Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli.

YcjN is a putative substrate binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 Å, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

Hydrolyzed collagen supplementation prior to resistance exercise augments collagen synthesis in a dose-response manner in resistance-trained, middle-aged men.

BACKGROUND: Resistance exercise (RE) increases collagen synthesis in young and older men, while hydrolyzed collagen (HC) ingestion improves this response to RE in a dose-response manner in young men. However, the collagen synthesis response to RE with and without HC in middle-aged men is unknown. METHODS: Eight resistance-trained men (age: 49±8 years; height: 1.78±0.02m; mass: 90±4kg) took part in this double-blind, crossover design study, and undertook 4×10 repetitions of lower-limb RE at maximum load, after consuming 0g, 15g, or 30g vitamin C-enriched HC. We analyzed venous blood samples for N-terminal propeptide of type 1 pro-collagen (PINP), ß-isomerized C-terminal telopeptide of type 1 collagen (ß-CTx) and 18 collagen amino acids throughout all three interventions. RESULTS: The serum PINP concentration×time area-under-the-curve (AUC) was higher following 30g (169±28 µg/mL×h) than 15g (134±23 µg/mL×h, P<0.05) HC ingestion, and both 15g and 30g were higher than 0g HC (96±23 µg/mL×h, P<0.05). RE with 0g HC showed no change in serum PINP concentration. The AUCs for glycine, proline, hydroxyproline, alanine, arginine, lysine, serine, leucine, valine and isoleucine were greater with 30g than 15g and 0g HC ingestion (P<0.05), and greater with 15g than 0g HC ingestion (P<0.05). Plasma ß-CTx concentration decreased after RE independently of HC dose. CONCLUSIONS: Our study suggests connective tissue anabolic resistance to RE in middle-aged men but ingesting 15g HC rescues the collagen synthesis response, and 30g augments that response further. This dose-response is associated with the increased bioavailability of collagen amino acids in the blood, which stimulate collagen synthesis.

Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli.

YcjN is a putative substrate-binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 Å, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

Residents' Communication With Attendings About Uncertainty: A Single-Site Longitudinal Survey.

OBJECTIVE: Managing uncertainty is a core competency of pediatric residents. However, discussing uncertainty with attending physicians can be challenging. Research is needed to understand residents' goals when communicating about uncertainty with attending physicians and how residents' perceptions of communication change during residency. Therefore, we assessed changes in residents' perceptions of their own ability to communicate uncertainty and their perceptions of attending physicians' willingness to discuss uncertainty effectively. We also identify residents' goals and challenges communicating uncertainty. METHODS: We conducted a 3-year (2018-2021) survey with 2 cohorts of residents at a US children's hospital. Of the 106 eligible residents, 100 enrolled and completed Phase I (94% response rate), 61 of the enrolled residents completed Phase II (61% response rate), and 53 completed Phase III (53% response rate). We employed hierarchical linear modeling to account for clustering of the data (Phases within residents) and to assess changes in communication efficacy and target efficacy over time. We coded open-ended responses to identify residents' communication goals and challenges. RESULTS: Communication efficacy and target efficacy significantly increased over time. Open-ended responses indicated that residents managed multiple task, identity, and relational goals. Residents described persistent challenges related to wanting to appear competent and working with attending physicians who were unwilling to discuss uncertainty. CONCLUSIONS: Although residents may grow more confident communicating uncertainty, such conversations are complex and can present challenges throughout residency. Our results support the value of training on communication about uncertainty, not only for residents, but also attending physicians.

Effects of resistance exercise, collagen ingestion and circulating oestrogen concentration on collagen synthesis in a female athlete: A case report.

We investigated the effects of resistance exercise (RE), hydrolysed collagen (HC) ingestion and circulating oestrogen concentration on collagen synthesis in a naturally menstruating female CrossFit athlete. In a double-blind, randomised cross-over design, the participant (36 years; height 1.61 m; mass 82.6 kg) consumed 0 or 30 g HC prior to performing back-squat RE when endogenous circulating oestrogen concentration was low (onset of menses, OM) and high (late follicular phase, LF) during two consecutive menstrual cycles. Ten 5-mL blood samples were collected during each of the four interventions to analyse concentrations of serum 17ß-oestradiol, and biomarkers of type I collagen turnover, that is serum procollagen type I N-terminal propeptide (PINP, a biomarker of collagen synthesis) and plasma ß-isomerised C-terminal telopeptide of type I collagen (ß-CTX, a biomarker of collagen breakdown), as well as the serum concentration of 18 collagen amino acids. 17ß-Oestradiol concentration was 5-fold higher at LF (891 ± 116 pmol L-1) than OM (180 ± 13 pmol L-1). The PINP concentration × time area under the curve (AUC) was higher in the 30 g HC OM intervention (201 µg L-1 h) than the 30 g HC LF (144 µg L-1 h), 0 g HC OM (151 µg L-1 h) and 0 g HC LF (122 µg L-1 h) interventions. ß-CTX concentration decreased 1.4-fold from pre-RE to 6 h post-RE in all interventions. Thus, high circulating oestrogen concentration was associated with lower collagen synthesis following RE in this female athlete. Ingesting 30 g HC, however, augmented the collagen synthesis response at LF and particularly at OM. HIGHLIGHTS: What is the central question of this study? Does resistance exercise-induced collagen synthesis vary according to circulating oestrogen concentration in a naturally menstruating female athlete, and if so, does hydrolysed collagen ingestion have any impact? What is the main finding and its importance? Exercise-induced collagen synthesis was low when circulating oestrogen concentration was high and vice versa. However, ingesting 30 g hydrolysed collagen prior to exercise reduced the negative effect of oestrogen on collagen synthesis. As high circulating oestrogen has been associated with greater injury risk in females, supplementing exercise with hydrolysed collagen may help protect these tissues from injury.

Barriers to Mental Health care in Canada Identified by Healthcare Providers: A Scoping Review.

The mental health treatment gap remains wide across the world despite mental illness being a significant cause of disability globally. Both end-user and healthcare provider perspectives are critical to understanding barriers to mental healthcare and developing interventions. However, the views of providers are relatively understudied. In this review, we synthesized findings from current literature regarding providers' perspectives on barriers to mental healthcare in Canada. We searched Medline, PsycINFO, Embase, and CINAHL for eligible Canadian studies published since 2000. Analysis and quality assessment were conducted on the included studies. Of 4,773 reports screened, 29 moderate-high quality studies were reviewed. Five themes of barriers emerged: health systems availability and complexity (reported in 72% of the studies), work conditions (55%), training/education (52%), patient accessibility (41%), and identity-based sensitivity (17%). Common barriers included lack of resources, fragmented services, and gaps in continuing education. Interestingly, clinicians often cited confusion in determining the ideal service for patients due to an overwhelming number of potential services without clear descriptions. These five domains of barriers present a synthesized review of areas of improvement for mental healthcare spanning both patients and clinicians. Canadian mental health systems face a need to improve capacity, clinician training, and in particular service navigability and collaboration.

Implementing ward-based practice books to increase the amount of practice completed during inpatient stroke rehabilitation: a mixed-methods process evaluation.

PURPOSE: Stroke survivors must complete large amounts of practice to achieve functional improvements but spend many hours inactive during their rehabilitation. We conducted a mixed methods process evaluation exploring factors affecting the success of a 6-month behaviour change intervention to increase use of ward-based practice books. METHODS: Audits of the presence, quality and use of ward based-practice books were conducted, alongside focus groups with staff (n = 19), and interviews with stroke survivors (n = 3) and family members (n = 4). Quantitative data were analysed descriptively. Focus group and interview transcripts were analysed using qualitative analysis. RESULTS: Personal (patient-related) factors (including severe weakness, cognitive and communication deficits of stroke survivors), staff coaching skills, understanding and beliefs about their role, affected practice book use. Staff turnover, nursing shift work and a lack of action planning reduced success of the behaviour change intervention. CONCLUSIONS: Staff with the necessary skills and understanding of their role in implementing ward practice overcame personal (patient-related) factors and assisted stroke survivors to successfully practice on the ward. To improve success of the intervention, repeated training of new staff is required. In addition to audit and feedback, team action planning is needed around the presence, quality, and use of ward practice books.

Ward-based practice books are one evidence-based strategy that can be used by rehabilitation teams to increase the amount of practice completed by stroke survivors during inpatient rehabilitation.Stroke survivors' personal factors (including severe weakness, cognitive and communication deficits), staff beliefs about their role and coaching skills, affected stroke survivors ability to practice on the ward using practice books.Staff with the necessary skills, understanding and belief about their role in implementing ward practice can overcome personal (patient related) factors (such as severe weakness) and assist stroke survivors to successfully practice on the ward.To increase the success of ward practice, repeated booster training of staff is required along with audit and feedback and team action planning on the presence, quality, and use of ward practice books.

A systematic review of early phase studies for children and young people with relapsed and refractory rhabdomyosarcoma: The REFoRMS-SR project.

Rhabdomyosarcoma is the commonest soft tissue sarcoma in children. Around one-third of children with rhabdomyosarcoma experience relapse or have refractory disease, which is associated with a poor prognosis. This systematic review of early phase studies in pediatric relapsed/refractory rhabdomyosarcoma was conducted to inform future research and provide accurate information to families and clinicians making difficult treatment choices. Nine databases and five trial registries were searched in June 2021. Early phase studies of interventions for disease control in patients under 18 years old with relapsed/refractory rhabdomyosarcoma were eligible. No language/geographic restrictions were applied. Studies conducted after 2000 were included. Survival outcomes, response rates, quality of life and adverse event data were extracted. Screening, data extraction and quality assessment (Downs and Black Checklist) were conducted by two researchers. Owing to heterogeneity in the included studies, narrative synthesis was conducted. Of 16,965 records screened, 129 published studies including over 1100 relapsed/refractory rhabdomyosarcoma patients were eligible. Most studies evaluated systemic therapies. Where reported, 70% of studies reported a median progression-free survival ≤6 months. Objective response rate was 21.6%. Adverse events were mostly hematological. One-hundred and seven trial registry records of 99 studies were also eligible, 63 of which report they are currently recruiting. Study quality was limited by poor and inconsistent reporting. Outcomes for children with relapsed/refractory rhabdomyosarcoma who enroll on early phase studies are poor. Improving reporting quality and consistency would facilitate the synthesis of early phase studies in relapsed/refractory rhabdomyosarcoma (PROSPERO registration: CRD42021266254).

Does gender still matter in the pursuit of a career in anaesthesia?

A survey sent to fellows of the Australian and New Zealand College of Anaesthetists (ANZCA) aimed to document issues affecting gender equity in the anaesthesia workplace. A response rate of 38% was achieved, with women representing a greater proportion of respondents (64.2%). On average women worked fewer hours than men and spent a larger percentage of time in public practice; however, satisfaction rates were similar between genders. There was a gender pay gap which could not be explained by the number of hours worked or years since achieving fellowship. The rates of bullying and harassment were high among all genders and have not changed in 20 years since the first gender equity survey by Strange Khursandi in 1998. Women perceived that they were more likely to be discriminated against particularly in the presence of other sources of discrimination, and highlighted the importance of the need for diversity and inclusion in anaesthetic workplaces. Furthermore, women reported higher rates of caregiving and unpaid domestic responsibilities, confirming that anaesthetists are not immune to the factors affecting broader society despite our professional status. The overall effect was summarised by half of female respondents reporting that they felt their gender was a barrier to a career in anaesthesia. While unable to be included in statistics due to low numbers, non-binary gendered anaesthetists responded and must be included in all future work. The inequities documented here are evidence that ANZCA's gender equity subcommittee must continue promoting and implementing policies in workplaces across Australia and New Zealand.

Twelve tips to aid interpretation of post-assessment psychometric reports.

Post-assessments psychometric reports are a vital component of the assessment cycle to ensure that assessments are reliable, valid and fair to make appropriate pass-fail decisions. Students' scores can be summarised by examination of frequency distributions, central tendency measures and dispersion measures. Item discrimination indicies to assess the quality of items, and distractors that differentiate between students achieving or not achieving the learning outcomes are key. Estimating individual item reliability and item validity indices can maximise test-score reliability and validity. Test accuracy can be evaluated by assessing test reliability, consistency and validity and standard error of measurement can be used to measure the variation. Standard setting, even by experts, may be unreliable and reality checks such as the Hofstee method, P values and correlation analysis can improve validity. The Rasch model of student ability and item difficulty assists in modifying assessment questions, pinpointing areas for additional instruction. We propose 12 tips to support test developers in interpreting structured psychometric reports, including analysis and refinement of flawed items and ensuring fair assessments with accurate and defensible marks.

Faculty Decision Making in Ad Hoc Entrustment of Pediatric Critical Care Fellows: A National Case-Based Survey.

Phenomenon: Ad hoc entrustment decisions reflect a clinical supervisor's estimation of the amount of supervision a trainee needs to successfully complete a task in the moment. These decisions have important consequences for patient safety, trainee learning, and preparation for independent practice. Determinants of these decisions have previously been described but have not been well described for acute care contexts such as critical care and emergency medicine. The ad hoc entrustment of trainees caring for vulnerable patient populations is a high-stakes decision that may differ from other contexts. Critically ill patients and children are vulnerable patient populations, making the ad hoc entrustment of a pediatric critical care medicine (PCCM) fellow a particularly high-stakes decision. This study sought to characterize how ad hoc entrustment decisions are made for PCCM fellows through faculty ratings of vignettes. The authors investigated how acuity, relationship, training level, and task interact to influence ad hoc entrustment decisions. Approach: A survey containing 16 vignettes that varied by four traits (acuity, relationship, training level, and task) was distributed to U.S. faculty of pediatric critical care fellowships in 2020. Respondents determined an entrustment level for each case and provided demographic data. Entrustment ratings were dichotomized by "high entrustment" versus "low entrustment" (direct supervision or observation only). The authors used logistic regression to evaluate the individual and interactive effects of the four traits on dichotomized entrustment ratings. Findings: One hundred seventy-eight respondents from 30 institutions completed the survey (44% institutional response rate). Acuity, relationship, and task all significantly influenced the entrustment level selected but did not interact. Faculty most frequently selected "direct supervision" as the entrustment level for vignettes, including for 24% of vignettes describing fellows in their final year of training. Faculty rated the majority of vignettes (61%) as "low entrustment." There was no relationship between faculty or institutional demographics and the entrustment level selected. Insights: As has been found in summative entrustment for pediatrics, internal medicine, and surgery trainees, PCCM fellows often rated at or below the "direct supervision" level of ad hoc entrustment. This may relate to declining opportunities to practice procedures, a culture of low trust propensity among the specialty, and/or variation in interpretation of entrustment scales.

The Collagen Synthesis Response to an Acute Bout of Resistance Exercise Is Greater when Ingesting 30 g Hydrolyzed Collagen Compared with 15 g and 0 g in Resistance-Trained Young Men.

BACKGROUND: Resistance exercise (RE) stimulates collagen synthesis in skeletal muscle and tendon but there is limited and equivocal evidence regarding an effect of collagen supplementation and exercise on collagen synthesis. Furthermore, it is not known if a dose-response exists regarding the effect of hydrolyzed collagen (HC) ingestion and RE on collagen synthesis. OBJECTIVE: To determine the HC dose-response effect on collagen synthesis after high-intensity RE in resistance-trained young men. METHODS: Using a double-blind, randomized crossover design, 10 resistance-trained males (age: 26 ± 3 y; height: 1.77 ± 0.04 m; mass: 79.7 ± 7.0 kg) ingested 0 g, 15 g, or 30 g HC with 50 mg vitamin C 1 h before performing 4 sets' barbell back squat RE at 10-repetition maximum load, after which they rested for 6 h. Blood samples were collected throughout each of the 3 interventions to analyze procollagen type Ⅰ N-terminal propeptide (PINP) and ß-isomerized C-terminal telopeptide of type I collagen (ß-CTX) concentration, and the concentration of 18 collagen amino acids. RESULTS: The serum PINP concentration × time area under the curve (AUC) was greater for 30 g (267 ± 79 µg·L-1·h) than for 15 g (235 ± 70 µg·L-1·h, P = 0.013) and 0 g HC (219 ± 88 µg·L-1·h, P = 0.002) but there was no difference between 0 and 15 g HC (P = 0.225). The AUCs of glycine and proline were greater for 30 g than for 15 and 0 g HC (P < 0.05). Plasma ß-CTX concentration decreased from -1 to +6 h (P < 0.05), with no differences between interventions. CONCLUSIONS: Ingesting 30 g HC before high-intensity RE augments whole-body collagen synthesis more than 15 g and 0 g HC in resistance-trained young males.

Innovations in assessment in health professions education during the COVID-19 pandemic: A scoping review.

BACKGROUND: After an initial period of uncertainty during the COVID-19 pandemic, medical universities responded to the pandemic by innovating their methods of teaching-learning and assessment. This scoping review aimed to identify innovations in assessment in medical, dental and nursing education at the undergraduate and postgraduate levels during the pandemic. METHODS: This review was conducted utilising Arksey and O'Malley's framework where three electronic databases-PubMed, Embase and Web of Science-were systematically searched in February 2022 for articles in English describing innovations in assessments published in or after January 2020. RESULTS: A total of 70 articles were included in the review. Among the primary research articles, 82.1% reported online assessment, 76.1% were related to medical speciality, 74.6% described assessment at the undergraduate level, 68.7% described summative assessment and 58.2% reported assessment of practical skills. All assessments of theoretical knowledge were done virtually with MCQ being the most used assessment tool and virtual OSCE being the most popular assessment tool to assess practical skills; however, it was acknowledged that the assessment of procedural skills was its limitation. Although the studies described the newer forms of assessments as feasible and acceptable, few studies reported the reliability and validity of the assessments. CONCLUSIONS: This scoping review identified several innovations in assessment during the COVID-19 pandemic with an overall shift towards virtual or hybrid forms of assessment. However, there was a dearth of evidence regarding the effectiveness of these assessment tools suggesting a need for more research before the tools can be implemented post-pandemic.

Determining intra-standard-setter inconsistency in the Angoff method using the three-parameter item response theory.

Objectives: To measure intra-standard-setter variability and assess the variations between the pass marks obtained from Angoff ratings, guided by the latent trait theory as the theoretical model. Methods: A non-experimental cross-sectional study was conducted to achieve the purpose of the study. Two knowledge-based tests were administered to 358 final-year medical students (223 females and 135 males) as part of their normal summative programme of assessments. The results of judgmental standard-setting using the Angoff method, which is widely used in medical schools, were used to determine intra-standard-setter inconsistency using the three-parameter item response theory (IRT). Permission for this study was granted by the local Research Ethics Committee of the University of Nottingham. To ensure anonymity and confidentiality, all identifiers at the student level were removed before the data were analysed. Results: The results of this study confirm that the three-parameter IRT can be used to analyse the results of individual judgmental standard setters. Overall, standard-setters behaved fairly consistently in both tests. The mean Angoff ratings and conditional probability were strongly positively correlated, which is a matter of inter-standard-setter validity. Conclusions: We recommend that assessment providers adopt the methodology used in this study to help determine inter and intra-judgmental inconsistencies across standard setters to minimise the number of false positive and false negative decisions.

Implementation of best practice goal-setting in five rehabilitation services: A mixed-methods evaluation study.

OBJECTIVE: This implementation study aimed to enhance the key elements of clinical practice goal-setting across 5 rehabilitation services. DESIGN: This study followed a participatory action research approach guided by the Knowledge to Action framework. METHODS: Medical record audits and structured client interviews were conducted prior to and following 12 weeks of implementation, in order to evaluate the success of the goal-setting implementation package. RESULTS: Medical record audits and interviews conducted pre-implementation (audits n = 132, interviews n = 64), post-implementation (audits n = 130, interviews n = 56) and at 3-month follow-up (audits n = 30) demonstrated varied success across sites. Following implementation 2 sites significantly improved their common goal focus (site 1 p ≤ 0.001, site 2 p = 0.005), these sites also demonstrated a significant increase in clients reporting that they received copies of their rehabilitation goals (site 1 p ≤ 0.001, site 2 p ≤ 0.001). Four sites improved client action planning, feedback and review, and 3 sites enhanced their specificity of goal-setting. At 3-month follow-up 4 sites had continued to improve their common goal focus; however, all sites decreased the specificity of their goal-setting. CONCLUSION: Elements of the implementation package were successful at enhancing the goal-setting process; however, how the package is implemented within the team may impact outcomes.

Collagen supplementation augments changes in patellar tendon properties in female soccer players.

We investigated the effect of collagen hydrolysate supplementation on changes in patellar tendon (PT) properties after 10 weeks' training in female soccer players from a Football Association Women's Super League Under 21 s squad. We pair-matched n = 17 players (age: 17 ± 0.9 years; height: 1.66 ± 0.06 m; mass: 58.8 ± 8.1 kg) for baseline knee extension (KE) maximum isometric voluntary contraction (MIVC) torque, age, height, and body mass, and randomly assigned them to collagen (COL) or placebo (PLA) groups (COL n = 8, PLA n = 9). Participants consumed 30 g collagen hydrolysate supplementation or energy-matched PLA (36.5 g maltodextrin, 8.4 g fructose) and plus both groups consumed 500 mg vitamin C, after each training session, which comprised bodyweight strength-, plyometric- and/or pitch-based exercise 3 days/week for 10 weeks in-season. We assessed KE MIVC torque, vastus lateralis muscle thickness and PT properties using isokinetic dynamometry and ultrasonography before and after 10 weeks' soccer training. KE MIVC torque, muscle thickness and tendon cross-sectional area did not change after training in either group. However, COL increased PT stiffness [COL, +18.0 ± 12.2% (d = 1.11) vs. PLA, +5.1 ± 10.4% (d = 0.23), p = 0.049] and Young's modulus [COL, +17.3 ± 11.9% (d = 1.21) vs. PLA, +4.8 ± 10.3% (d = 0.23), p = 0.035] more than PLA. Thus, 10 weeks' in-season soccer training with COL increased PT mechanical and material properties more than soccer training alone in high-level female soccer players. Future studies should investigate if collagen hydrolysate supplementation can improve specific aspects of female soccer performance requiring rapid transference of force, and if it can help mitigate injury risk in this under-researched population.