An apparatus for automatically training and collecting individualized behavioral data with socially housed rodents.

BACKGROUND: Conventional methods for individually housing, training, and testing rodents in behavioral assays can impose constraints that may limit some kinds of experimental external validity, preempt environmental enrichment, impose heavy experimenter time burdens that limit high-throughput data collection, and negatively impact animal welfare. NEW METHOD: To address these issues, we created a simple apparatus for automatically collecting individually identified data with rodents in social and/or enriched housing. RESULTS: We validated this "One Rat Turnstyle" (ORT) apparatus by utilizing it to automatically teach socially housed rats to individually press a lever without experimenter intervention or shaping. Results confirmed the feasibility and reliability of the apparatus, with almost all rats learning to move through the ORT and press a lever for sugar water by the end of the experiment. Rats had lower fecal cortisol when engaging with the ORT than with experimenter conducted daily behavioral training sessions. COMPARISON WITH EXISTING METHOD(S): The ORT is less electronically complex and more scalable compared to previous similar ideas. It requires only a 3d printer and the purchase of few parts. It is also designed to allow animals to quickly learn how to utilize it by minimizing passthrough time. CONCLUSIONS: Rats passed through the ORT both quickly and efficiently, self-administering reasonably timed behavioral sessions throughout the day. This success demonstrates that the ORT can enable the collection of both traditional and innovative, self-paced data in the context of socially housed animals, and may contribute to expanded, ecologically valid modelling.

Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Affimers as an alternative to antibodies for protein biomarker enrichment.

INTRODUCTION: Assessing the specificity of protein binders is an essential first step in protein biomarker assay development. Affimers are novel protein binders and can potentially replace antibodies in multiple protein capture-based assays. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions. Here we mimicked a typical initial screening of affimers and commercially available monoclonal antibodies against two non-related proteins, IL-37b and proinsulin, to assess the potential of affimers as alternative to antibodies. METHODS: Binding specificity of anti-IL-37b and anti-proinsulin affimers and antibodies was investigated via magnetic bead-based capture of their recombinant protein targets in human plasma. Captured proteins were analyzed using SDS-PAGE, Coomassie blue staining, Western blotting and LC-MS/MS-based proteomics. RESULTS: All affimers and antibodies were able to bind their target protein in human plasma. Gel and LC-MS/MS analysis showed that both affimer and antibody-based captures resulted in co-purified background proteins. However, affimer-based captures showed the highest relative enrichment of IL-37b and proinsulin. CONCLUSIONS: For both proteins tested, affimers show higher specificity in purifying their target proteins from human plasma compared to monoclonal antibodies. These results indicate that affimers are promising antibody-replacement tools for protein biomarker assay development.

Development and Utility of Quality Metrics for Ambulatory Pediatric Cardiology in Kawasaki Disease.

The Adult Congenital and Pediatric Cardiology (ACPC) Section of the American College of Cardiology sought to develop quality indicators/metrics for ambulatory pediatric cardiology practice. The objective of this study was to report the creation of metrics for patients with Kawasaki disease. Over a period of 5 months, 12 pediatric cardiologists developed 24 quality metrics based on the most relevant statements, guidelines, and research studies pertaining to Kawasaki disease. Of the 24 metrics, the 8 metrics deemed the most important, feasible, and valid were sent on to the ACPC for consideration. Seven of the 8 metrics were approved using the RAND method by an expert panel. All 7 metrics approved by the ACPC council were accepted by ACPC membership after an "open comments" process. They have been disseminated to the pediatric cardiology community for implementation by the ACPC Quality Network.

Variation in the use of pulmonary vasodilators in children and adolescents with pulmonary hypertension: a study using data from the MarketScan® insurance claims database.

Despite progress in pharmacotherapy in pediatric pulmonary hypertension, real-world patterns of directed pulmonary hypertension therapy have not been studied in the current era. A retrospective observational study of children (≤18 years) with pulmonary hypertension was performed using data from the MarketScan® Commercial and Medicaid claims databases. Associations between etiology of pulmonary hypertension and pharmaceutical regimen were evaluated, as were the associations between subject social and geographic characteristics (insurance-type, race, and/or census region) and regimen. Annualized costs of single- and multi-class regimens were calculated. In total, 873 subjects were studied, of which 94% received phosphodiesterase-5 inhibitors, 31% endothelin receptor antagonist, 9% prostacyclin analogs, and 7% calcium channel blockers. Monotherapy was used in 72% of subjects. Phosphodiesterase-5 inhibitors monotherapy was the most common regimen (93%). Subjects with idiopathic pulmonary hypertension, congenital heart disease, and unclassified pulmonary hypertension receive more than one agent and were more likely to receive both endothelin receptor antagonist and prostacyclin analogs than other forms of pulmonary hypertension. Compared to recipients of public insurance, subjects with commercial insurance were more likely to receive more intense therapy (p = 0.003), which was confirmed in multivariable analysis (OR: 1.4, p = 0.03). Receipt of commercial insurance was also associated with increased annual costs across all subjects (p < 0.001) and for the most common specific regimens. The majority of children with pulmonary hypertension receive phosphodiesterase monotherapy, followed by phosphodiesterase-endothelin receptor antagonist two drug regimens, and finally the addition of prostacyclin analogs for three-drug therapy. However, even after adjustment for measurable confounders, commercial insurance was associated with higher intensity care and higher costs (even within specific classes of pulmonary vasodilators). The effect of these associations on clinical outcome cannot be discerned from the current data set, but patterns of treatment deserve further attention.

Affimers as anti-idiotypic affinity reagents for pharmacokinetic analysis of biotherapeutics.

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.

Affimer-Enzyme-Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins.

Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an "active" enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme-inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-ß-lactamase and its inhibitor protein, such that target binding disrupts the enzyme-inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics.

A systematic review of the evidence on home care reablement services.

OBJECTIVE: To determine whether publically funded 'reablement services' have any effect on patient health or use of services. DESIGN: Systematic review of randomized controlled trials and non-randomized studies in which reablement interventions were compared with no care or usual care in people referred to public-funded personal care services. Data sources included: Cochrane Central Register of Controlled Trials, EPOC register of studies, trials registers, Medline, EMBASE, and CINHAL. Searches were from 2000 up to end February 2015. SETTING: Not applicable. PARTICIPANTS: Investigators' definition of the target population for reablement interventions. MAIN OUTCOME MEASURES: Use of publically funded personal care services and dependence in personal activities of daily living. RESULTS: We found no studies fulfilling our inclusion criteria that assessed the effectiveness of reablement interventions. We did note the lack of an agreed understanding of the nature of reablement. CONCLUSIONS: Reablement is an ill-defined intervention targeted towards an ill-defined and potentially highly heterogeneous population/patient group. There is no evidence to suggest it is effective at either of its goals; increasing personal independence or reducing use of personal care services.

Academic Dishonesty among Physical Therapy Students: A Descriptive Study.

PURPOSE: To examine academically dishonest behaviours based on physical therapy (PT) students' current practices and educators' prior behaviours as PT students. METHOD: A Web-based questionnaire was sent to 174 students and 250 educators from the PT programme at the University of Toronto. The questionnaire gathered data on demographics as well as on the prevalence of, seriousness of, and contributing factors to academic dishonesty (AD). RESULTS: In all, 52.4% of educators and 44.3% of students responded to the questionnaire over a 6-week data-collection period. Scenarios rated the most serious were the least frequently performed by educators and students. The impact of generation on attitudes and prevalence of AD was not significant. The factors most commonly reported as contributing to AD were school-related pressure, disagreement with evaluation methods, and the perception that "everyone else does it." CONCLUSION: This study parallels the findings of similar research conducted in other health care programmes: AD does occur within the PT curriculum. AD was more prevalent in situations associated with helping peers than in those associated with personal gain. The consistency in behaviours reported across generations suggests that some forms of cheating are accepted as the social norm and may be a function of the environment.

Objectif : Étudier les comportements malhonnêtes sur le plan académique à partir des pratiques actuelles des étudiants en physiothérapie (PT) et des comportements antérieurs des formateurs alors qu'ils étaient eux-mêmes étudiants en PT. Méthode : Un questionnaire sur le Web a été envoyé à 174 étudiants et à 250 formateurs dans des programmes de PT de l'Université de Toronto. Ce questionnaire a permis de recueillir des données démographiques ainsi que des données sur la prévalence, le degré de gravité et les facteurs contribuant à la malhonnêteté académique (MA). Résultats : Au total, 52 % des formateurs et 44,3 % des étudiants ont répondu au questionnaire durant la période de collecte des données, qui a duré six semaines. Les comportements considérés comme les plus graves ont été ceux que les formateurs et les étudiants ont le moins adoptés. L'influence de la génération à laquelle appartenaient les participants sur les attitudes par rapport à la MA et sur sa prévalence a été minime. Les facteurs contribuant à la MA le plus fréquemment évoqués sont les pressions liées aux études, un désaccord avec les méthodes d'évaluation et la perception que « tout le monde le fait ¼. Conclusion : Cette étude a permis de comparer les conclusions d'une étude similaire menée dans d'autres programmes de formation du secteur de la santé; il y a effectivement des cas de MA dans l'enseignement de la PT. Celle-ci est toutefois plus fréquente quand c'est pour aider des confrères ou consœurs que dans le seul intérêt personnel. La similitude des comportements des diverses générations de participants semble indiquer que certaines formes de tricherie sont acceptées parmi les normes sociales et peuvent être fonction de l'environnement.