Monitoring Replication Protein A (RPA) Dynamics in Homologous Recombination Through Site-specific Incorporation of Non-canonical Amino Acids

An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance

Activation of High and Low Affinity Dopamine Receptors Generates a Closed Loop that Maintains a Conductance Ratio and its Activity Correlate

Neuromodulators alter network output and have the potential to destabilize a circuit. The mechanisms maintaining stability in the face of neuromodulation are not well described. Using the pyloric network in the crustacean stomatogastric nervous system, we show that dopamine (DA) does not simply alter circuit output, but activates a closed loop in which DA-induced alterations in circuit output consequently drive a change in an ionic conductance to preserve a conductance ratio and its activity correlate. DA acted at low affinity type 1 receptors (D1Rs) to induce an immediate modulatory decrease in the transient potassium current (I A ) of a pyloric neuron. This, in turn, advanced the activity phase of that component neuron, which disrupted its network function and thereby destabilized the circuit. DA simultaneously acted at high affinity D1Rs on the same neuron to confer activity-dependence upon the hyperpolarization activated current (I h ) such that the DA- induced changes in activity subsequently reduced I h . This DA-enabled, activity-dependent, intrinsic plasticity exactly compensated for the modulatory decrease in I A to restore the I A :I h ratio and neuronal activity phase, thereby closing an open loop created by the modulator. Activation of closed loops to preserve conductance ratios may represent a fundamental operating principle neuromodulatory systems use to ensure stability in their target networks

LiveOcean: a daily forecast model of biogeochemistry in Washington marine waters

LiveOcean is a daily forecast model of ocean conditions for the coastal waters of Washington, Oregon, and Vancouver Island, as well as the Salish Sea. It is forced with realistic tides, winds, rivers, and ocean conditions. The model simulates biogeochemical properties including phytoplankton, nitrate, dissolved oxygen, dissolved inorganic carbon, and alkalinity, up to 3 days in the future. It is used for the prediction of ocean acidification events in coastal estuaries, and for harmful algal bloom events on coastal beaches. I will describe the model construction, comparisons with observations, uses, and future developments

Randomized controlled trial of a patient decision-making aid for orthodontics

INTRODUCTION: Patient decision-making aids (PDAs) are instruments that facilitate shared decision making and enable patients to reach informed, individual decisions regarding health care. The objective of this study was to assess the efficacy of a PDA compared with traditional information provision for adolescent patients considering fixed appliance orthodontic treatment. METHODS: Before treatment, orthodontic patients were randomly allocated into 2 groups: the intervention group received the PDA and standard information regarding fixed appliances, and the control group received the standard information only. Decisional conflict was measured using the Decisional Conflict Scale, and the levels of decisional conflict were compared between the 2 groups. RESULTS: Seventy-two patients were recruited and randomized in a ratio of 1:1 to the PDA and control groups. Seventy-one patients completed the trial (control group, 36; PDA group, 35); this satisfied the sample size calculation. The median total Decisional Conflict Scale score in the PDA group was lower than in the control group (15.63 and 19.53, respectively). However, this difference was not statistically significant (difference between groups, 3.90; 95% confidence interval of the difference, -4.30 to 12.11). Sex, ethnicity, age, and the time point at which patients were recruited did not have significant effects on Decisional Conflict Scale scores. No harm was observed or reported for any participant in the study. CONCLUSIONS: The results of this study showed that the provision of a PDA to adolescents before they consented for fixed appliances did not significantly reduce decisional conflict. There may be a benefit in providing a PDA for some patients, but it is not yet possible to say how these patients could be identified. REGISTRATION: This trial was registered with the Harrow National Research Ethics Committee (reference 12/LO/0279). PROTOCOL: The protocol was not published before trial commencement

Directional Phytoscreening: Contaminant Gradients in Trees for Plume Delineation

Tree Sampling Methods Have Been Used in Phytoscreening Applications to Delineate Contaminated Soil and Groundwater, Augmenting Traditional Investigative Methods that Are Time-Consuming, Resource-Intensive, Invasive, and Costly. in the Past Decade, Contaminant Concentrations in Tree Tissues Have Been Shown to Reflect the Extent and Intensity of Subsurface Contamination. This Paper Investigates a New Phytoscreening Tool: Directional Tree Coring, a Concept Originating from Field Data that Indicated Azimuthal Concentrations in Tree Trunks Reflected the Concentration Gradients in the Groundwater Around the Tree.To Experimentally Test This Hypothesis, Large Diameter Trees Were Subjected to Subsurface Contaminant Concentration Gradients in a Greenhouse Study. These Trees Were Then Analyzed for Azimuthal Concentration Gradients in Aboveground Tree Tissues, Revealing Contaminant Centroids Located on the Side of the Tree Nearest the Most Contaminated Groundwater. Tree Coring at Three Field Sites Revealed Sufficiently Steep Contaminant Gradients in Trees Reflected Nearby Groundwater Contaminant Gradients. in Practice, Trees Possessing Steep Contaminant Gradients Are Indicators of Steep Subsurface Contaminant Gradients, Providing Compass-Like Information About the Contaminant Gradient, Pointing Investigators toward Higher Concentration Regions of the Plume. © 2013 American Chemical Society