Characterization and preclinical evaluation of a protease activated receptor 2 (PAR2) monoclonal antibody as a preventive therapy for migraine.

AIM: Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models. METHODS: Flow cytometry, immunocytochemistry, calcium imaging, Homogeneous Time Resolved Technology (HTRF) epitope competition assay and serum pharmacokinetic (PK) assay in rats were performed to confirm the activity, specificity and in vivo stability of PAR650097, a novel anti- protease activated receptor 2 monoclonal antibody. In vivo assessment was performed in female C57BL/6J mice by evaluation of PAR650097 in preventing cutaneous allodynia elicited by (a) supradural injection of the protease activated receptor 2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide trifluoroacetate (SLIGRL), or calcitonin gene-related (CGRP) peptide, and (b) induction of latent sensitization by priming with three daily episodes of restraint stress followed by challenge with a subthreshold inhalational exposure to umbellulone (UMB), a transient receptor potential ankyrin 1 (TRPA1) agonist. PAR650097 was administered as a pretreatment prior to the first restraint stress, umbellulone exposure, SLIGRL or calcitonin gene-related peptide injection. Additionally, fremanezumab, a calcitonin gene-related peptide antibody was administered as pre-treatment prior to supradural administration of calcitonin gene-related peptide or SLIGRL. RESULTS: In vitro, PAR650097 demonstrated rapid interaction with protease activated receptor 2, enabling it to fully inhibit protease-induced protease activated receptor 2 activation, in human and mouse cells, with high potency. Furthermore, PAR650097 was highly selective for protease activated receptor 2, demonstrating no affinity for protease activated receptor 1 protein and no functional effect on the activation of cellular protease activated receptor 1 with thrombin. In addition, PAR650097 had an acceptable PK profile, compatible with testing the effects of selective protease activated receptor 2 inhibition in vivo. In vivo, PAR650097 blocked cutaneous allodynia induced by either supradural SLIGRL or calcitonin gene-related peptide. Fremanezumab abolished cutaneous allodynia induced by supradural CGRP, and partially attenuated cutaneous allodynia induced by SLIGRL. Administration of PAR650097, before the first restraint stress episode, did not prevent the acute stress-induced cutaneous allodynia or restraint stress priming revealed by cutaneous allodynia induced by inhalational umbellulone. In contrast, PAR650097 prevented expression of cutaneous allodynia when given before the umbellulone challenge in restraint stress-primed animals. CONCLUSION: PAR650097 specifically inhibits endogenously expressed protease activated receptor 2 in human and mouse cells with high potency. This antibody has an acceptable PK profile in rodents and effectively blocked SLIGR-induced cutaneous allodynia. PAR650097 additionally prevented cutaneous allodynia induced by supradural calcitonin gene-related peptide, indicating that the protease activated receptor 2 receptor is a downstream consequence of calcitonin gene-related peptide actions. Fremanezumab effectively blocked calcitonin gene-related peptide-induced cutaneous allodynia and only partially reduced cutaneous allodynia induced by a protease activated receptor 2 activator, suggesting both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine pain. While PAR650097 did not prevent stress-induced cutaneous allodynia or priming, it effectively prevented cutaneous allodynia induced by a TRPA1 agonist in animals with latent sensitization. Activation of protease activated receptor 2, therefore, contributes to both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine-like pain. Therapeutic targeting of protease activated receptor 2 receptors may represent an anti-migraine pain strategy with a potentially broad efficacy profile.

Decorative animations impair recall and are a source of extraneous cognitive load.

Working memory is critical for learning but has a limited capacity for processing new information in real time. Cognitive load theory is an evidence-based approach to education that seeks to minimize the extraneous (unnecessary) load on working memory to avoid overloading it. The "seductive details effect" postulates that extraneous load can come from instructional design materials that attract interest but are unrelated to, and impair, learning. Presentation packages, such as Microsoft PowerPoint, have built-in decorative animated "GIFs" that are designed to make presentations more visually appealing. The aim of the study was to investigate the effect of such "decorative" animations on learning and working memory performance. We found that students were less able to recall content presented in the presence of a decorative but relevant animation compared with a still image. This effect was found with two different topics (human physiology and enzyme kinetics). Compared with still images, students also found it harder to remember animations themselves, and the self-reported mental workload required to remember them was higher. These results show that decorative animations are seductive details and are thus a source of extraneous cognitive load.

Structural and functional characterization of a specific antidote for ticagrelor.

Ticagrelor is a direct-acting reversibly binding P2Y12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y12. Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 5'-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures.

The Educational Impact of Exposure to Clinical Psychiatry Early in an Undergraduate Medical Curriculum.

OBJECTIVE: The medical school at Swansea University provides compulsory early exposure to clinical education through short learning opportunities in the clinical setting (LOCS). These are 3-4-h sessions chosen by students from a list of over 900. Students are required to complete ten LOCS in each of their first 2 years of medical school, with at least one per year being in psychiatry. The objective of this study was to evaluate the educational experience of students undertaking LOCS in psychiatry, in part to understand whether this experience affects student understanding of psychiatry and the likelihood that they will pursue it as a career. METHODS: A mixed methods approach was used. Qualitative focus group discussions were conducted with medical students to explore perceptions of psychiatry and experiences of psychiatry LOCS. Findings informed the development of a structured quantitative survey aimed at a larger sample of students. RESULTS: Six qualitative themes emerged: (1) limited exposure to psychiatry, (2) organizational issues, (3) positive LOCS experiences, (4) stigma, (5) anticipated emotional burden, (6) psychiatry at odds with current understanding of medicine. Questionnaire data showed that psychiatry is not a popular future career choice when compared to other specialties. Psychiatry LOCS are extremely popular with students and have a positive effect on their understanding of the specialty but did little to influence their stated likelihood of pursuing psychiatry as a career. CONCLUSIONS: Early exposure to clinical psychiatry through LOCS gives students positive experiences, which improve understanding and awareness of psychiatry. They do not, however, affect stated career intentions for psychiatry as a profession.

Acquired equivalence and generalized suppression in a virtual reality environment.

Acquired equivalence was investigated using a virtual reality conditioned suppression task administered in a first-person-shooter game. Two visual cues, A1 and B1, were followed by a tone (O1), and another two cues, A2 and B2, were followed by another tone (O2). During differential Pavlovian conditioning, A1 was paired with an instructed unconditioned stimulus (US) consisting of a flashing white screen, whereas A2 was not. All cues and outcomes were then presented at test, in the absence of the US, and suppression ratios were calculated for multiple response topographies (shots, hits, and breaks). Clear evidence of the suppression of shots was seen for A1 and B1, with no suppression being seen for either A2 or B2. Presentations of O1 and O2 resulted in significant suppression of shots and hits, whereas only O1 led to the suppression of breaks. The US expectancy ratings were consistent with these behavioral results. The findings are discussed in the light of differing accounts of acquired equivalence.

Prevalence of the use of prescription stimulants as "study drugs" by UK university students: A brief report.

INTRODUCTION: There is media concern over students using prescription stimulants as "cognitive enhancers" to try and improve their academic performance. However, there is limited evidence about the prevalence of this behaviour in the United Kingdom, or whether it has increased in recent years. METHODS: We review survey studies on students' use of cognitive enhancers. RESULTS: Overall reported use is low, with some inconclusive evidence that it is increasing. Use of modafinil appears to be higher than that of methylphenidate or dexamphetamine. CONCLUSION: There is a clear need for large-scale research in this area, using representative sampling and survey methods that protect student anonymity.

Career Development Needs of Physician Associates in the United Kingdom: A Qualitative Study.

INTRODUCTION: The physician associate (PA) profession is relatively new to the United Kingdom (UK) with the first UK-trained PAs graduating in 2008. Unlike other UK health professions, there is currently no well-established career framework after graduating as a PA. This pragmatic research aimed primarily to provide useful information for the future development of a PA career framework that will best support the career development needs of the PA profession. METHODS: The current study used qualitative 1:1 interviews to understand senior PAs' aspirations, postgraduate education, career progression, development opportunities, and perceptions for a career framework. Where are they now? What are they doing? What are their expectations for the future? What subsequent changes do senior PAs think a career framework might bring to the profession? RESULTS: Most PAs support a career framework and the opportunity to highlight and facilitate the PA's unique ability to transfer specialties; both generalist and specialized PA experience should be recognized. All participants supported a postgraduate standardization of PA practice citing patient safety and equal opportunities for the PA workforce. Furthermore, although the PA profession was introduced to the UK with lateral rather than vertical progression, the current study demonstrates the existence of hierarchical roles within the PA workforce. DISCUSSION: A postqualification framework is needed in the UK, one that supports the current flexibility of the PA workforce.

Widening Access: what do dental school websites signal to prospective students?

Introduction Widening Access (WA) policies aim to ensure that a professional workforce reflects the community that it serves by facilitating the admission of applicants from under-represented demographics. WA has not been extensively studied in UK dental education. Website discourses are an important element in students' higher education choices and have the potential to engage those who might be under-represented.Methods Critical discourse analysis was used to investigate contents of the 16 UK dental school webpages in relation to WA, based on a previous study within medical education. Data were contextualised through identification of drivers and levers, as well as warrants of WA.Results Discourses emphasising social mobility, and the resultant advancement within social hierarchy of an individual, dominated the narrative rationalising WA as an initiative. WA was depicted as a mechanism to support applicants of high academic ability and exhibiting commitment to studying dentistry but who were unable to show their true potential due to their underprivileged backgrounds. This presentation portrayed dental schools as generous establishments, selectively granting career-advancement opportunities to disadvantaged students. Discourses on the benefits of WA for patient care and workforce diversification were largely absent.Conclusions Discourses representing WA on websites of UK dental schools are limited and do not portray applicants from deprived backgrounds or under-represented groups as individuals bringing unique positive benefits to dentistry and patient care. We encourage dental schools to consider alternate messages for attracting applicants from under-represented demographics.

What Happens to the Principles of Evidence-Based Practice When Clinicians Become Educators? A Case Study of the Learning Styles Neuromyth.

Introduction: The approach of matching teaching practice to individual student "Learning Styles" has been repeatedly shown to be ineffective, even harmful. Yet, it appears a majority of educators believe it to be an effective approach. The status of Learning Styles theory in health professions education is unclear. Method: We surveyed health professions educators to determine whether they believed that Learning Styles theory is effective and whether this belief translates to action. We also test knowledge of Learning Styles theory. Results: 87.4% of participants are familiar with Learning Styles, but knowledge about specific models varies. 69.9% of participants believed that Learning Styles theory is effective, but only one-third of them were actually using it. Discussion: More effort is required to emphasise the importance of evidence-based educational awareness and practice in the healthcare community. As is the case with clinical practice, a culture of promoting pedagogy validated by the scientific method should be the norm. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-023-01849-1.

A pragmatic evaluation of university student experience of remote digital learning during the COVID-19 pandemic, focusing on lessons learned for future practice.

The Covid-19 pandemic and subsequent national lockdowns resulted in drastic changes to the way that higher education was delivered. A mixed-methods research study was conducted to explore university students' perceptions of online learning during the 2020/21 academic year. Students from across all Welsh higher education institutions were invited to participate. First, a series of focus groups (n = 13) were conducted to explore students' experiences of online learning during the pandemic. Two were conducted in Welsh, the remaining eleven in English. Thematic analysis led researchers to develop eight key themes: Seeking the positives, Facilitators to learning, Barriers to learning, Lost sense of community, Let down by University, Workload, Assessment, and Health and well-being. These themes informed the design of a quantitative survey which was completed by 759 students. It was found that students were largely satisfied with the quality of online learning, however there were specific challenges associated with a lack of community, wellbeing concerns, and challenges with loneliness and isolation. Data from the focus groups and survey informed recommendations for practice in three key categories; teaching practice, institutional level recommendations, and student health and wellbeing considerations.

The biology of protein kinase C.

This review gives a basic introduction to the biology of protein kinase C, one of the first calcium-dependent kinases to be discovered. We review the structure and function of protein kinase C, along with some of the substrates of individual isoforms. We then review strategies for inhibiting PKC in experimental systems and finally discuss the therapeutic potential of targeting PKC. Each aspect is covered in summary, with links to detailed resources where appropriate.

What is the scope of teaching and training of undergraduate students and trainees in point of care testing in United Kingdom universities and hospital laboratories?

Point of care testing (POCT) is an analytical test performed by a healthcare professional outside of a conventional laboratory. The global POCT market was valued at US$ 23.16 billion in 2016 and is forecasted to grow to US$ 36.96 billion in 2021. This upward trend for POCT has increased workload for pathology departments who manage POCT. This research aims to characterize and analyse the teaching and training of POCT at United Kingdom (UK) universities on Institute of Biomedical Science (IBMS) accredited biomedical science degrees, and at UK hospital laboratories. A freedom of information (FOI) request was sent in 2018 to all 52 UK universities with an accredited IBMS Biomedical science degree to request information on teaching of POCT, with a 100% response rate. Further FOI requests were sent to all National Health Service (NHS) hospital pathology departments in the UK, regarding POCT training provided to trainee Biomedical scientists, with a 97% response rate. Twelve of the degrees contained no POCT teaching, with a further 9 having no specific POCT teaching. Sixty-six laboratories confirmed that there was no POCT training. The university teaching hours varied between 0 and 35 hours. The median time spent teaching POCT at university was 2 hours. The laboratory teaching hours varied between 0 and 450 hours The median time spent teaching POCT in hospital laboratories was 3 hours. A content analysis of the learning outcomes provided by 29 universities showed that only 61% (84/137) were measurable and 26% (36/137) of the learning outcomes used action verbs that have previously been listed to be avoided in learning outcome writing. Only 9% (13/137) of outcomes specifically described POCT, with 8 of these being measurable. The findings demonstrate that although this is a commonly required skill for biomedical scientists, there is a clear lack of POCT teaching and training in the UK. To meet the new Quality Assurance Agency for Higher Education (QAA) guidelines, but most importantly to ensure the workforce is fit for the needs of the current healthcare system, the quality and quantity of POCT teaching and training needs to improve.

Associations between Sleep Quality and Heart Rate Variability: Implications for a Biological Model of Stress Detection Using Wearable Technology.

INTRODUCTION: The autonomic nervous system plays a vital role in the modulation of many vital bodily functions, one of which is sleep and wakefulness. Many studies have investigated the link between autonomic dysfunction and sleep cycles; however, few studies have investigated the links between short-term sleep health, as determined by the Pittsburgh Quality of Sleep Index (PSQI), such as subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction, and autonomic functioning in healthy individuals. AIM: In this cross-sectional study, the aim was to investigate the links between short-term sleep quality and duration, and heart rate variability in 60 healthy individuals, in order to provide useful information about the effects of stress and sleep on heart rate variability (HRV) indices, which in turn could be integrated into biological models for wearable devices. METHODS: Sleep parameters were collected from participants on commencement of the study, and HRV was derived using an electrocardiogram (ECG) during a resting and stress task (Trier Stress Test). RESULT: Low-frequency to high-frequency (LF:HF) ratio was significantly higher during the stress task than during the baseline resting phase, and very-low-frequency and high-frequency HRV were inversely related to impaired sleep during stress tasks. CONCLUSION: Given the ubiquitous nature of wearable technologies for monitoring health states, in particular HRV, it is important to consider the impacts of sleep states when using these technologies to interpret data. Very-low-frequency HRV during the stress task was found to be inversely related to three negative sleep indices: sleep quality, daytime dysfunction, and global sleep score.

Peptide-YY3-36/glucagon-like peptide-1 combination treatment of obese diabetic mice improves insulin sensitivity associated with recovered pancreatic ß-cell function and synergistic activation of discrete hypothalamic and brainstem neuronal circuitries.

OBJECTIVE: Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. METHODS: In this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. RESULTS: Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous ß-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. CONCLUSIONS: These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous ß-cell function. These data also highlight the potential association between the gut-brain axis in control of metabolic homeostasis.

The substrates and binding partners of protein kinase Cepsilon.

The epsilon isoform of protein kinase C (PKCepsilon) has important roles in the function of the cardiac, immune and nervous systems. As a result of its diverse actions, PKCepsilon is the target of active drug-discovery programmes. A major research focus is to identify signalling cascades that include PKCepsilon and the substrates that PKCepsilon regulates. In the present review, we identify and discuss those proteins that have been conclusively shown to be direct substrates of PKCepsilon by the best currently available means. We will also describe binding partners that anchor PKCepsilon near its substrates. We review the consequences of substrate phosphorylation and discuss cellular mechanisms by which target specificity is achieved. We begin with a brief overview of the biology of PKCepsilon and methods for substrate identification, and proceed with a discussion of substrate categories to identify common themes that emerge and how these may be used to guide future studies.

The Learning Styles Neuromyth Is Still Thriving in Medical Education.

Learning Styles theory promises improved academic performance based on the identification of a personal, sensory preference for informational processing. This promise is not supported by evidence, and is in contrast to our current understanding of the neuroscience of learning. Despite this lack of evidence, prior research shows that that belief in the Learning Styles "neuromyth" remains high amongst educators of all levels, around the world. This perspective article is a follow up on prior research aimed at understanding why belief in the neuromyth of Learning Styles remains so high. We evaluated current research papers from the field of health professions education, to characterize the perspective that an educator would be given, should they search for evidence on Learning Styles. As in earlier research on Higher Education, we found that the use of Learning Style frameworks persist in education research for the health professions; 91% of 112 recent research papers published on Learning Styles are based upon the premise that Learning Styles are a useful approach to education. This is in sharp contrast to the fundamental principle of evidence-based practice within these professions. Thus any educator who sought out the research evidence on Learning Styles would be given a consistent but inaccurate endorsement of the value of a teaching technique that is not evidence based, possibly then propagating the belief in Learning Styles. Here we offer perspectives from both research and student about this apparent mismatch between educational practice and clinical practice, along with recommendations and considerations for the future.

Protein kinase C isozymes as regulators of sensitivity to and self-administration of drugs of abuse-studies with genetically modified mice.

Studies using targeted gene deletion in mice have revealed distinct roles for individual isozymes of the protein kinase C (PKC) family of enzymes in regulating sensitivity to various drugs of abuse. These changes in drug sensitivity are associated with altered patterns of drug self-administration. The purpose of this review is to summarize behavioral studies conducted on mice carrying targeted deletions of genes encoding specific PKC isozymes (namely the beta, gamma, delta, and epsilon isozymes), and to critically evaluate the possibility of using pharmacological inhibitors of specific PKC isozymes as modulators of the sensitivity to various drugs of abuse, as well as potential aids in the treatment of substance use disorders.

A blocker of N- and T-type voltage-gated calcium channels attenuates ethanol-induced intoxication, place preference, self-administration, and reinstatement.

There is a clear need for new therapeutics to treat alcoholism. Here, we test our hypothesis that selective inhibitors of neuronal calcium channels will reduce ethanol consumption and intoxication, based on our previous studies using knock-out mice and cell culture systems. We demonstrate that pretreatment with the novel mixed N-type and T-type calcium channel antagonist 1-(6,6-bis(4-fluorophenyl)hexyl)-4-(3,4,5-trimethoxybenzyl)piperazine (NP078585) reduced ethanol intoxication. NP078585 also attenuated the reinforcing and rewarding properties of ethanol, measured by operant self-administration and the expression of an ethanol conditioned place preference, and abolished stress-induced reinstatement of ethanol seeking. NP078585 did not affect alcohol responses in mice lacking N-type calcium channels. These results suggest that selective calcium channel inhibitors may be useful in reducing acute ethanol intoxication and alcohol consumption by human alcoholics.

Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.