Structural Characterization of Higher Order Complex Formation in Conjugative Transposition of an Antibiotic Resistance Carrying Mobile Genetic Element

The spread of antibiotic resistance genes (ARGs) has become one of the biggest health care challenges in the last decades. Conjugative transposons (CTns) are a major class of mobile genetic elements that can transfer antibiotic resistance genes between bacterial genomes. For CTn transposition, DNA cleavage and joining reactions are catalyzed by a transposon-encoded integrase enzyme (Int). However, transposition doesn’t involve only Int but requires rather complex machinery, which employs multiple CTn- and host-encoded factors assembled in distinct higher order protein-DNA complexes. These assemblies act as hubs to regulate recombination both spatially and temporally. Despite available biochemical and structural data about some CTn Int proteins, the coordination and regulation of the recombination reaction by higher order complex formation are not well understood. During my doctoral studies, I aimed to decipher the structural and functional principles of higher order nucleoprotein assemblies in the transposition of the GISul2 element, a wide host range CTn that propagates diverse antibiotic resistance genes in pathogenic Gram-negative bacteria. In the context of this work, several structures of transposon excision complexes formed at different steps of the reaction were determined using Cryo-EM, which revealed distinct molecular assemblies. First, the structure of the right transposon end (RE) complex showed that RE DNA is bent by integration host factor (IHF) to ~160°, which allows IntGISul2 to bridge its arm and core DNA sites. The formation of this complex strictly requires the presence of the host-encoded accessory protein IHF, indicating strong coordination between the host cell state and CTn mobilization. In the second part, I present the structural characterization of the left transposon end (LE) complex. The cryo-EM map revealed that seven excisionase (Xis) molecules bend the LE DNA to form a loop that is tethered by integrases. LE complex formation depends on DNA bending by the transposon-encoded Xis protein, a directionality factor of recombination, which is required for excision but inhibits integration. In the third part of the thesis, I describe structural insights into how right and left transposon ends come together to form a synaptic complex during transposon excision. This synaptic complex was captured using “suicide” DNA substrates, which can stall the recombination reaction after cleavage of the first DNA strand pair. Unexpectedly, this approach resulted in the formation of two different synaptic complexes, one including RE and LE, and the other one containing two RE molecules, which together elucidate the assembly process and catalytic activation of the native synaptic excision complex. Together our results shed light on the principles of how the CTn integrase cooperates with accessory DNA bending proteins to coordinate active nucleoprotein complex assembly during excision and integration. These insights offer new knowledge about the mechanisms of antibiotic resistance spreading in Gram-negative pathogens. I hope that the better understanding of the CTn movement will help develop new strategies for tackling antibiotic resistance spread in the future

The performance and durability of anti-reflective and anti-soiling coatings on solar cover glass

In recent years, huge advances have been made in the development of photovoltaic (PV) technology. Anti-reflective (AR) and anti-soiling (AS) coatings have demonstrably improved the energy output of PV modules and are establishing commercial viability. However, the durability of such coatings has not been sufficiently addressed and remains a key concern. In this thesis, a range of testing methods have been identified and evaluated for the purpose of assessing the performance, durability, environmental stability and wear resistance of AR and AS hydrophobic coatings. A comprehensive set of performance parameters and characterisation tools were identified to assess the degradation mechanisms of several coating formulations. Comparative results were generated for coatings before and after being subjected to a range of stress tests. New test rigs and procedures, including a sand impact and raindrop simulation test, were also designed and implemented to provide further insight into the degradation of coating performance following exposure to sand and rain water. The durability of two types of single layer (ARC1 and ARC2) and a multilayer anti-reflective (MAR) coatings were investigated. The reflectance obtained from single layer AR coatings after damp heat and abrasion tests suggest poor durability and inadequate useful lifetime despite an initial performance increase in transmittance when applied to PV cover glass. The test results on the MAR coating, designed and produced in CREST, showed excellent durability against all environmental and mechanical stresses. Six variants of a hydrophobic coating, ASC1 were stress tested during the development of an anti-soiling coating for PV cover glass application. The degradation mechanisms of each variant were used to improve the performance and durability of the coating. In addition, the ASC2 hydrophobic coating, that is commercially available for application on PV cover glass, was durability tested under same stresses as ASC1 coating for comparison. The ASC2 coating showed good resistance to mechanical stress and UV exposure. However, damp heat testing of the ASC2 coating lead to a significant performance drop revealing that it is not durable in high temperature and humidity conditions. The results indicate that better performance could be achieved if these types of coatings are designed and developed for specific climatic conditions. Three hydrophobic coatings commercially available for other applications such as on ophthalmic lenses and smart phone displays, ASC3, ASC4 and ASC5 coatings were subjected to a range of stress tests to demonstrate suitability for use in PV applications. The hydrophobic properties of ASC3 and ASC5 coatings showed good durability against mechanical abrasion, but both failed to perform well against exposure to UV light. There was no visual or performance degradation observed after both abrasion resistance and sand impact test on both ASC3 and ASC5 coatings. Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of ASC5 showed that the nanoparticles responsible for the hydrophobic properties of the coating degraded under UV light with the loss of fluorine. Nevertheless, the ASC5 coating showed excellent stability against damp heat and thermal cycling stress. Furthermore, the ASC4 coating showed good durability against temperature cycling and UV light exposure. The coating also retained its hydrophobicity after 1000 hours of damp heat exposure. In addition, the coating showed a variable abrasion resistance against different abrasive materials. The test results suggest that ASC4 coating has good adhesion to the glass substrate with good durability against cheesecloth and felt pad abrasion. However, a harder scour pad damaged the surface of ASC4 severely. The test results suggest that all three hydrophobic coatings (ASC3, ASC4 and ASC5) have their strengths and weaknesses to withstand certain environmental and mechanical stresses due to their design for different applications. The durability of these coatings against UV light and abrasion resistance would need to be improved if they are to be applied to PV cover glass

Assessment of in vitro Antioxidant and Antidiabetic Capacities of Medlar (Mespilus germanica)

The objective of the current study was to evaluate the antioxidant activity and enzyme inhibitory effect of different parts of medlar including fruit, leaf and flower bud by using various in vitro methods, and also determination of total phenolic and flavonoid content in the samples. Ethanol extracts of medlar parts were prepared and their antioxidant activities were determined using 1,1-diphenyl-2-picryl-hydrazil (DPPH•) scavenging and β-carotene bleaching methods. The leaf extract showed the strongest antioxidant activity. DPPHradical scavenging activity was in the order of BHA > leaf > bud > fruit. This ordering was the same for β-carotene bleaching activity, tocopherol > leaf > bud > fruit. The highest total phenolic (60.3 ± 1.69 mg GAE g-1 extract) and flavonoid (14.77 ± 1.15 mg QE g-1 extract) content were determined in leaf extract. For possible antidiabetic effects of extracts, α-amylase and α-glucosidase inhibitory activities were investigated, the bud extract showed the highest inhibition activities among the all extracts

Test methods for hydrophobic coatings on solar cover glass

The world market for solar energy continues to expand. However, to be competitive with traditional energy sources, photovoltaic (PV) modules must be capable of continuous and reliable high performance. Performance losses occur due to the soiling of the cover glass on modules. Soiling can be reduced by using hydrophobic coatings. These decrease surface energy and thus minimize adhesion to soiling. These coatings can help reduce maintenance and retain consistent electrical output. It is not yet clear, how hydrophobic coatings can be assessed and compared. In this paper, test methods to simulate the stresses that coatings experience in their life-time are assessed. These test methods help to predict the durability and useful lifetime of the coatings when applied to solar cover glass. Various test methods from different standards have been applied to hydrophobic coated glass surfaces and optimized to simulate real-outdoor conditions. A sand impact test and a water drop simulation test have been devised to study the effect of sand and rain on hydrophobic performance and durability

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

Testing of an anti-soiling coating for PV module cover glass

Soiling of solar module cover glass can significantly reduce the module power output. Coatings can be applied to the cover glass surface to reduce adhesion and make the surfaces easier to clean. These coatings should be resilient and resistant to environmental damage. A hydrophobic anti-soiling coating was exposed to a variety of environmental and abrasion stress tests. The hydrophobic performance of the coating was measured by monitoring the water contact angle and the water roll off angle after exposure to a range of environmental and mechanical stress tests. The coating was shown to be highly resistant to damp heat and thermal cycling. However, it was degraded by UV exposure and damaged during abrasion tests. The coating was also exposed to outdoor testing to compare the laboratory results with real performance degradation

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

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

Durable high-performance water-based anti-reflective coating for PV module glass

Without an antireflective coating, more than 4% of incident light is reflected from the standard front cover glass of photovoltaic (PV) modules. Module efficiency is one of the most important levers to impact the cost-per-watt of solar and recovering some of this reflected light with a simple anti-reflective coating (ARC) has become widespread. The types of ARC can vary in deposition method (roll coating, spray coating, sputtering, etc.) as well as composition and performance. The most widely adopted coatings today are based on a porous silica film with a thickness optimized for the solar spectrum. Current coatings, however, have room for improvement in both the performance and cost, giving manufacturers reason to seek new solutions that drive down the levelized cost of electricity (LCOE). In this work, we report the test results for a new AR coating from WattGlass showing significantly improved optical performance compared to the traditional AR coatings. The WattGlass AR coating takes advantage of water-based chemistry that is more environmentally friendly than the sol-gel process used in standard production coatings. This chemistry allows a high performance and durable coating to be deposited and cured at room temperature and is compatible with an industry standard glass tempering process. The samples under test in this work were subjected to extensive environmental and accelerated lifetime testing performed by CREST (Centre for Renewable Energy Systems Technology) at Loughborough University

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