The molecular mechanisms of streptococcus gordonii-platelet interactions involved in the pathogenesis of cardiovascular infection

Numerous studies have implicated bacteria in cardiovascular disease; however the mechanisms involved have yet to be elucidated. Infective Endocarditis (IE) is characterised by the formation of platelet-bacteria thrombi on a heart valve which, if untreated, can lead to valve failure or the formation of infected emboli. The oral bacterium, Streptococcus gordonii, is amongst the most common pathogens isolated from IE patients. S. gordonii is a member of the group of oral bacteria that occur primarily in dental plaque. The ability of S. gordonii to contribute to a growing platelet fibrin thrombus is a critical step in the development of IE, however the molecular mechanisms of rapid platelet recruitment and activation is poorly understood. Here we describe novel interactions between S. gordonii and human blood platelets. Previous studies have demonstrated that platelets roll on immobilised S. gordonii through a unique interaction between S. gordonii surface protein Hsa and platelet GPlba (Takahashi et al., 2004). Following rolling platelets eventually come to a stop and firmly adhere to the S. gordonii. The identity of the protein responsible for supporting firm adhesion is unknown. Here we describe the identification of a novel cell wall protein, platelet adherence protein A (PadA) which contains a domain homologous to the A1 domain of von Willebrand Factor. A PadA deficient mutant (ApadA) induced platelet aggregation to the same extent as the parent S. gordoniistrain DLI. However, the ability of ApadA to adhere to platelets was significantly reduced. Adherence of ApadA to immobilised glycocalicin (purified GPlba) was unaffected as compared to DL1 whereas adhesion to immobilised allbP3 was greatly reduced. Parent strain DL1 adhered to CHO cells stably transfected with allbP3, however ApadA failed to do so. Strain DL1 adhesion to CHO allbP3 cells was inhibited by an RGD peptide and abciximab. These results describe the identification of a novel S. gordonii cell wall protein PadA, which binds to platelet allbP3. lmmunoreceptor tyrosine-based activation motif containing proteins such as FcyRlla, have been shown to play an essential role in transmitting activating signals into platelets following firm adhesion in order to amplify and strengthen the interaction (Boylan et al., 2008). When platelets adhered to S. gordonii strong tyrosine phosphorylation of the ITAM region of FcyRlla, as well as phosphorylation of the downstream effectors, Syk and phospholipase Cy2 was observed. Inhibition of either allbP3 or FcyRlla ablated dense granule release and platelet spreading. These results suggest that when platelets adhere to immobilised S. gordonii it results in the generation of an outside-in signal that induces cytoplasmic-dependent phosphorylation of FcyRlla which leads to platelet spreading and dense granule release. S. gordonii maintains the ability to induce platelet aggregation following the deletion of Hsa and PadA from the bacterial surface, suggesting that other bacterial proteins present are capable of inducing platelet aggregation. Using a proteomic approach we identified several potential candidates of interest. Peptide searches identified streptococcal surface protein AIB (SspAIB) proteins present in the aggregating strain DL1 and missing in the nonaggregating strain Blackburn. Lactococcus lactis does not induce platelet aggregation, therefore acts as a good surrogate host for expression of proteins of interest. L. lactis expressing SspNB induced an allbP3d ependent platelet aggregation similar to that seen with DLI. L. lactis expressing SspNB failed to support platelet adhesion. These results highlight the specific nature of SspNB in inducing platelet aggregation. Another protein identified of particular interest was fructose bisphophate aldolase (FBA), as it has recently been shown to bind lung epithelial cells through flamingo cadherin. We purified recombinant FBA (rFBA) and expressed FBA in L. lactis. L. lactis FBA and rFBA did not induce platelet aggregation or support platelet adhesion, suggesting S. gordonii-induced platelet aggregation is a multifactorial event and FBA alone is not sufficient to induce platelet aggregation. In this thesis we describe several interactions between S. gordonii and platelets which we believe play critical roles in the development of a growing thrombus in IE patients. Unravelling the mechanisms involved in platelet bacterial interactions may aid in the development of novel or improved therapeutics for the treatment of blood borne disease

Leveraging Lean in construction: A case study of a BIM-based HVAC manufacturing process

The impetus towards efficiency in the AECO (Architecture, Engineering, Construction & Operations) sector is driving the implementation of Lean practices. BIM technologies and BIM processes provide methods by which this can be achieved. Major clients of building services contractors have begun to mandate the use of BIM and some are using BIM preparedness/experience as pre-tender qualification criteria. In this case study, an initial review has been conducted of the achievements of a major Irish M&E contractor in implementing BIM. The firm purpose-built a facility for the off-site manufacture of building services components. The operations of the plant are efficient and qualityassured through the use of an appropriately skilled workforce at all stages of manufacture, and tracking software that has developed as the knowledge of the contractor grew. Standardised processes have been developed which have resulted in greater efficiencies and lower costs for the contractor as a result of fewer requirements for onsite modifications (such as those caused by clashes), less waste, and greater flexibility. Despite some initial objections, the employees of the company are now more satisfied with their working conditions and are, as a result, more productive. Through investment in BIM-based, Lean processes, the contractor can now better compete when tenerding for large-scale projects in Ireland and worldwide, including the rapidly-increasing number where BIM experience and preparedness is mandated

Evaluating an automated machine learning model that predicts visual acuity outcomes in patients with neovascular age-related macular degeneration

PURPOSE: Neovascular age-related macular degeneration (nAMD) is a major global cause of blindness. Whilst anti-vascular endothelial growth factor (anti-VEGF) treatment is effective, response varies considerably between individuals. Thus, patients face substantial uncertainty regarding their future ability to perform daily tasks. In this study, we evaluate the performance of an automated machine learning (AutoML) model which predicts visual acuity (VA) outcomes in patients receiving treatment for nAMD, in comparison to a manually coded model built using the same dataset. Furthermore, we evaluate model performance across ethnic groups and analyse how the models reach their predictions. METHODS: Binary classification models were trained to predict whether patients' VA would be 'Above' or 'Below' a score of 70 one year after initiating treatment, measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. The AutoML model was built using the Google Cloud Platform, whilst the bespoke model was trained using an XGBoost framework. Models were compared and analysed using the What-if Tool (WIT), a novel model-agnostic interpretability tool. RESULTS: Our study included 1631 eyes from patients attending Moorfields Eye Hospital. The AutoML model (area under the curve [AUC], 0.849) achieved a highly similar performance to the XGBoost model (AUC, 0.847). Using the WIT, we found that the models over-predicted negative outcomes in Asian patients and performed worse in those with an ethnic category of Other. Baseline VA, age and ethnicity were the most important determinants of model predictions. Partial dependence plot analysis revealed a sigmoidal relationship between baseline VA and the probability of an outcome of 'Above'. CONCLUSION: We have described and validated an AutoML-WIT pipeline which enables clinicians with minimal coding skills to match the performance of a state-of-the-art algorithm and obtain explainable predictions

Assessment of Inactivating Stop Codon Mutations in Forty Saccharomyces cerevisiae Strains: Implications for [PSI+] Prion- Mediated Phenotypes

The yeast prion [PSI+] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI+] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI+] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI+]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI+]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth

Incidence of thromboembolism following detection by trans-oesophageal echocardiography of left atrial thrombus

Left atrial appendage (LAA) thrombus is an accepted risk factor for ischemic stroke. Following a literature review we were unable to identify a study that determined the incidence of ischemic stroke in patients with a confirmed LAA thrombus. The purpose of this study was to establish the incidence of ischemic stroke in patients with a LAA thrombus confirmed on trans-oesophageal echocardiography (TOE). A ten year retrospective single centre study was conducted for the period March 2005 to February 2014 in St. Vincents University Hospital, Ireland. All TOE studies performed during this period were reviewed. A chart review was carried out on any patient who had a LAA thrombus, left atrial (LA) thrombus or pre-thrombus state identified. Charts were reviewed for documented neurological deficits consistent with ischemic stroke or transient ischemic attack within six months following TOE study. Overall 1903 TOE studies were reviewed. A total of 67 TOE studies detected a LAA thrombus, LA thrombus or pre-thrombus state. In the days prior to TOE, an ischemic stroke had occurred in two of the patients. Following detection of thrombus or pre-thrombus state on TOE and optimization of oral anti-coagulation (OAC), no patient had an ischemic stroke over the subsequent six months. This is the only study to date that has looked at the incidence of ischemic stroke following a confirmed LAA thrombus, LA thrombus or pre-thrombus state. This single centre study found low stroke rates over a six month follow-up period in patients with a confirmed LAA thrombus, LA thrombus or pre-thrombus state and optimization of OAC. Larger studies would be required to confirm these findings

Incidence of thromboembolism following detection by trans-oesophageal echocardiography of left atrial thrombus

Background: Left atrial appendage (LAA) thrombus is an accepted risk factor for ischemic stroke. Following a literature review we were unable to identify a study that determined the incidence of ischemic stroke in patients with a confirmed LAA thrombus. The purpose of this study was to establish the incidence of ischemic stroke in patients with a LAA thrombus confirmed on trans-oesophageal echocardiography (TOE). Method: A ten year retrospective single centre study was conducted for the period March 2005 to February 2014 in St. Vincent's University Hospital, Ireland. All TOE studies performed during this period were reviewed. A chart review was carried out on any patient who had a LAA thrombus, left atrial (LA) thrombus or pre-thrombus state identified. Charts were reviewed for documented neurological deficits consistent with ischemic stroke or transient ischemic attack within six months following TOE study. Results: Overall 1903 TOE studies were reviewed. A total of 67 TOE studies detected a LAA thrombus, LA thrombus or pre-thrombus state. In the days prior to TOE, an ischemic stroke had occurred in two of the patients. Following detection of thrombus or pre-thrombus state on TOE and optimization of oral anti-coagulation (OAC), no patient had an ischemic stroke over the subsequent six months. Conclusion: This is the only study to date that has looked at the incidence of ischemic stroke following a confirmed LAA thrombus, LA thrombus or pre-thrombus state. This single centre study found low stroke rates over a six month follow-up period in patients with a confirmed LAA thrombus, LA thrombus or pre-thrombus state and optimization of OAC. Larger studies would be required to confirm these findings

Elacytarabine: lipid vector technology under investigation in acute myeloid leukemia

Cytosine arabinoside (cytarabine or Ara-C) has been one of the cornerstones of treatment of acute myeloid leukemia since its approval in 1969. Standard induction therapy worldwide for all patients deemed fit for treatment (excluding those with acute promyelocytic leukemia) remains unchanged for over 40 years and consists of Ara-C administered by continuous infusion in combination with a topoisomerase II inhibitor (e.g., daunorubicin, idarubicin and mitoxantrone). Despite decades of clinical investigation, the optimum dose of both agents still remains unclear. Although higher doses of Ara-C have been shown to improve response rates, the elderly poorly tolerate these regimens. Resistance mechanisms also develop or may be present at diagnosis resulting in poor outcomes. Elacytarabine (CP-4055), an elaidic acid ester of Ara-C, has been developed using lipid vector technology in an attempt to overcome these limitations. Clinical data are encouraging, with evidence suggesting that this novel agent is circumventing resistance mechanisms but retaining the potent antileukemic efficacy of Ara-C

EPHA3 as a novel therapeutic target in the hematological malignancies

The Eph receptors are the largest family of tyrosine kinases and are of increasing interest in developmental therapeutics. Their unique method of interaction with their ligands, the ephrins, via bidirectional signaling, and their variable expression in different tissues are well documented. Ephs are upregulated in, and critical to, embryological processes, most notably development of the neurological system. They are central in many processes involving cell motility and adhesion. Recent findings on elevated expression of Eph receptors in human malignancies as well as in stem cell environments are of particular interest. With increasing focus on molecularly targeted anticancer therapies, exploration of the potential of Eph receptors as therapeutic targets in both solid and hematologic malignancies has begun. The most promising of the Eph receptors in this regard is EPHA3, which is overexpressed in many hematologic malignancies. Preclinical data support the value of pursuing this target for further development, and lead compounds are now entering the clinic

Review of EU airport energy interests and priorities with respect to ICT, energy efficiency and enhanced building operation

This paper gives an overview on EU airport energy interests and priorities with respect to ICT, energy efficiency and enhanced building operation. To achieve this objective the paper begins with an overview on airports role on energy consumption, then novel review of airport energy consumption figures and energy efficiency actions at the EU level is presented. The research covers also interest and requirements of two Italian airports (MXP and FCO) in relation to enhanced operation which include: sub-metering and visualization needed to better understand the end energy use, data analysis for benchmarking and correlation with operational and weather data, action management for maintenance operation support.AcknowledgementsThis research was funded by the Irish Research Council for Science, Engineering & Technology (IRCSET), D'Appolonia s.p.a. and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 284920.peer-reviewe