Registered replication report: a large multilab cross-cultural conceptual replication of Turri, Buckwalter, & Blouw (2015)

According to the Justified True Belief account of knowledge (JTB), a person can only truly know something if they have a belief that is both justified and true (i.e., knowledge is justified true belief). This account was challenged by Gettier (1963), who argued that JTB does not explain knowledge attributions in certain situations, later called Gettier-type cases, wherein a protagonist is justified in believing something to be true, but their belief was only correct due to luck. Lay people may not attribute knowledge to protagonists with justified but only luckily true beliefs. While some research has found evidence for these so-called Gettier intuitions (e.g., Machery et al., 2017a), Turri et al. (2015) found no evidence that participants attributed knowledge in a counterfeit-object Gettier-type case differently than in a matched case of justified true belief. In a large-scale, cross-cultural conceptual replication of Turri and colleagues’ (2015) Experiment 1 (N = 4,724) using a within-participants design and three vignettes across 19 geopolitical regions, we did find evidence for Gettier intuitions; participants were 1.86 times more likely to attribute knowledge to protagonists in standard cases of justified true belief than to protagonists in Gettier-type cases. These results suggest that Gettier intuitions may be detectable across different scenarios and cultural contexts. However, the size of the Gettier intuition effect did vary by vignette, and the Turri et al. (2015) vignette produced the smallest effect, which was similar in size to that observed in the original study. Differences across vignettes suggest epistemic intuitions may also depend on contextual factors unrelated to the criteria of knowledge, such as the characteristics of the protagonist being evaluated

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (<i>K</i>_D = 190 μM) and association constants (<i>k</i>_on = 500 M^–1 s^–1, <i>k</i>_off = 0.2 s^–1) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21–37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells

Raising the value of research studies in psychological science by increasing the credibility of research reports: the transparent Psi Project

The low reproducibility rate in social sciences has produced hesitation among researchers in accepting published findings at their face value. Despite the advent of initiatives to increase transparency in research reporting, the field is still lacking tools to verify the credibility of research reports. In the present paper, we describe methodologies that let researchers craft highly credible research and allow their peers to verify this credibility. We demonstrate the application of these methods in a multi-lab replication of Bem’s Experiment 1 (2011) on extrasensory perception (ESP), which was co-designed by a consensus panel including both proponents and opponents of Bem’s original hypothesis. In the study we applied direct data deposition in combination with born-open data and real-time research reports to extend transparency to protocol delivery and data collection. We also used piloting, checklists, laboratory logs and video documented trial sessions to ascertain as-intended protocol delivery, and external research auditors to monitor research integrity. We found 49.89% successful guesses, while Bem reported 53.07% success rate, with the chance level being 50%. Thus, Bem’s findings were not replicated in our study. In the paper we discuss the implementation, feasibility, and perceived usefulness of the credibility-enhancing methodologies used throughout the project

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes

The revised Approved Instructional Resources score: An improved quality evaluation tool for online educational resources

BackgroundFree Open-Access Medical education (FOAM) use among residents continues to rise. However, it often lacks quality assurance processes and residents receive little guidance on quality assessment. The Academic Life in Emergency Medicine Approved Instructional Resources tool (AAT) was created for FOAM appraisal by and for expert educators and has demonstrated validity in this context. It has yet to be evaluated in other populations.ObjectivesWe assessed the AAT's usability in a diverse population of practicing emergency medicine (EM) physicians, residents, and medical students; solicited feedback; and developed a revised tool.MethodsAs part of the Medical Education Translational Resources: Impact and Quality (METRIQ) study, we recruited medical students, EM residents, and EM attendings to evaluate five FOAM posts with the AAT and provide quantitative and qualitative feedback via an online survey. Two independent analysts performed a qualitative thematic analysis with discrepancies resolved through discussion and negotiated consensus. This analysis informed development of an initial revised AAT, which was then further refined after pilot testing among the author group. The final tool was reassessed for reliability.ResultsOf 330 recruited international participants, 309 completed all ratings. The Best Evidence in Emergency Medicine (BEEM) score was the component most frequently reported as difficult to use. Several themes emerged from the qualitative analysis: for ease of use-understandable, logically structured, concise, and aligned with educational value. Limitations include deviation from questionnaire best practices, validity concerns, and challenges assessing evidence-based medicine. Themes supporting its use include evaluative utility and usability. The author group pilot tested the initial revised AAT, revealing a total score average measure intraclass correlation coefficient (ICC) of moderate reliability (ICC&nbsp;=&nbsp;0.68, 95% confidence interval [CI]&nbsp;=&nbsp;0 to 0.962). The final AAT's average measure ICC was 0.88 (95% CI&nbsp;= 0.77 to 0.95).ConclusionsWe developed the final revised AAT from usability feedback. The new score has significantly increased usability, but will need to be reassessed for reliability in a broad population