bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling Stream Channel Characteristics From Drainage‐Enforced DEMs in Puget Sound, Washington, USA1
Authors: Davies Jeremy R. , Lagueux Kerry M. , Sanderson Beth , Beechie Timothy J.
Published: March 2007

Abstract:  Mapping stream channels and their geomorphic attributes is an important step in many watershed research and management projects. Often insufficient field data exist to map hydromorphologic attributes across entire drainage basins, necessitating the application of hydrologic modeling tools to digital elevation models (DEMs) via a geographic information system (GIS). In this article, we demonstrate methods for deriving synthetic stream networks via GIS across large and diverse basins using drainage‐enforced DEMs, along with techniques for estimating channel widths and gradient on the reach scale. The two‐step drainage enforcement method we used produced synthetic stream networks that displayed a high degree of positional accuracy relative to the input streams. The accuracies of our estimated channel parameters were assessed with field data, and predictions of bankfull width, wetted width and gradient were strongly correlated with measured values (r2 = 0.92, r2 = 0.95, r2 = 0.88, respectively). Classification accuracies of binned channel attributes were also high. Our methodology allows for the relatively rapid mapping of stream channels and associated morphological attributes across large geographic areas. Although initially developed to provide salmon recovery planners with important salmon habitat information, we suggest these methodologies are relevant to a variety of research and management questions.
Poly(ethylene terephthalate) (PET) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(ethylene terephthalate) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed.
Nylon 6 (N6) Reference XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of nylon 6 recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon, oxygen, and nitrogen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some forty different polymers.
Poly(1,2 butadiene) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(1,2 butadiene) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss region of carbon peak are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some forty different polymers.
Poly(acrylic acid) (PAA) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(acrylic acid) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some forty different polymers.
Poly(caprolactone) (PCL) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(caprolactone) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some forty different polymers.
Poly(dimethyl siloxane) (PDMS) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(dimethyl siloxane) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon, oxygen, and silicon peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(ethyl methacrylate) (PEMA) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(ethyl methacrylate) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(ethylene) (PE) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(ethylene) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(ethylene oxide) (PEO) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(ethylene oxide) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(isobutyl methacrylate) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(isobutyl methacrylate) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(n-vinylpyrolidone) (PNVP) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(n-vinylpyrolidone) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(propylene) (PP) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(propylene) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(pyrrole) (PPY) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(pyrrole) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon, nitrogen and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(styrene) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(styrene) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some forty different polymers.
Polyimide XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of polyimide recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon, nitrogen, and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(butyl methacrylate) (PBMA) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(butyl methacrylate) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Poly(ethylene succinate) (PESU) XPS Reference Core Level and Energy Loss Spectra
Authors: Louette Pierre , Bodino Frederic , Pireaux Jean-Jacques
Published: March 2007

XPS measurements of poly(ethylene succinate) recorded with a SSX-100 spectrometer in standardized experimental conditions are presented: survey scan, high resolution core level spectra as well as the energy loss regions of carbon and oxygen peaks are analyzed. This is part of a contract work aiming to record spectra in the very same conditions of some 40 different polymers.
Founder Effects in the Assessment of HIV Polymorphisms and HLA Allele Associations
Authors: Bhattacharya Tanmoy , Daniels Marcus , Heckerman David , Foley Brian , Frahm Nicole , Kadie Carl , Carlson Jonathan , Yusim Karina , McMahon Ben , Gaschen Brian , Mallal Simon , Mullins James I. , Nickle David C. , Herbeck Joshua , Rousseau Christine , Learn Gerald H. , Miura Toshiyuki , Brander Christian , Walker Bruce , Korber Bette
Published: March 2007

Escape from T cell–mediated immune responses affects the ongoing evolution of rapidly evolving viruses such as HIV. By applying statistical approaches that account for phylogenetic relationships among viral sequences, we show that viral lineage effects rather than immune escape often explain apparent human leukocyte antigen (HLA)–mediated immune-escape mutations defined by older analysis methods. Phylogenetically informed methods identified immune-susceptible locations with greatly improved accuracy, and the associations we identified with these methods were experimentally validated. This approach has practical implications for understanding the impact of host immunity on pathogen evolution and for defining relevant variants for inclusion in vaccine antigens.
A Cytochrome c Oxidase Model Catalyzes Oxygen to Water Reduction Under Rate-Limiting Electron Flux
Authors: Collman James P. , Devaraj Neal K. , Decréau Richard A. , Yang Ying , Yan Yi-Long , Ebina Wataru , Eberspacher Todd A. , Chidsey Christopher E. D.
Published: March 2007

We studied the selectivity of a functional model of cytochrome c oxidase's active site that mimics the coordination environment and relative locations of Fe
a3
, Cu
B
, and Tyr
244
. To control electron flux, we covalently attached this model and analogs lacking copper and phenol onto self-assembled monolayer–coated gold electrodes. When the electron transfer rate was made rate limiting, both copper and phenol were required to enhance selective reduction of oxygen to water. This finding supports the hypothesis that, during steady-state turnover, the primary role of these redox centers is to rapidly provide all the electrons needed to reduce oxygen by four electrons, thus preventing the release of toxic partially reduced oxygen species.
Comment on "Global Genetic Change Tracks Global Climate Warming in
Drosophila subobscura
"
Authors: Rodríguez-Trelles Francisco , Rodríguez Miguel Ángel
Published: March 2007

Balanyà
et al
. (Reports, 22 September 2006, p. 1773) build on earlier claims that chromosomal inversion polymorphisms of
Drosophila subobscura
are rapidly evolving in response to global warming. However, that conclusion is not adequately buttressed by their data, because they overlooked the lag between calendar and climatological dates created by the progressive lengthening of the growing season in their sampling approach.
Long-Term Satellite Record Reveals Likely Recent Aerosol Trend
Authors: Mishchenko Michael I. , Geogdzhayev Igor V. , Rossow William B. , Cairns Brian , Carlson Barbara E. , Lacis Andrew A. , Liu Li , Travis Larry D.
Published: March 2007

Analysis of the long-term Global Aerosol Climatology Project data set reveals a likely decrease of the global optical thickness of tropospheric aerosols by as much as 0.03 during the period from 1991 to 2005. This recent trend mirrors the concurrent global increase in solar radiation fluxes at Earth's surface and may have contributed to recent changes in surface climate.
Resonant Amplification of Magnetic Domain-Wall Motion by a Train of Current Pulses
Authors: Thomas Luc , Hayashi Masamitsu , Jiang Xin , Moriya Rai , Rettner Charles , Parkin Stuart
Published: March 2007

The current-induced motion of magnetic domain walls confined to nanostructures is of interest for applications in magnetoelectronic devices in which the domain wall serves as the logic gate or memory element. The injection of spin-polarized current below a threshold value through a domain wall confined to a pinning potential results in its precessional motion within the potential well. We show that by using a short train of current pulses, whose length and spacing are tuned to this precession frequency, the domain wall's oscillations can be resonantly amplified. This makes possible the motion of domain walls with much reduced currents, more than five times smaller than in the absence of resonant amplification.
Arctic Air Pollution: Origins and Impacts
Authors: Law Kathy S. , Stohl Andreas
Published: March 2007

Notable warming trends have been observed in the Arctic. Although increased human-induced emissions of long-lived greenhouse gases are certainly the main driving factor, air pollutants, such as aerosols and ozone, are also important. Air pollutants are transported to the Arctic, primarily from Eurasia, leading to high concentrations in winter and spring (Arctic haze). Local ship emissions and summertime boreal forest fires may also be important pollution sources. Aerosols and ozone could be perturbing the radiative budget of the Arctic through processes specific to the region: Absorption of solar radiation by aerosols is enhanced by highly reflective snow and ice surfaces; deposition of light-absorbing aerosols on snow or ice can decrease surface albedo; and tropospheric ozone forcing may also be contributing to warming in this region. Future increases in pollutant emissions locally or in mid-latitudes could further accelerate global warming in the Arctic.
Attention-Like Processes in
Drosophila
Require Short-Term Memory Genes
Authors: van Swinderen Bruno
Published: March 2007

Although there is much behavioral evidence for complex brain functions in insects, it is not known whether insects have selective attention. In humans, selective attention is a dynamic process restricting perception to a succession of salient stimuli, while less relevant competing stimuli are suppressed. Local field potential recordings in the brains of flies responding to visual novelty revealed attention-like processes with stereotypical temporal properties. These processes were modulated by genes involved in short-term memory formation, namely
dunce
and
rutabaga
. Attention defects in these mutants were associated with distinct optomotor effects in behavioral assays.
Response to Comment on "Global Genetic Change Tracks Global Climate Warming in
Drosophila subobscura
"
Authors: Balanyà Joan , Oller Josep M. , Huey Raymond B. , Gilchrist George W. , Serra Luis
Published: March 2007

Rodríguez-Trelles and Rodríguez advocate standardizing old and new collections by climate rather than by calendar and also propose that some of our samples were biased by inappropriate timing. Their first suggestion applies to few species, and its implementation alters photoperiodic cues. Their second point is valid, but our conclusions are robust: Observed genetic changes reflect global warming, not sampling artifacts.
Critical Behavior of a Trapped Interacting Bose Gas
Authors: Donner T. , Ritter S. , Bourdel T. , Öttl A. , Köhl M. , Esslinger T.
Published: March 2007

The phase transition of Bose-Einstein condensation was studied in the critical regime, where fluctuations extend far beyond the length scale of thermal de Broglie waves. We used matter-wave interference to measure the correlation length of these critical fluctuations as a function of temperature. Observations of the diverging behavior of the correlation length above the critical temperature enabled us to determine the critical exponent of the correlation length for a trapped, weakly interacting Bose gas to be ν = 0.67 ± 0.13. This measurement has direct implications for the understanding of second-order phase transitions.
Perspectives on the Arctic's Shrinking Sea-Ice Cover
Authors: Serreze Mark C. , Holland Marika M. , Stroeve Julienne
Published: March 2007

Linear trends in arctic sea-ice extent over the period 1979 to 2006 are negative in every month. This ice loss is best viewed as a combination of strong natural variability in the coupled ice-ocean-atmosphere system and a growing radiative forcing associated with rising concentrations of atmospheric greenhouse gases, the latter supported by evidence of qualitative consistency between observed trends and those simulated by climate models over the same period. Although the large scatter between individual model simulations leads to much uncertainty as to when a seasonally ice-free Arctic Ocean might be realized, this transition to a new arctic state may be rapid once the ice thins to a more vulnerable state. Loss of the ice cover is expected to affect the Arctic's freshwater system and surface energy budget and could be manifested in middle latitudes as altered patterns of atmospheric circulation and precipitation.
Detection of Anti-Leishmania(Leishmania)chagasiImmunoglobulin G by Flow Cytometry for Cure Assessment following Chemotherapeutic Treatment of American Visceral Leishmaniasis
Authors: Lemos Elenice Moreira , Gomes Izabelle Teixeira , Carvalho Sílvio Fernando Guimarães , Rocha Roberta Dias Rodrigues , Pissinate Jauber Fornaciari , Martins-Filho Olindo Assis , Dietze Reynaldo
Published: March 2007

The residual serological reactivity observed in patients cured of visceral leishmaniasis (VL) represents the major factor underlying the low efficiency of most anti-Leishmaniaserological approaches to assess posttherapeutic cure in VL. Herein, we have described a detuned flow cytometry-based methodology to detect anti-live (FC-ALPA-immunoglobulin G [IgG]) and anti-fixed (FC-AFPA-IgG)L. chagasipromastigote IgG, along the titration curve (1:2,000 to 1:128,000), as a tool to assess late (12 months after treatment [12 mAT]) and early (2 and 6 mAT) posttherapeutic cure of pediatric American visceral leishmaniasis. Reactivities were reported as the percentage of positive fluorescent parasite (PPFP), using a PPFP of 50% as a cutoff to segregate positive and negative results. Our data demonstrated that both FC-ALPA-IgG at 1:4,000 and FC-ALPA-IgG at 1:32,000 are useful for late cure assessment in VL, with 100% specificity and outstanding likelihood ratio indices. Cure assessment at 6 mAT also showed promising performance indices, identifying 81% and 71.4% of the treated patients with negative results. However, new interpretation parameters were necessary to monitor cure at 2 mAT. We then introduced the differential PPFP (ΔPPFP) of 25% as a new cutoff for early cure assessment at specific serum dilutions to analyze IgG reactivity by FC-ALPA-IgG and FC-AFPA-IgG. Our data demonstrated that at 2 mAT, ΔPPFP was >25% in 60% and 57.1% of treated patients, whereas at 6 mAT, a ΔPPFP of >25% was observed in 100% and 95.2% of samples assayed by FC-ALPA-IgG and FC-AFPA-IgG, respectively. Together, our findings showed the potential of both FC-ALPA-IgG and FC-AFPA-IgG regarding their applicability to detect differential serological reactivity and further contribution to posttherapeutic cure assessment in VL.
Unusual Clinical Presentation of
Mycobacterium fortuitum
Infection in an Immunocompetent Woman
Authors: Serra Corrado , Loi Giovanni , Saddi Barbara , Pautasso Marisa , Manzin Aldo
Published: March 2007

The
Mycobacterium fortuitum
group of rapidly growing nontuberculous mycobacteria is an uncommon cause of renal infection, particularly in otherwise healthy hosts. We describe a case of nephritis due to
M. fortuitum
in an immunocompetent woman with a clinical and radiological diagnosis of renal tuberculosis.
Exceptionally High Representation of
Burkholderia cepacia
among
B. cepacia
Complex Isolates Recovered from the Major Portuguese Cystic Fibrosis Center
Authors: Cunha Mónica V. , Pinto-de-Oliveira Ana , Meirinhos-Soares Luís , Salgado Maria José , Melo-Cristino José , Correia Susana , Barreto Celeste , Sá-Correia Isabel
Published: March 2007

Burkholderia cepacia
, a species found infrequently in cystic fibrosis (CF), was isolated from 85% of patients infected with bacteria of the
B. cepacia
complex that visited the major Portuguese CF center, in Lisbon, during 2003 to 2005. A detailed molecular analysis revealed that this was mainly due to two
B. cepacia
clones. These clones were indistinguishable from two strains isolated from intrinsically contaminated nonsterile saline solutions for nasal application, detected during routine market surveillance by the Portuguese Medicines and Health Products Authority.
Risk Factors for the Presence of High-Level Shedders ofEscherichia coliO157 on Scottish Farms
Authors: Chase-Topping Margo E. , McKendrick Iain J. , Pearce Michael C. , MacDonald Peter , Matthews Louise , Halliday Jo , Allison Lesley , Fenlon Dave , Low J. Christopher , Gunn George , Woolhouse Mark E. J.
Published: March 2007

Escherichia coliO157 infections are the cause of sporadic or epidemic cases of often bloody diarrhea that can progress to hemolytic uremic syndrome (HUS), a systematic microvascular syndrome with predominately renal and neurological complications. HUS is responsible for most deaths associated withE. coliO157 infection. From March 2002 to February 2004, approximately 13,000 fecal pat samples from 481 farms with finishing/store cattle throughout Scotland were examined for the presence ofE. coliO157. A total of 441 fecal pats from 91 farms tested positive forE. coliO157. From the positive samples, a point estimate for high-level shedders was identified using mixture distribution analysis on counts ofE. coliO157. Models were developed based on the confidence interval surrounding this point estimate (high-level shedder, greater than 103or greater than 104CFU g−1feces). The mean prevalence on high-level-shedding farms was higher than that on low-level-shedding farms. The presence of a high-level shedder on a farm was found to be associated with a high proportion of low-level shedding, consistent with the possibility of a higher level of transmission. Analysis of risk factors associated with the presence of a high-level shedder on a farm suggested the importance of the pathogen and individual host rather than the farm environment. The proportion of high-level shedders of phage 21/28 was higher than expected by chance. Management-related risk factors that were identified included the type of cattle (female breeding cattle) and cattle stress (movement and weaning), as opposed to environmental factors, such as water supply and feed.
Comparison of Three Methods for Rapid Identification of Mycobacterial Clinical Isolates to the Species Level
Authors: Wu Xueqiong , Zhang Junxian , Liang Jianqin , Lu Yang , Li Hongmin , Li Chuihuan , Yue Jun , Zhang Lishui , Liu Zhihui
Published: March 2007

A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of
Mycobacterium
species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the
Mycobacterium
genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding
Mycobacterium
species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One
M. asia
isolate and one
M. neoaurum
isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from
M. intracellulare
reference strains had a negative hybridization signal with probe c, which is specific for
M. intracellulare.
The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.
Gamma Interferon Blocks Gammaherpesvirus Reactivation from Latency in a Cell Type-Specific Manner
Authors: Steed Ashley , Buch Thorsten , Waisman Ari , Virgin Herbert W.
Published: March 2007

Gammaherpesviruses are important pathogens whose lifelong survival in the host depends critically on their capacity to establish and reactivate from latency, processes regulated by both viral genes and the host immune response. Previous work has demonstrated that gamma interferon (IFN-γ) is a key regulator of chronic infection with murine gammaherpesvirus 68 (γHV68), a virus that establishes latent infection in B lymphocytes, macrophages, and dendritic cells. In mice deficient in IFN-γ or the IFN-γ receptor, γHV68 gene expression is altered during chronic infection, and peritoneal cells explanted from these mice reactivate more efficiently ex vivo than cells derived from wild-type mice. Furthermore, treatment with IFN-γ inhibits reactivation of γHV68 from latently infected wild-type peritoneal cells, and depletion of IFN-γ from wild-type mice increases the efficiency of reactivation of explanted peritoneal cells. These profound effects of IFN-γ on chronic γHV68 latency and reactivation raise the question of which cells respond to IFN-γ to control chronic γHV68 infection. Here, we show that IFN-γ inhibited reactivation of peritoneal cells and spleen cells harvested from mice lacking B lymphocytes, but not wild-type spleen cells, suggesting that IFN-γ may inhibit reactivation in a cell type-specific manner. To directly test this hypothesis, we expressed the diphtheria toxin receptor specifically on either B lymphocytes or macrophages and used diphtheria toxin treatment to deplete these specific cells in vivo and in vitro after establishing latency. We demonstrate that macrophages, but not B cells, are responsive to IFN-γ-mediated suppression of γHV68 reactivation. These data indicate that the regulation of gammaherpesvirus latency by IFN-γ is cell type specific and raise the possibility that cell type-specific immune deficiency may alter latency in distinct and important ways.
Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1
Authors: Jolly Clare , Mitar Ivonne , Sattentau Quentin J.
Published: March 2007

Human immunodeficiency virus type 1 (HIV-1) infection of CD4
+
T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.
Small Interfering RNAs against the TAR RNA Binding Protein, TRBP, a Dicer Cofactor, Inhibit Human Immunodeficiency Virus Type 1 Long Terminal Repeat Expression and Viral Production
Authors: Christensen Helen S. , Daher Aïcha , Soye Kaitlin J. , Frankel Lisa B. , Alexander Marina R. , Lainé Sébastien , Bannwarth Sylvie , Ong Chi L. , Chung Sean W. L. , Campbell Shahan M. , Purcell Damian F. J. , Gatignol Anne
Published: March 2007

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.
Tyrosine 3 of Poliovirus Terminal Peptide VPg(3B) Has an Essential Function in RNA Replication in the Context of Its Precursor Protein, 3AB
Authors: Liu Ying , Franco David , Paul Aniko V. , Wimmer Eckard
Published: March 2007

Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3D
pol
, but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3D
pol
. Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in
trans
or in
cis
could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in
cis
by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in
trans
, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.
Epstein-Barr Virus-Encoded Protein Kinase (BGLF4) Is Involved in Production of Infectious Virus
Authors: Gershburg Edward , Raffa Salvatore , Torrisi Maria Rosaria , Pagano Joseph S.
Published: March 2007

The Epstein-Barr virus (EBV) BGLF4 gene product is a protein kinase (PK). Although this kinase has been characterized and several of its targets have been identified, its biological role remains enigmatic. We have generated and assessed a BGLF4 knockdown phenotype by means of RNA interference and report the following: (i) BGLF4-targeting small interfering RNA effectively inhibited the expression of its product, the viral PK, during lytic reactivation, (ii) BGLF4 knockdown partially inhibited viral DNA replication and expression of selected late viral genes, (iii) the absence of EBV PK resulted in retention of the viral nucleocapsids in the nuclei, and (iv) as a result of the nuclear retention, release of infectious virions is significantly retarded. Our results provide evidence that EBV PK plays an important role in nuclear egress of the virus and ultimately is crucial for lytic virus replication.
Functional Analysis of
cis
- and
trans
-Acting Replication Factors of Porcine Circovirus Type 1
Authors: Steinfeldt Tobias , Finsterbusch Tim , Mankertz Annette
Published: March 2007

The replication proteins Rep and Rep′ of porcine circovirus type 1 (PCV1) are both capable of introducing and resealing strand discontinuities at the viral origin of DNA replication in vitro underlying genome amplification by rolling-circle replication. The PCV1 origin of replication encompasses the minimal binding site (MBS) of the Rep and Rep′ proteins and an inverted repeat with the potential to form a stem-loop. In this study, both elements of the PCV1 origin were demonstrated to be essential for viral replication in transfected cells. Furthermore, investigation of conserved amino acid motifs within Rep and Rep′ proteins revealed that the mutation of motifs I, II, and III and of the GKS box interfered with viral replication. In vitro studies demonstrated that motifs I to III were essential for origin cleavage, while the GKS box was dispensable for the initiation of viral replication. A covalent link between Rep/Rep′ and the DNA after origin cleavage was demonstrated, providing a mechanism for energy conservation for the termination of replication.
Both Virus and Tumor Necrosis Factor Alpha Are Critical for Endothelium Damage in a Mouse Model of Dengue Virus-Induced Hemorrhage
Authors: Chen Hsuen-Chin , Hofman Florence M. , Kung John T. , Lin Yang-Ding , Wu-Hsieh Betty A.
Published: March 2007

Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 × 10
9
PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 × 10
7
to 8 × 10
7
PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 × 10
7
to 8 × 10
7
PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-α) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-α in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-α
−/−
to IgH
−/−
, C5
−/−
, and wild-type mice, we found that TNF-α was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-α enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-α-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.
The E1^E4 Protein of Human Papillomavirus Interacts with the Serine-Arginine-Specific Protein Kinase SRPK1
Authors: Bell Ian , Martin Ashley , Roberts Sally
Published: March 2007

Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1^E4 protein during the productive phase of infection. However, the precise mechanisms of how E1^E4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1^E4. We map critical residues within an arginine-rich domain of HPV1 E1^E4, and in a region known to facilitate E1^E4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1^E4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1^E4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1^E4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1^E4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1^E4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.
New Adenovirus Species Found in a Patient Presenting with Gastroenteritis
Authors: Jones Morris Saffold , Harrach Balázs , Ganac Robert D. , Gozum Mary M. A. , dela Cruz Wilfred P. , Riedel Brian , Pan Chao , Delwart Eric L. , Schnurr David P.
Published: March 2007

An unidentified agent was cultured in primary monkey cells at the Los Angeles County Public Health Department from each of five stool specimens submitted from an outbreak of gastroenteritis. Electron microscopy and an adenovirus-specific monoclonal antibody confirmed this agent to be an adenovirus. Since viral titers were too low, complete serotyping was not possible. Using the DNase-sequence-independent viral nucleic acid amplification method, we identified several nucleotide sequences with a high homology to human adenovirus 41 (HAdV-41) and simian adenovirus 1 (SAdV-1). However, using anti-SAdV-1 sera, it was determined that this virus was serologically different than SAdV-1. Genomic sequencing and phylogenetic analysis confirmed that this new adenovirus was so divergent from the known human adenoviruses that it was not only a new type but also represented a new species (human adenovirus G). In a retrospective clinical study, this new virus was detected by PCR in one additional patient from a separate gastroenteritis outbreak. This study suggests that HAdV-52 may be one of many agents causing gastroenteritis of unknown etiology.
Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication
Authors: Nash Kevin , Chen Weijun , McDonald William F. , Zhou Xiaohuai , Muzyczka Nicholas
Published: March 2007

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase δ, but not polymerase ε or α, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase δ complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase δ, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase δ, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.
Unique Thermodynamic Response of Tipranavir to Human Immunodeficiency Virus Type 1 Protease Drug Resistance Mutations
Authors: Muzammil S. , Armstrong A. A. , Kang L. W. , Jakalian A. , Bonneau P. R. , Schmelmer V. , Amzel L. M. , Freire E.
Published: March 2007

Drug resistance is a major problem affecting the clinical efficacy of antiretroviral agents, including protease inhibitors, in the treatment of infection with human immunodeficiency virus type 1 (HIV-1)/AIDS. Consequently, the elucidation of the mechanisms by which HIV-1 protease inhibitors maintain antiviral activity in the presence of mutations is critical to the development of superior inhibitors. Tipranavir, a nonpeptidic HIV-1 protease inhibitor, has been recently approved for the treatment of HIV infection. Tipranavir inhibits wild-type protease with high potency (

K
i

= 19 pM) and demonstrates durable efficacy in the treatment of patients infected with HIV-1 strains containing multiple common mutations associated with resistance. The high potency of tipranavir results from a very large favorable entropy change (−
T
Δ
S
= −14.6 kcal/mol) combined with a favorable, albeit small, enthalpy change (Δ
H
= −0.7 kcal/mol, 25°C). Characterization of tipranavir binding to wild-type protease, active site mutants I50V and V82F/I84V, the multidrug-resistant mutant L10I/L33I/M46I/I54V/L63I/V82A/I84V/L90M, and the tipranavir in vitro-selected mutant I13V/V32L/L33F/K45I/V82L/I84V was performed by isothermal titration calorimetry and crystallography. Thermodynamically, the good response of tipranavir arises from a unique behavior: it compensates for entropic losses by actual enthalpic gains or by sustaining minimal enthalpic losses when facing the mutants. The net result is a small loss in binding affinity. Structurally, tipranavir establishes a very strong hydrogen bond network with invariant regions of the protease, which is maintained with the mutants, including catalytic Asp25 and the backbone of Asp29, Asp30, Gly48 and Ile50. Moreover, tipranavir forms hydrogen bonds directly to Ile50, while all other inhibitors do so by being mediated by a water molecule.
Enhanced Replication of Human T-Cell Leukemia Virus Type 1 in T Cells from Transgenic Rats Expressing Human CRM1 That Is Regulated in a Natural Manner
Authors: Takayanagi Ryo , Ohashi Takashi , Yamashita Eizaburo , Kurosaki Yohei , Tanaka Kumiko , Hakata Yoshiyuki , Komoda Yasumasa , Ikeda Satoru , Tsunetsugu-Yokota Yasuko , Tanaka Yuetsu , Shida Hisatoshi
Published: March 2007

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). To develop a better animal model for the investigation of HTLV-1 infection, we established a transgenic (Tg) rat carrying the human CRM1 (hCRM1) gene, which encodes a viral RNA transporter that is a species-specific restriction factor. At first we found that CRM1 expression is elaborately regulated through a pathway involving protein kinase C during lymphocyte activation, initially by posttranscriptional and subsequently by transcriptional mechanisms. This fact led us to use an hCRM1-containing bacterial artificial chromosome clone, which would harbor the entire regulatory and coding regions of the CRM1 gene. The Tg rats expressed hCRM1 protein in a manner similar to expression of intrinsic rat CRM1 in various organs. HTLV-1-infected T-cell lines derived from these Tg rats produced 100- to 10,000-fold more HTLV-1 than did T cells from wild-type rats, and the absolute levels of HTLV-1 were similar to those produced by human T cells. We also observed enhancement of the dissemination of HTLV-1 to the thymus in the Tg rats after intraperitoneal inoculation, although the proviral loads were low in both wild-type and Tg rats. These results support the essential role of hCRM1 in proper HTLV-1 replication and suggest the importance of this Tg rat as an animal model for HTLV-1.