i Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.C ellular differentiation is generally controlled by transcriptional activation or repression of specific genes. Consequently, a host of different molecular genetic events can shape the properties of cells during development. Recent evidence indicates that an evolutionarily conserved protein complex known as DREAM is capable of regulating diverse gene expression programs, thereby unifying many disparate events in development into a single molecular machine (1, 2).The DREAM complex was isolated, and its composition was determined from a number of different model organisms. Studies of aberrant growth factor signaling in Caenorhabditis elegans lead to the discovery of complementation groups that contribute to a multivulval (Muv) phenotype (3). Mutation of any two of the synthetic multivulval (synMuv) group A, B, or C genes resulted in worms with elevated numbers of vulvae (4). Group B contains a number of genes (the Lin-9, Lin-37, Lin-52, Lin-53/RBBP4, and Lin-54 genes) whose encoded proteins form the MuvB core complex (5-7). In addition, worm homologues of retinoblastoma protein (RB), E2F, and DP are also group B members (8, 9). The MuvB core was also found to interact with MYB in transcriptional control of cell cycle progression in fruit flies (6, 7). Isolation of MYB and RB revealed that they copurify with MuvB proteins, and this has formed the basis of the DREAM complex (Drosophila RB, E2F, and MuvB). In some organisms it also contains epigenetic readers and writers such as histone deacetylases (HDACs) and L3MBT (6,7,10). The model that has emerged is one in which the DREAM complex can confer both positive and negative regulation of transcripti...

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A–C2G, 13%–33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a ‘calcium-catching’ mechanism to respond to calcium influx during membrane repair.

ObjectiveSimulation plays an integral role in the Canadian healthcare system with applications in quality improvement, systems development, and medical education. High-quality, simulation-based research will ensure its effective use. This study sought to summarize simulation-based research activity and its facilitators and barriers, as well as establish priorities for simulation-based research in Canadian emergency medicine (EM).MethodsSimulation-leads from Canadian departments or divisions of EM associated with a general FRCP-EM training program surveyed and documented active EM simulation-based research at their institutions and identified the perceived facilitators and barriers. Priorities for simulation-based research were generated by simulation-leads via a second survey; these were grouped into themes and finally endorsed by consensus during an in-person meeting of simulation leads. Priority themes were also reviewed by senior simulation educators.ResultsTwenty simulation-leads representing all 14 invited institutions participated in the study between February and May, 2018. Sixty-two active, simulation-based research projects were identified (median per institution = 4.5, IQR 4), as well as six common facilitators and five barriers. Forty-nine priorities for simulation-based research were reported and summarized into eight themes: simulation in competency-based medical education, simulation for inter-professional learning, simulation for summative assessment, simulation for continuing professional development, national curricular development, best practices in simulation-based education, simulation-based education outcomes, and simulation as an investigative methodology.ConclusionThis study summarized simulation-based research activity in EM in Canada, identified its perceived facilitators and barriers, and built national consensus on priority research themes. This represents the first step in the development of a simulation-based research agenda specific to Canadian EM.

Objectives Checklists have been used to decrease adverse events associated with medical procedures. Simulation provides a safe setting in which to evaluate a new checklist. The objective of this study was to determine if the use of a novel peri-intubation checklist would decrease practitioners’ rates of omission of tasks during simulated airway management scenarios. Methods Fifty-four emergency medicine (EM) practitioners from two academic centers were randomized to either their usual approach or use of our checklist, then completed three simulated airway management scenarios. A minimum of two assessors documented the number of tasks omitted and the time until definitive airway management. Discrepancies between assessors were resolved by single assessor video review. Participants also completed a post-simulation survey. Results The average percentage of omitted tasks over three scenarios was 45.7% in the control group ( n = 25) and 13.5% in the checklist group ( n = 29)—an absolute difference of 32.2% (95% CI 27.8, 36.6%). Time to definitive airway management was longer in the checklist group in the first two of three scenarios (difference of 110.0 s, 95% CI 55.0 to 167.0; 83.0 s, 95% CI 35.0 to 128.0; and 36.0 s, 95% CI −18.0 to 98.0 respectively). Conclusions In this dual-center, randomized controlled trial, use of an airway checklist in a simulated setting significantly decreased the number of important airway tasks omitted by EM practitioners, but increased time to definitive airway management. Electronic supplementary material The online version of this article (10.1007/s43678-020-00010-w) contains supplementary material, which is available to authorized users.