bluuebunny/crossref_metadata_2025 huggingface dataset:
Clutter suppression and classification using twin inverted pulse sonar (TWIPS)
Authors: Leighton T. G. , Finfer D. C. , White P. R. , Chua G.-H. , Dix J. K.
Published: June 2010

This paper describes the detection and classification of targets against clutter by distinguishing between linear and nonlinear scatterers and, further, by distinguishing those nonlinear targets that scatter energy at the even-powered harmonics from those that scatter in the odd-powered harmonics. This is done using twin inverted pulse sonar (TWIPS), which can also, in some manifestations, require no range correction (and therefore does not require the
a priori
knowledge of the environment needed for most remote detection technologies). The method applies, in principle, to a range of sensor technologies, including the use of radar to distinguish between circuitry, metal and soil; Light Detection and Ranging (LIDAR) to detect combustion products; and Magnetic Resonance Imaging (MRI). A sonar application is demonstrated, detecting objects in bubbly water (including in the wake of a ship of 3953 gross register tonnage). A man-made sonar that can operate in bubbly water is relevant: Cold War sonar is not optimized for the shallow coastal waters that typify many current operations. The US Navy use dolphins in such waters. TWIPS arose as a demonstration that echolocation was possible in bubbly water in response to a video showing dolphins generating bubble nets when hunting: if echolocation were impossible in these nets, then during this hunt, the dolphins would have blinded their sonar.
Herbivore-Induced SABATH Methyltransferases of Maize That Methylate Anthranilic Acid Using S-Adenosyl-l-Methionine
Authors: Köllner Tobias G. , Lenk Claudia , Zhao Nan , Seidl-Adams Irmgard , Gershenzon Jonathan , Chen Feng , Degenhardt Jörg
Published: June 2010

Volatile methyl esters are common constituents of plant volatiles with important functions in plant defense. To study the biosynthesis of these compounds, especially methyl anthranilate and methyl salicylate, we identified a group of methyltransferases that are members of the SABATH enzyme family in maize (Zea mays). In vitro biochemical characterization after bacterial expression revealed three S-adenosyl-l-methionine-dependent methyltransferases with high specificity for anthranilic acid as a substrate. Of these three proteins, Anthranilic Acid Methyltransferase1 (AAMT1) appears to be responsible for most of the S-adenosyl-l-methionine-dependent methyltransferase activity and methyl anthranilate formation observed in maize after herbivore damage. The enzymes may also be involved in the formation of low amounts of methyl salicylate, which are emitted from herbivore-damaged maize. Homology-based structural modeling combined with site-directed mutagenesis identified two amino acid residues, designated tyrosine-246 and glutamine-167 in AAMT1, which are responsible for the high specificity of AAMTs toward anthranilic acid. These residues are conserved in each of the three main clades of the SABATH family, indicating that the carboxyl methyltransferases are functionally separated by these clades. In maize, this gene family has diversified especially toward benzenoid carboxyl methyltransferases that accept anthranilic acid and benzoic acid.
Disruption of Signaling in a Fungal-Grass Symbiosis Leads to Pathogenesis
Authors: Eaton Carla J. , Cox Murray P. , Ambrose Barbara , Becker Matthias , Hesse Uljana , Schardl Christopher L. , Scott Barry
Published: June 2010

Symbiotic associations between plants and fungi are a dominant feature of many terrestrial ecosystems, yet relatively little is known about the signaling, and associated transcriptome profiles, that define the symbiotic metabolic state. Using the Epichloë festucae-perennial ryegrass (Lolium perenne) association as a model symbiotic experimental system, we show an essential role for the fungal stress-activated mitogen-activated protein kinase (sakA) in the establishment and maintenance of this mutualistic interaction. Deletion of sakA switches the fungal interaction with the host from mutualistic to pathogenic. Infected plants exhibit loss of apical dominance, premature senescence, and dramatic changes in development, including the formation of bulb-like structures at the base of tillers that lack anthocyanin pigmentation. A comparison of the transcriptome of wild-type and sakA associations using high-throughput mRNA sequencing reveals dramatic changes in fungal gene expression consistent with the transition from restricted to proliferative growth, including a down-regulation of several clusters of secondary metabolite genes and up-regulation of a large set of genes that encode hydrolytic enzymes and transporters. Analysis of the plant transcriptome reveals up-regulation of host genes involved in pathogen defense and transposon activation as well as dramatic changes in anthocyanin and hormone biosynthetic/responsive gene expression. These results highlight the fine balance between mutualism and antagonism in a plant-fungal interaction and the power of deep mRNA sequencing to identify candidate sets of genes underlying the symbiosis.
Interference of redroot pigweed (Amaranthus retroflexus) in green bean (Phaseolus vulgaris)
Authors: MIRSHEKARI BAHRAM , JAVANSHIR AZIZ , ARBAT HAMDOLLAH KAZEMI
Published: June 2010

Weeds that emerge along with or immediately after crop plants usually can reduce the yield of those crops. Two randomized complete block design experiments were conducted during 2006 and 2007 in Tabriz, Iran to determine the critical period of redroot pigweed control in the green bean hybrid “Cantander”. The treatments were weed‐infested and weed‐free plots at 2, 4, 6, 8, 10, and 14 weeks after bean emergence (WABE). The green bean biomass was affected by the early emergence of redroot pigweed, but it was not reduced when redroot pigweed emerged at 10 weeks after crop emergence, along with crop emergence, and grew with green bean until 4 WABE. The redroot pigweed biomass decreased by 2.7 g m−2 per day when weed emergence was delayed. Each 100 g m−2 of weed biomass that was produced resulted in a 1.4 kg ha−1 loss in the green bean yield. When redroot pigweed interference lasted for ≥4 weeks after green bean emergence, the green bean yield was reduced significantly. Weeds, which emerged 2 weeks after green bean and thereafter were controlled, did not decrease crop productivity significantly. The highest crop yield was obtained when the weed emerged at 14 WABE. The critical period of redroot pigweed control, considering a 10% yield loss, was between 19 and 55 days after green bean emergence. Thus, weed control practises should be begun no later than 3 WABE and should continue until at least 8 WABE in order to obtain the maximum green bean yield.
Biological responses of a non‐target aquatic plant (Nasturtium officinale) to the herbicide, tribenuron‐methyl
Authors: DUMAN FATIH , UREY ERKAN , TEMIZGUL RIDVAN , BOZOK FUAT
Published: June 2010

To assess its response to the herbicide, tribenuron‐methyl, samples of Nasturtium officinale were exposed to 0, 0.01, 0.05, 0.1, and 0.5 mg L−1 of tribenuron‐methyl for 1, 2, 4 and 7 days. The influence of this herbicide on the relative growth rate, electrolyte leakage, lipid peroxidation, photosynthetic pigmentation, protein content, and performance of anti‐oxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate peroxidase (APX), was examined. The results indicated that tribenuron‐methyl, applied at 0.5 mg L−1, affected plant growth negatively. It also was determined that chlorophyll a is the most responsive photosynthetic pigment to tribenuron–methyl exposure. Under stress conditions, the anti‐oxidant enzymes were up‐regulated compared to the control. The SOD activity was significantly stimulated, while the activity of APX was inhibited. A significant correlation was found between lipid peroxidation and SOD activity. The exposure period and herbicide concentration had significant effects on the biological responses against tribenuron‐methyl stress. These results may be useful for clarifying the effect of herbicides on non‐target aquatic plants.
Genetic diversity and population genetic differentiation in the endangered annual weed, Bidens cernua (Compositae), and two common congeners in Japan
Authors: TANAHARA AI , MAKI MASAYUKI
Published: June 2010

In this study, we examined the genetic diversity in three populations of the critically endangered annual, Bidens cernua, in Japan by using inter‐simple sequence repeat markers and compared our data with those from two common congeners: Bidens radiata var. pinnatifida and Bidens tripartita. In contrast to our expectations, the degree of genetic diversity at the species level was higher in B. cernua than in B. radiata var. pinnatifida or B. tripartita. At the population level, the degree of genetic diversity was highest in B. cernua. These results may be ascribed to the mating system and method of seedbank formation in B. cernua. An analysis of molecular variance revealed relatively high genetic differentiation among the populations of all three species. We concluded that the distribution width could not be an index of genetic variability in Bidens examined in this study.
Estimates of milk constituents from lactating bonnet macaque (Macaca radiata) mothers between two and seven months post‐partum
Authors: Laudenslager Mark L. , Natvig Crystal , Cantwell Holly , Neville Margaret C. , Reite Martin L.
Published: June 2010

Background  The literature regarding milk composition in non‐human primates collected across offspring development is limited. We assayed milk samples from bonnet macaque (Macaca radiata) mothers as part of studies characterizing development of this species.

Methods  Milk was obtained when possible longitudinally from seven lactating bonnet macaque mothers. Samples were frozen until analysis. Individual samples were analyzed to determine the concentrations of electrolytes including sodium, potassium, calcium, chloride, and magnesium, as well as urea, protein, lipids, glucose, and lactose.

Results  A trend for increased lipids as well as protein percentage was noted with increasing infant age. Chloride and calcium showed an increase with age, whereas other electrolytes remained relatively stable across development.

Conclusions  The composition of the milk of this particular macaque species was similar to other Old World primates as well as humans. These data add to the limited information available on milk constituents among mammals.
A Proposal for Teaching Introductory and Intermediate Accounting in an Environment of International Financial Reporting Standards and Generally Accepted Accounting Principles for Private Enterprises*
Authors: PRIES FRED , BAKER RON
Published: June 2010

Teaching introductory and intermediate financial accounting in an environment of international financial reporting standards (IFRS) and generally accepted accounting principles for private enterprises (GAAP for PEs) is an issue facing faculty at Canadian universities and colleges. We present a number of options and then propose an approach that we believe provides a balanced treatment of accounting standards for both publicly accountable and private enterprises. This approach focuses on the concepts and principles that are common to both IFRS and GAAP for PEs. We argue that this approach encourages deep learning resulting in students’ better understanding of accounting standards and their application.
The Ontario Securities Commission on Accounting and Auditing from the 1960s to 2008 – Part 1: 1960s to 1985*
Authors: ZEFF STEPHEN A. , RADCLIFFE VAUGHAN S.
Published: June 2010

We present Part 1 of a historical review and analysis of the role played by the Ontario Securities Commission (OSC) in accounting and auditing from the 1960s onwards. Part 1 deals with the period from the 1960s to 1985. Parts 2 and 3 will treat the roles played by the first five Chief Accountants, from 1986 to 2008. As the principal Canadian stock exchange in recent times has been the Toronto Stock Exchange, the OSC has been the most important securities market regulator in Canada. Prior to this article, the academic and professional accounting literature has been largely barren on the OSC’s evolving role on accounting and auditing issues.
Pre‐ and postsynaptic modulation of monosynaptic reflex by GABAAreceptors on turtle spinal cord
Authors: Bautista Wendy , Aguilar Justo , Loeza‐Alcocer José Emanuel , Delgado‐Lezama Rodolfo
Published: June 2010

There is growing evidence that activation of high affinity extrasynaptic GABAAreceptors in the brain, cerebellum and spinal cord substantia gelatinosa results in a tonic inhibition controlling postsynaptic excitability. The aim of the present study was to determine if GABAAreceptors mediating tonic inhibition participate in the modulation of monosynaptic reflex (MSR) in the vertebrate spinal cord. Using anin vitroturtle lumbar spinal cord preparation, we show that conditioning stimulation of a dorsal root depressed the test monosynaptic reflex (MSR) at long condition–test intervals. This long duration inhibition is similar to the one seen in mammalian spinal cord and it is dependent on GABAAas it was completely blocked by 20 μmpicrotoxin (PTX) or bicuculline (BIC) or 1 μmgabazine, simultaneously depressing the dorsal root potential (DRP) without MSR facilitation. Interestingly 100 μmpicrotoxin or BIC potentiated the MSR, depressed the DRP, and produced a long lasting motoneurone after‐discharge. Furosemide, a selective antagonist of extrasynaptic GABAAreceptors, affects receptor subtypes with α4/6subunits, and in a similar way to higher concentrations of PTX or BIC, also potentiated the MSR but did not affect the DRP, suggesting the presence of α4/6GABAAreceptors at motoneurones. Our results suggest that (1) the turtle spinal cord has a GABAAmediated long duration inhibition similar to presynaptic inhibition observed in mammals, (2) GABAAreceptors located at the motoneurones and primary afferents might produce tonic inhibition of monosynaptic reflex, and (3) GABAAreceptors modulate motoneurone excitability reducing the probability of spurious and inappropriate activation.
The Incidence of Fire in Amazonian Forests with Implications for REDD
Authors: Aragão Luiz E. O. C. , Shimabukuro Yosio E.
Published: June 2010

Seeing REDD

Reducing emissions from deforestation and degradation (REDD) has become a key economic mechanism in the global strategy to reduce the rate of anthropogenic climate change. For Brazilian Amazonia,
Aragão and Shimabukuro
(p.
1275
) point out that the efficacy of REDD could depend not only on avoiding deforestation but also on tackling fire usage. A satellite-based time-series of Amazonian deforestation rates and fire incidence suggests an increased trend in fire incidence across most of the region, despite the overall decrease in deforestation rates. The survey also suggests that the introduction of managed agriculture, instead of traditional slash-and-burn land use in already deforested areas, may promote a reduction of in fire incidence in the Amazon.
Permissive Secondary Mutations Enable the Evolution of Influenza Oseltamivir Resistance
Authors: Bloom Jesse D. , Gong Lizhi Ian , Baltimore David
Published: June 2010

Influenza Escape Tricks

Tamiflu, or oseltamivir, has been extensively stockpiled by several governments in anticipation of a dangerous influenza pandemic. So far, its large-scale use has not been required, but, despite this, resistance has emerged in seasonal strains mediated by a single point mutation of histidine to tyrosine in the 274 residue (H274Y) of neuraminidase. When the resistant virus was first discovered in 1998, it grew poorly, but by 2008 the virus was reinvigorated and the mutation had spread worldwide in seasonal influenza. So what happened that improved viral fitness so radically?

Bloom
et al.

(p.
1272
; see the Perspective by

Holmes

) show that the H274Y mutation hinders the folding of the neuraminidase enzyme. In the more vigorous recent oseltamivir-resistant isolates, other mutations compensate for the deleterious effect of H274Y and restore fitness to the virus.
Operation Mechanism of a Molecular Machine Revealed Using Time-Resolved Vibrational Spectroscopy
Authors: Panman Matthijs R. , Bodis Pavol , Shaw Daniel J. , Bakker Bert H. , Newton Arthur C. , Kay Euan R. , Brouwer Albert M. , Buma Wybren Jan , Leigh David A. , Woutersen Sander
Published: June 2010

Not So Random Walk

In rotaxanes, a molecular ring can shuttle back and forth between docking sites along an axle.

Panman
et al.

(p.
1255
) traced the intricacies of this shuttling motion using vibrational spectroscopy. The kinetics were dominated by the slow scission of hydrogen bonds tying the ring to its starting site. Varying the length of the axle allowed the extraction of relative rates for forward and backward motion once the ring was free: Somewhat surprisingly, forward motion toward the destination site was slightly hindered relative to regression toward the starting place.
SphK1 Regulates Proinflammatory Responses Associated with Endotoxin and Polymicrobial Sepsis
Authors: Puneet Padmam , Yap Celestial T. , Wong Lingkai , Yulin Lam , Koh Dow Rhoon , Moochhala Shabbir , Pfeilschifter Josef , Huwiler Andrea , Melendez Alirio J.
Published: June 2010

Sepsis Protection

Sepsis is a serious medical condition characterized by an uncontrolled inflammatory response to infection. Sepsis often results in organ failure and/or death, and current treatments are not very effective.

Puneet
et al.

(p.
1290
) now show that the enzyme sphingosine kinase 1 (SphK1) may represent an important therapeutic target for the treatment of sepsis. SphK1 expression increased on human phagocytes in response to bacterial products and was also highly expressed on phagocytes from septic patients. Inhibition of SphK1 reduced the production of inflammatory mediators in vitro by human phagocytes stimulated with bacterial products. In vivo, pretreatment with small interfering RNA against SphK1 or a specific SphK1 inhibitor protected mice from death in two lethal models of sepsis. Protection was also seen when mice were treated with the SphK1 inhibitor up to 8 hours after sepsis induction, and this protection was enhanced if mice were given a broad-spectrum antibiotic.
Helping the Resistance
Authors: Holmes Edward C.
Published: June 2010

Phylogenetic analysis reveals mutations that led to the rise of drug resistance in seasonal H1N1 influenza.
Japanese Science in a Global World
Authors: Arai Ken-ichi
Published: June 2010

The beginning of 2010 brought good economic news to Japan, which has seen only lackluster growth for the past two decades. Although the country's debt load and deflation remain serious problems, the world's second largest economy reportedly grew in the last quarter of 2009. But that does not mean that Japan is on a route to long-term recovery. Although the science budget for fiscal year 2011 was not severely cut, a worrisome sign was the government's attempts to freeze investment in Japan's science infrastructure (for example, supercomputing) and reduce spending on earth sciences, cosmology, and other fields. These were avoided through the protests of prominent Japanese scientists. The lack of political interest in bolstering investment in science and technology indicates misguided thinking. There is therefore a growing awareness in the Japanese research community that scientists need to become more involved in formulating the country's science policy and in guiding young scientists into international networks that will support a successful global economy for Japan.
Louisiana Begins Controversial Engineering to Ward Off Oil Spill
Authors: Stokstad Erik
Published: June 2010

The magnitude of the disaster in the Gulf of Mexico—the largest oil spill in U.S. history—has inspired an unprecedented and untested idea for combating it: an extensive sand trap. But scientists are dubious about the project's chance of success.
No 'Smoking Gun' for Killer Oil
Authors: Schenkman Lauren
Published: June 2010

As turtle and dolphin corpses wash up on Gulf of Mexico beaches, scientists face a sleuthing challenge worthy of
CSI
: determining whether oil had a hand in the deaths.
Major Heart Disease Genes Prove Elusive
Authors: Couzin-Frankel Jennifer
Published: June 2010

So far, genome-wide association studies have not found common genes with a big impact on heart health; researchers hope that the low-effect genes they are finding will help identify pathways and drug targets.
An Unending Mission to Contain the Stuff of Nuclear Nightmares
Authors: Bhattacharjee Yudhijit
Published: June 2010

With new tools and painstaking analyses, researchers are helping to detect clandestine nuclear-weapons research and keep deadly material from going astray.
The Wild-Type Hepatitis C Virus Core Inhibits Initiation of Antigen-Specific T- and B-Cell Immune Responses in BALB/c Mice
Authors: Zhu Wenbo , Chang Yanzi , Wu Chunchen , Han Qingxia , Pei Rongjuan , Lu Mengji , Chen Xinwen
Published: June 2010

In this study, the effects of wild-type and deletion mutant hepatitis C virus (HCV) core proteins on the induction of immune responses in BALB/c mice were assessed. p2HA-C145-S23, encoding a core protein with the C-terminal 46 amino acids truncated, significantly produced stronger antibody and cellular responses than p2HA-C191-S23. The induction of immune responses by p2HA-C145-S23 was dose dependent. However, increasing the doses or repeated administration did not enhance immune responses by the wild-type core protein. In addition, p2HA-C191-S23 was apparently able to interfere with the priming of specific immune responses by p2HA-C145-S23 when the two were coadministered. These results demonstrated that the wild-type HCV core protein itself could inhibit the priming of immune responses in the course of a DNA vaccination, whereas the truncated HCV core protein could provide potential applications for the development of DNA- and peptide-based HCV vaccines.
Detection of Human Parechoviruses from Clinical Stool Samples in Aichi, Japan
Authors: Ito Miyabi , Yamashita Teruo , Tsuzuki Hideaki , Kabashima Yuka , Hasegawa Akiko , Nagaya Satoko , Kawaguchi Mariko , Kobayashi Shinichi , Fujiura Akira , Sakae Kenji , Minagawa Hiroko
Published: June 2010

Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5′ untranslated region (5′UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema, “hand, foot, and mouth disease,” aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.
Multiple Nucleotide Substitutions in the
Neisseria meningitidis
Serogroup C
ctrA
Gene Cause False-Negative Detection by Real-Time PCR
Authors: Cavrini Francesca , Liguori Giovanna , Andreoli Arianna , Sambri Vittorio
Published: June 2010

Two strains of
Neisseria meningitidis
serogroup C with disparate sequences of
ctrA
were isolated. The nucleotide substitutions did not alter the corresponding protein sequences, but they impeded the detection of these meningococcal isolates by real-time PCR.
Development of a Particle Agglutination Method with Soluble Virus Receptor for Identification of Poliovirus
Authors: Arita Minetaro , Masujima Souji , Wakita Takaji , Shimizu Hiroyuki
Published: June 2010

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples of patients with acute flaccid paralysis (AFP). In this study, we developed a particle agglutination (PA) method with a soluble human PV receptor (hPVR) in the form of an immunoadhesin (PVR-IgG2a) for the simple and rapid identification of PV. Sensitized gelatin particles with PVR-IgG2a showed specific agglutination with the culture fluid of PV-infected cells within 2 h of reaction in a one-step procedure. Detection limits for type 1, 2, and 3 PV(Sabin) strains were 1.5 × 10
6
50% cell culture infectious doses (CCID
50
), 5.3 × 10
5
CCID
50
, and 9.1 × 10
5
CCID
50
, respectively. Wild-type PVs and PV isolates from acute flaccid paralysis cases examined were identified correctly with this PA method, except for some samples with a mixture of different serotypes of PVs, where a minor population of PV failed to be detected. These results suggest that this PA method is useful for the simple and rapid identification of PV, although the sensitivity was not high enough to detect a minor population of PV (<1/10 of the major population) among mixed PVs.
Three-Hour Molecular Detection of
Campylobacter
,
Salmonella
,
Yersinia
, and
Shigella
Species in Feces with Accuracy as High as That of Culture
Authors: Cunningham Scott A. , Sloan Lynne M. , Nyre Lisa M. , Vetter Emily A. , Mandrekar Jayawant , Patel Robin
Published: June 2010

Campylobacter jejuni
and
Salmonella
,
Shigella
, and
Yersinia
species (along with Shiga toxin-producing
Escherichia coli
) are the most common causes of acute bacterial diarrheal disease in the United States. Current detection techniques are time-consuming, limiting usefulness for patient care. We developed and validated a panel of rapid PCR assays for the detection and identification of
C. jejuni
,
C. coli
,
Salmonella
, and
Yersinia
species and
Shigella
and enteroinvasive
E. coli
in stool samples. A total of 392 archived stool specimens, previously cultured for enteric pathogens, were evaluated by PCR. Overall, 104 stool specimens had been culture positive (
C. jejuni
/
coli
[
n
= 51],
Salmonella
species [
n
= 42],
Shigella
species [
n
= 6], and
Yersinia
species [
n
= 5]). Compared to culture, the overall sensitivity and specificity of PCR detection of these organisms were 92 and 98% (96/104 and 283/288), respectively, from fresh or Cary Blair stool (
P
= 0.41); 87 and 98% (41/47 and 242/246), respectively, from fresh stool (
P
= 0.53); and 96 and 98% (55/57 and 41/42), respectively, from Cary Blair stool (
P
= 0.56). For individual genera, PCR was as sensitive as the culture method, with the exception of
Salmonella
culture using selenite enrichment for which PCR was less sensitive than culture from fresh, but not Cary Blair (
P
= 0.03 and 1.00, respectively) stools. This PCR assay panel for the rapid diagnosis of acute infectious bacterial diarrheal pathogens has a sensitivity and specificity equivalent to that of culture for stools in Cary Blair transport medium. Paired with reflexive culture of stools testing positive, this should provide an improvement in care for patients with acute infectious diarrheal disease.
Unusual Distribution of
Burkholderia cepacia
Complex Species in Danish Cystic Fibrosis Clinics May Stem from Restricted Transmission between Patients
Authors: Nørskov-Lauritsen Niels , Johansen Helle Krogh , Fenger Mette G. , Nielsen Xiaohui C. , Pressler Tacjana , Olesen Hanne V. , Høiby Niels
Published: June 2010

Forty-four of 48
Burkholderia cepacia
complex strains cultured from Danish cystic fibrosis patients were
Burkholderia multivorans
, a distribution of species that has not been reported before. Although cases of cross infections were demonstrated, no major epidemic clone was found. The species distribution may represent the sporadic acquisition of bacteria from the environment.
Immediate Incubation Reduces Indeterminate Results for QuantiFERON-TB Gold In-Tube Assay
Authors: Herrera Victor , Yeh Ellen , Murphy Kelly , Parsonnet Julie , Banaei Niaz
Published: June 2010

In vitro
gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent
Mycobacterium tuberculosis
infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37°C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (
P
= 0.10) and 17.1% (7/41) (
P
= 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (
P
= 0.004) and 12 h (
P
< 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.
Monoclonal Antibody Kit for Identification of the Novel 2009 H1N1 Influenza A Virus
Authors: Higgins Angela D. , Shaw Carl J. , Johnson Jennifer G. , Navarro Adriana , Chapman Nathan A. , Ewers Steven D. , Stockwell James W. , Carpenter Julie M. , Olivo Paul D. , Miao Lynn Yihong
Published: June 2010

To develop an immunofluorescence assay for identification of the 2009 H1N1 influenza A virus, we generated a number of monoclonal antibodies (MAbs) by using inactivated H1N1 2009 virus (A/California/07/2009) as the immunogen. Two MAbs that target two different epitopes of the 2009 H1N1 hemagglutinin (HA) were selected to make the D
3
Ultra 2009 H1N1 Influenza A ID kit (2009 H1N1 ID kit; Diagnostic Hybrids, Inc., Athens, OH), which is intended for the identification of the 2009 H1N1 virus by indirect immunofluorescence assay (IFA). The kit does not detect any of 14 seasonal H1N1 or H3N2 prototype influenza virus strains and is also not reactive with seven other major respiratory viruses. Clinical respiratory specimens were evaluated using both the 2009 H1N1 ID kit and the CDC human influenza virus real-time reverse transcription-PCR swine flu panel (CDC rRT-PCR) and showed 100% agreement between the two assays. Four of these clinical specimens, however, were positive by the 2009 H1N1 ID kit but were identified as presumptively positive by the CDC rRT-PCR by virtue of showing threshold cycle (

C
T

) values only with universal InfA and swInfA primers, not with swH1 primers. Sequence analysis of the HA genes of these four specimens revealed point mutations in both the primer and probe regions. In addition, unlike the CDC rRT-PCR, the 2009 H1N1 ID kit can differentiate the 2009 H1N1 virus from a swine-derived H1 influenza A virus (A/New Jersey/8/76). The 2009 H1N1 ID kit offers clinical laboratories an alternative to RT-PCR for the identification of the 2009 H1N1 influenza A virus.
Detection of Large Numbers of Pneumococcal Virulence Genes in Streptococci of the Mitis Group
Authors: Johnston Calum , Hinds Jason , Smith Andrew , van der Linden Mark , Van Eldere Johan , Mitchell Tim J.
Published: June 2010

Seven streptococcal isolates from the mitis group were analyzed for the presence of pneumococcal gene homologues by comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of
Streptococcus pneumoniae
TIGR4 and R6. The diversity of pneumolysin (
ply
) and neuraminidase A (
nanA
) gene sequences was explored in more detail in a collection of 14
S. pseudopneumoniae
and 29 mitis group isolates, respectively. The mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), two
S. mitis
isolates from the nasopharynxes of children, one
S. mitis
isolate from a case of infective endocarditis, one
S. mitis
isolate from a dental abscess, and one
S. oralis
isolate and one
S. pseudopneumoniae
isolate from the nasopharynxes of children. The results of the microarray study showed that the 5
S. mitis
isolates had homologues to between 67 and 82% of pneumococcal virulence genes,
S. oralis
hybridized to 83% of pneumococcal virulence genes, and
S. pseudopneumoniae
hybridized to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (
mly
), and newly identified pseudopneumolysin (
pply
) gene sequences revealed that
mly
and
pply
genes are more closely related to each other than either is to
ply
. In contrast, the
nanA
gene sequences in the pneumococcus and streptococci from the mitis group are closely clustered together, sharing 99.4 to 99.7% sequence identity with pneumococcal
nanA
alleles.
Evaluation of Multiple-Locus Variable-Number Tandem-Repeat Analysis for Typing a Polyclonal Hospital-Acquired Methicillin-Resistant
Staphylococcus aureus
Population in an Area Where Such Infections Are Endemic
Authors: Rivero-Pérez Belinda , Pérez-Roth Eduardo , Méndez-Álvarez Sebastián
Published: June 2010

Multiple-locus variable-number tandem-repeat analysis (MLVA) was performed with 292 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates previously characterized by pulsed-field gel electrophoresis, multilocus sequence typing, and staphylococcal cassette chromosome
mec
typing. Quantitative correspondence analyses showed the best correlation between data when an ≥80% cutoff was applied to MLVA. We confirmed the validity of MLVA for identification of related strains in a polyclonal MRSA population.
Babesia lengausp. nov., a NovelBabesiaSpecies in Cheetah (Acinonyx jubatus, Schreber, 1775) Populations in South Africa
Authors: Bosman Anna-Mari , Oosthuizen Marinda C. , Peirce Michael A. , Venter Estelle H. , Penzhorn Barend L.
Published: June 2010

In a previous paper, we reported on a large number of cheetah blood specimens that gave positive signals only forBabesiaand/orTheileriagenus-specific probes on the reverse line blot (RLB) assay, indicating the presence of a novel species or variant of an existing species. Some of these specimens were investigated further by microscopic, serological, sequencing, and phylogenetic analyses. The near-full-length 18S rRNA genes of 13 samples, as well as the second internal transcribed spacer (ITS2) region, were amplified, cloned, and sequenced. A species-specific RLB probe, designed to target the hypervariable V4 region of the 18S rRNA gene for detection of the novelBabesiasp., was used to screen an additional 137 cheetah blood specimens for the presence of the species. The prevalence of infection was 28.5%. Here we describe the morphology and phylogenetic relationships of the novel species, which we have namedBabesia lengausp. nov.
Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation
Authors: Wang Manli , Tuladhar Era , Shen Shu , Wang Hualin , van Oers Monique M. , Vlak Just M. , Westenberg Marcel
Published: June 2010

The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the
Baculoviridae
family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a
p6.9
-null mutant of
Autographa californica
multiple nucleopolyhedrovirus (
Ac
MNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of
Alpha
baculovirus
species and one
Gammabaculovirus
species but not from the genus
Betabaculovirus
, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that
Ac
MNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing
p6.9
-null
Ac
MNPV, while a chimeric
Betabaculovirus
P6.9 containing the P6.9 C-terminal region of an
Alphabaculovirus
strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.
Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome
Authors: Somberg Monika , Schwartz Stefan
Published: June 2010

Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.
A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade
Authors: Aguilar Hector C. , Aspericueta Vanessa , Robinson Lindsey R. , Aanensen Karen E. , Lee Benhur
Published: June 2010

The deadly paramyxovirus Nipah virus (NiV) contains a fusion glycoprotein (F) with canonical structural and functional features common to its class. Receptor binding to the NiV attachment glycoprotein (G) triggers F to undergo a two-phase conformational cascade: the first phase progresses from a metastable prefusion state to a prehairpin intermediate (PHI), while the second phase is marked by transition from the PHI to the six-helix-bundle hairpin. The PHI can be captured with peptides that mimic F's heptad repeat regions, and here we utilized a NiV heptad repeat peptide to quantify PHI formation and the half-lives (t1/2) of the first and second fusion cascade phases. We found that ephrinB2 receptor binding to G triggered ∼2-fold more F than that triggered by ephrinB3, consistent with the increased rate and extent of fusion observed with ephrinB2- versus ephrinB3-expressing cells. In addition, for a series of hyper- and hypofusogenic F mutants, we quantified F-triggering capacities and measured the kinetics of their fusion cascade phases. Hyper- and hypofusogenicity can each be manifested through distinct stages of the fusion cascade, giving rise to vastly different half-lives for the first (t1/2, 1.9 to 7.5 min) or second (t1/2, 1.5 to 15.6 min) phase. While three mutants had a shorter first phase and a longer second phase than the wild-type protein, one mutant had the opposite phenotype. Thus, our results reveal multiple critical parameters that govern the paramyxovirus fusion cascade, and our assays should help efforts to elucidate other class I membrane fusion processes.
Cell-Based Quantification of Chronic Wasting Disease Prions
Authors: Bian Jifeng , Napier Dana , Khaychuck Vadim , Angers Rachel , Graham Catherine , Telling Glenn
Published: June 2010

Cell-based measurement of prion infectivity is currently restricted to experimental strains of mouse-adapted scrapie. Having isolated cell cultures with susceptibility to prions from diseased elk, we describe a modification of the scrapie cell assay allowing evaluation of prions causing chronic wasting disease, a naturally occurring transmissible spongiform encephalopathy. We compare this cervid prion cell assay to bioassays in transgenic mice, the only other existing method for quantification, and show this assay to be a relatively economical and expedient alternative that will likely facilitate studies of this important prion disease.
Clonal Expansion of Normal-Appearing Human Hepatocytes during Chronic Hepatitis B Virus Infection
Authors: Mason William S. , Liu Chen , Aldrich Carol E. , Litwin Samuel , Yeh Matthew M.
Published: June 2010

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A.102:1139-1144, 2005; W. S. Mason et al., J. Virol.83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.
Transcription Factor YY1 Interacts with Retroviral Integrases and Facilitates Integration of Moloney Murine Leukemia Virus cDNA into the Host Chromosomes
Authors: Inayoshi Yujin , Okino Yuuki , Miyake Katsuhide , Mizutani Akifumi , Yamamoto-Kishikawa Junko , Kinoshita Yuya , Morimoto Yusuke , Imamura Kazuhito , Morshed Mahboob , Kono Ken , Itoh Toshinari , Nishijima Ken-ichi , Iijima Shinji
Published: June 2010

Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and
in vitro
pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the
in vitro
integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration
in vivo
, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events
in vivo
. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.
Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion
Authors: Huang Kelly , Incognito Len , Cheng Xing , Ulbrandt Nancy D. , Wu Herren
Published: June 2010

Respiratory syncytial virus (RSV) is a major cause of virus-induced respiratory disease and hospitalization in infants. Palivizumab, an RSV-neutralizing monoclonal antibody, is used clinically to prevent serious RSV-related respiratory disease in high-risk infants. Motavizumab, an affinity-optimized version of palivizumab, was developed to improve protection against RSV. These antibodies bind RSV F protein, which plays a role in virus attachment and mediates fusion. Determining how these antibodies neutralize RSV is important to help guide development of new antibody drugs against RSV and, potentially, other viruses. This study aims to uncover the mechanism(s) by which palivizumab and motavizumab neutralize RSV. Assays were developed to test the effects of these antibodies at distinct steps during RSV replication. Pretreatment of virus with palivizumab or motavizumab did not inhibit virus attachment or the ability of F protein to interact with the target cell membrane. However, pretreatment of virus with either of these antibodies resulted in the absence of detectable viral transcription. These results show that palivizumab and motavizumab act at a point after F protein initiates interaction with the cell membrane and before virus transcription. Palivizumab and motavizumab also inhibited F protein-mediated cell-to-cell fusion. Therefore, these results strongly suggest that these antibodies block both cell-to-cell and virus-to-cell fusion, since these processes are likely similar. Finally, palivizumab and motavizumab did not reduce viral budding. Based on models developed from numerous studies of viral fusion proteins, our results indicate that these antibodies may prevent conformational changes in F protein required for the fusion process.