All hands on deck during the COVID-19 pandemic. Maintaining face-to-face medical education and clinical placements.

Medical students must have robust educational experiences, graduate and commence timely employment. Here, we describe how the Royal College of Surgeons in Ireland (RCSI) delivered clinical placements in medical programmes over the first three waves of the COVID-19 pandemic in Ireland, including a student-centred, holistic approach to students' educational, social and health needs with strong student involvement, re-organising the university's primary care team, developing COVID-19 PCR testing on site and re-focusing communications and student services. This resulted in re-arranging the students into learning communities, and students and staff electronically recorded their COVID-19 symptom status daily. In-person observed structured clinical examination and other clinical exams progressed. No lockdown of any campus occurred. Over the two senior years, 693 students completed 15,000 weeks of clinical and experiential learning across 104 sites, similar to previous years, including anatomy practicals, procedural skills training, simulated ward rounds and patient encounters, case-based presentations and small group tutorials. The compliance rate with the daily symptom tracker was 91%. The percentage response rate and the number of students providing feedback from October 2020 to April 2021 was as high as 50%. The overall response rate was 33%. By mid-May, 93-95% of students in the two senior years had had at least one dose of the SARS-CoV-2 vaccine, with 99% fully vaccinated by the start of the next academic year in autumn 2021. Over the period of testing for SARS-CoV-2, just over 22,000 samples were processed, of which 0.79% were positive; no medical student acquired COVID-19 or was associated with nosocomial transmission. The total investment by the RCSI in Dublin, was €9.3m (€1.2 in capital expenditure and €8.1 in operational expenses). Continuing face-to-face clinical placements during a pandemic was possible through a multi-model approach that prioritised two-way communication, compliance with national public health advice and student screening.

Molecular Cross-Talk between Integrins and Cadherins Leads to a Loss of Vascular Barrier Integrity during SARS-CoV-2 Infection.

The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins are emerging as possible receptor candidates for SARS-CoV-2, and their complex intracellular signaling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation by hijacking integrin αVß3, expressed on human endothelial cells. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin αVß3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein-through its RGD motif-binds to αVß3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.

Concordance between PCR-based extraction-free saliva and nasopharyngeal swabs for SARS-CoV-2 testing.

Introduction: Saliva represents a less invasive alternative to nasopharyngeal swab (NPS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. SalivaDirect is a nucleic acid extraction-free method for detecting SARS-CoV2 in saliva specimens. Studies evaluating the concordance of gold standard NPS and newly developed SalivaDirect protocols are limited. The aim of our study was to assess SalivaDirect as an alternative method for COVID-19 testing. Methods: Matching NPS and saliva samples were analysed from a cohort of symptomatic (n=127) and asymptomatic (n=181) participants recruited from hospital and university settings, respectively. RNA was extracted from NPS while saliva samples were subjected to the SalivaDirect protocol before RT-qPCR analysis. The presence of SARS-Cov-2 was assessed using RdRp and N1 gene targets in NPS and saliva, respectively. Results: Overall we observed 94.3% sensitivity (95% CI 87.2-97.5%), and 95.9% specificity (95% CI 92.4-97.8%) in saliva when compared to matching NPS samples. Analysis of concordance demonstrated 95.5% accuracy overall for the saliva test relative to NPS, and a very high level of agreement (κ coefficient = 0.889, 95% CI 0.833-0.946) between the two sets of specimens. Fourteen of 308 samples were discordant, all from symptomatic patients. Ct values were >30 in 13/14 and >35 in 6/14 samples. No significant difference was found in the Ct values of matching NPS and saliva sample ( p=0.860). A highly significant correlation (r = 0.475, p<0.0001) was also found between the Ct values of the concordant positive saliva and NPS specimens. Conclusions: Use of saliva processed according to the SalivaDirect protocol represents a valid method to detect SARS-CoV-2. Accurate and less invasive saliva screening is an attractive alternative to current testing methods based on NPS and would afford greater capacity to test asymptomatic populations especially in the context of frequent testing.

Staphylococcus aureus protein A causes osteoblasts to hyper-mineralise in a 3D extra-cellular matrix environment.

Osteomyelitis is an inflammatory bone infection that is caused most commonly by the opportunistic pathogen Staphylococcus aureus. Research into staphylococcal induced bone infection is typically conducted using traditional 2D in vitro culture settings, which is not fully representative of the dynamic in vivo environment. In this study we utilised a collagen glycosaminoglycan scaffold, previously developed for bone tissue engineering, as a representative 3D model of infection. The scaffold resisted degradation and retained its pore structure, which is important for cellular function and survival, when seeded with both cells and bacteria. Using this model, we showed that in the presence of S. aureus, osteoblast proliferation was reduced over 21 days. Interestingly however these cells were more metabolically active compared to the uninfected cells and demonstrated increased mineralisation. Protein A (SpA) is a virulence factor found on the surface of S. aureus and has been shown to interact with osteoblasts. When SpA was removed from the surface of S. aureus, the osteoblasts show comparable activity with the uninfected cells-demonstrating the importance of SpA in the interaction between bone cells and S. aureus. Our results suggest that infected osteoblasts are capable of over-compensating for bone loss and bone destruction by increasing mineralisation in a 3D environment, key elements required for ensuring bone strength. It also reinforces our previously established result that S. aureus SpA is a critical mediator in osteomyelitis and might be a potential novel drug target to treat osteomyelitis by preventing the interaction between S. aureus and osteoblasts.

Inhibition of Vascular Endothelial Cell Leak Following Escherichia coli Attachment in an Experimental Model of Sepsis.

OBJECTIVES: The vascular endothelium is a major target of sepsis-induced events, and endothelial activation accounts for much of the pathology of sepsis. Urinary tract infections and pneumonia caused by Escherichia coli are among of the most common infections causing sepsis in both community and hospital settings. Currently, there are no approved drugs on the market to treat the underlying pathophysiology of sepsis. The aim of this study is to elucidate the molecular mechanism by which E. coli induces endothelial injury as a result of attachment. DESIGN: Laboratory research using a hemodynamic perfusion ex vivo model. SETTING: Research Laboratories of Royal College of Surgeons in Ireland and Beaumont Hospital. PATIENTS: Ex vivo human vascular endothelial cells. INTERVENTIONS: Addition of αVß3 antagonist, cilengitide. MEASUREMENTS AND MAIN RESULTS: Clinical strains of E. coli isolated from patients with sepsis bound to sheared human endothelial cells under static and hemodynamic shear conditions. Binding was dependent on E. coli cell membrane protein outer membrane protein A attaching directly to endothelial cell integrin αVß3. Attachment resulted in disturbances in endothelial barrier integrity, as determined by loss of tight junction protein staining, permeability changes, and ultimately cell death by apoptosis. Using a low concentration of the αVß3 antagonist cilengitide or using a strain deficient in outer membrane protein A resulted in a significant reduction in endothelial dysfunction following infection. CONCLUSIONS: Inhibition of E. coli binding to endothelial cell αVß3 by cilengitide prevents endothelial dysfunction and may, therefore, present as a novel early therapeutic for the treatment of sepsis.

Electroconductive Biohybrid Collagen/Pristine Graphene Composite Biomaterials with Enhanced Biological Activity.

Electroconductive substrates are emerging as promising functional materials for biomedical applications. Here, the development of biohybrids of collagen and pristine graphene that effectively harness both the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching native cardiac tissue) obtainable with pristine graphene is reported. As well as improving substrate physical properties, the addition of pristine graphene also enhances human cardiac fibroblast growth while simultaneously inhibiting bacterial attachment (Staphylococcus aureus). When embryonic-stem-cell-derived cardiomyocytes (ESC-CMs) are cultured on the substrates, biohybrids containing 32 wt% graphene significantly increase metabolic activity and cross-striated sarcomeric structures, indicative of the improved substrate suitability. By then applying electrical stimulation to these conductive biohybrid substrates, an enhancement of the alignment and maturation of the ESC-CMs is achieved. While this in vitro work has clearly shown the potential of these materials to be translated for cardiac applications, it is proposed that these graphene-based biohybrid platforms have potential for a myriad of other applications-particularly in electrically sensitive tissues, such as neural and neural and musculoskeletal tissues.

The Evolving Role of MicroRNAs in Endothelial Cell Dysfunction in Response to Infection.

The microRNAs are short noncoding RNA molecules responsible for translational repression and silencing of target genes via binding to the mRNA. They are found in all eukaryotic cells and play a critical role in virtually all physiological processes, including within the cardiovascular system where they influence cellular development, differentiation, cardiovascular function, hemostasis, and programmed cell death. Dysregulated microRNA expression is associated with several conditions ranging from cancer and autoimmune disease to infection. Progressively, it has become increasingly clear that microRNAs are important components of the host response to microbes. The cardiovascular system, coupled with cells of the innate immune system, provide the initial interaction and first response to microbial infection, respectively. This review presents the current state of knowledge regarding the role of microRNAs with emphasis on their role in controlling endothelial cell function.

Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix.

A range of Streptococcus bacteria are able to interact with blood platelets to form a thrombus (clot). Streptococcus gordonii is ubiquitous within the human oral cavity and amongst the common pathogens isolated from subjects with infective endocarditis. Two cell surface proteins, Hsa and Platelet adherence protein A (PadA), in S. gordonii mediate adherence and activation of platelets. In this study, we demonstrate that PadA binds activated platelets and that an NGR (Asparagine-Glycine-Arginine) motif within a 657 amino acid residue N-terminal fragment of PadA is responsible for this, together with two other integrin-like recognition motifs RGT and AGD. PadA also acts in concert with Hsa to mediate binding of S. gordonii to cellular fibronectin and vitronectin, and to promote formation of biofilms. Evidence is presented that PadA and Hsa are each reliant on the other's active presentation on the bacterial cell surface, suggesting cooperativity in functions impacting both colonization and pathogenesis.

Multiple sites on Streptococcus gordonii surface protein PadA bind to platelet GPIIbIIIa.

Infective endocarditis is a life threatening disease caused by a bacterial infection of the endocardial surfaces of the heart. The oral pathogen, Streptococcus gordonii is amongst the most common pathogens isolated from infective endocarditis patients. Previously we identified a novel cell wall protein expressed on S. gordonii called platelet adherence protein A (PadA) that specifically interacts with platelet GPIIb/IIIa. The interaction between PadA and GPIIb/IIIa resulted in firm platelet adhesion, dense granule secretion and platelet spreading on immobilised S. gordonii. This study set out to identify specific motifs on the PadA protein that interacts with platelet GPIIb/IIIa. Proteomic analysis of the PadA protein identified two short amino acid motifs which have been previously shown to be important for fibrinogen binding to GPIIb/IIIa and contributing to the generation of outside-in signalling. Site directed mutagenesis on the PadA protein in which 454AGD was substituted to AAA, and the 383RGT was substituted to AAA suggests the RGT motif has no role in supporting platelet adhesion however plays a role in dense granule secretion and platelet spreading. In contrast to this the AGD motif has no role to play in supporting firm platelet adhesion or dense granule secretion however plays a role in platelet spreading. These results suggest that multiple sites on S. gordonii PadA interact with GPIIb/IIIa to mediate a number of platelet responses that likely contribute to the thrombotic complications of infective endocarditis.

Investigation of the interaction of biodegradable micro- and nanoparticulate drug delivery systems with platelets.

OBJECTIVES: Biodegradable micro- and nanoparticles are being increasingly investigated for drug delivery and targeting of therapeutics. The size and surface properties of these particles are important factors influencing their interaction and uptake by various cells, tissues and organs. Optimising these properties, to enhance cellular uptake, may increase their potential for interaction with other physiological components such as platelets resulting in platelet activation and inappropriate thrombus formation. The aim of this study was to investigate the potential interaction of particulates with platelets. METHODS: Biodegradable micro- and nanoparticles based on poly-lactide-co-glycolide (PLGA), poly-lactide-co-glycolide-macrogol (PLGA-macrogol) and chitosan were prepared using solvent evaporation, spray drying or solvent dispersion techniques. KEY FINDINGS: Microparticles formulated had a median diameter (D50%) of 2-9 µm, while nanoparticles had an average diameter of 100-500 nm. The surface morphology ranged from smooth and spherical to irregular depending on polymer and preparation method used. Particles, reconstituted in the concentration range of 0.1-500 µg/ml, were tested for their ability to induce or inhibit platelet aggregation. No effects on either induction of platelet activity or inhibition of aggregation were detected. CONCLUSIONS: None of the particles examined were found to alter platelet activity. These results suggested that the biodegradable micro- and nanoparticles tested were safe for use as potential drug carriers of therapeutic agents.

The effect of different strains of Helicobacter pylori on platelet aggregation.

BACKGROUND AND AIMS: Helicobacter pylori is the major causative agent in peptic ulcer disease and is strongly implicated in the development of gastric cancer. It has also been linked, less strongly, to cardiovascular disease. The mechanisms by which certain strains of H pylori induce platelet aggregation through interactions with platelet glycoprotein Ib have been previously described. METHODS: In the present study, 21 different strains of H pylori, varying in their vacuolating toxin gene, cytotoxic-associated gene A status and other pathogenicity factors, were tested for their ability to induce platelet aggregation. RESULTS: Ten of the 21 strains induced platelet aggregation, a response that appeared to be independent of their vacuolating toxin gene and cytotoxic-associated gene A status. CONCLUSIONS: Platelet aggregation has been suggested to be one of the possible mechanisms involved in the effects on the cardiovascular system induced by H pylori. Our results suggest that any putative role H pylori plays in cardiovascular disease may be strain dependent. Further work to identify the H pylori factors involved in induction of platelet aggregation may allow for identification of 'higher risk' strains for cardiovascular disease.

Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling.

Fibrinogen binding to integrin alphaIIbbeta3 mediates platelet aggregation and requires agonist-induced "inside-out" signals that increase alphaIIbbeta3 affinity. Agonist regulation of alphaIIbbeta3 also takes place in megakaryocytes, the bone marrow cells from which platelets are derived. To facilitate mechanistic studies of inside-out signaling, we describe here the generation of megakaryocytes in quantity from murine embryonic stem (ES) cells. Coculture of ES cells for 8-12 days with OP9 stromal cells in the presence of thrombopoietin, IL-6, and IL-11 resulted in the development of large, polyploid megakaryocytes that produced proplatelets. These cells expressed alphaIIbbeta3 and platelet glycoprotein Ibalpha but were devoid of hematopoietic stem cell, erythrocyte, and leukocyte markers. Mature megakaryocytes, but not megakaryocyte progenitors, specifically bound fibrinogen by way of alphaIIbbeta3 in response to platelet agonists. Retrovirus-mediated expression of the reporter gene, green fluorescent protein, in ES cell-derived megakaryocytes did not affect viability or alphaIIbbeta3 function. On the other hand, retroviral expression of CalDAG-GEFI, a Rap1 exchange factor identified by megakaryocyte gene profiling as a candidate integrin regulator, enhanced agonist-induced activation of Rap1b and fibrinogen binding to alphaIIbbeta3 (P < 0.01). These results establish that ES cells are a ready source of mature megakaryocytes for integrin studies and other biological applications, and they implicate CalDAG-GEFI in inside-out signaling to alphaIIbbeta3.