bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary dynamics of separate and combined exposure of Pseudomonas fluorescens SBW25 to antibiotics and bacteriophage
Authors: Escobar‐Páramo Patricia , Gougat‐Barbera Claire , Hochberg Michael E.
Published: February 2012

The use of bacteriophages against pathogenic bacteria in health care and in the food industry is now being advocated as an alternative to the use of antibiotics. But what is the evolutionary response for a bacterial population if both antibiotics and phages are used in combination? We employ an experimental evolution approach to address these questions and exposed Pseudomonas fluorescens SBW25 and a related hypermutator strain (mutS−) to the action of the antibiotic rifampicin and the lytic bacteriophage SBW25ϕ2. We then compared the densities, growth rates, and the mutations at the rpoB locus leading to rifampicin resistance of the evolved bacterial populations. We observed that the evolutionary response of populations under different treatments varied depending on the order in which the antimicrobials were added and whether the bacterium was a hypermutator. We found that wild‐type rifampicin‐resistant populations involved in biofilm formation often reverted to rifampicin sensitivity when stresses were added sequentially. In contrast, when the mortality agents were added simultaneously, phage populations frequently went extinct and the bacteria evolved antibiotic resistance. However, populations of the hypermutator mutS− converged to a single genotype at the rpoB locus. Future investigation on other bacteria and using different antibiotics and bacteriophage are needed to evaluate the generality of our findings.
Measurement of Circulating Cell‐Free DNA in Relation to 18F‐Fluorocholine PET/CT Imaging in Chemotherapy‐Treated Advanced Prostate Cancer
Authors: Kwee Sandi , Song Min‐Ae , Cheng Iona , Loo Lenora , Tiirikainen Maarit
Published: February 2012

Purpose: To examine the effects of chemotherapy on circulating cell‐free DNA (cfDNA) composition in relation to investigational whole‐body measurement of tumor activity by fluorine‐18 fluorocholine (FCH) positron emission tomography/computed tomography (PET/CT) in hormone‐refractory prostate cancer (HRPC).

Methods: Serial FCH PET/CT scans were performed in eight patients with HRPC receiving docetaxel‐based chemotherapy. Corresponding serial cfDNA samples were characterized by microfluidic electrophoresis, quantified by real‐time PCR, and compared with PET/CT results. Promoter methylation of two prostate cancer‐associated genes, GSTP1 and RARB2, was assessed by methylation‐specific PCR of bisulfite‐converted cfDNA.

Results: Plasma cfDNA concentrations increased significantly from 13.3 ng/mL at baseline to 46.8 ng/mL and 50.9 ng/mL after one and three treatment cycles, respectively (p= 0.001). GSTP1 and/or RARB2 promoter methylation was identified in all pretreatment samples. The appearance of large (200 bp–10.4 kb) cfDNA fragments was noted in posttreatment samples along with loss of methylation at GSTP1 and/or RARB2. Tumor activity on PET/CT correlated with cfDNA concentration (r=−0.50, p= 0.01). Patients meeting criteria for PET tumor response had significantly lower pretreatment cfDNA levels than those who did not (8.0 vs. 16.4 ng/mL, p= 0.03).

Conclusions: Chemotherapy is associated with significant changes in plasma cfDNA content and FCH PET/CT‐detected tumor activity. These interrelated measures are potential candidate markers of therapeutic response in HRPC. Clin Trans Sci 2012; Volume #: 1–6
Access to Core Facilities and Other Research Resources Provided by the Clinical and Translational Science Awards
Authors: Rosenblum Daniel
Published: February 2012

Principal investigators who received Clinical and Translational Science Awards created academic homes for biomedical research. They developed program‐supported websites to offer coordinated access to a range of core facilities and other research resources. Visitors to the 60 websites will find at least 170 generic services, which this review has categorized in the following seven areas: (1) core facilities, (2) biomedical informatics, (3) funding, (4) regulatory knowledge and support, (5) biostatistics, epidemiology, research design, and ethics, (6) participant and clinical interaction resources, and (7) community engagement. In addition, many websites facilitate access to resources with search engines, navigators, studios, project development teams, collaboration tools, communication systems, and teaching tools. Each of these websites may be accessed from a single site, http://www.CTSAcentral.org. The ability to access the research resources from 60 of the nation's academic health centers presents a novel opportunity for investigators engaged in clinical and translational research. Clin Trans Sci 2012; Volume #: 1–5
Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells
Authors: Filipp Fabian V. , Scott David A. , Ronai Ze’ev A. , Osterman Andrei L. , Smith Jeffrey W.
Published: February 2012

The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.
Delimitation and phylogeny of Aletris (Nartheciaceae) with implications for perianth evolution
Authors: ZHAO Yi‐Min , WANG Wei , ZHANG Shu‐Ren
Published: February 2012

Abstract  Aletris, containing approximately 21 species, is the largest genus in Nartheciaceae, and is disjunctively distributed in eastern Asia and eastern North America. Its delimitation has been controversial because it is uncertain whether Metanarthecium should be included in the genus. Although there are a few molecular phylogenetic studies on Aletris, the interspecific relationships within the genus have never been evaluated in a phylogenetic context. Here we used two cpDNA loci, matK and trnL‐F, to delimitate Aletris and discuss the phylogeny within the genus. Phylogenetic analyses showed Metanarthecium might be distantly related to Aletris. This is also supported by morphological, palynological, cytological, and phytochemical data. Therefore, Metanarthecium should be excluded from Aletris. Within Aletris, there are two major clades: A. farinosa and A. lutea of eastern North America and A. glabra of eastern Asia form clade A; and the remaining Asian species form clade B. The Asian clade includes three subclades: subclade I (two varieties of A. pauciflora, and A. glandulifera and A. megalantha), subclade II (three samples of A. laxiflora), and subclade III (all other sampled Asian species). Based on phylogenetic relationships, A. pauciflora var. khasiana deserves a specific status, and A. gracilipes, formerly a synonym of A. laxiflora, should be reinstated. The reconstruction of the perianth evolution indicates that perianth connate halfway and glabrous on abaxial surface are plesiomorphic for Aletris and Nartheciaceae. Farinose‐glutinous perianth is a diagnostic character for clade A.
Optical and electrical properties of Si-doped GaAs films grown on (631)-oriented substrates
Authors: Vázquez-Cortés D. , Cruz-Hernández E. , Méndez-García V. H. , Shimomura S. , López-López M.
Published: February 2012

This work studies the Si-doping of GaAs on (631)-oriented substrates as a function of the As4-beam equivalent pressure (PAs). The electrical properties obtained by Hall effect measurements show that the mobility of the layers grown on (631)-substrates present changes related to carrier compensation processes and the Si-doping changes from p- to n-type when PAs is increased. The optical properties of the samples, as observed by photoluminescence (PL) spectroscopy, modified according to the electrical characteristics. For (631)-samples, when PAs is increased the energy of the maximum intensity PL peak redshifts in the p-type region but, after the threshold of the conduction type conversion, the peak blueshifts in the n-type region. The variation of the PL excitation intensity also shifts the emission energy of the samples as a consequence of the increasing recombination rate for close pairs in donor acceptor pair recombination. Photoluminescence as a function of temperature shows that the activation energy of the PL lines transition is enhanced as PAs is increased.
A low-cost spatial light modulator for use in undergraduate and graduate optics labs
Authors: Huang Derek , Timmers Henry , Roberts Adam , Shivaram Niranjan , Sandhu Arvinder S.
Published: February 2012

Spatial light modulators (SLMs) are a versatile tool for teaching optics, but the cost associated with an SLM setup prevents its adoption in many undergraduate and graduate optics labs. We describe a simple method for creating a low-cost SLM by extracting components from a commercial LCD projector. We demonstrate the pedagogical applications of this SLM design by providing examples of its use in teaching diffraction and interference phenomena. We also discuss an SLM’s potential as a research tool in graduate labs. In particular, we demonstrate its use in holography and in the generation of optical vortices.
Embeddings and time evolution of the Schwarzschild wormhole
Authors: Collas Peter , Klein David
Published: February 2012

We show how to embed spacelike slices of the Schwarzschild wormhole (or Einstein-Rosen bridge) in R3. Graphical images of embeddings are given, including depictions of the dynamics of this nontraversable wormhole at constant Kruskal times up to and beyond the “pinching off” at Kruskal times ±1.
Quantitative investigation of Fresnel reflection coefficients by polarimetry
Authors: Salik Ertan
Published: February 2012

The phase shift that occurs upon reflection from a dielectric surface is an interesting yet elusive topic in undergraduate and graduate level optics courses. Many students learn the Fresnel equations, which describe the theory of reflection-induced phase shifts, but there are few experiments which enable direct measurements of these phase shifts. We describe several experiments based on polarimetry to measure the differential phase shifts which occur upon reflection from dielectric–dielectric interfaces. Comparison of the experimental results with the theory given in optics textbooks reveals apparent inconsistencies, which can be resolved by taking into account various conventions.
The cantilever beam magnetometer: A simple teaching tool for magnetic characterization
Authors: Adhikari Rajdeep , Kaundal Rakesh , Sarkar Anirban , Rana Pushpinder , Das Amal K.
Published: February 2012

We have designed and fabricated an ex situ optical cantilever beam magnetometer to measure the magnetization and the magnetostriction of ferromagnetic materials. We discuss the working principle of a cantilever beam magnetometer and derive the theory of the cantilever bending due to magnetic torque acting on the sample attached to the cantilever substrate. We also present the results for bulk iron obtained using the cantilever beam magnetometer.
A Season for Inquiry: Investigating Phenology in Local Campus Trees
Authors: Long Tammy , Wyse Sara
Published: February 2012

Campus Trees, the IBI Prize–winning module, uses local phenology to create authentic inquiry experiences in undergraduate biology.
Single-Molecule Fluorescence Experiments Determine Protein Folding Transition Path Times
Authors: Chung Hoi Sung , McHale Kevin , Louis John M. , Eaton William A.
Published: February 2012

A Fraction of Folding

An energy barrier has to be crossed as a protein transforms between folded and unfolded states. Molecular dynamic simulations have observed sharp transitions, with barrier crossing times of less than a microsecond, a fraction of the total folding time; however, this time range has been inaccessible to single-molecule experiments.

Chung
et al.

(p.
981
) described single-molecule fluorescence experiments that allowed measurement of the transition-path time for a fast-folding protein and to reduce the upper bound for a slow-folding protein. Although the folding rates differed by a factor of 10,000, the transition-path times differ by less than a factor of 5, pointing to energy landscape theory for the explanation.
Preserving Montreal Protocol Climate Benefits by Limiting HFCs
Authors: Velders Guus J. M. , Ravishankara A. R. , Miller Melanie K. , Molina Mario J. , Alcamo Joseph , Daniel John S. , Fahey David W. , Montzka Stephen A. , Reimann Stefan
Published: February 2012

With no impending global controls on HFCs, the Montreal Protocol offers a near-term path to preserve its climate benefits.
The Many Lives of Whales
The Sounding of the Whale
Science and Cetaceans in the Twentieth Century

by D. Graham Burnett

University of Chicago Press, Chicago, 2012. 823 pp. $45, £29. ISBN 9780226081304.
Authors: Mitman Gregg
Published: February 2012

Exploring the course and consequences of cetacean research through the 20th century, Burnett charts the interplay of science, conservation, and politics that substantially remade relationships between humans and whales.
Frictional Dissipation—Blame It on the Rain
Authors: Frierson Dargan M. W.
Published: February 2012

Satellite observations reveal the extent to which rainfall removes kinetic energy from the atmosphere, and thus its impact on circulation.
Grand Master of Reconstruction
Charles R. Knight
The Artist Who Saw Through Time

by Richard Milner

Abrams, New York, 2012. 180 pp., illus. $40, C$45, £24.99. ISBN 9780810984790.
Authors: Parrish Mary A.
Published: February 2012

Milner offers a beautifully illustrated survey of the life and work of the celebrated paleoartist Charles R. Knight.
Advocates Win 'Exceptional' Boost for Alzheimer's Research
Authors: Kaiser Jocelyn
Published: February 2012

The 2013 budget proposed by President Barack Obama last week gives special attention to one disease: Alzheimer's, which will receive $80 million in new research funding from a source outside NIH's budget.
Kansas Veterinary Biosecurity Lab Trampled in Spending Plan
Authors: Malakoff David
Published: February 2012

A flagship government biodefense laboratory set to be built in Kansas is facing critical funding troubles—in part because of fierce opposition from cattle ranchers the facility is supposed to help.
India Rising
Authors: Stone Richard
Published: February 2012

India excels in rocketry and nuclear science but has produced few breakthroughs in other fields. Now, free of sanctions and swimming in cash, the world's largest democracy is gunning for status as a scientific powerhouse.
India's Scholar-Prime Minister Aims for Inclusive Development
Authors: Bagla Pallava , Stone Richard
Published: February 2012

Indian Prime Minister Manmohan Singh plans to increase the government's R&D spending and create incentives for the private sector to increase spending on science and technology as well.
Crowd-Sourcing Drug Discovery
Authors: Bagla Pallava
Published: February 2012

The Open Source Drug Discovery network's army of volunteers is building a kind of Wikipedia on tuberculosis, which is the leading cause of death in India for those in the prime of life.
Drawing a Bead on India's Enigmatic Monsoon
Authors: Bagla Pallava
Published: February 2012

This year, India's Ministry of Earth Sciences is launching a 5-year, $75 million "monsoon mission" to improve the study of complex ocean-atmosphere interactions.
Lactobacillus acidophilus Induces Cytokine and Chemokine Production via NF-κB and p38 Mitogen-Activated Protein Kinase Signaling Pathways in Intestinal Epithelial Cells
Authors: Jiang Yujun , Lü Xuena , Man Chaoxin , Han Linlin , Shan Yi , Qu Xingguang , Liu Ying , Yang Shiqin , Xue Yuqing , Zhang Yinghua
Published: February 2012

Intestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses.Lactobacillus acidophilusNCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability ofL. acidophilusNCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed thatL. acidophilusNCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1β, CCL2, and CCL20 and that cytokines showed lower expression levels withL. acidophilusNCFM treatment than chemokines. Moreover,L. acidophilusNCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced byL. acidophilusNCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated byL. acidophilusNCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced byL. acidophilusNCFM.
Transcutaneous Immunization with a Vibrio cholerae O1 Ogawa Synthetic Hexasaccharide Conjugate following Oral Whole-Cell Cholera Vaccination Boosts Vibriocidal Responses and Induces Protective Immunity in Mice
Authors: Tarique A. A. , Kalsy A. , Arifuzzaman M. , Rollins S. M. , Charles R. C. , Leung D. T. , Harris J. B. , LaRocque R. C. , Sheikh A. , Bhuiyan M. S. , Saksena R. , Clements J. D. , Calderwood S. B. , Qadri F. , Kováč P. , Ryan E. T.
Published: February 2012

A shortcoming of currently available oral cholera vaccines is their induction of relatively short-term protection against cholera compared to that afforded by wild-type disease. We were interested in whether transcutaneous or subcutaneous boosting using a neoglycoconjugate vaccine made from a synthetic terminal hexasaccharide of the O-specific polysaccharide ofVibrio choleraeO1 (Ogawa) coupled to bovine serum albumin as a carrier (CHO-BSA) could boost lipopolysaccharide (LPS)-specific and vibriocidal antibody responses and result in protective immunity following oral priming immunization with whole-cell cholera vaccine. We found that boosting with CHO-BSA with immunoadjuvantative cholera toxin (CT) orEscherichia coliheat-labile toxin (LT) following oral priming with attenuatedV. choleraeO1 vaccine strain O395-NT resulted in significant increases in serum anti-V. choleraeLPS IgG, IgM, and IgA (P< 0.01) responses as well as in anti-Ogawa (P< 0.01) and anti-Inaba (P< 0.05) vibriocidal titers in mice. The LPS-specific IgA responses in stool were induced by transcutaneous (P< 0.01) but not subcutaneous immunization. Immune responses following use of CT or LT as an adjuvant were comparable. In a neonatal mouse challenge assay, immune serum from boosted mice was associated with 79% protective efficacy against death. Our results suggest that transcutaneous and subcutaneous boosting with a neoglycoconjugate following oral cholera vaccination may be an effective strategy to prolong protective immune responses againstV. cholerae.
Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68
Authors: Xu Jia-Nan , Chen Jian-Ping , Chen Da-Li
Published: February 2012

In order to identify immunodominant antigens ofMycobacterium tuberculosisthat may be used in the serodiagnosis of active tuberculosis (TB), we designed anM. tuberculosisfusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68′). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6×His tag were successfully overexpressed inEscherichia coliBL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10–ESAT-6–PPE68′ had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10–ESAT-6. In addition, the fusion protein CFP-10–ESAT-6–PPE68′ stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10–ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10–ESAT-6–PPE68′ were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10–ESAT-6. Thus, our study indicates that the fusion protein CFP-10–ESAT-6–PPE68′ may be useful as an immunodominant antigen for the serodiagnosis of active TB.
Rapid Direct Testing of Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin on Nutrient and Blood Agar in Resource-Starved Settings
Authors: Satti Luqman , Ikram Aamer , Coban Ahmet Yilmaz , Martin Anandi
Published: February 2012

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of
Mycobacterium tuberculosis
against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the “gold standard.” Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings.
Biomedical Mass Spectrometry in Today's and Tomorrow's Clinical Microbiology Laboratories
Authors: van Belkum Alex , Welker Martin , Erhard Marcel , Chatellier Sonia
Published: February 2012

Clinical microbiology is a conservative laboratory exercise where base technologies introduced in the 19th century remained essentially unaltered. High-tech mass spectrometry (MS) has changed that. Within a few years following its adaptation to microbiological diagnostics, MS has been introduced, embraced, and broadly accepted by clinical microbiology laboratories throughout the world as an innovative tool for definitive bacterial species identification. Herein, we review the current state of the art with respect to this exciting new technology and discuss potential future applications.
The First Locally Acquired Human Infection of Echinococcus multilocularis in The Netherlands
Authors: van Dommelen Laura , Stoot Jan H. M. B. , Cappendijk Vincent C. , Abdul Hamid Myrurgia A. , Stelma Foekje F. , Kortbeek Laetitia M. , van der Giessen Joke , Oude Lashof Astrid M. L.
Published: February 2012

In the northern part of Western Europe,
Echinococcus multilocularis
is primarily detected in and spreading among foxes. The present case marks
E. multilocularis
as an emerging pathogen for humans, as it describes the first human case of probably locally acquired
E. multilocularis
in The Netherlands, with various interesting clinical aspects.
Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Capripoxviruses
Authors: Das Amaresh , Babiuk Shawn , McIntosh Michael T.
Published: February 2012

Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited.
Molecular Characterization of the Processing of Arenavirus Envelope Glycoprotein Precursors by Subtilisin Kexin Isozyme-1/Site-1 Protease
Authors: Burri Dominique J. , Pasqual Giulia , Rochat Cylia , Seidah Nabil G. , Pasquato Antonella , Kunz Stefan
Published: February 2012

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/
cis
-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/
cis
-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.
High-Risk Human Papillomavirus E5 Oncoprotein Displays Channel-Forming Activity Sensitive to Small-Molecule Inhibitors
Authors: Wetherill Laura F. , Holmes Kristopher K. , Verow Mark , Müller Marietta , Howell Gareth , Harris Mark , Fishwick Colin , Stonehouse Nicola , Foster Richard , Blair G. Eric , Griffin Stephen , Macdonald Andrew
Published: February 2012

High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and therefore is responsible for significant morbidity and mortality worldwide. Cellular transformation is mediated directly by the expression of viral oncogenes, the least characterized of which, E5, subverts cellular proliferation and immune recognition processes. Despite a growing catalogue of E5-specific host interactions, little is understood regarding the molecular basis of its function. Here we describe a novel function for HPV16 E5 as an oligomeric channel-forming protein, placing it within the virus-encoded “viroporin” family. The development of a novel recombinant E5 expression system showed that E5 formed oligomeric assemblies of a defined luminal diameter and stoichiometry in membranous environments and that such channels mediated fluorescent dye release from liposomes. Hexameric E5 channel stoichiometry was suggested by native PAGE studies. In lieu of high-resolution structural information, established
de novo
molecular modeling and design methods permitted the development of the first specific small-molecule E5 inhibitor, capable of both abrogating channel activity
in vitro
and reducing E5-mediated effects on cell signaling pathways. The identification of channel activity should enhance the future understanding of the physiological function of E5 and could represent an important target for antiviral intervention.
Human CD4+T Cell Response to Human Herpesvirus 6
Authors: Nastke Maria-D. , Becerra Aniuska , Yin Liusong , Dominguez-Amorocho Omar , Gibson Laura , Stern Lawrence J. , Calvo-Calle J. Mauricio
Published: February 2012

Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4+T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easilyin vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4+T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4+T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.
Virion Architecture Unifies Globally Distributed Pleolipoviruses Infecting Halophilic Archaea
Authors: Pietilä Maija K. , Atanasova Nina S. , Manole Violeta , Liljeroos Lassi , Butcher Sarah J. , Oksanen Hanna M. , Bamford Dennis H.
Published: February 2012

Our understanding of the third domain of life,
Archaea
, has greatly increased since its establishment some 20 years ago. The increasing information on archaea has also brought their viruses into the limelight. Today, about 100 archaeal viruses are known, which is a low number compared to the numbers of characterized bacterial or eukaryotic viruses. Here, we have performed a comparative biological and structural study of seven pleomorphic viruses infecting extremely halophilic archaea. The pleomorphic nature of this novel virion type was established by sedimentation analysis and cryo-electron microscopy. These nonlytic viruses form virions characterized by a lipid vesicle enclosing the genome, without any nucleoproteins. The viral lipids are unselectively acquired from host cell membranes. The virions contain two to three major structural proteins, which either are embedded in the membrane or form spikes distributed randomly on the external membrane surface. Thus, the most important step during virion assembly is most likely the interaction of the membrane proteins with the genome. The interaction can be driven by single-stranded or double-stranded DNA, resulting in the virions having similar architectures but different genome types. Based on our comparative study, these viruses probably form a novel group, which we define as pleolipoviruses.
Roles of E4orf6 and VA I RNA in Adenovirus-Mediated Stimulation of Human Parvovirus B19 DNA Replication and Structural Gene Expression
Authors: Winter Kerstin , von Kietzell Kristina , Heilbronn Regine , Pozzuto Tanja , Fechner Henry , Weger Stefan
Published: February 2012

Despite its very narrow tropism for erythroid progenitor cells, human parvovirus B19 (B19V) has recently been shown to replicate and form infectious progeny virus in 293 cells in the presence of early adenoviral functions provided either by infection with adenovirus type 5 or by addition of the pHelper plasmid encoding the E2a, E4orf6, and VA RNA functions. In the present study we dissected the individual influence of these functions on B19V genome replication and expression of structural proteins VP1 and VP2. We show that, in the presence of the constitutively expressed E1A and E1B, E4orf6 alone is able to promote B19V DNA replication, resulting in a concomitant increase in VP expression levels. The stimulatory effects of E4orf6 require the integrity of the BC box motifs, which target cellular proteins such as p53 and the Mre11 DNA repair complex for proteosomal degradation through formation of an E3 ubiquitin ligase complex with E1B. VA RNA also strongly induces VP expression but, in contrast to E4orf6, in a replication-independent manner. This stimulation could be attributed exclusively to the VA I RNA transcript and does not involve major activating effects at the level of the B19V p6 promoter, but the nucleotide residues required for the well-defined pathway of VA I RNA mediated stimulation of translation through functional inactivation of protein kinase R. These data show that the cellular pathways regulating B19V replication may be very similar to those governing the productive cycle of the helper-dependent parvoviruses, the adeno-associated viruses.
Limited Transferrin Receptor Clustering Allows Rapid Diffusion of Canine Parvovirus into Clathrin Endocytic Structures
Authors: Cureton David K. , Harbison Carole E. , Cocucci Emanuele , Parrish Colin R. , Kirchhausen Tom
Published: February 2012

Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here we used fluorescence microscopy to directly visualize the association of single canine parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that most capsids associated with fewer than five TfRs and that ∼25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissociated from the cell surface with a half-life of ∼30 s. Together, our results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.
Fall in peptic ulcer mortality associated with increased consultant input, prompt surgery and use of high dependency care identified through peer-review audit
Authors: Aga Hiba , Readhead David , MacColl Gavin , Thompson Alastair
Published: February 2012

Objectives

Patients with peptic ulceration continue to present to surgeons with complications of bleeding or perforation and to die under surgical care. This study sought to examine whether improved consultant input, timely interventions and perioperative care could reduce mortality from peptic ulcer.

Design

Prospective collection of peer-review mortality data using Scottish Audit of Surgical Mortality methodologies (http://www.SASM.org) and analysed using SPSS.

Setting

Secondary care; all hospitals in Scotland, UK, admitting surgical patients over 13 years (1994–2006).

Participants

42 736 patients admitted (38 782 operative and 3954 non-operative) with peptic ulcer disease; 1952 patients died (1338 operative and 614 non-operative deaths) with a diagnosis of peptic ulcer.

Primary and secondary outcome measures

Adverse events; consultant presence at operation, operations performed within 2 h and high dependency/intensive therapy unit (HDU/ITU) use.

Results

Annual mortality fell from 251 in 1994 to 83 in 2006, proportionately greater than the reduction in hospital admissions with peptic ulcer. Adverse events declined over time and were rare for non-operative patients. Consultant surgeon presence at operation rose from 40.0% in 1994 to 73.4% in 2006, operations performed within 2 h of admission from 10.3% in 1994 to 28.1% in 2006 and HDU/ITU use from 52.7% in 1994 to 84.4% in 2006. Consultant involvement (p=0.005) and HDU/ITU care (p=0.026) were significantly associated with a reduction in operative deaths.

Conclusion

Patients with complications of peptic ulceration admitted under surgical care should be offered consultant surgeon input, timely surgery and HDU/ITU care.
Learning from near misses: from quick fixes to closing off the Swiss-cheese holes: Table 1
Authors: Jeffs Lianne , Berta Whitney , Lingard Lorelei , Baker G Ross
Published: February 2012

Introduction

The extent to which individuals in healthcare use near misses as learning opportunities remains poorly understood. Thus, an exploratory study was conducted to gain insight into the nature of, and contributing factors to, organisational learning from near misses in clinical practice.

Methods

A constructivist grounded theory approach was employed which included semi-structured interviews with 24 participants (16 clinicians and 8 administrators) from a large teaching hospital in Canada.

Results

This study revealed three scenarios for the responses to near misses, the most common involved ‘doing a quick fix’ where clinicians recognised and corrected an error with no further action. The second scenario consisted of reporting near misses but not hearing back from management, which some participants characterised as ‘going into a black hole’. The third scenario was ‘closing off the Swiss-cheese holes’, in which a reported near miss generated corrective action at an organisational level. Explanations for ‘doing a quick fix’ included the pervasiveness of near misses that cause no harm and fear associated with reporting the near miss. ‘Going into a black hole’ reflected managers' focus on operational duties and events that harmed patients. ‘Closing off the Swiss-cheese holes’ occurred when managers perceived substantial potential for harm and preventability. Where learning was perceived to occur, leaders played a pivotal role in encouraging near-miss reporting.

Conclusion

To optimise learning, organisations will need to determine which near misses are appropriate to be responded to as ‘quick fixes’ and which ones require further action at the unit and corporate levels.
Getting doctors to clean their hands: lead the followers
Authors: Haessler Sarah , Bhagavan Anju , Kleppel Reva , Hinchey Kevin , Visintainer Paul
Published: February 2012

Background

Despite ample evidence that hand hygiene (HH) can reduce nosocomial infections, physician compliance remains low. The authors hypothesised that attending physician role modelling and peer pressure among internal medicine teams would impact HH adherence.

Methods

Nine teams were covertly observed. Team member entry and exit order, and adherence to HH were recorded secretly. The mean HH percentage across encounters was estimated by compliance of the first person entering and exiting an encounter, and by the attending physician's HH compliance.

Results

718 HH opportunities prior to contact and 744 opportunities after contact were observed. If the first person entering a patient encounter performed HH, the mean compliance of other team members was 64%, but was only 45% if the first person failed to perform HH (p=0.002). When the attending physician performed HH upon entering the patient encounter, the mean HH compliance was 66%, but only 42% if the attending physician did not perform HH (p<0.001). Similar results were seen on exiting the room. The effects of the first person were not driven solely by the attending physician's HH behaviour because the attending physician was first or second to enter 57% of the encounters and exit 44% of the encounters.

Conclusions

If the first person entering a patient room performs HH, then others were more likely to perform HH too, implying that peer pressure impacts team member HH compliance. The attending physician's behaviour also influenced team members regardless of whether the attending physician was the first to enter or exit an encounter, implying that role modelling impacts the HH behaviour of learners. These findings should be used when designing HH improvement programmes targeting physicians.
Major trauma and urban cyclists: physiological status and injury profile
Authors: Manson Joanna , Cooper Sian , West Anita , Foster Elizabeth , Cole Elaine , Tai Nigel R M
Published: February 2012

Introduction

Pedal cycling in cities has the potential to deliver significant health and economic benefits for individuals and society. Safety is the main concern for potential cyclists although the statistical risk of death is low. Little is known about the severity of injuries sustained by city cyclists and their outcome.

Aim

The aim of this study was to characterise the physiological status and injury profile of cyclists admitted to our urban major trauma centre (MTC).

Methods

Database analysis of cyclist casualties between 2004 and 2009. The physiological parameters examined were admission systolic blood pressure (SBP), admission base deficit and prehospital Glasgow Coma Scale.

Results

265 cyclists required full trauma-team activation. 82% were injured during a collision with a motorised vehicle. The majority (73%) had collided with a car or a heavy goods vehicle (HGV). These casualties formed the cohort for further analysis. Cyclists who collided with an HGV were more severely injured and had a higher mortality rate. Low SBP and high base deficit indicate that haemorrhagic shock is a key feature of HGV casualties.

Conclusion

Collision with any vehicle can result in death or serious injury to a cyclist. Injury patterns vary with the type of vehicle involved. HGVs were associated with severe injuries and death as a result of uncontrollable haemorrhage. Awareness of this injury profile may aid prehospital management and expedite transfer to MTC care. Rapid haemorrhage control may salvage some, but not all, of these casualties. The need for continued collision prevention strategies and long-term outcome data collection in trauma patients is highlighted.
Tectonic evolution of Pliocene–Pleistocene wedge-top basins of the southern Apennines: new constraints from magnetic fabric analysis
Authors: Mazzoli Stefano , Szaniawski Rafal , Mittiga Francesco , Ascione Alessandra , Capalbo Andrea
Published: February 2012

In the last decades, multidiscipline investigation of the Apennine mountain belt of peninsular Italy has provided fundamental insights into the genesis and evolution of wedge-top basins. In this study, we focus on shallow-water to continental, Pliocene–Quaternary basins that formed on top of the southern Apennine allochthonous wedge after its emplacement onto a large foreland carbonate platform domain (Apulian Platform). The wedge-top basins analyzed in this study are mostly asymmetric, being bounded to the north by a normal fault showing evidence of synsedimentary activity. The basin successions are generally deformed by open folds and reverse faults resulting from shortening coeval with the sedimentation of the Lower Pliocene to lower–middle Pleistocene, upper part of the basin fill. Anisotropy of magnetic susceptibility data from the basin successions are consistent with a basin evolution characterized by an initial stage of tectonic subsidence controlled by roughly north-northwest–south-southeast-oriented extension, followed by southwest–northeast shortening active during subsequent basin filling. The latter shortening is marked by a dominant northwest–southeast mean trend of the magnetic lineation obtained from most sites in the study area and is interpreted as a result of upward propagation of deep-seated deformation (tectonic inversion) taking place within the buried Apulian Platform sitting in the footwall to the allochthonous wedge. Therefore, our results emphasize a complex interplay of different tectonic processes controlling wedge-top basin development.
The skull and appendicular skeleton of Gryposaurus latidens, a saurolophine hadrosaurid (Dinosauria: Ornithopoda) from the early Campanian (Cretaceous) of Montana, USA
Authors: Prieto-Márquez Albert
Published: February 2012

The osteology of the holotype and referred specimens of the saurolophine hadrosaurid, Gryposaurus latidens , from the lower Two Medicine Formation of Montana (USA), is described in detail. With an estimated early Campanian (Late Cretaceous) age, this dinosaur is one of the oldest known hadrosaurids. Gryposaurus latidens shows several autapomorphies, including dentary tooth crowns, with an apicobasal length/mesiodistal width ratio of 1.7–2.2, a single median carina, and large marginal denticles in sections of the dental battery; deep and oval depression on the posteroventral premaxillary surface; and, in the context of Hadrosauridae, a shallow lacrimal process of the rostral process of the jugal. Furthermore, G. latidens differs from G. notabilis and G. monumentensis in having a broadly arcuate anterolateral oral margin of the premaxilla, a posterior margin of the narial fenestra that is wider than the dorsoventral depth of the proximal region of the narial bar, with a nasal crest that rises above the level of the frontals (in adults) and is located above the posterior margin of the narial fenestra, and wide and shallow posteroventral margin of the rostral process of the jugal, among other characters. This study confirmed a previous hypothesis supporting G. latidens as the sister taxon to a clade composed of the other species of Gryposaurus .