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A new GIS-based screening tool to assess threats to shallow groundwater quality has been trialled in Glasgow, UK. The GRoundwater And Soil Pollutants (GRASP) tool is based on a British Standard method for assessing the threat from potential leaching of metal pollutants in unsaturated soil/superficial materials to shallow groundwater, using data on soil and Quaternary deposit properties, climate and depth to groundwater. GRASP breaks new ground by also incorporating a new Glasgow-wide soil chemistry dataset. GRASP considers eight metals, including chromium, lead and nickel at 1622 soil sample locations. The final output is a map to aid urban management, which highlights areas where shallow groundwater quality may be at risk from current and future surface pollutants. The tool indicated that 13% of soil sample sites in Glasgow present a very high potential threat to groundwater quality, due largely to shallow groundwater depths and high soil metal concentrations. Initial attempts to validate GRASP revealed partial spatial coincidence between the GRASP threat ranks (low, moderate, high and very high) and groundwater chemistry, with statistical correlation between areas of high soil and groundwater metal concentrations for both Cr and Cu (r 2>0.152; P <0.05). Validation was hampered by a lack of, and inconsistency in, existing groundwater chemistry data. To address this, standardised subsurface data collection networks have been trialled recently in Glasgow. It is recommended that, once available, new groundwater depth and chemistry information from these networks is used to validate the GRASP model further. [ABSTRACT FROM AUTHOR]

The 'othering' of ageing is linked to an integrated process of ageism and hinders planning for the future for both individuals and practitioners delivering housing and health services. This paper aims to explore how creative interventions can help personalise, exchange knowledge and lead to system changes that tackle the 'othering' of ageing. The Designing Homes for Healthy Cognitive Ageing (DesHCA) project offers new and creative insights through an innovative methodology utilising 'serious games' with a co-produced tool called 'Our House' that provides insights into how to deliver housing for older people for ageing well in place. In a series of playtests with over 128 people throughout the UK, the findings show that serious games allow interaction, integration and understanding of how ageing affects people professionally and personally. The empirical evidence highlights that the game mechanisms allowed for a more in-depth and nuanced consideration of ageing in a safe and creative environment. These interactions and discussions enable individuals to personalise and project insights to combat the 'othering' of ageing. However, the solutions are restrained as overcoming the consequences of ageism is a societal challenge with multilayered solutions. The paper concludes that serious gaming encourages people to think differently about the concept of healthy ageing-both physically and cognitively-with the consideration of scalable and creative solutions to prepare for ageing in place.

Background and Objectives: The term "intergenerational relationships" is widely used in gerontological literature and age-related policies. However, discussions of the term often tell us surprisingly little about what it means or why it matters. We suggest that this is due to a reductivism and instrumentalism in 2 main discourses within which intergenerational relationships are usually discussed. First, intergenerational relationships are often conceptualized through a binary "conflict/solidarity" lens, reinforcing an entrenched "generationalism". Second, they are predominantly constructed as a problem to be addressed within debates on how to tackle intergenerational segregation. Neither of these discourses provides much room for a more nuanced understanding of how intergenerational relationships are experienced or why they are meaningful. In this paper, we discuss how fictional narratives can introduce imagination and a richer vocabulary into discourses concerning how people of different ages relate to each other., Research Design and Methods: We present findings from reading groups where adults discussed novels depicting themes of older age, intergenerational relationships, and time., Results: In discussing the fictional narratives and characters, participants reflected on the significance and meaning of intergenerational relationships in ways that went beyond dichotomous and instrumentalist discourses. Drawing on the concept of lived ambivalence, we argue that fictional representations of intergenerational themes can elicit more meaningful reflections on the complexities and contradictions of relationships across age groups., Discussion and Implications: We conclude that a more nuanced understanding of intergenerational interaction can inform gerontological discourses and policy, but also that gerontological awareness of social challenges concerning age relations can inform interpretations of fictional narratives., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Amyloid plaques composed of Aβ fibrils are a hallmark of Alzheimer's disease (AD). However, the molecular architecture of amyloid plaques in the context of fresh mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the in situ molecular architecture of Aβ fibrils in the App NL-G-F familial AD mouse model containing the Arctic mutation and an atomic model of ex vivo purified Arctic Aβ fibrils. We show that in-tissue Aβ fibrils are arranged in a lattice or parallel bundles, and are interdigitated by subcellular compartments, extracellular vesicles, extracellular droplets and extracellular multilamellar bodies. The Arctic Aβ fibril differs significantly from an earlier App NL-F fibril structure, indicating a striking effect of the Arctic mutation. These structural data also revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises β-amyloid plaque pathology., (© 2023. The Author(s).)

Resolving atomic structures of isolated proteins has uncovered mechanisms and fundamental processes in biology. However, many functions can only be tested in the context of intact cells and tissues that are many orders of magnitude larger than the macromolecules on which they depend. Therefore, methods that interrogate macromolecular structure in situ provide a means of directly relating structure to function across length scales. Here, we developed several workflows using cryogenic correlated light and electron microscopy (cryoCLEM) and electron tomography (cryoET) that can bridge this gap to reveal the molecular infrastructure that underlies higher order functions within cells and tissues. We also describe experimental design considerations, including cryoCLEM labelling, sample preparation, and quality control, for determining the in situ molecular architectures within native, hydrated cells and tissues.

Gene therapy constitutes one of the most promising mode of disease treatments. Two key properties for therapeutic delivery vectors are its transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into unintended cells within the body). Here we developed an integrated bioinformatics and experimental pipeline that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. We further demonstrate that efficient AAV transduction observed in organoids is recapitulated in vivo in non-human primates. This library-on-library technology will be important for determining the safety and efficacy of therapeutic delivery vectors., (© 2022. The Author(s).)

The corneal endothelium located on the posterior corneal surface is responsible for regulating stromal hydration. This is contributed by a monolayer of corneal endothelial cells (CECs), which are metabolically active in a continuous fluid-coupled efflux of ions from the corneal stroma into the aqueous humor, preventing stromal over-hydration and preserving the orderly arrangement of stromal collagen fibrils, which is essential for corneal transparency. Mature CECs do not have regenerative capacity and cell loss due to aging and diseases results in irreversible stromal edema and a loss of corneal clarity. The current gold standard of treatment for this worldwide blindness caused by corneal endothelial failure is the corneal transplantation using cadaveric donor corneas. The top indication is Fuchs corneal endothelial dystrophy/degeneration, which represents 39% of all corneal transplants performed. However, the global shortage of transplantable donor corneas has restricted the treatment outcomes, hence instigating a need to research for alternative therapies. One such avenue is the CEC regeneration from endothelial progenitors, which have been identified in the peripheral endothelium and the adjacent transition zone. This review examines the evidence supporting the existence of endothelial progenitors in the posterior limbus and summarizes the existing knowledge on the microanatomy of the transitional zone. We give an overview of the isolation and ex vivo propagation of human endothelial progenitors in the transition zone, and their growth and differentiation capacity to the corneal endothelium. Transplanting these bioengineered constructs into in vivo models of corneal endothelial degeneration will prove the efficacy and viability, and the long-term maintenance of functional endothelium. This will develop a novel regenerative therapy for the management of corneal endothelial diseases.

Nuclear factor, erythroid 2 like 2 (Nrf2), is an oxidative stress induced transcription factor that regulates cytoprotective gene expression. Thus, Nrf2 is essential for cellular redox homeostasis. Loss or dysregulation of Nrf2 expression has been implicated in the pathogenesis of degenerative diseases, including diseases of the cornea. One of the most common diseases of the cornea in which Nrf2 is implicated is Fuchs' endothelial cornea dystrophy (FECD). FECD is the leading indication for corneal transplantation; and is associated with a loss of corneal endothelial cell (CEC) function. In this review, we propose that Nrf2 is an essential regulator of CEC function. Furthermore, we demonstrate that deficiency of Nrf2 function is a hallmark of FECD. In addition, we advocate that pharmacological targeting of Nrf2 as a possible therapy for FECD., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

As the cornea is one of the most transplanted tissues in the body it has placed a burden on the provision of corneas from cadaveric donors. Corneal endothelial dysfunction is the leading indication for cornea transplant. Therefore, tissue engineering is emerging as an alternative approach to overcome the global shortage of transplant-grade corneas. The propagation and expansion of corneal endothelial cells has been widely reported. However, one obstacle to overcome is the transport and storage of corneal endothelial cells. In this study we investigated whether tissue engineered corneal endothelial cells can be preserved in hypothermic conditions. Human corneal endothelial cells (HCEnCs) were exposed to various temperatures (4 °C, 23 °C, and 37 °C) in both adherent and suspension storage models. Optimal storage media and storage duration was tested along with post-storage viability. Following storage and subsequent recovery at 37 °C, cell phenotype was assessed by immunofluorescence, gene and protein expression, and proliferative capacity analysis. Functionality was also assessed within a rabbit model of bullous keratopathy. Our data support our hypothesis that functional HCEnCs can be preserved in hypothermic conditions.

Corneal transparency is maintained by a monolayer of corneal endothelial cells. Defects in corneal endothelial cells (CEnCs) can be rectified surgically through transplantation. Fuchs' endothelial corneal dystrophy (FECD) is the foremost cause of endothelial dysfunction and the leading indication for transplantation. Increased sensitivity of CEnCs to oxidative stress is thought to contribute to the pathogenesis of FECD through increased apoptosis. In part, this is thought to be due to loss of NRF2 expression: a global regulator of oxidative stress. We demonstrate that expression of the redox sensor, peroxiredoxin 1 (PRDX1) is selectively lost from CEnCs in FECD patient samples. We reveal that expression of PRDX1 is necessary to control the response of CEnCs to agents that cause lipid peroxidation. Iron-dependent lipid peroxidation drives non-apoptotic cell death termed ferroptosis. We establish that the inhibitor of ferroptosis, ferrostatin-1 rescues lipid peroxidation and cell death in CEnCs. Furthermore, we provide evidence that the transcription factor NRF2 similarly regulates lipid peroxidation in CEnCs., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The inner layer of the cornea, the corneal endothelium, is post-mitotic and unable to regenerate if damaged. The corneal endothelium is one of the most transplanted tissues in the body. Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal endothelial transplantation. FECD is thought to be an age-dependent disorder, with a major component related to oxidative stress. Prdx6 is an antioxidant with particular affinity for repairing peroxidised cell membranes. To address the role of Prdx6 in corneal endothelial cells, we used a combination of biochemical and functional studies. Our data reveal that Prdx6 is expressed at unusually high levels at the plasma membrane of corneal endothelial cells. RNAi-mediated knockdown of Prdx6 revealed a role for Prdx6 in lipid peroxidation. Furthermore, following induction of oxidative stress with menadione, Prdx6-deficient cells had defective mitochondrial membrane potential and were more sensitive to cell death. These data reveal that Prdx6 is compartmentalised in corneal endothelial cells and has multiple functions to preserve cellular integrity.

Alzheimer's disease (AD), an age-related neurodegenerative condition, is the most common cause of dementia among the elder people, but currently there is no treatment. A number of putative pathogenic events, particularly amyloid β peptide (Aβ) accumulation, are believed to be early triggers that initiate AD. However, thus far targeting Aβ generation/aggregation as the mainstay strategy of drug development has not led to effective AD-modifying therapeutics. Oxidative damage is a conspicuous feature of AD, but this remains poorly defined phenomenon and mechanistically ill understood. The TRPM2 channel has emerged as a potentially ubiquitous molecular mechanism mediating oxidative damage and thus plays a vital role in the pathogenesis and progression of diverse neurodegenerative diseases. This article will review the emerging evidence from recent studies and propose a novel 'hypothesis' that multiple TRPM2-mediated cellular and molecular mechanisms cascade Aβ and/or oxidative damage to AD pathologies. The 'hypothesis' based on these new findings discusses the prospect of considering the TRPM2 channel as a novel therapeutic target for intervening AD and age-related dementia., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Peroxiredoxin 6 (Prdx6) is the only mammalian 1-Cys member of the Prdx family, a group of enzymes which share the ability to reduce peroxides. In addition to its peroxidase function, Prdx6 also demonstrates phospholipase A 2 and lysophosphatidylcholine acyl transferase (LPCAT) activities. These enzymatic activities play an important role in regenerating oxidized membrane phospholipids and maintaining an appropriate balance of intracellular reactive oxygen species. Development of clinical pathologies, including those within the eye, have been linked to dysregulation of Prdx6 function. Interplay between external stressors like exposure to UV light, transforming growth factor β (TGF-β), and hyperglycemia in conjunction with diminished Prdx6 levels and loss of redox balance is associated with cellular changes in a variety of ophthalmic pathologies including cataracts, glaucoma, and retinal degeneration. Many of these cellular abnormalities can be rescued through supplementation with exogenous Prdx6. Additionally, corneal endothelial cells have been found to express high levels of Prdx6 in the plasma membrane. These findings highlight the importance of Prdx6 as an essential regulator of oxidative stress in the eye., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Residential homes encourage new residents to bring belongings with them, so that they can personalise their room and 'feel at home'. Existing literature on material culture in residential homes views objects as symbols and repositories of home and identity, which can facilitate a sense of belonging in residents through their display in residents' rooms. I suggest that this both misunderstands the processual and fluid nature of home and identity, and conceptualises objects as essentially passive. This article uses ethnographic data and theories of practice and relationality to argue that rather than the meaning of home being inherent in objects, or felt subjectively by residents, meaning is generated through ongoing, everyday interactions between the two. I show that residents became at home by acquiring new things -as well as displaying existing possessions - and also through interacting with mundane objects in everyday social and relational practices such as cleaning and hosting. I conclude that being at home in older people's residential homes need not be so different from being at home at other stages of the life course and in other settings. This challenges conceptualisations of older people's homes - and older age itself - as somehow unknowable and unfamiliar., (© 2018 Foundation for the Sociology of Health & Illness.)

Corneal tissue is the most transplanted of all body tissues. Currently, cadaveric donor tissues are used for transplantation. However, a global shortage of transplant grade material has prompted development of alternative, cell-based therapies for corneal diseases. Pluripotent stem cells are attractive sources of cells for regenerative medicine, because large numbers of therapeutically useful cells can be generated. However, a detailed understanding of how to differentiate clinically relevant cell types from stem cells is fundamentally required. Periocular mesenchyme (POM), a subtype of cranial neural crest, is vital for development of multiple cell types in the cornea, including clinically relevant cells such as corneal endothelium and stromal keratocytes. Herein, we describe protocols for differentiation of POM from pluripotent stem cells. Using defined media containing inhibitors of TGFβ and WNT signalling, we generated neural crest cells that express high levels of the POM transcription factors PITX2 and FOXC1. Furthermore, we identified cells resembling POM in the adult cornea, located in a niche between the trabecular meshwork and peripheral endothelium. The generation and expansion of POM is an important step in the generation of a number of cells types that could prove to be clinically useful for a number of diseases of the cornea., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Background: Online health forums provide peer support for a range of medical conditions including life-threatening and terminal illnesses. Trust is an important component of peer-to-peer support, although relatively little is known about how trust forms within online health forums., Objective: The aim of this paper is to examine how trust develops and influences sharing among users of an online breast cancer forum., Methods: An interpretive qualitative approach was adopted. Data were collected from forum posts from 135 threads on 9 boards on the UK charity, Breast Cancer Care (BCC). Semistructured interviews were conducted with 14 BCC forum users. Both datasets were analyzed thematically using Braun and Clarke's approach and combined to triangulate analysis., Results: Trust operates in 3 dimensions, structural, relational, and temporal, and these intersect with each other and do not operate in isolation. The structural dimension relates to how the affordances and formal rules of the site affected trust. The relational dimension refers to how trust was necessarily experienced in interactions with other forum users: it emerged within relationships and was a social phenomenon. The temporal dimension relates to how trust changed over time and was influenced by the length of time users spent on the forum., Conclusions: Trust is a process that changes over time and which is influenced by structural features of the forum, as well as informal but collectively understood relational interactions among forum users. The study provides a better understanding of how the intersecting structural, relational, and temporal aspects that support the development of trust facilitate sharing in online environments. These findings will help organizations developing online health forums., (©Melanie Lovatt, Peter A Bath, Julie Ellis. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 23.05.2017.)

The harmful effects of heavy drinking on health have been widely reported, yet public opinion on governmental responsibility for alcohol control remains divided. This study examines UK public attitudes towards alcohol policies, identifies underlying dimensions that inform these, and relationships with perceived effectiveness. A cross-sectional mixed methods study involving a telephone survey of 3477 adult drinkers aged 16-65 and sixteen focus groups with 89 adult drinkers in Scotland and England was conducted between September 2012 and February 2013. Principal components analysis (PCA) was used to reduce twelve policy statements into underlying dimensions. These dimensions were used in linear regression models examining alcohol policy support by demographics, drinking behaviour and perceptions of UK drinking and government responsibility. Findings were supplemented with a thematic analysis of focus group transcripts. A majority of survey respondents supported all alcohol policies, although the level of support varied by type of policy. Greater enforcement of laws on under-age sales and more police patrolling the streets were strongly supported while support for pricing policies and restricting access to alcohol was more divided. PCA identified four main dimensions underlying support on policies: alcohol availability, provision of health information and treatment services, alcohol pricing, and greater law enforcement. Being female, older, a moderate drinker, and holding a belief that government should do more to reduce alcohol harms were associated with higher support on all policy dimensions. Focus group data revealed findings from the survey may have presented an overly positive level of support on all policies due to differences in perceived policy effectiveness. Perceived effectiveness can help inform underlying patterns of policy support and should be considered in conjunction with standard measures of support in future research on alcohol control policies., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Public knowledge of the association between alcohol and cancer is reported to be low. We aimed to provide up-to-date evidence for England regarding awareness of the link between alcohol and different cancers and to determine whether awareness differs by demographic characteristics, alcohol use, and geographic region., Methods: A representative sample of 2100 adults completed an online survey in July 2015. Respondents were asked to identify which health outcomes, including specific cancers, may be caused by alcohol consumption. Logistic regressions explored whether demographic, alcohol use, and geographic characteristics predicted correctly identifying alcohol-related cancer risk., Results: Unprompted, 12.9% of respondents identified cancer as a potential health outcome of alcohol consumption. This rose to 47% when prompted (compared to 95% for liver disease and 73% for heart disease). Knowledge of the link between alcohol and specific cancers varied between 18% (breast) and 80% (liver). Respondents identified the following cancers as alcohol-related where no such evidence exists: bladder (54%), brain (32%), ovarian (17%). Significant predictors of awareness of the link between alcohol and cancer were being female, more highly educated, and living in North-East England., Conclusion: There is generally low awareness of the relationship between alcohol consumption and cancer, particularly breast cancer. Greater awareness of the relationship between alcohol and breast cancer in North-East England, where a mass media campaign highlighted this relationship, suggests that population awareness can be influenced by social marketing.

Background and Aims: The concept of national drinking culture is well established in research and policy debate, but rarely features in contemporary alcohol policy analysis. We aim to demonstrate the value of the alternative concept of social practices for quantitatively operationalizing drinking culture. We discuss how a practice perspective addresses limitations in existing analytical approaches to health-related behaviour before demonstrating its empirical application by constructing a statistical typology of British drinking occasions., Design: Cross-sectional latent class analysis of drinking occasions derived from retrospective 1-week drinking diaries obtained from quota samples of a market research panel. Occasions are periods of drinking with no more than 2 hours between drinks., Setting: Great Britain, 2009-11., Cases: A total of 187 878 occasions nested within 60 215 nationally representative adults (aged 18 + years)., Measurements: Beverage type and quantity per occasion; location, company and gender composition of company; motivation and reason for occasion; day, start-time and duration of occasion; and age, sex and social grade., Findings: Eight occasion types are derived based primarily on parsimony considerations rather than model fit statistics. These are mixed location heavy drinking (10.4% of occasions), heavy drinking at home with a partner (9.4%), going out with friends (11.1%), get-together at someone's house (14.4%), going out for a meal (8.6%), drinking at home alone (13.6%), light drinking at home with family (12.8%) and light drinking at home with a partner (19.6%)., Conclusions: An empirical model of drinking culture, comprising a typology of drinking practices, reveals the dominance of moderate drinking practices in Great Britain. The model demonstrates the potential for a practice perspective to be used in evaluation of how and why drinking cultures change in response to public health interventions., (© 2016 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Aims: To explore how the concept of lay epidemiology can enhance understandings of how drinkers make sense of current UK drinking guidelines., Methods: Qualitative study using 12 focus groups in four sites in northern England and four sites in central Scotland. Participants were 66 male and female drinkers, aged between 19 and 65 years, of different socio-economic backgrounds. Data were analysed thematically using a conceptual framework of lay epidemiology., Results: Current drinking guidelines were perceived as having little relevance to participants' drinking behaviours and were generally disregarded. Daily guidelines were seen as irrelevant by drinkers whose drinking patterns comprised heavy weekend drinking. The amounts given in the guidelines were seen as unrealistic for those motivated to drink for intoxication, and participants measured alcohol intake in numbers of drinks or containers rather than units. Participants reported moderating their drinking, but this was out of a desire to fulfil work and family responsibilities, rather than concerns for their own health. The current Australian and Canadian guidelines were preferred to UK guidelines, as they were seen to address many of the above problems., Conclusions: Drinking guidelines derived from, and framed within, solely epidemiological paradigms lack relevance for adult drinkers who monitor and moderate their alcohol intake according to their own knowledge and risk perceptions derived primarily from experience. Insights from lay epidemiology into how drinkers regulate and monitor their drinking should be used in the construction of drinking guidelines to enhance their credibility and efficacy., (© 2015 The Authors. Addiction published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Background: While previous research has suggested that health care assistants supporting palliative care work in the community regard the provision of emotional labour as a key aspect of their role, little research has explored the experiences of family carers who are the recipients of such support., Objective: To explore the emotional labour undertaken by health care assistants working in community palliative care from the perspectives of both health care assistants and bereaved family carers., Design: We conducted a qualitative interview study in 2011-2012 with bereaved family carers of cancer patients who had received the services of health care assistants in the community, and health care assistants who provided community palliative care services. Transcripts were coded and analysed for emergent themes using a constant comparative technique., Settings: Three different research sites in the United Kingdom, all providing community palliative care., Participants and Methods: Semi-structured interviews were conducted with 33 bereaved family carers and eight health care assistants., Results: Health care assistants view one of their key roles as providing emotional support to patients and their family carers, and family carers recognise and value this emotional support. Emotional support by health care assistants was demonstrated in three main ways: the relationships which health care assistants developed and maintained on the professional-personal boundary; the ability of health care assistants to negotiate clinical/domestic boundaries in the home; the ways in which health care assistants and family carers worked together to enable the patient to die at home., Conclusion: Through their emotional labour, health care assistants perform an important role in community palliative care which is greatly valued by family carers. While recent reports have highlighted potential dangers in the ambiguity of their role, any attempts to clarify the 'boundaries' of the health care assistant role should acknowledge the advantages health care assistants can bring in bridging potential gaps between healthcare professionals and family carers.

During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, β-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation., (© 2014. Published by The Company of Biologists Ltd.)

Malnutrition is a significant cause of morbidity and mortality, particularly among older people. Attention has focused on the inadequacies of food provision in institutions, yet the majority suffering from malnutrition live in the community. The aim of this study was to explore barriers and facilitators to food provision for older people receiving home care. It was a qualitative exploratory study using semi-structured interviews with nine home-care workers in June 2013 employed by independent agencies in a large city in northern England. Data were analysed thematically, based on the principles of grounded theory. Findings showed that significant time pressures limited home-care workers in their ability to socially engage with service users at mealtimes, or provide them with anything other than ready meals. Enabling choice was considered more important than providing a healthy diet, but choice was limited by food availability and reliance on families for shopping. Despite their knowledge of service users and their central role in providing food, home-care workers received little nutritional training and were not involved by healthcare professionals in the management of malnutrition. Despite the rhetoric of individual choice and importance of social engagement and nutrition for health and well-being, nutritional care has been significantly compromised by cuts to social care budgets. The potential role for home-care workers in promoting good nutrition in older people is undervalued and undermined by the lack of recognition, training and time dedicated to food-related care. This has led to a situation whereby good quality food and enjoyable mealtimes are denied to many older people on the basis that they are unaffordable luxuries rather than an integral component of fundamental care., (© 2014 John Wiley & Sons Ltd.)

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry-PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.

Background: The role of TCF/β-catenin signalling in T cell development is well established, but important roles in mature T cells have only recently come to light., Methodology/principal Findings: Here we have investigated the signalling pathways that are involved in the regulation of β-catenin in primary human T cells. We demonstrate that β-catenin expression is upregulated rapidly after T cell receptor (TCR) stimulation and that this involves protein stabilisation rather than an increase in mRNA levels. Similar to events in Wnt signalling, the increase in β-catenin coincides with an inhibition of GSK3, the kinase that is required for β-catenin degradation. β-catenin stabilisation in T cells can also be induced by the activation of PKC with phorbol esters and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PKC). Upon TCR signalling, β-catenin accumulates in the nucleus and, parallel to this, the ratio of TCF1 isoforms is shifted in favour of the longer β-catenin binding isoforms. However, phosphorylated β-catenin, which is believed to be inactive, can also be detected and the expression of Wnt target genes Axin2 and dickkopf is down regulated., Conclusions/significance: These data show that in mature human T cells, TCR signalling via PI3K and PKC can result in the stabilisation of β-catenin, allowing β-catenin to migrate to the nucleus. They further highlight important differences between β-catenin activities in TCR and Wnt signalling.

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21(ras) and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21(ras) -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21(ras) activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21(ras) activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55-/- primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21(ras) becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21(ras)-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion.

The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP(-/-) T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55(-/-) T cells retain ADAP expression yet show defects in beta1 and beta2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55(-/-) T cells showed the same general impairment of function as ADAP(-/-) T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP(-/-) mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal.

The src family kinases p56lck (Lck) and p59fyn (Fyn) are the most proximal signaling molecules to be activated downstream of the T-cell receptor. Using an inducible transgenic model, we can regulate the expression of Lck in primary T cells and ask how the signaling cascade and differentiation potential are affected by the absence or the presence of reduced levels of Lck. We show that in naïve T cells, Lck controls the threshold of activation by preferentially regulating multiple signaling pathways that result in the mobilization of Ca2+ through activation of phospholipase C-gamma and protein kinase C as well as activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Fyn is also able to stimulate the ERK/MAPK pathway in primary T cells but has little influence on the mobilization of Ca2+. Only Lck efficiently stimulates production of diacylglycerol and therefore RasGRP1 recruitment to the plasma membrane and phosphorylation of Shc, suggesting that Fyn activates ERK via a different upstream signaling route. Finally, we show that signals through Lck are essential for the development of T-cell-effector potential, particularly for effective cytokine transcription.

alpha beta T-cell development is restricted to the thymus. Interactions between developing lymphocytes and the thymic stroma, together with bone-marrow-derived monocytes and dendritic cells, are critical for proper development of the T-cell lineage. The developmental sequence through which T-cell progenitors pass on their way to maturity is well established, and can be followed by the sequential acquisition and/or removal of cell surface molecules. Using the combination of modern genetic manipulations, such as transgenesis, gene ablation (knockouts) and targeted mutagenesis (knock-ins), with the ever-improving conditional and inducible manipulation of gene expression, we are beginning to gain an understanding of how intercellular interactions can be relayed via intracellular signalling cascades to bring about nuclear re-organisation and the differentiated mature CD4(+) and CD8(+) subpopulations.

The src-family kinases p56lck (Lck) and p59fyn (Fyn) are expressed in T cells and are among the first signaling molecules to be activated downstream of the T cell receptor (TCR). Evidence is emerging that although closely related, these signaling molecules have discrete functions during development, maintenance and activation of peripheral T cells. For example, during thymopoiesis Lck is uniquely able to provide all the signals required for pre-TCRbeta selection, although Fyn can substitute for a subset of these. Positive selection of CD4 single-positive (SP) cells is also critically dependent on the expression of Lck but not Fyn, while differentiation of CD8 SP cells proceeds relatively efficiently in the absence of Lck. In naïve peripheral T cells either Lck or Fyn can transmit TCR-mediated survival signals, and yet only Lck is able to trigger TCR-mediated expansion signals under conditions of lymphopenia. Stimulation of naïve T cells by antigenic stimuli is also severely compromised in the absence of Lck, but more subtly impaired by the absence of Fyn. We discuss recent experiments addressing how these two src-kinase family members interface with downstream signaling pathways to regulate these diverse aspects of T cell behavior.

T cell recognition of antigenic peptides is thought to occur preferentially in the context of self-MHC. Here, we have tested the ability of four different K(b)-peptide combinations to stimulate self- and allo-restricted CTL responses in three different mouse stains. Responder T cells were primed in vitro with peptide-loaded stimulator cells, followed by limiting dilution assays to measure the number of peptide-specific cytotoxic T lymphocytes (CTL). For three peptides the number of CTL restricted by self-MHC was higher than for allo-MHC-restricted responses, although the difference was surprisingly small (3- to 5-fold). For the fourth peptide there was no detectable difference in the number of self- and allo-restricted CTL. Peptide titration experiments revealed that high avidity CTL were present in both the self- and allo-restricted setting. These data showed that the bias for preferred peptide recognition in the context of self-MHC imposed by positive thymic selection seems marginal. This raised the possibility that the TCR repertoire is inherently biased towards MHC restriction, independent of MHC-guided thymic selection. This was supported by the analysis of mature T cells generated from the thymus of MHC-deficient mice by lectin stimulation. K(b)-restricted CTL were found amongst these T cells at numbers similar to those of allo-restricted CTL. In summary, the data suggest that MHC-restricted peptide recognition is an inherent feature of the TCR repertoire and does not require thymic selection by MHC molecules.

The T lymphocyte-specific protein tyrosine kinase p56lck (Lck) is an essential component of the TCR-mediated signal transduction complex. Lck knockout mice have reduced numbers of double-positive thymocytes and very few mature single-positive cells, particularly of the CD4 lineage. Here we demonstrate the ability of a tetracycline-based tissue-specific inducible Lck transgene to restore expansion of early thymocytes and maturation of single-positive cells in Lckneg mice upon induction with doxycycline. Restoration of Lck expression is particularly important for positive selection to the CD4+ lineage but has a lesser impact on selection to the CD8+ lineage, suggesting activation of Lck is an important component of the signals involved in lineage choice during thymic differentiation.

MHC molecules are normally required for the development of thymocytes from the CD4(+)CD8(+) double-positive to the CD4 or CD8 single-positive stage. Here we show that mitogenic plant lectins can substitute for MHC molecules in driving the differentiation of phenotypically and functionally mature CD4 as well as CD8 T cells. Interestingly, lectin dosage determines whether CD4 or CD8 cells are generated, indicating that variation of cumulative signal strength (not necessarily signal quality) can result in an apparent switching of lineage preference. Thymocyte perception of differentiation-inducing signals is modulated by the cellular context, since stimuli that yield CD8 cells in the context of the thymic microenvironment fail to do so in suspension culture and generate CD4 progeny instead. Finally, we show that lectin-generated single-positive thymocytes retain the ability to respond to the ligands initially used to drive their differentiation. Our results call into question generalizations and predictions made from other experimental systems and reveal that thymocyte selection is considerably more flexible than had been anticipated.

T cell maturation requires the rearrangement of clonotypic T cell receptors (TCR) capable of interacting with major histocompatibility complex (MHC) ligands to initiate positive and negative selection. Only 3-5% of thymocytes mature to join the peripheral T cell pool. To investigate the basis for this low success rate, we have measured the frequency of preselection thymocytes capable of responding to MHC. As many as one in five MHC-naive thymocytes show upregulation of activation markers on exposure to MHC-expressing thymic stroma in short-term reaggregate culture. The majority of these cells display physiological changes consistent with entry into the selection process within 24 h. By exposing TCR transgenic thymocytes to a range of MHC-peptide complexes, we show that CD69 induction is indicative of thymocyte selection, positive or negative. Our data provide evidence that the fraction of thymocytes that qualify to enter the thymic selection process far exceeds the fraction that successfully complete it, and suggest that most MHC-reactive thymocytes are actively eliminated in the course of selection.

The energetics of dissociation of bovine insulin in aqueous solution have been investigated by sensitive dilution microcalorimetry. Cyclodextrins increase dissociation of insulin oligomers in a manner consistent with their interaction with protein side chains. For example, assuming monomer-dimer equilibrium, in the absence of cyclodextrins the calorimetric dilution data (25 degrees C, pH 2.5) indicate a dimer dissociation constant (K(diss)) of about 12 microM and an endothermic dissociation enthalpy (delta H(diss) of +41 kJ mol-1. Addition of methyl-beta-cyclodextrin (up to 200 mM) makes dissociation significantly more endothermic (delta H(diss) = 79 kJ mol-1) and reduces the apparent dimer dissociation constant by more than two orders of magnitude (K(diss) approximately 1.7 mM). Qualitatively similar results are observed with alpha-cyclodextrin and other beta-cyclodextrin derivatives. Cyclodextrin-induced insulin dissociation is also observed at pH 7.4.