bluuebunny/crossref_metadata_2025 huggingface dataset:
A study of pre- and inservice physics teachers’ understanding of photoelectric phenomenon as part of the development of a research-based quantum physics course
Authors: Asikainen Mervi A. , Hirvonen Pekka E.
Published: June 2009

We describe the development of a research-based quantum physics course for physics teachers. A case study approach is used to study the effect of the course on preservice and inservice teachers’ understanding of the photoelectric effect. Results offer new insights into the learning of the photoelectric effect by providing a detailed description of the participant understanding. The learning outcomes achieved indicate that the instructional approach and the teaching–learning procedure used in the course can help preservice and inservice teachers attain an in-depth understanding of key quantum physics concepts.
Exploring gender differences with different gain calculations in astronomy and biology
Authors: Willoughby Shannon D. , Metz Anneke
Published: June 2009

To investigate differences in learning gains by gender, we collected data in large introductory astronomy and biology courses. Male astronomy students had significantly higher pre- and post-test scores than female students on the astronomy diagnostic test. Male students also had significantly higher pretest and somewhat higher post-test scores than female students on a survey instrument designed for an introductory biology course. For both courses, males had higher learning gains than female students only when the normalized gain measure was utilized. No differences were found with any other measures, including other gain calculations, overall course grades, or individual exams. Implications for using different learning gain measures in science classrooms, as well as for research on learning differences by gender are discussed.
Comment on “Tail Reconnection Triggering Substorm Onset”
Authors: Lui A. T. Y.
Published: June 2009

Angelopoulos
et al
. (Research Articles, 15 August 2008, p. 931) reported that magnetic reconnection in Earth’s magnetotail triggered the onset of a magnetospheric substorm. We provide evidence that (i) near-Earth current disruption, occurring before the conventional tail reconnection signatures, triggered the onset; (ii) the observed auroral intensification and tail reconnection are not causally linked; and (iii) the onset they identified is a continuation of earlier substorm activities.
Silicon Carbide as a Platform for Power Electronics
Authors: Eddy C. R. , Gaskill D. K.
Published: June 2009

Methods for growing large, defect-free silicon carbide crystals have enabled the fabrication of devices that can operate at high power.
Oxygen-18 of O
2
Records the Impact of Abrupt Climate Change on the Terrestrial Biosphere
Authors: Severinghaus Jeffrey P. , Beaudette Ross , Headly Melissa A. , Taylor Kendrick , Brook Edward J.
Published: June 2009

Planted Evidence

To understand the spatial patterns and consequences of past climate change requires the identification of reliable proxies that reflect specific aspects of those changes, such as temperature or rainfall in a given location. Also of interest are proxies for broader categories of change, such as methane production or the sources of carbon dioxide.

Severinghaus
et al.

(p.
1431
) present a 100,000-year-long record of the oxygen isotopic composition of atmospheric O
2
(δ
18
O
atm
) extracted from air from polar ice cores. δ
18
O
atm
is a general measure of the strength of low-latitude terrestrial photosynthesis and thus of local rainfall because plant metabolism is controlled in large part by water availability. δ
18
O
atm
changes were related to Heinrich and Dansgaard-Oeschger events, two modes of abrupt climate change common over that interval, and δ
18
O
atm
was controlled mostly by the strength of the Asian and North African monsoons. The rapid changes observed should also help to synchronize ice core records.
Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab
Authors: Aoki Scott T. , Settembre Ethan C. , Trask Shane D. , Greenberg Harry B. , Harrison Stephen C. , Dormitzer Philip R.
Published: June 2009

Rotavirus Rumbled

Rotavirus infection is the primary cause of severe diarrhea in infants. For the virus to enter cells, a Ca
2+
-stabilized trimer of the outer layer protein VP7 must be dissociated.

Aoki
et al.

(p.
1444
) report the structure of the VP7 trimer in complex with the Fab fragment of a neutralizing monoclonal antibody. Based on the structure and an analysis of positions of neutralization escape mutations, the authors propose that many neutralizing antibodies inhibit cell entry by stabilizing the VP7 trimer even at low calcium concentrations. A disulfide-linked trimer was then produced that is a potential subunit immunogen.
Polarization Control of Electron Tunneling into Ferroelectric Surfaces
Authors: Maksymovych Peter , Jesse Stephen , Yu Pu , Ramesh Ramamoorthy , Baddorf Arthur P. , Kalinin Sergei V.
Published: June 2009

Ferroelectric Patterning with High Fields

Ferroelectric oxides have a net polarization that can switch direction upon application of a sufficiently high electric field. In principle, a ferroelectric thin film should be able to act as a polar switch—tunneling an electron through the film would effectively switch on or off depending on the direction of the polarization. In practice, the length scale needed for a sufficiently small tunneling barrier is nearly the same as the scale at which films no longer support ferroelectricity.

Maksymovych
et al.

(p.
1421
) now show that the tip of an atomic force microscope can be used to pattern polarization domains in a thin film of lead zirconate titanate in high electric fields similar to those for field emission tips.
Comprehensive Control of Atomic Motion
Authors: Raizen Mark G.
Published: June 2009

Cooling All Atoms

Laser cooling can be used to chill atoms to ultralow temperatures. However, the characteristics of the atoms and of the lasers limit the technique to a select few elements of the periodic table. Techniques are thus being explored that can be applied more generally.
Raizen
(p.
1403
) reviews recent work on the magnetic coilgun cooling technique—where a series of magnetic pulses are applied to a cloud of atoms or molecules and can cool them to millikelvin temperatures. The only requirement of the atoms is paramagnetism—a property of most of the elements in the periodic table.
Hyper-Recombination, Diversity, and Antibiotic Resistance in Pneumococcus
Authors: Hanage William Paul , Fraser Christophe , Tang Jing , Connor Thomas Richard , Corander Jukka
Published: June 2009

Recombining Resistance

Homologous recombination is frequent in many bacteria, but few studies have addressed whether subpopulations within a species are more or less likely to undergo this process and whether it has consequences for their evolution. Taking a large data set from the pathogen
Streptococcus pneumoniae
,

Hanage
et al.

(p.
1454
) discovered a group of strains characterized by an anomalous sequence of housekeeping genes. This sequence appeared to have been horizontally acquired from other pneumococci and related species and was associated with resistance to all classes of antibiotics for which data are available. Thus, hyper-recombination (in contrast to hypermutation) is important in the evolution and spread of antibiotic resistance and may play a role in determining the emergence of species clusters and the phenotypes associated with them.
Measuring the Charge State of an Adatom with Noncontact Atomic Force Microscopy
Authors: Gross Leo , Mohn Fabian , Liljeroth Peter , Repp Jascha , Giessibl Franz J. , Meyer Gerhard
Published: June 2009

Spotting Charges

Many nanoscale physical systems are sensitive to the position of isolated charges, such as single-electron transistors and molecular assemblies that separate charges with energy from photons. In order to probe the location of a charged atom, the most general methods would work on insulating surfaces.

Gross
et al.

(p.
1428
; see the Perspective by

Meyer and Glatzel
)

show that a tuning-fork atomic force microscope (AFM) operating in a noncontact mode at cryogenic temperatures can resolve the charge state of gold and silver atoms absorbed on a sodium chloride film. Charged atoms set up image charges in the AFM tip, which creates an electrostatic force not present with a neutral atom.
Herapathite
Authors: Kahr Bart , Freudenthal John , Phillips Shane , Kaminsky Werner
Published: June 2009

Discovered in 1852 and used for polarizing light, the crystal structure of iodoquinine sulfate has been solved.
Boom-and-Bust Development Patterns Across the Amazon Deforestation Frontier
Authors: Rodrigues Ana S. L. , Ewers Robert M. , Parry Luke , Souza Carlos , Veríssimo Adalberto , Balmford Andrew
Published: June 2009

Boom and Bust

The Brazilian Amazon is renowned for its biodiversity and for its influence on climate regulation and geochemical cycles. It is also one of the country's poorest regions. For decades, much economic development has been pursued through conversion of forest for agriculture and cattle-ranching.

Rodrigues
et al.

(p.
1435
) investigated whether this pattern of land use brings lasting prosperity by analyzing data on the economic development of nearly 300 municipalities across the deforestation frontier. Relative development, in terms of life expectancy, literacy, and standard of living, increases as deforestation begins but then declines again as the frontier passes through. As a result, pre- and postfrontier levels of development are similarly low, indicating a pattern of boom and bust.
Yield Stress Fluids Slowly Yield to Analysis
Authors: Bonn Daniel , Denn Morton M.
Published: June 2009

The behavior of a type of complex fluid (exemplified by mayonnaise and concrete) can depend on the sample's flow history.
Mr. Armstrong's Jersey and Mr. Rogers's Sweater
SuperSense
Why We Believe in the Unbelievable / From Superstition to Religion—the Brain Science of Belief

by Bruce M. Hood

HarperOne, San Francisco, 2009. 320 pp. $25.99. ISBN 9780061452642. Constable, London. 330 pp. Paper, £8.99. ISBN 9781849010306.
Authors: Shermer Michael
Published: June 2009

Discussing beliefs in the supernatural and irrational, Hood argues that they arise because human minds have been selected to "think there are unseen patterns, forces, and essences inhabiting the world"—the beliefs help people bind together.
Epidemics of Fear
Dread
How Fear and Fantasy Have Fueled Epidemics from the Black Death to Avian Flu

by Philip Alcabes

PublicAffairs, New York, 2009. 322 pp. $26.95, C$31. ISBN 9781586486181.
Authors: Foster Kenneth R.
Published: June 2009

Exploring biological, social, and narrative aspects of epidemics, Alcabes concludes that the common gap between the hype and the actual levels of damages reflects the crucial roles fear and imagination play in shaping our responses to diseases.
Novel Probes for Molecular Electronics
Authors: Meyer Ernst , Glatzel Thilo
Published: June 2009

An extension of noncontact atomic force microscopy allows detection and manipulation of the charge states of individual atoms.
Breaching the Cancer Fortress
Authors: Olson Peter , Hanahan Douglas
Published: June 2009

Pancreatic tumors are poorly vascularized, suggesting that new therapeutic strategies are needed.
Macau Launches Late Bid to Cure Its Pearl River Delta Blues
Authors: Stone Richard
Published: June 2009

Macau, a gambling haven known more for unbridled development than for ecological awareness, started down the road to sustainability this year with the creation of a wetland reserve and an Environmental Protection Bureau.
Science Windfall Stimulates High Hopes—and Political Maneuvering
Authors: Normile Dennis
Published: June 2009

A handful of stellar researchers could be in line for grants of as much as $90 million over 5 years, thanks to a new program included in a stimulus package approved by Japan's legislature on 29 May. But some scientists worry that a political selection process could steer money toward industry-favored projects.
U.S. Promises to Reduce Delays in Granting Visas for Scientists
Authors: Bhattacharjee Yudhijit
Published: June 2009

The U.S. government has streamlined procedures for processing visa applications from foreign students and researchers trying to enter the United States.
A Medical Mystery in Middle China
Authors: Stone Richard
Published: June 2009

China has launched a massive effort to stamp out Kashin-Beck disease, including moving populations from affected areas, but the cause of this crippling ailment remains elusive.
Russia's Polar Hero
Authors: Parfitt Tom
Published: June 2009

Artur Chilingarov has led researchers exploring the polar regions, the Arctic ocean floor, and the world's deepest lake. But is he promoting science or his homeland?
Diving Into the Sacred Sea
Authors: Parfitt Tom
Published: June 2009

Prompted by potential menaces to Lake Baikal, the largest, deepest, most ancient, and most biologically diverse freshwater lake on Earth, scientists from the Russian Academy of Sciences, led by oceanographer and explorer Artur Chilingarov, last year began a program of research using two deep-water
Mir
minisubmarines.
Following the Leaders
Authors: Koenig Robert
Published: June 2009

Several institutions have begun to imitate aspects of the Meyerhoff program at the University of Maryland, Baltimore County, and the Biology Scholars Program at the University of California, Berkeley (
see main text
). However, none has published comprehensive data on what has been accomplished.
Recombinant
Mycobacterium bovis
BCG Expressing the Chimeric Protein of Antigen 85B and ESAT-6 Enhances the Th1 Cell-Mediated Response
Authors: Xu Ying , Liu Wei , Shen Hongbo , Yan Jingran , Qu Di , Wang Honghai
Published: June 2009

The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant
Mycobacterium bovis
BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-A
N
-E-A
C
). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-γ) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4
+
and CD8
+
T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-A
N
-E-A
C
significantly increased the level of production of the major Th1 cytokines IFN-γ and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-A
N
-E-A
C
was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-A
N
-E-A
C
strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against
M. tuberculosis
.
Disappearance of Vaccine-Type Invasive Pneumococcal Disease and Emergence of Serotype 19A in a Minority Population with a High Prevalence of Human Immunodeficiency Virus and Low Childhood Immunization Rates
Authors: Tasslimi Azadeh , Sison Erica J. , Story Elizabeth , Alland David , Burday Michele , Morrison Susan , Nalmas Sandhya , Smith Stephen , Thomas Pauline A. , Wenger Peter , Sinha Anushua
Published: June 2009

We analyzed the epidemiology of invasive pneumococcal disease (IPD) following introduction of pneumococcal conjugated vaccine in an urban population with a 2% human immunodeficiency virus (HIV) prevalence and history of low childhood immunization rates. We observed near-elimination of vaccine-type IPD. Substantial disease remains due to non-vaccine-type pneumococci, highlighting the need to increase pneumococcal immunization among HIV-infected adults.
Evaluation of
Helicobacter pylori
Immunoglobulin G (IgG), IgA, and IgM Serologic Testing Compared to Stool Antigen Testing
Authors: She Rosemary C. , Wilson Andrew R. , Litwin Christine M.
Published: June 2009

The utility of
Helicobacter pylori
serology was evaluated in 4,722 specimens and compared to stool antigen detection. Immunoglobulin M (IgM) sensitivity (6.8%) was unacceptably low. Key performance differences were observed in IgG specificity, IgA sensitivity, and specificity between adults and children that may warrant differentiating optimal serologic cutoff values by age.
Natural Killer Cell Counts Are Not Different between Patients with Post-Lyme Disease Syndrome and Controls
Authors: Marques Adriana , Brown Margaret R. , Fleisher Thomas A.
Published: June 2009

It has been reported that patients with “chronic Lyme disease” have a decreased number of natural killer cells, as defined by the CD57 marker. We performed immunophenotyping in 9 individuals with post-Lyme disease syndrome, 12 who recovered from Lyme disease, and 9 healthy volunteers. The number of natural killer cells was not significantly different between the groups.
Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization
Authors: Weinberg Adriana , Song Lin-Ye , Wilkening Cynthia , Sevin Anne , Blais Bruce , Louzao Raul , Stein Dana , Defechereux Patricia , Durand Deborah , Riedel Eric , Raftery Nancy , Jesser Renee , Brown Betty , Keller M. Fran , Dickover Ruth , McFarland Elizabeth , Fenton Terence
Published: June 2009

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4
+
and CD8
+
T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.
T-Cell mRNA Expression in Response toMycobacterium bovisBCG Vaccination andMycobacterium bovisInfection of White-Tailed Deer
Authors: Thacker Tyler C. , Palmer Mitchell V. , Waters W. Ray
Published: June 2009

Understanding immune responses of white-tailed deer (WTD) to infection withMycobacterium bovisprovides insight into mechanisms of pathogen control and may provide clues to development of effective vaccine strategies. WTD were vaccinated with eitherM. bovisBCG strain Pasteur or BCG strain Danish. Both vaccinees and unvaccinated controls were subsequently inoculated with virulentM. bovisvia the intratonsillar route. Real-time PCR was used to assess T-cell mRNA expression in peripheral blood leukocytes (PBL) from animals following vaccination and infection. Recall T-cell responses were measured by assessing the relative expression of gamma interferon (IFN-γ), T-cell-specific T-box transcription factor (Tbet), interleukin 12p40 (IL-12p40), IL-12p35, IL-23p19, FoxP3, IL-17, and GATA3 in PBL stimulated in vitro with purified protein derivative (PPD) ofM. bovisor a recombinant fusion protein, ESAT6-CFP10. Animals vaccinated with BCG Danish expressed more IFN-γ and Tbet than either BCG Pasteur-vaccinated animals or unvaccinated controls. BCG Pasteur-vaccinated animals expressed more GATA3 than either group. After infection, unvaccinated controls expressed more Tbet and IL-12p40 than vaccinated animals. BCG Pasteur-vaccinated animals expressed more GATA3 than either the unvaccinated controls or the BCG Danish-vaccinated animals after infection. Animals were divided into pathology groups to correlate gene expression with severity of pathology. Animals in the visible lesion group expressed more Tbet and IFN-γ than animals that were culture negative, while Tbet and IFN-γ expression in the culture-positive, no-visible-lesion group was intermediate. GATA3 expression inversely correlated with pathology. Overall, expression of immune response genes correlated more closely with pathology than vaccination treatment.
Inaccuracy of Single-Target Sequencing for Discriminating Species of the
Mycobacterium abscessus
Group
Authors: Macheras Edouard , Roux Anne-Laure , Ripoll Fabienne , Sivadon-Tardy Valérie , Gutierrez Cristina , Gaillard Jean-Louis , Heym Beate
Published: June 2009

We determined nucleotide sequences of
rpoB
,
hsp65
, and
sodA
in 59 clinical isolates (from 58 patients) of the
M
ycobacterium abscessus
group. Identification to the species level, based on three target genes, was concordant for 44 isolates (25
M. abscessus
, 13
Mycobacterium massiliense
, and 6
Mycobacterium bolletii
isolates) and discordant for 15 isolates which had “interspecific composite patterns.” Sequence analysis of five housekeeping genes also showed composite patterns in 8 of these 15 isolates.
emmand C-Repeat Region Molecular Typing of Beta-Hemolytic Streptococci in a Tropical Country: Implications for Vaccine Development
Authors: Steer Andrew C. , Magor Graham , Jenney Adam W. J. , Kado Joseph , Good Michael F. , McMillan David , Batzloff Michael , Carapetis Jonathan R.
Published: June 2009

We designed a study to investigate the molecular epidemiology of group A streptococcal (GAS) and group C and G streptococcal (GCS and GGS) disease in Fiji, a country which is known to have a high burden of streptococcal disease. Molecular typing of the N-terminal portion (emmtyping) of the M protein was performed with 817 isolates (535 GAS and 282 GCS/GGS). We also performed genotyping of the C-repeat region in 769 of these isolates to identify J14 sequence types. The profile ofemmtypes for Fiji was very different from that found for the United States and Europe. There were no dominantemmtypes and a large number of overlapping types among clinical disease states. Commonly found GASemmtypes in industrialized countries, includingemm1,emm12, andemm28, were not found among GAS isolates from Fiji. Over 93% of GAS isolates and over 99% of GCS/GGS isolates that underwent J14 sequence typing contained either J14.0 or J14.1. Our data have implications for GAS vaccine development in developing countries and suggest that a vaccine based upon the conserved region of the M protein may be a feasible option for Fiji and potentially for other tropical developing countries.
Performance of MicroScan WalkAway and Vitek 2 for Detection of Oxacillin Resistance in a Set of Methicillin-Resistant
Staphylococcus aureus
Isolates with Diverse Genetic Backgrounds
Authors: Kaase Martin , Baars Barbara , Friedrich Susanne , Szabados Florian , Gatermann S. G.
Published: June 2009

Of 104 genotypically diverse methicillin-resistant
Staphylococcus aureus
(MRSA) isolates tested with the MicroScan WalkAway (Pos MIC 24 panel) and Vitek 2 (AST-P549 card) systems, 7 and 6 isolates, respectively, showed an oxacillin MIC of ≤2mg/liter. Most of these MRSA isolates were community acquired. However, if the cefoxitin screen of AST-P549 was also considered, MRSA detection failed for only one isolate.
Positive Selection Pressure Introduces Secondary Mutations at Gag Cleavage Sites in Human Immunodeficiency Virus Type 1 Harboring Major Protease Resistance Mutations
Authors: Banke Søren , Lillemark Marie R. , Gerstoft Jan , Obel Niels , Jørgensen Louise B.
Published: June 2009

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) specifically target the HIV-1 protease enzyme. Mutations in the enzyme can result in PI resistance (termed PI mutations); however, mutations in the HIV-1gagregion, the substrate for the protease enzyme, might also lead to PI resistance. We analyzedgagand pol sequence data from the following 313 HIV-1-infected patients: 160 treatment-naïve patients, 93 patients failing antiretroviral treatment that included a PI (with no major PI mutations), and 60 patients failing antiretroviral treatment that included a PI (with major PI mutations). Additional sequences from 13 patients were included for longitudinal analysis. We assessed positive selection pressure on thegag/protease region using a test for the overall influence of positive selection and a total of five tests to identify positively selected single codons. We found that positive selection pressure was the driving evolutionary force for thegagregion in all three patient groups. An increase in positive selection was observed ingagcleavage site regions p7/p1/p6 only after the acquisition of major PI mutations, suggesting that amino acids ingagcleavage sites under positive selection pressure could function as compensatory mutations for major PI mutations in the protease region. Isolatedgagmutations did not appear to confer PI resistance, but mutations in thegagcleavage sites could substitute for minor PI resistance mutations in the protease region.
The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection
Authors: Benedicto Ignacio , Molina-Jiménez Francisca , Bartosch Birke , Cosset François-Loïc , Lavillette Dimitri , Prieto Jesús , Moreno-Otero Ricardo , Valenzuela-Fernández Agustín , Aldabe Rafael , López-Cabrera Manuel , Majano Pedro L.
Published: June 2009

The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.
Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase
Authors: Jarahian Mostafa , Watzl Carsten , Fournier Philippe , Arnold Annette , Djandji Dominik , Zahedi Sarah , Cerwenka Adelheid , Paschen Annette , Schirrmacher Volker , Momburg Frank
Published: June 2009

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζlacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.
Vpu Directs the Degradation of the Human Immunodeficiency Virus Restriction Factor BST-2/Tetherin via a βTrCP-Dependent Mechanism
Authors: Douglas Janet L. , Viswanathan Kasinath , McCarroll Matthew N. , Gustin Jean K. , Früh Klaus , Moses Ashlee V.
Published: June 2009

The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein βTrCP. To identify additional cellular βTrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind βTrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of βTrCP in this process was confirmed by demonstrating that in the absence of βTrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via βTrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.
Heterosexual Transmission of Human Immunodeficiency Virus Type 1 Subtype C: Macrophage Tropism, Alternative Coreceptor Use, and the Molecular Anatomy of CCR5 Utilization
Authors: Isaacman-Beck Jesse , Hermann Emilia A. , Yi Yanjie , Ratcliffe Sarah J. , Mulenga Joseph , Allen Susan , Hunter Eric , Derdeyn Cynthia A. , Collman Ronald G.
Published: June 2009

Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.
Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein
Authors: Verma Dinesh , Ling Chen , Johannsen Eric , Nagaraja Tirumuru , Swaminathan Sankar
Published: June 2009

The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.
Ebola Virus VP35 Antagonizes PKR Activity through Its C-Terminal Interferon Inhibitory Domain
Authors: Schümann Michael , Gantke Thorsten , Mühlberger Elke
Published: June 2009

Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection.
Simian Virus 40 Small T Antigen Activates AMPK and Triggers Autophagy To Protect Cancer Cells from Nutrient Deprivation
Authors: Kumar Sravanth Hindupur , Rangarajan Annapoorni
Published: June 2009

As tumors grow larger, they often experience an insufficient supply of oxygen and nutrients. Hence, cancer cells must develop mechanisms to overcome these stresses. Using an in vitro transformation model where the presence of the simian virus 40 (SV40) small T (ST) antigen has been shown to be critical for tumorigenic transformation, we investigated whether the ST antigen has a role to play in regulating the energy homeostasis of cancer cells. We find that cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (−ST cells). Mechanistically, we find that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). The basal level of active, phosphorylated AMPK was higher in +ST cells than in −ST cells, and these levels increased further in response to glucose deprivation. Additionally, inhibition of AMPK in +ST cells increased the rate of cell death, while activation of AMPK in −ST cells decreased the rate of cell death, under conditions of glucose deprivation. We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation. Finally, we show that +ST cells exhibit a higher percentage of autophagy than −ST cells upon glucose deprivation. Thus, we demonstrate a novel role for the SV40 ST antigen in cancers, where it functions to maintain energy homeostasis during glucose deprivation by activating AMPK, inhibiting mTOR, and inducing autophagy as an alternate energy source.
A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Authors: Pejchal Robert , Gach Johannes S. , Brunel Florence M. , Cardoso Rosa M. , Stanfield Robyn L. , Dawson Philip E. , Burton Dennis R. , Zwick Michael B. , Wilson Ian A.
Published: June 2009

The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W670NWFDITN677) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens.
Transcription of the Herpes Simplex Virus Latency-Associated Transcript Promotes the Formation of Facultative Heterochromatin on Lytic Promoters
Authors: Cliffe Anna R. , Garber David A. , Knipe David M.
Published: June 2009

An important question in virology is the mechanism(s) by which persistent viruses such as the herpesviruses and human immunodeficiency virus (HIV) establish a latent infection in specific types of cells. In the case of herpesviruses, herpes simplex virus (HSV) infection of epithelial cells results in a lytic infection, whereas latent infection is established in sensory neurons. Recent studies have shown the importance of chromatin structure in the regulation of latent infection for both HSV and HIV. For HSV, we have shown previously that the viral latency-associated transcript (LAT) promotes lytic gene silencing and the association of one heterochromatin marker, dimethylation of lysine 9 on histone H3 (H3K9me2), with viral lytic genes. In this study, we further defined the structure of latent viral chromatin by examining the heterochromatin markers on histones associated with the HSV latent genome. We detected the H3K9me2, H3K9me3, and H3K27me3 modifications, with H3K27me3, which is indicative of facultative heterochromatin, exhibiting the highest enrichment on all viral promoters tested. A modification associated with cellular centromeric heterochromatin, H4K20me3, was not detected. A mutant virus containing a 1.8-kbp deletion within the LAT region showed reduced levels of the facultative heterochromatin marker (H3K27me3) along with H3K9me3 during latency, whereas a viral mutant defective for the LAT promoter showed a specific reduction in H3K27me3. Cellular long, noncoding RNAs induce facultative heterochromatin, and this study shows that transcription of a viral noncoding RNA can also induce facultative heterochromatin to promote lytic gene silencing during latency.
Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression
Authors: Ross P. Joel , Kennedy Michael A. , Parks Robin J.
Published: June 2009

Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.
Human VAP-C Negatively Regulates Hepatitis C Virus Propagation
Authors: Kukihara Hiroshi , Moriishi Kohji , Taguwa Shuhei , Tani Hideki , Abe Takayuki , Mori Yoshio , Suzuki Tetsuro , Fukuhara Takasuke , Taketomi Akinobu , Maehara Yoshihiko , Matsuura Yoshiharu
Published: June 2009

Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.
Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes
Authors: Phan Tung , Beran Rudolf K. F. , Peters Christopher , Lorenz Ivo C. , Lindenbach Brett D.
Published: June 2009

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.