bluuebunny/crossref_metadata_2025 huggingface dataset:
The Gene sml0013 of Synechocystis Species Strain PCC 6803 Encodes for a Novel Subunit of the NAD(P)H Oxidoreductase or Complex I That Is Ubiquitously Distributed among Cyanobacteria
Authors: Schwarz Doreen , Schubert Hendrik , Georg Jens , Hess Wolfgang R. , Hagemann Martin
Published: October 2013

The NAD(P)H oxidoreductase or complex I (NDH1) complex participates in many processes such as respiration, cyclic electron flow, and inorganic carbon concentration in the cyanobacterial cell. Despite immense progress in our understanding of the structure-function relation of the cyanobacterial NDH1 complex, the subunits catalyzing NAD(P)H docking and oxidation are still missing. The gene sml0013 of Synechocystis 6803 encodes for a small protein of unknown function for which homologs exist in all completely known cyanobacterial genomes. The protein exhibits weak similarities to the NDH-dependent flow6 (NDF6) protein, which was reported from Arabidopsis (Arabidopsis thaliana) chloroplasts as a NDH subunit. An sml0013 inactivation mutant of Synechocystis 6803 was generated and characterized. It showed only weak differences regarding growth and pigmentation in various culture conditions; most remarkably, it exhibited a glucose-sensitive phenotype in the light. The genome-wide expression pattern of the Ɗsml0013::Km mutant was almost identical to the wild type when grown under high CO2 conditions as well as after shifts to low CO2 conditions. However, measurements of the photosystem I redox kinetic in cells of the Ɗsml0013::Km mutant revealed differences, such as a decreased capability of cyclic electron flow as well as electron flow into respiration in comparison with the wild type. These results suggest that the Sml0013 protein (named NdhP) represents a novel subunit of the cyanobacterial NDH1 complex, mediating its coupling either to the respiratory or the photosynthetic electron flow.
A Numerical Study of Flow and Temperature Maldistribution in a Parallel Microchannel System for Heat Removal in Microelectronic Devices
Authors: Siva V. Manoj , Pattamatta Arvind , Kumar Das Sarit
Published: October 2013

A common assumption in basic heat exchanger design theory is that fluid is distributed uniformly at the inlet of the exchanger on each fluid side and throughout the core. However, in reality, uniform flow distribution is never achieved in a heat exchanger and is referred to as flow maldistribution. Flow maldistribution is generally well understood for the macrochannel system. But it is still unclear whether the assumptions underlying the flow distribution in conventional macrochannel heat exchangers hold good for microchannel system. In this regard, extensive numerical simulations are carried out in a “U” type parallel microchannel system in order to study flow and heat transfer maldistribution and validated with in-house experimental data. A detailed parametric analysis is carried out to characterize flow maldistribution in a microchannel system and to study the effect of geometrical factors such as number of channels, n, Area of cross section of the channel Ac, manifold cross section area Ap, and flow parameter such as Reynolds number, Re, on the pressure and temperature distribution. In order to minimize the variation in pressure and to reduce temperature hot spots in the microchannel, a response surface based surrogate approximation and a gradient based search algorithm are used to arrive at the best configuration of microchannel cooling system. A three level factorial design involving three parameters namely Ac/Ap, Re, n are considered. The results from the optimization indicate that the case of n = 7, Ac/Ap = 0.69, and Re = 100 is the best possible configuration to alleviate flow maldistribution and hotspot formation in microchannel cooling system.
Multiscale Transient Thermal, Hydraulic, and Mechanical Analysis Methodology of a Printed Circuit Heat Exchanger Using an Effective Porous Media Approach
Authors: Urquiza Eugenio , Lee Kenneth , Peterson Per F. , Greif Ralph
Published: October 2013

Printed circuit heat exchangers (PCHE) and the similar formed plate heat exchangers (FPHE) offer highly attractive economics due to their higher power densities when compared to more conventional shell-and-tube designs. However, their complex geometry makes them more vulnerable to damage from thermal stresses during transient thermal hydraulic conditions. Transient stresses far exceed those predicted from steady state analyses. Therefore, a transient, hydraulic, thermal, and structural analysis is needed to accurately simulate and design high performing PCHE. The overall length of the heat exchanger can be thousands of times larger than the characteristic length for the heat transfer and fluid flow. Furthermore, simulating the thermal hydraulics of the entire heat exchanger plate is very time consuming and computationally expensive. The proposed methodology mitigates this by using a multiscale analysis with local volume averaged (LVA) properties and a novel effective porous media (EPM) approach. This method is implemented in a new computer code named the compact heat exchanger explicit thermal and hydraulics (CHEETAH) code which solves the time-dependent, mass, momentum, and energy equations for the entire PCHE plate as well as hot and cold fluid streams using finite volume analysis (FVA). The potential of the method and code is illustrated with an example problem for a Heatric-type helium gas-to-liquid salt PCHE with offset strip fins (OSF). Given initial and boundary conditions, CHEETAH computes and plots transient temperature and flow data. A specially developed grid mapping code transfers temperature arrays onto adapted structural meshes generated with commercial FEA software. For the conditions studied, a multiscale stress analysis reveals mechanical vulnerabilities in the HX design. This integrated methodology using an EPM approach enables multiscale PCHE simulation. The results provide the basis for design improvements which can minimize flow losses while enhancing flow uniformity, thermal effectiveness, and mechanical strength.
Abrasive and Erosive Wear Performance of Rare Earth Oxide Doped Ni/WC Coatings
Authors: Purkayastha Surajit , Dwivedi D. K.
Published: October 2013

The effect of CeO2 modification on flame sprayed nickel-tungsten carbide (WC) coatings was investigated. The modified coatings exhibited smaller grain sizes of the ceramic phase due to enhanced dissolution of the WC phase. The rare earth doped coatings, especially Ni-WC +0.9% wt. CeO2, showed superior abrasive wear resistance with respect to the unmodified coating mainly due to enhanced hardness. Coating modified with 0.6% wt. CeO2 demonstrated superior erosion resistance at both impact angles, 30 deg and 90 deg, respectively, primarily due to low porosity levels. Microstructural examination showed different wear mechanisms in conventional and doped coatings.
Volumetric quantification of lung nodules in CT with iterative reconstruction (ASiR and MBIR)
Authors: Chen Baiyu , Barnhart Huiman , Richard Samuel , Robins Marthony , Colsher James , Samei Ehsan
Published: October 2013

Purpose:

Volume quantifications of lung nodules with multidetector computed tomography (CT) images provide useful information for monitoring nodule developments. The accuracy and precision of the volume quantification, however, can be impacted by imaging and reconstruction parameters. This study aimed to investigate the impact of iterative reconstruction algorithms on the accuracy and precision of volume quantification with dose and slice thickness as additional variables.

Methods:

Repeated CT images were acquired from an anthropomorphic chest phantom with synthetic nodules (9.5 and 4.8 mm) at six dose levels, and reconstructed with three reconstruction algorithms [filtered backprojection (FBP), adaptive statistical iterative reconstruction (ASiR), and model based iterative reconstruction (MBIR)] into three slice thicknesses. The nodule volumes were measured with two clinical software (A: Lung VCAR, B: iNtuition), and analyzed for accuracy and precision.

Results:

Precision was found to be generally comparable between FBP and iterative reconstruction with no statistically significant difference noted for different dose levels, slice thickness, and segmentation software. Accuracy was found to be more variable. For large nodules, the accuracy was significantly different between ASiR and FBP for all slice thicknesses with both software, and significantly different between MBIR and FBP for 0.625 mm slice thickness with Software A and for all slice thicknesses with Software B. For small nodules, the accuracy was more similar between FBP and iterative reconstruction, with the exception of ASIR vs FBP at 1.25 mm with Software A and MBIR vs FBP at 0.625 mm with Software A.

Conclusions:

The systematic difference between the accuracy of FBP and iterative reconstructions highlights the importance of extending current segmentation software to accommodate the image characteristics of iterative reconstructions. In addition, a calibration process may help reduce the dependency of accuracy on reconstruction algorithms, such that volumes quantified from scans of different reconstruction algorithms can be compared. The little difference found between the precision of FBP and iterative reconstructions could be a result of both iterative reconstructionˈs diminished noise reduction at the edge of the nodules as well as the loss of resolution at high noise levels with iterative reconstruction. The findings do not rule out potential advantage of IR that might be evident in a study that uses a larger number of nodules or repeated scans.
Quantifying Long-Term Scientific Impact
Authors: Wang Dashun , Song Chaoming , Barabási Albert-László
Published: October 2013

Citation Grabbers

Is there quantifiable regularity and predictability in citation patterns? It is clear that papers that have been cited frequently tend to accumulate more citations. It is also clear that, with time, even the most novel paper loses its currency. Some papers, however, seem to have an inherent “fitness” that can be interpreted as a community's response to the research.Wanget al.(p.127; see the Perspective byEvans) developed a mechanistic model to predict citation history. The model links a paper's ultimate impact, represented by the total number of citations the paper will ever receive, to a single measurable parameter inferred from its early citation history. The model was used to identify factors that influence a journal's impact factor.
Managing Forests and Fire in Changing Climates
Authors: Stephens S. L. , Agee J. K. , Fulé P. Z. , North M. P. , Romme W. H. , Swetnam T. W. , Turner M. G.
Published: October 2013

Policy focused on fire suppression only delays the inevitable.
Following Gene Duplication, Paralog Interference Constrains Transcriptional Circuit Evolution
Authors: Baker Christopher R. , Hanson-Smith Victor , Johnson Alexander D.
Published: October 2013

Two Are Not Necessarily Better Than One

Gene duplication is one of the major drivers of the evolution of gene and protein networks. However, specific examples of how genes change to maintain two paralogous gene copies within an organism are relatively rare.

Baker
et al.

(p.
104
) examined the functional divergence of the paralogs Mcm1 and Arg80, MADS-box transcription factors in the yeast
Saccharomyces cerevisiae
. The partitioning of ancestral functions in a fungal transcription factor appeared to be affected by competitive interference between the newly formed gene duplicates. Thus, in yeast, gene duplication has created a selective conflict between the paralogs, which appear to have driven the evolution of a novel protein function.
Specific Chemical Reactivities of Spatially Separated 3-Aminophenol Conformers with Cold Ca
+
Ions
Authors: Chang Yuan-Pin , Długołęcki Karol , Küpper Jochen , Rösch Daniel , Wild Dieter , Willitsch Stefan
Published: October 2013

Reactive Conformations

Most molecules manifest a fair amount of flexibility at room temperature, in particular through interconversion of rotational conformers—structures that differ by the relative orientation of groups on either side of a single covalent bond.

Chang
et al.

(p.
98
; see the Perspective by

Heaven

) devised a method to explore the comparative reactivities of different conformers. A mixture of the conformers was prepared in a molecular beam cold enough to preclude interconversion; then an electric field was used to push the different conformers apart, spatially resolving subsequent collisional interactions with a target of trapped ions.
Energizing ConsensusThe Power SurgeEnergy, Opportunity, and the Battle for America's Futureby Michael LeviOxford University Press, New York, 2013. 266 pp. $27.95, £18.99. ISBN 9780199986163.
Authors: Ali Saleem H.
Published: October 2013

Rejecting claims that we must choose between traditional fuels and clean energy, Levi argues both must be sought in new ways.
Scholarly Communication: Cultural Contexts, Evolving Models
Authors: Harley Diane
Published: October 2013

Despite predictions that emerging technologies will transform how research is conducted, disseminated, and rewarded, why do we see so little actual shift in how scholars in the most competitive and aspirant institutions actually disseminate their research? I describe research on faculty values and needs in scholarly communication that confirm a number of conservative tendencies in publishing. These tendencies, influenced by tenure and promotion requirements, as well as disciplinary cultures, have both positive and negative consequences. Rigorous research could inform development of good practices and policies in academic publishing, as well as counter rhetoric concerning the future of peer review and scholarly communication.
Social Factors in Epidemiology
Authors: Bauch Chris T. , Galvani Alison P.
Published: October 2013

The coupling of social and biological contagion in human populations can have positive or negative outcomes.

[Also see Perspective by
Kahan
]
Turn the Molecule This Way for a Faster Reaction
Authors: Heaven Michael C.
Published: October 2013

Molecular beams and ultracold atom-trapping methods resolve the different reaction rates of the cis and trans conformers of a flexible organic molecule.

[Also see Report by

Chang
et al.

]
Future Science
Authors: Evans James A.
Published: October 2013

Can predicting an article's success change science?

[Also see Report by

Wang
et al.

]
Improving Scientific Communication
Authors: McNutt Marcia
Published: October 2013

Even the most brilliant scientific discovery, if not communicated widely and accurately, is of little value. And with the explosion of science around the globe, the dissemination of scientific information, once the purview of learned societies and a handful of publishers, is now a growth industry. This growth has attracted new models and new providers of services. In the process, the standards for scientific communication are slipping (see the special section on Communication in Science beginning on p. 56). The science community must explore new ways to improve upon them.
Shutdown!
Authors: McNutt Marcia
Published: October 2013

On 1 October, more than 800,000 workers in the U.S. federal government received notices that they should not report for work “until further notice.” The federal government is effectively closed for business while the two branches of the U.S. Congress argue about whether a continuing resolution to fund the government until mid-November should defund health care reform in the process. Those laid-off workers include tens of thousands of government scientists deemed “nonessential.”
U.S. Shutdown Spares an 'Essential' Few
Authors: Kaiser Jocelyn , Malakoff David , Mervis Jeffrey , Pennisi Elizabeth , Servick Kelly
Published: October 2013

A U.S. government shutdown is wreaking havoc with research funding agencies and disrupting federal science projects, but a few "essential" scientists are still on the job.
A Stronger IPCC Report
Authors: Kerr Richard A.
Published: October 2013

Last week's climate assessment report substantially strengthened an already highly confident scientific consensus and took a moderate line on some contentious points.
Brain Stimulation Sparks 'Machiavellian' Choices
Authors: Culotta Elizabeth
Published: October 2013

A study establishes a specific brain region, the right lateral prefrontal cortex (rLPFC), as a crucial nerve center for fairness.
The Art of Eradicating Polio
Authors: Roberts Leslie
Published: October 2013

The world is close to wiping out the poliovirus, but Nigeria threatens to undo it all. Muhammad Ali Pate is on a mission to change that.
Who's Afraid of Peer Review?
Authors: Bohannon John
Published: October 2013

A spoof paper concocted by
Science
reveals little or no scrutiny at many open-access journals.
The Seer of Science Publishing
Authors: Rabesandratana Tania
Published: October 2013

Vitek Tracz was ahead of the pack on open access. Now he wants to rewrite the rules of peer review.
Cloak-and-Dagger Publishing
Authors: Malakoff David
Published: October 2013

Classified journals aim to solve a thorny problem: how to rigorously peer review and share sensitive government-funded findings that officials don't want sent to regular journals.
The Annual Meeting: Improving What Isn't Broken
Authors: Mervis Jeffrey
Published: October 2013

Annual meetings are moneymakers for most scientific societies, and scientists continue to flock to them. But as the world changes, how long can the status quo hold?
Great Presenters
Authors: Cohen Jon
Published: October 2013

Bonnie Bassler and Larry Smarr have a gift for enthralling audiences. They share advice on how to make powerful public presentations.
Gut Instinct
Authors: Cohen Jon
Published: October 2013

Bonnie Bassler and Larry Smarr have a gift for enthralling audiences. They share advice on how to make powerful public presentations.
Clinical Utility of Viral Load in Management of Cytomegalovirus Infection after Solid Organ Transplantation
Authors: Razonable Raymund R. , Hayden Randall T.
Published: October 2013

The negative impact of cytomegalovirus (CMV) infection on transplant outcomes warrants efforts toward improving its prevention, diagnosis, and treatment. During the last 2 decades, significant breakthroughs in diagnostic virology have facilitated remarkable improvements in CMV disease management. During this period, CMV nucleic acid amplification testing (NAT) evolved to become one of the most commonly performed tests in clinical virology laboratories. NAT provides a means for rapid and sensitive diagnosis of CMV infection in transplant recipients. Viral quantification also introduced several principles of CMV disease management. Specifically, viral load has been utilized (i) for prognostication of CMV disease, (ii) to guide preemptive therapy, (iii) to assess the efficacy of antiviral treatment, (iv) to guide the duration of treatment, and (v) to indicate the risk of clinical relapse or antiviral drug resistance. However, there remain important limitations that require further optimization, including the interassay variability in viral load reporting, which has limited the generation of standardized viral load thresholds for various clinical indications. The recent introduction of an international reference standard should advance the major goal of uniform viral load reporting and interpretation. However, it has also become apparent that other aspects of NAT should be standardized, including sample selection, nucleic acid extraction, amplification, detection, and calibration, among others. This review article synthesizes the vast amount of information on CMV NAT and provides a timely review of the clinical utility of viral load testing in the management of CMV in solid organ transplant recipients. Current limitations are highlighted, and avenues for further research are suggested to optimize the clinical application of NAT in the management of CMV after transplantation.
Investigational Antimicrobial Agents of 2013
Authors: Pucci Michael J. , Bush Karen
Published: October 2013

New antimicrobial agents are always needed to counteract the resistant pathogens that continue to be selected by current therapeutic regimens. This review provides a survey of known antimicrobial agents that were currently in clinical development in the fall of 2012 and spring of 2013. Data were collected from published literature primarily from 2010 to 2012, meeting abstracts (2011 to 2012), government websites, and company websites when appropriate. Compared to what was reported in previous surveys, a surprising number of new agents are currently in company pipelines, particularly in phase 3 clinical development. Familiar antibacterial classes of the quinolones, tetracyclines, oxazolidinones, glycopeptides, and cephalosporins are represented by entities with enhanced antimicrobial or pharmacological properties. More importantly, compounds of novel chemical structures targeting bacterial pathways not previously exploited are under development. Some of the most promising compounds include novel β-lactamase inhibitor combinations that target many multidrug-resistant Gram-negative bacteria, a critical medical need. Although new antimicrobial agents will continue to be needed to address increasing antibiotic resistance, there are novel agents in development to tackle at least some of the more worrisome pathogens in the current nosocomial setting.
Clinical Outcomes with Rapid Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Routine Blood Cultures
Authors: Nicolsen Nicole C. , LeCroy Nicholas , Alby Kevin , Laux Jeffrey , Lin Feng-Chang , Daniels Lindsay , Weber David J. , Miller Melissa B.
Published: October 2013

Staphylococcus aureus
is a common cause of bacteremia, with a substantial impact on morbidity and mortality. Because of increasing rates of methicillin-resistant
Staphylococcus aureus
, vancomycin has become the standard empirical therapy. However, beta-lactam antibiotics remain the best treatment choice for methicillin-susceptible strains. Placing patients quickly on the optimal therapy is one goal of antimicrobial stewardship. This retrospective, observational, single-center study compared 33 control patients utilizing only traditional full-susceptibility methodology to 22 case patients utilizing rapid methodology with CHROMagar medium to detect and differentiate methicillin-resistant and methicillin-susceptible
Staphylococcus aureus
strains hours before full susceptibilities were reported. The time to targeted therapy was statistically significantly different between control patients (mean, 56.5 ± 13.6 h) and case patients (44.3 ± 17.9 h) (
P
= 0.006). Intensive care unit status, time of day results emerged, and patient age did not make a difference in time to targeted therapy, either singly or in combination. Neither length of stay (
P
= 0.61) nor survival (
P
= 1.0) was statistically significantly different. Rapid testing yielded a significant result, with a difference of 12.2 h to targeted therapy. However, there is still room for improvement, as the difference in time to susceptibility test result between the full traditional methodology and CHROMagar was even larger (26.5 h). This study supports the hypothesis that rapid testing plays a role in antimicrobial stewardship by getting patients on targeted therapy faster.
Clinical Evaluation of the FilmArray Blood Culture Identification Panel in Identification of Bacteria and Yeasts from Positive Blood Culture Bottles
Authors: Altun Osman , Almuhayawi Mohammed , Ullberg Måns , Özenci Volkan
Published: October 2013

The FilmArray platform (FA; BioFire, Salt Lake City, UT) is a closed diagnostic system allowing high-order multiplex PCR analysis with automated readout of results directly from positive blood cultures in 1 h. In the present study, we evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel, which includes 19 bacteria, five yeasts, and three antibiotic resistance genes. In total, 206 blood culture bottles were included in the study. The FilmArray could identify microorganisms in 153/167 (91.6%) samples with monomicrobial growth. Thirteen of the 167 (7.8%) microorganisms were not covered by the FilmArray BCID panel. In 6/167 (3.6%) samples, the FilmArray detected an additional microorganism compared to blood culture. When polymicrobial growth was analyzed, the FilmArray could detect all target microorganisms in 17/24 (71%) samples. Twelve blood culture bottles that yielded a positive signal but showed no growth were also negative by FilmArray. In 3/206 (1.5%) bottles, the FilmArray results were invalid. The results of the FilmArray were reproducible, as demonstrated by the testing and retesting of five bottles in the same day and a longitudinal follow-up of five other blood cultures up to 4 weeks. The present study shows that the FilmArray is a rapid identification method with high performance in direct identification of bacteria and yeasts from positive blood culture bottles.
External Quality Assessment for Avian Influenza A (H7N9) Virus Detection Using Armored RNA
Authors: Sun Yu , Jia Tingting , Sun Yanli , Han Yanxi , Wang Lunan , Zhang Rui , Zhang Kuo , Lin Guigao , Xie Jiehong , Li Jinming
Published: October 2013

An external quality assessment (EQA) program for the molecular detection of avian influenza A (H7N9) virus was implemented by the National Center for Clinical Laboratories (NCCL) of China in June 2013. Virus-like particles (VLPs) that contained full-length RNA sequences of the hemagglutinin (HA), neuraminidase (NA), matrix protein (MP), and nucleoprotein (NP) genes from the H7N9 virus (armored RNAs) were constructed. The EQA panel, comprising 6 samples with different concentrations of armored RNAs positive for H7N9 viruses and four H7N9-negative samples (including one sample positive for only the MP gene of the H7N9 virus), was distributed to 79 laboratories in China that carry out the molecular detection of H7N9 viruses. The overall performances of the data sets were classified according to the results for the H7 and N9 genes. Consequently, we received 80 data sets (one participating group provided two sets of results) which were generated using commercial (
n
= 60) or in-house (
n
= 17) reverse transcription-quantitative PCR (qRT-PCR) kits and a commercial assay that employed isothermal amplification method (
n
= 3). The results revealed that the majority (82.5%) of the data sets correctly identified the H7N9 virus, while 17.5% of the data sets needed improvements in their diagnostic capabilities. These “improvable” data sets were derived mostly from false-negative results for the N9 gene at relatively low concentrations. The false-negative rate was 5.6%, and the false-positive rate was 0.6%. In addition, we observed varied diagnostic capabilities between the different commercially available kits and the in-house-developed assays, with the assay manufactured by BioPerfectus Technologies (Jiangsu, China) performing better than the others. Overall, the majority of laboratories have reliable diagnostic capacities for the detection of H7N9 virus.
Irreproducible and Uninterpretable Polymyxin B MICs for Enterobacter cloacae and Enterobacter aerogenes
Authors: Landman David , Salamera Julius , Quale John
Published: October 2013

Carbapenem-resistant
Enterobacter
species are emerging nosocomial pathogens. As with most multidrug-resistant Gram-negative pathogens, the polymyxins are often the only therapeutic option. In this study involving clinical isolates of
E. cloacae
and
E. aerogenes
, susceptibility testing methods with polymyxin B were analyzed. All isolates underwent testing by the broth microdilution (in duplicate) and agar dilution (in duplicate) methods, and select isolates were examined by the Etest method. Selected isolates were also examined for heteroresistance by population analysis profiling. Using a susceptibility breakpoint of ≤2 μg/ml, categorical agreement by all four dilution tests (two broth microdilution and two agar dilution) was achieved in only 76/114 (67%) of
E. cloacae
isolates (65 susceptible, 11 resistant). Thirty-eight (33%) had either conflicting or uninterpretable results (multiple skip wells, i.e., wells that exhibit no growth although growth does occur at higher concentrations). Of the 11 consistently resistant isolates, five had susceptible MICs as determined by Etest. Heteroresistant subpopulations were detected in eight of eight isolates tested, with greater percentages in isolates with uninterpretable MICs. For
E. aerogenes
, categorical agreement between the four dilution tests was obtained in 48/56 (86%), with conflicting and/or uninterpretable results in 8/56 (14%). For polymyxin susceptibility testing of
Enterobacter
species, close attention must be paid to the presence of multiple skip wells, leading to uninterpretable results. Susceptibility also should not be assumed based on the results of a single test. Until the clinical relevance of skip wells is defined, interpretation of polymyxin susceptibility tests for
Enterobacter
species should be undertaken with extreme caution.
Qualitative Analysis To Ascertain Genotypic Identity of or Differences between Mycobacterium tuberculosis Isolates in Laboratories with Limited Resources
Authors: Sislema-Egas Fernanda , Ruiz-Serrano María Jesús , Bouza Emilio , García-de-Viedma Darío
Published: October 2013

Mycobacterium tuberculosis
is currently genotyped using mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, although the high cost of this technique restricts its implementation in resource-limited settings. We designed a MIRU-VNTR format, MLP3 (MIRU-VNTR length polymorphism triplex), that is based on the qualitative comparison of 5 nonfluorescent 3-band fingerprints in conventional electrophoresis and minimizes costs and technical demands. MLP3 successfully resolved cross-contamination alerts, discriminated reinfections from reactivations, clarified suspected microepidemics, and tracked transmission events of high epidemiological interest.
Detection of Canine Pneumovirus in Dogs with Canine Infectious Respiratory Disease
Authors: Mitchell Judy A. , Cardwell Jacqueline M. , Renshaw Randall W. , Dubovi Edward J. , Brownlie Joe
Published: October 2013

Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% (n= 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% (n= 56/215 dogs) on the day of entry to 93.5% (n= 201/215 dogs) after 21 days (P<0001). Dogs that were seronegative on entry but seroconverted while in the kennel were 4 times more likely to develop severe respiratory disease than those that did not seroconvert (P< 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs (P< 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days (P< 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.
Characterization of Hepatitis C Virus Intra- and Intergenotypic Chimeras Reveals a Role of the Glycoproteins in Virus Envelopment
Authors: Steinmann Eike , Doerrbecker Juliane , Friesland Martina , Riebesehl Nina , Ginkel Corinne , Hillung Julia , Gentzsch Juliane , Lauber Chris , Brown Richard , Frentzen Anne , Pietschmann Thomas
Published: October 2013

Hepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intragenotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only a minimal impact on this process. In contrast, in the context of intergenotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-GT2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within intergenotypic chimeras, glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2, and NS2. However, it caused an accumulation of nonenveloped core protein and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment, which likely depends on a genotype-specific interplay with additional viral factors.
The DNA Virus White Spot Syndrome Virus Uses an Internal Ribosome Entry Site for Translation of the Highly Expressed Nonstructural Protein ICP35
Authors: Kang Shih-Ting , Wang Han-Ching , Yang Yi-Ting , Kou Guang-Hsiung , Lo Chu-Fang
Published: October 2013

Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (∼300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5′ untranslated region of
icp35
. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the
Renilla
luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the
icp35
dsRNA group was significantly reduced. Tunicamycin was used to show that the α subunit of eukaryotic initiation factor 2 is required for
icp35
IRES activity. We also found that the intercalating drug quinacrine significantly inhibited
icp35
IRES activity
in vitro
and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the
Spodoptera frugiperda
40S ribosomal protein RPS10 decreased
icp35
IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation.
Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses
Authors: Chirkova Tatiana , Boyoglu-Barnum Seyhan , Gaston Kelsey A. , Malik Fahad M. , Trau Steven P. , Oomens Antonius G. P. , Anderson Larry J.
Published: October 2013

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use anin vitromodel of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)2form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.
Spherical Influenza Viruses Have a Fitness Advantage in Embryonated Eggs, while Filament-Producing Strains Are Selected
In Vivo
Authors: Seladi-Schulman Jill , Steel John , Lowen Anice C.
Published: October 2013

Influenza viruses can take on two distinct morphologies: filamentous or spherical. While the functional significance of each virion type is unclear, filaments are generally observed in low-passage-number isolates, while an exclusively spherical morphology is seen in strains grown extensively in laboratory substrates. Previous studies have shown that filamentous morphology is lost upon passage in eggs. The fact that the filamentous morphology is maintained in nature but not in the laboratory suggests that filaments provide an advantage in the host that is not necessary for growth in laboratory substrates. To test this hypothesis and identify naturally occurring mutations that alter morphology, we examined the effect of serial adaptation in eggs, MDCK cells, and guinea pigs. Two filamentous strains, A/Netherlands/602/2009 (H1N1) and A/Georgia/M5081/2012 (H1N1), were passaged in eggs and MDCK cells. Conversely, the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) was passaged in guinea pigs. We found that although passage in eggs and MDCK cells can lead to a loss of filaments, an exclusively spherical morphology is not required for highly efficient growth in either substrate. We did, however, identify two point mutations in the matrix of egg passage 10 isolates that confer spherical morphology and increased growth in eggs. In contrast, serial passage in guinea pigs resulted in the selection of filament-forming variants. Sequencing revealed point mutations to the PR8 matrix that, when introduced individually, yielded filaments. These findings suggest a functional role for filaments in the infected host and expand the breadth of mutations known to affect influenza virus shape.
Cyclophilin A Binds to the Viral RNA and Replication Proteins, Resulting in Inhibition of Tombusviral Replicase Assembly
Authors: Kovalev Nikolay , Nagy Peter D.
Published: October 2013

Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a
Saccharomyces cerevisiae
model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional
in vitro
studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication
in vitro
due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.
Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress, Autophagy, and Apoptosis
Authors: Shai Ben , Schmukler Eran , Yaniv Roy , Ziv Naomi , Horn Galit , Bumbarov Velizar , Yadin Hagai , Smorodinsky Nechama I. , Bacharach Eran , Pinkas-Kramarski Ronit , Ehrlich Marcelo
Published: October 2013

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.
The
ha72
Core Gene of Baculovirus Is Essential for Budded Virus Production and Occlusion-Derived Virus Embedding, and Amino Acid K22 Plays an Important Role in Its Function
Authors: Huang Huachao , Wang Manli , Deng Fei , Hou Dianhai , Arif Basil M. , Wang Hualin , Hu Zhihong
Published: October 2013

ha72
of
Helicoverpa armigera
nucleopolyhedrovirus (a homologue of
ac78
) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.
Two Overlapping Regions within the N-Terminal Half of the Herpes Simplex Virus 1 E3 Ubiquitin Ligase ICP0 Facilitate the Degradation and Dissociation of PML and Dissociation of Sp100 from ND10
Authors: Perusina Lanfranca Mirna , Mostafa Heba H. , Davido David J.
Published: October 2013

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in sensory neurons and can reactivate from latency under stress conditions. To promote lytic infection, the virus must interact with specific cellular factors to evade the host's antiviral defenses. The HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), activates transcription of viral genes, in part, by mediating the degradation of certain cellular proteins that play a role in host antiviral mechanisms. One component of the cellular defenses that ICP0 disrupts is the suborganelle, nuclear domain 10 (ND10), by inducing the degradation and dissociation of the major organizer of ND10, a promyelocytic leukemia (PML) and ND10 constituent, Sp100. Because previously identified domains in ICP0 explain only partially how it directs the degradation and dissociation of PML and Sp100, we hypothesized that additional regions within ICP0 may contribute to these activities, which in turn facilitate efficient viral replication. To test this hypothesis, we used a series of ICP0 truncation mutants and examined PML protein levels and PML and Sp100 immunofluorescence staining in human embryonic lung cells. Our results demonstrate that two overlapping regions within the central N-terminal portion of ICP0 (residues 212 to 311) promoted the dissociation and degradation of PML and dissociation of Sp100 (residues 212 to 427). In conclusion, we have identified two additional regions in ICP0 involved in altering ND10 antiviral defenses in a cell culture model of HSV-1 infection.
Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2
Authors: Petrillo Jessica E. , Venter P. Arno , Short James R. , Gopal Radhika , Deddouche Safia , Lamiable Olivier , Imler Jean-Luc , Schneemann Anette
Published: October 2013

Flock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3′ end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in
Drosophila melanogaster
cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence
in situ
hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.