Project 1: Molecular interactions of AMIGO family members and Project 2: Adhesive interactions of mesenchymal stem cells with platelets and P-selectin

Project 1: The regenerative capacities between the peripheral and central nervous systems are contrasting; with axons in the peripheral nervous system being able to regenerate, unlike those in the central nervous system (CNS). Upon injury, the CNS responds by releasing myelin breakdown products, which are inhibitory to axon regrowth, and the formation of the glial scar which forms a physical barrier to regeneration. Whilst the formation of the glial scar takes days or weeks to form, the myelin breakdown products, which include myelin associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and Nogo, exert their inhibitory effects immediately. These products bind to the Nogo Receptor (NgR) complex which consists of the Nogo receptor (NgR), p75 neurotrophin receptor (p75NTR)/ (or an alternative protein known as TROY) and Lingo-1 which work together to activate RhoA, resulting in actin depolymerisation and growth cone collapse. The specific mechanisms by which this complex exhibits its effects remain unclear, particularly as recent unpublished data suggest that specific members of the Amigo family of proteins may be critical mediators of the inhibitory signaling pathway in vivo. This suggestion prompted us to clone NgR, p75NTR and Amigos 1, 2 and 3 in mammalian expression vectors and perform co-immunoprecipitation experiments with these constructs. These experiments suggest that Amigo-1 and -3 bind to NgR and/or p75NTR both independently or when expressed together. Although the approach was only semiquantitative at best, the experiments also suggest that Amigo 2 may bind to NgR and p75NTR independently and together, but to a much lesser extent. The results indicate that the Amigo family of proteins may form a component of the axon growth inhibitory complex, and potentially forming novel therapeutic targets for CNS injury. Project 2: Mesenchymal stem cells (MSCs) are non haematopoietic cells capable of differentiating into adipocytes, chondrocytes and osteoblasts they are positive for CD105, CD90 and CD73 and negative for CD34 and CD45 amongst others. MSCs are not immunogenic and do not stimulate alloreactivity and this characteristic along with their ability to differentiate into such a range of cell types make them attractive for use in regenerative therapies by systemic infusion of ex vivo expanded cells. Its not known how MSCs are recruited from the circulation, however they may employ mechanisms similar to those used by leukocytes and haematopoietic stem cells when they are transported from the circulation such as the employment of selectins and via the aid of platelets. We therefore wanted to determine if MSCs can bind to collagen or P selectin coated microslides under flow conditions and in parallel observe if platelets as a whole can bind to MSCs in a variety of conditions by staining MSC and blood samples with CD105 (MSC marker) and CD42b (platelet marker) and use flow cytometry to looking at the effects of anticoagulant, time and platelet activation. The results demonstrated that MSCs do not bind to collagen or P selectin under flow conditions and demonstrated that a small proportion of platelets can bind to MSCs under appropriate conditions. Further, the binding experiments revealed that platelet activation reduced the level of binding between MSCs and platelets

Local Scale Comparisons of Avian and Woody Vegetation Communities within Four Arkansas State Parks

Measuring the spatial distribution of biotic communities can provide useful data to wildlife managers on how and why species assemblages differ across a landscape. During 18 May – 7 August 2015, we conducted avian point counts and collected vegetation data in nested subplots at 4 Arkansas state parks. We then used a series of one-way ANOVAs and Kruskal-Wallis tests to examine differences in species richness, Simpson’s evenness, Simpson’s diversity, and Bray-Curtis similarity across the 4 parks. Mount Magazine State Park had the lowest avian evenness (F3,22 = 9.57 P = 0.003) and diversity (F3,22 = 17.8 P ≤ 0.001). Mount Magazine also had the lowest understory vegetation evenness (F3,22 = 9.41 P ≤ 0.001) and diversity (F3,22 = 17.8 P ≤ 0.001). Our analyses provided weak evidence supporting a possible relationship between avian and understory woody vegetation communities at Mount Magazine; however, this relationship was not observed in the remaining parks. Comparing biotic communities across 4 local state parks may aid park managers by providing a baseline of biotic data that can be used to better understand the collective effects acting on a specific park’s flora and fauna

Molecular interactions of AMIGO family members in the Nogo receptor complex

The capacity for regeneration in the central nervous system is limited, with little functional recovery following injury or disease. Upon injury, the CNS responds by releasing myelin associated inhibitory factors (MAIFs), including Nogo, MAG and OMgp, which act to suppress axonal regrowth by binding to the Nogo receptor (NgR) complex. The complex consists of the Nogo receptor (NgR), p75 neurotrophin receptor (p75NTR), (or an alternative protein, TROY) and LINGO-1. Signalling through the complex leads to activation of RhoA, resulting in growth cone collapse. However, the specific mechanisms by which the complex exhibits its effects remain unclear, particularly as more recent data suggest that specific members of the AMIGO family of proteins may be critical mediators of the inhibitory signaling pathway. Mammalian expression and subcellular fractionation and co-immunoprecipitation experiments established binding between both NgR and p75NTR with AMIGO-1, -2 and -3 and have shown the AMIGO proteins are expressed in the cell membrane as are NgR and p75NTR. Interestingly LINGO-1 was also shown to be expressed intracellularly. Furthermore, injection of soluble LINGO-1, AMIGO-1 and AMIGO-3 in surface plasmon resonance (SPR) approaches confirmed interaction with surface bound NgR. Finally, the structure of the human AMIGO-1 ectodomain, including LRR and Ig domains was determined to 1.9Å resolution. The protein formed a dimer in solution and crystal form and comparisons with structurally similar LRR and Ig domain containing proteins, combined with electrostatic properties and localisation of glycosylation sites, was used to ascertain likely ligand binding sites on the surface of the protein and AMIGO family in general. Collectively these results indicate that the AMIGO proteins may form a component of the axon growth inhibitory complex, and are potentially novel therapeutic targets for CNS injury

Long-term psychosocial impact of venous thromboembolism: a qualitative study in the community

Objectives: Venous thromboembolism is a serious, potentially traumatic, life-threatening condition and a major cause of mortality and morbidity. The aim of this study was to obtain detailed understandings of the impact of VTE and examine individual’s experiences over the first year since a first time VTE.Design: A longitudinal qualitative interview study using inductive thematic analysis. This study presents follow-up data for eleven participants, first interviewed six months following a first-time VTE.Setting: Outpatients recruited a community haematology clinic in a one UK District General Hospital.Intervention: Audio-recorded semi-structured interviews with a sample of eleven participants who experienced a first-time DVT or PE within the previous year and completed similar interviews three months previously. Interviews were transcribed and analysed using inductive thematic analysis.Results: Four over-arching themes were identified: life changing and forever changed, the trauma of care, ‘thrombo-neuroses’ and through adversity comes growth. Theme content varied according to age and developmental stage, presence of VTE symptoms and the experience of diagnosis.Conclusions: The data demonstrates the psychosocial impact of VTE and its diagnosis as physically and psychologically challenging, and individuals reported being forever changed by the experience. Participants’ reported continued high levels of trauma and anxietysymptoms, triggered by physical (e.g. symptoms) and psychological (e.g. health anxiety,negative emotions) reminders of VTE. Wider primary care service issues includingmisdiagnosis maintained negative emotions and health anxiety with implications fo relationships with professionals. Targeted clinical interventions to better identify and supportindividuals at risk of distress and enhance psychological well-being and reduce distress arediscusse

COVID-19 confessions: a qualitative exploration of healthcare workers experiences of working with COVID-19

Objectives To gain insight into the experiences and concerns of front-line National Health Service (NHS) workers while caring for patients with COVID-19. Design Qualitative analysis of data collected through an anonymous website (www.covidconfidential) provided a repository of uncensored COVID-19 experiences of front-line NHS workers, accessed via a link advertised on the Twitter feed of two high profile medical tweeters and their retweets. Setting Community of NHS workers who accessed this social media. Participants 54 healthcare workers, including doctors, nurses and physiotherapists, accessed the website and left a ‘story’. Results Stories ranged from 1 word to 10 min in length. Thematic analysis identified common themes, with a central aspect being the experience and psychological consequence of trauma. Specific themes were: (1) the shock of the virus, (2) staff sacrifice and dedication, (3) collateral damage ranging from personal health concerns to the long-term impact on, and care of, discharged patients and (4) a hierarchy of power and inequality within the healthcare system. Conclusions COVID-19 confidential gave an outlet for unprompted and uncensored stories of healthcare workers in the context of COVID-19. In addition to personal experiences of trauma, there were perceptions that many operational difficulties stemmed from inequalities of power between management and front-line workers. Learning from these experiences will reduce staff distress and improve patient care in the face of further waves of the pandemic

Longitudinal study of Asian elephants, Elephas maximus, indicates intermittent shedding of elephant endotheliotropic herpesvirus 1 during pregnancy

Introduction: EEHV-1 is a viral infection of elephants that has been associated with a fatal haemorrhagic syndrome in Asian elephants. Previous studies have suggested that pregnant animals may shed more virus than non-pregnant animals. Methods: This study examined whether pregnancy affected the frequency or magnitude of shedding of elephant endotheliotropic herpesvirus 1 (EEHV1) using Taq man real-time PCR on trunk washes from four female elephants from a UK collection over three time periods between 2011 and 2014. These periods included pregnancies in two animals (period 1 and period 3). Behavioural observations made by keepers were also assessed. Results: During period 1 there was a high degree of social hierarchical instability which led to a hierarchy change, and was associated with aggressive behaviour. Also during period 1 EEHV-1 shedding was of a higher magnitude and frequency than in the latter two time periods. Conclusions: These results suggest that there is no clear relationship between shedding and pregnancy, and that behavioural stressors may be related to an increase in EEHV-1 shedding

Pioglitazone and cause-specific risk of mortality in patients with type 2 diabetes: extended analysis from a European multidatabase cohort study.

OBJECTIVES: Describe and compare the risk of cardiovascular and non-cardiovascular mortality in patients whose antidiabetic therapy is modified to include pioglitazone compared with an alternative antidiabetic medication at the same stage of disease progression. RESEARCH DESIGN AND METHODS: This exploratory linked database cohort analysis used pooled health and mortality data from three European countries: Finland, Sweden and the UK. Propensity score together with exact matching was used to match 31 133 patients with type 2 diabetes first prescribed pioglitazone from 2000 to 2011, to 31 133 patients never prescribed pioglitazone. Exact matching variables were treatment stage, history of diabetes, diabetes complications and cardiovascular disease, and year of cohort entry. Mean follow-up time was 2.60 (SD 2.00) and 2.69 (SD 2.31) years in the pioglitazone and non-pioglitazone-exposed groups, respectively. Crude cause-specific mortality rates were ascertained. Association with pioglitazone use was estimated using Cox proportional hazards models adjusted a priori for country, age, sex, the propensity score quintile and time-dependent variables representing use of antidiabetic drugs. Stepwise testing identified no additional confounders to include in adjusted models. RESULTS: The crude mortality rate was lower in the pioglitazone-exposed group than the non-exposed group for both cardiovascular and non-cardiovascular mortality. Adjusted HRs comparing pioglitazone to alternative antidiabetic exposure were 0.58 (95% CI 0.52 to 0.63) and 0.63 (95% CI 0.58 to 0.68) for cardiovascular and non-cardiovascular mortality, respectively. A protective effect associated with pioglitazone was also found for all specific cardiovascular causes. CONCLUSIONS: This analysis suggests that pioglitazone is associated with a decrease in both cardiovascular and non-cardiovascular mortality. Results should be interpreted with caution due to the potential for residual confounding in this exploratory analysis. Further studies, specifically designed to test the association between pioglitazone use and patient-focused outcomes, are suggested. STUDY REGISTRATION NUMBER: European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP; EUPAS3626)

Discovery and Prevalence of Divergent RNA Viruses in European Field Voles and Rabbits

The advent of unbiased metagenomic virus discovery has revolutionized studies of virus biodiversity and evolution. Despite this, our knowledge of the virosphere, including in mammalian species, remains limited. We used unbiased metagenomic sequencing to identify RNA viruses in European field voles and rabbits. Accordingly, we identified a number of novel RNA viruses including astrovirus, rotavirus A, picorna-like virus and a morbilli-like paramyxovirus. In addition, we identified a sobemovirus and a novel luteovirus that likely originated from the rabbit diet. These newly discovered viruses were often divergent from those previously described. The novel astrovirus was most closely related to a virus sampled from the rodent-eating European roller bird (Coracias garrulous). PCR screening revealed that the novel morbilli-like paramyxovirus in the UK field vole had a prevalence of approximately 4%, and shared common ancestry with other rodent morbilli-like viruses sampled globally. Two novel rotavirus A sequences were detected in a UK field vole and a French rabbit, the latter with a prevalence of 5%. Finally, a highly divergent picorna-like virus found in the gut of the French rabbit virus was only ~35% similar to an arilivirus at the amino acid level, suggesting the presence of a novel viral genus within the Picornaviridae

Physician decision making in selection of second-line treatments in immune thrombocytopenia in children.

Immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder which presents with isolated thrombocytopenia and risk of hemorrhage. While most children with ITP promptly recover with or without drug therapy, ITP is persistent or chronic in others. When needed, how to select second-line therapies is not clear. ICON1, conducted within the Pediatric ITP Consortium of North America (ICON), is a prospective, observational, longitudinal cohort study of 120 children from 21 centers starting second-line treatments for ITP which examined treatment decisions. Treating physicians reported reasons for selecting therapies, ranking the top three. In a propensity weighted model, the most important factors were patient/parental preference (53%) and treatment-related factors: side effect profile (58%), long-term toxicity (54%), ease of administration (46%), possibility of remission (45%), and perceived efficacy (30%). Physician, health system, and clinical factors rarely influenced decision-making. Patient/parent preferences were selected as reasons more often in chronic ITP (85.7%) than in newly diagnosed (0%) or persistent ITP (14.3%, P = .003). Splenectomy and rituximab were chosen for the possibility of inducing long-term remission (P < .001). Oral agents, such as eltrombopag and immunosuppressants, were chosen for ease of administration and expected adherence (P < .001). Physicians chose rituximab in patients with lower expected adherence (P = .017). Treatment choice showed some physician and treatment center bias. This study illustrates the complexity and many factors involved in decision-making in selecting second-line ITP treatments, given the absence of comparative trials. It highlights shared decision-making and the need for well-conducted, comparative effectiveness studies to allow for informed discussion between patients and clinicians

Strategies to Support Recruitment of Patients With Life-Limiting Illness for Research: The Palliative Care Research Cooperative Group

The Palliative Care Research Cooperative group (PCRC) is the first clinical trials cooperative for palliative care in the United States