Evaluation of mRNA-1273 Vaccine in Children 6 Months to 5 Years of Age.

BACKGROUND: The safety, reactogenicity, immunogenicity, and efficacy of the mRNA-1273 coronavirus disease 2019 (Covid-19) vaccine in young children are unknown. METHODS: Part 1 of this ongoing phase 2-3 trial was open label for dose selection; part 2 was an observer-blinded, placebo-controlled evaluation of the selected dose. In part 2, we randomly assigned young children (6 months to 5 years of age) in a 3:1 ratio to receive two 25-µg injections of mRNA-1273 or placebo, administered 28 days apart. The primary objectives were to evaluate the safety and reactogenicity of the vaccine and to determine whether the immune response in these children was noninferior to that in young adults (18 to 25 years of age) in a related phase 3 trial. Secondary objectives were to determine the incidences of Covid-19 and severe acute respiratory syndrome coronavirus 2 infection after administration of mRNA-1273 or placebo. RESULTS: On the basis of safety and immunogenicity results in part 1 of the trial, the 25-µg dose was evaluated in part 2. In part 2, 3040 children 2 to 5 years of age and 1762 children 6 to 23 months of age were randomly assigned to receive two 25-µg injections of mRNA-1273; 1008 children 2 to 5 years of age and 593 children 6 to 23 months of age were randomly assigned to receive placebo. The median duration of follow-up after the second injection was 71 days in the 2-to-5-year-old cohort and 68 days in the 6-to-23-month-old cohort. Adverse events were mainly low-grade and transient, and no new safety concerns were identified. At day 57, neutralizing antibody geometric mean concentrations were 1410 (95% confidence interval [CI], 1272 to 1563) among 2-to-5-year-olds and 1781 (95% CI, 1616 to 1962) among 6-to-23-month-olds, as compared with 1391 (95% CI, 1263 to 1531) among young adults, who had received 100-µg injections of mRNA-1273, findings that met the noninferiority criteria for immune responses for both age cohorts. The estimated vaccine efficacy against Covid-19 was 36.8% (95% CI, 12.5 to 54.0) among 2-to-5-year-olds and 50.6% (95% CI, 21.4 to 68.6) among 6-to-23-month-olds, at a time when B.1.1.529 (omicron) was the predominant circulating variant. CONCLUSIONS: Two 25-µg doses of the mRNA-1273 vaccine were found to be safe in children 6 months to 5 years of age and elicited immune responses that were noninferior to those in young adults. (Funded by the Biomedical Advanced Research and Development Authority and National Institute of Allergy and Infectious Diseases; KidCOVE ClinicalTrials.gov number, NCT04796896.).

Analytical characterization of full, intermediate, and empty AAV capsids.

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation.

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D-pellet culture led to significant increases in the expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific -10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N-acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co-IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B-SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC-chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators.

Reference letters for subspecialty medicine residency positions: are they valuable for decision-making? Results from a Canadian study.

BACKGROUND: The letter of recommendation is currently an integral part of applicant selection for residency programs. Internal medicine residents will spend much time and expense completing sub-specialty away electives to obtain a letter of recommendation. The purpose of this study was 1) to examine a large sample of reference letters in order to define essential components of a high-quality letter, and 2) to elucidate the relationship between quality of reference letter and the letter writer. METHODS: We conducted a two-phase study. In phase one, a large sample of letters of recommendation was examined using an audit tool as a coding framework. A 5-point composite endpoint of high-quality letter components was subsequently developed. In phase two, program director letters were compared to non-program director home institution and non-home institution elective letters based on inclusion of components of the 5-point composite endpoint using Chi square testing. RESULTS: 715 letters were examined (398 non-program director home institution letters, 201 program director letters, and 116 non-home institution elective letters). High-quality letter components were: nature of relationship, duration of relationship, In Training Evaluation Report information, research involvement and comments on areas for improvement. Program director letters had a significantly higher proportion (10.4%) of all 5 high-quality components, compared to 0% in both non-program director home institution letters and elective letters (p < 0.001). A significantly higher proportion of program director letters had 4-5 high-quality components (62.5%) compared to 2% of non-program director home institution letters and 0% of elective letters (p < 0.0001). CONCLUSIONS: Letters of recommendation from elective rotations are of the poorest quality and such rotations should not be pursued for the sole purpose of obtaining a letter. The low quality of elective letters leads to the recommendation that writers should decline to write them, programs should not require them and trainees should not request them. Program directors write the highest quality letters and should be a resource for faculty development. Clinical supervisors can use the 5-point composite endpoint as a guide when writing letters for applicants.

Risk factors, costs and complications of delayed hospital discharge from internal medicine wards at a Canadian academic medical centre: retrospective cohort study.

BACKGROUND: Hospitalized patients are designated alternate level of care (ALC) when they no longer require hospitalization but discharge is delayed while they await alternate disposition or living arrangements. We assessed hospital costs and complications for general internal medicine (GIM) inpatients who had delayed discharge. In addition, we developed a clinical prediction rule to identify patients at risk for delayed discharge. METHODS: We conducted a retrospective cohort study of consecutive GIM patients admitted between 1 January 2015 and 1 January 2016 at a large tertiary care hospital in Canada. We compared hospital costs and complications between ALC and non-ALC patients. We derived a clinical prediction rule for ALC designation using a logistic regression model and validated its diagnostic properties. RESULTS: Of 4311 GIM admissions, 255 (6%) patients were designated ALC. Compared to non-ALC patients, ALC patients had longer median length of stay (30.85 vs. 3.95 days p < 0.0001), higher median hospital costs ($22,459 vs. $5003 p < 0.0001) and more complications in hospital (25.5% vs. 5.3% p < 0.0001) especially nosocomial infections (14.1% vs. 1.9% p < 0.0001). Sensitivity analyses using propensity score and pair matching yielded similar results. In a derivation cohort, seven significant risk factors for ALC were identified including age > =80 years, female sex, dementia, diabetes with complications as well as referrals to physiotherapy, occupational therapy and speech language pathology. A clinical prediction rule that assigned each of these predictors 1 point had likelihood ratios for ALC designation of 0.07, 0.25, 0.66, 1.48, 6.07, 17.13 and 21.85 for patients with 0, 1, 2, 3, 4, 5, and 6 points respectively in the validation cohort. CONCLUSIONS: Delayed discharge is associated with higher hospital costs and complication rates especially nosocomial infections. A clinical prediction rule can identify patients at risk for delayed discharge.

Creating Entrustable Professional Activities to Assess Internal Medicine Residents in Training: A Mixed-Methods Approach.

Background: Competency-based medical education has not advanced residency training as much as many observers expected. Some medical educators now advocate reorienting competency-based approaches to focus on a resident's ability to do authentic clinical work. Objective: To develop descriptions of clinical work for which internal medicine residents must gain proficiency to deliver meaningful patient care (for example, "Admit and manage a medical inpatient with a new acute problem"). Design: A modified Delphi process involving clinical experts followed by a conference of educational experts. Setting: The Royal College of Physicians and Surgeons of Canada. Participants: In phase 1 of the project, members of the Specialty Committee for Internal Medicine participated in a modified Delphi process to identify activities in internal medicine that represent the scope of the specialty. In phase 2 of the project, 5 experts who were scholars and leaders in competency-based medical education reviewed the results. Measurements: Phase 1 identified important activities, revised descriptions to improve accuracy and avoid overlap, and assigned activities to stages of training. Phase 2 compared proposed activity descriptions with published guidelines for their development and application in medical education. Results: The project identified 29 activities that qualify as entrustable professional activities. The project also produced a detailed description of each activity and guidelines for using them to assess residents. Limitation: These activities reflect the practice patterns of the developers and may not fully represent internal medicine practice in Canada. Conclusion: Identification of these activities is expected to facilitate modification of training and assessment programs for medical residents so that programs focus less on isolated skills and more on integrated tasks. Primary Funding Source: Southeastern Ontario Academic Medical Organization Endowed Scholarship and Education Fund and Queen's University Department of Medicine Innovation Fund.

Prehospital analysis of northern trauma outcome measures: the PHANTOM study.

OBJECTIVE: To compare the mortality and morbidity of traumatically injured patients who received additional prehospital care by a doctor and critical care paramedic enhanced care team (ECT), with those solely treated by a paramedic non-ECT. METHODS: A retrospective analysis of Trauma Audit and Research Network (TARN) data and case note review of all severe trauma cases (Injury Severity Score ≥9) in North East England from 1 January 2014 to 1 December 2017 who were treated by the North East Ambulance Service, the Great North Air Ambulance Service or both. TARN methods were used to calculate the number of unexpected survivors or deaths in each group (W score (Ws)). The Glasgow Outcome Scores were contrasted to evaluate morbidity. RESULTS: The ECT group treated 531 patients: there were 17 unexpected survivors and no unexpected deaths. The non-ECT group treated 1202 patients independently: there were no unexpected survivors and 31 unexpected deaths. The proportion of patients requiring critical care interventions differed between the two groups 49% versus 33% (CI for difference 12% to 20%). In the ECT group, the Ws was 3.22 (95% CI 0.79 to 5.64). In the non-ECT group, the Ws was -2.97 (95% CI -1.22 to -4.71). The difference between the Ws was 6.18 (95% CI 3.19 to 9.17). There was no evidence of worse morbidity in the ECT group. CONCLUSION: This is the first UK ECT service to demonstrate a risk-adjusted mortality benefit in trauma patients with no detriment in morbidity: our results demonstrate an additional 3.22 survivors per 100 severe trauma casualties when treated by an ECT. The authors encourage other ECT services to conduct similar research.

LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1ß, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1ß challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1-/- presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

Reducing the Incidence of Postoperative Nausea and Vomiting Begins With Risk Screening: An Evaluation of the Evidence.

Postoperative nausea and vomiting (PONV) is a daily concern for patients and perianesthesia nurses. PONV is experienced by approximately one third of all surgical patients. Identification of patients at risk for PONV through preoperative risk assessment is an effective means in reducing the incidence of PONV. Perianesthesia nurses are positioned to implement such risk assessments by using simplified risk scores to identify moderate to high-risk patients. Risk assessment allows for facilitation of targeted prophylaxis which positively impacts the patients' surgical outcome and experience. Targeted prophylaxis is efficacious in reducing the institutional incidence of PONV which decreases resource utilization and cost. The perianesthesia nurse is the crucial component in minimizing the PONV in the post-surgical patient. This evaluation of the evidence reveals that preoperative PONV risk screening leads to decreased incidence of PONV for the surgical patient, improves patient satisfaction and reduces postoperative complications.

A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFß deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

A food product as a potential serious cause of liver injury.

INTRODUCTION: Drug-induced liver injury can be challenging to diagnose, as it can develop following the use of many prescription and nonprescription medications, herbals, and dietary supplements. Food products may not be routinely considered as a potential cause of hepatotoxicity. We describe the clinical features of two cases of acute liver injury following consumption of a smoothie product. CASE PRESENTATIONS: Two patients independently presented to the hospital with epigastric pain and acute liver injury. Both patients had consumed a new smoothie product in the same month that they presented to the hospital, with a recurrence of acute liver injury with further consumption. A diagnosis of drug-induced liver injury was established after the evaluation excluded other causes of liver injury. It was thought that a natural ingredient in the smoothie, tara flour, was the cause of hepatotoxicity based on prior news reports. Both patients stopped drinking the smoothie product with subsequent normalization of liver enzyme activities and no further recurrence of epigastric pain. CONCLUSION: The diagnosis of drug-induced liver injury largely relies on a compatible history and exclusion of other causes of liver injury. We demonstrate the importance of considering new food products in the differential diagnosis of acute liver injury.

The association between geospatial and temporal factors and pre-hospital response to major trauma: a retrospective cohort study in the North of England.

BACKGROUND: Major trauma is a leading cause of premature death and disability worldwide, and many healthcare systems seek to improve outcomes following severe injury with provision of pre-hospital critical care. Much research has focussed on the efficacy of pre-hospital critical care and advanced pre-hospital interventions, but less is known about how the structure of pre-hospital critical care services may influence response to major trauma. This study assessed the association between likelihood of pre-hospital critical care response in major trauma and factors important in the planning and development of those services: geographic isolation, time of day, and tasking mechanism. METHODS: A local trauma registry, supported with data from the Trauma Audit and Research Network alongside additional information regarding pre-hospital management, identified patients sustaining major trauma admitted to Major Trauma Centres in the North of England. Data was extracted on location and time of incident, mechanism of injury, on-scene times, and presence or absence of pre-hospital critical care team. An isochrone map was constructed for 30-minute intervals to regional Major Trauma Centres, defining geographic isolation. Univariate logistic regression compared likelihood of pre-hospital critical care response to that of conventional ambulance response for varying degrees of geographic isolation, day or night period, and mechanism of injury, and multiple linear regression assessed the association between geographic isolation, service response and on-scene time. RESULTS: 2619 incidents were included, with 23.3% attended by pre-hospital critical care teams. Compared to conventional ambulance services, pre-hospital critical care teams were more likely to respond major trauma in areas of greater geographic isolation (OR 1.42, 95% CI 1.30-1.55, p < 0.005). There were significant differences in the mechanism of injury attended and no significant difference in response by day or night period. Pre-hospital critical care team response and increasing geographic isolation was associated with longer on-scene times (p < 0.005). CONCLUSION: Pre-hospital critical care teams are more likely to respond to major trauma in areas of greater geographic isolation. Enhanced pre-hospital care may mitigate geographic inequalities when providing advanced interventions and transport of severely injured patients. There may be an unmet need for pre-hospital critical care response in areas close to major hospitals.

Molecular recognition requires dimerization of a VHH antibody.

Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.

Directed differentiation of hPSCs through a simplified lateral plate mesoderm protocol for generation of articular cartilage progenitors.

Developmentally, the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced both LP derived prechondrocytes and preosteoblasts from human pluripotent stem cells (hPSC) through a common progenitor in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocyte- and preosteoblast-like cells can be generated. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to investigate further cartilage development and in the development of joint cartilage for potential treatments.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.

O data, where art thou? Revolutionizing data sharing to advance our sustainability goals through smart chemical innovation.

Antiquated and inefficient data-sharing practices represent one of the key obstacles to advancing sustainability goals through green chemistry. To this end, we need to robustly link data on chemical impacts with new chemical design strategies, which requires the development of next-generation data-sharing platforms to harmonize both data and efforts. These decentralized and interactive programs should be structured as live ecosystems for data generation and exchange, inviting conversations about the reliability and relevance of information used to make decisions regarding chemical performance and safety.

Intestinal Enteroendocrine Cell Signaling: Retinol-binding Protein 2 and Retinoid Actions.

Retinol-binding protein 2-deficient (Rbp2-/-) mice are more prone to obesity, glucose intolerance, and hepatic steatosis than matched controls. Glucose-dependent insulinotropic polypeptide (GIP) blood levels are dysregulated in these mice. The present studies provide new insights into these observations. Single cell transcriptomic and immunohistochemical studies establish that RBP2 is highly expressed in enteroendocrine cells (EECs) that produce incretins, either GIP or glucagon-like peptide-1. EECs also express an enzyme needed for all-trans-retinoic acid (ATRA) synthesis, aldehyde dehydrogenase 1 family member A1, and retinoic acid receptor-alpha, which mediates ATRA-dependent transcription. Total and GIP-positive EECs are significantly lower in Rbp2-/- mice. The plasma transport protein for retinol, retinol-binding protein 4 (RBP4) is also expressed in EECs and is cosecreted with GIP upon stimulation. Collectively, our data support direct roles for RBP2 and ATRA in cellular processes that give rise to GIP-producing EECs and roles for RBP2 and RBP4 within EECs that facilitate hormone storage and secretion.