Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

Thresholds of becoming: an exploration of threshold concepts as a means to support professional identity formation in competency-based curricula.

Inherent in every clinical preceptor's role is the ability to understand the learning needs of individual trainees, enabling them to meet their potential. Competency-based medical education frameworks have been developed to this end, but efforts to identify behaviours and activities that define competence are based on mapping knowledge, skills and ability, which can be difficult to integrate into a comprehensive picture of who the trainee is becoming. Professional identity formation, in contrast, prioritizes attention to who trainees are becoming, but provision of detailed guidance to preceptors on how to best support this form of development is challenging. The tension that results limits our ability to optimally support learners as strengths in competency development may mask professional identity development gaps and vice versa. To address this tension, this paper examines how the theory of threshold concepts - troublesome ideas that, once appreciated, fundamentally change how you understand and approach a particular activity - can shine light on professional identity formation and its relationship with developing competence. The recognition and identification of threshold concepts is offered as a means to improve our ability to identify, discuss and support behaviours and actions that impact the learner's capacity to act competently as they develop their identity at various stages of training.

Practicing Confidence: An Autoethnographic Exploration of the First Years as Physicians.

Phenomenon: Every year is heralded with a cohort of newly-minted medical school graduates. Through intense residency training and supervision, these learners gradually develop self-assurance in their newfound skills and ways of practice. What remains unknown, however, is how this confidence develops and on what it is founded. This study sought to provide an insider view of this evolution from the frontline experiences of resident doctors. Approach: Using an analytic collaborative autoethnographic approach, two resident physicians (Internal Medicine; Pediatrics) documented 73 real-time stories on their emerging sense of confidence over their first two years of residency. A thematic analysis of narrative reflections was conducted iteratively in partnership with a staff physician and a medical education researcher, allowing for rich, multi-perspective input. Reflections were analyzed and coded thematically and the various perspectives on data interpretation were negotiated by consensus discussion. Findings: In the personal stories and experiences shared, we take you through our own journey and development of confidence, which we have come to appreciate as a layered and often non-linear process. Key moments include fears in the face of the unknown; the shame of failures (real or perceived); the bits of courage gained by everyday and mundane successes; and the emergence of our personal sense of growth and physicianship. Insights: Through this work, we - as two Canadian resident physicians - have ventured to describe a longitudinal trajectory of confidence from the ground up. Although we enter residency with the label of 'physician,' our clinical acumen remains in its infancy. We graduate from residency still as physicians, but decidedly different in terms of our knowledge, attitudes, and skills. We sought to capitalize on the vulnerability and authenticity inherent in autoethnography to enrich our collective understanding of confidence acquisition in the resident physician and its implications for the practice of medicine.

Goal co-construction and dialogue in an internal medicine longitudinal coaching programme.

BACKGROUND: Longitudinal coaching in residency programmes is becoming commonplace and requires iterative and collaborative discussions between coach and resident, with the shared development of goals. However, little is known about how goal development unfolds within coaching conversations over time and the effects these conversations have. We therefore built on current coaching theory by analysing goal development dialogues within resident and faculty coaching relationships. METHODS: This was a qualitative study using interpretive description methodology. Eight internal medicine coach-resident dyads consented to audiotaping coaching meetings over a 1-year period. Transcripts from meetings and individual exit interviews were analysed thematically using goal co-construction as a sensitising concept. RESULTS: Two themes were developed: (i) The content of goals discussed in coaching meetings focused on how to be a resident, with little discussion around challenges in direct patient care, and (ii) co-construction mainly occurred in how to meet goals, rather than in prioritising goals or co-constructing new goals. CONCLUSIONS: In analysing goal development in the coach-resident relationships, conversations focused mainly around how to manage as a resident rather than how to improve direct patient care. This may be because academic coaching provides space separate from clinical work to focus on the stage-specific professional identity development of a resident. Going forward, focus should be on how to optimise longitudinal coaching conversations to ensure co-regulation and reflection on both clinical competencies and professional identity formation.

Genetic analyses of circulating PUFA-derived mediators identifies heritable dihydroxyeicosatrienoic acid species.

Estimates of heritability are the first step in identifying a trait with substantial variation due to genetic factors. Large-scale genetic analyses can identify the DNA variants that influence the levels of circulating lipid species and the statistical technique Mendelian randomisation can use these DNA variants to address potential causality of these lipids in disease. We estimated the heritability of plasma eicosanoids, octadecanoids and docosanoids to identify those lipid species with substantial heritability. We analysed plasma lipid mediators in 31 White British families (196 participants) ascertained for high blood pressure and deeply clinically and biochemically phenotyped over a 25-year period. We found that the dihydroxyeicosatrienoic acid (DHET) species, 11,12-DHET and 14,15-DHET, products of arachidonic acid metabolism by cytochrome P450 (CYP) monooxygenase and soluble epoxide hydrolase (sEH), exhibited substantial heritability (h2 = 33%-37%; Padj<0.05). Identification of these two heritable bioactive lipid species allows for future large-scale, targeted, lipidomics-genomics analyses to address causality in cardiovascular and other diseases.

Automated Digital Notification of COVID-19 Diagnoses Through Text and Email Messaging - North Carolina, December 2020-January 2021.

During October 3, 2020-January 9, 2021, North Carolina experienced a 400% increase in daily reported COVID-19 cases (1). To handle the increased number of cases and rapidly notify persons receiving a positive SARS-CoV-2 test result (patients), North Carolina state and local health departments moved from telephone call notification only to telephone call plus automated text and email notification (digital notification) beginning on December 24, 2020. Overall, among 200,258 patients, 142,975 (71%) were notified by telephone call or digital notification within the actionable period (10 days from their diagnosis date)* during January 2021, including at least 112,543 (56%) notified within 24 hours of report to North Carolina state and local health departments, a significantly higher proportion than the 25,905 of 175,979 (15%) notified within 24 hours during the preceding month (p<0.001). Differences in text notification by age, race, and ethnicity were observed. Automated digital notification is a feasible, rapid and efficient method to support timely outreach to patients, provide guidance on how to isolate, access resources, inform close contacts, and increase the efficiency of case investigation staff members.

Capturing the Moments: An Autoethnographic Exploration of Self-Preservation in Clerkship.

PhenomenonFor most medical students, clerkship represents a transitional phase into the 'real world' of medicine. This transition is often accompanied by significant mental stressors, burnout, and empathy decline. Educator led resilience curricula designed to support students during this critical period often focus on teaching generalized strategies to promote wellness and lack the student input and perspective in their development. Thus, they may be of minimal value when learners are faced with acute moments of challenge and distress in their day-to-day work. The following project seeks to provide an insider view on the experience, interpretation, and response to these moments of challenge and distress from the frontline perspective of clinical clerks. Approach: Using collaborative autoethnography, two medical students documented 85 reflections on their emerging professional identity over the course of a core clerkship year. A narrative analysis was conducted iteratively in partnership with a staff internist and a medical education researcher experienced in autoethnography, allowing for robust multi-perspective input. Reflections were analyzed and coded thematically; disagreements were resolved by consensus discussion. Findings: A key theme of the reflections was self-preservation, conceptualized within two principal contexts: (i) Clerk-patient relationships, wherein we found ourselves in emotionally difficult situations; and (ii) Clerk-preceptor relationships, in which self-preservation manifested through a series of self-protective mechanisms. Insights: The practice of self-preservation is understood as the conscious act of boundary-setting and psychological defense in situations that pose a real (or perceived) threat to the clerk's wellbeing. At best, self-preservation serves as a temporary compromise to the stressors and burnout of clerkship. We speculate, however, that, left unchecked, acts of self-preservation may lead to habitual selfishness and apathy, qualities that are in diametric opposition to those expected of future physicians, and may manifest later (when these learners progress through the hierarchy) as the unprofessional behaviors that perpetuate the cycle of the hidden curriculum.

Working in the dead of night: exploring the transition to after-hours duty.

CONTEXT: Transitions, although often difficult, represent integral components of medical training. New postgraduate trainees (first-year residents) find themselves in an especially challenging transition as they are expected to fulfil both learning and service expectations concurrently. Workplace learning theory has been suggested as a lens through which to understand this unique educational, yet service-oriented, role. This tension may be further amplified overnight when residents are on-call with little to no support. OBJECTIVES: The aims of this study were to explore the transition from medical student to resident with respect to the on-call experience, and to provide theory-based suggestions to enhance learning during this unique transition. METHODS: We conducted an interpretivist qualitative study by interviewing eight medical students and 10 first-year residents from six different specialty training programmes across four academic sites. Each semi-structured interview was transcribed verbatim and anonymised. Resident interview transcripts were initially coded for major themes, after which medical student interview transcripts were coded for consistencies and discrepancies. RESULTS: Four interrelated themes were identified in students' and residents' descriptions of on-call experiences: (i) shift in responsibility; (ii) supervisory support; (iii) contextual conditions, and (iv) clarity of expectations. Generally, students were not able to anticipate the challenges they would face as residents on-call, and residents perceived the transition as sudden with little emphasis placed on learning. CONCLUSIONS: First-year residents face multiple challenges during on-call, which may prevent optimal learning in this setting. These challenges are amplified by the large gap between the respective roles of medical students and residents. We identified promoters of and barriers to effective learning in this environment and, by using workplace learning theory, provide recommendations for how we might be able to enhance medical students' preparation for and first-year residents' learning during experiences of being on-call.

Your Goals, My Goals, Our Goals: The Complexity of Coconstructing Goals with Learners in Medical Education.

Phenomenon: Despite a long-standing recognition of the importance of learning goals in feedback, there has been relatively little research on how to address mismatches between learner goals and preceptor goals in medical education. Our study addresses this gap by reporting on challenges and strategies around goal coconstruction as identified by clinical educators who were learning and attempting to implement a goal-oriented feedback approach in their own teaching contexts. Approach: We employed a qualitative, design-based research methodology to study how 5 clinician educators incorporated goal-oriented feedback into their teaching practice. Participants attended workshops on goal-oriented feedback and reflective writing. They then narratively reflected over a 6-month period on their attempts with goal-oriented feedback and shared these reflections in periodic facilitated group discussions. Themes were developed using iterative thematic analysis of group discussions and individual exit interviews. Findings: Participants identified several benefits of goal setting in all environments. They perceived improved rapport with learners and developed empathy for the vastness of learner goals. However, they experienced several struggles especially when learner and preceptor goals did not match. These included (a) how to address learner goals that were not easily amenable to a coconstruction, (b) how to coconstruct goals while actively running a clinical practice, and (c) how to remain learner centered while raising preceptor goals based on perceived gaps. Insights: Mismatches between learner and preceptor goals are inevitable and frequent. Preceptors must find ways to coconstruct goals with learners in various learning environments. That said, in enacting goal-oriented feedback, preceptors are effectively using feedback throughout the teaching and learning interaction to coconstruct the learning environment, resulting in improved rapport with learners and emphasizing why it is important to focus feedback around goals.

Beyond hands-on and hands-off: supervisory approaches and entrustment on the inpatient ward.

CONTEXT: The concept of entrustment has garnered significant attention in medical specialties, despite variability in supervision styles and entrustment decisions. There is a need to further study the enactment of supervision on inpatient wards to inform competency-based assessment design. METHODS: Attending physicians, while supervising on clinical teaching inpatient wards, were invited to describe a recent moment of enacting supervision with an internal medicine resident. Constructivist grounded theory guided data collection and analysis. Interview transcripts were analysed in iterative cycles to inform data collection. Constant comparison was used to build a theory of supervision from the identified themes. RESULTS: In 2016-2017, 23 supervisors from two Canadian universities with supervision reputations ranging from very involved to less involved participated in one or two interviews (total: 28). Supervisors were not easily dichotomised into styles based on behaviour because all used similar oversight strategies. Supervisors described adjusting between 'hands-on' (e.g. detail oriented) and 'hands-off' (e.g. less visible on ward) styles depending on the context. All also contended with the competing roles of clinical teacher and care provider. Supervisors made a distinction between the terms `entrust' and `trust', and did not grant complete entrustment to senior residents. CONCLUSIONS: We propose that a supervisor's perceived responsibility for the ward underlies adjustments between 'hands-on' (i.e. personal ward responsibility) and 'hands-off' (i.e. shared ward responsibility) styles. Our approaches to clinical supervision model combines this responsibility tension with the tension between patient care and teaching to illustrate four supervisory approaches, each with unique priorities influencing entrustment. Given the fluidity in supervision, documenting changes in oversight strategies, rather than absolute levels of entrustment, may be more informative for assessment purposes. Research is needed to determine if there is sufficient association between the supervision provided, the entrustment decision made and the supervisor's trust in a trainee to use these as proxies in assessing a trainee's competence.

An autoethnographic exploration of the use of goal oriented feedback to enhance brief clinical teaching encounters.

Supervision in the outpatient context is increasingly in the form of single day interactions between students and preceptors. This creates difficulties for effective feedback, which often depends on a strong relationship of trust between preceptor and student. Building on feedback theories focusing on the relational and dialogic aspects of feedback, this study explored the use of goal-oriented feedback in brief encounters with learners. This study used autoethnography to explore one preceptor's feedback interactions over an eight-month period both in the ambulatory setting and on the wards. Data included written narrative reflections on feedback interactions with twenty-three learners informed by discussions with colleagues and repeated reading of feedback literature. Thematic and narrative analyses of data were performed iteratively. Data analysis emphasized four recurrent themes. (1) Goal discussions were most effective when initiated early and integrated throughout the learning experience. (2) Both learner and preceptor goals were multiple and varied, and feedback needed to reflect this complexity. (3) Negotiation or co-construction of goals was important when considering the focus of feedback discussions in order to create safer, more effective interactions. (4) Goal oriented interactions offer potential benefits to the learner and preceptor. Goal oriented feedback promotes dialogue as it requires both preceptor and learner to acknowledge and negotiate learning goals throughout their interaction. In doing so, feedback becomes an explicit component of the preceptor-learner relationship. This enhances feedback interactions even in relatively brief encounters, and may begin an early educational alliance that can be elaborated with longer interactions.

Autoethnography: introducing 'I' into medical education research.

CONTEXT: Autoethnography is a methodology that allows clinician-educators to research their own cultures, sharing insights about their own teaching and learning journeys in ways that will resonate with others. There are few examples of autoethnographic research in medical education, and many areas would benefit from this methodology to help improve understanding of, for example, teacher-learner interactions, transitions and interprofessional development. OBJECTIVES: We wish to share this methodology so that others may consider it in their own education environments as a viable qualitative research approach to gain new insights and understandings. METHODS: This paper introduces autoethnography, discusses important considerations in terms of data collection and analysis, explores ethical aspects of writing about others and considers the benefits and limitations of conducting research that includes self. RESULTS: Autoethnography allows medical educators to increasingly engage in self-reflective narration while analysing their own cultural biographies. It moves beyond simple autobiography through the inclusion of other voices and the analytical examination of the relationships between self and others. Autoethnography has achieved its goal if it results in new insights and improvements in personal teaching practices, and if it promotes broader reflection amongst readers about their own teaching and learning environments. CONCLUSIONS: Researchers should consider autoethnography as an important methodology to help advance our understanding of the culture and practices of medical education.

The Effects of poly-GA and poly-PR C9orf72 Dipeptide Repeats on Sleep Patterns in Drosophila melanogaster.

C9orf72 is the most common familial gene associated with amyotrophic lateral sclerosis (ALS). Dipeptide repeats (DPRs) encoded by an expanded nucleotide repeat sequence in the C9orf72 gene were found in the sleep-related neurons of patients, indicating a role of DPRs in ALS-associated sleep disruptions. Poly-GA or poly-PR DPRs were expressed in male Drosophila melanogaster to study their effect on sleep . Poly-PR expression caused sleep disruptions while poly-GA expression did not. This study validates the use of Drosophila as an in vivo model system for exploring the roles of DPRs in perturbing the underlying molecular mechanisms in sleep regulation.

A Dual-Adjuvanted Parenteral-Intranasal Subunit Nanovaccine generates Robust Systemic and Mucosal Immunity Against SARS-CoV-2 in Mice.

Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters can overcome the shortcomings of parenteral vaccines and enhance pre-existing systemic immunity. Here, a new protein subunit nanovaccine is developed by utilizing dual-adjuvanted (RIG-I: PUUC RNA and TLR-9: CpG DNA) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) along with SARS-CoV-2 S1 trimer protein, that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL subunit nanovaccine, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lungs and showed robust systemic humoral immunity. Interestingly, as a purely intranasal subunit vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. The data suggest that PUUC+CpG PAL subunit nanovaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.

A multiadjuvant polysaccharide-amino acid-lipid (PAL) subunit nanovaccine generates robust systemic and lung-specific mucosal immune responses against SARS-CoV-2 in mice.

Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, which are essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters could overcome the shortcomings of parenteral vaccines and enhance pre- existing systemic immunity. Here we present a new protein subunit nanovaccine using multiadjuvanted (e.g. RIG-I: PUUC, TLR9: CpG) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL- NPs, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lung, and showed robust systemic humoral immunity. Interestingly, as a purely intranasal vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. Our data suggest that PUUC+CpG PAL-NP subunit vaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons.

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.

Osteoarthritis-associated basic calcium phosphate crystals induce pro-inflammatory cytokines and damage-associated molecules via activation of Syk and PI3 kinase.

The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.