Dense and pleiotropic regulatory information in a developmental enhancer.

Changes in gene regulation underlie much of phenotypic evolution1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

This Free Life Campaign: Increasing Intention to Quit Among LGBTQ+ Young Adult Nondaily Smokers in Minneapolis.

PURPOSE: LGBTQ+ young adults smoke at disproportionately higher rates than their non-LGBTQ+ counterparts, but prevention efforts are limited. Furthermore, prior to This Free Life (TFL), no known campaigns target LGBTQ+ nondaily smokers. In this study Blue Cross and Blue Shield of Minnesota evaluated a local partnership extension of the Food and Drug Administration (FDA) Center for Tobacco Products' TFL campaign. METHODS: The intervention featured a variety of LGBTQ+-tailored events, social/digital media, and out-of-home media placed in locations with a high density of LGBTQ+ young adults. Cross-sectional surveys (n = 1215) were collected from LGBTQ+ young adult (18-26) nondaily smokers at 4 time points between 2016 and 2019. The national TFL campaign was tracked in a separate evaluation conducted by the FDA. RESULTS: 43.0% of nondaily LGBTQ+ smokers reported awareness of the campaign (n = 522), and 63.4% of those also engaged with TFL (n = 330). Engagement was highest for gay, lesbian and transgender participants, and for Asian and Black/African American participants. Each additional instance of campaign engagement increased participants' odds of intending to quit smoking by 20% (AOR = 1.20; 95% CI = 1.05, 1.36). The relationship between campaign engagement and intention to quit was fully mediated by the campaign's impact on attitudes against smoking and perceived normative trends, but not by perceived behavioral control. CONCLUSION: The local extension of TFL increased intentions to quit for LGBTQ+ nondaily smokers. Future research should further explore the relationship between perceived behavioral control and intentions to quit for nondaily smokers.

An open-source semi-automated robotics pipeline for embryo immunohistochemistry.

A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos. Central to this pipeline is a liquid handling robot, Flyspresso, which automates the steps of classical embryo fixation and staining. We provide the schematics and an overview of the technology for an engineer or someone equivalently trained to reproduce and further improve upon Flyspresso, and highlight the Drosophila embryo fixation and colorimetric or antibody staining protocols. Additionally, we provide a detailed overview and stepwise protocol for our adaptive-feedback pipeline for automated embryo imaging on confocal microscopes. We demonstrate the efficiency of this pipeline compared to classical techniques, and how it can be repurposed or scaled to other protocols and biological systems. We hope our pipeline will serve as a platform for future research, allowing a broader community of users to build, execute, and share similar experiments.

Potentiation of the early visual response to learned danger signals in adults and adolescents.

The reinforcing effects of aversive outcomes on avoidance behaviour are well established. However, their influence on perceptual processes is less well explored, especially during the transition from adolescence to adulthood. Using electroencephalography, we examined whether learning to actively or passively avoid harm can modulate early visual responses in adolescents and adults. The task included two avoidance conditions, active and passive, where two different warning stimuli predicted the imminent, but avoidable, presentation of an aversive tone. To avoid the aversive outcome, participants had to learn to emit an action (active avoidance) for one of the warning stimuli and omit an action for the other (passive avoidance). Both adults and adolescents performed the task with a high degree of accuracy. For both adolescents and adults, increased N170 event-related potential amplitudes were found for both the active and the passive warning stimuli compared with control conditions. Moreover, the potentiation of the N170 to the warning stimuli was stable and long lasting. Developmental differences were also observed; adolescents showed greater potentiation of the N170 component to danger signals. These findings demonstrate, for the first time, that learned danger signals in an instrumental avoidance task can influence early visual sensory processes in both adults and adolescents.