Twelve tips for using tactical decision games to teach non-technical skills.

Tactical decision games (TDGs) have been used in healthcare and other safety-critical industries to develop non-technical skills training (NTS). TDGs have been shown to be a realistic, feasible, and useful way of teaching NTS such as decision making, task prioritisation, situational awareness, and team working. Our 12-tips for using TDG to teach NTS are based on our experience of integrating them into an undergraduate medical and nursing programme. We cover how to design successful TDGs, how to facilitate and debrief them and how to integrate TDGs into curricula. We have found TDGs to be a cost-effective, low fidelity, and useful method of delivering NTS teaching, ideally as an adjunct to immersive simulation. Learners find them a useful way to be introduced to NTS in a safe and relaxed environment, with particular emphasis on critical decision making and prioritisation.

Rapid production of pure recombinant actin isoforms in Pichia pastoris.

Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.

The bromodomain protein Brd4 insulates chromatin from DNA damage signalling.

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.

Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine.

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.

A specialist, second-tier response to out-of-hospital cardiac arrest: setting up TOPCAT2.

BACKGROUND: Out-of-hospital cardiac arrest (OHCA) is the most common, immediately life-threatening, medical emergency faced by ambulance crews. Survival from OHCA is largely dependent on quality of prehospital resuscitation. Non-technical skills, including resuscitation team leadership, communication and clinical decision-making are important in providing high quality prehospital resuscitation. We describe a pilot study (TOPCAT2, TC2) to establish a second tier, expert paramedic response to OHCA in Edinburgh, Scotland. METHODS: Eight paramedics were selected to undergo advanced training in resuscitation and non-technical skills. Simulation and video feedback was used during training. The designated TC2 paramedic manned a regular ambulance service response car and attended emergency calls in the usual manner. Emergency medical dispatch centre dispatchers were instructed to call the TC2 paramedic directly on receipt of a possible OHCA call. Call and dispatch timings, quality of cardiopulmonary resuscitation and return-of-spontaneous circulation were all measured prospectively. RESULTS: Establishing a specialist, second-tier paramedic response was feasible. There was no overall impact on ambulance response times. From the first 40 activations, the TC2 paramedic was activated in a median of 3.2 min (IQR 1.6-5.8) and on-scene in a median of 10.8 min (8.0-17.9). Bimonthly team debrief, case review and training sessions were successfully established. OHCA attended by TC2 showed an additional trend towards improved outcome with a rate of return of spontaneous circulation of 22.5%, compared with a national average of 16%. CONCLUSIONS: Establishing a specialist, second-tier response to OHCA is feasible, without impacting on overall ambulance response times. Improving non-technical skills, including prehospital resuscitation team leadership, has the potential to save lives and further research on the impact of the TOPCAT2 pilot programme is warranted.

MAPKAP Kinase-2 phosphorylation of PABPC1 controls its interaction with 14-3-3 proteins after DNA damage: A combined kinase and protein array approach.

14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.

Postprandial heat production in skeletal muscle is associated with altered mitochondrial function and altered futile calcium cycling.

This study aimed to determine whether postprandial temperature excursions in skeletal muscle are consistent with thermogenesis or altered blood flow. Temperature probes were implanted into the vastus lateralis muscle of ovariectomized ewes, and blood flow was assessed using laser-Doppler flowmetry (tissue flow) and transit-time ultrasound flowmetry (femoral artery flow). The animals were program-fed between 1100 and 1600, and temperature and blood flow were measured during intravenous administration of either isoprenaline or phenylephrine and during feeding and meal anticipation. In addition, muscle biopsies were collected prefeeding and postfeeding to measure uncoupling protein (UCP) expression and mitochondrial function, as well as indices of calcium cycling (ryanodine 1 receptor: RyR1 and sarcoendoplasmic calcium-dependent ATPases SERCA1/ SERCA2a). Isoprenaline increased femoral artery blood flow, whereas phenylephrine reduced blood flow. At high doses only, isoprenaline treatment increased heat production in muscle. Phenylephrine treatment did not alter muscle temperature. Meal anticipation was evoked in fasted animals (previously program-fed) that were housed beside animals that were fed. Increases in muscle temperature were elicited by feeding and meal anticipation, without changes in blood flow during either paradigm. Analyses of respiration in isolated mitochondria indicated that the postprandial increase in heat production was associated with an increase in state 4 respiration, without increased UCP1, UCP2, or UCP3 expression. Feeding increased the expression of RyR1 and SERCA2a. We conclude that excursions in muscle temperature may occur independent of blood flow, suggesting that postprandial heat production is driven by altered mitochondrial function and changes in calcium cycling.

Identifying patients at risk of futile resuscitation: palliative care indicators in out-of-hospital cardiac arrest.

OBJECTIVES: Patients with indicators for palliative care, such as those with advanced life-limiting conditions, are at risk of futile cardiopulmonary resuscitation (CPR) if they suffer out-of-hospital cardiac arrest (OHCA). Patients at risk of futile CPR could benefit from anticipatory care planning (ACP); however, the proportion of OHCA patients with indicators for palliative care is unknown. This study quantifies the extent of palliative care indicators and risk of CPR futility in OHCA patients. METHODS: A retrospective medical record review was performed on all OHCA patients presenting to an emergency department (ED) in Edinburgh, Scotland in 2015. The risk of CPR futility was stratified using the Supportive and Palliative Care Indicators Tool. Patients with 0-2 indicators had a 'low risk' of futile CPR; 3-4 indicators had an 'intermediate risk'; 5+ indicators had a 'high risk'. RESULTS: Of the 283 OHCA patients, 12.4% (35) had a high risk of futile CPR, while 16.3% (46) had an intermediate risk and 71.4% (202) had a low risk. 84.0% (68) of intermediate-to-high risk patients were pronounced dead in the ED or ED step-down ward; only 2.5% (2) of these patients survived to discharge. CONCLUSIONS: Up to 30% of OHCA patients are being subjected to advanced resuscitation despite having at least three indicators for palliative care. More than 80% of patients with an intermediate-to-high risk of CPR futility are dying soon after conveyance to hospital, suggesting that ACP can benefit some OHCA patients. This study recommends optimising emergency treatment planning to help reduce inappropriate CPR attempts.

Calponin-homology domain mediated bending of membrane-associated actin filaments.

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that 'curly', a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Although F-actin is semi-flexible with a persistence length of ~10 µm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 µm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.

Method, Material, and Machine: A Review for the Surgeon Using Three-Dimensional Printing for Accelerated Device Production.

BACKGROUND: Physicians are at the forefront of identifying innovative targets to address current medical needs. 3D printing technology has emerged as a state-of-the-art method of prototyping medical devices or producing patient-specific models that is more cost-efficient, with faster turnaround time, in comparison to traditional prototype manufacturing. However, initiating 3D printing projects can be daunting due to the engineering learning curve, including the number of methodologies, variables, and techniques for printing from which to choose. To help address these challenges, we sought to create a guide for physicians interested in venturing into 3D printing. STUDY DESIGN: All commercially available, plug-and-play, material and stereolithography printers costing less than $15,000 were identified via web search. Companies were contacted to obtain quotes and information sheets for all printer models. The qualifying printers' manufacturer specification sheets were reviewed, and pertinent variables were extracted. RESULTS: We reviewed 309 commercially available printers and materials and identified 118 printers appropriate for clinicians desiring plug-and-play models for accelerated device production. We synthesized this information into a decision-making tool to choose the appropriate parameters based on project goals. CONCLUSIONS: There is a growing clinical need for medical devices to reduce costs of care and increase access to personalized treatments; however, the learning curve may be daunting for surgeons. In this review paper, we introduce the "3Ms of 3D printing" for medical professionals and provide tools and data sheets for selection of commercially available, affordable, plug-and-play 3D printers appropriate for surgeons interested in innovation.

Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.

Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.

Cell-Based High Content Analysis of Cell Proliferation and Apoptosis.

High content imaging-based cell cycle analysis allows multiplexing of various parameters including DNA content, DNA synthesis, cell proliferation, and other cell cycle markers such as phosho-histone H3. 5'-Ethynyl-2'-deoxyuridine (EdU) incorporation is a thymidine analog that provides a sensitive method for the detection of DNA synthesis in proliferating cells that is a more convenient method than the traditional BrdU detection by antibody. Caspase 3 is activated in programmed cell death induced by both intrinsic (mitochondrial) and extrinsic factors (death ligand). Cell cycle and apoptosis are common parameters studied in the phenotypic analysis of compound toxicity and anti-cancer drugs. In this chapter, we describe methods for the detection of s-phase cell cycle progression by EdU incorporation, and caspase 3 activation using the CellEvent caspase 3/7 detection reagent.

Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc.

Clinical applications of 3-dimensional printing in radiation therapy.

Three-dimensional (3D) printing is suitable for the fabrication of complex radiotherapy bolus. Although investigated from dosimetric and feasibility standpoints, there are few reports to date of its use for actual patient treatment. This study illustrates the versatile applications of 3D printing in clinical radiation oncology through a selection of patient cases, namely, to create bolus for photon and modulated electron radiotherapy (MERT), as well as applicators for surface high-dose rate (HDR) brachytherapy. Photon boluses were 3D-printed to treat a recurrent squamous cell carcinoma (SCC) of the nasal septum and a basal cell carcinoma (BCC) of the posterior pinna. For a patient with a mycosis fungoides involving the upper face, a 3D-printed MERT bolus was used. To treat an SCC of the nose, a 3D-printed applicator for surface brachytherapy was made. The structures' fit to the anatomy and the radiotherapy treatment plans were assessed. Based on the treatment planning computed tomography (CT), the size of the largest air gap at the interface of the 3D-printed structure was 3 mm for the SCC of the nasal septum, 3 mm for the BCC of the pinna, 2 mm for the mycosis fungoides of the face, and 2 mm for the SCC of the nose. Acceptable treatment plans were obtained for the SCC of the nasal septum (95% isodose to 99.8% of planning target volume [PTV]), the BCC of the pinna (95% isodose to 97.7% of PTV), and the mycosis fungoides of the face (90% isodose to 92.5% of PTV). For the latter, compared with a plan with a uniform thickness bolus, the one featuring the MERT bolus achieved relative sparing of all the organs at risk (OARs) distal to the target volume, while maintaining similar target volume coverage. The surface brachytherapy plan for the SCC of the nose had adequate coverage (95% isodose to 95.6% of clinical target volume [CTV]), but a relatively high dose to the left eye, owing to its proximity to the tumor. 3D printing can be implemented effectively in the clinical setting to create highly conformal bolus for photon and MERT, as well as applicators for surface brachytherapy.

Thymoma in a geriatric rabbit with hypercalcemia and periodic exophthalmos.

An 8-year-old rabbit was referred to an ophthalmologist because of intermittent bilateral exophthalmos and prolapse of the nictitating membranes. Both eyes could be retropulsed normally, and the exophthalmos was induced with ventroflexion. The rabbit had moderate hypercalcemia and a large mediastinal mass that could be seen on thoracic radiographs. The rabbit's condition was unchanged for 5 months. It was reexamined because of weight loss and paroxysmal coughing and, at that time, was thin and tachypneic, and had reduced thoracic compliance. Thoracotomy was performed, and a 5-cm-diameter encapsulated mass, subsequently determined histologically to be thymoma, was removed. The rabbit was euthanatized after surgery because of complications. The periodic exophthalmos and hypercalcemia in this rabbit were believed to be paraneoplastic syndromes.

Implementation of the Connective Tissue Screening Questionnaire in northeast Pennsylvania to identify comorbidities of connective tissue diseases in subjects with systemic lupus erythematosus.

Previous studies have described an increased risk of developing an additional connective tissue disease (CTD) when one such ailment is present. We examine here the likelihood that individuals with systemic lupus erythematosus (SLE) screen positive for one or more of the following five autoimmune CTDs: Sjögren's syndrome, scleroderma, rheumatoid arthritis, dermatomyositis/polymyositis, and mixed connective tissue disorder. Five hundred SLE-diagnosed subjects were asked to complete a CTD screening questionnaire (CSQ). The results were analyzed according to the set of diagnostic criteria given by the American College of Rheumatology to identify probable cases of each CTD. Significant standardized prevalence ratios and comorbidities indicate an increased risk for the other autoimmune CTDs. In all, 96% of the subjects screened positive for at least one additional CTD, and 13% screened positive for at least two additional CTDs. We see that the SLE-diagnosed population may benefit from further attention regarding the presence of additional CTDs, which may further inform treatment strategies. We also see the application of the CSQ as a potentially important tool for clinical practice, and we describe the present study's limitations along with possible ways that these can be addressed.

Differential effects of acute and chronic estrogen treatment on thermogenic and metabolic pathways in ovariectomized sheep.

Estrogen is protective against weight gain, but the underlying mechanisms are not fully elucidated. We sought to characterize the effects of estrogen on energy expenditure in skeletal muscle and adipose tissue in ovariectomized sheep. Temperature probes were implanted into sc (gluteal) and visceral (retroperitoneal) fat depots and skeletal muscle of the hind limb (vastus lateralis). Food was available from 1100-1600 h to entrain postprandial thermogenesis. We characterized the effects of single (50 µg estradiol benzoate, im) and repeated (25 µg estradiol-17ß, iv) injections as well as chronic (3 × 3 cm estradiol-17ß implants for 7 d) treatment on heat production. A single injection of estrogen increased heat production in visceral fat and skeletal muscle, without an effect on food intake. Increased heat production in skeletal muscle was sustained by repeated estradiol-17ß injections. On the other hand, continuous treatment reduced food intake but had no effect on thermogenesis. To determine possible mechanisms that underpin estradiol-17ß-induced heat production, we measured femoral artery blood flow, the expression of uncoupling protein (UCP) mRNA and the phosphorylation of AMP-activated protein kinase and Akt in fat and muscle. There was little effect of either single or repeated injections of estradiol-17ß on the expression of UCP1, -2, or -3 mRNA in visceral fat or skeletal muscle. Acute injection of estradiol-17ß increased the phosphorylation of AMP-activated protein kinase and Akt in muscle only. Estradiol-17ß treatment did not alter femoral artery blood flow. Thus, the stimulatory effect of estradiol-17ß on thermogenesis in female sheep is dependent upon a pulsatile pattern of treatment and not constant continuous exposure.