Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated. Here we assess the oligomerization behavior of three prototypical Class A GPCRs, the β1-ARs, β2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the β2-AR shows a slight degree of oligomerization. From a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86. Taken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels

Performance and durability of broadband antireflection coatings for thin film CdTe solar cells

Light reflection from the glass surface of a photovoltaic (PV) module is a significant source of energy loss for all types of PV devices. The reflection at the glass and air interface accounts for 4% of the total energy. Single layer antireflection coatings with sufficiently low refractive index have been used, such as those using magnesium fluoride or porous silica, but these are only effective over a narrow range of wavelengths. In this paper, the authors report on the design, deposition, and testing of multilayer broadband antireflection coatings. These coatings reduce the weighted average reflection over the wavelength range used by thin film CdTe devices to just 1.22%, resulting in a 3.6% relative increase in device efficiency. The authors have used multilayer stacks consisting of silica and zirconia layers deposited using reactive magnetron sputtering. Details of the stack design, sputter deposition process parameters, and the optical and microstructural properties of the layers are provided. Antireflection coatings on glass exposed to the outdoors must not degrade over the lifetime of the module. A comprehensive set of accelerated environmental durability tests has been carried out in accordance with IEC 61646 PV qualification tests. The durability tests confirmed no damage to the coatings or performance drop as a result of thermal cycling or damp heat. All attempts to perform pull tests resulted in either adhesive or substrate failure, with no damage to the coating itself. The coatings also passed acid attack tests. Scratch resistance, abrasion resistance, and adhesion tests have also been conducted. The optical performance of the coatings was monitored during these tests, and the coatings were visually inspected for any sign of mechanical failure. These tests provide confidence that broadband antireflection coatings are highly durable and will maintain their performance over the lifetime of the solar module. All dielectric metal-oxide multilayer coatings have better optical performance and superior durability compared with alternative single layer porous sol–gel coatings. Thin film CdTe devices are particularly problematic because the antireflection coating is applied to one side of the glass, while device layers are deposited directly on to the opposite glass surface in the superstrate configuration. In thin film CdTe production, the glass is exposed to high temperature processes during the absorber deposition and the cadmium chloride activation treatment. If glass precoated with a broadband antireflection coating is to be used, then the coating must withstand temperatures of up to 550 C. Surprisingly, our studies have shown that multilayer silica/zirconia antireflection coatings on soda lime glass remain unaffected by temperatures reaching 600 C, at which point mild crazing is observed. This is an important observation, demonstrating that low cost glass, which is preprocessed with a broadband antireflection coating, is directly useable in thin film CdTe module production

3D Finite Element Modelling of Cutting Forces in Drilling Fibre Metal Laminates and Experimental Hole Quality Analysis

Machining Glass fibre aluminium reinforced epoxy (GLARE) is cumbersome due to distinctively different mechanical and thermal properties of its constituents, which makes it challenging to achieve damage-free holes with the acceptable surface quality. The proposed work focuses on the study of the machinability of thin (~2.5 mm) GLARE laminate. Drilling trials were conducted to analyse the effect of feed rate and spindle speed on the cutting forces and hole quality. The resulting hole quality metrics (surface roughness, hole size, circularity error, burr formation and delamination) were assessed using surface profilometry and optical scanning techniques. A three dimensional (3D) finite-element (FE) model of drilling GLARE laminate was also developed using ABAQUS/Explicit to help understand the mechanism of drilling GLARE. The homogenised ply-level response of GLARE laminate was considered in the FE model to predict cutting forces in the drilling process

The performance and durability of single-layer sol-gel anti-reflection coatings applied to solar module cover glass

© 2018 A significant source of energy loss in photovoltaic (PV) modules is caused by reflection from the front cover glass surface. Reflection from the cover glass causes a loss of ~4% at the air-glass interface. Only a single air-glass interface can be coated on crystalline silicon solar modules as an ethylene-vinyl acetate (EVA) layer is inserted between the cover glass and the silicon absorber. A single-layer anti-reflection coating (ARC) on the outer surface of the cover glass is effective at reducing reflection losses over the wavelength range of most PV devices. The coating investigated in this work reduces the reflectance loss at the glass surface by 74%. However, the long-term durability of sol-gel coatings has not been established particularly for use in hot and humid climates. In this work, we investigate the damage resistance of a single-layer closed-surface hard coat ARC, deposited using sol-gel methods by applying a variety of accelerated weathering, scratch and abrasion test methods. The reflectance of the sol-gel ARC was measured and then the coating was put through a series of durability and environmental tests. The coating is resistant to damage from heating and can withstand temperatures higher than the phase change temperature of soda-lime glass. Scratch testing demonstrated that the sol-gel AR is relatively hard and difficult to remove from the substrate surface. Pull tests and cross-hatch testing also confirmed the strong adhesion of the coating. Weathering experiments show some degradation in weighted average reflectance, particularly an increase in reflectance of 0.6–0.9% after 1000 h of exposure to damp heat. Testing also showed a vulnerability to exposure to acid. These results indicate that the performance of this type of ARC could deteriorate and possibly delaminate in humid climate conditions The ARC had a low water contact angle, which means the coatings are hydrophilic and, therefore, hygroscopic increasing the risk of water damage over extended periods of time. This work shows that sol-gel anti-reflection coatings are currently unsuitable for use on PV and are unlikely to remain durable across the 25 year industry standard

Antioxidant and antimicrobial activities of white, green and black tea extracts

In the present study, the antioxidant and antimicrobial activities of three tea (Camellia sinensis) types (white tea — WT, green tea — GT, and black tea — BT) were compared and the relationships between total phenolic, tannin and flavonoid contents were determined. Regardless of the assays used, the highest total phenolic content (313.3±1.41 μg GAE/mg extract), total flavonoid (16.98±0.27 μg QE/mg extract) and total tannin content (266.79±2.59 μg TAE/mg extract) were determined in green tea extract, which also demonstrated the highest antioxidant capacity. Black tea extract showed the lowest phenolic content and antioxidant capacity. The EC50 value of DPPH scavenging activity was in the order of: ascorbic acid >GT>BHA>WT>BT>BHT. While the tea extracts exhibited antibacterial activity against Staphylococcus aureus, no inhibitory effects were observed against Escherichia coli and Salmonella enteritidis. All extracts exhibited antifungal activity against two afl atoxigenic moulds Aspergillus parasiticus NRRL 2999 and NRRL 465. The antibacterial activity of tea extracts decreased in the following order: GT>WT>BT DPPH scavenging activity strongly correlated with total phenolic content, reducing power, antimicrobial activity against S. aureus, A. parasiticus NRRL 2999, A. parasiticus NRRL 465 (P<0.05). These data suggest that green tea extract is more effective than white and black tea extracts as a potential source of natural antioxidants

Structural and functional characterization of allatostatin receptor type-C of Thaumetopoea pityocampa revealed the importance of Q271(6.55) residue in G protein-dependent activation pathway

Insect neuropeptide receptors are among the potential targets for designing next-generation pesticides. Activation of allatostatin receptor type C (AstR-C), a G Protein-coupled receptor (GPCR), upon stimulation with its endogenous ligand, allatostatin C (AST-C), leads to the inhibition of juvenile hormone (JH) secretion that consequently regulates physiology of insects. Here we conducted in silico and in vitro approaches to characterize the structure and function of AstR-C of Thaumetopoea pityocampa (T.pit), a well-known pest in Mediterranean countries. The sequence of AstR-C and AST-C were derived from whole genome sequencing (WGS) data. Resonance energy transfer (RET) methods were used to investigate the downstream effectors of the receptor and the temporal kinetics of G protein activation. Three-dimensional (3D) structure of AstR-C constructed via homology modeling methods was subjected to molecular dynamics (MD) simulations and docking studies to identify the orthosteric pocket. Our results showed that T.pit AstR-C couples to Gαi/o subtype of G proteins at sub-nanomolar ranges of the the ligand with the G protein recruitment and activation kinetics of ~4 and 6 seconds, respectively, when 1 nM AST-C is administered. At the increasing concentration of native ligand, βarrestin was shown to be recruited at nanomolar ranges the ligand. Docking and MD simulation studies revealed the importance of extracellular loop 2 (ECL2) in T.pit AstRC/AST-C interaction, and combination of in silico and in vitro methods supported the accuracy of the built model and the predicted orthosteric pocket. Q271(6.55) (Ballesteros-Weinstein generic numbering) was found to have a substantial role in G protein dependent activation of AstR-C possibly via contributing to the flexibility of the structure

Effect of Ulexite on Mechanical, Thermal, and Flame Properties of Halogen-Free Fire Retardant Polypropylene

8th International Advances in Applied Physics and Materials Science Congress and Exhibition (APMAS) -- APR 24-30, 2018 -- Fethiye, TURKEYWOS: 000481717400079In this study it was aimed to improve the fire retardant performance of halogen free flame-retardant polypropylene by increasing limiting oxygen index value. Ammonium polyphosphate, pentaerythritol, and ulexite were used to improve flame retardancy of polypropylene. Ulexite powder filled (1, 2, and 4 wt%) polypropylene composites were obtained by using a twin-screw extruder and injection molding method. Thermal, mechanical, and flame retardant performances of polypropylene composites were investigated by several characterization techniques. Limiting oxygen index value was obtained higher than 45% when ulexite was added into polypropylene at 1-4 wt%. It was observed that flame retardant additives do not change the mechanical properties considerably. The effect of ulexite addition on melting and crystallization temperatures of polypropylene-based composite is not pronounced.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [Teydeb 7160061]The authors would like to thank the Scientific and Technological Research Council of Turkey (TUBITAK) for financial support (Project No: Teydeb 7160061)

Degradation of Hydrophobic, Anti-Soiling Coatings for Solar Module Cover Glass

Soiling of solar module cover glass is a serious problem for solar asset managers. It causes a reduction in power output due to attenuation of the incident light, and reduces the return on investment. Regular cleaning is required to mitigate the effect but this is a costly procedure. The application of transparent hydrophobic, anti-soiling coatings to the cover glass is a promising solution. These coatings have low surface energy and contaminants do not adhere well. Even if soiling does remain on the coated surface, it is much more easily removed during cleaning. The performance of the coatings is determined using the water contact angle and roll-off angle measurements. However, although hydrophobic coatings hold out great promise, outdoor testing revealed degradation that occurs surprisingly quickly. In this study, we report on results using laboratory-based damp heat and UV exposure environmental tests. We used SEM surface imaging and XPS surface chemical analysis to study the mechanisms that lead to coating degradation. Loss of surface fluorine from the coatings was observed and this appeared to be a major issue. Loss of nanoparticles was also observed. Blistering of surfaces also occurs, leading to loss of coating material. This was probably due to the movement of retained solvents and was caused by insufficient curing. This mechanism is avoidable if care is taken for providing and carrying out carefully specified curing conditions. All these symptoms correlate well with observations taken from parallel outdoor testing. Identification of the mechanisms involved will inform the development of more durable anti-soiling, hydrophobic coatings for solar application