bluuebunny/crossref_metadata_2025 huggingface dataset:
In situ beam drift detection using a two-dimensional electron-beam position monitoring system for multiple-electron-beam–direct-write lithography
Authors: Chen Sheng-Yung , Tsai Kuen-Yu , Ng Philip C. W. , Ng Hoi-Tou , Liu Chun-Hung , Shen Yu-Tian , Kuan Chieh-Hsiung , Chen Yung-Yaw , Kuo Yi-Hung , Wu Cheng-Ju , Yen Jia-Yush
Published: August 2011

Electron-beam lithography is one of the promising candidates to replace optical projection lithography due to its high resolution and maskless direct-write capability. In order to achieve the throughput requirement for high-volume manufacturing, miniaturized electro-optics elements are utilized to drive massively parallel beams simultaneously. In high-throughput multiple-electron-beam systems, beam positioning drift problems can become quite serious due to several factors such as thermal distortion and fabrication errors of electron optics. In single-beam systems, periodic recalibration with reference markers on the wafer can be utilized to achieve beam placement accuracy. This technique is not easy for multiple-beam systems. In this article, an innovative in situ two-dimensional electron-beam position monitoring system for multiple-electron-beam lithography is studied. An array of miniaturized electron detectors to measure scattered electrons from the substrate is placed above the wafer. It is assumed that the detector array signals are correlated with the distribution of electron trajectories, and the change of trajectory distortion due to the beam drift can be predicted by Monte Carlo electron-scattering simulation. A standard quadrant detection (SQD) method and a linear least-squares (LLS) method are used to estimate the beam drift from the detector array signals. Simulation results indicate that while the estimation uncertainty of both methods can be reduced substantially when the number of detected electrons is large enough. The LLS method always outperforms the SQD one regardless the detected electron numbers.
Incorporation of hydrogen in CuInSe2: Improvements of the structure
Authors: Yakushev M. V. , Ogorodnikov I. I. , Volkov V. A. , Mudryi A. V.
Published: August 2011

CuInSe2 single crystals were ion implanted with a dose of 3 × 1016 cm−2 by 2.5 keV H+ at 150 and 250 °C. Before and after the implantation the crystals were analyzed by Rutherford backscattering/channeling (RBS/C) along the 〈112〉 axis using 2 MeV He+. The RBS/C spectra indicate that the implantation at 150 °C introduces a layer of radiation damage, whereas after the implantation at 250 °C no structural deterioration of the lattice can be seen. Quite the contrary, the RBS/C spectra reveal a considerable decrease in the dechanneling parameters suggesting improvements in the lattice structural quality attributed to the incorporation of hydrogen.
Current transport mechanisms of InGaN metal-insulator-semiconductor photodetectors
Authors: Shao Z. G. , Chen D. J. , Liu B. , Lu H. , Xie Z. L. , Zhang R. , Zheng Y. D.
Published: August 2011

The authors report on InGaN metal-insulator-semiconductor (MIS) photodetectors with two different insulating layers of Si3N4 and Al2O3 deposited via plasma-enhanced chemical vapor deposition and atomic layer deposition, respectively. The photoresponse spectra show that the metal-Al2O3-InGaN photodetector exhibits an approximately threefold higher photoelectric responsivity and a larger spectral rejection ratio as compared to the metal-Si3N4-InGaN photodetector at a 1 V reverse bias. The current transport mechanisms in MIS photodetectors were investigated in order to determine the difference in photoresponse. The results show that the space charge limited current is a dominant leakage conduction mechanism in the InGaN MIS photodetectors, but this mechanism is mediated by the exponential trap distribution in the metal-Si3N4-InGaN photodetector. This indicates a higher density of trap states in the Si3N4 bulk. A bidirectional Fowler–Nordheim tunneling effect was observed in the metal-Si3N4-InGaN photodetector, which indicates high trap states in the Si3N4 bulk and the Si3N4–InGaN interface. These traps increase the probability of photogenerated carrier recombination in the bulk of the dielectrics and at the interface of dielectric-InGaN, and hence the photoelectric responsivity is lower.
Long range node‐strut analysis of trabecular bone microarchitecture
Authors: Schmah Tanya , Marwan Norbert , Thomsen Jesper Skovhus , Saparin Peter
Published: August 2011

Purpose:

We present a new morphometric measure of trabecular bone microarchitecture, calledmean node strength (NdStr), which is part of a newly developed approach called long range node‐strut analysis. Our general aim is to describe and quantify the apparent “latticelike” microarchitecture of the trabecular bone network.

Methods:

Similar in some ways to the topological node‐strut analysis introduced by Garrahanet al. [J. Microsc. 142, 341–349 (1986)], our method is distinguished by an emphasis on long‐range trabecular connectivity. Thus, while the topological classification of a pixel (after skeletonization) as a node, strut, or terminus, can be determined from the 3 × 3 neighborhood of that pixel, our method, which does not involve skeletonization, takes into account a much larger neighborhood. In addition, rather than giving a discrete classification of each pixel as a node, strut, or terminus, our method produces a continuous variable, node strength. The node strength is averaged over a region of interest to produce the mean node strength of the region.

Results:

We have applied our long range node‐strut analysis to a set of 26 high‐resolution peripheral quantitative computed tomography (pQCT) axial images of human proximal tibiae acquired 17 mm below the tibial plateau. We found that NdStr has a strong positive correlation with trabecular volumetric bone mineral density (BMD). After an exponential transformation, we obtain a Pearson's correlation coefficient ofr = 0.97. Qualitative comparison of images with similar BMD but with very different NdStr values suggests that the latter measure has successfully quantified the prevalence of the “latticelike” microarchitecture apparent in the image. Moreover, we found a strong correlation (r = 0.62) between NdStr and the conventional node‐terminus ratio (Nd/Tm) of Garrahan et al. The Nd/Tm ratios were computed using traditional histomorphometry performed on bone biopsies obtained at the same location as the pQCT scans.

Conclusions:

The newly introduced morphometric measure allows a quantitative assessment of the long‐range connectivity of trabecular bone. One advantage of this method is that it is based on pQCT images that can be obtained noninvasively from patients, i.e., without having to obtain a bone biopsy from the patient.
Value-Added Measures and the Future of Educational Accountability
Authors: Harris Douglas N.
Published: August 2011

Research has yet to show whether new estimates of teacher performance can be used to improve teaching and learning.
Nest Inheritance Is the Missing Source of Direct Fitness in a Primitively Eusocial Insect
Authors: Leadbeater Ellouise , Carruthers Jonathan M. , Green Jonathan P. , Rosser Neil S. , Field Jeremy
Published: August 2011

Fitness benefits from the inheritance of breeding resources may explain why
Polistes
wasps cooperate with nonrelatives.
Viviparity and K-Selected Life History in a Mesozoic Marine Plesiosaur (Reptilia, Sauropterygia)
Authors: O’Keefe F. R. , Chiappe L. M.
Published: August 2011

Plesiosaurs gave birth to a single, live large offspring and may have engaged in maternal care.
Moving Knowledge

How Well Do Facts Travel? The Dissemination of Reliable Knowledge
Peter Howlett and Mary S. Morgan, Eds.

Cambridge University Press, Cambridge, 2011. 485 pp. $95, £60. ISBN 9780521196543. Paper, $29.99, £19.99. ISBN 9780521159586.
Authors: Porter Theodore M.
Published: August 2011

In this series of essays, the authors explore how facts sometimes gain an "independent life" that allows them to be used in fields removed from their original location.
Interaction-Driven Spectrum Reconstruction in Bilayer Graphene
Authors: Mayorov A. S. , Elias D. C. , Mucha-Kruczynski M. , Gorbachev R. V. , Tudorovskiy T. , Zhukov A. , Morozov S. V. , Katsnelson M. I. ,   V. I. Fal’ko, Geim A. K. , Novoselov K. S.
Published: August 2011

A correlated-electron phase was observed at low temperatures in suspended graphene bilayers with high carrier mobilities.
We Need a Deeper Sense of Time
Deep Future
The Next 100,000 Years of Life on Earth

by Curt Stager

Thomas Dunne Books, New York, 2011. 300 pp. $25.99. ISBN 9780312614621.
Authors: Wing Scott L.
Published: August 2011

Discussing examples of climate change in Earth's past, Stager furnishes a balanced look at how humans should weigh their long-term effects on the planet's future.
Moving Toward Global Science
Authors: Suresh Subra
Published: August 2011

We are experiencing a new era in the advancement of science. More nations recognize that innovation, driven by science and engineering (S&E), is the fuel for economic growth, prosperity, and social well-being. Global annual investment in R&D has doubled since 1996 to $1.1 trillion. There is strong determination in countries large and small to be aggressively competitive in R&D growth, given the lure of innovation, the sway of geopolitical events, and tighter economic interdependence. Relative to their respective gross domestic products, China, India, Korea, Singapore, and Qatar are some of the nations that have substantially increased investments in S&E research and education, with a focus on the strategic development of their S&E workforce. Relative precollege student achievement rankings, intellectual property registrations, and authorship of scientific papers highlight a dynamic global balance. At the same time, the world has become highly interconnected, so that local economic shifts hinge not only on long-term support for scientific research in each country but also on creative global collaboration. Cooperation in this context requires new thinking and an auspicious environment in which to cultivate and fortify this synergy. What strategies will move us there?
The Plant-Fungal Marketplace
Authors: Selosse Marc-André , Rousset François
Published: August 2011

Plant-fungal symbiosis is stabilized by the extent of reciprocal nutrient supply.
Catching a Moving Target
Authors: Wang Taia T. , Palese Peter
Published: August 2011

Broadly neutralizing antibodies could help create an influenza vaccine that would not have to be updated.
Landmark Study Reveals an Oil Quagmire
Authors: Malakoff David
Published: August 2011

Last week, the United Nations Environment Programme reported that oil pollution in Nigeria's oil-rich and politically volatile Ogoniland region has "penetrated further and deeper than many have supposed."
Rejected Salmon Plan Could Bring Changes to U.S. Dams
Authors: Service Robert F.
Published: August 2011

A federal judge has said that the U.S. government needs to do more to improve the habitat of endangered salmon and steelhead in the Columbia River Basin.
Despite Data Gaps, U.S. Moves Closer to Drilling in Arctic Ocean
Authors: Schmidt Charles
Published: August 2011

Last week, the Department of the Interior conditionally approved Shell Oil Co.'s plans to begin drilling four exploratory wells in the Arctic Ocean in 2012.
Mayhem on the Mekong
Authors: Stone Richard
Published: August 2011

Hydropower dams planned for the lower Mekong could damage ecosystems and erode food security across the river basin.
Trying to Reset the Clock on Type 1 Diabetes
Authors: Couzin-Frankel Jennifer
Published: August 2011

Researchers dream of permanently halting or reversing a major autoimmune disease; new drug studies show that, despite some promising signs, the goal is still far off.
Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans
Authors: Dangoudoubiyam Sriveny , Vemulapalli Ramesh , Ndao Momar , Kazacos Kevin R.
Published: August 2011

Baylisascarislarva migrans is an important zoonotic disease caused byBaylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although aB. procyonisexcretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinantB. procyonisantigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis ofBaylisascarislarva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinicalBaylisascarislarva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies toToxocarainfection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology ofBaylisascarislarva migrans by ELISA.
Epithelial Interleukin-8 Responses to Oral Bacterial Biofilms
Authors: Peyyala R. , Kirakodu S. , Novak K. F. , Ebersole J. L.
Published: August 2011

Anin vitromodel of bacterial biofilms on rigid gas-permeable contact lenses (RGPLs) was developed to challenge oral epithelial cells. This novel model provided seminal data on oral biofilm-host cell interactions, and with selected bacteria, the biofilms were more effective than their planktonic counterparts at stimulating host cell responses.
The Signal Peptide Sequence Impacts the Immune Response Elicited by a DNA Epitope Vaccine
Authors: Vatakis Dimitrios , McMillan Minnie
Published: August 2011

We examined the effect of two leader sequences, one from a transmembrane molecule (H2-Ld) and another from a secreted molecule (rat KC chemokine), on the immunogenicity of DNA epitope vaccines. The chemokine leader enhanced vaccine immunogenicity, thus underscoring the importance of the leader sequence in DNA epitope vaccine design.
Systematic Review and Meta-Analysis of Antigen Detection Tests for the Diagnosis of Tuberculosis
Authors: Flores L. L. , Steingart K. R. , Dendukuri N. , Schiller I. , Minion J. , Pai M. , Ramsay A. , Henry M. , Laal S.
Published: August 2011

Tests that detect
Mycobacterium tuberculosis
antigens in clinical specimens could provide rapid direct evidence of active disease. We performed a systematic review to assess the diagnostic accuracy of antigen detection tests for active tuberculosis (TB) according to standard methods and summarized test performance using bivariate random effects meta-analysis. Overall, study quality was a concern. For pulmonary TB (47 studies, 5,036 participants), sensitivity estimates ranged from 2% to 100% and specificity from 33% to 100%. Lipoarabinomannan (LAM) was the antigen most frequently targeted (23 studies, 49%). The pooled sensitivity of urine LAM was higher in HIV-infected than HIV-uninfected individuals (47%; 95% confidence interval [CI], 26 to 68% versus 14%; 95% CI, 4 to 38%); pooled specificity estimates were similar: 96%; 95% CI, 81 to 100% and 97%; 95% CI, 86 to 100%, respectively. For extrapulmonary TB (21 studies, 1,616 participants), sensitivity estimates ranged from 0% to 100% and specificity estimates from 62% to 100%. Five studies targeting LAM, ESAT-6, Ag85 complex, and the 65-kDa antigen in cerebrospinal fluid, when pooled, yielded the highest sensitivity (87%; 95% CI, 61 to 98%), but low specificity (84%; 95% CI, 60 to 95%). Because of the limited number of studies targeting any specific antigen other than LAM, we could not draw firm conclusions about the overall clinical usefulness of these tests. Further studies are warranted to determine the value of LAM detection for TB meningitis in high-HIV-prevalence settings. Considering that antigen detection tests could be translated into rapid point-of-care tests, research to improve their performance is urgently needed.
Effect of the Modified Live Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccine on European and North American PRRSV Shedding in Semen from Infected Boars
Authors: Han Kiwon , Seo Hwi Won , Shin Jeoung Hwa , Oh Yeonsu , Kang Ikjae , Park Changhoon , Chae Chanhee
Published: August 2011

The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 100.1to 101.0viral genome copies per ml and 3.63 to 101.150% tissue culture infective doses (TCID50)/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 100.2to 104.7viral genome copies per ml and 1.14 to 103.07TCID50/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 100.1to 104.57viral genome copies per ml and 1.66 to 103.10TCID50/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 100.3to 105.14viral genome copies per ml and 1.69 to 103.17TCID50/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge.
Vaccine and Monoclonal Antibody That Enhance Mouse Resistance to Candidiasis
Authors: Xin Hong , Cutler Jim E.
Published: August 2011

Previously we showed that antibodies specific for the glycan β-1,2-mannotriose [β-(Man)3] on the cell surface ofCandida albicansprotect mice against disseminated candidiasis (H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, Proc. Natl. Acad. Sci. U. S. A. 105:13526–13531, 2008). Furthermore, six 14-mer peptides that are within the N-terminal portion ofC. albicanswall proteins were conjugated to the glycan in an attempt to create immunogenic glycopeptide conjugates. By a dendritic cell (DC)-based immunization approach, all were immunogenic and three of the six conjugates induced a high degree of protection in mice. Interestingly, whereas all six peptides induced antibody responses when used alone to pulse DCs for subsequent immunizations, three peptides induced protection, and one in particular, peptide Fba (derived fromfructose-bisphosphatealdolase), induced robust protective responses and is the focus of the current work. Fba peptide is not restricted by the major histocompatibility complex class II (MHC-II), as it induced anti-Fba antibodies in mice of different H-2 haplotypes and in rabbits. Furthermore, the peptide induced protection against disease caused by differentC. albicansstrains. Partial protection was achieved when alum was used in place of DCs for Fba immunizations. The passive transfer of immune sera from Fba-vaccinated mice, but not immune serum preabsorbed with fungal cells, conferred protection in naïve mice. This result, along with our finding that a monoclonal antibody specific for the peptide, E2-9 (IgM), protected mice against candidiasis, provide strong evidence that antibodies contribute to protection. Our work demonstrates the utility of cell wall peptides alone or as glycopeptides in vaccines designed for the induction of immunity against candidiasis and monoclonal antibodies as a rapid immunoprotective approach against the disease.
Identification of the Immunogenic Outer Membrane Protein A Antigen of Haemophilus parasuis by a Proteomics Approach and Passive Immunization with Monoclonal Antibodies in Mice
Authors: Tian Huabin , Fu Fang , Li Xuesong , Chen Xin , Wang Wei , Lang Yuekun , Cong Feng , Liu Changjun , Tong Guangzhi , Li Xi
Published: August 2011

Monoclonal antibodies (MAbs) againstHaemophilus parasuiswere generated by fusing spleen cells from BALB/c mice immunized with whole bacterial cells with SP2/0 murine myeloma cells. Desirable hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Neutralizing MAb 1D8 was selected in protection assays. ELISA results demonstrated that 1D8 can react with all 15 serotypes ofH. parasuisand field isolateH. parasuisHLJ-018. Passive immunization studies showed that mice inoculated intraperitoneally with 1D8 had significantly reduced prevalence ofH. parasuiscolonization in the blood, lung, spleen, and liver and had prolonged survival time compared to that of the control group. Furthermore, the passive transfer experiment indicated that MAb 1D8 can protect mice from both homologous and heterologous challenges withH. parasuis. Using two-dimensional gel electrophoresis (2-DE), the immunoreactive protein target for MAb 1D8 was identified. The data presented confirm the protective role of MAb 1D8 and identify OmpA as the target of the protective monoclonal antibody. The data suggest that OmpA is a promising candidate for a subunit vaccine againstH. parasuis.
A Multivalent Mannheimia-Bibersteinia Vaccine Protects Bighorn Sheep against Mannheimia haemolytica Challenge
Authors: Subramaniam Renuka , Shanthalingam Sudarvili , Bavananthasivam Jegarubee , Kugadas Abirami , Potter Kathleen A. , Foreyt William J. , Hodgins Douglas C. , Shewen Patricia E. , Barrington George M. , Knowles Donald P. , Srikumaran Subramaniam
Published: August 2011

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) toMannheimia haemolyticapneumonia. Although both species carryM. haemolyticaas a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalentMannheimia-Bibersteiniavaccine will protect them uponM. haemolyticachallenge. Four BHS were vaccinated with a culture supernatant vaccine prepared fromM. haemolyticaserotypes A1 and A2 andBibersteinia trehalosiserotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens againstM. haemolyticawere 1:160 and 1:4,000, respectively. Following intranasal challenge withM. haemolyticaA2 (1 × 105CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic ofM. haemolyticainfection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to surviveM. haemolyticachallenge which is likely to reduce the BHS population decline due to pneumonia.
Bloodstream Infection Due to Brachyspira pilosicoli in a Patient with Multiorgan Failure
Authors: Prim Núria , Pericas Roser , Español Montse , Rivera Alba , Mirelis Beatriz , Coll Pere
Published: August 2011

Brachyspira pilosicoli
is an etiological agent of human intestinal spirochetosis. Bloodstream infection due to this microorganism is rare. We report a case of
B. pilosicoli
bacteremia in a 70-year-old patient who presented with multiorgan failure.
DNA Extracted from Stained Sputum Smears Can Be Used in the MTBDR
plus
Assay
Authors: Dubois Cauwelaert Natasha , Ramarokoto Herimanana , Ravololonandriana Pascaline , Richard Vincent , Rasolofo Voahangy
Published: August 2011

We examined the feasibility of using DNA extracted from stained sputum smears for the detection of rifampin and isoniazid resistance with the commercial MTBDR
plus
assay from Hain Lifescience GmbH, Nehren, Germany. Overall sensitivity was initially low (70.0%) but increased to 96.7% when a multiplex PCR preamplification step was added. We then tested stored
Mycobacterium tuberculosis
-positive stained smears prepared from 297 patients' sputum samples. Species identification and drug susceptibility testing (DST) had been performed at the Institut Pasteur de Madagascar. Overall, the performance of the MTBDR
plus
assay applied to slide DNA was similar to that obtained in other studies with DNA extracted from clinical specimens. With the ready availability of stained smears in routine diagnostic laboratories and their easy transport and storage at room temperature, this approach should be useful for optimizing the treatment of multidrug-resistant tuberculosis and for conducting resistance surveys aimed at identifying hot-spot regions and breaking chains of transmission.
Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis
Authors: Chen Xiaoyou , Kong Fanrong , Wang Qinning , Li Chuanyou , Zhang Jianyuan , Gilbert Gwendolyn L.
Published: August 2011

A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in
Mycobacterium tuberculosis
by targeting resistance-associated mutations in the
katG
,
mabA-inhA
promoter,
rpoB
, and
gyrA
genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical
M. tuberculosis
isolates was selected for development and evaluation of HRMA. PCR amplicons from the
katG
,
mabA-inhA
promoter,
rpoB
, and
gyrA
genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified
katG
and/or
mabA-inhA
promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates,
rpoB
mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and
gyrA
mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in
M. tuberculosis
which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.
High-Resolution Melting Analysis for Identification of the Cryptococcus neoformans-Cryptococcus gattii Complex
Authors: Gago Sara , Zaragoza Óscar , Cuesta Isabel , Rodríguez-Tudela Juan L. , Cuenca-Estrella Manuel , Buitrago María J.
Published: August 2011

We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the
Cryptococcus
species complex (
Cryptococcus neoformans
var.
grubii
,
Cryptococcus neoformans
var.
neoformans
, and
Cryptococcus gattii
). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main
Cryptococcus
species.
A Comprehensive Evaluation of the Proficiency Testing Program for the HIV-1 BED Incidence Assay
Authors: Dobbs Trudy , Liu Xin , Anderson Rebecca , Nkengasong John , Parekh Bharat S.
Published: August 2011

The HIV-1 BED incidence assay was developed at the Centers for Disease Control and Prevention and since 2005 has been available as a commercial kit for use in HIV-1 incidence surveillance. A BED-specific proficiency testing (PT) program was initiated in 2006 that included a panel of eight coded specimens (six unique and two duplicates) to participating laboratories. The number of participating laboratories increased from 12 to 38 from 2006 to 2009. Overall, 96.1% of the laboratories reported results, and 95.4% of those reporting achieved a 100% score. The observed mean normalized optical density (OD-n) values of all participants correlated well with the expected OD-n values for all specimens (
R
2
= 0.98) used for seven PT rounds. BED testing demonstrated high reproducibility among all laboratories, with an agreement of 99.3% (574/578) between initial and confirmatory classification and regression statistics of
R
2
= 0.96, slope = 1.022, and intercept = 0.0066. Reproducibility among duplicate specimens was very high during each PT round, with mean deviation of 1.8%. Analysis of controls and calibrator specimen for all 343 runs showed a coefficient of variation of ca. 20% for raw ODs in the dynamic range, which was reduced to <10% when the OD was normalized (OD-n). Most laboratories that failed the PT assessment had transcriptional errors, kit reagent problems, or specimen handling errors. Thus, the BED-specific PT program enabled us to track performance of different laboratories conducting the BED assay while identifying areas for improvements. This program will also serve as a template for future PT programs for new incidence assays as they become available.
Molecular Epidemiology of Aspergillus fumigatus: an In-Depth Genotypic Analysis of Isolates Involved in an Outbreak of Invasive Aspergillosis
Authors: Guinea Jesús , García de Viedma Darío , Peláez Teresa , Escribano Pilar , Muñoz Patricia , Meis Jacques F. , Klaassen Corné H. W. , Bouza Emilio
Published: August 2011

We recently reported an outbreak of invasive aspergillosis in the major heart surgery unit of Hospital Gregorio Marañón, Madrid, Spain (T. Peláez, P. Muñoz, J. Guinea, M. Valerio, M. Giannella, C. H. W. Klaassen, and E. Bouza, Clin. Infect. Dis., in press).
Aspergillus fumigatus
was isolated from clinical samples from 10 patients admitted to the unit during the outbreak period (surgical wound invasive aspergillosis,
n
= 2; probable pulmonary invasive aspergillosis,
n
= 4; colonization,
n
= 4). In the study described here, we have studied the genotypic diversity of the
A. fumigatus
isolates found in the air and clinical samples. We used short tandem repeats of
A. fumigatus
(STR
Af
) typing to analyze the genotypes found in the 168 available
A. fumigatus
isolates collected from the clinical samples (
n
= 109) from the patients and from the environmental samples taken from the air of the unit (
n
= 59). The genotypic variability of
A. fumigatus
was higher in environmental than in clinical samples. Intrasample variability was also higher in environmental than in clinical samples: 2 or more different genotypes were found in 26% and 89% of clinical and environmental samples, respectively. We found matches between environmental and clinical isolates in 3 of the 10 patients: 1 patient with postsurgical invasive aspergillosis and 2 patients with probable pulmonary invasive aspergillosis. A total of 7 genotypes from 3 different patients and the air grouped together in 2 clusters. Clonally related genotypes and microvariants were detected in both clinical and environmental samples. STR
Af
typing proved to be a valuable tool for identifying the source of invasive aspergillosis outbreaks and for studying the genotypic diversity of clinical and environmental
A. fumigatus
isolates.
Bead-Based Multiplex Genotyping of 58 Cutaneous Human Papillomavirus Types
Authors: Michael K. M. , Forslund O. , Bacevskij O. , Waterboer T. , Bravo I. G. , Pawlita M. , Schmitt M.
Published: August 2011

Cutaneous human papillomaviruses (HPVs) are a heterogeneous, nonmonophyletic assembly, comprising about 50 characterized types and at least 133 isolates putatively representing new types. Their natural history of infection and potential association with nonmelanoma skin cancer are not well understood. Several PCR systems have been developed that amplify a broad spectrum of cutaneous HPVs. However, amplicon genotyping by sequencing or reverse line blot assays are complex and not well suited for high-throughput analyses. We developed a novel multiplex cutaneous papillomavirus genotyping (McPG) assay for 38 defined and 20 putative cutaneous HPVs of the beta, gamma, mu, and nu genera. Viral DNA was amplified by the use of a modified single-tube nested “hanging-droplet” FAP PCR. The amplifiable papillomavirus (PV) spectrum was enlarged by the use of 9 outer and 13 inner primers. Biotinylated PCR products were hybridized to type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads and analyzed using Luminex technology. Analytical sensitivity was analyzed for 38 defined HPVs and was ≤100 genome copies for all types. Integrated β-globin primers allow for simultaneous DNA quality control. McPG is characterized by high reproducibility (κ= 0.84, 95% confidence interval = 0.79 to 0.88), good concordance with the original nested FAP PCR, followed by sequencing (70.2% complete or partial agreement) when 322 skin biopsy DNA samples were analyzed, and improved ability to detect multiple infections (on average 2.5 HPV types per HPV-positive sample compared to 1.7 HPV types with nested FAP-PCR). In conclusion, McPG is a powerful tool for genotyping multiple cutaneous HPVs in a high-throughput format and is thus suitable for large-scale epidemiological studies.
Evaluation of Two Magnetic-Bead-Based Viral Nucleic Acid Purification Kits and Three Real-Time Reverse Transcription-PCR Reagent Systems in Two TaqMan Assays for Equine Arteritis Virus Detection in Semen
Authors: Miszczak Fabien , Shuck Kathleen M. , Lu Zhengchun , Go Yun Young , Zhang Jianqiang , Sells Stephen , Vabret Astrid , Pronost Stéphane , Fortier Guillaume , Timoney Peter J. , Balasuriya Udeni B. R.
Published: August 2011

This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.
Extent of Antigenic Cross-Reactivity among Highly Pathogenic H5N1 Influenza Viruses
Authors: Ducatez Mariette F. , Cai Zhipeng , Peiris Malik , Guan Yi , Ye Zhiping , Wan Xiu-Feng , Webby Richard J.
Published: August 2011

Highly pathogenic H5N1 avian influenza viruses emerged in 1996 and have since evolved so extensively that a single strain can no longer be used as a prepandemic vaccine or diagnostic reagent. We therefore sought to identify the H5N1 strains that may best serve as cross-reactive diagnostic reagents. We compared the cross-reactivity of 27 viruses of clades 0, 1, 2.1, 2.2, 2.3, and 4 and of four computationally designed ancestral H5N1 strains by hemagglutination inhibition (HI) and microneutralization (MN) assays. Antigenic cartography was used to analyze the large quantity of resulting data. Cartographs of HI titers with chicken red blood cells were similar to those of MN titers, but HI with horse red blood cells decreased antigenic distances among the H5N1 strains studied. Thus, HI with horse red blood cells seems to be the assay of choice for H5N1 diagnostics. Whereas clade 2.2 antigens were able to detect antibodies raised to most of the tested H5N1 viruses (and clade 2.2-specific antisera detected most of the H5N1 antigens), ancestral strain A exhibited the widest reactivity pattern and hence was the best candidate diagnostic reagent for broad detection of H5N1 strains.
Comparison of Multilocus Variable-Number Tandem-Repeat Analysis and Multilocus Sequence Typing for Differentiation of Hemolytic-Uremic Syndrome-Associated Escherichia coli (HUSEC) Collection Strains
Authors: Jenke Christian , Lindstedt Björn Arne , Harmsen Dag , Karch Helge , Brandal Lin Thorstensen , Mellmann Alexander
Published: August 2011

Multilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic
Escherichia coli
strains. Although MLVA—like MLST—was highly discriminatory (index of diversity, 0.988 versus 0.984), a low level of concordance demonstrated the limited ability of MLVA to reflect long-term evolutionary events.
Myxoma Virus Induces Type I Interferon Production in Murine Plasmacytoid Dendritic Cells via a TLR9/MyD88-, IRF5/IRF7-, and IFNAR-Dependent Pathway
Authors: Dai Peihong , Cao Hua , Merghoub Taha , Avogadri Francesca , Wang Weiyi , Parikh Tanvi , Fang Chee-Mun , Pitha Paula M. , Fitzgerald Katherine A. , Rahman Masmudur M. , McFadden Grant , Hu Xiaoyu , Houghton Alan N. , Shuman Stewart , Deng Liang
Published: August 2011

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the
Leporipoxvirus
genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-β, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.
Infection of Nonhost Species Dendritic CellsIn Vitrowith an Attenuated Myxoma Virus Induces Gene Expression That Predicts Its Efficacy as a Vaccine Vector
Authors: Top S. , Foulon E. , Pignolet B. , Deplanche M. , Caubet C. , Tasca C. , Bertagnoli S. , Meyer G. , Foucras G.
Published: August 2011

Recombinant myxoma virus (MYXV) can be produced without a loss of infectivity, and its highly specific host range makes it an ideal vaccine vector candidate, although careful examination of its interaction with the immune system is necessary. Similar to rabbit bone marrow-derived dendritic cells (BM-DCs), ovine dendritic cells can be infected by SG33, a MYXV vaccine strain, and support recombinant antigen expression. The frequency of infected cells in the nonhost was lower and the virus cycle was abortive in these cell types. Among BM-DC subpopulations, Langerhans cell-like DCs were preferentially infected at low multiplicities of infection. Interestingly, ovine BM-DCs remained susceptible to MYXV after maturation, although apoptosis occurred shortly after infection as a function of the virus titer. When gene expression was assessed in infected BM-DC cultures, type I interferon (IFN)-related and inflammatory genes were strongly upregulated. DC gene expression profiles were compared with the profiles produced by other poxviruses in interaction with DCs, but very few commonalities were found, although genes that were previously shown to predict vaccine efficacy were present. Collectively, these data support the idea that MYXV permits efficient priming of adaptive immune responses and should be considered a promising vaccine vector along with other poxviruses.
Rab5 and Class III Phosphoinositide 3-Kinase Vps34 Are Involved in Hepatitis C Virus NS4B-Induced Autophagy
Authors: Su Wen-Chi , Chao Ti-Chun , Huang Yih-Leh , Weng Shih-Che , Jeng King-Song , Lai Michael M. C.
Published: August 2011

Autophagy has been shown to facilitate replication or production of hepatitis C virus (HCV); nevertheless, how HCV induces autophagy remains unclear. Here, we demonstrate that HCV nonstructural protein 4B (NS4B) alone can induce autophagy signaling; amino acid residues 1 to 190 of NS4B are sufficient for this induction. Further studies showed that the phosphorylation levels of S6K and 4E-BP1 were not altered, suggesting that the mTOR/S6 kinase pathway and mTOR/4E-BP1 pathway did not contribute to NS4B- or HCV-induced autophagy. Inhibition of Rab5 function by silencing Rab5 or overexpressing dominant-negative Rab5 mutant (S34N) resulted in significant reduction of NS4B- or HCV-induced autophagic vesicle formation. Moreover, the autophagy induction was impaired by inhibition of class III phosphoinositide 3-kinase (PI 3-kinase) Vps34 function. Finally, the coimmunoprecipitation assay indicated that NS4B formed a complex with Rab5 and Vps34, supporting the notion that Rab5 and Vps34 are involved in NS4B-induced autophagy. Taken together, these results not only reveal a novel role of NS4B in autophagy but also offer a clue to the mechanism of HCV-induced autophagy.
Adenovirus Type 5 Rupture of Lysosomes Leads to Cathepsin B-Dependent Mitochondrial Stress and Production of Reactive Oxygen Species
Authors: McGuire Kathleen A. , Barlan Arlene U. , Griffin Tina M. , Wiethoff Christopher M.
Published: August 2011

In response to viral infection, reactive oxygen species (ROS) mediate innate immune signaling or generate danger signals to activate immune cells. The mechanisms of virally induced ROS are poorly defined, however. We demonstrate that ROS are produced within minutes of adenovirus type 5 (Ad5) infection of macrophages and that oxidative stress supports Ad5-induced cytokine secretion. We show that short hairpin RNA (shRNA) knockdown of TLR9 has no effect on ROS production despite observed decreases in Ad-induced cytokine secretion. A major source of ROS in macrophages is NADPH oxidase. However, shRNA knockdown of the NADPH oxidase subunit NOX2 does not attenuate Ad-induced ROS. Induction of ROS is not observed in cells infected with a temperature-sensitive mutant of Ad2,ts1, which is defective in endosomal membrane penetration during cell entry. Further, Ad5, but notts1, induces the release of lysosomal cathepsin B into the cytoplasm of infected cells. In agreement with this finding, we observe a loss of mitochondrial membrane potential upon Ad infection which requires Ad endosomal membrane penetration and cathepsin B activity. Overexpression of Bcl-2 attenuates Ad5-induced ROS, further supporting the role for mitochondrial membrane destabilization as the source of ROS in response to Ad5 infection. Together, these data suggest that ROS produced in response to Ad5 infection depends on the virally induced endosomal membrane rupture to release lysosomal cathepsins. Furthermore, the release of cathepsins leads to mitochondrial membrane disruption and thus the release of ROS from the mitochondria.
Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function
Authors: Andrabi Shaida , Hwang Justin H. , Choe Jennifer Kean , Roberts Thomas M. , Schaffhausen Brian S.
Published: August 2011

Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but murine polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not.
Structural Basis for Antiviral Inhibition of the Main Protease, 3C, from Human Enterovirus 93
Authors: Costenaro Lionel , Kaczmarska Zuzanna , Arnan Carme , Janowski Robert , Coutard Bruno , Solà Maria , Gorbalenya Alexander E. , Norder Heléne , Canard Bruno , Coll Miquel
Published: August 2011

Members of the
Enterovirus
genus of the
Picornaviridae
family are abundant, with common human pathogens that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically processed into mature proteins at Gln↓Gly cleavage sites by the 3C protease (3C
pro
), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3C
pro
an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3C
pro
from an enterovirus B, EV-93, a recently identified pathogen, alone and in complex with the anti-HRV molecules compound 1 (AG7404) and rupintrivir (AG7088) at resolutions of 1.9, 1.3, and 1.5 Å, respectively. The EV-93 3C
pro
adopts a chymotrypsin-like fold with a canonically configured oxyanion hole and a substrate binding pocket similar to that of rhino-, coxsackie- and poliovirus 3C proteases. We show that compound 1 and rupintrivir are both active against EV-93 in infected cells and inhibit the proteolytic activity of EV-93 3C
pro
in vitro
. These results provide a framework for further structure-guided optimization of the tested compounds to produce antiviral drugs against a broad range of EV species.