosting. These trends were observed with different vaccine formulations of the porins. Our results show for the first time that B-cell subpopulations involved in a successfully maturated antibody response against a clinically relevant vaccine antigen are maintained at smaller population sizes than those associated with poor affinity maturation. This bears consequences for the interpretation of immunological memory data in clinical vaccine trials.
Root Canal Microbiota of Teeth with Chronic Apical Periodontitis
Authors: Rôças I. N. , Siqueira J. F.
Published: September 2008

Samples from infected root canals of 43 teeth with chronic apical periodontitis were analyzed for the presence and relative levels of 83 oral bacterial species and/or phylotypes using a reverse-capture checkerboard hybridization assay. Associations between the most frequently detected taxa were also recorded. The most prevalent taxa were
Olsenella uli
(74%),
Eikenella corrodens
(63%),
Porphyromonas endodontalis
(56%),
Peptostreptococcus anaerobius
(54%), and
Bacteroidetes
oral clone X083 (51%). When prevalence was considered only for bacteria present at levels >10
5
,
Bacteroidetes
clone X083 was the most frequently isolated bacterium (37%), followed by
Parvimonas micra
(28%),
E. corrodens
(23%), and
Tannerella forsythia
(19%). The number of target taxa per canal was directly proportional to the size of the apical periodontitis lesion, with lesions >10 mm in diameter harboring a mean number of approximately 20 taxa. Several positive associations for the most prevalent taxa were disclosed for the first time and may have important ecological and pathogenic implications. In addition to strengthening the association of several cultivable named species with chronic apical periodontitis, the present findings using a large-scale analysis allowed the inclusion of some newly named species and as-yet-uncultivated phylotypes in the set of candidate pathogens associated with this disease.
Phenotypic and Genotypic Characterization of Verotoxin-ProducingEscherichia coliO103:H2 Isolates from Cattle and Humans
Authors: Karama Musafiri , Johnson Roger P. , Holtslander Robert , Gyles Carlton L.
Published: September 2008

Characterization of important non-O157 verotoxin-producingEscherichia coli(VTEC) has lagged considerably behind that of O157:H7 strains. This study characterized 91 VTEC O103:H2 strains from bovine and human sources and of North American and European origins by virulence or putative virulence genes, pulsed-field gel electrophoresis (PFGE) patterns, plasmid profiles, antimicrobial resistance, and colicin production. All strains were positive forvt1andeae-ε; 97% were positive forehxA; and all were negative forhlyA. Two strains carriedvt2. There were 66 PFGE patterns grouped in six clusters, and there were 25 different plasmid profiles. Plasmid-encodedkatPandetpgenes were significantly more frequent in European than in North American human strains. The distribution of selected phenotypes was as follows: enterohemorrhagicE. coli(EHEC) hemolysin, 95%; colicin production, 38%; antimicrobial resistance, 58%. All the strains were negative for the alpha-hemolytic phenotype. In conclusion, the VTEC O103:H2 strains were diverse, as shown by PFGE, plasmid profiles, virulence markers, and antimicrobial resistance patterns, and all strains showed an EHEC hemolytic phenotype instead of the alpha-hemolytic phenotype that has been shown previously.
Fatal Outcome of Hepatitis A Virus (HAV) Infection in a Traveler with Incomplete HAV Vaccination and Evidence of Rift Valley Fever Virus Infection
Authors: Oltmann Anke , Kämper Stephanie , Staeck Oliver , Schmidt-Chanasit Jonas , Günther Stephan , Berg Thomas , Frank Christina , Krüger Detlev H. , Hofmann Jörg
Published: September 2008

Hepatitis A virus (HAV) infection is rarely fatal except in patients with chronic liver disease. In the case reported here, an elderly women died of HAV infection 12 years after incomplete HAV vaccination. The possible role of a concordant Rift Valley fever virus infection acquired in Kenya is discussed.
Escherichia hermannii
as the Sole Isolate from a Patient with Purulent Conjunctivitis
Authors: Poulou Aggeliki , Dimitroulia Evangelia , Markou Fani , Tsakris Athanassios
Published: September 2008

Escherichia hermannii
was isolated in pure culture from a patient with acute purulent conjunctivitis after a minor ocular injury. This is the first report of
E. hermannii
isolated as the sole pathogen from an infected site without prior antibiotic exposure, confirming the pathogenic potential of the microorganism.
Lentivirus Vector Can Be Readministered to Nasal Epithelia without Blocking Immune Responses
Authors: Sinn Patrick L. , Arias Ariadna C. , Brogden Kim A. , McCray Paul B.
Published: September 2008

For many envisioned applications of lentivirus vectors as tools in respiratory biology and therapeutic gene delivery, the efficiency of gene transfer must be improved. We previously demonstrated stable, persistent (>11 months) in vivo expression following a single application of a feline immunodeficiency virus (FIV)-based lentivirus vector (GP64-FIV) to murine nasal epithelia. Here we investigate the efficacy of repeated administration of lentivirus vectors to the airways. Using quantitative bioluminescent imaging, we found that consecutive daily dosing achieved a linear increase in gene expression and greatly increased the number of epithelial cells targeted. Surprisingly, reporter gene expression also increased additively following each of seven doses of FIV delivered over consecutive weeks (1 dose/week), without the development of systemic or local neutralizing antibodies. This approach enhanced expression of both reporter and therapeutic transgenes. Transduction efficiency achieved following a single dose of FIV expressing mouse erythropoietin was insufficient to increase hematocrit, whereas seven consecutive daily doses significantly increased hematocrit. These unexpected results contrast strikingly with findings reported for adenovirus vectors. Prolonged gene expression has been observed in vivo following a single dose of virus vector; however, depending on the application, repeated administration of vector may be necessary to achieve stable, therapeutic gene expression.
Hepatitis C Virus Infection Induces Apoptosis through a Bax-Triggered, Mitochondrion-Mediated, Caspase 3-Dependent Pathway
Authors: Deng Lin , Adachi Tetsuya , Kitayama Kikumi , Bungyoku Yasuaki , Kitazawa Sohei , Ishido Satoshi , Shoji Ikuo , Hotta Hak
Published: September 2008

We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome
c
from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).
Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry
Authors: Funk Anneke , Mhamdi Mouna , Hohenberg Heinz , Heeren Jörg , Reimer Rudolph , Lambert Carsten , Prange Reinhild , Sirma Hüseyin
Published: September 2008

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.
Mutations at Alternative 5′ Splice Sites of M1 mRNA Negatively Affect Influenza A Virus Viability and Growth Rate
Authors: Chiang Chiayn , Chen Guang-Wu , Shih Shin-Ru
Published: September 2008

Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5′ splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5′ splice site), C51G (at the 3′ end of the exon of the M2 mRNA 5′ splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3′ end of exon of the mRNA3 5′ splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3′ end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5′ splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.
Measles Virus-Induced Block of Transendothelial Migration of T Lymphocytes and Infection-Mediated Virus Spread across Endothelial Cell Barriers
Authors: Dittmar Sandra , Harms Harry , Runkler Nicole , Maisner Andrea , Kim Kwang Sik , Schneider-Schaulies Jürgen
Published: September 2008

In order to analyze whether measles virus (MV) is transported via transmigrating leukocytes across endothelial barriers or whether virus spreads via infection of endothelial cells and basolateral release, we investigated the migratory behavior of infected human primary T lymphocytes across polarized cell layers of human brain microvascular endothelial cells. We found that the capacity of lymphocytes to migrate through filter pores was only slightly affected by wild-type MV infection, whereas their capacity to migrate through endothelial barriers was drastically reduced. MV infection stimulated the expression and activation of the leukocyte integrins LFA-1 and VLA-4, mediating a strong adherence to the surface of endothelial cells. Furthermore, the formation of engulfing membrane protrusions by endothelial cells, so-called transmigratory cups, was induced, but transmigration was impaired. As a consequence of this close cell-cell contact, MV infection was transmitted from lymphocytes to the endothelium. MV envelope proteins were expressed on the apical and basolateral surfaces of infected polarized endothelial cells, and virus was released from both sides. Wild-type MV infection did not induce the formation of syncytia, suggesting virus spread from cell to cell via cell processes and contacts. Our data indicate that transendothelial migration of infected T cells is strongly inhibited, whereas virus can cross endothelial barriers by productive infection of the endothelium and subsequent bipolar virus release.
Transactivation of the Hepatitis B Virus Core Promoter by the Nuclear Receptor FXRα
Authors: Ramière Christophe , Scholtès Caroline , Diaz Olivier , Icard Vinca , Perrin-Cocon Laure , Trabaud Mary-Anne , Lotteau Vincent , André Patrice
Published: September 2008

Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand-activated transcription factors, via
cis
-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXRα) in modulating transcription from the HBV core promoter. FXRα is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXRα). Electrophoretic mobility shift assays demonstrated that FXRα-RXRα heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXRα response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXRα. Moreover, using a greater-than-genome-length HBV construct, we showed that FXRα also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXRα is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.
Identification of Monomorphic and Divergent Haplotypes in the 2006-2007 Norovirus GII/4 Epidemic Population by Genomewide Tracing of Evolutionary History
Authors: Motomura Kazushi , Oka Tomoichiro , Yokoyama Masaru , Nakamura Hiromi , Mori Hiromi , Ode Hirotaka , Hansman Grant S. , Katayama Kazuhiko , Kanda Tadahito , Tanaka Tomoyuki , Takeda Naokazu , Sato Hironori
Published: September 2008

Our norovirus (NoV) surveillance group reported a >4-fold increase in NoV infection in Japan during the winter of 2006-2007 compared to the previous winter. Because the increase was not linked to changes in the surveillance system, we suspected the emergence of new NoV GII/4 epidemic variants. To obtain information on viral changes, we conducted full-length genomic analysis. Stool specimens from 55 acute gastroenteritis patients of various ages were collected at 11 sites in Japan between May 2006 and January 2007. Direct sequencing of long PCR products revealed 37 GII/4 genome sequences. Phylogenetic study of viral genome and partial sequences showed that the two new GII/4 variants in Europe, termed 2006a and 2006b, initially coexisted as minorities in early 2006 in Japan and that 2006b alone had dominated over the resident GII/4 variants during 2006. A combination of phylogenetic and entropy analyses revealed for the first time the unique amino acid substitutions in all eight proteins of the new epidemic strains. These data and computer-assisted structural study of the NoV capsid protein are compatible with a model of antigenic drift with tuning of the structure and functions of multiple proteins for the global outgrowth of new GII/4 variants. The availability of comprehensive information on genome sequences and unique protein changes of the recent global epidemic variants will allow studies of diagnostic assays, molecular epidemiology, molecular biology, and adaptive changes of NoV in nature.
CD40 Engagement on Dendritic Cells, but Not on B or T Cells, Is Required for Long-Term Control of Murine Gammaherpesvirus 68
Authors: Giannoni Francesca , Shea Ashley , Inglis Chandra , Lee Lian Ni , Sarawar Sally R.
Published: September 2008

CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII−/−) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII−/−dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII−/−recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40+/+or CD40−/−mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.
Mutation of Mapped TIA-1/TIAR Binding Sites in the 3′ Terminal Stem-Loop of West Nile Virus Minus-Strand RNA in an Infectious Clone Negatively Affects Genomic RNA Amplification
Authors: Emara Mohamed M. , Liu Hsuan , Davis William G. , Brinton Margo A.
Published: September 2008

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3′ minus-strand stem-loop [WNV3′(−)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3′(−)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3′(−)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3′ (−)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3′(−)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.
Mode of Transmission Affects the Sensitivity of Human Immunodeficiency Virus Type 1 to Restriction by Rhesus TRIM5α
Authors: Richardson Max W. , Carroll Richard G. , Stremlau Matthew , Korokhov Nikolay , Humeau Laurent M. , Silvestri Guido , Sodroski Joseph , Riley James L.
Published: September 2008

Rhesus TRIM5α (rhTRIM5α), but not human TRIM5α (huTRIM5α), potently inhibits human immunodeficiency virus (HIV) infection and is thus a potentially valuable therapeutic tool. Primary human CD4 T cells engineered to express rhTRIM5α were highly resistant to cell-free HIV type 1 (HIV-1) infection. However, when cocultured with unmodified T cells, rhTRIM5α-expressing cells became highly permissive to HIV-1 infection. Physical separation of rhTRIM5α-expressing cells and unmodified cells revealed that rhTRIM5α efficiently restricts cell-free but not cell-associated HIV transmission. Furthermore, we observed that HIV-infected human cells could infect rhesus CD4 T cells by cell-to-cell contact, but the infection was self-limiting. Subsequently, we noted that a spreading infection ensued when HIV-1-infected rhTRIM5α-expressing human cells were cultured with huTRIM5α- but not rhTRIM5α-expressing cells. Our results suggest that cell-associated HIV transmission in humans is blocked only when both donor and recipient cells express rhTRIM5α. These studies further define the role of rhTRIM5α in cell-free and cell-associated HIV transmission and delineate the utility of rhTRIM5α in anti-HIV therapy.
Glutamine Deprivation Causes Enhanced Plating Efficiency of a Herpes Simplex Virus Type 1 ICP0-Null Mutant
Authors: Bringhurst Ryan M. , Dominguez Antonia A. , Schaffer Priscilla A.
Published: September 2008

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0
−
) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0
−
virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0
−
virus.
Transactivation, Dimerization, and DNA-Binding Activity of White Spot Syndrome Virus Immediate-Early Protein IE1
Authors: Liu Wang-Jing , Chang Yun-Shiang , Wang Hao-Ching , Leu Jiann-Horng , Kou Guang-Hsiung , Lo Chu-Fang
Published: September 2008

Immediate-early proteins from many viruses function as transcriptional regulators and exhibit transactivation activity, DNA binding activity, and dimerization. In this study, we investigated these characteristics in white spot syndrome virus (WSSV) immediate-early protein 1 (IE1) and attempted to map the corresponding functional domains. Transactivation was investigated by transiently expressing a protein consisting of the DNA binding domain of the yeast transactivator GAL4 fused to full-length IE1. This GAL4-IE1 fusion protein successfully activated the
Autographa californica
multicapsid nucleopolyhedrovirus
p35
basal promoter when five copies of the GAL4 DNA binding site were inserted upstream of the TATA box. A deletion series of GAL4-IE1 fusion proteins suggested that the transactivation domain of WSSV IE1 was carried within its first 80 amino acids. A point mutation assay further showed that all 12 of the acidic residues in this highly acidic domain were important for IE1's transactivation activity. DNA binding activity was confirmed by an electrophoresis mobility shift assay using a probe with
32
P-labeled random oligonucleotides. The DNA binding region of WSSV IE1 was located in its C-terminal end (amino acids 81 to 224), but mutation of a putative zinc finger motif in this C-terminal region suggested that this motif was not directly involved in the DNA binding activity. A homotypic interaction between IE1 molecules was demonstrated by glutathione
S
-transferase pull-down assay and a coimmunoprecipitation analysis. A glutaraldehyde cross-linking experiment and gel filtration analysis showed that this self-interaction led to the formation of stable IE1 dimers.
The Interferon Antagonist ML Protein of Thogoto Virus Targets General Transcription Factor IIB
Authors: Vogt Carola , Preuss Ellen , Mayer Daniel , Weber Friedemann , Schwemmle Martin , Kochs Georg
Published: September 2008

The ML protein of Thogoto virus, a tick-transmitted orthomyxovirus, is a splice variant of the viral matrix protein and antagonizes the induction of antiviral type I interferon (IFN). Here we identified the general RNA polymerase II transcription factor IIB (TFIIB) as an ML-interacting protein. Overexpression of TFIIB neutralized the inhibitory effect of ML on IRF3-mediated promoter activation. Moreover, a recombinant virus expressing a mutant ML protein unable to bind TFIIB was severely impaired in its ability to suppress IFN induction. We concluded that TFIIB binding is required for the IFN antagonist effect exerted by ML. We further demonstrate that the ML-TFIIB interaction has surprisingly little impact on gene expression in general, while a strong negative effect is observed for IRF3- and NF-κB-regulated promoters.
Eastern and Venezuelan Equine Encephalitis Viruses Differ in Their Ability To Infect Dendritic Cells and Macrophages: Impact of Altered Cell Tropism on Pathogenesis
Authors: Gardner Christina L. , Burke Crystal W. , Tesfay Mulu Z. , Glass Pamela J. , Klimstra William B. , Ryman Kate D.
Published: September 2008

Eastern and Venezuelan equine encephalitis viruses (EEEV and VEEV, respectively) cause severe morbidity and mortality in equines and humans. Like other mosquito-borne viruses, VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues, preferentially infecting osteoblasts. Here, we demonstrate that infectivity of EEEV for myeloid lineage cells including DCs and macrophages was dramatically reduced compared to that of VEEV, whereas both viruses replicated efficiently in mesenchymal lineage cells such as osteoblasts and fibroblasts. We determined that EEEV infection of myeloid lineage cells was restricted after attachment, entry, and uncoating of the genome. Using replicon particles and translation reporter RNAs, we found that translation of incoming EEEV genomes was almost completely inhibited in myeloid, but not mesenchymal, lineage cells. Alpha/beta interferon (IFN-α/β) responses did not mediate the restriction, as infectivity was not restored in the absence of double-stranded RNA-dependent protein kinase, RNase L, or IFN-α/β receptor-mediated signaling. We confirmed these observations in vivo, demonstrating that EEEV is compromised in its ability to replicate within lymphoid tissues, whereas VEEV does so efficiently. The altered tropism of EEEV correlated with an almost complete avoidance of serum IFN-α/β induction in vivo, which may allow EEEV to evade the host's innate immune responses and thereby enhance neurovirulence. Taken together, our data indicate that inhibition of genome translation restricts EEEV infectivity for myeloid but not mesenchymal lineage cells in vitro and in vivo. In this regard, the tropisms of EEEV and VEEV differ dramatically, likely contributing to observed differences in disease etiology.
Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA
Authors: Oh Jangsuk , Chang Kevin W. , Hughes Stephen H.
Published: September 2008

The two ends of RSV linear DNA are independently inserted into host DNA by integrase in vivo. We previously showed that the range of U3 sequences that are acceptable substrates for integrase appeared to be greater than the range of acceptable U5 sequences in vivo. We have done additional experiments to determine which U3 sequences are good integrase substrates. On the U3 end, there does not appear to be a stringent requirement for the canonical CA, integrase can efficiently remove three nucleotides, and six nucleotides are sufficient to allow integration with reasonable, albeit reduced, efficiency.
Artificial MicroRNAs Highly Accessible to Targets Confer Efficient Virus Resistance in Plants
Authors: Duan Cheng-Guo , Wang Chun-Han , Fang Rong-Xiang , Guo Hui-Shan
Published: September 2008

Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3′ untranslated region (UTR) of the
Cucumber mosaic virus
(CMV) genome. Transiently expressed 3′UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3′UTR of the CMV-infected wild-type
Arabidopsis
to those of the triple
dcl2/3/4
mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3′UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.
Methods for Sampling of Airborne Viruses
Authors: Verreault Daniel , Moineau Sylvain , Duchaine Caroline
Published: September 2008

To better understand the underlying mechanisms of aerovirology, accurate sampling of airborne viruses is fundamental. The sampling instruments commonly used in aerobiology have also been used to recover viruses suspended in the air. We reviewed over 100 papers to evaluate the methods currently used for viral aerosol sampling. Differentiating infections caused by direct contact from those caused by airborne dissemination can be a very demanding task given the wide variety of sources of viral aerosols. While epidemiological data can help to determine the source of the contamination, direct data obtained from air samples can provide very useful information for risk assessment purposes. Many types of samplers have been used over the years, including liquid impingers, solid impactors, filters, electrostatic precipitators, and many others. The efficiencies of these samplers depend on a variety of environmental and methodological factors that can affect the integrity of the virus structure. The aerodynamic size distribution of the aerosol also has a direct effect on sampler efficiency. Viral aerosols can be studied under controlled laboratory conditions, using biological or nonbiological tracers and surrogate viruses, which are also discussed in this review. Lastly, general recommendations are made regarding future studies on the sampling of airborne viruses.
Getting a Handle on the Role of Coenzyme M in Alkene Metabolism
Authors: Krishnakumar Arathi M. , Sliwa Darius , Endrizzi James A. , Boyd Eric S. , Ensign Scott A. , Peters John W.
Published: September 2008

Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO
2
to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism
Xanthobacter autotrophicus
Py2. A four-step pathway requiring CoM converts propylene and CO
2
to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.