Recruitment of Eph receptors into signaling clusters does not require ephrin contact

Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Efficiency of a borehole seal by means of pre-compacted bentonite blocks

The backfilling and sealing of shafts and galleries is an essential part of the design of underground repositories for high-level radioactive waste. Part of the EC funded project RESEAL studied the feasibility of sealing off a borehole in plastic Boom Clay by means of pre-compacted bentonite blocks. Two bentonites, namely the FoCa and Serrata clay, have been used. Based on laboratory tests, the bentonite blocks had an initial dry density of about 1.8 g/cm3 to obtain a swelling pressure of about 4.4 MPa, corresponding to the in situ lithostatic stress, at full saturation. The set-up was equipped with several sensors to follow-up the behaviour of the seal and the surrounding host rock during hydration. Full saturation was reached after five months and was mainly reached by natural hydration. Swelling pressure was lower than originally foreseen due to the slow reconsolidation of the host rock. Later on, the efficiency of the seal with respect to water, gas and radionuclide migration was tested. The in situ measured permeability of the seals was about 5 × 10-13 m/s. A gas breakthrough experiment did not show any preferential gas migration through the seal. No evidences of a preferential pathway could be detected from 125I tracer test result

Formation and quenching mechanisms of the electron beam pumped (XeRb)+ ionic excimer in different buffer gases

The ionic excimer molecule XeRb+ is formed in an electron beam excited gas mixture of Xe, Rb, and a buffer gas. The formation and quenching mechanisms of ionic excimers are investigated by measuring the XeRb+fluorescence as a function of the gas composition and gas pressure. The formation of XeRb+ is achieved by a three‐body association reaction between Xe+, Rb, and a buffer gas atom. For the buffer gases He, Ne, or Ar the values of the important formationrate constants are determined from the observed fluorescence signal decay

Efficient 3D trilateration algorithm for visible light positioning

© 2019 IOP Publishing Ltd. This letter presents an efficient algorithm for estimating the three-dimensional (3D) location of a photodiode (PD) receiver via visible light positioning. It solely works on measured powers from different light-emitting diode (LED) sources and does not require any prior knowledge of the PD receiver height. It is found that four LEDs are required that are not on the same circle, in order to unambiguously determine the 3D location. The algorithm is optimized towards a minimized calculation time in view of real-time operation on energy-constrained lightweight and mobile devices such as drones

What do patients and dermatologists prefer regarding low-risk basal cell carcinoma follow-up care? A discrete choice experiment

Background Follow-up after low-risk basal cell carcinoma (BCC) is being provided more frequently than recommended by guidelines. To design an acceptable strategy to successfully reduce this ‘low-value’ care, it is important to obtain insights into the preferences of patients and dermatologists. Objective To determine the preferences and needs of patients and dermatologists to reduce low-risk BCC follow-up care, and the trade-offs they are willing to make. Methods A questionnaire including a discrete choice experiment was created, containing attributes regarding amount of follow-up, continuity of care, method of providing addition information, type of healthcare provider, duration of follow-up visits and skin examination. In total, 371 BCC patients and all Dutch dermatologists and dermatology residents (n = 620) were invited to complete the questionnaire. A panel latent class model was used for analysis. Results Eighty-four dermatologists and 266 BCC patients (21% and 72% response rates respectively) completed the discrete choice experiment. If the post-treatment visit was performed by the same person as treatment provider and a hand-out was provided to patients containing personalised information, the acceptance of having no additional follow-up visits (i.e. following the guidelines) would increase from 55% to 77% by patients. Female patients and older dermatologists, however, are less willing to accept the guidelines and prefer additional follow-up visits. Limitations The low response rate of dermatologists. Conclusion This discrete choice experiment revealed a feasible strategy to substantially reduce costs, while maintaining quality of care, based on the preferences and needs of BCC patients, which is supported by dermatologists.Q1Revista Internacional - Indexad

A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation.

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose

Where there’s smoke, there’s more smoke: The social settings and friendship interactions that encourage young adults to smoke cigarettes

Despite widespread public health campaigns in Western countries people continue to smoke cigarettes and more worryingly, young people continue to take up the habit. In this thesis it is argued that cigarette smoking practices for young adults can be understood in terms of contributing to their sense of identity construction through friendship interactions and sociability. Data collected from email administered surveys and snowball sampling techniques, alongside secondary data from the National Drug Strategy Household Survey (2007), inform the research undertaken in this thesis and the results support the social benefits hypothesis in explaining why young adults smoke cigarettes. This study thereby suggests that in order for anti-smoking initiatives to be more successful in tackling the smoking habits of young adults additional research is required in identity formation, interactive factors and sociability factors that affect cigarette smoking practices of young adults

RESEAL II: a large-scale in situ demonstration for repository sealing in an argillaceous host rock: phase II

Final ReportPreprin