Membrane Topology of Pestiviral Non-Structural Protein 2 and determination of the minimal autoprotease domain.

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae A distinctive feature of the Flaviviridae is the importance of non-structural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes.In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the Substituted Cysteine Accessibility Method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the ER lumen and is followed by three transmembrane segments (TM) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM segment was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to the one of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2.ImportancePestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports virion morphogenesis. For a better understanding of the underlying molecular interactions, topological and structural data are required. The topology-based determination of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as non-structural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.

Scaffolds in a shell-a new approach combining one-photon and two-photon polymerization.

We report on a laser system combining one-photon and two-photon polymerization for precise and fast fabrication of macroscopic three-dimensional structures featuring microscale and nanoscale characteristics. This single-stage process significantly reduces the production time as demonstrated by scaffolds in a shell application. Porous scaffolds with different pore sizes are surrounded by a ring so that cells can be seeded directly to the scaffolds kept in a shell and do not spread over the whole substrate expecting a saving of cell suspension, faster growth on the scaffolds, and a more controllable environment. Compared to a two-photon polymerization process, the ring is fabricated about 500 times faster using one-photon polymerization. The presented hybrid process qualifies for further applications illustrated by a fluidic system.

Probing Sizes and Shapes of Nobelium Isotopes by Laser Spectroscopy.

Until recently, ground-state nuclear moments of the heaviest nuclei could only be inferred from nuclear spectroscopy, where model assumptions are required. Laser spectroscopy in combination with modern atomic structure calculations is now able to probe these moments directly, in a comprehensive and nuclear-model-independent way. Here we report on unique access to the differential mean-square charge radii of ^{252,253,254}No, and therefore to changes in nuclear size and shape. State-of-the-art nuclear density functional calculations describe well the changes in nuclear charge radii in the region of the heavy actinides, indicating an appreciable central depression in the deformed proton density distribution in ^{252,254}No isotopes. Finally, the hyperfine splitting of ^{253}No was evaluated, enabling a complementary measure of its (quadrupole) deformation, as well as an insight into the neutron single-particle wave function via the nuclear spin and magnetic moment.

Self-consistent absorption correction for quantifying very noisy X-ray maps: group III nitride nanowires as an example.

Energy-dispersive X-ray mapping in a scanning transmission electron microscope is a method to visualize the spatial distribution of chemical elements in a sample. Quantification of the signal intensities depends on proper background elimination and correction of the self-absorption and fluorescence of X-ray lines in the sample. The latter become particularly relevant for slightly thicker and rough samples, small take-off angles and low-energetic X-ray lines, for which we have recently introduced a self-consistent absorption correction based on effective k*-factors selected automatically from curves simulated as function of a K/L line intensity ratio of one of the heavier elements in the sample for ranges of different compositions. This has been shown to work well for thick and for rough samples. Correcting the background intensity to sub-pixel accuracy is necessary for reliable quantification of very noisy maps. In this study, we show how this self-consistent absorption correction method can be applied to InGaN and AlGaN layers in GaN nanowires, the net maps of which can be so noisy the layers can hardly be seen by the eye (a few counts per pixel) and the background is below a single count in each spectrum channel. The result indicates that background estimation for the Ga L-line intensity using fractional counts from an interpolation of maps from neighbouring X-ray lines of elements that are not actually present in the specimen is critical for quantification. The nanowires studied were between 66 and 375 nm thick. LAY DESCRIPTION: Energy-dispersive X-ray mapping in a scanning transmission electron microscope is a method to visualize the spatial distribution of chemical elements in a sample. Quantification of the signal intensities depends on proper background elimination and correction of the self-absorption of X-ray lines in the sample. Here we show that our previously developed method of self-consistent effective absorption factors works well even with extremely noise elemental maps of a few net counts only where the human eye can hardly discern any pattern and the background signal is typically less than a single count in each spectrum channel. Correcting the background intensity to sub-pixel accuracy is then necessary for reliable quantification.

Taming the sphinx: Mechanisms of cellular sphingolipid homeostasis.

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Transmission electron microscopy of AlGaAs/GaAs quantum cascade laser structures.

Quantum cascade lasers can be efficient infrared radiation sources and consist of several hundreds of very thin layers arranged in stacks that are repeated periodically. Both the thicknesses of the individual layers as well as the period lengths need to be monitored to high precision. Different transmission electron microscopy methods have been combined to analyse AlGaAs/GaAs quantum cascade laser structures in cross-section. We found a small parabolic variation of the growth rate during deposition, affecting the stack periodicity and a reduced aluminium content of the AlGaAs barriers, whereas their widths as well as those of the GaAs quantum wells agreed with the nominal values within one atomic layer. Growth on an offcut substrate led to facets and steps at the interfaces.

Effective absorption correction for energy dispersive X-ray mapping in a scanning transmission electron microscope: analysing the local indium distribution in rough samples of InGaN alloy layers.

We have applied our previous method of self-consistent k*-factors for absorption correction in energy-dispersive X-ray spectroscopy to quantify the indium content in X-ray maps of thick compound InGaN layers. The method allows us to quantify the indium concentration without measuring the sample thickness, density or beam current, and works even if there is a drastic local thickness change due to sample roughness or preferential thinning. The method is shown to select, point-by-point in a two-dimensional spectrum image or map, the k*-factor from the local Ga K/L intensity ratio that is most appropriate for the corresponding sample geometry, demonstrating it is not the sample thickness measured along the electron beam direction but the optical path length the X-rays have to travel through the sample that is relevant for the absorption correction.

Comparison of cross-sectional transmission electron microscope studies of thin germanium epilayers grown on differently oriented silicon wafers.

We compare transmission electron microscopical analyses of the onset of islanding in the germanium-on-silicon (Ge/Si) system for three different Si substrate orientations: (001), (11¯0) and (11¯1)Si. The Ge was deposited by reduced pressure chemical vapour deposition and forms islands on the surface of all Si wafers; however, the morphology (aspect ratio) of the deposited islands is different for each type of wafer. Moreover, the mechanism for strain relaxation is different for each type of wafer owing to the different orientation of the (111) slip planes with the growth surface. Ge grown on (001)Si is initially pseudomorphically strained, yielding small, almost symmetrical islands of high aspect ratio (clusters or domes) on top interdiffused SiGe pedestals, without any evidence of plastic relaxation by dislocations, which would nucleate later-on when the islands might have coalesced and then the Matthews-Blakeslee limit is reached. For (11¯0)Si, islands are flatter and more asymmetric, and this is correlated with plastic relaxation of some islands by dislocations. In the case of growth on (11¯1)Si wafers, there is evidence of immediate strain relaxation taking place by numerous dislocations and also twinning. In the case of untwined film/substrate interfaces, Burgers circuits drawn around certain (amorphous-like) regions show a nonclosure with an edge-type a/4[1¯12] Burgers vector component visible in projection along [110]. Microtwins of multiples of half unit cells in thickness have been observed which occur at the growth interface between the Si(11¯1) buffer layer and the overlying Ge material. Models of the growth mechanisms to explain the interfacial configurations of each type of wafer are suggested.

Temporal trends in transcatheter and surgical aortic valve replacement : An analysis of aortic valve replacements in Germany during 2012-2014.

This analysis of the data of the obligatory quality assurance programme AQUA shows the perioperative risk as well as the procedural outcome evaluated by the observed versus expected in-hospital mortality ratio (O/E ratio) of 62,872 patients undergoing isolated surgical (sAVR), transcatheter transvascular (TV-), or transapical (TA-) aortic valve replacement (TAVI) from 2012 to 2014 in Germany. The number of TAVI procedures increased from 9,352 in 2012 to 13,278 in 2014, whereas the number of sAVR remained constant (2012: 9,949; 2014: 9,953). Between 2012 and 2014, the number of TAVI implanted in patients with a logistic EuroScore I (logESI) of ≤10 % (2012: 21 %; 2014: 26 %) as well as with a logESI <20 % (2012: 57 %; 2014: 64 %) increased. In-hospital mortality in TAVI patients decreased from 5.2 % (TV: 5.0 %; TA: 7.4 %) in 2012 to 4.2 % (TV: 3.8 %; TA: 5.5 %) in 2014, whereas it was stable for sAVR patients (2012: 2.8 %; 2014: 2.6 %). The O/E ratio of TAVI patients decreased from 0.91 (TV: 0.79; TA: 1.2) to 0.73 (TV: 0.69; TA: 0.89), whereas this ratio remained constant for sAVR patients (2012: 0.92; 2014: 0.93). In summary, estimated surgical risk, in-hospital mortality, as well as the O/E ratio for patients undergoing TAVI declined constantly during the last 3 years.

Green tea reduces body fat via upregulation of neprilysin.

BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

Self-consistent method for quantifying indium content from X-ray spectra of thick compound semiconductor specimens in a transmission electron microscope.

Based on Monte Carlo simulations of X-ray generation by fast electrons we calculate curves of effective sensitivity factors for analytical transmission electron microscopy based energy-dispersive X-ray spectroscopy including absorption and fluorescence effects, as a function of Ga K/L ratio for different indium and gallium containing compound semiconductors. For the case of InGaN alloy thin films we show that experimental spectra can thus be quantified without the need to measure specimen thickness or density, yielding self-consistent values for quantification with Ga K and Ga L lines. The effect of uncertainties in the detector efficiency are also shown to be reduced.

What environmental transmission electron microscopy measures and how this links to diffusivity: thermodynamics versus kinetics.

Environmental or in situ electron microscopy means the observation of material in its native environment, which can be gaseous or liquid, as compared to more traditional post-mortem electron microscopy carried out under (ultra) high vacuum conditions. Experiments can be performed on bulk samples in scanning electron microscopes or on thinned samples in transmission (scanning) electron microscopes. In the latter, the movement, in real time and in situ, of nanoparticles, clusters or even single atoms on the surfaces of thinned material or within a liquid can be observed. It is argued here that due to the changes that a specimen typically undergoes during in situ observation, electron irradiation effects are difficult to evaluate and so thermodynamic parameters, such as activation energies for diffusion and segregation, which are governed by movements of only a minority of atoms in the specimen, cannot be reliably determined because of the potentially high energy transfer by the irradiating electron beam to some atoms in the sample. In order to measure diffusivities reliably, radiation effects and surface diffusion need to be excluded or kept minimal so as not to disturb the measurements, which can be checked by repeating experiments and comparing results as function of time and dose for the same position, at different positions or for different specimen thicknesses. Kinetic measurements of nucleation and growth phenomena, such as Ostwald ripening, are possibly influenced to a far lesser degree by irradiation effects, as a majority of atoms actively participate in these processes and if a small fraction of them will get extra energy from the irradiation process then their influence on the overall kinetics may be rather minor.

Electron microscopy of quantum dots.

This brief review describes the different types of semiconductor quantum dot systems, their main applications and which types of microscopy methods are used to characterize them. Emphasis is put on the need for a comprehensive investigation of their size distribution, microstructure, chemical composition, strain state and electronic properties, all of which influence the optical properties and can be measured by different types of imaging, diffraction and spectroscopy methods in an electron microscope.

New pathways for improved quantification of energy-dispersive X-ray spectra of semiconductors with multiple X-ray lines from thin foils investigated in transmission electron microscopy.

Theoretical approaches to quantify the chemical composition of bulk and thin-layer specimens using energy-dispersive X-ray spectroscopy in a transmission electron microscope are compared to experiments investigating (In)GaAs and Si(Ge) semiconductors. Absorption correctors can be improved by varying the take-off angle to determine the depth of features within the foil or the samples thickness, or by definition of effective k-factors that can be obtained from plots of k-factors versus foil thickness or, preferably, versus the K/L intensity ratio for a suitable element. The latter procedure yields plots of self-consistent absorption corrections that can be used to determine the chemical composition, iteratively for SiGe using a set of calibration curves or directly from a single calibration curve for InGaAs, for single X-ray spectra without knowledge of sample thickness, density or mass absorption coefficients.

Dynamics of water-alcohol mixtures: insights from nuclear magnetic resonance, broadband dielectric spectroscopy, and triplet solvation dynamics.

We combine (2)H nuclear magnetic resonance (NMR), broadband dielectric spectroscopy (BDS), and triplet solvation dynamics (TSD) to investigate molecular dynamics in glass-forming mixtures of water and propylene glycol in very broad time and temperature ranges. All methods yield consistent results for the α process of the studied mixtures, which hardly depends on the composition and shows Vogel-Fulcher temperature dependence as well as Cole-Davidson spectral shape. The good agreement between BDS and TDS data reveals that preferential solvation of dye molecules in microheterogeneous mixtures does not play an important role. Below the glass transition temperature T(g), NMR and BDS studies reveal that the ß process of the mixtures shows correlation times, which depend on the water concentration, but exhibit a common temperature dependence, obeying an Arrhenius law with an activation energy of E(a) = 0.54 eV, as previously reported for mixtures of water with various molecular species. Detailed comparison of NMR and BDS correlation functions for the ß process unravels that the former decay faster and more stretched than the latter. Moreover, the present NMR data imply that propylene glycol participates in the ß process and, hence, it is not a pure water process, and that the mechanism for molecular dynamics underlying the ß process differs in mixtures of water with small and large molecules.

Homogeneous array of nanowire-embedded quantum light emitters.

The potential for scale-up coupled with minimized system size is likely to be a major determining factor in the realization of applicable quantum information systems. Nanofabrication technology utilizing the III-V semiconductor system provides a path to scalable quantum bit (qubit) integration and a materials platform with combined electronic/photonic functionality. Here, we address the key requirement of qubit-site and emission energy control for scale-up by demonstrating uniform arrays of III-V nanowires, where each nanowire contains a single quantum dot. Optical studies of single nanowire quantum dots reveal narrow linewidth exciton and biexciton emission and clear state-filling at higher powers. Individual nanowire quantum dots are shown to emit nonclassically with clear evidence of photon antibunching. A model is developed to explain unexpectedly large excited state separations as revealed by photoluminescence emission spectra. From measurements of more than 40 nanowire quantum dots, we find emission energies with an ensemble broadening of 15 meV. The combination of deterministic site control and the narrow distribution in ensemble emission energy results in a system readily capable of scaling for multiqubit quantum information applications.

Aortic stenosis in high-risk patients. Surgical therapy.

Conventional aortic valve replacement is the standard approach for treating aortic stenosis, it is performed via a full or partial sternotomy, and is associated with low risks for patients and with excellent long-term outcomes. This also holds true for octogenarians, if they present without relevant comorbidities. After resection of the calcified native leaflets, biological prostheses with good functionality and durability are implanted. Elderly patients with an increasing risk profile, however, should be treated by a heart team using transcatheter approaches including cardiac surgery.

[Myocardial revascularization]. / Myokardrevaskularisation.

Coronary artery disease (CAD) is a leading cause of morbidity and mortality in western countries and is of significant socio-economic importance due to its increasing prevalence. Until percutaneous coronary interventions (PCI) were established, CAD could only be treated by surgical revascularization or pharmacological therapy. In-stent restenosis remains a major problem after stent implantation. However, the use of new materials and stent coatings have led to a significant reduction in in-stent restenosis. Thus, surgical revascularization and PCI are currently of equal value for the treatment of CAD. The decision-making for PCI or surgical revascularization depends on various factors such as number of diseased vessels, complexity of the coronary stenoses, concomitant diseases, and the patient's general condition. The therapeutic regime of every patient should be adjusted to the recommendations of the European and German Society for Cardiology, while controversial and complex cases should be discussed in an interdisciplinary case conference ("heart team").

[Transcatheter aortic valve implantation (TAVI)]. / Transkatheteraortenklappenersatz (TAVI).

Aortic valve stenosis (AS) is the most common acquired valve disease in the industrialized countries. Most patients--especially young and low-risk patients--can be safely and effectively operated with aortic valve replacement. Due to increasing life expectancy, however, the number of elderly patients with AS and various concomitant diseases will increase in the coming decades. For those elderly high-risk patients, transfemoral or transapical aortic valve implantation (TAVI) has evolved as a valuable alternative to conventional surgery. The TAVI approach has been shown to be superior to standard medical treatment in these high-risk patients. All patients considered for TAVI should be discussed in a consensus conference consisting of cardiac surgeons and cardiologists (heart team). Furthermore, for successful treatment with transcatheter techniques, sophisticated pre-interventional imaging is required to screen patients. Available data on TAVI from randomized trials and large-scale registries demonstrate that this method is very promising.

[Transcutaneous aortic valve implantation]. / Kathetergestützte Aortenklappenimplantation.

Due to increasing life expectancy in the coming decades the number of elderly patients with aortic valve stenosis (AS) and various concomitant diseases will increase. Conventional surgical aortic valve replacement represents the treatment of choice in patients with severe and symptomatic AS. Transfemoral and transapical aortic valve implantation (T-AVI) has evolved as a standard procedure for patients with severe AS who are technically inoperable or at very high risk for surgical valve replacement. The T-AVI approach has been shown to be superior to the standard medical treatment in these high-risk patients. All patients to be considered for T-AVI should be discussed in a consensus conference consisting of cardiac surgeons and cardiologists (heart team).