Graduate Research Fellowship Research Articles

Understanding the Structure and Speciation of Molten LiCl-KCl Using Raman and Ultraviolet-Visible-Near IR Spectroscopic Techniques

Pyrochemical reprocessing (pyroprocessing) of used nuclear fuel (UNF) is a promising pathway towards closing the nuclear fuel cycle by recycling actinides into new fuel and providing the means to generate durable wasteforms for portions of the UNF that are not able to be recycled. One of the most significant challenges in the advancement of this technology is developing a more complete understanding of the chemistry of the electrolyte molten salts used in the electrorefining process, where fission products, transuranic elements and fuel elements are anodically dissolved into a LiCl-KCl electrolyte, forming a chemically complex mixture. To enhance our understanding of the chemistry of the mixture of molten salt electrolyte and UNF we are studying the structure and speciation of lanthanide and actinide elements dissolved in LiCl-KCl using Raman and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. These techniques are starting to provide information on the coordination, symmetry, and electronic structure of the dissolved species, which will result in a more complete understanding of the chemistry of these solutions. Results of a Raman spectroscopy study of mixtures of NdCl3, SmCl3, CeCl3, and EuCl3 with LiCl-KCl and LiCl-KCl-UCl3 and the development of a system to conduct Raman and UV-Vis-NIR spectroscopy and spectroelectrochemistry will be presented.Acknowledgements: This work was performed under the auspices of the Department of Energy (DOE) under contracts DE-NE0008889, and the US Nuclear Regulatory Commission (NRC) under contracts NRC-HQ-13-G-38-0027 and 31310018M0032. Dr. Kenny Osborne and Ms. Nancy Hebron-Isreal serve as the program managers for the DOE and NRC awards, respectively. For support of furnace design and fabrication work at INL, RG and WCP acknowledge the Laboratory Directed Research & Development (LDRD) Program under DOE Idaho Operations Office Contract DE-AC07-05ID14517 and a subcontract under LDRD (INL) to University of Nevada, Reno via award number 204471 (UNR award AWD-01-00001719); this design served as starting iteration for this work. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1447692. One of the authors, JM, acknowledges the Graduate Research Fellowship from the US National Science Foundation.

(Invited) Understanding the Corrosion of SS316L in Molten LiCl-Li2O-Li Using 3D Transmission X-Ray Microscopy and 3D XANES

Pyrochemical reprocessing (pyroprocessing) of used nuclear fuel (UNF) is a promising pathway towards closing the nuclear fuel cycle and providing the means to generate durable wasteforms for portions of the UNF that are not able to be recycled. To incorporate commercial UNF (UO2) into the pyroprocessing scheme, the ceramic UNF must be converted to a metallic form at the head end of the process, which can be accomplished via electrolytic reduction of the UNF in LiCl-Li2O molten salt electrolyte. During the electrolytic reduction of UO2 at the cathode, metallic Li is inevitably co-reduced from the electrolyte due to the proximity of the reduction potential of the U4+ in UO2 and Li+ in the electrolyte. Metallic Li disperses into the LiCl electrolyte and can then interact with the process vessel and other apparatus in contact with the electrolyte, altering the rate and mechanism of the degradation of those materials.Our group has previously shown that the presence of metallic Li in the electrolyte creates a reducing condition where the surface oxide layer of SS316 is removed, and extensive intergranular corrosion takes place. To further the understanding of the mechanism and morphology of the intergranular corrosion, transmission X-ray microscopy (TXM) at Brookhaven National Laboratory’s National Synchrotron Light Source II (NSLSII) beamline 18-ID was used to examine ~20 μm diameter focused-ion beam (FIB) milled pillars of the cross-section of SS316L exposed to LiCl-Li2O-Li with 0 wt.%, 0.3 wt.%, 0.6 wt.% and 1 wt.% Li. Computed Tomographic (CT) images were obtained at X-ray energies above and below the absorption edges of alloying elements of interest, providing insight into the distribution of alloying elements after exposure to the molten salt. 3D X-ray absorption near edge spectroscopy (XANES) tomography at the Cr K-edge was also attempted. This analysis provided additional evidence of the formation of Cr-rich intermetallic phases and 3D CT images of the void structure of the exposed samples, strengthening the evidence that the sensitization of SS316L at process temperatures and the subsequent dissolution of the Cr carbide precipitates into the electrolyte is the primary driver of the deep intergranular corrosion of SS316L in LiCl-Li2O-Li. To our knowledge, this is the first application of this FIB milling technique and TXM imaging of the FIB-milled pillars to corrosion samples and lessons learned regarding the sample preparation and analysis will be presented.Acknowledgements: The authors thank Zachary Karmiol, research scientist at Materials Characterization Nevada, for technical assistance with the FIB sample preparation. This work was performed under the auspices of the Department of Energy (DOE) under contracts DE- NE0008236, and the US Nuclear Regulatory Commission (USNRC) under contract NRC-HQ-13-G-38-0027. Dr. Kenny Osborne and Ms. Nancy Hebron-Isreal serve as the program managers for the DOE and NRC awards, respectively. This research used beamline 18-ID of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. This work was supported as part of the Molten Salts in Extreme Environments Energy Frontier Research Center, funded by the U.S. Department of Energy (US-DOE), Office of Science, Basic Energy Sciences, at Idaho National Laboratory under contract DE-AC07-05ID14517. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1447692. One of the authors, JM, acknowledges the Graduate Research Fellowship from the US National Science Foundation.

Impact of Solvent on PFSA Properties from Dispersion to Cast Film

Perfluorosulfonic-acid (PFSA) ionomers are a critical part of proton-exchange-membrane (PEM) fuel-cell catalyst layers (CLs). They serve both to bind the catalyst particles together, lending mechanical support, and provide pathways for ion transport. The CL fabrication process has garnered significant attention in recent years, with research efforts focused on understanding the properties of the CL ink (colloidal dispersions of solvent(s), catalyst particles, and ionomer), and how those properties translate to the cast CL structure.1-2 The PFSA/solvent interaction is a crucial and complex piece of the larger multi-component interaction phenomena. Recent work has shown the strong impact of solvent composition on dispersion properties3 and structure,4 and eventual CL performance,1 but there is little understanding of how these initial (dispersion) and final (dried film) states are connected. How do different dispersion properties as a result of varying ionomer/solvent interactions control cast structure and behavior? Do these interactions persist upon drying? This information is vital to understand, predict, and control PFSA film and CL structure and properties, instead of relying on current empirical approaches to the formation process.In this presentation, we investigate these questions by studying the impact of solvent on PFSA properties over the course of film preparation. In-situ grazing incidence x-ray scattering (GISAXS)5 experiments on ionomer dispersions are systematically performed over a range of water to n-propanol composition to explore the evolution of film structure from dispersion to dried state. Dispersion properties (particle size, viscosity, structure) and film properties (structure, water uptake, conductivity, etc.) are evaluated within the context of the GISAXS data, connecting properties across the range of PFSA states, and lending insight into interaction/property relationships. These results provide important understanding of the impact of solvent during film and CL manufacturing processes, and reveal that final properties are a strong function of ionomer/solvent interactions in the dispersion state that persist long past formation and annealing.AcknowledgementsScattering work was conducted at the Advanced Light Source (ALS), beamline 7.3.3, which is supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy (Contract No. DE-AC02-05CH11231). This work was mainly funded under the Fuel Cell Performance and Durability Consortium (FC-PAD), by the Hydrogen and Fuel Cell Technologies Office (HFTO), of the office of the Energy Efficiency and Renewable Energy (EERE), of the U. S. Department of Energy under contract number DE-AC02-05CH11231. S.B. acknowledges support from the Graduate Research Fellowship Program by the National Science Foundation under Grant No. DGE 1752814.References Van Cleve, T.; Khandavalli, S.; Chowdhury, A.; Medina, S.; Pylypenko, S.; Wang, M.; More, K. L.; Kariuki, N.; Myers, D. J.; Weber, A. Z.; Mauger, S. A.; Ulsh, M.; Neyerlin, K. C. ACS Appl. Mater. Interfaces 2019, 11 (50), 46953-46964.Hatzell, K. B.; Dixit, M. B.; Berlinger, S. A.; Weber, A. Z. J. Mater. Chem. A 2017, 5 (39), 20527-20533.Berlinger, S. A.; McCloskey, B. D.; Weber, A. Z. J. Phys. Chem. B. 2018, 122 (31), 7790-7796.Welch, C.; Labouriau, A.; Hjelm, R.; Orler, B.; Johnston, C.; Kim, Y. S. ACS Macro. Lett. 2012, 1 (12), 1403-1407.Dudenas, P. J.; Kusoglu, A. Macromolecules 2019, 52 (20), 7779-7785.

Accessible Surface Area in Porous Carbon Electrodes and Its Impact on Redox Flow Battery Performance

Carbon-fiber electrodes exhibit many desirable material properties including conductivity, porosity, and chemical stability, and, accordingly, are employed in a range of electrochemical technologies.1 Such electrodes are particularly important in redox flow battery (RFB) systems, as they serve multiple critical functions including providing surface area for electrochemical reactions, distributing liquid electrolytes, and conducting electrons and heat. While commercially available materials possess suitable permeability and conductivity, the performance of pristine substrates is limited, thus oxidative treatment prior to use is common practice.2,3 Partial oxidation of the electrode surface has been shown to add a range of oxygen functional groups, which improve hydrophilicity and, in some cases, catalyze redox reactions, as well as to increase available surface area for the electrochemical reactions.4,5 Both effects are posited to improve electrode performance but their relative importance and effectiveness remains unclear.6 Here, we critically assess the nature of the surface area generated by thermal pretreatment in air and its role on RFB electrode performance. Using binder-free Freundenberg H23 as a model substrate, we systematically vary pretreatment time and temperature to create different surface morphologies and develop structure-function relations. First, we use Brauner Emmanuel Teller gas adsorption (e.g., N2, Kr, Ar/CO2) and mercury porosimetry to estimate physical surface area and pore size distribution. In parallel, we use voltammetry and impedance spectroscopy to determine electrochemically active surface area with different electrolyte compositions. In general, we find that only a fraction of the physical surface area is accessible for electrochemical redox reactions due, in large part, to the nanoscopic dimensions of the added surface area. Second, we develop a simple mathematical model to assess the effectiveness of the available surface area of carbon fibers for Faradaic reactions. We find that, under typical RFB operating conditions, external mass transfer limitations govern performance, limiting the utilization of recessed areas generated during pretreatment. Ultimately, these results highlight the potential limits of performance improvement strategies based on increasing surface area of fibrous substrates and motivate new approaches for electrode development. Acknowledgements The authors acknowledge the financial support of the Joint Center for Energy Storage Research, an Energy Innovation Hub funded by the United States Department of Energy. K.V.G acknowledges additional funding from the National Science Foundation Graduate Research Fellowship. The authors acknowledge the Center for Nanoscale Systems and the NSF’s National Nanotechnology Infrastructure Network (NNIN) for the use of Nanoscale Analysis facility for electrode property characterization. References M. H. Chakrabarti et al., Journal of Power Sources, 253, 150–166 (2014).T. J. Rabbow, M. Trampert, P. Pokorny, P. Binder, and A. H. Whitehead, Electrochimica Acta, 173, 17–23 (2015).N. Pour et al., The Journal of Physical Chemistry C, 119, 5311–5318 (2015).B. Sun and M. Skyllas-Kazacos, Electrochim. Acta, 37, 8 (1992).T. J. Rabbow and A. H. Whitehead, Carbon, 111, 782–788 (2017).K. V. Greco, A. Forner-Cuenca, A. Mularczyk, J. Eller, and F. R. Brushett, ACS Applied Materials & Interfaces, 10, 44430–44442 (2018).

Employing Neural Network Regression Models to Screen the Electrode Design Space for Redox Flow Batteries

Redox flow batteries (RFBs) are a promising electrochemical technology for integrating renewable energies into the current grid infrastructure, but further cost reductions are needed for widespread deployment.1 Porous electrodes are an integral component of the RFB reactor providing heterogeneous active sites for electrochemical reactions, a solid-void matrix that facilitates electrolyte dispersion, and a conductive solid for transporting electrons throughout the reaction zones. Many electrodes employed in RFBs use gas diffusion layers (GDLs)—such as carbon papers, cloths, and felts—that are repurposed from fuel cell applications. While GDLs possess some favorable properties, they are not broadly tailored to support electrochemical reactions and forced liquid-phase flow. As such, these materials are suboptimal for RFB applications, and, consequently, research has focused on post-process modifications to improve electrochemical and fluid dynamic performance.2,3 In contrast, the ideal electrode would be a bottom-up design with a property profile specific to the particular redox chemistry and operating conditions. Unfortunately, manufacturing limitations and time-intensive experimentations stymie broad screening of electrode designs. This motivates the use of computational tools.The advances of computational fluid dynamics and multi-physics solvers enable a platform to construct novel electrode designs and investigate resulting structure-performance relationships. By incorporating physical properties and operating conditions, the electrode can be modeled at a variety of length scales.4 Simulation domains can be used to span a wide variety of electrode and system parameter values allowing for the generation of large data sets for statistical and sensitivity analysis. Further, the current epoch of machine learning algorithms engender opportunities to build regressive models for predicting RFB electrochemical and fluid dynamic performance that can be subsequently used for optimization protocols.In this presentation, we use an experimentally-validated RFB model to generate >8,000 electrochemical simulations across a range of electrode properties. We then use feature engineering to reduce data complexity for building a robust and accurate neural network to predict output current density. Subsequently, we use this trained and validated neural network in conjunction with metaheuristics to search the electrode design space and report optimal morphological properties. This approach—as shown in the figure below—represents a first step to exploring the electrode design space for advanced RFBs and may ultimately support performance optimization of existing battery installation, materials selection for prototypes, or new approaches to electrode design.AcknowledgmentsThis work was funded by the Joint Center for Energy Storage Research, an Energy Innovation Hub of the U.S. Department of Energy, Office of Science, Basic Energy (De-AC02-06CH11357). K.M.T. recognizes additional support from the U.S. NSF Graduate Research Fellowship (1122374).References A. Z. Weber et al., J Appl Electrochem, 41, 1137 (2011).K. J. Kim, Y.-J. Kim, J.-H. Kim, and M.-S. Park, Materials Chemistry and Physics, 131, 547–553 (2011).K. V. Greco, A. Forner-Cuenca, A. Mularczyk, J. Eller, and F. R. Brushett, ACS Appl. Mater. Interfaces, 10, 44430–44442 (2018).A. Gayon Lombardo, B. A. Simon, O. Taiwo, S. J. Neethling, and N. P. Brandon, Journal of Energy Storage, 24, 100736 (2019). Figure 1

Modeling the Impact of Sequential Two-Electron Transfer Compounds on Redox Flow Battery Performance

Redox flow batteries (RFBs) are promising candidates for grid-scale energy storage, but further cost reductions are needed for widespread use.1 As such, redox-active organic molecules and metal-coordination complexes are emerging as charge storage materials considering their redox potential, solubility, and stability can be tailored through molecular functionalization.2,3 Moreover, a number of these compounds can support multiple electron transfers, affording dramatic increases in energy storage capacity.4,5 However, the nature of the electron transfer events, concerted or sequential, plays a key role in determining whether the added capacity can be accessed efficiently. Specifically, depending on molecular features and cell operation, voltaic inefficiencies may be incurred which, in turn, offset any benefits of increased capacity. Therefore, we sought to quantify these losses and their relationship to molecular- and cell-level parameters to better understand the feasibility of multi-electron transfer in RFBs.In this presentation, we use a zero-dimensional electrochemical model, accounting for thermodynamic, kinetic, and mass transfer, to simulate the performance of two-electron compounds in redox flow cells. First, using a single half-cell, we show that voltaic efficiency is significantly influenced by the average redox potential and potential difference between the redox events. We also discuss the effects of different mass transfer coefficients and comproportionation reaction rates. Second, using a full cell, we consider the effects of utilizing two-electron compounds in both half-cells and subsequent effects of charge imbalance, both resulting in further inefficiencies. By understanding the operational tradeoffs of multi-electron compounds, we can develop design criterion to guide molecular engineering efforts towards more efficient high energy density electrolytes for RFBs.AcknowledgementsThis work was funded by the National Science Foundation (NSF) under Award Number 1805566. B.J.N gratefully acknowledges the NSF Graduate Research Fellowship Program under Grant Number 1122374. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF.References(1) Chalamala, B. R.; Soundappan, T.; Fisher, G. R.; Anstey, M. R.; Viswanathan, V. V.; Perry, M. L. Redox Flow Batteries: An Engineering Perspective. Proceedings of the IEEE 2014, 102 (6), 976–999.(2) Hogue, R. W.; Toghill, K. E. Metal Coordination Complexes in Nonaqueous Redox Flow Batteries. Current Opinion in Electrochemistry 2019.(3) Ding, Y.; Zhang, C.; Zhang, L.; Zhou, Y.; Yu, G. Molecular Engineering of Organic Electroactive Materials for Redox Flow Batteries. Chem. Soc. Rev. 2018, 47 (1), 69–103.(4) Kowalski, J. A.; Casselman, M. D.; Kaur, A. P.; Milshtein, J. D.; Elliott, C. F.; Modekrutti, S.; Attanayake, N. H.; Zhang, N.; Parkin, S. R.; Risko, C.; Brushett, F. R.; Odom, S. A. A Stable Two-Electron-Donating Phenothiazine for Application in Nonaqueous Redox Flow Batteries. J. Mater. Chem. A 2017, 5 (46), 24371–24379.(5) Sevov, C. S.; Fisher, S. L.; Thompson, L. T.; Sanford, M. S. Mechanism-Based Development of a Low-Potential, Soluble, and Cyclable Multielectron Anolyte for Nonaqueous Redox Flow Batteries. J. Am. Chem. Soc. 2016, 138 (47), 15378–15384.

Existence of stationary stochastic Burgers evolutions on R 2 and R 3 * *The author was partially supported by the NSF Graduate Research Fellowship Program under Grant DGE-1147470.

We prove that the stochastic Burgers equation on R d , d < 4, forced by gradient noise that is white in time and smooth in space, admits spacetime-stationary solutions. These solutions are thus the gradients of solutions to the KPZ equation on R d with stationary gradients. The proof works by proving tightness of the time-averaged laws of the solutions in an appropriate weighted space.

AI, quantum information emphasized for NSF graduate student funding

The National Science Foundation’s Graduate Research Fellowship Program (GRFP) is one of the most high-profile awards for graduate students, providing 1,600 students a year with their own funding as...

Fundamental Radiative Processes in Near-Zero-Index Media of Various Dimensionalities

R.W.B., N.E., and E.M. acknowledge support from the Defense Advanced Research Projects Agency (DARPA) Defense Sciences Office (DSO) Nascent program and from the U.S. Army Research Office. This work was performed while M.L. was a recipient of a Fellowship of the Belgian American Educational Foundation. O.R. acknowledges the support of the Banting Postdoctoral Fellowship of the Natural Sciences and Engineering Research Council of Canada (NSERC). E.N.K. is supported by the National Science Foundation Graduate Research Fellowship Program under Grant Nos. DGE1144152 and DGE1745303.

Scientists' political behaviors are not driven by individual-level government benefits.

Is it appropriate for scientists to engage in political advocacy? Some political critics of scientists argue that scientists have become partisan political actors with self-serving financial agendas. However, most scientists strongly reject this view. While social scientists have explored the effects of science politicization on public trust in science, little empirical work directly examines the drivers of scientists’ interest in and willingness to engage in political advocacy. Using a natural experiment involving the U.S. National Science Foundation Graduate Research Fellowship (NSF-GRF), we causally estimate for the first time whether scientists who have received federal science funding are more likely to engage in both science-related and non-science-related political behaviors. Comparing otherwise similar individuals who received or did not receive NSF support, we find that scientists’ preferences for political advocacy are not shaped by receiving government benefits. Government funding did not impact scientists’ support of the 2017 March for Science nor did it shape the likelihood that scientists donated to either Republican or Democratic political groups. Our results offer empirical evidence that scientists’ political behaviors are not motivated by self-serving financial agendas. They also highlight the limited capacity of even generous government support programs to increase civic participation by their beneficiaries.

Activation of the Exocyst Tethering Complex for SNARE Complex Regulation and Membrane Fusion

A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high resolution structures of large, multisubunit tethering complexes (MTCs) that spatially and temporally control intracellular membrane fusion. Exocyst is a hetero‐octameric protein complex, proposed to tether secretory vesicles at the plasma membrane, and to provide quality control of SNARE‐mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence and single‐particle cryo‐EM, are providing critical insights into the structure and function of the exocyst. We are investigating how the yeast exocyst interacts with SNARE proteins to control SNARE complex formation and membrane fusion. Intriguingly, fully assembled exocyst interacts weakly with the individual SNAREs, SNARE complexes, and the SNARE regulator Sec1, despite previously observed robust SNARE binding with recombinant proteins. Using an auxin‐inducible degradation system and mutant yeast strains, we purified exocyst subcomplexes and mutant complexes and showed that several have increased affinities for the different SNAREs. Negative stain EM was used to visualize the structure of an exocyst subcomplex, as well as the 3D structure of an activated mutant exocyst complex. Comparison of the negative stain images to the cryo‐EM structure of fully assembled exocyst revealed that several subunits become more dynamic and accessible for SNARE interactions. We propose that exocyst needs to become activated and undergo a conformational change, in order to efficiently interact with and regulate the SNAREs for membrane tethering and fusion.Support or Funding InformationWork in our laboratories is supported by US National Institutes of Health grants GM068803 (M.M.), GM054712 (P.B), F32GM123704 (D.L.) GM127673 (A.F.), DP2GM110772 (A.F.), A.F. is an American Asthma Foundation Scholar, an HHMI Faculty Scholar, and a Chan Zuckerberg Biohub Investigator. L.R.K. was supported by a graduate research fellowship from the NSF.

Mechanistic Insights Into RNA Splicing As A Trigger For Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that causes sudden death in young adults and athletes. ARVC is termed a “disease of the desmosome” as 40% of mutations in ARVC patients are found in the cardiac desmosomal components, with plakophilin‐2 (PKP2) being the most frequently mutated desmosomal gene in ARVC. Evidence from humans suggests that altered RNA splicing may be a critical mechanism through which PKP2 patient genetics drive ARVC. Studies have suggested that approximately one third of all human disease‐causing mutations are a result of defects in RNA splicing; highlighting the importance of testing the relevance of this mechanism in human ARVC. However, there are no models and limited mechanistic insights into how mutations in RNA splicing impact ARVC disease pathogenesis. We hypothesize that RNA splicing mutations in PKP2 are sufficient to recapitulate classic disease features associated with human ARVC and can provide valuable mechanistic insight into how altered RNA splicing impacts disease. Through CRISPR‐Cas9 we generated a novel knock‐in mouse model harboring a human equivalent PKP2 mutation (IVS10‐1 G&gt;C) that impacts RNA splicing and is sufficient to recapitulate all classic ARVC disease features. PKP2 homozygous mutant (PKP2 Hom) mice are viable at birth yet display adult hallmarks of ARVC including ventricular arrhythmias, right and left ventricular dysfunction, and fibro‐fatty replacement of myocardium leading to sudden death. RNA and sequencing analyses of exons spanning the PKP2 mutation site reveals a larger PKP2 transcript that retains a 54 base pair portion of the intronic sequence in PKP2 Hom hearts. However, RNA analysis of exons outside the PKP2 mutation site reveals PKP2 RNA transcripts at similar levels to wild type PKP2, suggesting that total RNA levels are not impacted by the mutation. Instead, we show that PKP2 Hom hearts express a higher molecular weight mutant PKP2 protein in the absence of endogenous PKP2. Analysis of PKP2 Hom neonatal hearts and cardiomyocytes reveal early desmosomal protein loss coupled with a susceptibility to baseline arrhythmias in the absence of overt cardiac disease features at this stage (e.g., no changes in heart weight/body weight ratios, no upregulation of cardiac stress and fibrotic markers). We provide a novel mouse model that highlights the sufficiency and molecular consequences of a PKP2 RNA splicing mutation that triggers all of the classic early and adult onset disease features associated with human ARVC.Support or Funding InformationNational Science Foundation (Graduate Research Fellowship Program), National Institutes of Health (NIH R01, HL142251‐A1)

Fetal Exposure to the Synthetic‐Progesterone Levonorgestrel (LNG) targets the brain resulting in hyperactive behavior using the Zebrafish (Danio rerio) as a model

Anxiety‐related disorders are a rising health concern in adolescent and young adults. Evidence suggests that disrupting normal neuronal development (neurogenesis) in the fetal brain could play a role in the manifestation of mental health disorders later in life. Growing literature suggests a role in hormone receptor (HR)‐regulated brain development, particularly in the hypothalamus, the neuroendocrine section of the brain and part of the limbic center. Yet, this link has not been thoroughly investigated. Levonorgestrel (LNG), a synthetic steroid used in contraceptive pills in adults, may also pose a threat to the developing brain by targeting HRs. Furthermore, this endocrine disrupting chemical (EDC) has been found at physiologically relevant levels in potable water sources worldwide providing an alternative route of exposure for unknowing individuals. Therefore, we investigated the effects embryonic exposure to LNG had on neurogenesis and hyperactivity using the zebrafish (Danio rerio) as neurobiological model. First, computational modeling using Quantitative structure‐activity relationships (QSAR), determined the predicted bioavailability, physiological half‐life and overall potential efficacy of LNG as a neuroendocrine disruptor. Next, Zebrafish were bred and fertilized embryos were collected and individually placed in 96‐well plates. Embryos were exposed to LNG levels (at doses ranging from 5–1000ng/L) or a vehicle control starting at 3 hours post fertilization (hpf) to 5 days pf (dpf), the time encompassing neurogenesis. There were no observable differences in fertilization ratios, hatching or morphology between LNG treated groups and vehicle controls. On 5 dpf, all treated and controls were assayed for changes in locomotor activity (i.e. hyperactivity busts). Larval movement were analyzed using computational locomotor activity software. Interestingly, the 5ng dose of LNG significantly (p&lt;0.05) increased hyperactive behavior compared to vehicle controls. HR expression and neuronal marker α‐HuC were used to document neurogenesis. Our data suggests that the observed LNG‐induced hyperactivity represents anxiety‐like behavior as a manifestation of aberrant neurogenesis. This is the first study to show that exposure to low environmentally relevant levels of LNG causes hyperactivity and thus, potentially alters HR regulated neuronal development increasing the risk of other mental issues later in life.Support or Funding InformationThis project was supported in part by UTRGV College of Science (COS) Research Enhancement Seed Grants Program to RKD, the UTRGV Presidential Graduate Research Fellowship to AF and GH, the UTRGV COS Dean's Fellowship to EZ, and the UTRGV Engaged Scholars Award to BHS.

Subcellular Location‐Dependent Activation and Signaling of the Delta‐Opioid Receptor

G protein‐coupled receptor (GPCR) signaling from intracellular compartments is an emerging idea in the field. Classically, GPCRs have been thought to signal mainly from the plasma membrane. However, GPCRs localized to endosomes and the Golgi are a critical component of receptor signaling. GPCR location affects signaling at multiple levels, from generation of second messengers to transcriptional responses, and even physiological responses. One physiologically relevant receptor that shows location‐dependent signaling is the delta‐opioid receptor (DOR), an attractive alternative target for pain management. DOR is localized primarily to intracellular compartments, including the Golgi, in neuronal cells. Relocation of these receptors to the cell surface increases the ability of DOR agonists to relieve pain, suggesting that the subcellular location and signaling of this receptor are closely linked and important for receptor pharmacology. However, the mechanisms by which DOR relocation to the plasma membrane from Golgi membranes influences receptor pharmacology are poorly understood. Live cell imaging with nanobody and mini‐G protein biosensors indicates receptor localization influences ligand‐induced recruitment of active‐conformation biosensors. Additionally, we used cAMP and calcium sensors to study how DOR localization influences receptor signaling. Together, our data support a model in which DOR activation and signaling are spatially regulated. This spatial regulation may underlie the complexity of physiological effects associated with DOR activation and provide new ways to modulate receptor signaling.Support or Funding InformationM.A.P. was supported by NIHGM117425 and NSF1517776. S.E.C was supported by NSF Graduate Research Fellowship DGE1256260.

Effect of Continuous‐Flow Left Ventricular Assist Device Support on Coronary Artery Endothelial Function in Ischemic and Non‐Ischemic Cardiomyopathy

The coronary vasculature encounters a reduction in pulsatility after implementing durable continuous‐flow left ventricular assist device (CF‐LVAD) circulatory support. Evidence exists that appropriate pulsatility is required to maintain endothelial cell homeostasis. We hypothesized that coronary artery endothelial function would be impaired after CF‐LVAD intervention. Coronary arteries from patients with end‐stage heart failure caused by ischemic (ICM, n=16) or non‐ischemic (NICM, n=22) cardiomyopathy were isolated from the LV apical core which was removed for the CF‐LVAD implantation. In 11 of these patients, paired coronary arteries were obtained from an adjacent region of myocardium after the CF‐LVAD intervention (n = 6 ICM, 5 NICM). Vascular function was assessed ex vivo using isometric tension procedures in these patients and in 7 non‐failing donor controls. Maximal endothelium‐dependent vasorelaxation to bradykinin (BK, 10−6 to 10−10 M) was blunted (p&lt;0.05) in arteries from patients with ICM compared to NICM and donor controls, whereas responses to sodium nitroprusside (SNP, 10−4 to 10−9 M) were similar among the groups. Contrary to our hypothesis, vasorelaxation responses to BK and SNP were similar before and 219±37 days after CF‐LVAD support. Of these patients, an exploratory subgroup analysis revealed that BK‐induced coronary artery vasorelaxation was greater (p&lt;0.05) after (87±6%) vs. before (54±14%) CF‐LVAD intervention in ICM patients, while SNP‐evoked responses were similar. Coronary artery endothelial function is not impaired by durable CF‐LVAD support, and in ICM patients appears to be improved. Investigating coronary endothelial function using in vivo approaches in a larger patient population is warranted.Support or Funding InformationJDS (AHA16GRNT31050004, NIH RO3AGO52848, NHLBI RO1 HL141540); LD, ND, TB (University of Utah Undergraduate Research Opportunities Program); JMC (University of Utah Graduate Research Fellowship); SGD (AHA Heart Failure Strategically Focused Research Network 16SFRN29020000, NHLBI RO1 HL135121, NHLBI RO1 HL132067).

Vasoreactivity of the Murine External Jugular Vein and Carotid Artery

Few studies have characterized vasoreactivity of the murine external jugular vein. This segment often is used in preclinical models to evaluate vascular remodeling in response to arteriovenous fistula (AVF) maturation. When an AVF is established, the venous segment is exposed to arterial pressures and disturbed flow patterns that have strong potential to impact vasoreactivity. Before testing this hypothesis in the context of AVF, we sought to establish procedures to comprehensively characterize endothelium‐dependent and vascular smooth muscle function of the carotid artery and external jugular vein from healthy mice. External jugular veins and ipsilateral common carotid arteries were isolated from five‐month‐old male C57BL/6 mice and vasomotor responses were evaluated using isometric tension procedures. External jugular veins developed tension (p&lt;0.05) to the voltage‐gated Ca2+ channel opener potassium chloride (KCl) and the thromboxane A2 receptor agonist U‐46619, but not to the α‐1 receptor agonist phenylephrine (PE), whereas common carotid arteries responded to all three vasocontractile agents in a more robust (p&lt;0.05) manner. While maximal responses (~70%) to the endothelium‐dependent vasodilator acetylcholine (ACh) were similar between the venous and arterial segments, the dose required to achieve this value was lower (p&lt;0.05) in the artery versus the vein. Nitric oxide synthase (NOS) inhibition using L‐NMMA attenuated (p&lt;0.05) but did not abolish ACh‐evoked vasorelaxation in both vascular segments. Endothelium‐independent vasorelaxation to sodium nitroprusside (SNP) was similar in the artery and the vein. Endothelium‐dependent and vascular smooth muscle function can be reliably assessed in the murine external jugular vein of healthy male C57Bl/6 mice. Compared to common carotid arteries, external jugular veins are resistant to PE‐evoked vasocontraction, less responsive to KCl and U‐46619‐evoked vasocontraction, less sensitive to ACh‐evoked vasorelaxation, and equally responsive to NOS inhibition and guanylate cyclase activation via SNP. Ongoing studies will determine if and how external jugular vein function is altered by arterial pressures and disturbed flow patterns that are encountered subsequent to establishing an AVF.Support or Funding InformationJMC (University of Utah Graduate Research Fellowship); YTS (R01DK100505; I01BX004133); JDS (AHA16GRNT31050004, NIH RO3AGO52848, RO1HL141540); TL (R44DK109789, R01HL139692, I01BX003387).

To bee inside or outside? Impacts of overwintering environment on gene expression of the alfalfa leafcutting bee, Megachile rotundata

Diapause is a non‐feeding stage that many insects go through to survive the winter months. How nutrition is regulated during these months of overwintering is still unclear. Diapausing insects are exposed to environmental stressors including temperature stress, but responding can be energetically costly. With fixed resources, overall metabolism and insulin signaling are maintained at low levels, but it is unclear if those pathways change in response to seasonal temperature fluctuations. Our hypothesis is that insulin signaling is responsible for allocating energy in response to fluctuations in temperature during overwintering months. To test this hypothesis, we overwintered alfalfa leafcutting bees, Megachile rotundata, in either a lab setting at a constant 4°C or in the field in naturally fluctuating temperatures, collected samples monthly throughout the winter, and then measured expression of genes in the insulin pathway using nCounter analysis (NanoString Technologies Inc.). nCounter is a highly multiplexed single molecule counting technique. A Dunn’s test was performed to analyze the effects of time and location for all possible comparisons. Our results showed that several genes were differentially expressed over time and between locations. However, more changes were seen in field samples, possibly in response to environmental cues. With a hierarchical cluster, field samples showed three distinct month clades, possibly correlating with different developmental stages: diapause, post‐diapause quiescence, and direct development. Although several genes were differentially expressed, many of them were downstream of the insulin pathway (Foxo, MAPK, GSK3β). This could mean that components of the insulin pathway are being utilized to respond to temperature fluctuations in the field. Along with evidence from other studies, our results suggest that insects may use different mechanisms for diapause regulation in response to their environment. This plasticity in regulation may be achieved by using targets of other pathways such as that of the insulin pathway. There is no denying that pollinators are crucial for the health of our ecosystem, but before we can save the bees, we need a better understanding of how their overall physiology is impacted in a drastically changing environment.Support or Funding InformationThis research was funded, in part, by grants from the National Science Foundation Graduate Research Fellowship and an NSF Graduate Research Internship with the USDA‐ARS Fargo, ND, and NSF EPSCoR‐1826834 to KJG.

Recognition Motif of Protein Kinase C and Protein Phosphatase 2A

Protein phosphorylation plays a central role in transducing intracellular signaling and the balance between phosphorylation and dephosphorylation must be exquisitely controlled to maintain cellular homeostasis. Two central effectors of protein phosphorylation, protein kinase C (PKC) and protein phosphatase 2A (PP2A), are themselves regulated by protein phosphorylation but the mechanism of their interaction is not known. Here we identify a putative binding motif on the PKC kinase domain that is predicted to recruit PP2A via its B56 regulatory subunit. These two enzymes regulate the phosphorylation state of each other, with PP2A opposing constitutive phosphates on PKC required for structural integrity, and PKC proposed to phosphorylate B56, controlling its interaction with PP2A substrates. While many kinase consensus phosphorylation sequences are well‐characterized, emerging research into phosphatase‐recognition of substrates via short linear motifs (SLiMs) has uncovered a PP2A‐B56 SLiM comprised of L/F‐x‐x‐I/V‐x‐E. Sequence analysis of PKC reveals a potential PP2A‐B56 binding site that is conserved in conventional PKC isozymes (α, β, and γ); indeed the Glu is required for binding PP2A‐B56 and is present in a majority of the PKC family. This sequence is located on the C‐lobe of the kinase in a region involved in substrate binding. This study characterizes the effect of mutation on this motif on PP2A‐B56 binding and function, and on the phosphorylation state of PKC. Attesting to the importance of this motif, several cancer‐associated mutations have been identified in the motif, including PKCα‐E552Q and PKCβ‐E555K. Our study demonstrates that phosphatase SLiMs are able to inform new kinase/phosphatase relationships and may be a valuable predictive tool for studying signaling cascades in disease.Support or Funding InformationThis work was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), National Science Foundation Graduate Research Fellowship Program, and NIH R35 GM122523.

Structural and Functional Characterization of the Integrase HK022

There is great demand for sophisticated molecular tools for precise genome editing and engineering. The current state of the art for this purpose is to use site‐specific nucleases to generate targeted double‐stranded breaks (DSBs) that are repaired by the cell. Though effective, nucleases often display off‐target cleavage and generate heterogeneous repair products via operations such as non‐homologous end joining. In contrast, tyrosine site‐specific recombinases (SSRs), like Cre and FLP which function biologically to drive integration of phage/viral genomes, generate precise repair outcomes. These outcomes include genetic insertions, deletions or inversions and are performed with considerably lower off‐target activity. HK022 is a relatively under‐studied tyrosine SSR with considerable sequence similarity to the well‐studied lambda phage integrase. In contrast to that enzyme, HK022 displays a recognition profile that is appropriate for targeting disease‐causing mutations, is highly site‐specific, and does not require auxiliary proteins for its function. Additionally, HK022 has the potential to recognize and recombine non‐identical DNA sites which provides the ability to selectively promote recombination in one direction, instead of an equilibrium between non‐recombined and recombined forms. The flexibility and small size of the DNA recognition site, combined with its ability to utilize large DNA cassette inserts make HK022 a promising candidate for optimization for genome engineering. Three forms of HK022 (optimized for synaptic, post‐strand exchange, and Holliday junction structures with DNA) have successfully been expressed and purified for co‐crystallization with unique DNA of interest. Given the success with the lambda integrase, we suspect that HK022 will be equally amenable to crystallization. Solving the structure of HK022 in its many forms provides the ability to compare its recognition pattern to lambda integrase to further understand the diversity of this enzyme class. In addition to generating the first crystal structure of HK022, we wish to enhance the overall stability and activity of the enzyme and optimize it for therapeutic and biotechnological purposes.Support or Funding InformationNational Science Foundation Graduate Research Fellowship Program (NSF GRFP) DGE‐1762114. National Institute of Health (NIH) R01 GM105691.

Gαs (GNAS) suppression of the p53 genomic‐stability checkpoint unleashes RAS‐driven oncogenesis

As one of the most frequently mutated oncogenes, mutant RAS has been well established to drive proliferative cell programs that are exploited by cancer. Interestingly, RAS, like many oncogenes, has been found in benign tumors that never progress to cancer. Although seemingly counterintuitive, RAS mutant tumors require an additional mutational insult to progress to malignancy. Using an unbiased, pan‐cancer analysis from patient sequencing data, we discovered that RAS mutations significantly co‐occur with mutations in GNAS, the gene encoding the Gαs subunit of G protein couple receptors (GPCRs). Given the established tractability of GPCRs as drug targets, we aimed to explore GNAS as a potential co‐driver in RAS mutant cancers. To first validate the ability of mutations in RAS and GNAS to cooperate to induce malignancy, we expressed the constitutive active mutations, KRASG12D and GNASR201C in the skin of adult mice. These double mutant mice rapidly developed skin papillomas, while mice with either mutation alone had no phenotype. Furthermore, when GNASR201C mice were treated with the chemical carcinogen DMBA, known to induce Hras mutations, mice rapidly developed squamous cell carcinoma in only 3 weeks. Analysis of the lesions revealed a surprising lack of mutations in the Trp53 tumor suppressor gene, which controls genomic stability. In vitro experiments revealed that activation of signaling through Gαs, or its downstream kinase PKA, was sufficient to block DNA damage‐induced p53 protein accumulation. Analysis of the underlying mechanisms demonstrated that MDM2, the cognate p53 E3 ubiquitin ligase, is a direct substrate of PKA, which phosphorylates MDM2 on S166 resulting in enhanced ligase activity. In vitro ubiquitination assays revealed that activation of Gαs or PKA increased MDM2‐mediated p53 ubiquitination and degradation. In patients, appendix cancer is the best example of the GNAS‐KRAS co‐driver state, with 70% of patients harboring this co‐mutation. Ongoing experiments are aimed at validating the mechanism of Gαs‐PKA regulation of MDM2‐p53 growth checkpoints in relevant murine and human intestinal organoid models. In summary, we have uncovered a significant co‐occurrence of RAS and GNAS mutations in cancer and demonstrated that these mutations cooperate to initiate carcinogenesis in vivo. Mechanistically, the cooperation of RAS and GNAS can be explained by the ability of Gαs to functionally repress p53, through PKA mediated phosphorylation of MDM2, releasing the normal brakes that check aberrant RAS‐driven growth.Support or Funding InformationDana Steffen was supported by the National Science Foundation Graduate Research Fellowship Program (NSF GRFP) and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752). This study is supported in part by the San Diego NCI Cancer Centers Council (C3) Collaborative Translational Cancer Research Pilot Grant.