bluuebunny/crossref_metadata_2025 huggingface dataset:
Storage Effects on Dielectric Properties of Eggs from 10 to 1800 MHz
Authors: Guo W. , Trabelsi S. , Nelson S.O. , Jones D.R.
Published: June 2007

ABSTRACT:  The dielectric constant and loss factor of egg albumen and egg yolk over the frequency range from 10 to 1800 MHz were measured at 24 °C at weekly intervals during 5‐wk storage at 15 °C. Moisture and ash contents of albumen and yolk, as well as Haugh unit and yolk index, were also measured. The dielectric constant and loss factor of albumen were higher than those of yolk. Linear relationships were evident between the log of frequency, below about 1000 MHz, and the log of loss factor of albumen as well as that of yolk. The dielectric constants of albumen and yolk at 10 MHz were lower than those of fresh albumen and yolk when eggs were stored at 15 °C for 1 wk. However, after 2 wk in storage these dielectric constants rose and remained at higher levels for the rest of the 5‐wk period. At frequencies of 100 MHz and higher, the dielectric constant was essentially constant during the entire storage period. Storage had much less influence on the loss factor of either albumen or yolk. In general, the moisture content and ash content of albumen and yolk decreased slightly as eggs aged. The moisture content of yolk increased somewhat with storage, and there was a corresponding decrease in albumen moisture content. The freshness qualities, Haugh unit and yolk index, also decreased as eggs aged. No obvious correlation between dielectric properties and moisture content, ash content, Haugh unit, or yolk index was observed.
Characterization of Protein Fractions from Immature Alaska Walleye Pollock (Theragra chalcogramma) Roe
Authors: Bechtel P.J. , Chantarachoti J. , Oliveira A.C.M. , Sathivel S.
Published: June 2007

ABSTRACT:  There are good markets for mature pollock roe; however, immature pollock roe is underutilized. The physical and nutritional properties of immature pollock roe (IPR) have not been reported, which limits its potential use as a food ingredient. The objective of this study was to evaluate the physical and nutritional properties of immature pollock roe and soluble and insoluble protein powders made from the immature roe. IPR samples were obtained during the spring pollock harvest from a seafood processing plant in Kodiak, Alaska. Soluble (SP) and insoluble protein (IP) fractions were produced by heating IPR, separating by centrifugation and freeze drying. The protein contents of freeze‐dried IPR, SP, and IP were 81.7%, 63.2%, and 83.0%, respectively. The amino acid contents of IPR and IP were similar except for isoleucine and valine. However, the amino acid contents of IPR and IP were different from values for SP. Lipid contents of IPR, SP, and IP were 9.2%, 9.3%, and 11.1%, respectively. Palmitic acid (C16:0; 21.2%), DHA (C22:6ω3; 21.2%), and EPA (C20:5ω3; 19.0%) were the 3 most abundant fatty acids in fresh IPR. Fat adsorption capacity value for SP was significantly higher than IPR and IP (P < 0.05). SDS electrophoresis indicated a major protein band with molecular weight of 103 KDa in all samples. Results indicate that IPR can be utilized to make a number of unique food ingredients with good nutritional characteristics and functional properties.
Wnt Induces LRP6 Signalosomes and Promotes Dishevelled-Dependent LRP6 Phosphorylation
Authors: Bilić Josipa , Huang Ya-Lin , Davidson Gary , Zimmermann Timo , Cruciat Cristina-Maria , Bienz Mariann , Niehrs Christof
Published: June 2007

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor–related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane–associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and β-catenin stabilization.
The Vaccine Adjuvant Monophosphoryl Lipid A as a TRIF-Biased Agonist of TLR4
Authors: Mata-Haro Verónica , Cekic Caglar , Martin Michael , Chilton Paula M. , Casella Carolyn R. , Mitchell Thomas C.
Published: June 2007

The inflammatory toxicity of lipopolysaccharide (LPS), a component of bacterial cell walls, is driven by the adaptor proteins myeloid differentiation factor 88 (MyD88) and Toll-interleukin 1 receptor domain–containing adapter inducing interferon-β (TRIF), which together mediate signaling by the endotoxin receptor Toll-like receptor 4 (TLR4). Monophosphoryl lipid A (MPLA) is a low-toxicity derivative of LPS with useful immunostimulatory properties, which is nearing regulatory approval for use as a human vaccine adjuvant. We report here that, in mice, the low toxicity of MPLA's adjuvant function is associated with a bias toward TRIF signaling, which we suggest is likely caused by the active suppression, rather than passive loss, of proinflammatory activity of this LPS derivative. This finding may have important implications for the development of future vaccine adjuvants.
Crystal Structures of Human MD-2 and Its Complex with Antiendotoxic Lipid IVa
Authors: Ohto Umeharu , Fukase Koichi , Miyake Kensuke , Satow Yoshinori
Published: June 2007

Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD-2 and Toll-like receptor 4. Crystal structures of human MD-2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS have been determined at 2.0 and 2.2 angstrom resolutions, respectively. MD-2 shows a deep hydrophobic cavity sandwiched by two β sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. These structures suggest that MD-2 plays a principal role in endotoxin recognition and provide a basis for antiseptic drug development.
The Mass of Dwarf Planet Eris
Authors: Brown Michael E. , Schaller Emily L.
Published: June 2007

The discovery of dwarf planet Eris was followed shortly by the discovery of its satellite, Dysnomia, but the satellite orbit, and thus the system mass, was not known. New observations with the Keck Observatory and the Hubble Space Telescopes show that Dysnomia has a circular orbit with a radius of 37,350 ± 140 (1-σ) kilometers and a 15.774 ± 0.002 day orbital period around Eris. These orbital parameters agree with expectations for a satellite formed out of the orbiting debris left from a giant impact. The mass of Eris from these orbital parameters is 1.67 × 10
22
± 0.02 × 10
22
kilograms, or 1.27 ± 0.02 that of Pluto.
The Release 5.1 Annotation of
Drosophila melanogaster
Heterochromatin
Authors: Smith Christopher D. , Shu ShengQiang , Mungall Christopher J. , Karpen Gary H.
Published: June 2007

The repetitive DNA that constitutes most of the heterochromatic regions of metazoan genomes has hindered the comprehensive analysis of gene content and other functions. We have generated a detailed computational and manual annotation of 24 megabases of heterochromatic sequence in the Release 5
Drosophila melanogaster
genome sequence. The heterochromatin contains a minimum of 230 to 254 protein-coding genes, which are conserved in other Drosophilids and more diverged species, as well as 32 pseudogenes and 13 noncoding RNAs. Improved methods revealed that more than 77% of this heterochromatin sequence, including introns and intergenic regions, is composed of fragmented and nested transposable elements and other repeated DNAs.
Drosophila
heterochromatin contains “islands” of highly conserved genes embedded in these “oceans” of complex repeats, which may require special expression and splicing mechanisms.
Neural Responses to Taxation and Voluntary Giving Reveal Motives for Charitable Donations
Authors: Harbaugh William T. , Mayr Ulrich , Burghart Daniel R.
Published: June 2007

Civil societies function because people pay taxes and make charitable contributions to provide public goods. One possible motive for charitable contributions, called “pure altruism,” is satisfied by increases in the public good no matter the source or intent. Another possible motive, “warm glow,” is only fulfilled by an individual's own voluntary donations. Consistent with pure altruism, we find that even mandatory, tax-like transfers to a charity elicit neural activity in areas linked to reward processing. Moreover, neural responses to the charity's financial gains predict voluntary giving. However, consistent with warm glow, neural activity further increases when people make transfers voluntarily. Both pure altruism and warm-glow motives appear to determine the hedonic consequences of financial transfers to the public good.
Metal Chlorides in Ionic Liquid Solvents Convert Sugars to 5-Hydroxymethylfurfural
Authors: Zhao Haibo , Holladay Johnathan E. , Brown Heather , Zhang Z. Conrad
Published: June 2007

Replacing petroleum feedstocks by biomass requires efficient methods to convert carbohydrates to a variety of chemical compounds. We report the catalytic conversion of sugars giving high yield to 5-hydroxymethylfurfural (HMF), a versatile intermediate. Metal halides in 1-alkyl-3-methylimidazolium chloride are catalysts, among which chromium (II) chloride is found to be uniquely effective, leading to the conversion of glucose to HMF with a yield near 70%. A wide range of metal halides is found to catalyze the conversion of fructose to HMF. Only a negligible amount of levulinic acid is formed in these reactions.
Origin of the Low Rigidity of the Earth's Inner Core
Authors: Belonoshko Anatoly B. , Skorodumova Natalia V. , Davis Sergio , Osiptsov Alexander N. , Rosengren Anders , Johansson Börje
Published: June 2007

Earth's solid-iron inner core has a low rigidity that manifests itself in the anomalously low velocities of shear waves as compared to shear wave velocities measured in iron alloys. Normally, when estimating the elastic properties of a polycrystal, one calculates an average over different orientations of a single crystal. This approach does not take into account the grain boundaries and defects that are likely to be abundant at high temperatures relevant for the inner core conditions. By using molecular dynamics simulations, we show that, if defects are considered, the calculated shear modulus and shear wave velocity decrease dramatically as compared to those estimates obtained from the averaged single-crystal values. Thus, the low shear wave velocity in the inner core is explained.
Tuning the Quantum Stability and Superconductivity of Ultrathin Metal Alloys
Authors: Özer Mustafa M. , Jia Yu , Zhang Zhenyu , Thompson James R. , Weitering Hanno H.
Published: June 2007

Quantum confinement of itinerant electrons in atomically smooth ultrathin lead films produces strong oscillations in the thickness-dependent film energy. By adding extra electrons via bismuth alloying, we showed that both the structural stability and the superconducting properties of such films can be tuned. The phase boundary (upper critical field) between the superconducting vortex state and the normal state indicates an anomalous suppression of superconducting order just below the critical temperature,
T
c
. This suppression varies systematically with the film thickness and the bismuth content and can be parametrized in terms of a characteristic temperature,
T
c
* (less than
T
c
), that is inversely proportional to the scattering mean free path. The results indicate that the isotropic nature of the superconductive pairing in bulk lead-bismuth alloys is altered in the quantum regime.
Cross-Reactive T-Cell Responses to the Nonstructural Regions of Dengue Viruses among Dengue Fever and Dengue Hemorrhagic Fever Patients in Malaysia
Authors: Appanna Ramapraba , Huat Tan Lian , See Lucy Lum Chai , Tan Phoay Lay , Vadivelu Jamuna , Devi Shamala
Published: June 2007

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3422-431were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3422-431and NS5563-571in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3422-431. A large number of patients' T cells also responded to the NS2B97-106region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.
Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies
Authors: Lalonde Matthew S. , Troyer Ryan M. , Syed Aslam R. , Bulime Stanley , Demers Korey , Bajunirwe Francis , Arts Eric J.
Published: June 2007

This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of ∼0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population.
Development of Real-Time PCR Assays for Genotyping of
Chlamydia trachomatis
Authors: Jalal Hamid , Stephen Hannah , Alexander Sarah , Carne Christopher , Sonnex Christopher
Published: June 2007

We have developed and validated a nested real-time PCR (NRT-PCR) for the genotyping of
Chlamydia trachomatis
and used it specifically for the typing of either eight genovars from D to K or three genovars of lymphogranuloma venereum (LGV). The 11 probes used in the NRT-PCR correctly identified the DNA from D to K and LGV reference strains and did not cross-react with the DNA from 26 strains representing the bacterial pathogens and commensals of the oropharynx, genital tract, and rectum. The NRT-PCR had a 95% probability of detection at four genome copies (confidence interval, three to six copies) of
C. trachomatis
per reaction. One hundred cervical and urethral swab specimens containing
C. trachomatis
DNA from 63 women and 37 men were used to validate the method. The results from the NRT-PCR and the DNA sequencing of amplicons generated from the
omp1
gene showed 100% correlation for these samples. The assay also identified the LGV-II genotype in 24 of 48 rectal swab specimens containing
C. trachomatis
DNA that were obtained from men having sex with men. The Sexually Transmitted Bacteria Reference Laboratory, London, independently confirmed these results using group-specific LGV real-time PCR and restriction fragment length polymorphism analysis. Compared with the NRT-PCR, non-NRT-PCR was found to be less sensitive: it typed
C. trachomatis
DNA in only 80% of the genital samples and 90% of the rectal swab samples. This is the first successful demonstration of the use of real-time PCR for the genotype-specific typing of
C. trachomatis
strains that cause sexually transmitted diseases.
Detection of
Clostridium difficile
Toxin: Comparison of Enzyme Immunoassay Results with Results Obtained by Cytotoxicity Assay
Authors: Musher Daniel M. , Manhas Atisha , Jain Pranav , Nuila Franziska , Waqar Amna , Logan Nancy , Marino Bernard , Graviss Edward A.
Published: June 2007

Several kinds of laboratory techniques are available to detect
Clostridium difficile
toxin in fecal samples. Because questions have been raised about the reliability of immunoassays compared to the accepted standard, cytotoxicity assay, we studied three enzyme immunoassays (EIAs) and one rapid EIA, which demonstrated relatively good sensitivities and specificities compared to cytotoxicity assay.
Desulfovibrio fairfieldensis
Bacteremia Associated with Choledocholithiasis and Endoscopic Retrograde Cholangiopancreatography
Authors: Pimentel Jason D. , Chan Raymond C.
Published: June 2007

Desulfovibrio fairfieldensis
is a gram-negative, curved, motile, anaerobic bacillus.
D. fairfieldensis
has been isolated only from human specimens and is considered a normal resident of the human gastrointestinal tract. We report the second case of
Desulfovibrio
bacteremia associated with choledocholithiasis and review the other reported cases of
D. fairfieldensis
bacteremia.
Streptococcus mutans
Clonal Variation Revealed by Multilocus Sequence Typing
Authors: Nakano Kazuhiko , Lapirattanakul Jinthana , Nomura Ryota , Nemoto Hirotoshi , Alaluusua Satu , Grönroos Lisa , Vaara Martti , Hamada Shigeyuki , Ooshima Takashi , Nakagawa Ichiro
Published: June 2007

Streptococcus mutans
is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis.
S. mutans
strains have been classified into four serotypes (
c
,
e
,
f
, and
k
). However, little is known about the
S. mutans
population, including the clonal relationships among strains of
S. mutans
, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for
S. mutans
. Eight housekeeping gene fragments were sequenced from each of 102
S. mutans
isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 × 10
10
sequence types. We identified 92 sequence types in these 102 isolates, indicating that
S. mutans
contains a diverse population. Whereas serotype
c
strains were widely distributed in the dendrogram, serotype
e
,
f
, and
k
strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of
S. mutans
was serotype
c
. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (
cnm
) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for
S. mutans
may have important practical implications.
Population-Based Study of Salivary Carriage of Periodontal Pathogens in Adults
Authors: Könönen Eija , Paju Susanna , Pussinen Pirkko J. , Hyvönen Mari , Di Tella Paulo , Suominen-Taipale Liisa , Knuuttila Matti
Published: June 2007

Large, general population-based data on carriage rates of periodontal pathogens hardly exist in the current literature. The objectives of the present study were to examine the salivary detection of
Aggregatibacter
(formerly
Actinobacillus
)
actinomycetemcomitans
,
Campylobacter rectus
,
Porphyromonas gingivalis
,
Prevotella intermedia
,
Tannerella forsythensis
, and
Treponema denticola
in a representative sample of the adult population living in southern Finland and to clarify which determinants are associated with the presence of these pathogens in saliva. 16S rRNA-based PCR methods with species-specific primers were employed to determine the presence of the six target bacteria in stimulated saliva samples, which were available from 1,294 subjects aged ≥30 years. The age group, gender, level of education, marital status, smoking history, number of teeth, and number of teeth with deepened pockets were included in the statistical analysis. In general, the carriage of periodontal pathogens was common, since at least one of the examined pathogens was found in 88.2% of the subjects. In descending order, the total detection rates were 56.9%, 38.2%, 35.4%, 31.3%, 20.0%, and 13.9% for
T. forsythensis
,
T. denticola
,
P. gingivalis
,
C. rectus
,
A. actinomycetemcomitans
, and
P. intermedia
, respectively. Age per se was strongly associated with the carriage of
P. gingivalis
(
P
= 0.000), and the level of education with that of
T. denticola
(
P
= 0.000). There was an association between the number of teeth with deepened pockets and carriage of
P. gingivalis
(
P
= 0.000),
P. intermedia
(
P
= 0.000),
T. denticola
(
P
= 0.000), and
A. actinomycetemcomitans
(
P
= 0.004). The data suggest that distinct species have a different carriage profile, depending on variables such as age, educational level, and periodontal status.
Local Expression of Secondary Lymphoid Tissue Chemokine Delivered by Adeno-Associated Virus within the Tumor Bed Stimulates Strong Anti-Liver Tumor Immunity
Authors: Liang Chun-min , Zhong Cui-ping , Sun Rui-xia , Liu Bin-bin , Huang Cheng , Qin Jie , Zhou Shuang , Shan Junling , Liu Yin-kun , Ye Sheng-long
Published: June 2007

Development of an effective antitumor immune response depends on the appropriate interaction of effector and target cells. Thus, the expression of chemokines within the tumor may induce a more potent antitumor immune response. Secondary lymphoid tissue chemokine (SLC) is known to play a critical role in establishing a functional microenvironment in secondary lymphoid tissues. Its capacity to attract dendritic cells (DCs) and colocalize them with T cells makes it a good therapeutic candidate against cancer. In this study, we used SLC as a treatment for tumors established from a murine hepatocellular carcinoma model. SLC was encoded by recombinant adeno-associated virus (rAAV), a system chosen for the low host immunity and high efficiency of transduction, enabling long-term expression of the gene of interest. As a result, rAAV-SLC induced a significant delay of tumor progression, which was paralleled by a profound infiltration of DCs and activated CD4+T cells and CD8+T cells (CD3+CD69+cells) into the tumor site. In addition, rAAV-SLC treatment was also found to reduce tumor growth in nude mice, most likely due to inhibition of neoangiogenesis. In conclusion, local expression of SLC by rAAV represents a promising approach to induce immune-mediated regression of malignant tumors.
Retrograde Axonal Transport: a Major Transmission Route of Enterovirus 71 in Mice
Authors: Chen Che-Szu , Yao Yi-Chuan , Lin Shin-Chao , Lee Yi-Ping , Wang Ya-Fang , Wang Jen-Ren , Liu Ching-Chuan , Lei Huan-Yao , Yu Chun-Keung
Published: June 2007

Inoculation of enterovirus 71 (EV71) by the oral (p.o.), intramuscular (i.m.), or intracranial route resulted in brain infection, flaccid paralysis, pulmonary dysfunction, and death of 7-day-old mice. The lag time of disease progression indicated that neuroinvasion from the inoculation sites was a prerequisite for the development of the clinical signs. Although EV71 p.o. inoculation led to a persistent viremia and a transient increase in blood-brain barrier permeability at the early stage of the infection, only low levels of virus, which led to neither severe infection nor clinical illness, could be detected in the brain, suggesting that hematogenous transport might not represent a major transmission route. In the spinal cord, following both p.o. and hind limb i.m. inoculation, the virus first appeared and increased rapidly in the lower segments, especially at the anterior horn areas, and then spread to the upper segments and brain in the presence of viremia. A reverse pattern, with the virus being first detected in the upper segment, was observed when the virus was i.m. inoculated in the forelimb. Colchicine, a fast axonal transport inhibitor, but not sciatic nerve transection reduced EV71 neuroinvasion in a dose-dependent manner, indicating a neuronal transmission of the virus.
Early Production of Type I Interferon during West Nile Virus Infection: Role for Lymphoid Tissues in IRF3-Independent Interferon Production
Authors: Bourne Nigel , Scholle Frank , Silva Maria Carlan , Rossi Shannan L. , Dewsbury Nathan , Judy Barbara , De Aguiar Juliana B. , Leon Megan A. , Estes D. Mark , Fayzulin Rafik , Mason Peter W.
Published: June 2007

Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I interferon (IFN-α/β). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor 3 null (IRF3−/−) mice, indicating that IFN production was independent of the IRF3 pathways to IFN gene transcription, consistent with the IFN type produced (predominantly α).
Upregulation of PD-1 Expression on Circulating and Intrahepatic Hepatitis C Virus-Specific CD8+T Cells Associated with Reversible Immune Dysfunction
Authors: Golden-Mason Lucy , Palmer Brent , Klarquist Jared , Mengshol John A. , Castelblanco Nicole , Rosen Hugo R.
Published: June 2007

Infection with hepatitis C virus (HCV) is associated with persistence in the majority of individuals. We demonstrate here that the inhibitory molecule programmed death-1 (PD-1) is significantly upregulated on total and HCV-specific CD8+cytotoxic T lymphocytes (CTLs) in the peripheral blood and livers of patients with chronic infection compared to subjects with spontaneous HCV resolution, patients with nonviral liver disease, and normal controls. PD-1 expression on cytomegalovirus-specific CTLs also varies according to HCV status and is highest in patients with chronic infection. HCV-specific CTLs that are PD-1highexpress higher levels of the senescence marker CD57 than PD-1lowCTLs, and CD57 expression is greater in chronic than in resolved infection. In vitro blockade of PD-1 by monoclonal antibodies specific to its ligands (PD-L1 and PD-L2) results in restoration of functional competence (proliferation and gamma interferon and interleukin-2 secretion) of HCV-specific CTLs, including those residing in the liver. This reversal of CTL exhaustion is evident even in individuals who lack HCV-specific CD4+T-cell help. Our data indicate that the PD-1/PD-L pathway is critical in persistent HCV infection in humans and represents a potential novel target for restoring function of exhausted HCV-specific CTLs.
Hepatitis C Virus Nonstructural Protein 5A Modulates the Toll-Like Receptor-MyD88-Dependent Signaling Pathway in Macrophage Cell Lines
Authors: Abe Takayuki , Kaname Yuuki , Hamamoto Itsuki , Tsuda Yoshimi , Wen Xiaoyu , Taguwa Shuhei , Moriishi Kohji , Takeuchi Osamu , Kawai Taro , Kanto Tatsuya , Hayashi Norio , Akira Shizuo , Matsuura Yoshiharu
Published: June 2007

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase 1 to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.
Viral Phenotypes and Antibody Responses in Long-Term Survivors Infected with Attenuated Human Immunodeficiency Virus Type 1 Containing Deletions in thenefand Long Terminal Repeat Regions
Authors: Verity Erin E. , Zotos Dimitra , Wilson Kim , Chatfield Catherine , Lawson Victoria A. , Dwyer Dominic E. , Cunningham Anthony , Learmont Jennifer , Dyer Wayne , Sullivan John , Churchill Melissa , Wesselingh Steven L. , Gabuzda Dana , Gorry Paul R. , McPhee Dale A.
Published: June 2007

The Sydney Blood Bank Cohort (SBBC) consists of eight blood transfusion recipients infected withnef-attenuated human immunodeficiency virus type 1 (HIV-1) acquired from a single donor. Here, we show that viral phenotypes and antibody responses differ considerably between individual cohort members, despite the single source of infection. Replication of isolated virus varied from barely detectable to similar to that of the wild-type virus, and virus isolated from five SBBC members showed coreceptor usage signatures unique to each individual. Higher viral loads and stronger neutralizing antibody responses were associated with better-replicating viral strains, and detectable viral replication was essential for the development of strong and sustained humoral immune responses. Despite the presence of strong neutralizing antibodies in a number of SBBC members, disease progression was not prevented, and each cohort member studied displayed a unique outcome of infection withnef-attenuated HIV-1.
Differential Polymerase Activity in Avian and Mammalian Cells Determines Host Range of Influenza Virus
Authors: Gabriel G. , Abram M. , Keiner B. , Wagner R. , Klenk H.-D. , Stech J.
Published: June 2007

As recently shown, mutations in the polymerase genes causing increased polymerase activity in mammalian cells are responsible for the adaptation of the highly pathogenic avian influenza virus SC35 (H7N7) to mice (G. Gabriel et al., Proc. Natl. Acad. Sci. USA 102:18590-18595, 2005). We have now compared mRNA, cRNA, and viral RNA levels of SC35 and its mouse-adapted variant SC35M in avian and mammalian cells. The increase in levels of transcription and replication of SC35M in mammalian cells was linked to a decrease in avian cells. Thus, the efficiency of the viral polymerase is a determinant of both host specificity and pathogenicity.
The Early Protective Thymus-Independent Antibody Response to Foot-and-Mouth Disease Virus Is Mediated by Splenic CD9+B Lymphocytes
Authors: Ostrowski Matias , Vermeulen Monica , Zabal Osvaldo , Zamorano Patricia I. , Sadir Ana M. , Geffner Jorge R. , Lopez Osvaldo J.
Published: June 2007

Infection of mice with cytopathic foot-and-mouth disease virus (FMDV) induces a rapid and specific thymus-independent (TI) neutralizing antibody response that promptly clears the virus. Herein, it is shown that FMDV-infected dendritic cells (DCs) directly stimulate splenic innate-like CD9+B lymphocytes to rapidly (3 days) produce neutralizing anti-FMDV immunoglobulin M antibodies without T-lymphocyte collaboration. In contrast, neither follicular (CD9−) B lymphocytes from the spleen nor B lymphocytes from lymph nodes efficiently respond to stimulation with FMDV-infected DCs. The production of these protective neutralizing antibodies is dependent on DC-derived interleukin-6 (IL-6) and on CD9+cell-derived IL-10 secretion. In comparison, DCs loaded with UV-inactivated FMDV are significantly less efficient in directly stimulating B lymphocytes to secrete TI antibodies. A critical role of the spleen in the early production of anti-FMDV antibodies in infected mice was also demonstrated in vivo. Indeed, either splenectomy or functional disruption of the marginal zone of the spleen delays and reduces the magnitude of the TI anti-FMDV antibody response in infected mice. Together, these results indicate that in addition to virus localization, the FMDV-mediated modulation of DC functionality is a key parameter that collaborates in the induction of a rapid and protective TI antibody response against this virus.
Downregulation of Class I Major Histocompatibility Complex Surface Expression by Varicella-Zoster Virus Involves Open Reading Frame 66 Protein Kinase-Dependent and -Independent Mechanisms
Authors: Eisfeld Amie J. , Yee Michael B. , Erazo Angela , Abendroth Allison , Kinchington Paul R.
Published: June 2007

We show here that the varicella-zoster virus (VZV) open reading frame 66 (ORF66) protein kinase is one mechanism employed to reduce class I major histocompatibility complex (MHC-I) surface expression in VZV-infected cells. Cells expressing enhanced green fluorescent protein-tagged functional and inactivated ORF66 (GFP-66 and GFP-66kd) from replication-defective adenovirus vectors revealed that ORF66 reduced MHC-I surface levels in a manner dependent on kinase activity. Cells infected with recombinant VZV expressing GFP-66 exhibited a significantly greater reduction in MHC-I surface expression than that observed in cells infected with VZV disrupted in GFP-66 expression. MHC-I maturation was delayed in its transport from the endoplasmic reticulum through the Golgi in both adenovirus-transduced cells expressing only GFP-66 and in VZV-infected cells expressing high levels of GFP-66, and this was predominantly kinase dependent. MHC-I levels were reduced in VZV-infected cells, and analyses of intracellular MHC-I revealed accumulation of folded MHC-I in the Golgi region, irrespective of ORF66 expression. Thus, the ORF66 kinase is important for VZV-mediated MHC-I downregulation, but additional mechanisms also may be involved. Analyses of the VZV ORF9a protein, the ortholog of the bovine herpesvirus 1 transporter associated with antigen processing inhibitor UL49.5 revealed no effects on MHC-I. These results establish a new role for viral protein kinases in immune evasion and suggest that VZV utilizes unique mechanisms to inhibit antigen presentation.
Heterosubtypic Immunity to Influenza A Virus Infection Requires a Properly Diversified Antibody Repertoire
Authors: Nguyen Huan H. , Zemlin Michael , Ivanov Ivaylo I. , Andrasi Judit , Zemlin Cosima , Vu Huong L. , Schelonka Robert , Schroeder Harry W. , Mestecky Jiri
Published: June 2007

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT−/−) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (DH) with an altered DHsegment (namely, ΔD-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT−/−mice compared to those in ΔD-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.
Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication
Authors: Belov George A. , Habbersett Courtney , Franco David , Ehrenfeld Ellie
Published: June 2007

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.
Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells
Authors: Wang Jian-Hua , Janas Alicia M. , Olson Wendy J. , Wu Li
Published: June 2007

Dendritic cells (DCs) potently stimulate the transmission of human immunodeficiency virus type 1 (HIV-1) to CD4+T cells. Immature DCs (iDCs) located in submucosal tissues can capture HIV-1 and migrate to lymphoid tissues, where they become mature DCs (mDCs) for effective antigen presentation. DC maturation promotes HIV-1 transmission; however, the underlying mechanisms remain unclear. Here we have compared monocyte-derived iDCs and mDCs for their efficiencies and mechanisms of HIV-1 transmission. We have found that mDCs significantly facilitate HIV-1 endocytosis and efficiently concentrate HIV-1 at virological synapses, which contributes to mDC-enhanced viral transmission, at least in part. mDCs were more efficient than iDCs in transferring HIV-1 to various types of target cells independently of C-type lectins, which partially accounted for iDC-mediated HIV-1 transmission. Efficient HIV-1trans-infection mediated by iDCs and mDCs required contact between DCs and target cells. Moreover, rapid HIV-1 degradation occurred in both iDCs and mDCs, which correlated with the lack of HIV-1 retention-mediated long-term viral transmission. Our results provide new insights into the mechanisms underlying DC-mediated HIV-1 transmission, suggesting that HIV-1 exploits mDCs to facilitate its dissemination within lymphoid tissues.
Fusion Promotion by a Paramyxovirus Hemagglutinin-Neuraminidase Protein: pH Modulation of Receptor Avidity of Binding Sites I and II
Authors: Palermo Laura M. , Porotto Matteo , Greengard Olga , Moscona Anne
Published: June 2007

Paramyxoviruses, including the childhood respiratory pathogen human parainfluenza virus type 3 (HPIV3), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving (neuraminidase), as well as receptor-binding, activity. HN is a type II transmembrane glycoprotein, present on the surface of the virus as a tetramer composed of two dimers. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane. This initial step of viral entry occurs at the host cell surface at neutral pH. The HN molecule carries out these three different critical activities at specific points in the process of viral entry, and understanding the regulation of these activities is key for the design of strategies that block infection. One bifunctional site (site I) on the HN of HPIV3 possesses both receptor binding and neuraminidase activities, and we recently obtained experimental evidence for a second receptor binding site (site II) on HPIV3 HN. Mutation of HN at specific residues at this site, which is next to the HN dimer interface, confers enhanced fusion properties, without affecting neuraminidase activity or receptor binding at neutral pH. We now demonstrate that mutations at this site II, as well as at site I, confer pH dependence on HN′s receptor avidity. These mutations permit pH to modulate the binding and fusion processes of the virus, potentially providing regulation at specific stages of the viral life cycle.
Ebola Virus VP30 Is an RNA Binding Protein
Authors: John Sinu P. , Wang Tan , Steffen Scott , Longhi Sonia , Schmaljohn Connie S. , Jonsson Colleen B.
Published: June 2007

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in
trans
with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn
2+
, but not Mg
2+
, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.
An Attenuated Strain of Enterovirus 71 Belonging to Genotype A Showed a Broad Spectrum of Antigenicity with Attenuated Neurovirulence in Cynomolgus Monkeys
Authors: Arita Minetaro , Nagata Noriyo , Iwata Naoko , Ami Yasushi , Suzaki Yuriko , Mizuta Katsumi , Iwasaki Takuya , Sata Tetsutaro , Wakita Takaji , Shimizu Hiroyuki
Published: June 2007

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3′)], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3′), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3′). Monkeys inoculated with EV71(S1-3′) showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3′), two monkeys were intravenously inoculated with EV71(S1-3′), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3′) was isolated only from the spinal cord. These results indicate that EV71(S1-3′) acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.