Mechanism of resonant electron emission from the deprotonated GFP chromophore and its biomimetics

The Green Fluorescent Protein (GFP), which is widely used in bioimaging, is known to undergo light-induced redox transformations. Electron transfer is thought to occur resonantly through excited states of its chromophore; however, a detailed understanding of the electron gateway states of the chromophore is still missing. Here, we use photoelectron spectroscopy and high-level quantum chemistry calculations to show that following UV excitation, the ultrafast electron dynamics in the chromophore anion proceeds via an excited shape resonance strongly coupled to the open continuum. The impact of this state is found across the entire 355–315 nm excitation range, from above the first bound–bound transition to below the opening of higher-lying continua. By disentangling the electron dynamics in the photodetachment channels, we provide an important reference for the adiabatic position of the electron gateway state, which is located at 348 nm, and discover the source of the curiously large widths of the photoelectron spectra that have been reported in the literature. By introducing chemical modifications to the GFP chromophore, we show that the detachment threshold and the position of the gateway state, and hence the underlying excited-state dynamics, can be changed systematically. This enables a fine tuning of the intrinsic electron emission properties of the GFP chromophore and has significant implications for its function, suggesting that the biomimetic GFP chromophores are more stable to photooxidation

A conceptual framework and practical guide for assessing fitness-to-operate in the offshore oil and gas industry

The paper outlines a systemic approach to understanding and assessing safety capability in the offshore oil and gas industry. We present a conceptual framework and assessment guide for understanding fitness-to-operate (FTO) that builds a more comprehensive picture of safety capability for regulators and operators of offshore facilities. The FTO framework defines three enabling capitals that create safety capability: organizational capital, social capital, and human capital. For each type of capital we identify more specific dimensions based on current theories of safety, management, and organizational processes. The assessment guide matches specific characteristics to each element of the framework to support assessment of safety capability. The content and scope of the FTO framework enable a more comprehensive coverage of factors that influence short-term and long-term safety outcomes

ortho and para chromophores of green fluorescent protein: controlling electron emission and internal conversion

Green fluorescent protein (GFP) continues to play an important role in the biological and biochemical sciences as an efficient fluorescent probe and is also known to undergo light-induced redox transformations. Here, we employ photoelectron spectroscopy and quantum chemistry calculations to investigate how the phenoxide moiety controls the competition between electron emission and internal conversion in the isolated GFP chromophore anion, following photoexcitation with ultraviolet light in the range 400–230 nm. We find that moving the phenoxide group from the para position to the ortho position enhances internal conversion back to the ground electronic state but that adding an additional OH group to the para chromophore, at the ortho position, impedes internal conversion. Guided by quantum chemistry calculations, we interpret these observations in terms of torsions around the C–C–C bridge being enhanced by electrostatic repulsions or impeded by the formation of a hydrogen-bonded seven-membered ring. We also find that moving the phenoxide group from the para position to the ortho position reduces the energy required for detachment processes, whereas adding an additional OH group to the para chromophore at the ortho position increases the energy required for detachment processes. These results have potential applications in tuning light-induced redox processes of this biologically and technologically important fluorescent protein

Photoelectron spectroscopy of isolated luciferin and infraluciferin anions in vacuo: competing photodetachment, photofragmentation and internal conversion

The electronic structure and excited-state dynamics of the ubiquitous bioluminescent probe luciferin and its furthest red-shifted analogue infraluciferin have been investigated using photoelectron spectroscopy and quantum chemistry calculations. In our electrospray ionization source, the deprotonated anions are formed predominantly in their phenolate forms and are directly relevant to studies of luciferin and infraluciferin as models for their unstable oxyluciferin and oxyinfraluciferin emitters. Following photoexcitation in the range 357–230 nm, we find that internal conversion from high-lying excited states to the S1(1ππ*) state competes efficiently with electron detachment. In infraluciferin, we find that decarboxylation also competes with direct electron detachment and internal conversion. This detailed spectroscopic and computational study defines the electronic structure and electronic relaxation processes of luciferin and infraluciferin and will inform the design of new bioluminescent systems and applications

Deterministic control of magnetic vortex wall chirality by electric field

Concepts for information storage and logical processing based on magnetic domain walls have great potential for implementation in future information and communications technologies. To date, the need to apply power hungry magnetic fields or heat dissipating spin polarized currents to manipulate magnetic domain walls has limited the development of such technologies. The possibility of controlling magnetic domain walls using voltages offers an energy efficient route to overcome these limitations. Here we show that a voltage-induced uniaxial strain induces reversible deterministic switching of the chirality of a magnetic vortex wall. We discuss how this functionality will be applicable to schemes for information storage and logical processing, making a significant step towards the practical implementation of magnetic domain walls in energy efficient computing

COVID-19 vaccine-induced antibody and T cell responses in immunosuppressed patients with inflammatory bowel disease after the third vaccine dose

Background: COVID-19 vaccine-induced antibody responses are reduced in patients with inflammatory bowel disease (IBD) taking infliximab or tofacitinib after two vaccine doses. We sought to determine whether immunosuppressive treatments were associated with reduced antibody and T cell responses after a third vaccine dose. Methods: 352 adults (72 healthy controls and 280 IBD) from the prospectively recruited study cohort were sampled 28-49 days after a third dose of SARS-CoV-2 vaccine. IBD medications studied included thiopurines (n=65), infliximab (n=46), thiopurine/infliximab combination therapy (n=49), ustekinumab (n=44), vedolizumab (n=50) or tofacitinib (n=26). SARS-CoV-2 spike antibody binding and T cell responses were measured. Findings: Geometric mean [geometric SD] anti-S1 RBD antibody concentrations increased in all study groups following a third dose of vaccine, but were significantly lower in patients treated with infliximab (2736.8 U/mL [4.3]; P<0.0001), infliximab and thiopurine combination (1818.3 U/mL [6.7]; P<0.0001) and tofacitinib (8071.5 U/mL [3.1]; P=0.0018) compared to controls (16774.2 U/ml [2.6]). There were no significant differences in anti-S1 RBD antibody concentrations between control subjects and thiopurine (12019.7 U/mL [2.2]; P=0.099), ustekinumab (11089.3 U/mL [2.8]; P=0.060), nor vedolizumab treated patients (13564.9 U/mL [2.4]; P=0.27). In multivariable modelling, lower anti-S1 RBD antibody concentrations were independently associated with infliximab (Geometric mean ratio 0.15, 95% CI 0.11-0.21, P<0.0001), tofacitinib (0.52, 95% CI 0.31-0.87, P=0.012) and thiopurine (0.69, 95% CI 0.51-0.95, P=0.021), but not with ustekinumab (0.64, 95% CI 0.39-1.06, P=0.083), or vedolizumab (0.84, 95% CI 0.54-1.30, P=0.43). Previous SARS-CoV-2 infection (1.58, 95% CI 1.22-2.05, P=0.00056) and older age (0.88, 95% CI 0.80-0.97, P=0.0073) were independently associated with higher and lower anti-S1 antibody concentrations respectively. However, antigen specific T cell responses were similar in IBD patients in all treatment groups studied, except for recipients of tofacitinib without evidence of previous infection, where T cell responses were significantly reduced relative to healthy controls (p=0.021). Interpretation: A third dose of COVID-19 vaccine induced a boost in antibody binding in immunosuppressed patients with IBD, but these responses were reduced in patients taking infliximab, infliximab/thiopurine combination and tofacitinib therapy. Tofacitinib was also associated with reduced T cell responses. These findings support continued prioritisation of immunosuppressed groups for further booster dosing, particularly those on Janus Kinase (JAK) inhibitors who have attenuation of both serological and cell-mediated vaccine-induced immunity. Funding: Financial support was provided as a Research Grant by Pfizer Ltd

Undergraduate medical research: the student perspective

Background: Research training is essential in a modern undergraduate medical curriculum. Our evaluation aimed to (a) gauge students&#x2019; awareness of research activities, (b) compare students&#x2019; perceptions of their transferable and research-specific skills competencies, (c) determine students&#x2019; motivation for research and (d) obtain students&#x2019; personal views on doing research. Methods: Undergraduate medical students (N=317) completed a research skills questionnaire developed by the Centre for Excellence in Teaching and Learning in Applied Undergraduate Research Skills (CETL-AURS) at Reading University. The questionnaire assessed students&#x2019; transferable skills, research-specific skills (e.g., study design, data collection and data analysis), research experience and attitude and motivation towards doing research. Results: The majority of students are motivated to pursue research. Graduate entrants and male students appear to be the most confident regarding their research skills competencies. Although all students recognise the role of research in medical practice, many are unaware of the medical research activities or successes within their university. Of those who report no interest in a career incorporating research, a common perception was that researchers are isolated from patients and clinical practice. Discussion: Students have a narrow definition of research and what it entails. An explanation for why research competence does not align more closely with research motivation is derived from students&#x2019; lack of understanding of the concept of translational research, as well as a lack of awareness of the research activity being undertaken by their teachers and mentors. We plan to address this with specific research awareness initiatives

Violence against primary school children with disabilities in Uganda: a cross-sectional study.

BACKGROUND: 150 million children live with disabilities globally, and a recent systematic review found 3 to 4 times the levels of violence versus non-disabled children in high income countries. However, almost nothing is known about violence against disabled children in lower income countries. We aim to explore the prevalence, patterns and risk factors for physical, sexual and emotional violence among disabled children attending primary school in Luwero District, Uganda. METHODS: We performed a secondary analysis of data from the baseline survey of the Good Schools Study. 3706 children and young adolescents aged 11-14 were randomly sampled from 42 primary schools. Descriptive statistics were computed and logistic regression models fitted. RESULTS: 8.8% of boys and 7.6% of girls reported a disability. Levels of violence against both disabled and non-disabled children were extremely high. Disabled girls report slightly more physical (99.1% vs 94.6%, p = 0.010) and considerably more sexual violence (23.6% vs 12.3%, p = 0.002) than non-disabled girls; for disabled and non-disabled boys, levels are not statistically different. The school environment is one of the main venues at which violence is occurring, but patterns differ by sex. Risk factors for violence are similar between disabled and non-disabled students. CONCLUSIONS: In Uganda, disabled girls are at particular risk of violence, notably sexual violence. Schools may be a promising venue for intervention delivery. Further research on the epidemiology and prevention of violence against disabled and non-disabled children in low income countries is urgently needed

Microarray identifies ADAM family members as key responders to TGF-β1 in alveolar epithelial cells

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-β1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-β1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-β1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-β1, suggesting a potential role for these molecules in ECM accumulation in IPF

Cognitive behaviour therapy (CBT) for anxiety and depression in adults with mild intellectual disabilities (ID): a pilot randomised controlled trial

Background: Several studies have showed that people with intellectual disabilities (ID) have suitable skills to undergo cognitive behavioural therapy (CBT). Case studies have reported successful use of cognitive behavioural therapy techniques (with adaptations) in people with ID. Modified cognitive behavioural therapy may be a feasible and effective approach for the treatment of depression, anxiety, and other mood disorders in ID. To date, two studies have reported group-based manaulised cognitive behavioural treatment programs for depression in people with mild ID. However, there is no individual manualised programme for anxiety or depression in people with intellectual disabilities. The aims of the study are to determine the feasibility of conducting a randomised controlled trial for CBT in people with ID. The data will inform the power calculation and other aspects of carrying out a definitive randomised controlled trial.Methods: Thirty participants with mild ID will be allocated randomly to either CBT or treatment as usual (TAU). The CBT group will receive up to 20 hourly individual CBT over a period of 4 months. TAU is the standard treatment which is available to any adult with an intellectual disability who is referred to the intellectual disability service (including care management, community support, medical, nursing or social support). Beck Youth Inventories (Beck Anxiety Inventory & Beck Depression Inventory) will be administered at baseline; end of treatment (4 months) and at six months to evaluate the changes in depression and anxiety. Client satisfaction, quality of life and the health economics will be secondary outcomes.Discussion: The broad outcome of the study will be to produce clear guidance for therapists to apply an established psychological intervention and identify how and whether it works with people with intellectual disabilities