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An alternative approach to focused-ion-beam (FIB)-based fabrication of magnetic nanostructures suitable for utilizing and probing intrinsic properties of magnetic nanostructures in both the in-plane and out-of-plane configurations is presented. It allows the fabrications of nanostencil mask templates for fast exploration of spin transfer torque effects in magnetic thin films in both the current in plane (CIP) and current perpendicular to the plane (CPP) geometry in the sub-100-nm size range, without the limitations and concerns usually associated with FIB milling.

Research comparing the growth of highly textured CrO2 films on Al2O3(0001) and TiO2(100) substrates is presented. It was found that the magnitude of the Lorentz magnetoresonance and the integrain tunneling magnetoresonance depends heavily on the defect density of the films.

Fe(110) films were epitaxially grown on sapphire substrates using a Mo(110) buffer layer in an ultrahigh-vacuum molecular-beam epitaxy system. The magnetic properties were examined ex situ by Brillouin light scattering and superconducting quantum interference device magnetometry. To determine the magnetic anisotropy constants the frequency of the Damon-Eshbach [J. Phys. Chem. Solids 19, 308 (1961)] surface spin-wave mode was measured as a function of the in-plane angle between the external magnetic field and the Fe[001] crystal axis. The angle-dependent frequency was fitted by a spin-wave model. We found that the easy axis of the cubic magnetocrystalline anisotropy K1 and an additional uniaxial in-plane anisotropy K∥(2) are aligned parallel to the in-plane Fe[001] axis for Fe-layer thicknesses from 0.8 to 37 nm, with K1 increasing and K∥(2) decreasing with increasing Fe thickness. Possible origins of the observed uniaxial anisotropy are discussed. [ABSTRACT FROM AUTHOR]

The magnetic properties of Fe(110) films epitaxially grown on sapphire substrate using a Mo(110) buffer layer in an ultrahigh-vacuum molecular- beam epitaxy system were examined. The easy axis of the cubic magnetocrystalline anisotropy K(sub 1) and an additional uniaxial in-plane anisotropy K(sub II)(super 2) are aligned parallel to the inplane Fe[001] axis for Fe-layer thickness from 0.8 to 37nm, with K(sub 1) increasing and K(sub II)(super 2) decreasing with increasing Fe thickness.

The growth of (010)-oriented CrO[sub 2] thin films on Al[sub 2]O[sub 3](0001) substrates leads to a higher grain boundary density than the growth of (100)-oriented CrO[sub 2] thin films on isostructural TiO[sub 2](100) substrates. For both types of films an intrinsic linear contribution to the high field magnetoresistance (MR) due to spin disorder has been determined at T=300 K. This contribution does not depend on the crystalline quality of the films and supports the suggested intrinsic double exchange mechanism for CrO[sub 2]. At low temperature (T=10 K) intergrain tunneling MR and Lorentz MR appear, which strongly depend on the crystalline properties of the CrO[sub 2] films. © 2001 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Background: Inpatient nurses identified the need to recognize clinical deterioration earlier, including rapid response team (RRT) activations and transfers to the intensive care unit. Surveys identified the need for better interdisciplinary communication., Local Problem: A system was needed to detect early clinical deterioration with structured responses promoting multidisciplinary collaboration., Methods: An early warning score (EWS) identified patients at risk. The system ensured timely and accurate actions were taken when scores reached 3 or above., Interventions: Collaborative, graded responses to EWSs and nurse-led rounds promoted communication and timely interventions., Results: Mortality decreased (-27%) as did multiple RRT activations on a single patient (-15%). Nurses are aware of early vital sign changes (+45%) and resident responsiveness has improved. There has been an increase in nurse/resident communication satisfaction (+31%)., Conclusions: The system implemented reinforces the importance of patient assessment, collaboration among the multidisciplinary team, and promotes early interventions., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Quality Assurance (QA) programs are essential to evaluate performance in diagnostics laboratories., Objectives: We present the results from the first QA for HIV-1 genotypic tropism testing, organized and coordinated by the Institute of Virology at the University of Cologne., Study Design: 12 cell culture-derived viral strains of different HIV-1 clades from the NIH AIDS Reagent Program were sent to the participants to be processed with their standard diagnostic methods Fasta files containing the V3 region sequence were centrally analyzed at the Institute of Virology, Cologne. All samples were sent in parallel for phenotypic testing., Results: 36 laboratories from 16 countries reported genotypic results. The sequence-generation efficacy was 95.1%, while the phenotypic assays ESTA ® and PhenXR only achieved results for 58.3% of the samples. All four X4 samples were identified by 31/36 laboratories, two laboratories amplified 3/4×4 samples, and three detected 2/4×4 samples. There was high concordance among the genotypic and phenotypic results, although differences in FPR values were detected. Most deficiencies in sequence editing did not result in wrong classification of X4 viruses as R5, with the exception of sample NRZ05 by laboratory 38, but in an overestimation of CXCR4 use., Conclusions: This demonstrates that genotypic tropism prediction is a safe procedure for clinical purposes. As we used homogeneous cell culture samples and all sequence fasta files were centrally analyzed, variations in FPR values can only be attributed to sample preparation, RT-PCR or sequence editing protocols., (Copyright © 2017. Published by Elsevier B.V.)

The face of hepatitis C virus (HCV) therapy is changing dramatically. Direct-acting antiviral agents (DAAs) specifically targeting HCV proteins have been developed and entered clinical practice in 2011. However, despite high sustained viral response (SVR) rates of more than 90%, a fraction of patients do not eliminate the virus and in these cases treatment failure has been associated with the selection of drug resistance mutations (RAMs). RAMs may be prevalent prior to the start of treatment, or can be selected under therapy, and furthermore they can persist after cessation of treatment. Additionally, certain DAAs have been approved only for distinct HCV genotypes and may even have subtype specificity. Thus, sequence analysis before start of therapy is instrumental for managing DAA-based treatment strategies. We have created the interpretation system geno2pheno[HCV] (g2p[HCV]) to analyse HCV sequence data with respect to viral subtype and to predict drug resistance. Extensive reviewing and weighting of literature related to HCV drug resistance was performed to create a comprehensive list of drug resistance rules for inhibitors of the HCV protease in non-structural protein 3 (NS3-protease: Boceprevir, Paritaprevir, Simeprevir, Asunaprevir, Grazoprevir and Telaprevir), the NS5A replicase factor (Daclatasvir, Ledipasvir, Elbasvir and Ombitasvir), and the NS5B RNA-dependent RNA polymerase (Dasabuvir and Sofosbuvir). Upon submission of up to eight sequences, g2p[HCV] aligns the input sequences, identifies the genomic region(s), predicts the HCV geno- and subtypes, and generates for each DAA a drug resistance prediction report. g2p[HCV] offers easy-to-use and fast subtype and resistance analysis of HCV sequences, is continuously updated and freely accessible under http://hcv.geno2pheno.org/index.php. The system was partially validated with respect to the NS3-protease inhibitors Boceprevir, Telaprevir and Simeprevir by using data generated with recombinant, phenotypic cell culture assays obtained from patients' virus variants.

Background: Entecavir is an efficient inhibitor of HBV reverse transcriptase (RT) and widely used for therapy of chronic hepatitis B. Entecavir treatment of HBV patients with lamivudine-resistant viral strains, however, often fails, but the mechanism of cross-resistance development is not fully understood., Methods: Using non-linear regression models, dose-response curves of cloned HBV strains from patients pre-treated with RT inhibitors were established in human hepatoma cell lines after transfection with HBV genomes containing HBV polymerase genes from patient isolates. 50% and 90% inhibitory concentrations (IC50 and IC90) and corresponding antiviral resistance factors (RF50 and RF90) were calculated., Results: The entecavir dose-response curve of lamivudine-resistant HBV RT mutants rtM204 for the replication of HBV decreased less than expected with increasing drug dose. Remarkably, due to the flat dose-response curves, RF90 values against entecavir of samples with rtM204 substitutions were up to 30× higher than their RF50 values., Conclusions: The unexpectedly high IC90 indicates a strong residual replication capacity of lamivudine-resistant HBV rtM204 variants under entecavir therapy, although IC50 values are initially within the therapeutic range of entecavir. This characteristic favours rapid selection of additional mutants with overt resistance against entecavir. Thus, the current phenotypic resistance assays should include determination of IC90.

Drug resistance testing, genotype analysis, and the determination of immune and vaccine escape variants support personalized antiviral treatment for hepatitis B patients. As phenotypic drug resistance testing for hepatitis B virus (HBV) is especially labor-intensive, due to the lack of simple cell culture systems, genotypic methods play a very pronounced role. The genetic structure of HBV allows the simultaneous analysis of two different genes by examination of a single region in the genome of HBV. Nevertheless, the overlapping open reading frames of the hepatitis B surface antigen (HBsAg) and the reverse transcriptase (RT) have to be interpreted separately. In diagnostic procedures, standard Sanger type sequencing (mostly performed as a dye-dideoxynucleotide terminator system) is still the most commonly used method. This allows using established techniques for interpreting those types of genetic information. Besides reviewing the foundation of drug resistance interpretation for HBV, different interpretation systems, either commercially available (TRUGENE, Abbott, ViroScore) or free to use (geno2pheno[HBV], HIV-GRADE HBV tool), are compared in this article., (© 2014 S. Karger AG, Basel.)

Background: The fully automated and closed LIAISON(®)XL platform was developed for reliable detection of infection markers like hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab) or human immunodeficiency virus (HIV)-Ag/Ab. To date, less is known about the diagnostic performance of this system in direct comparison to the common Abbott ARCHITECT(®) platform., Objectives: We compared the diagnostic performance and usability of the DiaSorin LIAISON(®)XL with the commonly used Abbott ARCHITECT(®) system., Study Design: The qualitative performance of the above mentioned assays was compared in about 500 sera. Quantitative tests were performed for HBsAg-positive samples from patients under therapy (n=289) and in vitro expressed mutants (n=37). For HCV-Ab, a total number of 155 selected samples from patients chronically infected with different HCV genotypes were tested., Results: The concordance between both systems was 99.4% for HBsAg, 98.81% for HCV-Ab, and 99.6% for HIV-Ab/Ag. The quantitative LIAISON(®)XL murex HBsAg assay detected all mutants in comparable amounts to the HBsAg wild type and yielded highly reliable HBsAg kinetics in patients treated with antiviral drugs. Dilution experiments using the 2nd International Standard for HBsAg (WHO) showed a high accuracy of this test. HCV-Ab from patients infected with genotypes 1-3 were equally detected in both systems. Interestingly, S/CO levels of HCV-Ab from patients infected with genotype 3 seem to be relatively low using both systems., Conclusions: The LIAISON(®)XL platform proved to be an excellent system for diagnostics of HBV, HCV, and HIV with equal performance compared to the ARCHITECT(®) system., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Direct Sanger sequencing of viral genome populations yields multiple ambiguous sequence positions. It is not straightforward to derive linkage information from sequencing chromatograms, which in turn hampers the correct interpretation of the sequence data. We present a method for determining the variants existing in a viral quasispecies in the case of two nearby ambiguous sequence positions by exploiting the effect of sequence context-dependent incorporation of dideoxynucleotides. The computational model was trained on data from sequencing chromatograms of clonal variants and was evaluated on two test sets of in vitro mixtures. The approach achieved high accuracies in identifying the mixture components of 97.4% on a test set in which the positions to be analyzed are only one base apart from each other, and of 84.5% on a test set in which the ambiguous positions are separated by three bases. In silico experiments suggest two major limitations of our approach in terms of accuracy. First, due to a basic limitation of Sanger sequencing, it is not possible to reliably detect minor variants with a relative frequency of no more than 10%. Second, the model cannot distinguish between mixtures of two or four clonal variants, if one of two sets of linear constraints is fulfilled. Furthermore, the approach requires repetitive sequencing of all variants that might be present in the mixture to be analyzed. Nevertheless, the effectiveness of our method on the two in vitro test sets shows that short-range linkage information of two ambiguous sequence positions can be inferred from Sanger sequencing chromatograms without any further assumptions on the mixture composition. Additionally, our model provides new insights into the established and widely used Sanger sequencing technology. The source code of our method is made available at http://bioinf.mpi-inf.mpg.de/publications/beggel/linkageinformation.zip.

Introduction: The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification., Methods: 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay., Results: Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate., Conclusions: Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)