Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems.

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each

Їдемо з Новóї Каховки до Асканії-Новóї

A number of universities worldwide have created new courses and degrees or modified existing ones, as a response to the increasing interest by companies to hire sustainability literate graduates. However, many of such courses have been developed with a focus on 'hard' technocentric or managerial issues. The examples that have been published in academic journal have tended to be descriptive, and in only a limited number of cases have they been based on theories of teaching and learning. This paper presents the process of designing and delivering a new course on organisational change management for sustainability for the BA Environment and Business degree at the University of Leeds. The course was developed based on holism and a constructivist position to help deal with the complexities of sustainability and organisational change management. The course objective was to educate students as sustainability change agents by dealing with the complexities of sustainability and 'soft' issues in organisational change management. The process had three key elements: (1) the learning outcomes; (2) the course delivery; and (3) the course assessment (including feedback). During the process a number of challenges had to be overcome. The paper provides a more complete, systematic, robust, and focused approach to education for sustainable development, specifically on course design and delivery, by using theories of teaching and learning and linking the course aims, delivery, and assessment. The paper integrates education for sustainability development and corporate sustainability into a relatively new discipline, organisational change management for sustainability

Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7 angstrom range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.Peer reviewe

The sensory features of a food cue influence its ability to act as an incentive stimulus and evoke dopamine release in the nucleus accumbens core

The sensory properties of a reward-paired cue (a Conditioned Stimulus; CS) may impact the motivational value attributed to the cue, and in turn influence the form of the conditioned response (CR) that develops. A cue with multiple sensory qualities, such as a moving lever-CS, may activate numerous neural pathways that process auditory and visual information, resulting in CRs that vary both within and between individuals. For example, CRs include approach to the lever-CS itself (rats that “sign-track;” ST), approach to the location of reward delivery (rats that “goal-track;” GT), or an “intermediate” combination of these behaviors. We found that the multimodal sensory features of the lever-CS were important to the development and expression of sign-tracking. When the lever-CS was covered, and thus could only be heard moving, STs continued to approach the lever location, but also started to approach the food cup during the CS period. While still predictive of reward, the auditory component of the lever-CS was a much weaker conditioned reinforcer than the visible lever-CS. This plasticity in behavioral responding observed in STs closely resembled behaviors normally seen in rats classified as “intermediates.” Furthermore, the ability of both the lever-CS and reward-delivery to evoke dopamine release in the nucleus accumbens was also altered by covering the lever – dopamine signaling in STs resembled neurotransmission observed in rats that normally only GT. These data suggest that while the visible lever-CS was attractive, wanted, and had incentive value for STs, when presented in isolation the auditory component of the cue was simply predictive of reward, lacking incentive salience. Therefore, the specific sensory features of cues may differentially contribute to responding and ensure behavioral flexibility

Demonstration of radon removal from SF6 using molecular sieves

The gas SF6 has become of interest as a negative ion drift gas for use in directional dark matter searches. However, as for other targets in such searches, it is important that radon contamination can be removed as this provides a source of unwanted background events. In this work we demonstrate for the first time filtration of radon from SF6 gas by using a molecular sieve. Four types of sieves from Sigma-Aldrich were investigated, namely 3Å, 4Å, 5Å and 13X. A manufactured radon source was used for the tests. This was attached to a closed loop system in which gas was flowed through the filters and a specially adapted Durridge RAD7 radon detector. In these measurements, it was found that only the 5Å type was able to significantly reduce the radon concentration without absorbing the SF6 gas. The sieve was able to reduce the initial radon concentration of 3875 ± 13 Bqm−3 in SF6 gas by 87% when cooled with dry ice. The ability of the cooled 5Å molecular sieve filter to significantly reduce radon concentration from SF6 provides a promising foundation for the construction of a radon filtration setup for future ultra-sensitive SF6 gas rare-event physics experiments

Lowering the energy threshold in COSINE-100 dark matter searches

COSINE-100 is a dark matter detection experiment that uses NaI(Tl) crystal detectors operating at the Yangyang underground laboratory in Korea since September 2016. Its main goal is to test the annual modulation observed by the DAMA/LIBRA experiment with the same target medium. Recently DAMA/LIBRA has released data with an energy threshold lowered to 1 keV, and the persistent annual modulation behavior is still observed at 9.5$\sigma$. By lowering the energy threshold for electron recoils to 1 keV, COSINE-100 annual modulation results can be compared to those of DAMA/LIBRA in a model-independent way. Additionally, the event selection methods provide an access to a few to sub-GeV dark matter particles using constant rate studies. In this article, we discuss the COSINE-100 event selection algorithm, its validation, and efficiencies near the threshold

Quantification of the unsharp masking technique of image enhancement

The technique of unsharp masking is described and its use as an image enhancement technique discussed. A mathematical model for the masking process is developed; experimental testing and MTF measurements of the masked and sharpened images are made to test the validity of the mathematical model as a predictor of the mask and final image characteristics. The effect of contrast, mask unsharpness, and source spread function size on the resulting MTF are presented. Subjective evaluations are used to determine the visually optimum image. It is shown that the visually best image is not necessarily the one with the largest MTF value or area; suggestions are made for adjusting existing image quality specifications to incorporate the results of unsharp masking techniques