Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide

Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities co..

Designing Photoswitchable Peptides Using the AsLOV2 Domain

Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Ja helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark, but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49-fold and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light dependent co-localization, which we demonstrate with photoactivable gene transcription in yeast

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Photoactivatable proteins are powerful tools for studying biological processes. Light-induced dimers are especially useful because they can be turned on and off with high spatial and temporal resolution in living systems, allowing for control of protein localization and activity. Here, we develop and apply methods for identifying mutations that improve the effectiveness of a light-induced dimer. The engineered switch is modular, can be used in most organisms, has more than 50-fold change in binding affinity upon light stimulation, and can be used to initiate signaling pathways in a specific region of a cell

Field Measurements of Terrestrial and Martian Dust Devils

Surface-based measurements of terrestrial and martian dust devils/convective vortices provided from mobile and stationary platforms are discussed. Imaging of terrestrial dust devils has quantified their rotational and vertical wind speeds, translation speeds, dimensions, dust load, and frequency of occurrence. Imaging of martian dust devils has provided translation speeds and constraints on dimensions, but only limited constraints on vertical motion within a vortex. The longer mission durations on Mars afforded by long operating robotic landers and rovers have provided statistical quantification of vortex occurrence (time-of-sol, and recently seasonal) that has until recently not been a primary outcome of more temporally limited terrestrial dust devil measurement campaigns. Terrestrial measurement campaigns have included a more extensive range of measured vortex parameters (pressure, wind, morphology, etc.) than have martian opportunities, with electric field and direct measure of dust abundance not yet obtained on Mars. No martian robotic mission has yet provided contemporaneous high frequency wind and pressure measurements. Comparison of measured terrestrial and martian dust devil characteristics suggests that martian dust devils are larger and possess faster maximum rotational wind speeds, that the absolute magnitude of the pressure deficit within a terrestrial dust devil is an order of magnitude greater than a martian dust devil, and that the time-of-day variation in vortex frequency is similar. Recent terrestrial investigations have demonstrated the presence of diagnostic dust devil signals within seismic and infrasound measurements; an upcoming Mars robotic mission will obtain similar measurement types

Alcohol consumption in tertiary education students

Background Heavy alcohol consumption among adolescents and young adults is an issue of significant public concern. With approximately 50% of young people aged 18-24 attending tertiary education, there is an opportunity within these settings to implement programs that target risky drinking. The aim of the current study was to survey students and staff within a tertiary education institution to investigate patterns of alcohol use, alcohol-related problems, knowledge of current National Health and Medical Research Council (NHMRC) guidelines for alcohol consumption and intentions to seek help for alcohol problems. Methods Students of an Australian metropolitan university (with staff as a comparison group) participated in a telephone interview. Questions related to knowledge of NHMRC guidelines, drinking behaviour, alcohol-related problems and help-seeking intentions for alcohol problems. Level of psychological distress was also assessed. Results Of the completed interviews, 774 (65%) were students and 422 (35%) were staff. While staff were more likely to drink regularly, students were more likely to drink heavily. Alcohol consumption was significantly higher in students, in males and in those with a history of earlier onset drinking. In most cases, alcohol-related problems were more likely to occur in students. The majority of students and staff had accurate knowledge of the current NHMRC guidelines, but this was not associated with lower levels of risky drinking. Psychological distress was associated with patterns of risky drinking in students. Conclusions Our findings are consistent with previous studies of tertiary student populations, and highlight the disconnect between knowledge of relevant guidelines and actual behaviour. There is a clear need for interventions within tertiary education institutions that promote more effective means of coping with psychological distress and improve help-seeking for alcohol problems, particularly among young men

Combining motivational and volitional approaches to reducing excessive alcohol consumption in pre-drinkers: A theory-based intervention protocol

Background: Pre-drinking refers to the consumption of alcohol at home or a private residence prior to attending a subsequent social event. We present the study protocol of an online theory-based intervention to reduce pre-drinking and related harm in pre-drinking undergraduates, using behavior change techniques targeting the motivational and volitional phases of behaviour. Design: A fully randomized 2 (autonomy support: present vs. absent) x 2 (implementation intention: present vs. absent) between-participants design will be used to ascertain the effectiveness of the intervention in reducing pre-drinking alcohol consumption and alcohol-related harm. Participants will complete a range of theory-based measures prior to being allocated to one of the four experimental conditions. Four weeks later, participants will complete a follow-up questionnaire comprised of theoretical and behavioral measures. Analyses: The main and interactive effects of the intervention components in reducing our primary dependent variables, namely, pre-drinking alcohol consumption and alcohol-related harm at four-week follow-up will be tested. Baseline alcohol consumption and demographic information will be included in the analysis as covariates. Discussion: This online intervention is the first to be developed to reduce pre-drinking alcohol consumption, a behaviour linked to increased risk of alcohol-related harm. The intervention targets motivational and volitional components of the behaviour change process and is therefore likely to lead to greater reductions in pre-drinking alcohol consumption and experience of alcohol-related harm compared to either approach in isolation. If successful, the intervention can be implemented across various contexts and in populations where pre-drinking is prevalent. © 2016 Caudwell et al

Human and mouse essentiality screens as a resource for disease gene discovery.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD