Recombinant expression and bioinformatic analysis of the Hepatitis B virus X protein

There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer. The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer and has been reported to manipulate several critical cellular pathways. These include the cell cycle, the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of animal models and infectable cell lines together with solubility and stability issues related to the HBx protein have made progress difficult. The reliance on approximate cellular and animal models has yielded many discordant studies that have confounded our interpretations of the role of HBx. There have been no novel approaches attempting to express HBx at a quantity and quality sufficient for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be responsible for these challenges have not been reported. This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus (SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed HBx. This result was despite the positive visualisation of HBx using the same antibodies and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity of HBx. Furthermore these findings supported the decision to develop eukaryotic-based HBx expression vectors as results suggested structural differences between eukaryote and prokaryote expressed protein. HBx was subsequently detected and purified in a soluble and active form using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point from which further work at optimising HBx expression and structural elucidation can occur. Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly regulated and thus experimental systems employing overexpression methodologies may encourage cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding synonymous and non-synonymous substitutions at the overlapping and non-overlapping domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution occurred in the overlapping partner gene. The presence of sites under positive, neutral and negative selection were identified across the length of HBx. The different genotypes showed positive selection indicating selective pressures unique to each, thus offering a contributing explanation for the variable disease severity observed between the subtypes. Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and mammalian cells. These methods, together with an increased understanding of the nature of HBx sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the progression to liver cancer

Results from an interlaboratory comparison of areal surface texture parameter extraction from X-ray computed tomography of additively manufactured parts

This paper presents the results of the CT-STARR (CT-Surface Texture for Additive Round Robin) interlaboratory comparison. The study compares the results obtained for the extraction of areal surface texture data per ISO 25178-2 from five X-ray computed tomography (XCT) volume measurements from each of four laboratories. To reduce the number of process variables, all participants utilise a Nikon XCT machine, either an XT H 225 industrial CT or an MCT225 metrology CT. Measurement process parameters, such as physical X-ray filtering, acceleration voltage and filament current, are set at similar values for all machines. All data processing and computation to extract, align, crop, filter and generate surface texture parameter information and deviation analysis results from the measurement volumes is performed by one participant. Two Ti6Al4V ELI (extra low interstitial) components are included in each of the XCT acquisitions. The first component is an additively manufactured cube built on an Arcam Q10 electron beam melting machine. Surface texture data is extracted from XCT scans of this part. The second component is a machined artefact designed for XCT scaling and surface determination analysis and verification. The data extracted from XCT measurements of these components is compared with measurements from coordinate measuring machine, focus variation and stylus instruments. The effect of scaling correction and XCT surface determination on extracted surface texture data, as well as measurement repeatability and reproducibility, are discussed

A Systematic Review and Meta-Analysis Exploring Effects of Third-Wave Psychological Therapies on Hearing-Related Distress, Depression, Anxiety, and Quality of Life in People With Audiological Problems

Purpose: There is growing evidence supporting the use of third-wave psychological therapies, such as mindfulness-based interventions (MBIs) and acceptance and commitment therapy (ACT), for people with long-term or chronic physical health conditions. We conducted a systematic review and meta-analysis to critically evaluate the effectiveness of third-wave interventions for improving hearing-related distress and psychological well-being in people with audiological problems. / Method: We searched online bibliographic databases and assessed study quality. We conducted random-effects meta-analyses if at least two randomized controlled trials (RCTs) examined hearing-related distress, depression, anxiety, or quality of life in people with audiological problems. Findings of pre–post studies were summarized narratively. / Results: We identified 15 studies: six RCTs and nine pre–post studies. The methodological quality of studies was mostly poor to moderate, and sample sizes were typically small (overall n = 750). Most studies focused on tinnitus (n = 12), MBIs (n = 8), and ACT (n = 6). Statistically significant improvements in hearing-related distress were found with ACT and MBIs versus controls and other treatments at post-intervention in people with tinnitus, while improvements in depression and anxiety were only found for ACT versus controls at post-intervention. However, gains were either not maintained or not examined at follow-up, and there was no evidence for improvements in quality of life. / Conclusions: At present, there is insufficient evidence to recommend the use of third-wave interventions for improving hearing-related distress or psychological well-being in people with audiological problems. There is some evidence that ACT and MBIs may be useful in addressing hearing-related distress in people with tinnitus, but only in the short term. However, findings should be interpreted with caution given the small number of studies with generally small sample sizes and mostly poor-to-moderate methodological quality. More high-quality, adequately powered, double-blind RCTs, particularly in audiological problems other than tinnitus, are needed to draw firm conclusions and meaningful clinical recommendations

Results from an interlaboratory comparison of areal surface texture parameter extraction from X-ray computed tomography of additively manufactured parts

This paper presents the results of the CT-STARR (CT-Surface Texture for Additive Round Robin) interlaboratory comparison. The study compares the results obtained for the extraction of areal surface texture data per ISO 25178-2 from five X-ray computed tomography (XCT) volume measurements from each of four laboratories. To reduce the number of process variables, all participants utilise a Nikon XCT machine, either an XT H 225 industrial CT or an MCT225 metrology CT. Measurement process parameters, such as physical X-ray filtering, acceleration voltage and filament current, are set at similar values for all machines. All data processing and computation to extract, align, crop, filter and generate surface texture parameter information and deviation analysis results from the measurement volumes is performed by one participant. Two Ti6Al4V ELI (extra low interstitial) components are included in each of the XCT acquisitions. The first component is an additively manufactured cube built on an Arcam Q10 electron beam melting machine. Surface texture data is extracted from XCT scans of this part. The second component is a machined artefact designed for XCT scaling and surface determination analysis and verification. The data extracted from XCT measurements of these components is compared with measurements from coordinate measuring machine, focus variation and stylus instruments. The effect of scaling correction and XCT surface determination on extracted surface texture data, as well as measurement repeatability and reproducibility, are discussed

After the storm, solar comes out : a new service model for children and adolescent mental health

Aim: Existing children and adolescent mental health services in the United Kingdom have many gaps, such as reduced access to community‐based services, and a lack of early intervention, prevention, and 24/7 crisis care. These gaps prevent timely access to appropriate levels of care, decrease children and young people's engagement with providers, and lead to increased pressures on urgent and emergency care. In this paper, we outline a newly created 0‐19 model and its crisis service, which have been transformed into a fully integrated, “joint partnership” service, in line with the recommendations from the recent UK policies that aim to meet the aforementioned challenges. Method: The “Solar” service is described as a case study of a 0‐19 service model. We cover the national and local contexts of the service, in addition to its rationale, aims, organizational structure, strengths and limitations. Results: The presented model is a fully integrated and innovative example of a service model that operates without tiers, and helps to create an inclusive, compassionate, stigma‐free and youth‐friendly environment. Additionally, the model aims to prioritize recovery, early intervention, prevention and the development of resilience. Conclusion: The 0‐19 model is a result of the recent transformation of children and youth mental health services in the United Kingdom. The ongoing evaluation of the 0‐19 model and its crisis component will investigate the model's effectiveness, accessibility and acceptability, as well as understanding the potential of the model to contribute towards solving numerous gaps in the existing mental health service provision within the United Kingdom

Understanding the presence of mental fatigue in English academy soccer players

Research has demonstrated that induced mental fatigue impairs soccer-specific technical, tactical and physical performance in soccer players. The findings are limited by the lack of elite players and low ecological validity of the tasks used to induce mental fatigue, which do not resemble the cognitive demands of soccer. The current study collected survey data from English academy soccer players (n = 256; age groups - U14 – U23), with questions comprising of five themes (descriptors of physical and mental fatigue, travel, education, match-play and fixture congestion). The survey consisted of multiple choice responses, checkboxes and blinded/unblinded (for duration based questions) 0-100 arbitrary unit (AU) slider scales. Listening to music (81.6% of players), using social media (58.3%) and watching videos (34.3%) were the most common pre-match activities. Pre-match subjective mental fatigue was low (18.7±18.8 AU), and most frequently reported at the end of a match (47±26 AU) and remained elevated 24-hours post-match (36±27 AU). Travel (29±24 AU), fixture congestion (44±25 AU) and education (30±26 AU) demonstrated a low to moderate presence of subjective mental fatigue. These findings provide an overview of activities performed by English academy soccer players pre-match, and demonstrate that mental fatigue is experienced as a result of match-play

An interlaboratory comparison of X-ray computed tomography measurement for texture and dimensional characterisation of additively manufactured parts

Abstract This paper presents the results of the CT-STARR (CT-Surface Texture for Additive Round Robin) Stage 1 interlaboratory comparison. The study compared the results obtained for the extraction of areal surface texture data per ISO 25178-2 from five X-ray computed tomography (CT) volume measurements from each of four laboratories. Two Ti6Al4V ELI (extra-low interstitial) components were included in each of the CT acquisitions. The first component was an additively manufactured (AM) cube manufactured using an Arcam Q10 electron beam melting (EBM) machine. Surface texture data was extracted from CT scans of this part. The values of selected parameters per ISO 25178-2 are reported, including Sa, the arithmetic mean height, for which the values from the Nikon MCT 225 metrology CT measurements were all within 0.5% of the mean reference focus variation measurement. CT resolution requirements are discussed. The second component was a machined dimensional test artefact designed to facilitate independent analysis of CT global voxel scaling errors and surface determination errors. The results of mathematical global scaling and surface determination correction of the dimensional artefact data is reported. The dimensional test artefact errors for the XT H 225 commercial CT for length, outside diameter and inside diameter reduced from -0.27%, -0.83% and -0.54% respectively to less than 0.02% after performing mathematical correction. This work will assist the development of surface texture correction protocols, help define surface-from-CT measurement envelope limits and provide valuable information for an expanded Stage 2 interlaboratory comparison, which will include a more diverse range of CT systems and technologies, further expanding the surface-from-CT knowledge base

Fast and Facile Synthesis Route to Epitaxial Oxide Membrane Using a Sacrificial Layer

The advancement in thin-film exfoliation for synthesizing oxide membranes has opened up new possibilities for creating artificially-assembled heterostructures with structurally and chemically incompatible materials. The sacrificial layer method is a promising approach to exfoliate as-grown films from a compatible material system, allowing their integration with dissimilar materials. Nonetheless, the conventional sacrificial layers often possess intricate stoichiometry, thereby constraining their practicality and adaptability, particularly when considering techniques like Molecular Beam Epitaxy (MBE). This is where easy-to-grow binary alkaline earth metal oxides with a rock salt crystal structure are useful. These oxides, which include (Mg, Ca, Sr, Ba)O, can be used as a sacrificial layer covering a much broader range of lattice parameters compared to conventional sacrificial layers and are easily dissolvable in deionized water. In this study, we show the epitaxial growth of single-crystalline perovskite SrTiO3 (STO) on sacrificial layers consisting of crystalline SrO, BaO, and Ba1-xCaxO films, employing a hybrid MBE method. Our results highlight the rapid (< 5 minutes) dissolution of the sacrificial layer when immersed in deionized water, facilitating the fabrication of millimeter-sized STO membranes. Using high-resolution x-ray diffraction, atomic-force microscopy, scanning transmission electron microscopy, impedance spectroscopy, and scattering-type near-field optical microscopy (SNOM), we demonstrate epitaxial STO membranes with bulk-like intrinsic dielectric properties. The employment of alkaline earth metal oxides as sacrificial layers is likely to simplify membrane synthesis, particularly with MBE, thus expanding research possibilities.Comment: 36 pages, 4 figure

Bespoke contrast-matched diblock copolymer nanoparticles enable the rational design of highly transparent Pickering double emulsions

We report the preparation of highly transparent oil-in-water Pickering emulsions using contrast-matched organic nanoparticles. This is achieved via addition of judicious amounts of either sucrose or glycerol to an aqueous dispersion of poly(glycerol monomethacrylate)56–poly(2,2,2-trifluoroethyl methacrylate)500 [PGMA–PTFEMA] diblock copolymer nanoparticles prior to high shear homogenization with an equal volume of n-dodecane. The resulting Pickering emulsions comprise polydisperse n-dodecane droplets of 20–100 μm diameter and exhibit up to 96% transmittance across the visible spectrum. In contrast, control experiments using non-contrast-matched poly(glycerol monomethacrylate)56–poly(benzyl methacrylate)300 [PGMA56–PBzMA300] diblock copolymer nanoparticles as a Pickering emulsifier only produced conventional highly turbid emulsions. Thus contrast-matching of the two immiscible phases is a necessary but not sufficient condition for the preparation of highly transparent Pickering emulsions: it is essential to use isorefractive nanoparticles in order to minimize light scattering. Furthermore, highly transparent oil-in-water-in-oil Pickering double emulsions can be obtained by homogenizing the contrast-matched oil-in-water Pickering emulsion prepared using the PGMA56–PTFEMA500 nanoparticles with a contrast-matched dispersion of hydrophobic poly(lauryl methacrylate)39–poly(2,2,2-trifluoroethyl methacrylate)800 [PLMA39–PTFEMA800] diblock copolymer nanoparticles in n-dodecane. Finally, we show that an isorefractive oil-in-water Pickering emulsion enables fluorescence spectroscopy to be used to monitor the transport of water-insoluble small molecules (pyrene and benzophenone) between n-dodecane droplets. Such transport is significantly less efficient than that observed for the equivalent isorefractive surfactant-stabilized emulsion. Conventional turbid emulsions do not enable such a comparison to be made because the intense light scattering leads to substantial spectral attenuation