Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators.

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D = 67 nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.

Autopodial development is selectively impaired by misexpression of chordin-like 1 in the chick limb.

Chordin-like 1 (CHRDL1) is a secreted bone morphogenetic protein (BMP) antagonist expressed in mesenchymal tissues whose function in development of the skeleton has not been examined in detail. Here we show Chrdl1 is dynamically expressed in the early distal limb bud mesenchyme, with expression becoming downregulated as development proceeds. Chrdl1 expression is largely excluded from the critical signaling center of the posterior limb bud, the Zone of Polarizing Activity (ZPA), as has been described for the BMP antagonist Gremlin (GREM1) (Scherz et al., 2004, Science, 305, 396-399). Unlike Grem1, Chrdl1 is expressed in the hindlimb by a small subset of ZPA cells and their descendants suggesting divergent regulation and function between the various BMP antagonists. Ectopic expression of Chrdl1 throughout the avian limb bud using viral misexpression resulted in an oligodactyly phenotype with loss of digits from the anterior limb, although the development of more proximal elements of the zeugopod and stylopod were unaffected. Overgrowths of soft tissue and syndactyly were also observed, resulting from impaired apoptosis and failure of the anterior mesenchyme to undergo SOX9-dependent chondrogenesis, instead persisting as an interdigital-like soft tissue phenotype. Sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling were upregulated and persisted later in development, however these changes were only detected late in limb development at timepoints when endogenous Grem1 would normally be downregulated and increasing BMP signaling would cause termination of Shh and Fgf expression. Our results suggest that the early stages of the GREM1-SHH-FGF signaling network are resistant to Chrdl1-overexpression, leading to normal formation of proximal limb structures, but that later Bmp expression, impaired by ectopic CHRDL1, is essential for formation of the correct complement of digits.

Exploring the recovery journey of COVID-19 critical care survivors during the first year after hospital discharge.

Objectives: To explore the longitudinal recovery of patients admitted to critical care following COVID-19 over the year following hospital discharge. To understand the important aspects of the patients' recovery process and key elements of their caregivers' experiences during this time. Design: A longitudinal qualitative study using semi-structured interviews. Setting: Two acute hospitals in South East England and follow-up in the community. Participants: Six COVID-19 critical care survivors from the first wave of the pandemic (March-May 2020) and five relatives were interviewed 3 months after hospital discharge. The same six survivors and one relative were interviewed again at 1 year. Interviews were transcribed verbatim, anonymised and a reflexive thematic analysis was conducted. Results: Three themes were developed: (1) 'The cycle of guilt, fear and stigma'; (2) 'Facing the uncertainties of recovery' and (3) 'Coping with lingering symptoms - the new norm'. The first theme highlights survivors' reluctance to share their experiences associated with contracting the disease. The second theme, explores challenges faced by the survivors and their relatives in navigating the recovery process, given the unknown nature of the illness. The final theme illustrates the mechanisms survivors develop to come to terms with the remnants of their illness and critical care stay. Conclusions: The longitudinal nature of the study highlighted the persisting symptoms of long COVID-19, their impact on survivors and coping methods amidst the ongoing pandemic. Further research into the experiences of those affected in the first and subsequent waves of the COVID-19 pandemic, is desirable to help guide the formulation of the optimally supported recovery pathways.

Assessing Serious Spinal Pathology Using Bayesian Network Decision Support: Development and Validation Study.

BACKGROUND: Identifying and managing serious spinal pathology (SSP) such as cauda equina syndrome or spinal infection in patients presenting with low back pain is challenging. Traditional red flag questioning is increasingly criticized, and previous studies show that many clinicians lack confidence in managing patients presenting with red flags. Improving decision-making and reducing the variability of care for these patients is a key priority for clinicians and researchers. OBJECTIVE: We aimed to improve SSP identification by constructing and validating a decision support tool using a Bayesian network (BN), which is an artificial intelligence technique that combines current evidence and expert knowledge. METHODS: A modified RAND appropriateness procedure was undertaken with 16 experts over 3 rounds, designed to elicit the variables, structure, and conditional probabilities necessary to build a causal BN. The BN predicts the likelihood of a patient with a particular presentation having an SSP. The second part of this study used an established framework to direct a 4-part validation that included comparison of the BN with consensus statements, practice guidelines, and recent research. Clinical cases were entered into the model and the results were compared with clinical judgment from spinal experts who were not involved in the elicitation. Receiver operating characteristic curves were plotted and area under the curve were calculated for accuracy statistics. RESULTS: The RAND appropriateness procedure elicited a model including 38 variables in 3 domains: risk factors (10 variables), signs and symptoms (17 variables), and judgment factors (11 variables). Clear consensus was found in the risk factors and signs and symptoms for SSP conditions. The 4-part BN validation demonstrated good performance overall and identified areas for further development. Comparison with available clinical literature showed good overall agreement but suggested certain improvements required to, for example, 2 of the 11 judgment factors. Case analysis showed that cauda equina syndrome, space-occupying lesion/cancer, and inflammatory condition identification performed well across the validation domains. Fracture identification performed less well, but the reasons for the erroneous results are well understood. A review of the content by independent spinal experts backed up the issues with the fracture node, but the BN was otherwise deemed acceptable. CONCLUSIONS: The RAND appropriateness procedure and validation framework were successfully implemented to develop the BN for SSP. In comparison with other expert-elicited BN studies, this work goes a step further in validating the output before attempting implementation. Using a framework for model validation, the BN showed encouraging validity and has provided avenues for further developing the outputs that demonstrated poor accuracy. This study provides the vital first step of improving our ability to predict outcomes in low back pain by first considering the problem of SSP. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/21804.

A Bayesian Network Decision Support Tool for Low Back Pain Using a RAND Appropriateness Procedure: Proposal and Internal Pilot Study.

BACKGROUND: Low back pain (LBP) is an increasingly burdensome condition for patients and health professionals alike, with consistent demonstration of increasing persistent pain and disability. Previous decision support tools for LBP management have focused on a subset of factors owing to time constraints and ease of use for the clinician. With the explosion of interest in machine learning tools and the commitment from Western governments to introduce this technology, there are opportunities to develop intelligent decision support tools. We will do this for LBP using a Bayesian network, which will entail constructing a clinical reasoning model elicited from experts. OBJECTIVE: This paper proposes a method for conducting a modified RAND appropriateness procedure to elicit the knowledge required to construct a Bayesian network from a group of domain experts in LBP, and reports the lessons learned from the internal pilot of the procedure. METHODS: We propose to recruit expert clinicians with a special interest in LBP from across a range of medical specialties, such as orthopedics, rheumatology, and sports medicine. The procedure will consist of four stages. Stage 1 is an online elicitation of variables to be considered by the model, followed by a face-to-face workshop. Stage 2 is an online elicitation of the structure of the model, followed by a face-to-face workshop. Stage 3 consists of an online phase to elicit probabilities to populate the Bayesian network. Stage 4 is a rudimentary validation of the Bayesian network. RESULTS: Ethical approval has been obtained from the Research Ethics Committee at Queen Mary University of London. An internal pilot of the procedure has been run with clinical colleagues from the research team. This showed that an alternating process of three remote activities and two in-person meetings was required to complete the elicitation without overburdening participants. Lessons learned have included the need for a bespoke online elicitation tool to run between face-to-face meetings and for careful operational definition of descriptive terms, even if widely clinically used. Further, tools are required to remotely deliver training about self-identification of various forms of cognitive bias and explain the underlying principles of a Bayesian network. The use of the internal pilot was recognized as being a methodological necessity. CONCLUSIONS: We have proposed a method to construct Bayesian networks that are representative of expert clinical reasoning for a musculoskeletal condition in this case. We have tested the method with an internal pilot to refine the process prior to deployment, which indicates the process can be successful. The internal pilot has also revealed the software support requirements for the elicitation process to model clinical reasoning for a range of conditions. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/21804.

Standard setting anchor statements: a double cross-over trial of two different methods.

This article was migrated. The article was marked as recommended. Context: We challenge the philosophical acceptability of the Angoff method, and propose an alternative method of standard setting based on how important it is for candidates to know the material each test item assesses, and not how difficult it is for a subgroup of candidates to answer each item. Methods: The practicalities of an alternative method of standard setting are evaluated here, for the first time, with direct comparison to an Angoff method. To negate bias due to any leading effects, a prospective cross-over design was adopted involving two groups of judges (n=7 and n=8), both of which set the standards for the same two 100 item multiple choice question tests, by the two different methods. Results: Overall, we found that the two methods took a similar amount of time to complete. The alternative method produced a higher cut-score (by 12-14%), and had a higher degree of variability between judges' cut-scores (by 5%). When using the alternative method, judges reported a small, but statistically significant, increase in their confidence to decide accurately the standard (by 3%). Conclusion: This is a new approach to standard setting where the quantitative differences are slight, but there are clear qualitative advantages associated with use of the alternative method.

Lubricin restoration in a mouse model of congenital deficiency.

OBJECTIVE: Congenital deficiency of the principal boundary lubricant in cartilage (i.e., lubricin, encoded by the gene PRG4) increases joint friction and causes progressive joint failure. This study was undertaken to determine whether restoring lubricin expression in a mouse model would prevent, delay, or reverse the disease process caused by congenital deficiency. METHODS: Using genetically engineered lubricin-deficient mice, we restored gene function before conception or at ages 3 weeks, 2 months, or 6 months after birth. The effect of restoring gene function (i.e., expression of lubricin) on the tibiofemoral patellar joints of mice was evaluated histologically and by ex vivo biomechanical testing. RESULTS: Restoring gene function in mice prior to conception prevented joint disease. In 3-week-old mice, restoring gene function improved, but did not normalize, histologic features of the articular cartilage and whole-joint friction. In addition, cyclic loading of the joints produced fewer activated caspase 3-containing chondrocytes when lubricin expression was restored, as compared to that in littermate mice whose gene function was not restored (nonrestored controls). Restoration of lubricin expression in 2-month-old or 6-month-old mice had no beneficial effect on histopathologic cartilage damage, extent of whole-joint friction, or activation of caspase 3 when compared to nonrestored controls. CONCLUSION: When boundary lubrication is congenitally deficient and cartilage becomes damaged, the window of opportunity for restoring lubrication and slowing disease progression is limited.

Lubricin protects the temporomandibular joint surfaces from degeneration.

The temporomandibular joint (TMJ) is a specialized synovial joint essential for the mobility and function of the mammalian jaw. The TMJ is composed of the mandibular condyle, the glenoid fossa of the temporal bone, and a fibrocartilagenous disc interposed between these bones. A fibrous capsule, lined on the luminal surface by the synovial membrane, links these bones and retains synovial fluid within the cavity. The major component of synovial fluid is lubricin, a glycoprotein encoded by the gene proteoglycan 4 (Prg4), which is synthesized by chondrocytes at the surface of the articular cartilage and by synovial lining cells. We previously showed that in the knee joint, Prg4 is crucial for maintenance of cartilage surfaces and for regulating proliferation of the intimal cells in the synovium. Consequently, the objective of this study was to determine the role of lubricin in the maintenance of the TMJ. We found that mice lacking lubricin have a normal TMJ at birth, but develop degeneration resembling TMJ osteoarthritis by 2 months, increasing in severity over time. Disease progression in Prg4-/- mice results in synovial hyperplasia, deterioration of cartilage in the condyle, disc and fossa with an increase in chondrocyte number and their redistribution in clusters with loss of superficial zone chondrocytes. All articular surfaces of the joint had a prominent layer of protein deposition. Compared to the knee joint, the osteoarthritis-like phenotype was more severe and manifested earlier in the TMJ. Taken together, the lack of lubricin in the TMJ causes osteoarthritis-like degeneration that affects the articular cartilage as well as the integrity of multiple joint tissues. Our results provide the first molecular evidence of the role of lubricin in the TMJ and suggest that Prg4-/- mice might provide a valuable new animal model for the study of the early events of TMJ osteoarthritis.

Gene expression of catabolic inflammatory cytokines peak before anabolic inflammatory cytokines after ACL injury in a preclinical model.

BACKGROUND: The response of the joint to anterior cruciate ligament (ACL) injury has not been fully characterized. In particular, the characterization of both catabolic factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), and markers of ongoing tissue damage (CRP), and anabolic factors, including vascular endothelial growth factor (VEGF), transforming growth factor ß-induced (TGFßI), and the presence of CD163+ macrophages, have not been well defined. In this study, we hypothesized ACL injury would catalyze both catabolic and anabolic processes and that these would have different temporal profiles of expression. METHODS: Adolescent Yucatan minipigs were subjected to ACL transection. Within the joint, gene expression levels of IL-6, IL-8, VEGF, and TGFßI were quantified in the synovium, ligament, and provisional scaffold located between the torn ligament ends at days 1, 5, 9, and 14 post-injury. Macrophage infiltration was also assessed in the joint tissues over the two week period. Serum C-reactive protein (CRP) levels were measured at multiple time points between 1 hour to 14 days after injury. RESULTS: Increases in IL-6 and IL-8 gene expression peaked at day 1 after injury in the synovium and ligament. CRP levels were significantly increased at day 3 before returning to pre-injury levels. VEGF and TGFßI gene expression did not significantly increase until day 9 in the synovium and were unchanged in the other tissues. CD163+ macrophages increased in the ligament and synovium until day 9. CONCLUSION: Taken together, these results suggest that the response within the joint is primarily catabolic in the first three days after injury, switching to a more anabolic phase by nine days after injury. The effect of medications which alter these processes may thus depend on the timing of administration after injury.

Golgi disruption and early embryonic lethality in mice lacking USO1.

Golgins are a family of long rod-like proteins characterized by the presence of central coiled-coil domains. Members of the golgin family have important roles in membrane trafficking, where they function as tethering factors that capture transport vesicles and facilitate membrane fusion. Golgin family members also have essential roles in maintaining the organization of the Golgi apparatus. Knockdown of individual golgins in cultured cells resulted in the disruption of the Golgi structure and the dispersal of Golgi marker proteins throughout the cytoplasm. However, these cellular phenotypes have not always been recapitulated in vivo. For example, embryonic development proceeds much further than expected and Golgi disruption was observed in only a subset of cell types in mice lacking the ubiquitously expressed golgin GMAP-210. Cell-type specific functional compensation among golgins may explain the absence of global cell lethality when a ubiquitously expressed golgin is missing. In this study we show that functional compensation does not occur for the golgin USO1. Mice lacking this ubiquitously expressed protein exhibit disruption of Golgi structure and early embryonic lethality, indicating that USO1 is indispensable for early embryonic development.

Comprehensive approaches to school health promotion: how to achieve broader implementation?

The Health Promoting School (HPS) and Comprehensive School Health Program (CSHP) initiatives have been proposed as a means of going beyond some of the limitations associated with health promotion initiatives aimed at school-aged children. This involves moving beyond practices that rely mainly on classroom-based health education models, to a more comprehensive, integrated approach of health promotion that focuses both on child-youth attitudes and behaviors, and their environment. Despite the tremendous potential of these initiatives in terms of health and educational gains, only rarely are they actually put into practice. This article briefly reviews the features of these initiatives, as well as the extent of their implementation and current benefits. Against that backdrop, the authors identify some issues to consider and propose four conditions with a view to achieving broader practical application of these approaches. These issues, which are discussed from the standpoint of potential avenues of further study and courses of action, relate to the comprehensive, integrated nature of the intervention, the school/family/community partnership, political and financial support from policy makers, and, finally, evaluative research as a support to implementation.