Starter Grant Research Articles

Experience of an NIHR Clinical Lectureship (medical/dental) and the determining factors for a clinical academic career post lectureship: a mixed-method evaluation

ObjectivesThe objective of this study is to investigate early-to-late postdoctoral clinical academic progression and the experiences of NIHR Clinical Lectureship (CL) fellows, considering enablers and barriers to success, and identifying...

44-OR: NFAT Inhibition Reduces Retinal Vascular Leakage and Inflammation in Diabetic Retinopathy

Purpose: Macular edema resulting from retinal vascular leakage is the leading cause of vision loss in diabetic retinopathy (DR) . The nuclear factor of activated T-cells (NFAT) family of transcription factors is known to upregulate inflammatory cytokines in response to high glucose. This study explored the effect of NFAT inhibition on retinal vascular leakage and inflammation in DR in vivo and in vitro. Methods: The streptozocin-induced (STZ) model was used as the mouse model of diabetic retinopathy. Hyperglycemia was induced at 4 weeks in C57BL/6J mice and confirmed when fasting plasma glucose became persistently elevated (>300 mg/dL) . Experiments were performed at weeks of hyperglycemia. STZ mice were injected intravitreally with the NFAT signaling inhibitor INCA-6 (25 uM) or vehicle (0.1% DMSO in PBS) . Uninjected eyes of both STZ and control mice served as additional controls. Evans blue dye (EBD) was used to measure retinal vascular leakage. Primary mouse retinal microvascular endothelial cells (MRMECs) were used for in vitro experiments. MRMEC monolayers were treated with INCA-6 (2.5 uM) or with vehicle and cell permeability was induced with mouse VEGF165. The transendothelial electrical resistance (TEER) was measured with the electrical cell-substrate impedance sensing method. MRMEC IL-6 production was investigated by ELISA. Results: In the STZ model of diabetic retinopathy, EBD vascular leakage was significantly reduced in INCA-6-treated eyes (INCA-6: 0.85 ± 0.57, n=5; vehicle: 1.78 ± 0.38, n= 7; P=0.007) , and in INCA-6 treated eyes compared to untreated eyes (untreated: 2.83 ± 1.63, n=4, P=0.04) . In the VEGF-induced MRMEC monolayer permeability model, INCA-6 significantly preserved TEER compared to vehicle (n=12, P<0.05) and decreased the VEGF-induced production of IL-6 (n=5, P<0.05) . Conclusions: NFAT inhibition has therapeutic potential to prevent and treat retinal vascular leakage and inflammation in DR. Disclosure I.De la huerta: None. J.L.Nunez: None. J.M.Poloway: None. V.Reddy: None. J.Penn: None. Funding National Institutes of Health (EY032620) International Retinal Research Foundation Grant Knights Templar Foundation Career Starter Grant

Productive Aging During COVID-19 - Lessons from WH SeniorLink

<h3>Introduction</h3> According to the National Academies of Science, Medicine, and Engineering, even before the pandemic, 24% of Americans 65+ were considered socially isolated, and 43% of adults 60+ were lonely. Both experiences are associated with serious physical and mental health problems, including increased risks of dementia, stroke, depression, and suicidal ideation. As older adults engage in stricter social distancing to protect themselves from COVID-19, their risk of social isolation and loneliness is heightened. According to research by the Kaiser Family Foundation, 46% of adults 65+ reported that the worry and stress caused by pandemic has had a negative impact on their mental health. In order to combat this unique challenge, we must employ innovative, flexible solutions that adapt to the shifting circumstances and an uncertain future. <h3>Methods</h3> WH SeniorLink has developed an innovative program for integrating friendly visiting, mutual aid, needs assessment and case management, all delivered remotely by trained volunteers. The program was conceived, developed and executed since the beginning of COVID-19 restrictions and has grown to become a fully fledged 501(c)(3) organization, and is a model of flexible service delivery during this unusual time. The program operates through a volunteerism-mutual aid model. Volunteers are trained using online modules and quizzes and supervised by a licensed social worker, including training on needs and risk assessment. Volunteers are then matched with an older adult with similar interests and language preferences to provide friendly weekly phone calls, care packages and letters. Older adults with higher needs are referred to WH SeniorLink's service navigation program which operates using the online platform, Apricot, and is staffed by masters level social work interns and supervised by licensed social workers. Service navigation involves conducting comprehensive assessments and identifying resources and referrals. Older adults who express interest are also paired with each other to provide social support. Barriers to program development have included identifying consistent funding sources and volunteer attrition. <h3>Results</h3> Data shows 169% growth in contacts made with older adults between May and October, with 2.5 times more older adults served in November than in May. Initial responses to the Dejong Gierveld Loneliness Scale and qualitative data collection reflect that older adults continue to feel lonely and isolated in the midst of the pandemic, but that WH SeniorLink is helping. By placing emphasis on empowering older adults to form sustained relationships with volunteers and their peers, WH SeniorLink encourages reciprocal relationships through which older adult participants are valued for their experiences and contributions. <h3>Conclusions</h3> The mission of WH SeniorLink is to strengthen community ties and improve health outcomes among older New Yorkers by providing social-emotional support and connection to essential services. WH SeniorLink was founded during the COVID-19 pandemic, in response to the myriad of new challenges faced by older adults including greater risk of social isolation and increased disconnection from essential services. However, we recognize that the pandemic has only exacerbated a problem that began long before, and older adults are at risk of being left behind in the coming months and beyond. <h3>Funding</h3> WH SeniorLink is funded by a starter grant from Columbia School of Social Work and subsequent crowdfunded donations.

Pax2a Regulates Endothelial Cell Invasion into the Optic Fissure Resulting in Degradation of the Basement Membrane by mmp2

Epithelial Sheet Fusion (ESF), a process by which two populations of epithelial cells fuse to create a continuous sheet, is a crucial aspect of embryogenesis. ESF can be observed in the development of many tissues, including fusion of the optic fissure (OF) during retinal development in vertebrates. Failure in fusion of the OF results in congenital coloboma, a leading cause of pediatric blindness. Unfortunately, majority of coloboma cases lack a definitive genetic cause. This is likely reflected by the fact that the molecular mechanisms driving OF ESF are still poorly understood. This study aims to identify the molecular machinery regulating ESF during OF fusion in zebrafish. To do so, we utilized a well characterized mutant line of zebrafish carrying a mutation in the Pax2a gene. Pax2a is a transcription factor known to be required for OF fusion and has been linked to cases of coloboma in many species. The mutant pax2anoi/noi line of zebrafish display a completely penetrant coloboma phenotype. To assess transcriptional differences between normal and abnormal OF fusion, RNA sequencing was performed comparing WT and pax2anoi/noi whole eye transcriptomes. The results indicated a down‐regulation of hyaloid vasculature regulators. Examining retinal vasculature in Tg[kdrl:mCherry], pax2anoi/noi mutants revealed a significant reduction and malformation of the hyaloid vasculature. Subsequently, OF fusion was analyzed in the absence of vasculature. Treatment with a pharmacological inhibitor of VEGF signaling, DMH‐4, completely inhibited the establishment of hyaloid vasculature in the retina. In the presence of DMH4, the OF failed to fuse. In order to understand the mechanism behind the reduction of vasculature in pax2noi/noi embryos we returned to the transcriptomic data and analyzed for angiogenic regulators. This analysis identified adamts1, an anti‐angiogenic protease which sequesters VEGF ligands, to be up‐regulated in pax2noi/noi embryos. As validation of our findings, overexpression of adamts1 mRNA in WT embryos successfully phenocopied the pax2noi/noi associated failure of OF fusion. Lastly, in order to determine the functional role of hyaloid vasculature cells during OF fusion we examined basement membrane remodeling. Matrix metalloproteinases (MMP’s) have the ability to degrade the BM. After characterizing expression of all zebrafish mmp’s, mmp2, mmp14a and mmp14b were found to be expressed in the OF. Using two‐color in‐situ hybridization we confirmed that mmp2 and mmp14 expression in OF is confined to the hyaloid vasculature cells. Furthermore, mmp2 and mmp14 expression is decreased in the OF in both DMH‐4 treated and Pax2 mutant embryos. Treatment with a pharmacological inhibitor specific to mmp2, ARP101 resulted in a failure of OF BM remodeling and subsequently fusion. Overall, our results show that pax2a negatively regulates adamts1 expression to enable proper vascularization of the developing retina. Vascularization is subsequently necessary for optic fissure fusion as hyaloid vasculature cells provide mmp2 necessary for the degradation of the OF BM and thus OF fusion.Support or Funding InformationLyman T. Johnson Fellowship, Knights Templar Career Starter Grant

A Comparative Analysis of the Vascular Effects of Clopidogrel Metabolites

Dual antiplatelet therapy using low‐dose aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most common preventative method for arterial thrombosis. Clopidogrel, a P2Y12 antagonist, effectively inhibits ADP‐induced platelet aggregation. However, it also increases adverse bleeding side effects which do not correlate with the antiplatelet effect. Of patients who have taken clopidogrel for at least one year, 31% have cerebral microbleeds and 47% of those patients also develop intracerebral hemorrhage. Through clopidogrel’s complex metabolism only 5% of the active metabolite responsible for the antiplatelet effect is generated. In order to avoid the complex metabolism associated with clopidogrel, DT678 was developed. DT678, a conjugate of the clopidogrel active metabolite, is completely converted into the active metabolite to inhibit platelet aggregation. We have previously reported that DT678 is equally as effective in preventing platelet aggregation however, it has a reduced bleeding risk compared to clopidogrel in a rabbit model. A more complete mechanistic understanding of the cellular pathways affected by clopidogrel and its metabolites are required in order to explain the difference in bleeding risk.The purpose of this study was to determine the effects of clopidogrel on spontaneous myogenic tone generation, vasoconstriction or vasodilation in middle cerebral arteries (MCA). Rabbits were anesthetized and the jugular vein was cannulated for blood collection and intravenous drug administration of various doses of DT678, clopidogrel or vehicle. Blood collections were taken at baseline and two‐hour post IV drug administration. The blood was then used in a platelet aggregometry assay to determine the inhibition of platelet aggregation. Two‐hours post treatment the brain was collected and placed in Ca2+ free physiological salt solution. The MCA was isolated and cannulated on an arteriography chamber. The cannulated artery, in Ca2+ physiological salt solution, was pressurized to 80mmHg with a flow rate of 0.25ml/min through the lumen at 37°C. Spontaneous myogenic tone was allowed to develop before assessing the vascular effects of increasing concentrations (10−9–10−5M) of ADP or specific purinergic receptor agonists (P2Y1: MRS 2365; P2Y2 MRS 2768; P2Y4 MRS 4062; P2Y6: MRS 2693; P2Y11: NF 546; or P2Y14: MRS 2905). If spontaneous myogenic tone was not generated the MCA was preconstricted with 10−6M serotonin. Spontaneous myogenic tone and ADP‐induced vasodilation was inhibited in clopidogrel‐treated, but not DT678‐treated middle cerebral arteries. These data suggest that clopidogrel has non‐platelet mediated effects on purinergic receptors expressed in the MCA and may explain the cerebral bleeding side effects observed.Support or Funding InformationThis work was supported in part by MSU PHM/TOX and CVM Startup Funds, Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation Research Starter Grant and NIH/NHLBI R43HL139380.

A Tribute to Richard M. Crooks on the Occasion of His 65th Birthday

A Tribute to Richard M. Crooks on the Occasion of His 65th Birthday

P3109Coronary vulnerable plaque development is promoted by multidirectional wall shear stress

Abstract Aim Wall shear stress (WSS) has been widely associated with plaque development and destabilization. However, the multidirectionality of WSS, induced by the pulsatile nature of blood flow in combination with the arterial geometry, is rarely taken into account. The purpose of this study was to investigate the influence and predictive value of five (multidirectional) WSS parameters for coronary plaque progression and composition. Methods Familial hypercholesterolemic pigs (n=10, castrated male, 3 years old) were put on a high-fat diet and underwent imaging (near infrared spectroscopy-intravascular ultrasound (NIRS-IVUS), optical coherence tomography (OCT) and CT) of the three main coronary arteries at baseline and 10/12 months follow-up. IVUS-based lumen contours were combined with the centerline of artery as extracted from CT to obtain a 3D reconstruction of the coronary artery. Local flow velocity measurements were combined with the 3D-geometry of the coronary arterial lumen to calculate five multidirectional WSS parameters including: time average wall shear stress (TAWSS), oscilatory shear index (OSI), relative residence time (RRT), trans wall shear stress (TransWSS), cross flow index (CFI). For the analysis, arteries were divided into 3mm/45° sectors (n=3627). Per vessel each segment was classified as low, mid or high for each WSS metric based on the average value in the sector. Changes in wall thickness (WT) and plaque composition were assessed with NIRS-IVUS and OCT, and histology and related to the local (multidirectional) WSS. Results Half of the pigs developed lumen intruding, complex, lipid-rich plaques. In these pigs, coronary segments exposed to low baseline levels of TAWSS exhibited a significantly larger plaque growth per month compared to regions with either mid or high TAWSS (p&lt;0.05, see figure). Furthermore, plaque growth correlated positively with the multidirectional WSS metrics OSI, RRT and CFI, with high multidirectionality resulting in the largest plaque growth (see figure). The positive predictive values (PPV) of the WSS metrics for plaque presence (WT&gt;0.5mm) at follow up were 50% (low TAWSS), 48% (high OSI), 49% (high RRT), 47% (high CFI) and 43% (high transWSS). The PPVs for the presence of a fibroatheroma observed with histology were 61% (low TAWSS), 58% (high OSI), 61% (high RRT), 59% (high CFI) and 49% (high transWSS). Plaque growth vs. multidirectional WSS Conclusions This study shows that both low and multidirectional WSS promote the development of large and complex coronary atherosclerotic plaques with vulnerable characteristics. The high predictive values for fibrous cap atheroma development demonstrate the potential of multidirectional WSS metrics as a predictive clinical marker for vulnerable disease. Acknowledgement/Funding ERC- starter grant (grant agreement 310457)

5222A distinct LDL profile to predict the risk of cardiovascular disease in familial hypercholesterolemia subjects: initial pre-clinical results

Abstract Aim Cardiovascular disease development is highly variable between patients with familial hypercholesterolemia (FH). Since current risk prediction methods fail to estimate the risk of individual patients, all patients are treated with high-intensity lipid-lowering medication like statins and PCSK9-inhibitors. To prevent overtreatment (with its associated costs and side effects) of patients with a low risk, reliable biomarkers are urgently needed. While studying atherosclerosis development in an FH porcine model, we discovered a specific LDL profile that was directly associated with the severity of atherosclerosis development. Methods 10 adult pigs with an LDLR mutation received a high-fat diet for 12 months. Atherosclerosis development in the three main coronary arteries was monitored with intravascular ultrasound (IVUS) and optical coherence tomography at three time points. After sacrifice, tissue was harvested for detailed histological analysis of the coronary plaque composition. Size-exclusion chromatography (SEC, n=10) and LC-MSMS (n=4) were used to assess the plasma lipoprotein profile, and the sphingolipid content of LDL, relative to cholesterol. Results Imaging and histology revealed a marked difference in pigs that developed large, lumen intruding plaques (IVUS-based plaque burden 13–77%) within 9 months (n=5) and pigs (n=5) that only developed early lesions (IVUS-based plaque burden 8–34%), even after 12 months of follow-up. The plaques seen in the fast responding pigs possessed distinct characteristics of advanced disease (i.e. heavy macrophage infiltration, large lipid-rich necrotic cores, neovascularisation, micro- and macrocalcifications and intraplaque haemorrhage, see figure). In these fast responders, fibrous cap atheroma occupied 34% of the total artery. SEC revealed two distinct LDL subclasses: regular and “larger” LDL particles. Fast responding pigs with advanced atherosclerosis displayed a significantly higher ratio in cholesterol concentration of regular/'larger' LDL than slow responding pigs (1.7 (1.3–1.9) vs. 0.8 (0.6–1.2); (p=0.004), see figure). Compared to regular LDL, “larger” LDL contained relatively more sphingolipids in the fast responding than in the slow responding animals (regular LDL/'larger' LDL: S1P 0.5 (0.5–0.5) vs. 1.0 (0.8–1.2); Cer16:0 0.70 (0.67–0.73) vs. 1.04 (0.95–1.13); Cer18:0 0.60 (0.58–0.61) vs. 1.15 (1.13–1.16); Cer20:0 0.73 (0.73–0.74) vs. 0.94 (0.94–0.94)). “Larger” LDL particles and comparable sphingolipid ratios were also observed in FH patients. Cardiovascular data from our FH patient cohort, coupled to the LDL subclass distribution, will provide more insight into the potential of this novel biomarker. Conclusion A distinct difference in LDL subclasses, including a new “larger” LDL particle, was found in fast versus slow responding FH animals. This finding can potentially be used to identify FH patients at the highest risk of CVD to avoid overtreatement of low risk patients. Acknowledgement/Funding ERC- starter grant (grant agreement 310457)

The opioid receptor positive allosteric modulator BMS‐986187 enhances some, but not all, delta‐opioid receptor mediated behaviors

Activation of the delta opioid receptor (DOR) by orthosteric agonists produces antinociception, antihyperalgesia, antidepressant‐like effects, and convulsions in animal models. Positive allosteric modulators (PAMs) bind to a receptor site distinct from the orthosteric binding site and can augment the activity of the endogenous system. Therefore, a DOR PAM could be an effective strategy for eliciting potential therapeutic effects without convulsions. To evaluate this hypothesis, we investigated the ability of the opioid receptor PAM BMS‐986187 to elicit DOR‐mediated behaviors in mice alone or in combination with the DOR orthosteric agonist SNC80 and evaluated the potential contribution of multiple opioid receptor subtypes to the observed behavioral effects. Antidepressant‐like effects were evaluated in the mouse forced swim test and convulsions were evaluated by continuous observation. The contribution of DORs and mu‐opioid receptors (MORs) was evaluated through pharmacological and genetic manipulation of the receptor subtypes. BMS‐986187 alone produced antidepressant‐like effects without convulsions. These antidepressant‐like effects were blocked by the DOR antagonist naltrindole and absent in DOR knockout mice, indicating a DOR‐mediated effect. The antidepressant‐like effects of BMS‐986187 were enhanced in MOR knockout mice and following betaFNA pretreatment, suggesting that activation of MORs functionally inhibited DOR‐mediated antidepressant‐like effects. BMS‐986187 increased the potency of the orthosteric DOR agonist SNC80 to decrease immobility in the forced swim test but not to elicit convulsions. In addition, SNC80, but not BMS‐986187, enhanced the convulsive effects of pentylenetetrazol. Overall, these findings suggest that BMS‐986187 has activity at DORs and MORs in vivo and enhances some, but not all, behavioral effects of DOR agonists.Support or Funding InformationThis work was funded in part by a Research Starter Grant from the PhRMA Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cerebrospinal Fluid Transcriptional Analyses Reveals Upregulation of IL-17, Type 1 Interferon Transcriptional Pathways and Neutrophil Persistence Genes Associated with Increased Mortality from Pneumococcal Meningitis in Adults

Background: Improving outcomes from pneumococcal meningitis (PM), particularly in populations with high HIV prevalence, requires better understanding of host inflammatory responses to infection. Methods: We compared the transcriptome in pre-antibiotic cerebrospinal fluid (CSF) and blood from Malawian adults with PM using RNA sequencing. We used network analyses and cellular/process deconvolution of the transcriptome to identify important patho-physiological associations with outcome. Findings: Blood transcriptional profiles were obtained in 28 patients (21 HIV co-infected; median age 33 years [26-66]; median CSF WCC 28 cells/mm3 [0-3660]; median bacterial load 4.7x106 copies/ml CSF [671-2x109]; in-hospital mortality 64%), paired CSF profiles were obtained in 13. Marked differences in gene expression by outcome were confined to the CSF. In non-survivors, differentially expressed genes in the CSF were co-correlated in a network of pro-inflammatory gene-clusters enriched for collagen degradation and platelet degranulation. In contrast, CSF gene expression networks from surviving patients were dominated by DNA repair, transcriptional regulation and immunological signalling. CSF expression of gene response-modules for IL-17, Type 1 interferons and IL-10 were enriched in non-survivors, expression of cell-specific response-modules did not differ by outcome. However, genes for neutrophil chemotaxis and persistence were highly over-expressed in non-survivors. Interpretation: These data suggest poor outcome in PM is associated with over-expression of IL-17 and T1-IFN associated pro-inflammatory responses in the CSF and suggest a role for neutrophil-mediated inflammation. These responses are unlikely to be effected by current adjunctive treatments. Improving poor outcomes from PM will require better-targeted interventions. Registration Number: (Current Controlled Trials registration ISRCTN96218197). Funding Statement: This study was funded by a Clinical Lecturer Starter Grant from the Academy of Medical Sciences (UK), by a PhD Fellowship in Global Health to EW from the Wellcome Trust (089671/B/09/Z) and by a Postdoctoral Clinical Research Fellowship to GP from the Wellcome Trust (WT101766). The Malawi-Liverpool-Wellcome Trust Clinical Research Programme is supported by a core grant from the Wellcome Trust (101113/Z/13/Z). This work was undertaken at UCLH/UCL who received a proportion of funding from the National Institute for Health Research University College London Hospitals Department of Health’s NIHR Biomedical Research Centre. JSB, MN and CV are supported by the Centre’s funding scheme. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data and the final responsibility to submit for publication. Declaration of Interests: All authors have no conflicts of interest to declare. Ethics Approval Statement: All participants or nominated guardians gave written informed consent for inclusion. Ethical approval for the transcriptomics study was granted by both the College of Medicine Research and Ethics Committee (COMREC), University of Malawi, (P.01/10/980, January 2011), and the Liverpool School of Tropical Medicine Research Ethics Committee, UK (P10.70, November 2010) Committee, Liverpool, UK

The influence of hippocampal atrophy on the cognitive phenotype of dementia with Lewy bodies.

ObjectiveThe level of hippocampal atrophy in dementia with Lewy bodies (DLB) is typically less than that observed in Alzheimer's disease (AD). However, it is not known how the cognitive phenotype of DLB is influenced by hippocampal atrophy or the atrophy of adjacent medial temporal lobe structures.MethodsDementia with Lewy bodies (n = 65), AD (n = 76) and control (n = 63) participants underwent 3T magnetic resonance imaging and cognitive Cambridge Cognitive Examination and Mini‐Mental State Examination (CAMCOG and MMSE) assessments. Hippocampal volume, and parahippocampal, entorhinal and temporal pole cortical thickness, was compared between groups. Regression models were used to investigate whether hippocampal volume and cortical thickness associated with global cognition and cognitive subdomains.ResultsDementia with Lewy bodies, AD and control participants showed significantly different hippocampal, parahippocampal and entorhinal cortical thinning, where atrophy was greatest in AD and intermediate in DLB. Temporal pole thickness was reduced in DLB and AD compared with control participants. In DLB, but not AD, hippocampal volume associated with total CAMCOG, CAMCOG memory and MMSE scores. In DLB, parahippocampal, entorhinal and temporal pole thickness associated with total CAMCOG and CAMCOG memory scores, parahippocampal thickness associated with MMSE scores, and entorhinal thickness associated with CAMCOG executive function scores.ConclusionsIn this large sample, these results are in agreement with other studies indicating that hippocampal atrophy is less severe in DLB than AD. Hippocampal atrophy and medial temporal lobe cortical thickness were associated with the severity of cognitive symptoms, suggesting that atrophy in these structures, as a potential proxy of AD pathology, may partly mediate specific DLB cognitive symptoms. © 2017 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd.

ALS and Ubiquilin‐2: Effects of ALS Mutations on Ubiquilin‐2 Structure and Function

Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases are caused by malfunctions in ubiquitin‐mediated protein degradation pathways. Ubiquilin‐2 is a 624‐amino acid multi‐domain adaptor protein critical for maintaining protein homeostasis through the ubiquitin‐proteasome system, the endoplasmic reticulum‐associated protein degradation (ERAD) pathway and autophagy. Mutations in ubiquilin‐2 have recently been shown to cause dominant x‐linked inheritance of ALS and ALS/dementia. Moreover, wild‐type ubiquilin‐2 is present in protein inclusions found in patients diagnosed with either familial or sporadic ALS, further linking ubiquilin‐2 to the pathogenesis of ALS. Interestingly, most ALS‐linked mutations are localized to the proline‐rich repeat (Pxx) region that is unique to ubiquilin‐2 and not present in the other members of the ubiquilin protein family. The consequences of these mutations (specifically to residues P497, P506, P509, P525 and P533) on ubiquilin‐2's structure and function remain unknown. Towards that end, we are using biomolecular NMR spectroscopy and other biophysical techniques to characterize the Pxx region of ubiquilin‐2. 13C‐detect NMR experiments have allowed for the direct and necessary observation of the prolines as well as aided in resonance assignment for a significant portion of this ubiquilin‐2 region. Significantly, even though the Pxx region is intrinisically disordered, we observed sequence‐distant perturbations in several ALS‐linked mutants. Serendipitously, similar to some other ALS‐linked proteins such as TDP‐43 and hnRNPA1, our ubiquilin‐2 constructs exhibit salt and temperature dependent liquid‐like phase‐separation, a behavior that is also affected by ALS mutations. Our data suggest that the C‐terminal region of ubiquilin‐2 is important for modulating phase separation behavior. We are working towards modeling the structure of ubiquilin‐2 containing the Pxx region, as well as identifying protein‐binding surfaces involving the Pxx region.Support or Funding InformationALS Association Starter Grant to C.A.C.

Mining Human Prostate Cancer Datasets: The “camcAPP” Shiny App

Core CRUK funding: MD, AGL, ADL. Academy of Medical Sciences Clinical Lecturer Starter Grant SGCL11 (prinicipal funder of this work): ADL.

Starter grants and building your pilot data

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Novel circulating placental markers prokineticin-1, soluble fms-like tyrosine kinase-1, soluble endoglin and placental growth factor and association with late miscarriage.

Are novel circulating placental markers prokineticin-1 (PK-1), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng) and placental growth factor (PlGF) associated with late miscarriage in asymptomatic first trimester pregnant women? Increased serum sFlt-1 or PlGF, but not sEng or PK-1, were significantly associated with reduced miscarriage risk after adjustment for age, BMI, gestational age, smoking and blood pressure. Abnormal placental development is observed in two-thirds of miscarriages. Identifying women at high risk of late miscarriage could help diagnose potentially treatable causes of miscarriage such as infection, thrombosis or immunological disease. Recently, the circulating placental markers PK-1, sFlt-1, sEng and PlGF have been identified; however, it is not known if circulating levels of these markers are associated with late miscarriage. A single-centre observational cohort study with prospectively collected data was carried out at a tertiary care centre 2010-2012, in 993 asymptomatic pregnant women. Plasma PK-1, and serum sEng, sFlt-1 and PlGF were measured once in each patient during the antenatal booking visit, and pregnancy outcome was monitored prospectively. Less than 1% of patients were lost to follow-up. Multiples of median (MOM) levels were calculated to adjust for gestational age. Nine-hundred and ninety-three asymptomatic pregnant women attending antenatal clinic for a routine booking antenatal appointment were recruited to the study, of whom 12 were lost to follow-up and excluded from analysis. Of the cohort, 50 of the remaining 981 women suffered late miscarriage. Gestation-adjusted sEng, sFlt-1 and PlGF levels were 11% (P<0.01), 36% (P<0.001) and 30% (P<0.001), respectively, lower in women who later suffered miscarriage compared with unaffected pregnancies, while PK-1 did not differ significantly. Logistic regression modelling suggested that increased sFlt-1 (odds ratio (OR) 0.15 95% confidence interval [0.08-0.26], P=0.0001) and PlGF (OR 0.02 [0.01-0.05], P=0.0001), but not sEng, were associated with reduced miscarriage risk after adjustment for age, BMI, gestational age, smoking and blood pressure. The combination of sFlt-1 and PlGF did not improve the diagnostic accuracy beyond the use of sFlt-1. First trimester levels of sFlt-1 and PlGF, but not sEng or PK-1, were associated with late miscarriage risk in asymptomatic women. However, a new prospective study is now required to investigate the utility of these markers to predict early (<10 weeks) and late miscarriage, as well as to predict other complications of pregnancy. Our data suggest that circulating sFlt-1 and PlGF, but not sEng or PK-1, are independently associated with late miscarriage risk in asymptomatic pregnant women attending their antenatal visit. Therefore, sFlt-1 and PlGF may represent novel markers of placental viability. These data further our understanding of placental function, and have important potential implications for utilizing novel hormonal markers to detect adverse clinical outcomes during pregnancy. The authors have no competing interests. The Section of Investigative Medicine is funded by grants from the MRC, BBSRC, NIHR, an Integrative Mammalian Biology (IMB) Capacity Building Award, an FP7-HEALTH-2009-241592 EuroCHIP grant and is supported by the NIHR Imperial Biomedical Research Centre Funding Scheme. This project was funded by an NIHR grant (reference: CDF-2009-02-05). The following authors are also funded as follows: CNJ is supported by an NIHR Clinical Lectureship and AMS/ Wellcome Starter Grant for Clinical Lecturers. AA and ANC are supported by NIHR academic clinical lectureships. CI-E is supported by an Imperial College Healthcare NHS Trust Charity Research Fellowship. WSD is supported by an NIHR Career Development Fellowship. Q0406/80.

The changing funding environment for clinical academics

The changing funding environment for clinical academics

Computational scoring system to predict HLA immunogenicity

BackgroundOur preliminary work has indicated that donor HLA immunogenicity in kidney and haemopoietic stem-cell transplantation might be predicted by assessment of their aminoacid sequence and physicochemical properties. We have now created a novel computational algorithm to quantify structural and surface electrostatic potential differences between donor and recipient HLA and applied it to predict alloantibody responses in a unique patient cohort. MethodsWe examined 141 patients undergoing treatment for infertility with lymphocyte immunotherapy. Patients were injected subcutaneously with partners' lymphocytes, and serum samples were collected before and after immunotherapy to assess sensitisation. After four-digit HLA typing and HLA structural modelling, donor–recipient HLA comparisons were performed to determine electrostatic mismatch score (EMS-3D) and assess its ability to predict development of donor-specific alloantibody and overall sensitisation to HLA after immunotherapy. Overall sensitisation was expressed as calculated reaction frequency (cRF), a measure that denotes incompatibility with a UK pool of 10 000 organ donors. FindingsDonor EMS-3D was a strong predictor of overall HLA class I and class II sensitisation risk (cRF >15%) (odds ratio [OR] 5·68 per unit increase, 95% CI 1·84–17·49; p=0·002) with a receiver operating characteristic area under curve score of 0·68 for cRF of more than 15% (p=0·0046) and 0·71 for cRF more than 85% (p<0·0001). Donor HLAs with the highest EMS-3D (fourth quartile) were associated with a higher risk of development of donor-specific alloantibody both for HLA class I (OR 2·90, 95% CI 1·62–5·23; p=0·031) and for HLA class II (16·07, 9·11–142·68; p<0·0001). Physicochemical differences between donor and recipient HLA-DQ were strong predictors of developing HLA-DQ donor-specific alloantibody (30·00, 6·33–87·73; p<0·0001). InterpretationOur study provides compelling evidence that donor HLA immunogenicity can be predicted by assessment of their structural and physicochemical disparities to recipient HLA. If confirmed in larger studies, our findings might enable better assessment of transplant immunological risk before transplantation and could inform future policies on allocation of deceased-donor kidney and haemopoietic stem-cell transplants to maximise the benefits of transplantation. FundingRoyal College of Surgeons of England Research Fellowship, Kiel University Starter Grant, Evelyn Trust, Academy of Medical Sciences.

Role of Eros, a novel transmembrane protein, in regulation of host defence

BackgroundReactive oxygen species (ROS), generated via the phagocyte NADPH oxidase cytochrome b558, are essential for effective immune responses to common and serious pathogens. The phagocyte NADPH oxidase is a multisubunit protein complex and deficiency of either the membrane bound or cytoplasmic components leads to chronic granulomatous disease, a serious and often fatal illness characterised by recurrent infections and autoimmunity. Moreover, abnormal generation of ROS has been implicated in the pathogenesis of multigenic autoimmune diseases such as systemic lupus erythematosus. Eros (essential for reactive oxygen species), encoded by bc017643, is a novel transmembrane protein that is highly expressed in the immune system and highly conserved in evolution but has no previously identified function. Eros is an orthologue of the plant protein Ycf4, necessary for expression of proteins of the photosynthetic photosystem 1 complex, an NADPH oxio-reductase complex. We elucidated its role in infection in mice. MethodsROS are essential for host defence against the serious bacterial pathogen Salmonella enterica serovar Typhimurium. We screened individual knockout mice (Wellcome Trust Knockout mouse project) for susceptibility to salmonella infection. Having identified mice deficient in Eros as being highly susceptible to salmonella, we used ex-vivo approaches including reactive oxygen burst assays and western blot, to characterise their defect further. FindingsWe found that Eros was essential for host defence to infection. Eros was crucial for generating reactive oxygen species through regulation of the essential NADPH oxidase components, gp91 and p22. Eros-deficient mice expressed almost no gp91 and p22 in neutrophils and macrophages secondary to accelerated degradation in the absence of Eros. As a result Eros-deficient mice died rapidly after infection with salmonella or listeria. Eros also regulated the ROS-dependent formation of neutrophil extracellular traps and melanoma metastases. InterpretationWe have found a a key role for Eros in regulating host defence. The finding that Eros-deficient mice lack gp91 and p22 at the protein, though not mRNA, level shows how these key components of the reatcive oxygen burst are protected from degradation and furthers our understanding of reactive oxygen burst biology. Eros is highly conserved between mouse and man so it is likely that it also has a crucial role in human immunity. FundingWellcome Trust, Academy of Medical Sciences starter grant.

Urinary congophilia in women with pre-eclampsia and chronic kidney disease

BackgroundPre-eclampsia remains a major cause of fetal and maternal morbidity. Researchers have reported that urinary congophilia could be a useful test in the diagnosis and prediction of the disorder. Congophilia is thought to indicate the presence of amyloid protein, an aggregate of misfolded proteins implicated through placental endoplasmic reticulum stress in pre-eclampsia. We aimed to determine whether congophilia is a specific diagnostic test for pre-eclampsia and superimposed pre-eclampsia. MethodsWe analysed samples from women with pre-eclampsia (n=23), gestational hypertension (10), chronic kidney disease (28), chronic hypertension (14), chronic kidney disease with superimposed pre-eclampsia (5), chronic hypertension with superimposed pre-eclampsia (12), and healthy pregnant controls (31). Samples from healthy non-pregnant controls (n=10) and non-pregnant women with systemic lupus erythematosus either with (25) or without lupus nephritis (14) were analysed. Urinary congophilia was quantified by measurement of congo red retention (CRR) and compared between groups. FindingsA significant increase in congophilia was detected in urine from women with pre-eclampsia (median CRR 47%, IQR 22–68) compared with healthy pregnant controls (16, 13–21; p=0·002), women with gestational hypertension (20, 13–27; p=0·008), or women with chronic hypertension (17, 12–28; p=0·01). Congophilia was also present in pregnant women with chronic kidney disease and was not significantly different from those with pre-eclampsia or women with chronic kidney disease and superimposed pre-eclampsia. Non-pregnant women with lupus nephritis had significantly higher congophilia retention than did non-pregnant controls (38%, 17–73 vs 9, 7–11; p<0·0001) and non-pregnant women with systemic lupus erythematosus but no lupus nephritis (13, 11–17; p=0·001). InterpretationThis study confirms the presence of urinary congophilia, as assessed by CRR, in women with pre-eclampsia but demonstrates that it is similarly present in other conditions associated with renal impairment in pregnant and non-pregnant women. FundingThis work was supported by funding from Tommy's Charity UK, an Academy of Medical Sciences UK Starter Grant for Clinical Lecturers, and the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London. FPM is funded by a NIHR Clinical Academic Fellowship.

Delivery of endothelial colony-forming cells in dermal scaffolds to promote vascularisation and wound healing

BackgroundWound healing requires a complex interplay of processes, with development of a functional vascular supply central to achieving healing. Delivery of stem cells requires both the appropriate cell type and a suitable method to support cells and protect them in the potentially hostile wound environment until vasculogenesis has been established. We aimed to investigate the efficacy of a characterised subpopulation of stem cells to promote wound healing, when delivered in a dermal scaffold. MethodsEndothelial colony-forming cells (CD31+, CD105+, vWF+, VEGFR-2+, CD14–, CD45–) were labelled and seeded onto a commercially available dermal substitute (MatriDerm (R), MedSkin Solutions Dr Suwelack, Billerbeck, Germany). After 72 h in culture, microtubular structures were formed. Full thickness wounds (6 mm diameter, two wounds per animal) were created on the dorsum of athymic nude mice. Control animals received no treatment. Treatment groups had application of either the scaffold alone or the scaffold-cell construct to the wounds. Wound closure was determined by measurement of serial images, and expressed as percentage closure. Wound blood flow was determined with laser doppler imaging, and expressed as a percentage of the baseline (day 0) measurement of blood flow. FindingsWound healing, expressed as the percentage closure of the wound, was similar between the control group (no scaffold) and the group treated with scaffold alone, with no difference in percentage wound healing up to 28 days after wounding. There was a significant increase in wound healing in the group treated with the scaffold-cell construct compared with scaffold-only control at day 2 (mean percentage closure 33·6 [SE 6·6] vs 12·4 [1·49]), day 4 (39·3 [6·7] vs 17·3 [2·2]), and day 6 post wounding (68·8 [2·2] vs 40·6 [5·1]) (p<0·0001, two-way ANOVA grouped analysis). Preliminary results show that wound blood flow was significantly increased 2 days after wounding (p=0·0134, unpaired t test) in wounds treated with scaffold-cell constructs, compared with a control group (mean blood flow in scaffold-cell group 150% of baseline [SE 7·1] vs 108% of baseline [10·1]). Wound blood flow was similar between groups at day 4 and day 6 post wounding. InterpretationThese early results indicate that wound healing can be promoted by introducing endothelial colony-forming cells in a dermal scaffold, and that this scaffold could be a useful adjunct to wound healing. We plan to further investigate the efficacy of using a scaffold-tubule construct to promote healing in skin grafts over complex wounds. FundingAcademy of Medical Sciences Starter Grant.