Single step, direct fluorescence immunoassays based on metal enhanced fluorescence (MEF-FIA) applicable as micro plate-, array-, multiplexing- or point of care-format.

Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lower sub-picomolar concentrations. We hereby present a new assay platform based on metal enhanced fluorescence (MEF), that remedies these problems since it eliminates the need for washing steps, for using enzyme labels and allows detection of analytes down to sub-picomolar concentrations. In addition this technology is fully compatible to standard fluorescence reader equipment as it is found in many laboratories nowadays. Since our present work is focused on single biomarker evaluation, we chose a 96 well plate format for convenience, but any other formate like antibody arrays, strip-like point of care devices etc. is feasible too.

RADIATION DOSE MEASUREMENTS FOR HIGH-INTENSITY LASER INTERACTIONS WITH SOLID TARGETS AT SLAC.

A systematic study of photon and neutron radiation doses generated in high-intensity laser-solid interactions is underway at SLAC National Accelerator Laboratory. These laser-solid experiments are being performed using a 25 TW (up to 1 J in 40 fs) femtosecond pulsed Ti:sapphire laser at the Linac Coherent Light Source's (LCLS) Matter in Extreme Conditions (MEC) facility. Radiation measurements were performed with passive and active detectors deployed at various locations inside and outside the target chamber. Results from radiation dose measurements for laser-solid experiments at SLAC MEC in 2014 with peak intensity between 1018 and 7.1 × 1019 W cm-2 are presented.

Separation of active laccases from Pleurotus sapidus culture supernatant using aqueous two-phase systems in centrifugal partition chromatography.

For the production of bio active compounds, e.g., active enzymes or antibodies, a conserved purification process with a minimum loss of active compounds is necessary. In centrifugal partition chromatography (CPC), the separation effect is based on the different distribution of the components to be separated between two immiscible liquid phases. Thereby, one liquid phase is kept stationary in chambers by a centrifugal field and the mobile phase is pumped through via connecting ducts. Aqueous two phase systems (ATPS) are known to provide benign conditions for biochemical products and seem to be promising when used in CPC for purification tasks. However, it is not known if active biochemical compounds can "survive" the conditions in a CPC where strong shear forces can occur due to the two-phasic flow under centrifugal forces. Therefore, this aspect has been faced within this study by the separation of active laccases from a fermentation broth of Pleurotus sapidus. After selecting a suitable ATPS and operating conditions, the activity yield was calculated and the preservation of the active enzymes could be observed. Therefore, CPC could be shown as potentially suitable for the purification of bio-active compounds.

Biomolecular interaction analysis in functional proteomics.

To understand the function of highly complex eukaryotic tissues like the human brain, in depth knowledge about cellular protein networks is required. Biomolecular interaction analysis (BIA), as a part of functional proteomics, aims to quantify interaction patterns within a protein network in detail. We used the cAMP dependent protein kinase (PKA) as a model system for the binding analysis between small natural ligands, cAMP and cAMP analogues, with their physiological interaction partner, the regulatory subunit of PKA. BIA comprises a variety of methods based on physics, biochemistry and molecular biology. Here we compared side by side real time SPR (surface plasmon resonance, Biacore), a bead based assay (AlphaScreen), a fluorescence based method (Fluorescence polarisation) and ITC (isothermal titration calorimetry). These in vitro methods were complemented by an in cell reporter assay, BRET(2) (bioluminescence resonance energy transfer), allowing to test the effects of cAMP analogues in living cells.

Advantage of three dimensional animated teaching over traditional surgical videos for teaching ophthalmic surgery: a randomised study.

BACKGROUND/AIM: Owing to the complex topographical aspects of ophthalmic surgery, teaching with conventional surgical videos has led to a poor understanding among medical students. A novel multimedia three dimensional (3D) computer animated program, called "Ophthalmic Operation Vienna" has been developed, where surgical videos are accompanied by 3D animated sequences of all surgical steps for five operations. The aim of the study was to assess the effect of 3D animations on the understanding of cataract and glaucoma surgery among medical students. METHOD: Set in the Medical University of Vienna, Department of Ophthalmology, 172 students were randomised into two groups: a 3D group (n=90), that saw the 3D animations and video sequences, and a control group (n=82), that saw only the surgical videos. The narrated text was identical for both groups. After the presentation, students were questioned and tested using multiple choice questions. RESULTS: Students in the 3D group found the interactive multimedia teaching methods to be a valuable supplement to the conventional surgical videos. The 3D group outperformed the control group not only in topographical understanding by 16% (p<0.0001), but also in theoretical understanding by 7% (p<0.003). Women in the 3D group gained most by 19% over the control group (p<0.0001). CONCLUSIONS: The use of 3D animations lead to a better understanding of difficult surgical topics among medical students, especially for female users. Gender related benefits of using multimedia should be further explored.

Benchmark studies of induced radioactivity produced in LHC materials, Part I: Specific activities.

Samples of materials which will be used in the LHC machine for shielding and construction components were irradiated in the stray radiation field of the CERN-EU high-energy reference field facility. After irradiation, the specific activities induced in the various samples were analysed with a high-precision gamma spectrometer at various cooling times, allowing identification of isotopes with a wide range of half-lives. Furthermore, the irradiation experiment was simulated in detail with the FLUKA Monte Carlo code. A comparison of measured and calculated specific activities shows good agreement, supporting the use of FLUKA for estimating the level of induced activity in the LHC.

Benchmark studies of induced radioactivity produced in LHC materials, Part II: Remanent dose rates.

A new method to estimate remanent dose rates, to be used with the Monte Carlo code FLUKA, was benchmarked against measurements from an experiment that was performed at the CERN-EU high-energy reference field facility. An extensive collection of samples of different materials were placed downstream of, and laterally to, a copper target, intercepting a positively charged mixed hadron beam with a momentum of 120 GeV c(-1). Emphasis was put on the reduction of uncertainties by taking measures such as careful monitoring of the irradiation parameters, using different instruments to measure dose rates, adopting detailed elemental analyses of the irradiated materials and making detailed simulations of the irradiation experiment. The measured and calculated dose rates are in good agreement.

Comparison of synchrotron radiation calculations between analytical codes (STAC8, photon) and Monte Carlo codes (FLUKA, EGS4).

STAC8 is a significant improvement upon its predecessor PHOTON and is a valuable analytical code for quick and conservative beamline shielding designs for synchrotron radiation (SR) facilities. In order to check the applicability, accuracy and limitations of STAC8, studies were conducted to compare the results of STAC8 with those of PHOTON with calculations using the FLUKA and EGS4 Monte Carlo codes. Doses and spectra for scattered SR in a few beam-target-shield geometries were calculated, with and without photon linear polarisation effects. Areas for expanding the STAC8 capabilities, for example, features of the mirror-reflected lights and double-Compton light calculations and the use of monochromatic light have been identified. Some of these features have been implemented and benchmarked against Monte Carlo calculations. Reasonable agreements were found between the STAC8 and Monte Carlo calculations.

Phase resetting and phase locking in hybrid circuits of one model and one biological neuron.

To determine why elements of central pattern generators phase lock in a particular pattern under some conditions but not others, we tested a theoretical pattern prediction method. The method is based on the tabulated open loop pulsatile interactions of bursting neurons on a cycle-by-cycle basis and was tested in closed loop hybrid circuits composed of one bursting biological neuron and one bursting model neuron coupled using the dynamic clamp. A total of 164 hybrid networks were formed by varying the synaptic conductances. The prediction of 1:1 phase locking agreed qualitatively with the experimental observations, except in three hybrid circuits in which 1:1 locking was predicted but not observed. Correct predictions sometimes required consideration of the second order phase resetting, which measures the change in the timing of the second burst after the perturbation. The method was robust to offsets between the initiation of bursting in the presynaptic neuron and the activation of the synaptic coupling with the postsynaptic neuron. The quantitative accuracy of the predictions fell within the variability (10%) in the experimentally observed intrinsic period and phase resetting curve (PRC), despite changes in the burst duration of the neurons between open and closed loop conditions.

Interaction effects in conductivity of Si inversion layers at intermediate temperatures.

We compare the temperature dependence of resistivity rho(T) of Si-metal-oxide-semiconductor field-effect transistors with the recent theory by Zala et al. In this comparison, the effective mass m* and g* factor for mobile electrons have been determined from independent measurements. An anomalous increase of rho with temperature, which has been considered as a signature of the "metallic" state, can be described quantitatively by the interaction effects in the ballistic regime. The in-plane magnetoresistance rho(B(axially)) is only qualitatively consistent with the theory; the lack of quantitative agreement indicates that the magnetoresistance is more sensitive to sample-specific effects than rho(T).

Low-density spin susceptibility and effective mass of mobile electrons in Si inversion layers.

We studied the Shubnikov-de Haas (SdH) oscillations in high-mobility Si-MOS samples over a wide range of carrier densities n approximately (1-50)x10(11) cm(-2), which includes the vicinity of the apparent metal-insulator transition in two dimensions (2D MIT). Using a novel technique of measuring the SdH oscillations in superimposed and independently controlled parallel and perpendicular magnetic fields, we determined the spin susceptibility chi(*), the effective mass m(*), and the g(*) factor for mobile electrons. These quantities increase gradually with decreasing density; near the 2D MIT, we observed enhancement of chi(*) by a factor of approximately 4.7.

Weak anisotropy and disorder dependence of the in-plane magnetoresistance in high-mobility (100) Si-inversion layers.

We report studies of the magnetoresistance (MR) in a two-dimensional electron system in (100) Si-inversion layers, for perpendicular and parallel orientations of the current with respect to the magnetic field in the 2D plane. The magnetoresistance is almost isotropic; this result does not support the suggestion of its orbital origin. In the hopping regime, however, the MR contains a weak anisotropic component that is nonmonotonic in the magnetic field. We found that the field, at which the MR saturates, varies for different samples by a factor of 2 at a given carrier density. Therefore, the saturation of the MR cannot be identified with the complete spin polarization of free carriers.

Exclusion of quantum coherence as the origin of the 2D metallic state in high-mobility silicon inversion layers.

The temperature and density dependence of the phase coherence time tau(phi) in high-mobility silicon inversion layers was determined from the magnetoresistivity due to weak localization. The upper temperature limit for single-electron quantum interference effects was delineated by comparing tau(phi) with the momentum relaxation time tau. A comparison between the density dependence of the borders for quantum interference effects and the strong resistivity drop reveals that these effects are not related to each other. As the strong resistivity drop occurs in the Drude regime, the apparent metallic behavior cannot be caused by quantum coherent effects.

CD4(+) T cell frequencies and Th1/Th2 cytokine patterns expressed in the acute and memory response to respiratory syncytial virus I-E(d)-restricted peptides.

The respiratory syncytial virus (RSV)-specific frequencies and cytokine expression patterns of acute and memory CD4(+) T cells from RSV strain-A- and strain-B-infected BALB/c mice were determined following restimulation with a panel of 14 predicted RSV I-E(d) peptides from NSP-2, M, SH, F, and L proteins. Ten of fourteen peptides stimulated intracellular Th1 and/or Th2 cytokines in CD4(+) T cells from the mediastinal lymph nodes (MLN) and spleens of RSV strain-A- or strain-B-immune BALB/c mice. Spleen cells exhibited a predominant Th2 cytokine expression pattern after peptide stimulation, whereas MLN cells exhibited a mixed Th1/Th2 cytokine pattern. For a few peptides, there were differences in the Th1/Th2 cytokine response to peptides from the homologous versus heterologous RSV group. None of the 10 peptides induced both Th1 and Th2 cytokines in cells from similarly immunized mice. The frequency and breadth of cytokine expression by I-E(d)-restricted CD4(+) T cells to peptide stimulation was diminished in the memory response.

Sec61p is the main ribosome receptor in the endoplasmic reticulum of Saccharomyces cerevisiae.

A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

Electrical synapses by guided growth of cultured neurons from the snail Lymnaea stagnalis.

The ability to assemble neuronal networks with designed topology would allow uniquely defined experiments on neurocomputing. We describe a fundamental step, the controlled formation of synapses by guided outgrowth, in vitro for the first time combining simple neuritic geometry with predefined connectivity. We used neurons from the A-clusters in the pedal ganglia of the snail Lymnaea stagnalis. They were cultured on a substrate with linear patterns made by adsorption of brain-derived conditioning factors and photolithography. We induced and observed the frontal collision of two growth cones on narrow lanes. Following such encounters. individual electrical synapses formed that were sometimes strong enough for prolonged presynaptic stimulation to reach the threshold of postsynaptic firing.

Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA.

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the protein-conducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved.

Plant diversity and productivity experiments in european grasslands

At eight European field sites, the impact of loss of plant diversity on primary productivity was simulated by synthesizing grassland communities with different numbers of plant species. Results differed in detail at each location, but there was an overall log-linear reduction of average aboveground biomass with loss of species. For a given number of species, communities with fewer functional groups were less productive. These diversity effects occurred along with differences associated with species composition and geographic location. Niche complementarity and positive species interactions appear to play a role in generating diversity-productivity relationships within sites in addition to sampling from the species pool.