bluuebunny/crossref_metadata_2025 huggingface dataset:
RADIOGRAPHIC CHARACTERIZATION OF PRIMARY LUNG TUMORS IN 74 DOGS
Authors: Barrett Laura E. , Pollard Rachel E. , Zwingenberger Allison , Zierenberg‐Ripoll Alexandra , Skorupski Katherine A.
Published: March 2014

Primary pulmonary neoplasia is well recognized in dogs and prognosis depends upon the tumor type. The purpose of this retrospective study was to characterize the radiographic appearance of different primary lung tumors with the goal of establishing imaging criteria to separate the different types. Three‐view thoracic radiographs of 74 dogs with histologically confirmed pulmonary anaplastic carcinoma (n = 2), adenocarcinoma (n = 31), bronchioalveolar carcinoma (n = 19), histiocytic sarcoma (n = 21), and squamous cell carcinoma (n = 1) were evaluated. Radiographs were assessed for tumor volume, affected lobe, location within lobe, overall pulmonary pattern, presence of cavitation, mineralization, air bronchograms, lymphadenomegaly, and pleural fluid. Histiocytic sarcomas were significantly larger than other tumor types (271 cm3; P = 0.009) and most likely to be found in the left cranial (38%; 8/21) and right middle (43%; 9/21) lung lobes, whereas adenocarcinomas were most likely to be found in the left caudal (29%; 9/31) lung lobe. Fifty‐seven percent (12/21) of histiocytic sarcomas had an internal air bronchogram. Findings indicate that a large mass in the periphery or affecting the whole lobe of the right middle or left cranial lung lobe with an internal air bronchogram is likely to be an histiocytic sarcoma.
A Large‐scale, Rapid Public Health Response to Rabies in an Organ Recipient and the Previously Undiagnosed Organ Donor
Authors: Wallace R. M. , Stanek D. , Griese S. , Krulak D. , Vora N. M. , Pacha L. , Kan V. , Said M. , Williams C. , Burgess T. H. , Clausen S. S. , Austin C. , Gabel J. , Lehman M. , Finelli L. N. , Selvaggi G. , Joyce P. , Gordin F. , Benator D. , Bettano A. , Cersovsky S. , Blackmore C. , Jones S. V. , Buchanan B. D. , Fernandez A. I. , Dinelli D. , Agnes K. , Clark A. , Gill J. , Irmler M. , Blythe D. , Mitchell K. , Whitman T. J. , Zapor M. J. , Zorich S. , Witkop C. , Jenkins P. , Mora P. , Droller D. , Turner S. , Dunn L. , Williams P. , Richards C. , Ewing G. , Chapman K. , Corbitt C. , Girimont T. , Franka R. , Recuenco S. , Blanton J. D. , Feldman K. A.
Published: March 2014

This article describes and contrasts the public health response to two human rabies cases: one organ recipient diagnosed within days of symptom onset and the transplant donor who was diagnosed 18 months post‐symptom onset. In response to an organ‐transplant‐related rabies case diagnosed in 2013, organ donor and recipient investigations were conducted by multiple public health agencies. Persons with potential exposure to infectious patient materials were assessed for rabies virus exposure. An exposure investigation was conducted to determine the source of the organ donor's infection. Over 100 persons from more than 20 agencies spent over 2700 h conducting contact investigations in healthcare, military and community settings. The 564 persons assessed include 417 healthcare workers [5.8% recommended for post‐exposure prophylaxis (PEP)], 96 community contacts (15.6% recommended for PEP), 30 autopsy personnel (50% recommended for PEP), and 21 other persons (4.8% recommended for PEP). Donor contacts represented 188 assessed with 20.2% recommended for PEP, compared with 5.6% of 306 recipient contacts recommended for PEP. Human rabies cases result in substantial use of public health and medical resources, especially when diagnosis is delayed. Although rare, clinicians should consider rabies in cases of encephalitis of unexplained aetiology, particularly for cases that may result in organ donation.
Superior Performance of Nanofluids in an Automotive
Radiator
Authors: Ray Dustin R. , Das Debendra K.
Published: March 2014

This study compares the performance of three different nanofluids containing aluminum oxide, copper oxide, and silicon dioxide nanoparticles dispersed in the same base fluid, 60:40 ethylene glycol and water by mass, as coolant in automobile radiators. The computational scheme adopted here is the effectiveness-number of transfer unit (ε − NTU) method encoded in matlab. Appropriate correlations of thermophysical properties for these nanofluids developed from measurements are summarized in this paper. The computational scheme has been validated by comparing the results of pumping power, convective heat transfer coefficients on the air and coolant side, overall heat transfer coefficient, effectiveness and NTU, reported by other researchers. Then the scheme was adopted to compute the performance of nanofluids. Results show that a dilute 1% volumetric concentration of nanoparticles performs better than higher concentration. It is proven that at optimal conditions of operation of the radiator, under the same heat transfer basis, a reduction of 35.3% in pumping power or 7.4% of the surface area can be achieved by using the Al2O3 nanofluid. The CuO nanofluid showed slightly lower magnitudes than the Al2O3 nanofluid, with 33.1% and 7.2% reduction for pumping power or surface area respectively. The SiO2 nanofluid showed the least performance gain of the three nanofluids, but still could reduce the pumping power or area by 26.2% or 5.2%. The analysis presented in this paper was used for an automotive radiator but can be extended to any liquid to gas heat exchanger.
Three-Dimensional Thermohydrodynamic Morton Effect Simulation—Part I: Theoretical Model
Authors: Suh Junho , Palazzolo Alan
Published: March 2014

The present study is focused on accurate prediction of the Morton effect problem including journal asymmetric heating and the corresponding long period amplitude oscillations using a nonlinear time transient rotor-dynamic simulation. This paper presents a theoretical model of thermal induced synchronous instability problems in a nonlinear rotor–bearing system, and suggests a new computational algorithm for the nonlinear transient analysis of the Morton effect where the dynamic and thermal problems are combined. For the analysis of the Morton effect problem, a variable viscosity Reynolds equation and a 3D energy equation are coupled via temperature and viscosity, and solved simultaneously. Three-dimensional heat transfer equations of bearing and shaft are modeled by a finite element method, and thermally coupled with the fluid film via a heat flux boundary condition. Asymmetric heat flux into the synchronously whirling rotor is solved by the orbit time averaged heat flux from fluid film to the spinning shaft surface. The journal orbit is calculated by the nonlinear transient dynamic analysis of a rotor–bearing system with a variable time step numerical integration scheme. For the computation time reduction, modal coordinate transformation is adopted in dynamic and thermal transient analysis. Thermal bow effect makes a significant change to the dynamic behavior of a rotor–bearing system, and a thermal hysteresis bode plot, that is one of the characteristics of the Morton effect problem, is presented with time varying spin speed.
Low-Cycle Fatigue of Pressurized Steel Elbows Under In-Plane Bending
Authors: Varelis George E. , Karamanos Spyros A.
Published: March 2014

The present study examines the mechanical behavior of steel process piping elbows, subjected to strong cyclic loading conditions. The work is numerical, supported by experimental data on elbow specimens subjected to in-plane cyclic bending, with or without internal pressure, resulting in failure in the low-cycle-fatigue range. The investigation of elbow behavior is conducted using rigorous finite element analysis accounting for measured elbow geometry and the actual material properties. An advanced cyclic plasticity material model is employed for the simulation of the tests. Emphasis is given on the value of local strain and its accumulation at the critical elbow location where cracking occurs. Based on the cyclic stress–strain curve of the material and the strain-based fatigue curve from the test data, the use of Neuber's formula leads to a fatigue analysis and design methodology, offering a simple and efficient tool for predicting elbow fatigue life.
Subwavelength Focalization of Acoustic Waves Using Time Reversal. Yes We Can!
Authors: El Abed Mohamed
Published: March 2014

By superimposing two sound waves of the same wavelength, propagating in the opposite direction, we can create an intensity pattern having a characteristic scale equal to half a wavelength: it is the diffraction limit. Recently a group from the Institut Laue-Langevin in Paris1 has shown that it is possible to go beyond this limit by focusing sound waves on a spot whose size is much shorter than half of a wavelength. We explain here how to reproduce, at an elementary level, this experiment that illustrates fundamental properties of waves.
The Confirmation of the Inverse Square Law Using Diffraction Gratings
Authors: Papacosta Pangratios , Linscheid Nathan
Published: March 2014

Understanding the inverse square law, how for example the intensity of light or sound varies with distance, presents conceptual and mathematical challenges. Students know intuitively that intensity decreases with distance. A light source appears dimmer and sound gets fainter as the distance from the source increases. The difficulty is in understanding why the intensity decreases as 1/r2 rather than as 1/r or 1/r3, or even as 1/ √r, where r is the distance from the source. This paper describes a simple experiment that verifies the inverse square law using a laser pointer, a pair of diffraction gratings, and a ruler. The method examines how the rectangular area outlined by bright spots produced by a laser beam going through a pair of diffraction gratings varies with the distance to the screen. These bright spots are the result of diffraction and interference caused by the diffraction grating assembly. The relationship between the area marked by these bright spots and the diffraction gratings-screen distance is an indirect confirmation of the inverse square law.
Quantum Plasmon Resonances Controlled by Molecular Tunnel Junctions
Authors: Tan Shu Fen , Wu Lin , Yang Joel K.W. , Bai Ping , Bosman Michel , Nijhuis Christian A.
Published: March 2014

Controlling Quantum Plasmonics

Electron tunneling across cavities could potentially induce a quantum mechanical plasmon mode that would be important in nano-electronics, catalysis, nonlinear optics, or single-molecule sensing, but has been expected to occur only at length scales beyond the reach of current state-of-the-art technology. Using a system of plasmonic dimers comprising silver nanocubes bridged by a molecular self-assembled monolayer,

Tan
et al.

(p.
1496
; see the Perspective by

Nordlander

) observed quantum plasmonic tunneling between the resonators and were able to tune the frequency of this tunneling plasmon resonance via selection of the molecular tunnel junctions. Moreover, the effects were observed at length scales that are technologically accessible.
The Resolution Revolution
Authors: Kühlbrandt Werner
Published: March 2014

Advances in detector technology and image processing are yielding high-resolution electron cryo-microscopy structures of biomolecules.

[Also see Report by

Amunts
et al.

]
The Axis of Tolerance
Authors: Aychek Tegest , Jung Steffen
Published: March 2014

Communication between different immune cells of the intestinal mucosa acts as a rheostat that maintains tolerance to gut microbes.

[Also see Research Article by

Mortha
et al.

]
Breast Cancer: A World of Differences
Authors: Servick Kelly
Published: March 2014

Advances in detecting and treating breast cancer offer an ever brightening outlook for women diagnosed in wealthy countries, but the recent progress has been far from uniform, and in poor countries mortality remains disproportionately high.
Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses
Authors: Jawale Chetan V. , Lee John Hwa
Published: March 2014

TheEscherichia coliheat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant.Salmonella entericaserovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins.S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB inS. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with eitherS. Enteritidis-LTB ghosts orS. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n= 20 for each): group A, nonvaccinated control; group B, immunized withS. Enteritidis-LTB ghosts; and group C, immunized withS. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3+CD4+subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization withS. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization withS. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis.
Activation of Innate Immune Responses by Haemophilus influenzae Lipooligosaccharide
Authors: Choi Joshua , Cox Andrew D. , Li Jianjun , McCready William , Ulanova Marina
Published: March 2014

A Gram-negative pathogenHaemophilus influenzaehas a truncated endotoxin known as lipooligosaccharide (LOS). Recent studies onH. influenzaeLOS highlighted its structural and compositional implications for bacterial virulence; however, the role of LOS in the activation of innate and adaptive immunity is poorly understood. THP-1 monocytes were stimulated with either lipopolysaccharide (LPS) fromEscherichia colior LOS compounds derived fromH. influenzaeEagan, Rd, and Rdlic1 lpsAstrains. Cell surface expression of key antigen-presenting, costimulatory, and adhesion molecules, as well as gene expression of some cytokines and pattern recognition receptors, were studied. Eagan and Rd LOS had a lower capacity to induce the expression of ICAM-1, CD40, CD58, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) compared to LPS. In contrast, antigen-presenting (HLA-ABC or HLA-DR) and costimulatory (CD86) molecules and NOD2 were similarly upregulated in response to LOS and LPS. LOS from a mutant Rd strain (Rdlic1 lpsA) consistently induced higher expression of innate immune molecules than the wild-type LOS, suggesting the importance of phosphorylcholine and/or oligosaccharide extension in cellular responses to LOS. An LOS compound with a strong ability to upregulate antigen-presenting and costimulatory molecules combined with a low proinflammatory activity may be considered a vaccine candidate to immunize againstH. influenzae.
Antibodies against Mycobacterial Proteins as Biomarkers for HIV-Associated Smear-Negative Tuberculosis
Authors: Siev Michael , Wilson Douglas , Kainth Supreet , Kasprowicz Victoria O. , Feintuch Catherine M. , Jenny-Avital Elizabeth R. , Achkar Jacqueline M.
Published: March 2014

Serology data are limited for patients with sputum smear-negative HIV-associated active tuberculosis (TB). We evaluated the serum antibody responses against the mycobacterial proteins MPT51, MS, and echA1 and the 38-kDa protein via enzyme-linked immunosorbent assay (ELISA) in South African (S.A.) HIV-positive (HIV
+
) smear-negative TB patients (
n
= 56), U.S. HIV

+

controls with a positive tuberculin skin test (TST
+
;
n
= 21), and S.A. HIV-negative (HIV
−
) (
n
= 18) and HIV
+
(
n
= 24) controls. TB patients had positive antibody reactivity against MPT51 (73%), echA1 (59%), MS (36%), and the 38-kDa protein (11%). Little reactivity against MPT51 and echA1 was observed in control groups at low risk for TB, i.e., S.A. HIV
−
(0% and 6%, respectively), and at moderate risk for TB development, i.e., U.S. HIV
+
TST
+
controls (14% and 10%, respectively). By contrast, more reactivity was detected in the S.A. HIV
+
control group at higher risk for TB (25% and 45%, respectively). Our data hold promise that antibody detection against MPT51 and echA1 might have adjunctive value in the detection of HIV
+
smear-negative TB and might reflect increasing
Mycobacterium tuberculosis
infection activity in asymptomatic HIV
+
individuals.
Evaluation of the Remel RapID NF Plus Rapid Biochemical Method for Identification of Burkholderia pseudomallei
Authors: Maloney Samuel , Engler Cathy , Norton Robert
Published: March 2014

Typical methods for the identification of
Burkholderia pseudomallei
colonies produce results in 18 h. The Remel RapID NF Plus kit produces results in 4 h. We used the kit for 190 stored
B. pseudomallei
isolates and correctly identified 189 of them. This kit produces consistent results for known
B. pseudomallei
isolates.
Beta-Hemolytic, Multi-Lancefield Antigen-Agglutinating Enterococcus durans from a Pregnant Woman, Mimicking Streptococcus agalactiae
Authors: Savini Vincenzo , Franco Alessia , Gherardi Giovanni , Marrollo Roberta , Argentieri Angela Valentina , Pimentel de Araujo Fernanda , Amoruso Roberta , Battisti Antonio , Fazii Paolo , Carretto Edoardo
Published: March 2014

A beta-hemolytic Lancefield antigen A-, B-, C-, D-, F-, and G-positive
Enterococcus durans
strain was cultivated from the rectovaginal swab of a pregnant woman who underwent antenatal screening for
Streptococcus agalactiae
. The isolate raised concern as to what extent similar strains are misrecognized and lead to false diagnosis of group B streptococci.
Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses
Authors: Kommedal Øyvind , Wilhelmsen Marianne Thulin , Skrede Steinar , Meisal Roger , Jakovljev Aleksandra , Gaustad Peter , Hermansen Nils Olav , Vik-Mo Einar , Solheim Ole , Ambur Ole Herman , Sæbø Øystein , Høstmælingen Christina Teisner , Helland Christian
Published: March 2014

Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture- and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications.
Aggregatibacter aphrophilus
,
Fusobacterium nucleatum
, and
Streptococcus intermedius
or combinations of them were found in all spontaneous polymicrobial abscesses.
F. nucleatum
was systematically detected in samples with anaerobic flora. The increased detection rate for
Actinomyces
spp. and facultative Gram-negative rods further revealed several species associations. We suggest that
A. aphrophilus
,
F. nucleatum
, and
S. intermedius
are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition,
F. nucleatum
seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop.
In Vitro
Antimicrobial Susceptibility of Aerococcus urinae
Authors: Humphries Romney M. , Hindler Janet A.
Published: March 2014

Aerococcus urinae
may cause urinary tract infections, bacteremia, and endocarditis. No standardized susceptibility test methods or interpretive criteria have been proposed for this organism. This study reports the MIC results for 128
A. urinae
isolates tested by broth microdilution. The isolates had low MICs to amoxicillin, cefotaxime, ceftriaxone, doxycycline, linezolid, meropenem, penicillin, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. However, 55% of the isolates had MICs to clindamycin of >0.25 μg/ml, 44% had MICs to erythromycin of >0.25 μg/ml, and 16% had MICs to levofloxacin of >2 μg/ml.
Ureaplasma parvum Prosthetic Joint Infection Detected by PCR
Authors: Farrell John J. , Larson Joshua A. , Akeson Jeffrey W. , Lowery Kristin S. , Rounds Megan A. , Sampath Rangarajan , Bonomo Robert A. , Patel Robin
Published: March 2014

We describe the first reported case of
Ureaplasma parvum
prosthetic joint infection (PJI) detected by PCR.
Ureaplasma
species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material).
U. parvum
is a relatively new species name for certain serovars of
Ureaplasma urealyticum
, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected
U. parvum
in all samples.
U. parvum
-specific PCR testing was performed on all synovial fluid samples to confirm the
U. parvum
detection.
Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants Coding for Carbapenemases with Different Activities by Use of a PCR-Based
In Vitro
Protein Expression Method
Authors: Huang Li , Hu Xiumei , Zhou Man , Yang Yinmei , Qiao Jinjuan , Wang Dianbing , Yu Junping , Cui Zongqiang , Zhang Zhiping , Zhang Xian-En , Wei Hongping
Published: March 2014

New Delhi metallo-β-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on β-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based
in vitro
protein expression (PCR-P) method was used to detect
bla
NDM-1
and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with
in vitro
cell-free expression to convert the
bla
NDM-1
amplicons into NDM for carbapenemase assay. The method could screen for
bla
NDM-1
within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent
bla
NDM-1
variants, 2 functional variants,
bla
NDM-4
and
bla
NDM-5
, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing
bla
NDM-1
. This novel method provides the first integrated approach for rapidly detecting the full-length
bla
NDM-1
and revealing its functional variants in clinical isolates.
NEMO Is Essential for Kaposi's Sarcoma-Associated Herpesvirus-Encoded vFLIP K13-Induced Gene Expression and Protection against Death Receptor-Induced Cell Death, and Its N-Terminal 251 Residues Are Sufficient for This Process
Authors: Tolani Bhairavi , Matta Hittu , Gopalakrishnan Ramakrishnan , Punj Vasu , Chaudhary Preet M.
Published: March 2014

Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 was originally believed to protect virally infected cells against death receptor-induced apoptosis by interfering with caspase 8/FLICE activation. Subsequent studies revealed that K13 also activates the NF-κB pathway by binding to the NEMO/inhibitor of NF-κB (IκB) kinase gamma (IKKγ) subunit of an IKK complex and uses this pathway to modulate the expression of genes involved in cellular survival, proliferation, and the inflammatory response. However, it is not clear if K13 can also induce gene expression independently of NEMO/IKKγ. The minimum region of NEMO that is sufficient for supporting K13-induced NF-κB has not been delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis remains to be determined. In this study, we used microarray analysis on K13-expressing wild-type and
NEMO
-deficient cells to demonstrate that NEMO is required for modulation of K13-induced genes. Reconstitution of
NEMO
-null cells revealed that the N-terminal 251 amino acid residues of NEMO are sufficient for supporting K13-induced NF-κB but fail to support tumor necrosis factor alpha (TNF-α)-induced NF-κB. K13 failed to protect
NEMO
-null cells against TNF-α-induced cell death but protected those reconstituted with the NEMO mutant truncated to include only the N-terminal 251 amino acid residues [the NEMO(1-251) mutant]. Taken collectively, our results demonstrate that NEMO is required for modulation of K13-induced genes and the N-terminal 251 amino acids of NEMO are sufficient for supporting K13-induced NF-κB. Finally, the ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB.

IMPORTANCE
Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptor-induced apoptosis and to activate the NF-κB pathway by binding to adaptor protein NEMO/IKKγ. However, whether K13 can also induce gene expression independently of NEMO and the minimum region of NEMO that is sufficient for supporting K13-induced NF-κB remain to be delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis is not clear. We demonstrate that NEMO is required for modulation of K13-induced genes and its N-terminal 251 amino acids are sufficient for supporting K13-induced NF-κB. The ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. Our results suggest that K13-based gene therapy approaches may have utility for the treatment of patients with NEMO mutations and immunodeficiency.
A Complex Comprising Phosphatidylinositol 4-Kinase IIIβ, ACBD3, and Aichi Virus Proteins Enhances Phosphatidylinositol 4-Phosphate Synthesis and Is Critical for Formation of the Viral Replication Complex
Authors: Ishikawa-Sasaki Kumiko , Sasaki Jun , Taniguchi Koki
Published: March 2014

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754–766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the
in vitro
kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated.

IMPORTANCE
The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus
Kobuvirus
, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication and individual viral proteins enhance the level of cellular PI4P, and suggested that the viral protein/ACBD3/PI4KB complex plays an important role in forming a functional replication complex. Thus, the present study provides a new example of modulation of cellular lipid metabolism by viruses to support the replication of their genomes.
Lysophosphatidylcholine Reversibly Arrests Pore Expansion during Syncytium Formation Mediated by Diverse Viral Fusogens
Authors: Ciechonska Marta , Duncan Roy
Published: March 2014

Using lysophosphatidylcholine, a curvature-inducing lysolipid, we have isolated a reversible, “stalled pore” phenotype during syncytium formation induced by the p14 fusion-associated small transmembrane (FAST) protein and influenza virus hemagglutinin (HA) fusogens. This is the first evidence that lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores.
An Allometric Relationship between the Genome Length and Virion Volume of Viruses
Authors: Cui Jie , Schlub Timothy E. , Holmes Edward C.
Published: March 2014

Virions vary in size by at least 4 orders of magnitude, yet the evolutionary forces responsible for this enormous diversity are unknown. We document a significant allometric relationship, with an exponent of approximately 1.5, between the genome length and virion volume of viruses and find that this relationship is not due to geometric constraints. Notably, this allometric relationship holds regardless of genomic nucleic acid, genome structure, or type of virion architecture and therefore represents a powerful scaling law. In contrast, no such relationship is observed at the scale of individual genes. Similarly, after adjusting for genome length, no association is observed between virion volume and the number of proteins, ruling out protein number as the explanation for the relationship between genome and virion sizes. Such a fundamental allometric relationship not only sheds light on the constraints to virus evolution, in that increases in virion size but not necessarily structure are associated with concomitant increases in genome size, but also implies that virion sizes in nature can be broadly predicted from genome sequence data alone.

IMPORTANCEViruses vary dramatically in both genome and virion sizes, but the factors responsible for this diversity are uncertain. Through a comparative and quantitative investigation of these two fundamental biological parameters across diverse viral taxa, we show that genome length and virion volume conform to a simple allometric scaling law. Notably, this allometric relationship holds regardless of the type of virus, including those with both RNA and DNA genomes, and encompasses viruses that exhibit more than 3 logs of genome size variation. Accordingly, this study helps to reveal the basic rules of virus design.
NTPDASE4
Gene Products Cooperate with the Adenovirus E4orf4 Protein through PP2A-Dependent and -Independent Mechanisms and Contribute to Induction of Cell Death
Authors: Avital-Shacham Meirav , Sharf Rakefet , Kleinberger Tamar
Published: March 2014

The adenovirus E4orf4 protein induces nonclassical apoptosis in mammalian cells through at least two complementing pathways regulated by the interactions of E4orf4 with protein phosphatase 2A (PP2A) and Src kinases. In
Saccharomyces cerevisiae
cells, which do not express Src, E4orf4 induces PP2A-dependent toxicity. The yeast Golgi apyrase Ynd1 was found to contribute to E4orf4-mediated toxicity and to interact with the PP2A-B55α regulatory subunit. In addition, a mammalian Ynd1 orthologue, the
NTPDASE4
gene product Golgi UDPase, was shown to physically interact with E4orf4. Here we report that knockdown of
NTPDASE4
suppressed E4orf4-induced cell death. Conversely, overexpression of the
NTPDASE4
gene products Golgi UDPase and LALP70 enhanced E4orf4-induced cell killing. We found that similarly to results obtained in yeast, the apyrase activity of mammalian UDPase was not required for its contribution to E4orf4-induced toxicity. The interaction between E4orf4 and UDPase had two consequences: a PP2A-dependent one, resulting in increased UDPase levels, and a PP2A-independent outcome that led to dissociation of large UDPase-containing protein complexes. The present report extends our findings in yeast to E4orf4-mediated death of mammalian cells, and combined with previous results, it suggests that the E4orf4-NTPDase4 pathway, partly in association with PP2A, may provide an alternative mechanism for the E4orf4-Src pathway to contribute to the cytoplasmic death function of E4orf4.

IMPORTANCE
The adenovirus E4orf4 protein contributes to regulation of the progression of virus infection from the early to the late phase, and when expressed alone, it induces a unique caspase-independent programmed cell death which is more efficient in cancer cells than in normal cells. The interactions of E4orf4 with cellular proteins that mediate its functions, such as PP2A and Src kinases, are highly conserved in evolution. The results presented here reveal that the
NTPDASE4
gene product Golgi UDPase, first discovered to contribute to E4orf4 toxicity in
Saccharomyces cerevisiae
, associates with E4orf4 and plays a role in induction of cell death in mammalian cells. Details of the functional interaction between E4orf4, PP2A, and the UDPase are described. Identification of the evolutionarily conserved mechanisms underlying E4orf4 activity will increase our understanding of the interactions between the virus and the host cell and will contribute to our grasp of the unique mode of E4orf4-induced cell death.
The Rad6/18 Ubiquitin Complex Interacts with the Epstein-Barr Virus Deubiquitinating Enzyme, BPLF1, and Contributes to Virus Infectivity
Authors: Kumar Ravindra , Whitehurst Christopher B. , Pagano Joseph S.
Published: March 2014

Epstein-Barr virus (EBV) encodes BPLF1, a lytic cycle protein with deubiquitinating activity that is contained in its N-terminal domain and conserved across the
Herpesviridae
. EBV replication is associated with cellular DNA replication and repair factors, and initiation of EBV lytic replication induces a DNA damage response, which can be regulated at least in part by BPLF1. The cellular DNA repair pathway, translesion synthesis (TLS), is disrupted by BPLF1, which deubiquitinates the DNA processivity factor, PCNA, and inhibits the recruitment of the TLS polymerase, polymerase eta (Pol eta), after damage to DNA by UV irradiation. Here we showed that the E3 ubiquitin ligase, which activates TLS repair by monoubiquitination of PCNA, is also affected by BPLF1 deubiquitinating activity. First, BPLF1 interacts directly with Rad18, and overexpression of BPLF1 results in increased levels of the Rad18 protein, suggesting that it stabilizes Rad18. Next, expression of functionally active BPLF1 caused relocalization of Rad18 into nuclear foci, which is consistent with sites of cellular DNA replication that occur during S phase. Also, levels of Rad18 remain constant during lytic reactivation of wild-type virus, but reactivation of BPLF1 knockout virus resulted in decreased levels of Rad18. Finally, the contribution of Rad18 levels to infectious virus production was examined with small interfering RNA (siRNA) targeting Rad18. Results demonstrated that reducing levels of Rad18 decreased production of infectious virus, and infectious titers of BPLF1 knockout virus were partially restored by overexpression of Rad18. Thus, BPLF1 interacts with and maintains Rad18 at high levels during lytic replication, which assists in production of infectious virus.

IMPORTANCE
Characterization of EBV BPLF1's deubiquitinating activity and identification of its targets and subsequent functional effects remain little studied. All members of the
Herpesviridae
contain BPLF1 homologs with conserved enzymatic activity, and findings discovered with EBV BPLF1 are likely applicable to other members of the family. Discovery of new targets of BPLF1 will point to cellular pathways and viral processes regulated by the enzymatic activity of the EBV-encoded deubiquitinating enzyme. Here we determined the importance of the cellular ubiquitin ligase Rad18 in these processes and how it is affected by BPLF1. Our findings demonstrate that EBV can co-opt Rad18 as a novel accessory factor in the production of infectious virus.
The Rhinovirus Subviral A-Particle Exposes 3′-Terminal Sequences of Its Genomic RNA
Authors: Harutyunyan Shushan , Kowalski Heinrich , Blaas Dieter
Published: March 2014

Enteroviruses, which represent a large genus within the family
Picornaviridae
, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (+)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that
in vitro
acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ∼700 bases. However, even after hours of incubation at pH 5.2, 5′-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment
in vivo
. This finding suggests that the exposed 3′-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded.

IMPORTANCE
For host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification
in vitro
results in RNA exit starting from the 3′-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.
Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown
Authors: Galvis Alvaro E. , Fisher Hugh E. , Nitta Takayuki , Fan Hung , Camerini David
Published: March 2014

Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different factors and occur in different intracellular locations.

IMPORTANCE
This study shows that HIV-1 reverse transcription starts in the cytoplasm but is completed in or on the surface of the nucleus. Moreover, we show that nuclear reverse transcription is dependent on the activity of the human RNA lariat debranchng enzyme (DBR1), while cytoplasmic reverse transcription is not. These findings may provide new avenues for inhibiting HIV-1 replication and therefore may lead to new medicines for treating HIV-1-infected individuals.
Identification of a Novel Rhabdovirus in Spodoptera frugiperda Cell Lines
Authors: Ma Hailun , Galvin Teresa A. , Glasner Dustin R. , Shaheduzzaman Syed , Khan Arifa S.
Published: March 2014

The Sf9 cell line, derived from
Spodoptera frugiperda
, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order
Mononegavirales
in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the
S. frugiperda
rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the
Mononegavirales
from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus
Cytorhabdovirus
. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-
5
B
1-4
(Tn
5
; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses.

IMPORTANCE
The
Spodoptera frugiperda
Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms,
de novo
assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results.
The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity
Authors: Masante Cyril , El Najjar Farah , Chang Andres , Jones Angela , Moncman Carole L. , Dutch Rebecca Ellis
Published: March 2014

Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection.

IMPORTANCEHuman metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified.