bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust LBB Analyses for Atucha II Nuclear Plant
Authors: Wilkowski G. , Brust B. , Zhang T. , Hattery G. , Kalyanam S. , Shim D.-J. , Kurth E. , Hioe Y. , Uddin M. , Johnson J. J. , Maslenikov O. R. , Gu¨rpinar A. , Asfura A. P. , Sumodobila B. , Betervide A. A. , Mazzantini O.
Published: May 2012

The Atucha II nuclear power plant is a unique pressurized heavy water reactor being constructed in Argentina. The original plant design was by KWU in the 1970’s using the then German methodology of break preclusion, which assumed that the largest break-opening area would be 10-percent of the cross-sectional area of the largest pipe diameter. That philosophy was used for the design of the emergency core cooling system in the 1970’s. The plant construction was halted for several decades, but a recent need for power was the driver for restarting the construction. The construction is progressing with initial start-up in 2011. Since the 10-percent of the cross-sectional area is a smaller ECCS design requirement than the normally assumed double-ended-guillotine break, the safety evaluation of the plant for beyond design basis seismic loading of the nuclear plant was a regulatory requirement. This overview paper describes a Robust LBB Evaluation that was conducted in great detail to assess the safety aspects of the piping system under beyond design basis seismic loading and the implications to the ECCS. Key aspects involved: • Static and dynamic material property testing, • Determination of weld residual stresses, • Determination of crack sizes that might evolve by worst case SCC growth rates under weld residual stresses and normal operating stresses, • Determination of leakage rates as a function of time with the upper-bounding crack growth rates, • Development of seismic hazard curves for the site, • Development of FE models of the containment building and primary NSSS system within the building, • Determination of normal operating stresses, SSE stresses and 10−6 seismic stresses using worst case soil foundation assumptions, • Evaluation of flaw behavior for circumferential cracks using the shapes from the natural crack growth. • Evaluation of margins on the critical flaw size and times to leakage, and • Standard LBB analyses, as well as Transition Break Size evaluations. The key result from this effort was that even with all the normal operating plus 10−6 seismic event loading, the pipe system behaved more like it was displacement-controlled than load-controlled. The displacement-controlled behavior made the pipe much more flaw tolerant, and it was found that a DEGB was not possible because the flaw could never reach the critical flaw size without greatly surpassing the leakage and water make-up capacity of the plant. Since there are many details in this multi-year effort, only the key points will be summarized in this paper while other details will be the topics of other papers.
PWSCC Crack Growth Modeling Approaches
Authors: Brust F. W. , Shim D.-J. , Wilkowski G. , Rudland D.
Published: May 2012

Flaw indications have been found in some dissimilar metal (DM) nozzle to stainless steel piping welds and reactor pressure vessel heads (RPVH) in pressurized water reactors (PWR) throughout the world. The nozzle welds usually involve welding ferritic (often A508) nozzles to 304/316 stainless steel pipe) using Alloy 182/82 weld metal. The welds may become susceptible to a form of corrosion cracking referred to as primary water stress corrosion cracking (PWSCC). It can occur if the temperature is high enough (usually >300C) and the water chemistry in the PWR is typical of operating plants. The weld residual stresses (WRS) induced by the welds are a main driver of PWSCC. Several mechanical mitigation methods to control PWSCC have been developed for use on a nozzle welds in nuclear PWR plants. These methods consist of applying a weld overlay repair (WOR), using a method called mechanical stress improvement process (MSIP), and applying an inlay to the nozzle ID. The purpose of a mitigation method is to reduce the probability that PWSCC will occur in the nozzle joint. The key to assessing the effectiveness of mitigation is to determine the crack growth time to leak with and without the mitigation. Indeed, for WOR and MSIP, the weld residual stresses are often reduced after application while for inlay they are actually increased. However, all approaches reduce crack growth rates if applied properly. Procedures for modeling PWSCC growth tend to vary between organizations performing the analyses. Currently, the prediction of PWSCC crack growth is based on the stress intensity factors at the crack tips. Several methods for evaluating the stress intensity factor for modeling the crack growth through these WRS fields are possible, including using analytical, natural crack growth using finite element methods, and using the finite element alternating method. This paper will summarize the methods used, critique the procedures, and provide some examples for crack growth with and without mitigation. Suggestions for modeling such growth will be provided.
New Trends in Accurate Simulations for the Verification of Safety Margins in the Nuclear Power Plant Industry: Application of Virtual Performance Solution™ for the Response of Immersed Structures Subjected to Earthquakes
Authors: Tramec¸on Alain , Kuhnert Jorg , Mouchette Laurent , Perrin Morgane
Published: May 2012

Constraints on the safety of nuclear power plant components have increased recently along with the necessity to extend the lifespan of existing plants. For example, the acceleration levels to be sustained by the plant equipment during an earthquake have been increased many folds by the safety regulation agencies. Industrial and economic requirements plead for a verification of unknown safety margins, by accurate and physics based models taking into account all non-linear effects (for example contacts and fluid structure interaction). These effects are only approximately represented by standard linear analysis tools. Virtual Performance Solution (VPS), developed by ESI Group, includes (among other capabilities) a structural finite element software for non-linear, high velocity, dynamic simulations (PAM-CRASH), as well as a coupled, mesh free CFD module, FPM (Finite Point Method), developed in partnership with Fraunhofer ITWM. This solution accurately predicts fluid structure interactions, taking into account non-linear structural effects (contacts, friction, damping…) as well as complex fluid influences.
A Perspective on the Failure Rates of Long Seam-Welded Low Alloy Steel High Energy Piping
Authors: Foulds Jude , Shingledecker John
Published: May 2012

For over three decades, the long seam-welded low alloy steel, Grades 11 and 22, high energy piping in fossil power plants has been considered at risk of premature damage and failure. The experience with piping damage and failures has been documented and extensively studied, but there remains a lack of perspective on how the overall experience with such piping, including that of the large “survivor” population, compares with what one may expect with the design rules used in their construction. Such a perspective can be useful in helping decide on suitable design rules for this class of piping. This paper focuses on an aggregate, global, semi-quantitative evaluation of the damage and failure experience in fossil plant low alloy steel long seam-welded piping in terms of a rate of failure measured against the performance of the overall population. A key aspect of the evaluation is the consideration of the survivor population, particularly important since the documented cases of failure and damage represent a very small fraction of the population of relevant components. The damage and failure rates have been derived from the Electric Power Research Institute database, using an exposure parameter represented by the product of operating time and length of piping. The rates are viewed against the backdrop of the statistical scatter band of base metal stress rupture data used in development of the ASME Code design allowable stresses and against the weld strength reduction factors recently adopted by the ASME Boiler Pressure Vessel Code, Section I and the Power Piping Code, B31.1.
Evaluation of the Influence of Residual Stresses on Ductile Fracture
Authors: Bolinder Tobias , Sattari-Far Iradj , Faleskog Jonas
Published: May 2012

In this work the significance of residual stresses for ductile fracture was investigated. The treatment of residual stresses as expressed in fracture assessment procedures such as the R6 method is believed to be very conservative for ductile materials, when fracture occurs at high primary loads. Earlier numerical studies have reinforced this belief. Tests on notched 3PB specimens with and without residual stresses were conducted on two ferritic steels. The residual stresses were introduced by applying a compressive pre-load on notched specimens. The tests were designed to achieve crack initiation at load levels around the limit load. The crack growth in the tests was measured by a compliance method and by colour marking of the crack surface. The crack-tip driving force J was evaluated numerically for specimens with and without residual stresses. The experimental results show that the residual stresses clearly contribute to J at low primary loads. However, this contribution diminishes as the primary loads increase. The experimental results were also compared with results evaluated using the R6 procedure. These comparisons revealed an overly high conservativeness in R6 for cases with residual stresses compared to the conservativeness for cases without residual stresses.
A parametric study of error in the parabolic approximation of focused axisymmetric ultrasound beams
Authors: Soneson Joshua E.
Published: May 2012

The parabolic approximation results in a tractible model for studying ultrasound beams, but the limits of validity of the approximation are often presented only qualitatively. In this work the most common model for axisymmetric ultrasound beam propagation, the Kuznetsov–Zabolotskaya–Khokhlov equation, is directly compared with the more general Westervelt equation with regard to diffractive and absorptive effects in continuous wave beams. The parametric study compares the solutions of the two models as a function of source frequency and focusing geometry using peak focal pressure, the axial location at which that peak occurs, and the loss due to absorption as metrics.
From “Science in Europe” to “European Science”
Authors: Nedeva Maria , Stampfer Michael
Published: May 2012

Early impacts of the European Research Council suggest shifts toward competition and excellence in EU-wide basic science.
Linking Crystallographic Model and Data Quality
Authors: Karplus P. Andrew , Diederichs Kay
Published: May 2012

Finessing Crystal Analysis

Protein crystallography has revolutionized our understanding of a whole variety of biological processes (see the Perspective by

Evans

). In crystallography, the measure of agreement between the data and the calculated model is not on the same scale as the measure of data quality, making it challenging to choose an optimal high resolution limit beyond which the data should be discarded. Now,
Karplus and Diederichs
(p.
1030
) introduce a statistical model that assesses agreement of model and data accuracy on the same scale. Determining the structures of biological macromolecules by x-ray crystallography requires solving the phase problem. The two techniques that dominate phase evaluation (multi- and single-wavelength anomalous diffraction) rely on element-specific scattering from incorporated heavy atoms.

Liu
et al.

(p.
1033
) present procedures for routine structure determination of native proteins with no heavy atom incorporation. The technique, which relies on combining data from multiple crystals, was used to determine the structures of four native proteins, including a 1200-residue complex.
Metabolite Profiling Identifies a Key Role for Glycine in Rapid Cancer Cell Proliferation
Authors: Jain Mohit , Nilsson Roland , Sharma Sonia , Madhusudhan Nikhil , Kitami Toshimori , Souza Amanda L. , Kafri Ran , Kirschner Marc W. , Clish Clary B. , Mootha Vamsi K.
Published: May 2012

More Glycine, Please

To better characterize metabolic properties of cancer cells,Jainet al.(p.1040; see the Perspective byTomita and Kami) measured systematically the concentrations of hundreds of metabolites in cell culture medium in which 60 different cancer cell lines were growing. The fastest growing cancer cells tended to consume glycine, whereas more slowly growing cells excreted some glycine. The rapidly growing cancer cells appeared to need glycine for synthesis of purine nucleotides required for continued synthesis of DNA. Interfering with glycine metabolism slowed growth of the rapidly proliferating cancer cells. Thus, an increased dependence on glycine by rapidly growing cancer cells could potentially provide a target for therapeutic intervention.
Structures from Anomalous Diffraction of Native Biological Macromolecules
Authors: Liu Qun , Dahmane Tassadite , Zhang Zhen , Assur Zahra , Brasch Julia , Shapiro Lawrence , Mancia Filippo , Hendrickson Wayne A.
Published: May 2012

Finessing Crystal Analysis

Protein crystallography has revolutionized our understanding of a whole variety of biological processes (see the Perspective by

Evans

). In crystallography, the measure of agreement between the data and the calculated model is not on the same scale as the measure of data quality, making it challenging to choose an optimal high resolution limit beyond which the data should be discarded. Now,
Karplus and Diederichs
(p.
1030
) introduce a statistical model that assesses agreement of model and data accuracy on the same scale. Determining the structures of biological macromolecules by x-ray crystallography requires solving the phase problem. The two techniques that dominate phase evaluation (multi- and single-wavelength anomalous diffraction) rely on element-specific scattering from incorporated heavy atoms.

Liu
et al.

(p.
1033
) present procedures for routine structure determination of native proteins with no heavy atom incorporation. The technique, which relies on combining data from multiple crystals, was used to determine the structures of four native proteins, including a 1200-residue complex.
Resolving Some Old Problems in Protein Crystallography
Authors: Evans Phil
Published: May 2012

Two methods improve the quality and ease of structural modeling by showing how to include diffraction data that are often thrown away.
Genetically Modified Strains of Ralstonia eutropha H16 with β-Ketothiolase Gene Deletions for Production of Copolyesters with Defined 3-Hydroxyvaleric Acid Contents
Authors: Lindenkamp Nicole , Volodina Elena , Steinbüchel Alexander
Published: May 2012

β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] biosynthesis in bacteria by condensation of two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA and also take part in the degradation of fatty acids. During growth on propionate or valerate,
Ralstonia eutropha
H16 produces the copolymer poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) [poly(3HB-
co
-3HV)]. In
R. eutropha
, 15 β-ketothiolase homologues exist. The synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) could be significantly reduced in an 8-fold mutant (Lindenkamp et al., Appl. Environ. Microbiol. 76:5373–5382, 2010). In this study, a 9-fold mutant deficient in nine β-ketothiolase gene homologues (
phaA
,
bktB
, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, and
pcaF
) was generated. In order to examine the polyhydroxyalkanoate production capacity when short- or long-chain and even- or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-type
R. eutropha
and the mutants. In contrast, during the growth on oleate of a multiple mutant lacking
phaA
,
bktB
, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The β-ketothiolase activity toward acetoacetyl-CoA in H16Δ
phaA
and all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents.
Engineering of a Xylose Metabolic Pathway in Rhodococcus Strains
Authors: Xiong Xiaochao , Wang Xi , Chen Shulin
Published: May 2012

The two metabolically versatile actinobacteriaRhodococcus opacusPD630 andR. jostiiRHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway ofd-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes,xylA, encoding xylose isomerase, andxylB, encoding xylulokinase fromStreptomyces lividansTK23, under the control of thetacpromoter with anEscherichia coli-Rhodococcusshuttle vector. The recombinantR. jostiiRHA1 bearingxylAcould grow on xylose as the sole carbon source, and additional expression ofxylBfurther improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strainR. opacusPD630 by expression ofxylAandxylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants ofR. jostiiRHA1 andR. opacusPD630 carryingxylAandxylBrepresented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.
Evaluation of Commonly Used Serological Tests for Detection of Coxiella burnetii Antibodies in Well-Defined Acute and Follow-Up Sera
Authors: Wegdam-Blans M. C. A. , Wielders C. C. H. , Meekelenkamp J. , Korbeeck J. M. , Herremans T. , Tjhie H. T. , Bijlmer H. A. , Koopmans M. P. G. , Schneeberger P. M.
Published: May 2012

In this study, we comparedCoxiella burnetiiIgG phase I, IgG phase II, and IgM phase II detection among a commercially available enzyme-linked immunosorbent assay (ELISA) (Virion/Serion), an indirect fluorescent antibody test (IFAT) (Focus Diagnostics), and a complement fixation test (CFT) (Virion/Serion). For this, we used a unique collection of acute- and convalescent-phase sera from 126 patients with acute Q fever diagnosed by positiveCoxiella burnetiiPCR of blood. We were able to establish a reliable date of onset of disease, since DNA is detectable within 2 weeks after the start of symptoms. In acute samples, att= 0, IFAT demonstrated IgM phase II antibodies in significantly more sera than did ELISA (31.8% versus 19.7%), although the portion of solitary IgM phase II was equal for IFAT and for ELISA (18.2% and 16.7%, respectively). Twelve months after the diagnosis of acute Q fever, 83.5% and 62.2% of the sera were still positive for IgM phase II with IFAT and ELISA, respectively. At 12 months IFAT IgG phase II showed the slowest decline. Therefore, definitive serological evidence of acute Q fever cannot be based on a single serum sample in areas of epidemicity and should involve measurement of both IgM and IgG antibodies in paired serum. Based on IgG phase II antibody detection in paired samples (at 0 and 3 months) from 62 patients, IFAT confirmed more cases than ELISA and CFT, but the differences were not statically significant (100% for IFAT, 95.2% for ELISA, and 96.8% for CFT). This study demonstrated that the three serological tests are equally effective in diagnosing acute Q fever within 3 months of start of symptoms. In follow-up sera, more IgG antibodies were detected by IFAT than by ELISA or CFT, making IFAT more suitable for prevaccination screening programs.
A Lysine-Rich Region within Fungal BAG Domain-Containing Proteins Mediates a Novel Association with Ribosomes
Authors: Verghese Jacob , Morano Kevin A.
Published: May 2012

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that assists in the folding of nascent chains and the repair of unfolded proteins through iterative cycles of ATP binding, hydrolysis, and nucleotide exchange tightly coupled to polypeptide binding and release. Cochaperones, including nucleotide exchange factors (NEFs), modulate the rate of ADP/ATP exchange and serve to recruit Hsp70 to distinct processes or locations. Among three nonrelated cytosolic NEFs in
Saccharomyces cerevisiae
, the Bag-1 homolog
SNL1
is unique in being tethered to the endoplasmic reticulum (ER) membrane. We demonstrate here a novel physical association between Snl1 and the intact ribosome. This interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. Additionally, we demonstrate a ribosome association with the
Candida albicans
Snl1 homolog and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. We therefore propose that the Snl1 family of NEFs serves a previously unknown role in fungal protein biogenesis based on the coincident recruitment of ribosomes and Hsp70 to the ER membrane.
Novel
l
-Cysteine-Dependent Maleylpyruvate Isomerase in the Gentisate Pathway of Paenibacillus sp. Strain NyZ101
Authors: Liu Ting-Ting , Zhou Ning-Yi
Published: May 2012

Glutathione- and mycothiol-dependent maleylpyruvate isomerases are known to be involved, respectively, in gentisate catabolism in Gram-negative and high G+C Gram-positive strains. In the present study, a low-G+C Gram-positive
Paenibacillus
sp. strain, NyZ101, was isolated and shown to degrade 3-hydroxybenzoate via gentisate. A 6.5-kb fragment containing a conserved region of gentisate 1,2-dioxygenase genes was cloned and sequenced, and four genes (
bagKLIX
) were shown to encode the enzymes involved in the catabolism to central metabolites of 3-hydroxybenzoate via gentisate. The Bag proteins share moderate identities with the reported enzymes in the 3-hydroxybenzoate catabolism, except BagL that had no obvious homology with any functionally characterized proteins. Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration. BagL was demonstrated to be a novel thiol-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate with
l
-cysteine, cysteinylglycine, or glutathione, as its cofactor. The

K
m

values of these three thiols for BagL were 15.5, 8.4, and 552 μM, respectively. Since cysteine and coenzyme A were reported to be abundant in low-G+C Gram-positive strains, BagL should utilize
l
-cysteine as its physiological cofactor
in vivo
. The addition of Ni
2+
increased BagL activity, and site-directed mutagenesis experiments indicated that three conserved histidines in BagL were associated with binding to Ni
2+
ion and were necessary for its enzyme activity. BagL is the first characterized
l
-cysteine-dependent catabolic enzyme in microbial metabolism and is likely a new and distinct member of DinB family, with a four-helix-bundle topology, as deduced by sequence analysis and homology modeling.
MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis
Authors: Murdeshwar Maya S. , Chatterji Dipankar
Published: May 2012

In the noninfectious soil saprophyte
Mycobacterium smegmatis
, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel
Msm
. This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The

rel
Msm

knockout strain of
M. smegmatis
(Δ

rel
Msm

) is expected to show a (p)ppGpp null [(p)ppGpp
0
] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp
in vivo
. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in
M. smegmatis
. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel
Msm
in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as
d
omain of
u
nknown
f
unction 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active
in vivo
and behaves like a monofunctional (p)ppGpp synthetase
in vitro
. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an
in vivo
role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.
The
bhuQ
Gene Encodes a Heme Oxygenase That Contributes to the Ability of Brucella abortus 2308 To Use Heme as an Iron Source and Is Regulated by Irr
Authors: Ojeda Jenifer F. , Martinson David A. , Menscher Evan A. , Roop R. Martin
Published: May 2012

The
Brucella
BhuQ protein is a homolog of the
Bradyrhizobium japonicum
heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of
Brucella abortus
2308 to use heme as an iron source, an isogenic
bhuQ
mutant was constructed and its phenotype evaluated. Although the
Brucella abortus bhuQ
mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity
in vitro
, this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a
bhuQ
mutation into the
B. abortus dhbC
mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the
dhbC bhuQ
double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl
3
, but not heme, to the growth medium. The
bhuQ
gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that
B. abortus
2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the
rirA-bhuQ
operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source.
Evaluation of the New NucliSENS EasyQ KPC Test for Rapid Detection of Klebsiella pneumoniae Carbapenemase Genes (
bla
KPC
)
Authors: Spanu Teresa , Fiori Barbara , D'Inzeo Tiziana , Canu Giulia , Campoli Serena , Giani Tommaso , Palucci Ivana , Tumbarello Mario , Sanguinetti Maurizio , Rossolini Gian Maria
Published: May 2012

KPC-type carbapenemases are emerging in
Klebsiella pneumoniae
and other Gram-negative pathogens worldwide. Rapid and sensitive detection of these resistance determinants has become relevant to clinical management and infection control. We evaluated the bioMérieux EasyQ real-time PCR assay for
bla
KPC
detection with 300 members of the
Enterobacteriaceae
, including 29 control strains producing known carbapenemases and 271 nonreplicate clinical isolates. The EasyQ assay correctly detected all of the 111 isolates harboring
bla
KPC
genes, with no false positives, and results were available within 2 h.
Nonclinical Selection Criteria for Maximizing Yield of Nucleic Acid Amplification Tests in Tuberculosis Diagnosis
Authors: Han Linda L. , Elvin Paul , Bernardo John
Published: May 2012

In spite of the excellent performance of rapid tuberculosis (TB) nucleic acid amplification (NAA) tests and the clear benefits of immediate diagnosis of TB disease, NAA tests frequently are not used in the diagnosis of pulmonary TB cases, particularly TB cases with smear-negative sputa. Public health laboratories primarily perform TB NAA tests only on a targeted subset of specimens, usually including those that are smear positive and those for which a clinician has specifically requested NAA testing. As an alternative to targeted testing, some laboratories use TB NAA tests universally for all respiratory specimens, though this practice can be prohibitively costly and can be associated with an increased frequency of false-positive results due to testing of lower-risk patients. We propose a strategy for identifying individuals for NAA testing on the basis of nonclinical risk criteria that are routinely provided on the test requisition form, such as type of health care facility from which the specimen is received and patient age group. Use of this strategy at the Massachusetts Department of Public Health Laboratory would allow for NAA test identification of approximately 54 (74%) of 72 culture-positive pulmonary TB cases over a 1-year period while requiring NAA testing for only 933 (17%) of 5,469 individuals submitting respiratory specimens. We demonstrate that use of nonclinical NAA test selection criteria is an effective strategy for maximizing the number of TB cases that can be rapidly identified while minimizing the number of specimens that must be tested.
Coinfection of Leptomonas seymouri and Leishmania donovani in Indian Leishmaniasis
Authors: Ghosh Susmita , Banerjee Priyanka , Sarkar Avijit , Datta Simanti , Chatterjee Mitali
Published: May 2012

Leishmania donovaniis considered the causative organism of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). Testing of 4/29 DNA samples from VL and PKDL patients as well as 2/7 field isolates showed an aberrant internal transcribed spacer 1 (ITS1) restriction fragment length polymorphism (RFLP) pattern, which upon sequencing strongly matchedLeptomonas seymouri, thus confirming its presence in Indian leishmaniasis.
Cellular GCN5 Is a Novel Regulator of Human Adenovirus E1A-Conserved Region 3 Transactivation
Authors: Ablack Jailal N. G. , Cohen Michael , Thillainadesan Gobi , Fonseca Gregory J. , Pelka Peter , Torchia Joe , Mymryk Joe S.
Published: May 2012

The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (
hat/hat
) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.
Metagenomic Characterization of Airborne Viral DNA Diversity in the Near-Surface Atmosphere
Authors: Whon Tae Woong , Kim Min-Soo , Roh Seong Woon , Shin Na-Ri , Lee Hae-Won , Bae Jin-Woo
Published: May 2012

Airborne viruses are expected to be ubiquitous in the atmosphere but they still remain poorly understood. This study investigated the temporal and spatial dynamics of airborne viruses and their genotypic characteristics in air samples collected from three distinct land use types (a residential district [RD], a forest [FR], and an industrial complex [IC]) and from rainwater samples freshly precipitated at the RD site (RD-rain). Viral abundance exhibited a seasonal fluctuation in the range between 1.7 × 106and 4.0 × 107viruses m−3, which increased from autumn to winter and decreased toward spring, but no significant spatial differences were observed. Temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. Metagenomic analysis of air viromes amplified by rolling-circle phi29 polymerase-based random hexamer priming indicated the dominance of plant-associated single-stranded DNA (ssDNA) geminivirus-related viruses, followed by animal-infecting circovirus-related sequences, with low numbers of nanoviruses and microphages-related genomes. Particularly, the majority of the geminivirus-related viruses were closely related to ssDNA mycoviruses that infect plant-pathogenic fungi. Phylogenetic analysis based on the replication initiator protein sequence indicated that the airborne ssDNA viruses were distantly related to known ssDNA viruses, suggesting that a high diversity of viruses were newly discovered. This research is the first to report the seasonality of airborne viruses and their genetic diversity, which enhances our understanding of viral ecology in temperate regions.
Identification of Novel Host Cell Binding Partners of Oas1b, the Protein Conferring Resistance to Flavivirus-Induced Disease in Mice
Authors: Courtney S. C. , Di H. , Stockman B. M. , Liu H. , Scherbik S. V. , Brinton M. A.
Published: May 2012

Oas1b was previously identified as the product of the
Flv

r

allele that confers flavivirus-specific resistance to virus-induced disease in mice by an uncharacterized, RNase L-independent mechanism. To gain insights about the mechanism by which Oas1b specifically reduces the efficiency of flavivirus replication, cellular protein interaction partners were identified and their involvement in the Oas1b-mediated flavivirus resistance mechanism was analyzed. Initial difficulties in getting the two-hybrid assay to work with full-length Oas1b led to the discovery that this Oas protein uniquely has a C-terminal transmembrane domain that targets it to the endoplasmic reticulum (ER). Two peptides matching to oxysterol binding protein-related protein 1L (ORP1L) and ATP binding cassette protein 3, subfamily F (ABCF3), were identified as Oas1b interaction partners in yeast two-hybrid assays, and both
in vitro
-transcribed/translated peptides and full-length proteins in mammalian cell lysates coimmunoprecipitated with Oas1b. Knockdown of a partner involved in Oas1b-mediated antiflavivirus activity would be expected to increase flavivirus replication but not that of other types of viruses. However, RNA interference (RNAi) knockdown of ORP1L decreased the replication of the flavivirus West Nile virus (WNV) as well as that of other types of RNA viruses. This virus-nonspecific effect may be due to the recently reported dysregulation of late endosome movement by ORP1L knockdown. Knockdown of ABCF3 protein levels increased the replication of WNV but not that of other types of RNA viruses, and this effect on WNV replication was observed only in Oas1b-expressing cells. The results suggest that Oas1b is part of a complex located in the ER and that ABCF3 is a component of the
Flv

r

-mediated resistance mechanism.
Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus
Authors: Fuchs Walter , Backovic Marija , Klupp Barbara G. , Rey Felix A. , Mettenleiter Thomas C.
Published: May 2012

Glycoprotein H (gH) is an envelope protein conserved in the
Herpesviridae
. Together with glycoprotein B (gB), the heterodimeric complex of gH and glycoprotein L (gL) mediates penetration and direct viral cell-to-cell spread. In herpes simplex and pseudorabies virus (PrV), coexpression of gH/gL, gB, and gD induces membrane fusion to form polykaryocytes. The recently determined crystal structure of a core fragment of PrV gH revealed marked structural similarity to other gH proteins (M. Backovic et al., Proc. Natl. Acad. Sci. U. S. A. 107:22635–22640, 2010). Within the membrane-proximal part (domain IV), a conserved negatively charged surface loop (flap) is flanked by intramolecular disulfide bonds. Together with an N-linked carbohydrate moiety, this flap covers an underlying patch of hydrophobic residues. To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry.
Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein
Authors: Kemler Iris , Saenz Dyana , Poeschla Eric
Published: May 2012

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.
A Novel Bat Herpesvirus Encodes Homologues of Major Histocompatibility Complex Classes I and II, C-Type Lectin, and a Unique Family of Immune-Related Genes
Authors: Zhang Huajun , Todd Shawn , Tachedjian Mary , Barr Jennifer A. , Luo Minhua , Yu Meng , Marsh Glenn A. , Crameri Gary , Wang Lin-Fa
Published: May 2012

Herpesviruses or herpesviral sequences have been identified in various bat species. Here, we report the isolation, cell tropism, and complete genome sequence of a novel betaherpesvirus from the bat
Miniopterus schreibersii
(MsHV). In primary cell culture, MsHV causes cytopathic effects (CPE) and reaches peak virus production 2 weeks after infection. MsHV was found to infect and replicate less efficiently in a feline kidney cell, CRFK, and failed to replicate in 13 other cell lines tested. Sequencing of the MsHV genome using the 454 system, with a 224-fold coverage, revealed a genome size of 222,870 bp. The genome was extensively analyzed in comparison to those of related viruses. Of the 190 predicted open reading frames (ORFs), 40 were identified as herpesvirus core genes. Among 93 proteins with identifiable homologues in tree shrew herpesvirus (THV), human cytomegalovirus (HCMV), or rat cytomegalovirus (RCMV), most had highest sequence identities with THV counterparts. However, the MsHV genome organization is colinear with that of RCMV rather than that of THV. The following unique features were discovered in the MsHV genome. One predicted protein, B125, is similar to human herpesvirus 6 (HHV-6) U94, a homologue of the parvovirus Rep protein. For the unique ORFs, 7 are predicted to encode major histocompatibility complex (MHC)-related proteins, 2 to encode MHC class I homologues, and 3 to encode MHC class II homologues; 4 encode the homologues of C-type lectin- or natural killer cell lectin-like receptors;, and the products of a unique gene family, the b149 family, of 16 members, have no significant sequence identity with known proteins but exhibit immunoglobulin-like beta-sandwich domains revealed by three-dimensional (3D) structural prediction. To our knowledge, MsHV is the first virus genome known to encode MHC class II homologues.
Complete Genome Sequence of a Novel Marine Siphovirus, pVp-1, Infecting Vibrio parahaemolyticus
Authors: Kim Ji Hyung , Jun Jin Woo , Choresca Casiano H. , Shin Sang Phil , Han Jee Eun , Park Se Chang
Published: May 2012

Among the abundant bacteriophages that belong to the order
Caudovirales
in the ocean, the genome sequences of marine siphoviruses are poorly investigated in comparison to those of myo- or podoviruses. Here we report the complete genome sequence of
Vibrio
phage pVP-1, which belongs to the family
Siphoviridae
and infects
Vibrio parahaemolyticus
ATCC 33844.
Unexpected Maintenance of Hepatitis C Viral Diversity following Liver Transplantation
Authors: Gray Rebecca R. , Strickland Samantha L. , Veras Nazle M. , Goodenow Maureen M. , Pybus Oliver G. , Lemon Stanley M. , Fried Michael W. , Nelson David R. , Salemi Marco
Published: May 2012

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis in up to 20% of individuals, often requiring liver transplantation. Although the new liver is known to be rapidly reinfected, the dynamics and source of the reinfecting virus(es) are unclear, resulting in some confusion concerning the relationship between clinical outcome and viral characteristics. To clarify the dynamics of liver reinfection, longitudinal serum viral samples from 10 transplant patients were studied. Part of the E1/E2 region was sequenced, and advanced phylogenetic analysis methods were used in a multiparameter analysis to determine the history and ancestry of reinfecting lineages. Our results demonstrated the complexity of HCV evolutionary dynamics after liver transplantation, in which a large diverse population of viruses is transmitted and maintained for months to years. As many as 30 independent lineages in a single patient were found to reinfect the new liver. Several later posttransplant lineages were more closely related to older pretransplant viruses than to viruses detected immediately after transplantation. Although our data are consistent with a number of interpretations, the persistence of high viral genetic variation over long periods of time requires an active mechanism. We discuss possible scenarios, including frequency-dependent selection or variation in selective pressure among viral subpopulations, i.e., the population structure. The latter hypothesis, if correct, could have relevance to the success of newer direct-acting antiviral therapies.
Defective Antiviral Responses of Induced Pluripotent Stem Cells to Baculoviral Vector Transduction
Authors: Chen Guan-Yu , Hwang Shiaw-Min , Su Hung-Ju , Kuo Chien-Yi , Luo Wen-Yi , Lo Kai-Wei , Huang Cheng-Chieh , Chen Chiu-Ling , Yu Sheng-Han , Hu Yu-Chen
Published: May 2012

Genetic engineering of induced pluripotent stem cells (iPSCs) is important for their clinical applications, and baculovirus (BV) holds promise as a gene delivery vector. To explore the feasibility of using BV for iPSCs transduction, in this study we first examined how iPSCs responded to BV. We determined that BV transduced iPSCs efficiently, without inducing appreciable negative effects on cell proliferation, apoptosis, pluripotency, and differentiation. BV transduction slightly perturbed the transcription of 12 genes involved in the Toll-like receptor (TLR) signaling pathway, but at the protein level BV elicited no well-known cytokines (e.g., interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α], and beta interferon [IFN-β]) except for IP-10. Molecular analyses revealed that iPSCs expressed no TLR1, -6, -8, or -9 and expressed merely low levels of TLR2, -3, and -4. In spite of evident expression of such RNA/DNA sensors as RIG-I and AIM2, iPSCs barely expressed MDA5 and DAI (DNA-dependent activator of IFN regulatory factor [IRF]). Importantly, BV transduction of iPSCs stimulated none of the aforementioned sensors or their downstream signaling mediators (IRF3 and NF-κB). These data together confirmed that iPSCs responded poorly to BV due to the impaired sensing and signaling system, thereby justifying the transduction of iPSCs with the baculoviral vector.
Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21
Authors: Adeyemi Richard O. , Pintel David J.
Published: May 2012

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G
2
/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK.