bluuebunny/crossref_metadata_2025 huggingface dataset:
BRAF V600E expression and distribution in desmoplastic infantile astrocytoma/ganglioglioma
Authors: Koelsche C. , Sahm F. , Paulus W. , Mittelbronn M. , Giangaspero F. , Antonelli M. , Meyer J. , Lasitschka F. , von Deimling A. , Reuss D.
Published: July 2013

Aims

Desmoplastic infantile astrocytoma/ganglioglioma (DIA/DIG) is a rare primary neuroepithelial brain tumour typically affecting paediatric patients younger than 24 months. Knowledge about genetic alterations in DIA/DIG is limited. However, a previous study on BRAF V600E mutation in paediatric glioma revealed a BRAF mutation in one of two tested DIAs/DIGs. The limited number of cases in that study did not allow any conclusion about mutation frequency of BRAF in this tumour entity.

Methods

We collected a series of 18 DIAs/DIGs for testing BRAF V600E mutational status by BRAF V600E immunohistochemistry (clone VE1). Cases with sufficient DNA were tested for BRAF V600E mutation by pyrosequencing.

Results

Three out of 18 DIAs/DIGs presented with VE1 binding. A considerable proportion of BRAF V600E mutated tumour cells was detected in the cortical tumour component, whereas the pronounced leptomeningeal tumoural stroma was predominantly negative for VE1 binding. Pyrosequencing confirmed BRAF V600E mutation in two of three VE1‐positive cases.

Conclusion

BRAF V600E mutation affects a subset of DIAs/DIGs and offers new therapeutic opportunities.
Magnetic Resonance Imaging in Patients with Recently Implanted Pacemakers
Authors: FRIEDMAN HANNAH L. , ACKER NANCY , DALZELL CONNIE , SHEN WIN K. , ASIRVATHAM SAMUEL J. , CHA YONG‐MEI , HODGE DAVID , FELMLEE JOEL , WATSON ROBERT , FRIEDMAN PAUL A.
Published: July 2013

Background

Recent studies have shown that magnetic resonance imaging (MRI) of patients with pacemakers can be safely performed under careful monitoring, but they excluded patients with recently implanted devices. Patients with recent implants may be at a greater risk for complications during MRI imaging due to lack of lead and wound maturity.

Methods

We implemented a clinical protocol for MRI imaging of patients with implanted cardiac devices, and prospectively collected data. For this study, we retrospectively analyzed two groups of patients: those with recently implanted (≤42 days) and nonrecently implanted (>42 days) leads at the time of MRI scanning. All devices were interrogated before and after scanning, and were reprogrammed during the scan as per protocol.

Results

Of the 219 scans (in 171 patients), eight included patients with recently implanted (range: 7–36 days) and 211 with only nonrecently implanted pacemaker leads. During the scan, there were no complications in the early or late group. In one patient imaged 79 days postimplant, frequent premature ventricular complexes were noted during the scan, requiring no action. No patient reported pain during or immediately after the procedure. No clinically significant changes in function were seen at subsequent follow up (average 104 days post‐MRI). Compared to patients with nonrecently implanted leads, there was no difference in any parameter between the two groups.

Conclusions

With a strong clinical indication and with careful monitoring, MRI imaging is feasible in patients with recently implanted pacemakers, although experience is limited.
Why Bad People Can't be Good Friends
Authors: Elder Alexis
Published: July 2013

Must the best friends necessarily be good people? On the one hand, as Aristotle puts it, ‘people think that the same people are good and also friends’. But on the other hand, friendship sometimes seems to require that one behave badly. For example, a normally honest person might lie to corroborate a friend's story. What I will call closeness, which I take to include sensitivity to friends' subjective values and concerns as well as an inclination to take their subjective interests as reasons for action, is characteristic of friendship. But this seems to require that good friends should be morally flexible, more so than is compatible with a virtuous character. This would imply tension between ideals of friendship and ideals of character. But there is an important connection between virtue and friendship which arises precisely from friends' closeness, when concern for wellbeing, another important feature of friendship, is also taken into account. This helps mitigate the tension and shows how friendship and virtue are interconnected. The connection in turn provides friendship‐based reason to think the best friends must be good people, even though concerns of friendship may occasionally clash with other moral concerns.
Doa PosterioriPhysicalists Get Our Phenomenal Concepts Wrong?
Authors: Diaz‐Leon E.
Published: July 2013

A posterioriphysicalism is the combination of two appealing views: physicalism (i.e. the view that all facts are either physical or entailed by the physical), and conceptual dualism (i.e. the view that phenomenal truths are not entaileda prioriby physical truths). Recently, some philosophers such as Goff (2011), Levine (2007) and Nida‐Rümelin (2007), among others, have suggested thata posterioriphysicalism cannot explain how phenomenal concepts can reveal the nature of phenomenal properties. In this paper, I wish to defenda posterioriphysicalism from this new and interesting challenge, by arguing thata posterioriphysicalists have the resources to explain how phenomenal concepts can reveal at least something of what it would take for the corresponding phenomenal property to be instantiated.
Relation between soil P test values and mobilization of dissolved and particulate P from the plough layer of typical Danish soils from a long‐term field experiment with applied P fertilizers
Authors: Glæsner N. , Kjaergaard C. , Rubæk G. H. , Magid J.
Published: July 2013

Accumulation of phosphorus (P) in agricultural topsoils can contribute to leaching of P which may cause eutrophication of surface waters. An understanding of P mobilization processes in the plough layer is needed to improve agricultural management strategies. We compare leaching of total dissolved and particulate P through the plough layer of a typical Danish sandy loam soil subjected to three different P fertilizer regimes in a long‐term field experiment established in 1975. The leaching experiment used intact soil columns (20 cm diameter, 20 cm high) during unsaturated conditions. The three soils had small to moderate labile P contents, expressed by water‐extractable P (3.6–10.7 mg/kg), Olsen P (11–28 mg/kg) and degree of P saturation (DPS) (25–34%). Mobilization of total dissolved P (TDP) increased significantly (P < 0.05) from the intact soil columns with increasing labile P, whereas the increase in particulate P (PP) with increasing labile P content was modest and statistically insignificant. We found concentrations up to 1.5 mg TP/L for the plough layer of this typical Danish sandy loam soil. This highlights that even a moderate labile P content can be a potential source of TDP from the plough layer, and that a lower concentration margin of optimum agronomic P levels should be considered.
Teaching with Complicating Views: Beyond the Survey, Behind the Pro and Con
Authors: Locklin Reid B.
Published: July 2013

In this article I propose a method of selecting and assigning readings in the religious studies or theology classroom, such that these readings complicate one another, rather than standing in opposition or as simple alternatives. Such a strategy emulates key pedagogical insights of twelfth‐century sentence collection, an activity at the very heart of the earliest universities in Europe. It also draws support from the theories of intellectual development advanced by William G. Perry, Jr. and the Women's Ways of Knowing Collaborative. Both precedents suggest a principle of “complicating views” that can be flexibly employed in a variety of ways and diverse pedagogical contexts, as illustrated by examples from several classes. Such strategies aim to avoid reinforcing intellectual patterns of dualism or undifferentiated relativism; instead, they attempt to promote students' ability to integrate discordant voices and to appreciate diverse points of view, while also staking their own claims relative to them.
Distribution of Megastigmus aculeatus (Hymenoptera: Torymidae) and the levels of seed predation of Rosa multiflora (Rosaceae)
Authors: Jesse Laura , Collyer Michael , Moloney Kirk , Obrycki John J.
Published: July 2013

Rosa multiflora Thunb. (Rosaceae), an invasive plant that currently infests millions of hectares in the eastern half of the USA, was planted initially in the 1940s as a “living fence”, cover for game animals and for erosion control. The larvae of Megastigmus aculeatus var. nigroflavus Hoffmeyer (Hymenoptera: Torymidae) feed on the developing R. multiflora seeds and have the potential to reduce the seed output of R. multiflora. Rosehips were collected from 49 sites across eastern and southern Iowa, USA, to determine the presence and distribution of M. aculeatus, the larvae of which were found in 266 of the 979 (27%) rosehips that were dissected and at 31 of the 49 sites (63%) that were sampled. The levels of M. aculeatus infestation over 2–3 years were determined at three selected sites and it was documented that more seeds were aborted than were viable or contained wasp larvae. A negative relationship was observed between the numbers of both the aborted and the viable seeds and the probability of a rosehip being attacked by M. aculeatus, indicating that M. aculeatus females are not preferentially selecting rosehips with higher numbers of viable or aborted seeds for oviposition. There is a significant reduction in both the viable seeds and the aborted seeds in hips that have been attacked by M. aculeatus. Detailed knowledge of R. multiflora demography is necessary to determine the level of seed predation that is required to reduce the recruitment of new individuals into the population.
Modulation of Diastolic Filling Using an Epicardial Diastolic Recoil Device
Authors: Snowden Timothy , Biswas Saurabh , Criscione John
Published: July 2013

Diastolic dysfunction likely contributes to all cases of congestive heart failure and is solely responsible for many. Existing cardiac support devices largely ignore diastolic dysfunction and may exacerbate it. Current diastolic devices in development rely on either extensive extraventricular fixation or intraventricular implantation with complications associated with blood contact. A diastolic recoil device is proposed that pneumatically locks to the outside of the heart wall. The end-diastolic total biventricular pressure-volume relationship (EDTBPVR) was used to evaluate, in vitro, the ability of a recoil device to modulate filling mechanics through pneumatic locking as the method of fixation. The pressure in a model heart was incremented and the corresponding volume changes were measured. The heart model and device were pneumatically locked together using a vacuum sac to model the pericardium. The diastolic recoil component shifted the EDTBPVR towards lower pressures at low volumes, providing up to 0.9 kPa (9 cm H2O) of suction, demonstrating enhanced diastolic recoil at beginning diastole. We conclude that pneumatic locking appears to be a viable method for a recoil device to engage the heart.
Development of a Bimodal Ankle-Foot Prosthesis for Walking and Standing/Swaying
Authors: Hansen Andrew H. , Nickel Eric A.
Published: July 2013

The human ankle-foot system conforms to a circular effective rocker shape for walking, but to a much flatter effective shape for standing and swaying. Many persons with lower limb amputations have impaired balance and reduced balance confidence, and may benefit from prostheses designed to provide flatter effective rocker shapes during standing and swaying tasks. This paper describes the development and testing of an ankle-foot prosthesis prototype that provides distinctly different mechanical properties for walking and standing/swaying. The prototype developed was a single-axis prosthetic foot with a lockable ankle for added stability during standing and swaying. The bimodal ankle-foot prosthesis prototype was tested on pseudoprostheses (walking boots with prosthetic feet beneath) for walking and standing/swaying loads, and was compared to an Otto Bock single-axis prosthetic foot and to able-bodied data collected in a previous study. The height-normalized radius of the effective rocker shape for walking with the bimodal ankle-foot prototype was equal to that found earlier for able-bodied persons (0.17); the standing and swaying effective shape had a lower height-normalized radius (0.70) compared with that previously found for able-bodied persons (1.11). The bimodal ankle-foot prosthesis prototype had a similar radius as the Otto Bock single-axis prosthetic foot for the effective rocker shape for walking (0.17 for both), but had a much larger radius for standing and swaying (0.70 for bimodal, 0.34 for single-axis). The results suggest that the bimodal ankle-foot prosthesis prototype provides two distinct modes, including a biomimetic effective rocker shape for walking and an inherently stable base for standing and swaying. The radius of the prototype's effective rocker shape for standing/swaying suggests that it may provide inherent mechanical stability to a prosthesis user, since the radius is larger than the typical body center of mass’s distance from the floor (between 50–60% of height). Future testing is warranted to determine if the bimodal ankle-foot prosthesis will increase balance and balance confidence in prosthesis users.
A Video-Oculography Based Telemedicine System for Automated Nystagmus Identification
Authors: Swart Wayne , Scheffer Cornie , Schreve Kristiaan
Published: July 2013

This paper presents the research and results for an automated jerk-type nystagmus identification system that makes use of an efficient, low cost video-oculography (VOG) device, designed for telemedicine applications. The pupil position is estimated by a hybrid tracking algorithm from the captured VOG images. It is also shown that wavelet analysis with an appropriate mother wavelet, coupled with well-defined geometric constraints can provide a reliable and robust nystagmus identification algorithm. Some original research regarding robust analysis for signals with mixed content is also presented.
Design of an Experimental Set-Up to Study the Behavior of a Flexible Surgical Instrument Inside an Endoscope
Authors: Khatait Jitendra P. , Brouwer Dannis M. , Soemers Herman M. J. R. , Aarts Ronald G. K. M. , Herder Just L.
Published: July 2013

The success of flexible instruments in surgery requires high motion and force fidelity and controllability of the tip. However, the friction and the limited stiffness of such instruments limit the motion and force transmission of the instrument. In a previous study, we developed a flexible multibody model of a surgical instrument inside an endoscope in order to study the effect of the friction, bending and rotational stiffness of the instrument and clearance on the motion hysteresis and the force transmission. In this paper, we present the design and evaluation of an experimental setup for the validation of the flexible multibody model and the characterization of the instruments. A modular design was conceived based on three key functionalities: the actuation from the proximal end, the displacement measurement of the distal end, and the measurement of the interaction force. The exactly constrained actuation module achieves independent translation and rotation of the proximal end. The axial displacement and the rotation of the distal end are measured contactless via a specifically designed air bearing guided cam through laser displacement sensors. The errors in the static measurement are 15 μm in translation and 0.15 deg in rotation. Six 1-DOF load cell modules using flexures measure the interaction forces and moments with an error of 0.8% and 2.5%, respectively. The achieved specifications allow for the measurement of the characteristic behavior of the instrument inside a curved rigid tube and the validation of the flexible multibody model.
Probing ultrathin film continuity and interface abruptness with x-ray photoelectron spectroscopy and low-energy ion scattering
Authors: Zhang Wenyu , Nahm Rambert K. , Ma Paul F. , Engstrom James R.
Published: July 2013

The authors have examined ultrathin (≤10 Å) tantalum nitride (TaNx) thin films deposited by atomic layer deposition (ALD) on three surfaces relevant to interconnect layers in microelectronic devices: thermally grown SiO2; a Cu thin film grown by physical vapor deposition, and a carbon-doped SiO2 porous low-κ thin film. The authors have employed ex situ angle-resolved x-ray photoelectron spectroscopy (ARXPS), low-energy ion scattering spectroscopy (LEISS), and atomic force microscopy (AFM) to determine the continuity of these thin films, and by implication, the abruptness of the thin film/substrate interface. On SiO2 and low-κ, the authors find similar results: both ARXPS and AFM indicate that smooth, uniform thin films are deposited, consistent with nearly layer-by-layer growth of TaNx on these surfaces. Examination of these films using LEISS reveals that while the 10 Å TaNx thin films are continuous, the 5 Å TaNx thin films are not continuous and may possess on the order of ∼10% exposed substrate in the form of small subnanometer inclusions. On Cu, the situation is quite different. The TaNx thin films on these surfaces are not continuous, and our results point to a mixed layer of TaNx and Cu forming during ALD. In all cases, if one were to rely solely on results from ARXPS, the picture would be incomplete as the results from LEISS are ultimately decisive concerning thin film continuity.
Fabrication of superconducting tantalum nitride thin films using infrared pulsed laser deposition
Authors: Chaudhuri Saumyadip , Maasilta Ilari J. , Chandernagor Lucie , Ging Marion , Lahtinen Manu
Published: July 2013

The authors report the successful fabrication of superconducting tantalum nitride (TaN) thin films using a pulsed laser deposition technique with 1064 nm radiation. Films with thickness ∼100 nm deposited on MgO (100) single crystals and on oxidized silicon (SiO2) substrates exhibited a superconducting transition temperature of ∼8 K and 6 K, respectively. The topography of these films were investigated using atomic force and scanning electron microscopy, revealing fairly large area particulate free and smooth surfaces, while the structure of the films were investigated using θ-2θ and glancing angle x-ray diffraction techniques. For films grown on MgO a face-centered cubic phase of TaN was observed, while films grown on SiO2 exhibited the face-centered cubic as well as a mononitride hexagonal phase. The transition temperature of the TaN deposited on SiO2 was found to be more sensitive to the nitrogen pressure during deposition as compared to the TaN deposited on MgO.
Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay
Authors: Molina Daniel Martinez , Jafari Rozbeh , Ignatushchenko Marina , Seki Takahiro , Larsson E. Andreas , Dan Chen , Sreekumar Lekshmy , Cao Yihai , Nordlund Pär
Published: July 2013

Drug Targeting

Drug efficacy depends on the extent of binding to a cellular target (often a protein) with adverse effects caused by excessive target binding or by off-target binding. Engagement of a target protein inside cells is influenced by the effective drug concentration and by factors that regulate the protein conformation, making it difficult to predict efficacy based on in vitro affinity studies.

Martinez Molina
et al.

(p.
84
) took advantage of the shift in protein thermal stability caused by drug binding to directly monitor target protein-drug interactions in cells. The method was used to monitor drug target engagement in cancer cells and in mouse livers and kidneys.
Bringing Ecosystem Services into Economic Decision-Making: Land Use in the United Kingdom
Authors: Bateman Ian J. , Harwood Amii R. , Mace Georgina M. , Watson Robert T. , Abson David J. , Andrews Barnaby , Binner Amy , Crowe Andrew , Day Brett H. , Dugdale Steve , Fezzi Carlo , Foden Jo , Hadley David , Haines-Young Roy , Hulme Mark , Kontoleon Andreas , Lovett Andrew A. , Munday Paul , Pascual Unai , Paterson James , Perino Grischa , Sen Antara , Siriwardena Gavin , van Soest Daan , Termansen Mette
Published: July 2013

Monitoring Land Use

Land-use decisions are based largely on agricultural market values. However, such decisions can lead to losses of ecosystem services, such as the provision of wildlife habitat or recreational space, the magnitude of which may overwhelm any market agricultural benefits. In a research project forming part of the UK National Ecosystem Assessment,

Bateman
et al.

(p.
45
) estimate the value of these net losses. Policies that recognize the diversity and complexity of the natural environment can target changes to different areas so as to radically improve land use in terms of agriculture and greenhouse gas emissions, recreation, and wild species habitat and diversity.
Emergence of Agriculture in the Foothills of the Zagros Mountains of Iran
Authors: Riehl Simone , Zeidi Mohsen , Conard Nicholas J.
Published: July 2013

Early Farmers?

What was the role of the eastern Fertile Crescent (which includes southeastern Turkey, northeastern Iraq, and western Iran) in the transition from foraging to farming?

Riehl
et al.

(p.
65
; see the Perspective by

Willcox

) investigated an archaeobotanical assemblage from Chogha Golan, in modern Iran over an apparently continuous occupation of the site over 2000 years, which captures the transition from foraging to farming. The rich archaeobotanical remains suggest the use of a wide array of plant species, including the progenitors of key crop plants (wheat, barley, and large-seeded legumes). Residents of the eastern Fertile Crescent thus appear to have been involved in plant management and possibly in the domestication of wild crop plants.
Being Modern in Revolutionary Russia
We Modern People
Science Fiction and the Making of Russian Modernity

by Anindita Banerjee

Wesleyan University Press, Middletown, CT, 2012. 218 pp. $75. ISBN 9780819573339. Paper, $24.95. ISBN 9780819573346.
Authors: Siddiqi Asif
Published: July 2013

Banerjee explores links among science, technology, science popularization, and modernization in early-20th-century Russia.
Seabirds—Individuals in Colonies
Authors: Weimerskirch Henri
Published: July 2013

Seabirds from nearby colonies forage in different locations rather than directly competing for food.

[Also see Report by

Wakefield
et al.

]
Can Oompa-Loompas Save Us from Perebor?
The Golden Ticket
P, NP, and the Search for the Impossible

by Lance Fortnow

Princeton University Press, Princeton, NJ, 2013. 188 pp. $26.95, £18.95. ISBN 9780691156491.
Authors: Elser Veit
Published: July 2013

Using stories and examples, Fortnow surveys the P versus NP problem and discusses how computer scientists deal with hard problems.
Effect of Prolonged Incubation Time on Results of the QuantiFERON TB Gold In-Tube Assay for Diagnosis of Latent Tuberculosis Infection
Authors: Min Joo-Won , Lee Ha-Youn , Lee Ji Sun , Lee Jinwoo , Chung Jae Ho , Han Sung Koo , Yim Jae-Joon
Published: July 2013

Previous reports have shown that the sensitivity of the 6-day lymphocyte stimulation test is much higher than those of commercially available gamma interferon release assays (IGRAs). The aim of this study was to elucidate the effect of prolonged incubation on the results of the QuantiFERON TB Gold in-tube (QFT-GIT) assay. Patients aged >20 years with suspected tuberculosis (TB) were recruited prospectively from 1 May 2009 to 31 December 2010. In addition, healthy volunteers with no history of TB treatment were included as controls. For each participant, three sets of the QFT-GIT assay were performed using 24-, 48-, and 72-h incubation tests, and the results were compared. Thirty-seven patients with suspected pulmonary TB and 33 healthy controls were enrolled in the study. Of the 37 patients with suspected TB, the QFT-GIT assay results were positive for 28 (75.7%) after a 24-h incubation period. After prolonged incubation, the results differed in four (10.8%) of the 37 patients suspected of having TB. Among 27 patients with culture-confirmed TB, the sensitivities of the QFT-GIT assay after the 24-, 48-, and 72-h incubation tests were 85.2%, 81.5%, and 81.5%, respectively. Among the 33 healthy controls, the QFT-GIT assay results were positive in two (6.1%) after a 24-h incubation period. The results changed for two (6.1%) of the 33 healthy controls after prolonged incubation. The specificities of the QFT-GIT assay after 24, 48, and 72 h of incubation were 93.9%, 87.9%, and 90.9%, respectively. Prolonging the incubation time did not increase the sensitivity of the QFT-GIT assay. The manufacturer-recommended incubation time of 16 to 24 h should be respected because prolonged incubation can cause indeterminate or false-positive results.
Rapid Antifungal Susceptibility Testing by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis
Authors: Vella Antonietta , De Carolis Elena , Vaccaro Luisa , Posteraro Patrizia , Perlin David S. , Kostrzewa Markus , Posteraro Brunella , Sanguinetti Maurizio
Published: July 2013

The widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of
Candida albicans
after a 3-h incubation in the presence of “breakpoint” level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) μg/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65
C. albicans
isolates, including 13
fks1
mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 μg/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 μg/ml. In this way, the drug resistance of
C. albicans
isolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant
FKS1
genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.
Identification of Salmonella enterica Serovar Typhi Genotypes by Use of Rapid Multiplex Ligation-Dependent Probe Amplification
Authors: Pham Thanh Duy , Tran Vu Thieu Nga , Tran Thuy Chau , Lodén Martin , Tuin Kiki , Campbell James I. , Van Minh Hoang Nguyen , Voong Vinh Phat , Farrar Jeremy J. , Holt Kathryn E. , Dougan Gordon , Baker Stephen
Published: July 2013

Salmonella enterica
serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the
S
. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for
S
. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene
gyrA
and the topoisomerase gene
parC
with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic.
Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins
Authors: Payne Angela M. , Zorman Julie , Horton Melanie , Dubey Sheri , ter Meulen Jan , Vora Kalpit A.
Published: July 2013

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities ofClostridium difficiletoxins A (TcdA) and B (TcdB) and binary toxin (CDTa-CDTb),Corynebacterium diphtheriaetoxin (DT), andPseudomonas aeruginosaexotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity againstC. difficiletoxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.
Rapid Differentiation of Cryptic Species within Cryptococcus gattii by a Duplex PCR Assay
Authors: Feng Xiaobo , Fu Xiaohua , Ling Bo , Wang Lei , Liao Wanqing , Pan Weihua , Yao Zhirong
Published: July 2013

Cryptococcus gattii
consists of four cryptic species, VGI, VGII, VGIII, and VGIV. Herein, a duplex PCR assay using two primer pairs targeting the vacuolar membrane gene and the intergenic spacer region was developed. It successfully distinguished the cryptic species according to the distinct size of the amplicons.
Expanded Cocirculation of Stable Subtypes, Emerging Lineages, and New Sporadic Reassortants of Porcine Influenza Viruses in Swine Populations in Northwest Germany
Authors: Harder Timm C. , grosse Beilage Elisabeth , Lange Elke , Meiners Carolin , Döhring Stefanie , Pesch Stefan , Noé Thomas , Grund Christian , Beer Martin , Starick Elke
Published: July 2013

The emergence of the human 2009 pandemic H1N1 (H1N1pdm) virus from swine populations refocused public and scientific attention on swine as an important source of influenza A viruses bearing zoonotic potential. Widespread and year-round circulation of at least four stable lineages of porcine influenza viruses between 2009 and 2012 in a region of Germany with a high-density swine population is documented here. European avian influenza virus-derived H1N1 (H1N1av) viruses dominated the epidemiology, followed by human-derived subtypes H1N2 and H3N2. H1N1pdm viruses and, in particular, recently emerging reassortants between H1N1pdm and porcine HxN2 viruses (H1pdmN2) were detected in about 8% of cases. Further reassortants between these main lineages were diagnosed sporadically. Ongoing diversification both at the phylogenetic and at the antigenic level was evident for the H1N1av lineage and for some of its reassortants. The H1avN2 reassortant R1931/11 displayed conspicuously distinct genetic and antigenic features and was easily transmitted from pig to pig in an experimental infection. Continuing diverging evolution was also observed in the H1pdmN2 lineage. These viruses carry seven genome segments of the H1N1pdm virus, including a hemagglutinin gene that encodes a markedly antigenically altered protein. The zoonotic potential of this lineage remains to be determined. The results highlight the relevance of surveillance and control of porcine influenza virus infections. This is important for the health status of swine herds. In addition, a more exhaustive tracing of the formation, transmission, and spread of new reassortant influenza A viruses with unknown zoonotic potential is urgently required.
Identification of a Suppressor Mutation That Improves the Yields of Hexon-Modified Adenovirus Vectors
Authors: Bruder Joseph T. , Chen Ping , Semenova Elena , Thomas Charlie A. , Konovalova Svetlana , Ekberg Greg , Ettyreddy Damodar , McVey Duncan , Gall Jason G. , King C. Richter , Brough Douglas E.
Published: July 2013

We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.
Mutational Analysis of the Binding Pockets of the Diketo Acid Inhibitor L-742,001 in the Influenza Virus PA Endonuclease
Authors: Stevaert Annelies , Dallocchio Roberto , Dessì Alessandro , Pala Nicolino , Rogolino Dominga , Sechi Mario , Naesens Lieve
Published: July 2013

The influenza virus PA endonuclease, which cleaves capped host pre-mRNAs to initiate synthesis of viral mRNA, is a prime target for antiviral therapy. The diketo acid compound L-742,001 was previously identified as a potent inhibitor of the influenza virus endonuclease reaction, but information on its precise binding mode to PA or potential resistance profile is limited. Computer-assisted docking of L-742,001 into the crystal structure of inhibitor-free N-terminal PA (PA-Nter) indicated a binding orientation distinct from that seen in a recent crystallographic study with L-742,001-bound PA-Nter (R. M. DuBois et al., PLoS Pathog. 8:e1002830, 2012). A comprehensive mutational analysis was performed to determine which amino acid changes within the catalytic center of PA or its surrounding hydrophobic pockets alter the antiviral sensitivity to L-742,001 in cell culture. Marked (up to 20-fold) resistance to L-742,001 was observed for the H41A, I120T, and G81F/V/T mutant forms of PA. Two- to 3-fold resistance was seen for the T20A, L42T, and V122T mutants, and the R124Q and Y130A mutants were 3-fold more sensitive to L-742,001. Several mutations situated at noncatalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes reconstituted in cell culture, consistent with the less conserved nature of these PA residues. Our data provide relevant insights into the binding mode of L-742,001 in the PA endonuclease active site. In addition, we predict some potential resistance sites that should be taken into account during optimization of PA endonuclease inhibitors toward tight binding in any of the hydrophobic pockets surrounding the catalytic center of the enzyme.
Cell Susceptibility to Baculovirus Transduction and Echovirus Infection Is Modified by Protein Kinase C Phosphorylation and Vimentin Organization
Authors: Turkki Paula , Makkonen Kaisa-Emilia , Huttunen Moona , Laakkonen Johanna P. , Ylä-Herttuala Seppo , Airenne Kari J. , Marjomäki Varpu
Published: July 2013

Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.
Alphacoronavirus Protein 7 Modulates Host Innate Immune Response
Authors: Cruz Jazmina L. G. , Becares Martina , Sola Isabel , Oliveros Juan Carlos , Enjuanes Luis , Zúñiga Sonia
Published: July 2013

Innate immune response is the first line of antiviral defense resulting, in most cases, in pathogen clearance with minimal clinical consequences. Viruses have developed diverse strategies to subvert host defense mechanisms and increase their survival. In the transmissible gastroenteritis virus (TGEV) as a model, we previously reported that accessory gene 7 counteracts the host antiviral response by associating with the catalytic subunit of protein phosphatase 1 (PP1c). In the present work, the effect of the absence of gene 7 on the host cell, during infection, was further analyzed by transcriptomic analysis. The pattern of gene expression of cells infected with a recombinant mutant TGEV, lacking gene 7 expression (rTGEV-Δ7), was compared to that of cells infected with the parental virus (rTGEV-wt). Genes involved in the immune response, the interferon response, and inflammation were upregulated during TGEV infection in the absence of gene 7. An exacerbated innate immune response during infection with rTGEV-Δ7 virus was observed bothin vitroandin vivo. An increase in macrophage recruitment and activation in lung tissues infected with rTGEV-Δ7 virus was observed compared to cells infected with the parental virus. In summary, the absence of protein 7 bothin vitroandin vivoled to increased proinflammatory responses and acute tissue damage after infection. In a porcine animal model, which is immunologically similar to humans, we present a novel example of how viral proteins counteract host antiviral pathways to determine the infection outcome and pathogenesis.
Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability
In Vivo
Authors: Redrejo-Rodríguez Modesto , Rodríguez Javier M. , Suárez Cristina , Salas José , Salas María L.
Published: July 2013

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information.
Increased Acid Stability of the Hemagglutinin Protein Enhances H5N1 Influenza Virus Growth in the Upper Respiratory Tract but Is Insufficient for Transmission in Ferrets
Authors: Zaraket Hassan , Bridges Olga A. , Duan Susu , Baranovich Tatiana , Yoon Sun-Woo , Reed Mark L. , Salomon Rachelle , Webby Richard J. , Webster Robert G. , Russell Charles J.
Published: July 2013

Influenza virus entry is mediated by the acidic-pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in the HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) (VN1203) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (K to I at position 58) (activation pH 5.5). The VN1203-HA2-K58I virus had replication kinetics similar to those of wild-type VN1203 in MDCK and normal human bronchial epithelial cells and yet had reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar levels of morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naive contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract, and yet it was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer α(2,6) receptor binding specificity and remove a critical N-linked glycosylation site. The information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals in the background of a non-laboratory-adapted virus provides essential information for the surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.
Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein
Authors: Klasse Per Johan , Depetris Rafael S. , Pejchal Robert , Julien Jean-Philippe , Khayat Reza , Lee Jeong Hyun , Marozsan Andre J. , Cupo Albert , Cocco Nicolette , Korzun Jacob , Yasmeen Anila , Ward Andrew B. , Wilson Ian A. , Sanders Rogier W. , Moore John P.
Published: July 2013

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.
Endothelial Cell Stimulation Overcomes Restriction and Promotes Productive and Latent HIV-1 Infection of Resting CD4
+
T Cells
Authors: Shen Anding , Baker Jacob J. , Scott Geoffrey L. , Davis Yelena P. , Ho Yen-Yi , Siliciano Robert F.
Published: July 2013

Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4
+
T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4
+
T cells, and latent infection in those cells may arise when infected CD4
+
T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4
+
T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells
in vivo
. Our study proposes a new mechanism for infection of resting CD4
+
T cells
in vivo
and a new mechanism for latent infection in resting CD4
+
T cells.