bluuebunny/crossref_metadata_2025 huggingface dataset:
Numerical Analysis of Self-Pierce Riveting of Magnesium Alloys
Authors: Moraes J. F. C. , Jordon J. B.
Published: April 2014

Regulations all over the world have been pushing vehicle manufacturers to increase fuel economies and decrease green house gas emissions. An effective way to meet these new regulations is to reduce automobile weight through the use of lightweight metals. Magnesium alloys have received recent interest due to its high strength-to-weight ratio. However, conventional fusion joining methods such as resistance spot welding are not effective for magnesium alloys. As such, an attractive joining technique for these lightweight metals is self-pierce riveting (SPR) which is fast, fumeless and does not melt the material. However, SPR must be performed at elevated temperatures because of the low ductility of magnesium alloys at room temperature. Even though the SPR joining process has been established on magnesium alloys, this joining process is not optimized. As such, this study establishes the first attempt at simulating the SPR of magnesium alloys through the use of the finite element method. An internal state variable (ISV) plasticity and damage material model was employed and comparison to experimental results show good results. The results of this study show that the ISV material model is ideally suited for modeling the SPR in magnesium alloys.
Multiphase-Field Modeling of Micropore Formation in Metallic Alloys
Authors: Meidani Hossein , Rappaz Michel , Jacot Alain
Published: April 2014

A multiphase-field model has been developed in order to study the evolution of micropores constrained to grow in a solid network, with a so-called pinching effect. The model accounts for the pressure difference due to capillarity between liquid and gas, the equilibrium condition at triple (solid-liquid-pore) lines, the partitioning and diffusion of dissolved gases such as hydrogen in aluminum alloys.

The model is used to study the growth of a pore in a solid network, reconstructed from X-ray tomography observations. It was observed that such calculations can help to better understand the experimental observations, which have a limited temporal and spatial resolution. The predicted morphology is also compared with the experimental observation.
Simulations of the Effects of Mold Properties on Directional Solidification
Authors: Lauer Mark A. , Poirier David R. , Erdmann Robert G. , Johnson Luke , Tewari Surendra N.
Published: April 2014

The mold geometry and its thermal properties greatly influence the solidification process. Finite element simulations of directional solidification in various molds are presented. These simulations were performed using volume averaged properties in the mushy zone in order to model the convection, transport of solute and energy, and phase change occurring during solidification. These simulations show the interactions of the mold and alloy with the resultant solidification phenomena, including steepling. Mold geometries can cause macrosegregation because of shrinkage flows, by interrupting the development of the mushy zone, and by causing or influencing thermosolutal convection. Mold materials with different thermal properties result in different macrosegregation patterns even for the same geometries. Changes in cross section and the thermal properties of the mold also affect the gradients and solidification rates obtained in the alloy, as opposed to those measured on the mold wall. Simulations are compared qualitatively to a verification experiment of directionally solidifying a hypoeutectic Al-7wt%Si alloy in a mold with changing cross sections.
Optical characteristics of nanocrystalline AlxGa1−xN thin films deposited by hollow cathode plasma-assisted atomic layer deposition
Authors: Goldenberg Eda , Ozgit-Akgun Cagla , Biyikli Necmi , Kemal Okyay Ali
Published: April 2014

Gallium nitride (GaN), aluminum nitride (AlN), and AlxGa1−xN films have been deposited by hollow cathode plasma-assisted atomic layer deposition at 200 °C on c-plane sapphire and Si substrates. The dependence of film structure, absorption edge, and refractive index on postdeposition annealing were examined by x-ray diffraction, spectrophotometry, and spectroscopic ellipsometry measurements, respectively. Well-adhered, uniform, and polycrystalline wurtzite (hexagonal) GaN, AlN, and AlxGa1−xN films were prepared at low deposition temperature. As revealed by the x-ray diffraction analyses, crystallite sizes of the films were between 11.7 and 25.2 nm. The crystallite size of as-deposited GaN film increased from 11.7 to 12.1 and 14.4 nm when the annealing duration increased from 30 min to 2 h (800 °C). For all films, the average optical transmission was ∼85% in the visible (VIS) and near infrared spectrum. The refractive indices of AlN and AlxGa1−xN were lower compared to GaN thin films. The refractive index of as-deposited films decreased from 2.33 to 2.02 (λ = 550 nm) with the increased Al content x (0 ≤ x ≤ 1), while the extinction coefficients (k) were approximately zero in the VIS spectrum (>400 nm). Postdeposition annealing at 900 °C for 2 h considerably lowered the refractive index value of GaN films (2.33–1.92), indicating a significant phase change. The optical bandgap of as-deposited GaN film was found to be 3.95 eV, and it decreased to 3.90 eV for films annealed at 800 °C for 30 min and 2 h. On the other hand, this value increased to 4.1 eV for GaN films annealed at 900 °C for 2 h. This might be caused by Ga2O3 formation and following phase change. The optical bandgap value of as-deposited AlxGa1−xN films decreased from 5.75 to 5.25 eV when the x values decreased from 1 to 0.68. Furthermore, postdeposition annealing did not affect the bandgap of Al-rich films.
Energetics of Shuttle Runs: The Effects of Distance and Change of Direction
Authors: Zamparo Paola , Zadro Ivan , Lazzer Stefano , Beato Marco , Sepulcri Luigino
Published: April 2014

Shuttle runs can be used to study the physiological responses in sports (such as basketball) characterized by sprints (accelerations/decelerations) and changes of direction.

Purpose:

To determine the energy cost (C) of shuttle runs with different turning angles and over different distances (with different acceleration/deceleration patterns).

Methods:

Nine basketball players were asked to complete 6 intermittent tests over different distances (5, 10, 25 m) and with different changes of direction (180° at 5 and 25 m; 0°, 45°, 90°, and 180° at 10 m) at maximal speed (v ≍ 4.5 m/s), each composed by 10 shuttle runs of 10-s duration and 30-s recovery; during these runs oxygen uptake (VO2), blood lactate (Lab), and C were determined.

Results:

For a given shuttle distance (10 m) no major differences where observed in VO2 (~33 mL · min−1 · kg−1), Lab (~3.75 mM), and C (~21.2 J · m−1 · kg−1) when the shuttle runs were performed with different turning angles. For a given turning angle (180°), VO2 and Lab were found to increase with the distance covered (VO2 from 26 to 35 mL · min−1 · kg−1; Lab from 0.7 to 7.6 mM) while C was found to decrease with it (from 29.9 to 10.6 J · m−1 · kg−1); the relationship between C and d (m) is well described by C = 92.99 × d0.656, R2 = .971.

Conclusions:

The metabolic demands of shuttle tests run at maximal speeds can be estimated based on the running distance, while the turning angle plays a minor role in determining C.
Toward Strategic and Authentic Corporate Social Responsibility in Professional Sport: A Case Study of the Detroit Lions
Authors: Heinze Kathryn L. , Soderstrom Sara , Zdroik Jennifer
Published: April 2014

The rise and institutionalization of corporate social responsibility (CSR) in sport is captured in a growing body of work in sport management. This literature suggests professional teams should be strategic in their approaches—matching internal resources with external needs—but we lack an understanding of the processes and mechanisms in the evolution to more strategic CSR, as well as specific practices that characterize these approaches. Further, by focusing on broad trends in how and why teams are adopting CSR, we miss the opportunity to learn from teams with innovative and authentic CSR approaches. To address these gaps, this article uses a qualitative case-study approach to examine how one professional team in the U.S.—the Detroit Lions—evolved their CSR to a more strategic and authentic partnership-focused model. Our findings point to key process steps and mechanisms in the decision making around, and implementation of, this approach, including the role of organizational structure, leadership, and community partnerships. We draw out themes around these central partnerships and highlight best practices. In offering a more nuanced understanding of professional sport CSR process and practice, we contribute to the literature on CSR in sport, sport-community partnerships, and sport and city revitalization.
A 12-Million-Year Temperature History of the Tropical Pacific Ocean
Authors: Zhang Yi Ge , Pagani Mark , Liu Zhonghui
Published: April 2014

Old Gradients

The surface ocean temperature gradient between the warmer Western Equatorial Pacific and the cooler Eastern Equatorial Pacific is smaller during El Niño episodes than during neutral periods or during La Niñas. Some reconstructions of Pacific Ocean sea surface temperatures (SST) covering periods before ∼3 million years ago have suggested a permanent El Niño–like state.

Zhang
et al.

(p.
84
; see the Perspective by

Lea

) present data from a biomarker-derived proxy for SST that indicate a sizable east-west gradient has existed for the past 12 million years, contradicting the concept of a permanent El Niño–like state existed.
Construction of a Vertebrate Embryo from Two Opposing Morphogen Gradients
Authors: Xu Peng-Fei , Houssin Nathalie , Ferri-Lagneau Karine F. , Thisse Bernard , Thisse Christine
Published: April 2014

Designer Embryo

Numerous signaling pathways have been implicated in controlling early vertebrate embryogenesis.

P.-F. Xu
et al.

(p.
87
) identify the minimal set of factors necessary to get uncommitted cells to organize a complete embryo. Two opposing gradients of the growth factors Nodal and Bone Morphogenetic Protein were sufficient to instruct zebrafish embryonic pluripotent cells to organize a complete embryo, not only in vivo but also in vitro. These findings may provide guidance for regenerative medicine studies aimed at constructing tissues and organs in vitro from cultured pluripotent cells.
Optical Control of Muscle Function by Transplantation of Stem Cell–Derived Motor Neurons in Mice
Authors: Bryson J. Barney , Machado Carolina Barcellos , Crossley Martin , Stevenson Danielle , Bros-Facer Virginie , Burrone Juan , Greensmith Linda , Lieberam Ivo
Published: April 2014

Optogenetics Applied to Motorneuron Control

Nerves damaged by disease or injury do not always regenerate. In such cases, therapies involving transplanted stem cells show some promise. However, the new neurons derived from transplanted cells cannot communicate with the central control systems that would normally regulate movement. To avoid the need for such communication, in a proof-of-principle study,

Bryson
et al.

(p.
94
; see the Perspective by

Iyer and Delp

) added optogenetic control to differentiation and transplantation of motor neurons. In the mouse, these engineered neurons were able to reestablish connections within a damaged sciatic nerve and, when activated by localized light stimulation, could drive muscle contractions.
Science Funding and Short-Term Economic Activity
Authors: Weinberg Bruce A. , Owen-Smith Jason , Rosen Rebecca F. , Schwarz Lou , Allen Barbara McFadden , Weiss Roy E. , Lane Julia
Published: April 2014

Expenditures from grant funds support many different types of workers and vendors across the nation.
Complex Interplay of Islam and Science

Reading Darwin in Arabic, 1860–1950
by Marwa Elshakry

University of Chicago Press, Chicago, 2014. 447 pp., illus. $45, £31.50. ISBN 9780226001302.
Authors: Küçük B. Harun
Published: April 2014

Through her exploration of the impact of Darwinian theories on the intellectual life of Ottoman Syria and Egypt, Elshakry illuminates the development of science-and-religion issues in the region.
Inside Earth Runs Hot and Cold
Authors: Kelley Katherine A.
Published: April 2014

Mid-ocean ridge volcanoes sample a mantle that varies in temperature and composition.

[Also see Report by

Dalton
et al.

]
RIKEN Panel Finds Misconduct in Controversial Paper
Authors: Normile Dennis
Published: April 2014

Fabrication and falsification raise further concerns about claims for a new and easy way to create stem cells.
Steroids Alone or as Adjunctive Therapy with Doxycycline Fail To Improve Oviduct Damage in Mice Infected with Chlamydia muridarum
Authors: Corr Tammy E. , Sullivan Jeanne , Frazer Lauren C. , Andrews Charles W. , O'Connell Catherine M. , Darville Toni
Published: April 2014

In women,
Chlamydia trachomatis
can ascend from the cervix to the fallopian tubes, where an overly aggressive host inflammatory response can cause scarring that leads to chronic pelvic pain, infertility, or ectopic pregnancy. Although screening and treatment programs for women have resulted in decreased rates of sequelae, morbidities associated with oviduct scarring continue to occur. Since corticosteroids have anti-inflammatory and antifibrotic effects, we tested the ability of dexamethasone to inhibit inflammation and prevent oviduct scarring in mice genitally infected with
Chlamydia muridarum
. The administration of 1 or 2.5 mg/kg of body weight of dexamethasone on days 7 to 21 of infection resulted in reduced accumulation of inflammatory cells in the oviducts compared to that in controls. However, a concomitant increase in bacterial burden was observed, and chronic oviduct disease was not reduced. Adjunctive administration of a prolonged (21-day) or short (3-day) course of dexamethasone in combination with the antibiotic doxycycline also failed to reduce chronic oviduct pathology compared to antibiotic treatment alone. Steroids administered alone or adjunctively with antibiotics failed to prevent oviduct damage in this murine model of
C. trachomatis
infection.
Lymphoproliferative and Gamma Interferon Responses to Stress-Regulated Mycobacterium avium subsp. paratuberculosis Recombinant Proteins
Authors: Gurung Ratna B. , Begg Douglas J. , Purdie Auriol C. , de Silva Kumudika , Bannantine John P. , Whittington Richard J.
Published: April 2014

Johne's disease in ruminants is a chronic infection of the intestines caused byMycobacterium aviumsubsp.paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinantM. aviumsubsp.paratuberculosisproteins known to be upregulated underin vitrostress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with liveMycobacterium aviumsubsp.paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis.
Three Clustered Cases of Candidemia Caused by Candida quercitrusa and Mycological Characteristics of This Novel Species
Authors: Xiao Meng , Wang He , Lu Juan , Chen Sharon C.-A. , Kong Fanrong , Ma Xiao-Jun , Xu Ying-Chun
Published: April 2014

We investigated three nosocomial
Candida quercitrusa
candidemia cases occurring within 2 months in a Chinese hospital. Isolates were identifiable only by DNA sequencing of the rRNA genes. Genetic (via random amplified polymorphic DNA [RAPD]) and protein mass spectral (via matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS]) analyses yielded identical profiles suggesting an outbreak. The fluconazole MICs of all the strains were 16 to 32 μg/ml.
Antifungal Drug Susceptibility and Phylogenetic Diversity among Cryptococcus Isolates from Dogs and Cats in North America
Authors: Singer Lisa M. , Meyer Wieland , Firacative Carolina , Thompson George R. , Samitz Eileen , Sykes Jane E.
Published: April 2014

Molecular types of the
Cryptococcus neoformans/Cryptococcus gattii
species complex that infect dogs and cats differ regionally and with host species. Antifungal drug susceptibility can vary with molecular type, but the susceptibility of
Cryptococcus
isolates from dogs and cats is largely unknown.
Cryptococcus
isolates from 15 dogs and 27 cats were typed using
URA5
restriction fragment length polymorphism analysis (RFLP), PCR fingerprinting, and multilocus sequence typing (MLST). Susceptibility was determined using a microdilution assay (Sensititre YeastOne; Trek Diagnostic Systems). MICs were compared among groups. The 42 isolates studied comprised molecular types VGI (7%), VGIIa (7%), VGIIb (5%), VGIIc (5%), VGIII (38%), VGIV (2%), VNI (33%), and VNII (2%), as determined by
URA5
RFLP. The VGIV isolate was more closely related to VGIII according to MLST. All VGIII isolates were from cats. All sequence types identified from veterinary isolates clustered with isolates from humans. VGIII isolates showed considerable genetic diversity compared with other
Cryptococcus
molecular types and could be divided into two major subgroups. Compared with
C. neoformans
MICs,
C. gattii
MICs were lower for flucytosine, and VGIII MICs were lower for flucytosine and itraconazole. For all drugs except itraconazole,
C. gattii
isolates exhibited a wider range of MICs than
C. neoformans
. MICs varied with
Cryptococcus
species and molecular type in dogs and cats, and MICs of VGIII isolates were most variable and may reflect phylogenetic diversity in this group. Because sequence types of dogs and cats reflect those infecting humans, these observations may also have implications for treatment of human cryptococcosis.
Detection and Differentiation of Herpes Simplex Viruses by Use of the Viper Platform: Advantages, Limitations, and Concerns
Authors: Lang Amanda L. S. , Roberts Catherine , Mazzulli Tony , Hatchette Todd F. , LeBlanc Jason J.
Published: April 2014

The Viper HSV-Q
x
assay was evaluated for the detection of herpes simplex virus 1 (HSV-1) and HSV-2 in specimens from oral, anogenital, and other miscellaneous sites. The HSV-Q
x
assay was found to be highly sensitive and accurate; however, a gray zone may be required for specimens with values falling between 50 and 800 maximum relative fluorescence units.
Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry
Authors: Moller-Tank Sven , Albritton Lorraine M. , Rennert Paul D. , Maury Wendy
Published: April 2014

T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs.

IMPORTANCE
T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral membrane. While it is known that the PtdSer binding is essential for the PVEER function of TIM-1, TIM-3 shares this binding activity but does not enhance virus entry. No comprehensive studies have been done to characterize the other domains of TIM-1. In this study, using a variety of chimeric proteins and deletion mutants, we define the features necessary for a functional PVEER. With these features in mind, we generated a TIM-1 mimic using functionally similar domains from other proteins. This mimic, like TIM-1, effectively enhanced transduction. These studies provide insight into the key features necessary for PVEERs and will allow for more effective identification of unknown PVEERs.
Innate Nuclear Sensor IFI16 Translocates into the Cytoplasm during the Early Stage of
In Vitro
Human Cytomegalovirus Infection and Is Entrapped in the Egressing Virions during the Late Stage
Authors: Dell'Oste Valentina , Gatti Deborah , Gugliesi Francesca , De Andrea Marco , Bawadekar Mandar , Lo Cigno Irene , Biolatti Matteo , Vallino Marta , Marschall Manfred , Gariglio Marisa , Landolfo Santo
Published: April 2014

Intrinsic immune mechanisms mediated by constitutively expressed proteins termed “restriction factors” provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the
in vitro
infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established.

IMPORTANCE
Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.
Imaging the Alphavirus Exit Pathway
Authors: Martinez Maria Guadalupe , Snapp Erik-Lee , Perumal Geoffrey S. , Macaluso Frank P. , Kielian Margaret
Published: April 2014

Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites.

IMPORTANCE
Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.
The Impact of the Hepatitis B Virus Polymerase rtA181T Mutation on Replication and Drug Resistance Is Potentially Affected by Overlapping Changes in Surface Gene
Authors: Ahn Sung Hyun , Park Yong Kwang , Park Eun-Sook , Kim Jeong Han , Kim Doo Hyun , Lim Keo-Heun , Jang Moon Sun , Choe Won Hyeok , Ko Soon Young , Sung In-Kyung , Kwon So Young , Kim Kyun-Hwan
Published: April 2014

The emergence of drug-resistant hepatitis B virus (HBV) is a major problem for antiviral treatment in chronic hepatitis B infection. In this study, we analyzed the evolution of drug-resistant mutations and characterized the effects of the rtA181T and rtI233V mutations on viral replication and drug resistance. We performed a clonal analysis of the HBV polymerase gene from serum samples during viral breakthrough treated with antiviral agents. A series of mutant clones containing rtA181T and/or rtI233V mutations were constructed and determined the effect of these mutations on the replication ability and drug resistance. Anin vitrostudy revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. Compared to the rtA181T surface missense mutation (rtA181T/sW172S), the introduction of rtA181T surface nonsense mutation (rtA181T/sW172*) resulted in decreased viral replication and increased drug resistance. Complementation assay revealed that the truncated PreS1 is responsible for reduced replication of rtA181T/sW172* mutant. Moreover, the rtA181T/sW172* mutant exhibited a defect in viral particle secretion. The rtI233V mutation that emerged during adefovir therapy reduced viral replication and conferred resistance to adefovir. Our data suggest that the impact of the rtA181T mutation on replication and drug resistance differs based on the mutation status of the corresponding surface gene. The rtI233V mutation also affects replication ability and drug resistance. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation.

IMPORTANCEThe emergence of drug-resistant HBV that are no longer susceptible to nucleos(t)ide analogues is a major problem for antiviral treatment in chronic hepatitis B infection. Among drug-resistant mutations, the single rtA181T mutation is known to confer cross-resistance to antiviral drugs. This mutation causes intermediate or reduced susceptibility to tenofovir. Moreover, the clinical occurrence of the rtA181T mutation during antiviral therapy is also high. Our study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. We believe that our study will not only extend the understanding of the drug resistance mechanism, but it will also ultimately provide new treatment options for patients with multidrug resistant HBV.
Protein-Protein Interactions Leading to Recruitment of the Host DNA Sliding Clamp by the Hyperthermophilic Sulfolobus islandicus Rod-Shaped Virus 2
Authors: Gardner Andrew F. , Bell Stephen D. , White Malcolm F. , Prangishvili David , Krupovic Mart
Published: April 2014

Viruses infecting hyperthermophilic archaea typically do not encode DNA polymerases, raising questions regarding their genome replication. Here, using a yeast two-hybrid approach, we have assessed interactions between proteins of
Sulfolobus islandicus
rod-shaped virus 2 (SIRV2) and the host-encoded proliferating cell nuclear antigen (PCNA), a key DNA replication protein in archaea. Five SIRV2 proteins were found to interact with PCNA, providing insights into the recruitment of host replisome for viral DNA replication.
Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity
Authors: Cuddington Breanne P. , Mossman Karen L.
Published: April 2014

Oncolytic viruses (OVs) are attractive avenues of cancer therapy due to the absence of toxic side effects often seen with current treatment modalities. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines. To gain a better understanding of the oncolytic breadth of BHV-1, the NCI panel of established human tumor cell lines was screened for sensitivity to the virus. Overall, 72% of the panel is permissive to BHV-1 infection, with corresponding decreases in cellular viability. This sensitivity is in comparison to a sensitivity of only 32% for a herpes simplex virus 1 (HSV-1)-based oncolytic vector. Strikingly, while 35% of the panel supports minimal or no BHV-1 replication, significant decreases in cellular viability still occur. These data suggest that BHV-1 is an OV with tropism for multiple tumor types and is able to induce cytotoxicity independent of significant virus replication. In contrast to other species-specific OVs, cellular sensitivity to BHV-1 does not correlate with type I interferon (IFN) signaling; however, mutations in KRAS were found to correlate with high levels of virus replication. The knockdown or overexpression of KRAS in human tumor cell lines yields modest changes in viral titers; however, overexpression of KRAS in normal primary cells elicits permissivity to BHV-1 infection. Together, these data suggest that BHV-1 is a broad-spectrum OV with a distinct mechanism of tumor targeting.

IMPORTANCE
Cancer remains a significant health issue, and novel treatments are required, particularly for tumors that are refractory to conventional therapies. Oncolytic viruses are a novel platform given their ability to specifically target tumor cells while leaving healthy cells intact. For this strategy to be successful, a fundamental understanding of virus-host interactions is required. We previously identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy.
Restoration of MAGI-1 Expression in Human Papillomavirus-Positive Tumor Cells Induces Cell Growth Arrest and Apoptosis
Authors: Kranjec Christian , Massimi Paola , Banks Lawrence
Published: April 2014

The cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins target a number of cellular proteins that contain PDZ domains. However, the role of many of these interactions in either the HPV life cycle or in HPV-induced malignancy remains to be defined. Previous studies had shown that MAGI-1 was one of the most strongly bound PDZ domain-containing substrates of E6, and one consequence of this interaction appeared to facilitate the perturbation of tight junctions (TJs) by E6. In this study, we describe the generation of a mutation, K499E, within the MAGI-1 PDZ1 domain, which is resistant to E6 targeting. This mutant allows restoration of MAGI-1 expression in HPV-positive cells and defines additional activities of MAGI-1 that are overcome as a consequence of the association with E6. The reexpression of MAGI-1 in HPV-positive cells results in an increased recruitment of ZO-1 and PAR3 to sites of cell-cell contact, repression of cell proliferation, and induction of apoptosis. While the K499E mutation does not significantly affect these intrinsic activities of MAGI-1 in HPV-negative cells, its resistance to E6 targeting in an HPV-positive setting results in more cells expressing the mutant MAGI-1 than the wild-type MAGI-1, with a corresponding increase in TJ assembly, induction of apoptosis, and reduction in cell proliferation. These studies provide compelling evidence of a direct role for the perturbation of MAGI-1 function by E6 in the HPV life cycle and in HPV-induced malignancy.

IMPORTANCEIt is clear that the targeting of PDZ-containing substrates by E6 is important for the normal viral life cycle and for the progression to malignancy. Nevertheless, which of these PDZ domain-containing proteins is relevant for HPV pathology is still elusive. In a previous study, we provided evidence that MAGI-1 is a sensitive proteolytic substrate for both the HPV-16 and HPV-18 E6 oncoproteins; however, the biological consequences associated with loss of MAGI-1 expression in HPV-positive cervical cancer cells are still poorly understood. Using a mutant MAGI-1, resistant to E6-mediated degradation, we show that its expression in cervical cancer cells promotes membrane recruitment of the tight junction-associated proteins ZO-1 and PAR3, represses cell proliferation, and promotes apoptosis. These findings suggest that E6-mediated inhibition of MAGI-1 function contributes to HPV pathology by perturbing tight junction assembly with concomitant stimulation of proliferation and inhibition of apoptosis.
Human Papillomavirus Type 16 E6* Induces Oxidative Stress and DNA Damage
Authors: Williams Vonetta M. , Filippova Maria , Filippov Valery , Payne Kimberly J. , Duerksen-Hughes Penelope
Published: April 2014

High-risk types of human papillomavirus (HPV) are the causative agents of virtually all cases of cervical cancer and a significant proportion of other anogenital cancers, as well as both oral and pharyngeal cancers. The high-risk types encode two viral oncogenes, E6 and E7, which work together to initiate cell transformation. Multiple steps involving the activities and interactions of both viral and cellular proteins are involved in the progression from HPV infection to cell transformation to cancer. The E6 oncoprotein is expressed as several isoforms: a full-length variant referred to as E6 and a few shorter isoforms collectively referred to as E6*. In this study, we found that expression of E6* increased the level of reactive oxygen species (ROS) in both HPV-positive and HPV-negative cells. This increased oxidative stress led to higher levels of DNA damage, as assessed by the comet assay, quantification of 8-oxoguanine, and poly(ADP-ribose) polymerase 1. The observed increase in ROS may be due to a decrease in cellular antioxidant activity, as we found that E6* expression also led to decreased expression of superoxide dismutase isoform 2 and glutathione peroxidase. These studies indicate that E6* may play an important role in virus-induced mutagenesis by increasing oxidative stress and DNA damage.

IMPORTANCE
Our findings demonstrate for the first time that an HPV gene product, E6*, can increase ROS levels in host cells. This ability may play a significant role both in the viral life cycle and in cancer development, because an increase in oxidative DNA damage may both facilitate HPV genome amplification and increase the probability of HPV16 DNA integration. Integration, in turn, is thought to be an important step in HPV-mediated carcinogenesis.
CCR2 Deficiency Promotes Exacerbated Chronic Erosive Neutrophil-Dominated Chikungunya Virus Arthritis
Authors: Poo Yee Suan , Nakaya Helder , Gardner Joy , Larcher Thibaut , Schroder Wayne A. , Le Thuy T. , Major Lee D. , Suhrbier Andreas
Published: April 2014

Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2−/−mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2−/−mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2−/−mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2−/−mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2+monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides.

IMPORTANCEHere we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.
Neurovirulence and Immunogenicity of Attenuated Recombinant Vesicular Stomatitis Viruses in Nonhuman Primates
Authors: Clarke David K. , Nasar Farooq , Chong Siew , Johnson J. Erik , Coleman John W. , Lee Margaret , Witko Susan E. , Kotash Cheryl S. , Abdullah Rashed , Megati Shakuntala , Luckay Amara , Nowak Becky , Lackner Andrew , Price Roger E. , Little Peter , Kalyan Narender , Randolf Valerie , Javadian Ali , Zamb Timothy J. , Parks Christopher L. , Egan Michael A. , Eldridge John , Hendry Michael , Udem Stephen A.
Published: April 2014

In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on
trans
expression of G protein for
in vitro
propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased
in vivo
attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation.

IMPORTANCE
The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the
Rhabdoviridae
and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.
Innominate artery injury: a catastrophic complication of tracheostomy, operative procedure revisited
Authors: Maruti Pol Manjunath , Gupta Amit , Kumar Subodh , Mishra Biplab
Published: April 2014

A patient presented with profuse bleeding from the oronasal cavity following orofaciomaxillary trauma associated with tracheolaryngeal injury and suspected cervical-spine injury due to collapse of a wall on the face, neck and upper chest. The patient was gasping, coughing blood and was unable to speak. Threatened airway was diagnosed. Inability to maintain oxygenation on cricothyroidotomy, forced emergency department surgeons to shift the patient to the operating room for definitive airway. During tracheostomy a major vessel was injured. Application of vascular clamp in the event of achieving haemostasis resulted in disappearance of saturation and pulse in the right upper limb, thus we suspected innominate artery (IA) injury. High tracheostomy performed and endotracheal tube passed into the trachea after removing clot and overcoming compromised narrow tracheal lumen. The injured IA was repaired and the patient survived for 14 days. On postoperative day 14 he died following profound bleeding into the tracheobronchial tree and asphyxia/apnoea. Tracheoinnominate artery fistula was detected at autopsy.
Iliac artery pseudoaneurysm: a rare complication following allograft nephrectomy
Authors: Borges Lisa , Oliveira Nelson , Dias Emanuel , Cássio Isabel
Published: April 2014

Our aim is to present a case of a common iliac artery pseudoaneurysm, which complicated an allograft nephrectomy. A 27-year-old woman presented with acute abdominal pain and a palpable pulsatile mass in the right iliac fossa, 1 year after a right pelvic allograft nephrectomy. An iliac pseudoaneurysm was suspected and confirmed on triplex ultrasound and CT angiography. The patient underwent a pseudoaneurysm resection with direct repair of the previous allograft Carrell patch suture dehiscence. The intervention and recovery were uneventful and after a follow-up of 6 months, the patient remains asymptomatic with no clinical or imaging recurrence of the pseudoaneurysm.Vascular complications following allograft nephrectomy are rare but may present significant morbidity and mortality. Endovascular exclusion is currently the preferred option for the management of pseudoaneurysms following allograft nephrectomy; however, open surgical approach remains an alternative for selected patients.
Retroperitoneal Castleman's disease: benign in the midst of malignancy
Authors: Jayant Kumar , Agrawal Swati , Agrawal Rajendra
Published: April 2014

A 32-year-old asymptomatic woman presented with localised mass on the left side of the abdomen for 8 months. An abdominal ultrasound and CT scan revealed a solid heterogenous mass located just below the left kidney in the retroperitoneum. Provisional diagnosis of soft tissue sarcoma was made. Following this, surgery was planned and open biopsy (frozen section) was performed, which demonstrated localised hyaline-vascular type of Castleman's disease, which was then excised completely. Thus, intraoperative diagnosis avoided extensive resection and radiation therapy, which reduced the patient's morbidity and expenses. Postoperative period was uneventful and no recurrence was seen even after 3 years of follow-up.