Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml

Mask-based dual-axes tomoholography using soft x-rays

We explore tomographic mask-based Fourier transform x-ray holography with respect to the use of a thin slit as a reference wave source. This imaging technique exclusively uses the interference between the waves scattered by the object and the slit simplifying the experimental realization and ensuring high data quality. Furthermore, we introduce a second reference slit to rotate the sample around a second axis and to record a dual-axes tomogram. Compared to a single-axis tomogram, the reconstruction artifacts are decreased in accordance with the reduced missing data wedge. Two demonstration experiments are performed where test structures are imaged with a lateral resolution below 100 nm

Moregrasp: Restoration of Upper Limb Function in Individuals with High Spinal Cord Injury by Multimodal Neuroprostheses for Interaction in Daily Activities

The aim of the MoreGrasp project is to develop a noninvasive, multimodal user interface including a brain-computer interface (BCI) for intuitive control of a grasp neuroprosthesis to support individuals with high spinal cord injury (SCI) in everyday activities. We describe the current state of the project, including the EEG system, preliminary results of natural movements decoding in people with SCI, the new electrode concept for the grasp neuroprosthesis, the shared control architecture behind the system and the implementation of a user-centered design

Reference shape effects on Fourier transform holography

Soft-x-ray holography which utilizes an optics mask fabricated in direct contact with the sample, is a widely applied x-ray microscopy method, in particular, for investigating magnetic samples. The optics mask splits the x-ray beam into a reference wave and a wave to illuminate the sample. The reconstruction quality in such a Fourier-transform holography experiment depends primarily on the characteristics of the reference wave, typically emerging from a small, high-aspect-ratio pinhole in the mask. In this paper, we study two commonly used reference geometries and investigate how their 3D structure affects the reconstruction within an x-ray Fourier holography experiment. Insight into these effects is obtained by imaging the exit waves from reference pinholes via high-resolution coherent diffraction imaging combined with three-dimensional multislice simulations of the x-ray propagation through the reference pinhole. The results were used to simulate Fourier-transform holography experiments to determine the spatial resolution and precise location of the reconstruction plane for different reference geometries. Based on our findings, we discuss the properties of the reference pinholes with view on application in soft-x-ray holography experiments

North to south: ecosystem features determine seagrass community response to sea otter foraging

We compared sea otter recovery in California (CA) and British Columbia (BC) to determine how key ecosystem properties shape top-down effects in seagrass communities. Potential ecosystem drivers of sea otter foraging in CA and BC seagrass beds that we examined include the role of coastline complexity and environmental stress on sea otter effects. In BC, we found greater species richness across seagrass trophic assemblages. Furthermore, Cancer spp. crabs, an important link in the seagrass trophic cascade observed in CA, was less common. Additionally, the more recent reintroduction of sea otters, more complex coastline, and reduced environmental stress in BC seagrass habitats supported the hypothesis that sea otter foraging pressure is currently reduced in more northern latitudes. In order to manage the ecosystem features that lead to regional differences in top predator effects in seagrass communities, we review our findings, their spatial and temporal constraints, and present a social-ecological framework for future re- search

Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view

Background: Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. Results: We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Conclusions: Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time

Nanoscale Imaging of High‐Field Magnetic Hysteresis in Meteoritic Metal Using X‐Ray Holography

Stable paleomagnetic information in meteoritic metal is carried by the “cloudy zone”: ~1–10 μm‐wide regions containing islands of ferromagnetic tetrataenite embedded in a paramagnetic antitaenite matrix. Due to their small size and high coercivity (theoretically up to ~2.2 T), the tetrataenite islands carry very stable magnetic remanence. However, these characteristics also make it difficult to image their magnetic state with the necessary spatial resolution and applied magnetic field. Here, we describe the first application of X‐ray holography to image the magnetic structure of the cloudy zone of the Tazewell IIICD meteorite with spatial resolution down to ~40 nm and in applied magnetic fields up to ±1.1 T, sufficient to extract high‐field hysteresis data from individual islands. Images were acquired as a function of magnetic field applied both parallel and perpendicular to the surface of a ~100 nm‐thick slice of the cloudy zone. Broad distributions of coercivity are observed, including values that likely exceed the maximum applied field. Horizontal offsets in the hysteresis loops indicate an interaction field distribution with half width of ~100 mT between the islands in their room temperature single‐domain state, providing a good match to first‐order reversal curve diagrams. The results suggest that future models of remanence acquisition in the cloudy zone should take account of strong interactions in order to extract quantitative estimates of the paleofield.EC/FP7/320750/EU/Nanopaleomagnetism: a multiscale approach to paleomagnetic analysis of geological materials/NanoPaleoMagEC/FP7/312284/EU/Coordinated Access to Lightsources to Promote Standards and Optimization/CALIPS

Breaking Traditions:An Isotopic Study on the Changing Funerary Practices in the Dutch Iron Age (800-12 bc)

Urnfields in the Dutch river area were replaced by cemeteries with a mixture of cremation and inhumation graves around the sixth century bc. This study provides the first biogeochemical evidence that the Iron Age communities were heterogeneous in terms of geological origins. The high percentage of non-locally born individuals (~48%) supports the hypothesis that the change in burial practice was the result of the influx of foreign people, who were being allowed to keep their own burial customs, whereas some of the local inhabitants adapted the burial rites of foreign cultures, leading to a heterogeneous burial rite for some centuries

Field-free deterministic ultra fast creation of skyrmions by spin orbit torques

Magnetic skyrmions are currently the most promising option to realize current-driven magnetic shift registers. A variety of concepts to create skyrmions were proposed and demonstrated. However, none of the reported experiments show controlled creation of single skyrmions using integrated designs. Here, we demonstrate that skyrmions can be generated deterministically on subnanosecond timescales in magnetic racetracks at artificial or natural defects using spin orbit torque (SOT) pulses. The mechanism is largely similar to SOT-induced switching of uniformly magnetized elements, but due to the effect of the Dzyaloshinskii-Moriya interaction (DMI), external fields are not required. Our observations provide a simple and reliable means for skyrmion writing that can be readily integrated into racetrack devices