A role for the thiol-dependent reductase ERp57 in the assembly of MHC class I molecules

AbstractAn important mammalian defence strategy against intracellular pathogens is the presentation of cytoplasmically derived short peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes. MHC class I molecules assemble in the endoplasmic reticulum (ER) with chaperones, including calnexin and calreticulin, before binding to the transporter associated with antigen processing (TAP). We show here that the thiol-dependent reductase ERp57 (also known as ER60 protease) is involved in MHC class I assembly. ERp57 co-purified with the rat TAP complex (comprising TAP1 and TAP2), and associated with MHC class I molecules at an early stage in their biosynthesis. This association was sensitive to castanospermine, which inhibits the processing of glycoproteins. Human MHC class I molecules were also found to associate with ERp57. We conclude that ERp57 is a newly identified component of the MHC class I pathway, and that it appears to interact with MHC class I molecules before they associate with TAP

Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in <em>Saccharomyces cerevisiae</em>

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism

Believing is achieving: a longitudinal study of self-efficacy and positive affect in resettled refugees

Research has shown that self-efficacy can play an important role in recovery from trauma (Benight and Bandura 2004). We hypothesised that for refugees, whose (often traumatic) experiences pre- and post-resettlement have been linked to a decrease in their wellbeing (e.g., Aspinall and Watters 2010), self-efficacy would play a key role in improving wellbeing. This paper investigates the link between self-efficacy and positive affect among resettled refugees (N = 180). Research used mixed methods. The longitudinal survey with three time points confirmed that higher levels of general self-efficacy were consistently associated with better positive affect at later time points. The reverse effects, from positive affect to later self-efficacy, were not significant. In addition, qualitative interviews with a subsample provide suggestions as to how self-efficacy of refugees might be improved: that is, by improving access to employment and language classes, by clarifying how British social and cultural systems work, including the practical information necessary to navigate daily life, and by providing more opportunities to increase social networks, all suggesting the necessity of a proactive role of the receiving society

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then also migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 - but not its homologues LPP1 or LPP2 - diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes melanoma cells' ability to invade. Our results demonstrate that LPP3 is the key enzyme in melanoma cells' breakdown of LPA, and confirm the importance of attractant breakdown in LPA-mediated cell steering

Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets

Effect of increasing fruit and vegetable intake by dietary intervention on nutritional biomarkers and attitudes to dietary change : a randomised trial

This work was funded by The Scottish Government Rural and Environmental Science and Analytical Sciences Division (RESAS) and supported by the Rank Prize Funds.Peer reviewedPublisher PD

"More than just a medical student”: a mixed methods exploration of a structured volunteering programme for undergraduate medical students

Background As a result of the COVID-19 pandemic Imperial College School of Medicine developed a structured volunteering programme involving 398 medical students, across eight teaching hospitals. This case study aims to illuminate the experiences of volunteers, mechanisms of learning and draw lessons for future emergencies and curriculum improvements. Methods Using an illuminative approach to evaluation we invited all volunteers and supervisors to complete a mixed-methods survey. This gathered nominal demographic information and qualitative data related to motivations, experiences, insights into learning, processual and contextual factors. Qualitative responses were coded, thematically organised, and categorised into an overarching framework. Mann-Whitney U tests determined whether volunteers’ overall rating of the experience varied according to demographic features and modulating factors. Spearman’s rank correlation assessed the relationship between aspects of induction and supervision, and overall volunteering rating. Follow up interviews were carried out with students to check back findings and co-create conclusions. Results Modulating factors identified through thematic analysis include altruistic motivation, engaged induction and supervision, feeling valued, having responsibility and freedom from the formal curriculum. Statistically significant positive correlations are identified between volunteers overall rating and being a year 1 or 2 student, ability to discuss role and ask questions during induction, being male, and having regular meetings and role support from supervisors. Qualitatively reported impacts include improved wellbeing, valuable contribution to service and transformative learning. Transformative learning effects included reframing of role within the multidisciplinary team, view of effective learning and view of themselves as competent clinicians. The number of weeks, number of shifts per week, and the role the volunteers performed, did not significantly impact experiences. Conclusions While acknowledging the uniqueness of the situation presented by the first wave COVID-19, we suggest the features of a successful service-learning programme include: a learner-centred induction, engaged and appreciative supervisors, and the entrustment of students with meaningful work with reciprocal benefits to services. Programmes in similar settings may find that 1) volunteering is best appreciated in years 1 or 2, 2) students with altruistic motivations and meaningful work may flourish without formal outcomes and assessments, and 3) that female volunteers may experience emergency learning differently to men

Bring a plate: facilitating experimentation in the Welcome Dinner Project

Drawing on in-depth empirical research, we explore a project called The Welcome Dinner (WDP). The WDP aims to bring together ‘newly arrived’ people and ‘established Australians’ to meet and ‘share stories’ over a potluck meal in ‘the comfort of their own home’. The purpose is to create meaningful connections, new friendships and social solidarities. In this paper, we focus on the micro-contexts of the dinners and the minute activities and techniques that facilitators use in hosting. Our aim is not to analyse the effects of the project but rather the design and meaning of the activities. As a form of ‘designed everyday multiculturalism’, focused on welcoming new arrivals to Australia, it takes effort, skill and labour to manage the contact between different cultural groups over organised meals. Thus, facilitators take over the hosting of the lunches and dinners to run activities, which are imagined to lubricate social dynamics and relations, and produce convivial commensal affects and behaviours. Drawing on theories of training activities as embodied and cognitive experimentations, which enable new knowledge practices and social relations, we analyse field notes and interviews about the facilitation, structure and activities at the WDP home dinners

Vacuum Cherenkov radiation and bremsstrahlung from disformal couplings

The simplest way to modify gravity is to extend the gravitational sector to include an additional scalar degree of freedom. The most general metric that can be built in such a theory includes disformal terms, so that standard model fields move on a metric which is the sum of the space time metric and a tensor constructed from first derivatives of the scalar. In such a theory gravitational waves and photons can propagate at different speeds, and these can in turn be different from the maximum speed limit for matter particles. In this work we show that disformal couplings can cause charged particles to emit Cherenkov radiation and bremsstrahlung apparently in vacuum, depending on the background evolution of the scalar field. We discuss the implications of this for observations of cosmic rays, and the constraints that arise for models of dark energy with disformal couplings

The flood pulse in a semi-arid riparian forest:metabolic and biogeochemical responses to inter-flood interval

Flood pulse inundation of riparian forests alters rates of nutrient retention and organic matter processing in the aquatic ecosystems formed in the forest interior. Along the Middle Rio Grande (New Mexico, USA), impoundment and levee construction have created riparian forests that differ in their inter-flood intervals (IFIs) because some floodplains are still regularly inundated by the flood pulse (i.e., connected), while other floodplains remain isolated from flooding (i.e., disconnected). This research investigates how ecosystem responses to the flood pulse relate to forest IFI by quantifying nutrient and organic matter dynamics in the Rio Grande floodplain during three years of experimental flooding of the disconnected floodplain and during a single year of natural flooding of the connected floodplain. Surface and subsurface conditions in paired sites (control, flood) established in the two floodplain types were monitored to address metabolic and biogeochemical responses. Compared to dry controls, rates of respiration in the flooded sites increased by up to three orders of magnitude during the flood pulse. In the disconnected forest, month-long experimental floods produced widespread anoxia of four-week duration during each of the three years of flooding. In contrast, water in the connected floodplain remained well oxygenated (3-8 ppm). Material budgets for experimental floods showed the disconnected floodplain to be a sink for inorganic nitrogen and suspended solids, but a potential source of dissolved organic carbon (DOC). Compared to the main stem of the Rio Grande, flood-water on the connected floodplain contained less nitrate, but comparable concentrations of DOC, phosphate-phosphorus, and ammonium-nitrogen. Results suggest that floodplain IFI drives metabolic and biogeochemical responses during the flood pulse. Impoundment and fragmentation have altered floodplains from a mosaic of patches with variable IFI to a bimodal distribution. Relatively predictable flooding occurs in the connected forest, while inundation of the disconnected forest occurs only as the result of managed application of water. In semiarid floodplains, water is scarce except during the flood pulse. Ecosystem responses to the flood pulse are related to the IFI and other measures of flooding history that help describe spatial variation in ecosystem function