bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjoint Measurement with Constraints on Attribute Levels: A Mixture–Amount Model Approach
Authors: Raghavarao Damaraju , Wiley James B.
Published: July 2009

Traditional and choice‐based conjoint analyses (CAs) have used full or fractional factorial designs to generate concept profile descriptions. However, these designs confound two factors when costs are associated with attributes: first is the total cost of the concept profile, and second is the allocation of costs among the attributes. Both factors may influence consumers' preferences. So far, these issues have not been separated in the CA literature. The present paper shows how mixture–amount designs used in industrial experiments may be used to separate amount and mixture effects in traditional CA. The extension to choice‐based CA using balance incomplete block (BIB) designs is also given.
Spatiotemporal changes in the genetic diversity in French bloom‐forming populations of the toxic cyanobacterium, Microcystis aeruginosa
Authors: Sabart Marion , Pobel David , Latour Delphine , Robin Joel , Salençon Marie‐J. , Humbert Jean‐F.
Published: July 2009

Microcystis aeruginosa is a toxic cyanobacterium, which is able to bloom in a wide range of freshwater ecosystems. By sequencing the Internal Transcribed Spacer (ITS) of the ribosomal operon, we compared the genetic composition of several French bloom‐forming M. aeruginosa populations from two reservoirs located on the Loire River, at two sampling points located between these reservoirs, and finally in two ponds closely linked to this river. No significant difference was found in the genetic diversity of the six Microcystis populations but we evidenced a strong genetic differentiation between most of these populations. Indeed, the Microcystis population in the Grangent reservoir was genetically differentiated from the other three populations sampled further downstream, implying that no massive transfer of population occurs from this reservoir to downstream segments. We also found genetic differentiation between the populations from the two ponds, and between these populations and those from the Loire River. On the other hand, the same dominant genotype was found in the populations sampled both in the river and in the Villerest reservoir, suggesting the selection of a distinct genotype adapted to river conditions and also an accumulation of this genotype in the downstream reservoir. Finally, by comparing our ITS sequences with those available in the GenBank, no biogeographical differentiation could be detected at a global scale, suggesting that most of the Microcystis genotypes seem to be ubiquitous.
Role of Layer 6 of V2 Visual Cortex in Object-Recognition Memory
Authors: López-Aranda Manuel F. , López-Téllez Juan F. , Navarro-Lobato Irene , Masmudi-Martín Mariam , Gutiérrez Antonia , Khan Zafar U.
Published: July 2009

Modulating Visual Memory

Layer 3 of the secondary visual cortical area V2 plays a role in visual information processing. However, in contrast to layer 3, layer 6 of visual cortex is composed of many types of neurons and their response to visual stimuli is more complex. The importance of layer 6 in visual information processing remains an enigma.

López-Aranda
et al.

(p.
87
; see the Perspective by

Saksida

) investigated its role in the rat visual cortex in the processing of both short- and long-term visual memory. Elimination of neurons from layer 6 of area V2 led to complete loss of normal memory. However, overexpression of a protein called RGS-14 in the same area boosted the visual memory capacity in such a way that the animals retained the visual information for many months, rather than the normal 45 minutes.
Dissociable Components of Rule-Guided Behavior Depend on Distinct Medial and Prefrontal Regions
Authors: Buckley Mark J. , Mansouri Farshad A. , Hoda Hassan , Mahboubi Majid , Browning Philip G. F. , Kwok Sze C. , Phillips Adam , Tanaka Keiji
Published: July 2009

Card Sorting Monkeys

Single-neuron studies in primates help to establish a detailed understanding of cognitive processing and to provide an experimental base for understanding the cognitive deficits incurred by patients who have suffered damage to areas of the brain.

Buckley
et al.

(p.
52
) present the results of an intensive behavioral analysis of a group of monkeys bearing lesions to distinct areas of the prefrontal lobe. The Wisconsin Card Sorting Task is widely used in the clinic to assess the flexible learning of abstract rules. In the primates, a functional dissociation was observed across three regions: the principal sulcus, the orbitofrontal cortex, and the anterior cingulate cortex. This set of results contributes to the ongoing discussion of goal-directed behavior and serves to bridge neuropsychological studies in human patients and neurophysiological studies in primates.
A Coherent Single-Hole Spin in a Semiconductor
Authors: Brunner Daniel , Gerardot Brian D. , Dalgarno Paul A. , Wüst Gunter , Karrai Khaled , Stoltz Nick G. , Petroff Pierre M. , Warburton Richard J.
Published: July 2009

A Hole New Approach

Quantum dots can behave as artificial atoms, exhibiting a ladder of quantized energy levels with the number of electrons added to the dot being controllable. They are thus being extensively studied for application in the likes of quantum information processing strategies. However, the electrons interact with their environment and quickly lose their coherence properties.

Brunner
et al.

(p.
70
; see the Perspective by

Kolodrubetz and Petta

) now show that if the charge of the dot is manipulated so that it is positive; that is, populated with a single hole, then the coherence properties of the dot can be extended. The strategy of using holes instead of electrons may provide a solution to the decoherence problem.
Serengeti Birds Maintain Forests by Inhibiting Seed Predators
Authors: Sharam Gregory J. , Sinclair A. R. E. , Turkington Roy
Published: July 2009

Fruit-eating birds inhibit seed predation by beetles, a mechanism that is destabilized when disturbance opens the forest canopy.
Green Life Through Time
Paleobotany
The Biology and Evolution of Fossil Plants. 2nd ed.

by Thomas N. Taylor, Edith L. Taylor, and Michael Krings

Academic (Elsevier), Burlington, MA, 2009. 1252 pp. $125, £62.99, €91.95. ISBN 9780123739728.
Authors: Wilson Jonathan P.
Published: July 2009

An expansion and update of Taylor and Taylor's classic 1993 book, this lavishly illustrated volume offers a comprehensive survey of fossil plants and fungi.
Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing
Authors: Webby Celia J. , Wolf Alexander , Gromak Natalia , Dreger Mathias , Kramer Holger , Kessler Benedikt , Nielsen Michael L. , Schmitz Corinna , Butler Danica S. , Yates John R. , Delahunty Claire M. , Hahn Phillip , Lengeling Andreas , Mann Matthias , Proudfoot Nicholas J. , Schofield Christopher J. , Böttger Angelika
Published: July 2009

Modifying the Modifier

Covalent modification of proteins provides an important means whereby their function is regulated. Hydroxylation, catalyzed by oxygenase enzymes, plays an important role in the response to hypoxia, for example. The human protein Jmjd6 has been thought to act as an oxygenase, catalyzing the demethylation of histone H3 at arginine-2 and histone H4 at arginine-3.

Webby
et al.

(p.
90
) now show that Jmjd6 interacts with the messenger
RNA
splicing factor U2AF65 and acts to hydroxylate this protein at lysine residues, modifications also seen in vivo. Furthermore, Jmjd6 modulates the alternative splicing of both an endogenous gene and an introduced mini-gene.
Coherent Holes in a Semiconductor Quantum Dot
Authors: Kolodrubetz Michael H. , Petta Jason R.
Published: July 2009

Quantum states of positive charge carriers may be more stable to information loss than those of electron-based systems.
Predicting El Niño's Impacts
Authors: Holland Greg J.
Published: July 2009

Insights into El Niño's spatial structure may help to predict its effect on Atlantic tropical cyclones.
Insect Conservation
Authors: Settele Josef , Kühn Elisabeth
Published: July 2009

Understanding of ecosystem interactions and management has led to a major advance in the conservation of specialized insects.
Remembering Outside the Box
Authors: Saksida Lisa M.
Published: July 2009

The mammalian brain may be organized in terms of multipurpose representations rather than psychologically defined modules.
House Vote Seen as Big Step Toward Cooling the Greenhouse
Authors: Kerr Richard A.
Published: July 2009

Its praises have been sung as an energy security bill and a jobs bill. But the American Clean Energy and Security Act passed last Friday by the U.S. House of Representatives is in fact milestone greenhouse legislation.
Ferrets Shed Light on New Virus's Severity and Spread
Authors: Enserink Martin
Published: July 2009

Two groups have infected ferrets with the new pandemic A(H1N1) influenza strain, and their papers, published online by
Science
this week, confirm that the new virus is a bit more pathogenic than seasonal influenza but nowhere near as dangerous as the 1918 pandemic virus or H5N1 avian influenza.
Researchers Fight Against Bigger Slice to Small Business
Authors: Mervis Jeffrey
Published: July 2009

A coalition of U.S. scientific societies and university organizations is urging Congress not to expand a $2.3 billion research program for small businesses. To succeed, however, the coalition must overcome one of the most influential interest groups in Washington and mend fences with legislators still smarting from a recent tweak to the program.
A Hot Competition for a Cold Contract
Authors: Mervis Jeffrey
Published: July 2009

The usually low-key competition for the nearly $2 billion contract to provide logistical support for the National Science Foundation's $300-million-a-year Antarctic research program is heating up this year, with bidders running ads in cars and stations in the Washington, D.C., Metro system.
House Panel Cuts DOE Education Program
Authors: Mervis Jeffrey
Published: July 2009

A House spending panel has rejected a comprehensive $115 million science education initiative from the Department of Energy (DOE) to train a new cadre of scientists pursuing basic and applied research on clean energy.
Betting on Biotech to Transform Guangdong, China's Export Hub
Authors: Stone Richard
Published: July 2009

Local officials are betting that biotech will help wean Guangdong Province off its heavy dependence on cheap exports.
Archaeologists Seek New Clues to the Riddle of Emperor Qin's Terra-Cotta Army
Authors: Stone Richard
Published: July 2009

Archaeologists hope to penetrate lingering puzzles about the Qin Dynasty in a new excavation at Emperor Qin Shihuang's tombs, where life-size terra-cotta soldiers and horses were discovered 35 years ago.
Still Seeking Peking Man
Authors: Stone Richard
Published: July 2009

Last week, work commenced on a new excavation here in the cave-riddled hills of Zhoukoudian, 50 kilometers southwest of Beijing, where early last century scientists discovered Peking Man: a trove of
Homo erectus
fossils as well as rudimentary tools and the bones of woolly rhinos and other Ice Age fauna.
Behavioral Geneticist Celebrates Twins, Scorns PC Science
Authors: Holden Constance
Published: July 2009

Twins researcher Thomas Bouchard spoke with
Science
at the Behavior Genetics Association meeting last month; his comments have been edited for clarity and brevity.
Private Money, Public Disclosure
Authors: Kaiser Jocelyn
Published: July 2009

A Senate investigation is forcing federally funded medical institutions to reveal exactly how much money their researchers receive from industry.
Senate Probe of Research Psychiatrists
Authors: Kaiser Jocelyn
Published: July 2009

Science
has compiled a table of cases made public by Senator Charles Grassley (R–IA) involving NIH-funded researchers—all psychiatrists, many of whom allegedly received consulting income from companies whose drugs they were studying.
China Searches for an 11th-Hour Lifesaver for a Dying Discipline
Authors: Jiao Li
Published: July 2009

With hundreds of thousands of collected specimens unidentified and many more undiscovered, China hopes to prop up its taxonomists before vital skills slip away.
Performance of the Epstein-Barr Virus and Herpes Simplex Virus Immunoglobulin M Assays on the Liaison Platform with Sera from Patients Displaying Acute Parvovirus B19 Infection
Authors: Costa Elisa , Tormo Nuria , Clari María Ángeles , Bravo Dayana , Muñoz-Cobo Beatriz , Navarro David
Published: July 2009

Acute parvovirus B19 infection has been reported to cause false-positive results frequently in the Epstein-Barr (EBV) and herpes simplex virus (HSV) immunoglobulin M (IgM) assays from DiaSorin performed on the Liaison platform. We tested 65 sera from patients with a presumptive or conclusive diagnosis of acute parvovirus B19 infection in both assays and obtained no false-positive results in the EBV IgM test and 10.4% nonspecific reactivities in the HSV IgM assay. Our data support the specificity of both assays in this clinical setting.
An Oral
Mycobacterium bovis
BCG Vaccine for Wildlife Produced in the Absence of Animal-Derived Reagents
Authors: Cross Martin L. , Lambeth Matthew R. , Aldwell Frank E.
Published: July 2009

Cultures of
Mycobacterium bovis
BCG, comprising predominantly single-cell bacilli, were prepared in broth without animal-derived reagents. When formulated into a vegetable-derived lipid matrix, the vaccine was stable in vitro and was immunogenic in vivo upon feeding it to mice. This formulation could be useful for oral vaccination of wildlife against tuberculosis, where concern over transmissible prions may preclude the field use of vaccines containing animal products.
Genetic Characterization ofClostridium botulinumAssociated with Type B Infant Botulism in Japan
Authors: Umeda Kaoru , Seto Yoshiyuki , Kohda Tomoko , Mukamoto Masafumi , Kozaki Shunji
Published: July 2009

The 15 proteolyticClostridium botulinumtype B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis ofboNT/Bgene sequences, genotyping, and determination of theboNT/Bgene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a uniqueboNT/Bgene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that theboNT/Bgene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.
Evaluation of a New Rapid Antigen Test Using Immunochromatography for Detection of Human Metapneumovirus in Comparison with Real-Time PCR Assay
Authors: Matsuzaki Yoko , Takashita Emi , Okamoto Michiko , Mizuta Katsumi , Itagaki Tsutomu , Katsushima Fumio , Katsushima Yuriko , Nagai Yukio , Nishimura Hidekazu
Published: July 2009

A new rapid human metapneumovirus (hMPV) detection kit using immunochromatography (SAS hMPV test) was compared to real-time PCR for 224 nasal swab specimens, 96.4% of which were obtained from children of <15 years of age. The overall sensitivity and specificity were 82.3% and 93.8%, respectively, suggesting that this test is useful for pediatricians to diagnose hMPV infection in a clinical setting.
Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of
Escherichia coli
Authors: Santiso Rebeca , Tamayo María , Fernández José Luis , del Carmen Fernández María , Molina Francisca , Villanueva Rosa , Gosálvez Jaime , Bou Germán
Published: July 2009

We recently reported a simple new in situ diffusion assay, developed as a kit, to visualize DNA fragmentation in single bacterial cells. Use of this assay in a collection of 95 genetically unrelated
Escherichia coli
clinical strains resulted in correct identification of all of the isolates as resistant or susceptible to ciprofloxacin, consistent with the MIC results. This relevant information is obtained in 80 min.
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Fast and Reliable Identification of Clinical Yeast Isolates
Authors: Marklein G. , Josten M. , Klanke U. , Müller E. , Horré R. , Maier T. , Wenzel T. , Kostrzewa M. , Bierbaum G. , Hoerauf A. , Sahl H.-G.
Published: July 2009

The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid routine identification of clinical yeast isolates. A total of 18 type collection strains and 267 recent clinical isolates of
Candida
(
n
= 250),
Cryptococcus
,
Saccharomyces
,
Trichosporon
,
Geotrichum
,
Pichia
, and
Blastoschizomyces
spp. were identified by MALDI-TOF MS. The results were compared with those obtained by conventional phenotyping and biochemical tests, including the API ID 32C system (bioMérieux, Nürtingen, Germany). Starting with cells from single colonies, accurate species identification by MALDI-TOF MS was achieved for 247 of the clinical isolates (92.5%). The remaining 20 isolates required complementation of the reference database with spectra for the appropriate reference strains which were obtained from type culture collections or identified by 26S rRNA gene sequencing. The absence of a suitable reference strain from the MALDI-TOF MS database was clearly indicated by log(score) values too low for the respective clinical isolates; i.e., no false-positive identifications occurred. After complementation of the database, all isolates were unambiguously identified. The established API ID 32C biochemical diagnostic system identified 244 isolates in the first round. Overall, MALDI-TOF MS proved a most rapid and reliable tool for the identification of yeasts and yeast-like fungi, with the method providing a combination of the lowest expenditure of consumables, easy interpretation of results, and a fast turnaround time.
Evaluation of Quinolones for Use in Detection of Determinants of Acquired Quinolone Resistance, Including the New Transmissible Resistance Mechanisms
qnrA
,
qnrB
,
qnrS
, and
aac
(6′)
Ib-cr
, in
Escherichia coli
and
Salmonella enterica
and Determinations of Wild-Type Distributions
Authors: Cavaco L. M. , Aarestrup F. M.
Published: July 2009

Fluoroquinolone resistance in members of the
Enterobacteriaceae
family is mostly due to mutations in the quinolone resistance-determining regions of the topoisomerase genes. However, transferable genes encoding quinolone resistance have recently been described. The current methods for susceptibility testing are not adapted to the detection of new resistance determinants, which confer low levels of resistance. The aim of this study was to compare the ability of the screening of the different quinolones by disk diffusion assays and MIC determinations to detect fluoroquinolone resistance. Sixty-nine
Escherichia coli
strains and 62
Salmonella
strains, including strains fully susceptible to quinolones, nalidixic acid-resistant strains, strains with resistance to fluoroquinolones (resistant to nalidixic acid), and strains showing low-level resistance to fluoroquinolones conferred by transferable quinolone resistance genes, including
qnrA
,
qnrB
,
qnrS
, and
aac
(6′)
Ib-cr
, were selected. Disk diffusion assays and MIC determinations by the agar dilution method were performed, according to CLSI standards, with nalidixic acid, flumequine, oxolinic acid, ciprofloxacin, enrofloxacin, marbofloxacin, norfloxacin, ofloxacin, and levofloxacin. The MIC of levofloxacin was determined by an Etest. The results showed a trimodal distribution of the MICs for both
E. coli
and
Salmonella
. The MIC distributions for the isolates varied with the compounds tested. Screening for nalidixic acid resistance by MIC testing or disk diffusion assay was not efficient for the detection of some of the isolates carrying
qnr
and
aac
(6′)
Ib-cr
. Transferable resistance genes would best be detected by testing for the MIC of ciprofloxacin or norfloxacin, as testing for the MICs of the other compounds would fail to detect isolates carrying
aac
(6′)
Ib-cr
because the enzyme produced is able to reduce the activities of these two compounds only due to their chemical structures. In conclusion, screening with nalidixic acid is efficient for the detection of mutants, but it is not so efficient for the detection of
qnr
and
aac
(6′)
Ib-cr
. Detection would be maximized by screening with either ciprofloxacin or norfloxacin by both MIC determination and disk diffusion assays. Furthermore, a low concentration of ciprofloxacin (1 μg) in the disks seemed to increase the sensitivity of the disk diffusion assay.
Utility of a Commercially Available Multiplex Real-Time PCR Assay To Detect Bacterial and Fungal Pathogens in Febrile Neutropenia
Authors: von Lilienfeld-Toal Marie , Lehmann Lutz E. , Raadts Ansgar D. , Hahn-Ast Corinna , Orlopp Katjana S. , Marklein Günter , Purr Ingvill , Cook Gordon , Hoeft Andreas , Glasmacher Axel , Stüber Frank
Published: July 2009

Infection is the main treatment-related cause of mortality in cancer patients. Rapid and accurate diagnosis to facilitate specific therapy of febrile neutropenia is therefore urgently warranted. Here, we evaluated a commercial PCR-based kit to detect the DNA of 20 different pathogens (SeptiFast) in the setting of febrile neutropenia after chemotherapy. Seven hundred eighty-four serum samples of 119 febrile neutropenic episodes (FNEs) in 70 patients with hematological malignancies were analyzed and compared with clinical, microbiological, and biochemical findings. In the antibiotic-naïve setting, bacteremia was diagnosed in 34 FNEs and 11 of them yielded the same result in the PCR. Seventy-three FNEs were negative in both systems, leading to an overall agreement in 84 of 119 FNEs (71%). During antibiotic therapy, positivity in blood culture occurred only in 3% of cases, but the PCR yielded a positive result in 15% of cases. In six cases the PCR during antibiotic treatment detected a new pathogen repetitively; this was accompanied by a significant rise in procalcitonin levels, suggestive of a true detection of infection. All patients with probable invasive fungal infection (IFI;
n
= 3) according to the standards of the European Organization for Research and Treatment of Cancer had a positive PCR result for
Aspergillus fumigatus
; in contrast there was only one positive result for
Aspergillus fumigatus
in an episode without signs and symptoms of IFI. Our results demonstrate that the SeptiFast kit cannot replace blood cultures in the diagnostic workup of FNEs. However, it might be helpful in situations where blood cultures remain negative (e.g., during antimicrobial therapy or in IFI).
Emergence of a Community-Associated Methicillin-Resistant
Staphylococcus aureus
Strain with a Unique Resistance Profile in Southwest Nigeria
Authors: Ghebremedhin B. , Olugbosi M. O. , Raji A. M. , Layer F. , Bakare R. A. , König B. , König W.
Published: July 2009

Phenotypic, genotypic, and toxin gene analyses have not yet been done all in one for the Nigerian
Staphylococcus aureus
population. This study provides a comprehensive overview of the molecular epidemiology and genetic diversity of
S. aureus
strains at the largest university clinic in Ibadan, Nigeria. From 1,300 patients' clinical samples collected at the University Teaching Hospital in Ibadan, Nigeria, during a 1-year-surveillance in 2007, 346 nonduplicate
S. aureus
isolates were obtained. All isolates underwent antibiotic susceptibility testing, toxin gene analysis, multilocus sequence typing,
agr
group typing, and
spa
typing. For methicillin (meticillin)-resistant
S. aureus
(MRSA), staphylococcal cassette chromosome
mec
(SCC
mec
) typing was also performed. Of the 346 isolates, 20.23% were methicillin resistant. Thirty-three patients' isolates (47.15%) fulfilled the definition criteria for community-associated MRSA (CA-MRSA) according to a review of the medical charts. The majority of MRSA strains analyzed were isolated from surgical or pediatric patients. The commonest types of MRSA infection identified were surgical-site infections (>70%), whereas those for CA-MRSA were conjunctivitis and otitis (19 patients [57.6%]) and accidental skin and subcutaneous tissue infections (14 patients [42.4%]). The methicillin-susceptible
S. aureus
strains (ST1, ST5, ST15, ST7, ST8, ST25, ST30, ST72, ST80, ST121, and ST508) were heterogeneous by phenotypic and genotypic analyses. The first report of a Panton-Valentine leukocidin-positive ST88 strain (
agr
III, SCC
mec
IV) in Nigeria, as well as genetic analyses of this strain, is presented in this study. The ST88 strain was resistant to trimethoprim-sulfamethoxazole as well as to penicillin and oxacillin. CA-MRSA infections are increasing rapidly among young patients with ophthalmologic and auricular infections. Urban regions with populations of lower socioeconomic status and evidence of overcrowding appear to be at high risk for the emergence of this clone.
A Trilocus Sequence Typing Scheme for Hospital Epidemiology and Subspecies Differentiation of an Important Nosocomial Pathogen,
Enterococcus faecalis
Authors: Chowdhury Shahreen A. , Arias Cesar A. , Nallapareddy Sreedhar R. , Reyes Jinnethe , Willems Rob J. L. , Murray Barbara E.
Published: July 2009

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes,
ace
and
salA
(encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance,
lsa
(encoding a putative ABC transporter), for subspecies differentiation of
Enterococcus faecalis
. Each of the alleles was analyzed using 50
E. faecalis
isolates representing 42 diverse multilocus sequence types (ST
M
; based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.
Two Cases of
Mycobacterium microti
-Induced Culture-Negative Tuberculosis
Authors: de Jong Eefje , Rentenaar Rob J. , van Pelt Rob , de Lange Wiel , Schreurs Wim , van Soolingen Dick , Sturm Patrick D. J.
Published: July 2009

We present descriptions of two human
Mycobacterium microti
infections: one of a patient with pulmonary disease and one of a patient with osteomyelitis of the hip. Both patients had acid-fast bacilli and a positive
Mycobacterium tuberculosis
complex PCR from clinical specimens, but mycobacterial cultures remained negative. The microbiological diagnosis was established by molecular methods.
Characterization of Mycobacteria from a Major Brazilian Outbreak Suggests that Revision of the Taxonomic Status of Members of the
Mycobacterium chelonae
-
M. abscessus
Group Is Needed
Authors: Leao Sylvia Cardoso , Tortoli Enrico , Viana-Niero Cristina , Ueki Suely Yoko Mizuka , Lima Karla Valeria Batista , Lopes Maria Luiza , Yubero Jesus , Menendez Maria Carmen , Garcia Maria Jesus
Published: July 2009

An outbreak of postsurgical infections caused by rapidly growing mycobacteria has been ongoing in Brazil since 2004. The degrees of similarity of the
rpoB
and
hsp65
sequences from the clinical isolates and the corresponding sequences from both the
Mycobacterium massiliense
and the
M. bolletii
type strains were above the accepted limit for interspecies variability, leading to conflicting identification results. Therefore, an extensive characterization of members of the
M. chelonae-M. abscessus
group was carried out. The
M. abscessus
,
M. chelonae
,
M. immunogenum
,
M. massiliense
, and
M. bolletii
type strains and a subset of clinical isolates were analyzed by biochemical tests, high-performance liquid chromatography, drug susceptibility testing, PCR-restriction enzyme analysis of
hsp65
(PRA-
hsp65
),
rpoB
, and
hsp65
gene sequencing and analysis of phylogenetic trees, DNA-DNA hybridization (DDH), and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (RFLP-16S rRNA). The clinical isolates and the
M. abscessus
,
M. massiliense
, and
M. bolletii
type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. The results of DDH also confirmed the >70% relatedness of the clinical isolates and the
M. abscessus
,
M. massiliense
, and
M. bolletii
type strains; and indistinguishable RFLP-16S rRNA patterns were obtained. On the contrary, the separation of clinical isolates and the
M. abscessus
,
M. massiliense
, and
M. bolletii
type strains from
M. chelonae
and
M. immunogenum
was supported by the results of PRA-
hsp65
, DDH, and RFLP-16S rRNA and by the
rpoB
and
hsp65
phylogenetic trees. Taken together, these results led to the proposition that
M. abscessus
,
M. massiliense
, and
M. bolletii
represent a single species, that of
M. abscessus.
Two subspecies are also proposed,
M. abscessus
subsp.
abscessus
and
M. abscessus
subsp.
massiliense
, and these two subspecies can be distinguished by two different PRA-
hsp65
patterns, which differ by a single HaeIII band, and by differences in their
rpoB
(3.4%) and
hsp
65 (1.3%) sequences.
Identification of 20 Common Human Enterovirus Serotypes by Use of a Reverse Transcription-PCR-Based Reverse Line Blot Hybridization Assay
Authors: Zhou Fei , Kong Fanrong , McPhie Kenneth , Ratnamohan Mala , Donovan Linda , Zeng Frank , Gilbert Gwendolyn L. , Dwyer Dominic E.
Published: July 2009

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3′ half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.
WHO Global Salm-Surv External Quality Assurance System for Serotyping of
Salmonella
Isolates from 2000 to 2007
Authors: Hendriksen Rene S. , Mikoleit Matthew , Carlson Valeria P. , Karlsmose Susanne , Vieira Antonio R. , Jensen Arne B. , Seyfarth Anne Mette , DeLong Stephanie M. , Weill François-Xavier , Lo Fo Wong Danilo Marino Armando , Angulo Frederick J. , Wegener Henrik C. , Aarestrup Frank M.
Published: July 2009

An international external quality assurance system (EQAS) for the serotyping of
Salmonella
species was initiated in 2000 by WHO Global Salm-Surv to enhance the capacity of national reference laboratories to obtain reliable data for surveillance purposes worldwide. Seven EQAS iterations were conducted between 2000 and 2007. In each iteration, participating laboratories submitted serotyping results for eight
Salmonella
isolates. A total of 249 laboratories in 96 countries participated in at least one EQAS iteration. A total of 756 reports were received from the participating laboratories during the seven EQAS iterations. Cumulatively, 76% of participating laboratories submitted data for all eight strains, and 82% of strains were correctly serotyped. In each iteration, 84% to 96% of the laboratories correctly serotyped the
Salmonella enterica
serovar Enteritidis isolate that was included as an internal quality control strain. Regional differences in performance were observed, with laboratories in Central Asia and the Middle East performing less well overall than those in other regions. Errors that resulted in incorrect serovar identification were typically caused by difficulties in the detection of the phase two flagellar antigen or in differentiation within antigen complexes; some of these errors are likely related to the quality of the antisera available. The results from the WHO Global Salm-Surv EQAS, the largest of its kind in the world, show that most laboratories worldwide are capable of correctly serotyping
Salmonella
species. However, this study also indicates a continuing need for improvement. Future training efforts should be aimed at enhancing the ability to detect the phase two flagellar antigen and at disseminating information on where to purchase high-quality antisera.
Simian Virus 40 T-Antigen-Mediated Gene Regulation in Enterocytes Is Controlled Primarily by the Rb-E2F Pathway
Authors: Rathi Abhilasha V. , Sáenz Robles M. Teresa , Cantalupo Paul G. , Whitehead Robert H. , Pipas James M.
Published: July 2009

Simian virus 40 large T antigen (TAg) contributes to cell transformation, in part, by targeting two well-characterized tumor suppressors, pRb and p53. TAg expression affects the transcriptional circuits controlled by Rb and by p53. We have performed a microarray analysis to examine the global change in gene expression induced by wild-type TAg (TAg
wt
) and TAg mutants, in an effort to link changes in gene expression to specific transforming functions. For this analysis we have used enterocytes from the mouse small intestine expressing TAg. Expression of TAg
wt
in the mouse intestine results in hyperplasia and dysplasia. Our analysis indicates that practically all gene expression regulated by TAg in enterocytes is dependent upon its binding and inactivation of the Rb family proteins. To further dissect the role of the Rb family in the induction of intestinal hyperplasia, we have screened several lines of transgenic mice expressing a truncated TAg (TAg
N136
), which is able to interfere with the Rb pathway but lacks the functions associated with the carboxy terminus of the protein. This analysis confirmed the pivotal association between the Rb pathway and the induction of intestinal hyperplasia and revealed that upregulation of p53 target genes is not associated with the tumorigenic phenotype. Furthermore, we found that TAg
N136
was sufficient to induce intestinal hyperplasia, although the appearance of dysplasia was significantly delayed.
Human Immunodeficiency Virus Type 1 Escape from Cyclotriazadisulfonamide-Induced CD4-Targeted Entry Inhibition Is Associated with Increased Neutralizing Antibody Susceptibility
Authors: Vermeire Kurt , Van Laethem Kristel , Janssens Wouter , Bell Thomas W. , Schols Dominique
Published: July 2009

Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV-1-infected patients.
The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells
Authors: Alvisi Gualtiero , Roth Daniela Martino , Camozzi Daria , Pari Gregory S. , Loregian Arianna , Ripalti Alessandro , Jans David A.
Published: July 2009

Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP
174
]), which has been proposed to be directly involved in ppUL44's interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Δloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Δloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Δloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Δloop fails to transcomplement HCMV
ori
Lyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.
A Function Essential to Viral Entry Underlies the Hepatitis B Virus “a” Determinant
Authors: Salisse Jessica , Sureau Camille
Published: July 2009

The hepatitis B virus (HBV) particles bear a receptor-binding site located in the pre-S1 domain of the large HBV envelope protein. Using the hepatitis delta virus (HDV) as a surrogate of HBV, a second infectivity determinant was recently identified in the envelope proteins antigenic loop (AGL), and its activity was shown to depend upon cysteine residues that are essential for the structure of the HBV immunodominant “a” determinant. Here, an alanine-scanning mutagenesis approach was used to precisely map the AGL infectivity determinant to a set of conserved residues, which are predicted to cluster together with cysteines in the AGL disulfide bridges network. Several substitutions suppressed both infectivity and the “a” determinant, whereas others were infectivity deficient with only a partial impact on antigenicity. Interestingly, G145R, a substitution often arising under immune pressure selection and detrimental to the “a” determinant, had no effect on infectivity. Altogether, these findings indicate that the AGL infectivity determinant is closely related to, yet separable from, the “a” determinant. Finally, a selection of HDV entry-deficient mutations were introduced at the surface of HBV virions and shown to also abrogate infection in the HBV model. Therefore, a function can at last be assigned to the orphan “a” determinant, the first-discovered marker of HBV infection. The characterization of the AGL functions at viral entry may lead to novel approaches in the development of antivirals against HBV.
Subcellular Localization of the
Barley Stripe Mosaic Virus
Triple Gene Block Proteins
Authors: Lim Hyoun-Sub , Bragg Jennifer N. , Ganesan Uma , Ruzin Steven , Schichnes Denise , Lee Mi Yeon , Vaira Anna Maria , Ryu Ki Hyun , Hammond John , Jackson Andrew O.
Published: July 2009

Barley stripe mosaic virus
(BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used
Agrobacterium tumefaciens
-mediated protein expression in
Nicotiana benthamiana
leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.
Maintenance of an Old World Betasatellite by a New World Helper Begomovirus and Possible Rapid Adaptation of the Betasatellite
Authors: Nawaz-ul-Rehman Muhammad Shah , Mansoor Shahid , Briddon Rob W. , Fauquet Claude M.
Published: July 2009

Begomoviruses (family
Geminiviridae
) cause major losses to crops throughout the tropical regions of the world. Begomoviruses originating from the New World (NW) and the Old World (OW) are genetically distinct. Whereas the majority of OW begomoviruses have monopartite genomes and whereas most of these associate with a class of symptom-modulating satellites (known as betasatellites), the genomes of NW begomoviruses are exclusively bipartite and do not associate with satellites. Here, we show for the first time that a betasatellite (cotton leaf curl Multan betasatellite [CLCuMuB]) associated with a serious disease of cotton across southern Asia is capable of interacting with a NW begomovirus. In the presence of CLCuMuB, the symptoms of the NW cabbage leaf curl virus (CbLCuV) are enhanced in
Nicotiana benthamiana
. However, CbLCuV was unable to interact with a second betasatellite, chili leaf curl betasatellite. Although CbLCuV can transreplicate CLCuMuB, satellite accumulation levels in plants were low. However, progeny CLCuMuB isolated after just one round of infection with CbLCuV contained numerous mutations. Reinoculation of one such progeny CLCuMuB with CbLCuV to
N. benthamiana
yielded infections with significantly higher satellite DNA levels. This suggests that betasatellites can rapidly adapt for efficient transreplication by a new helper begomovirus, including begomoviruses originating from the NW. Although the precise mechanism of transreplication of betasatellites by begomoviruses remains unknown, an analysis of betasatellite mutants suggests that the sequence(s) required for maintenance of CLCuMuB by one of its cognate begomoviruses (cotton leaf curl Rajasthan virus) differs from the sequences required for maintenance by CbLCuV. The significance of these findings and, particularly, the threat that betasatellites pose to agriculture in the NW, are discussed.
Random Insertion Mutagenesis of Sindbis Virus Nonstructural Protein 2 and Selection of Variants Incapable of Downregulating Cellular Transcription
Authors: Frolov Ilya , Garmashova Natalia , Atasheva Svetlana , Frolova Elena I.
Published: July 2009

Sindbis virus nonstructural protein 2 (SINV nsP2) is an important determinant of virus pathogenesis and downregulation of virus-induced cell response. This protein efficiently inhibits transcription of cellular messenger and ribosomal RNAs and, thus, is capable of inhibiting the activation of genes whose products are involved in development of the antiviral response. Alphavirus nsP2 has a number of predicted functional domains, some of which were confirmed by crystal structure. Our current study demonstrated that none of the putative or known structural domains alone or their combinations was capable of functioning in transcription inhibition. By using random, transposon-mediated mutagenesis, we generated a library of SINV nsP2 variants having short peptide insertions and selected those that lost the ability to inhibit cellular transcription and cause a cytopathic effect. Insertions abrogating the nuclear functions of the protein were found in the three different functional nsP2 domains. Some of the mutated protein variants retained the enzymatic functions required for replication of the viral genome. Such viruses were capable of efficient, productive replication in cells defective in interferon (IFN) signaling but were attenuated and incapable of spreading in cells with an intact type I IFN response. These results revealed new information about the structure of SINV nsP2 and interaction of its domains.