Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

SummaryCardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic αF-αG loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase

Structural and Functional Characterization of Hyper-Phosphorylated GRK5 Protein Expressed From E. coli

G protein-coupled receptor (GPCR) kinases (GRKs) are proteins in the cell responsible for regulating GPCRs located on the cell membrane. GRKs regulate active GPCRs by phosphorylating them at certain sites which causes them to stop normal signaling on the membrane. This ultimately affects how the cell responds to its environment. GRK5 is a kinase of particular interest due to its involvement in the pathology of diseases such as cardiac failure, cancers, and diabetes. Understanding the structure and function of GRK5 is essential for discovering ways to manipulate its behavior with these diseases, but not much is known about how GRK5 interacts with GPCRs. Although past studies used mammalian and insect cells to produce GRK5, this study aims to use E. coli cells to discover more about GRK5’s structure and function. Previous studies revealed E. coli produce a hyper-phosphorylated version of the GRK5 protein. We attempted to crystalize this GRK5 produced from E. coli to reveal its conformation in a phosphorylated state that we hypothesize to be similar to its form when bound to GPCRs. We also tested the functionality of this GRK5 to reveal the effects of phosphorylation. We genetically edited the GRK5 gene in multiple E. coli samples to create GRK5 with less phosphorylation sites and tested activity levels by measuring the phosphorylation of GPCRs mediated by each GRK5 variant. Successfully creating an E. coli system for structural and functional analysis of GRK5 would help reduce time and costs for GRK5 research, and it could speed up the full understanding of the interactions between GRK5 and GPCRs

Room temperature ferromagnetic-like behavior in Mn-implanted and post-annealed InAs layers deposited by Molecular Beam Epitaxy

We report on the magnetic and structural properties of Ar and Mn implanted InAs epitaxial films grown on GaAs (100) by Molecular Beam Epitaxy (MBE) and the effect of Rapid Thermal Annealing (RTA) for 30 seconds at 750C. Channeling Particle Induced X- ray Emission (PIXE) experiments reveal that after Mn implantation almost all Mn atoms are subsbtitutional in the In-site of the InAs lattice, like in a diluted magnetic semiconductor (DMS). All of these samples show diamagnetic behavior. But, after RTA treatment the Mn-InAs films exhibit room-temperature magnetism. According to PIXE measurements the Mn atoms are no longer substitutional. When the same set of experiments were performed with As as implantation ion all of the layers present diamagnetism without exception. This indicates that the appearance of room-temperature ferromagnetic-like behavior in the Mn-InAs-RTA layer is not related to lattice disorder produce during implantation, but to a Mn reaction produced after a short thermal treatment. X-ray diffraction patterns (XRD) and Rutherford Back Scattering (RBS) measurements evidence the segregation of an oxygen deficient-MnO2 phase (nominally MnO1.94) in the Mn-InAs-RTA epitaxial layers which might be on the origin of room temperature ferromagnetic-like response observed.Comment: 16 pages, 5 figures. Acepted in J. Appl. Phy

Generating Interpretable Fuzzy Controllers using Particle Swarm Optimization and Genetic Programming

Autonomously training interpretable control strategies, called policies, using pre-existing plant trajectory data is of great interest in industrial applications. Fuzzy controllers have been used in industry for decades as interpretable and efficient system controllers. In this study, we introduce a fuzzy genetic programming (GP) approach called fuzzy GP reinforcement learning (FGPRL) that can select the relevant state features, determine the size of the required fuzzy rule set, and automatically adjust all the controller parameters simultaneously. Each GP individual's fitness is computed using model-based batch reinforcement learning (RL), which first trains a model using available system samples and subsequently performs Monte Carlo rollouts to predict each policy candidate's performance. We compare FGPRL to an extended version of a related method called fuzzy particle swarm reinforcement learning (FPSRL), which uses swarm intelligence to tune the fuzzy policy parameters. Experiments using an industrial benchmark show that FGPRL is able to autonomously learn interpretable fuzzy policies with high control performance.Comment: Accepted at Genetic and Evolutionary Computation Conference 2018 (GECCO '18

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

Unlubricated sliding properties of ion beam and excimer laser mixed Fe-Ti-C multilayered films

Multilayered Fe-Ti-C films consisting of eleven sublayers were vacuum deposited onto an AISI 304 stainless steel substrate and subsequently mixed using either 400 keV Xe ions or an excimer laser, operating at a wavelength of 308 nm. Ion mixing was accomplished in a two step process: the multilayers were first irradiated with 1 /times/ 10/sup 17/ Xe/cm/sup 2/ at 520 C, after which half of the sample was irradiated with 5 /times/10/sup 15/ Xe/cm/sup 2/ at O C. Laser mixing was carried out at both 1.1 and 1.7 J/cm/sup 2/ with the number of pulses varied between 1 and 10. Pin-on-disc studies revealed only slight differences between the two kinds of ion beam mixed samples, whereas the dry sliding properties of laser mixed samples were strongly dependent on the total fluence used. In the optimum conditions similar friction coefficients were obtained on both kinds of samples. 13 refs., 4 figs

Chemical History with a Nuclear Microprobe

A nuclear microprobe cannot give direct information on the chemical state of an element, but the spatial distribution of elements in a specimen is often determined by the chemical history of the sample. Fuel cells and minerals are examples of complex systems whose elemental distributions are determined by past chemical history. The distribution of catalyst in used fuel cell electrodes provides direct information on the chemical stability of dispersed catalysts under operating conditions. We have used spatially resolved Rutherford backscattering to measure the migration of platinum and vanadium from intermetallic catalysts and to determine their suitability for use under the extreme operating conditions found in phosphoric acid fuel cells. Geologic materials are complex, heterogeneous samples with small mineral grains. The trace element distribution within the individual mineral grains and between different mineral phases is sensitive to the details of the mineral formation and history. The spatial resolution and sub-100-ppm sensitivity available with a nuclear microprobe open up several new classes of experiments to the geochemist. Geochemistry and electrochemistry are two areas proving particularly fruitful for application of the nuclear microprobe

Nitrogen and boron ion implantation into electrodeposited hard chrome

Electrodeposited hard chrome was ion implanted with N alone, B alone, and a combination. Separate N and B implantation was done at 75 keV and incident doses of 2, 4, and 8x10{sup 17} at/cm{sup 2}. Samples with both N/B implants used 75 keV and incident dose levels of 4x10{sup 17} N- and B-at/cm{sup 2}. Beam-line system was used. Retained dose was measured using ion beam analysis, which indicated most of the incident dose was retained. Surface hardness, wear coefficient, and friction coefficient were determined by nanohardness indentation and pin-on-disk wear. At a depth of 50 nm, surface hardness increased from 18{+-}1 GPa (unimplanted) to a max of 23{+-}4 GPa for B implant and 26{+-}1 GPa for N implant. the wear coefficient was reduced by 1.3x to 7.4x, depending on implantation. N implant results in lower wear coefficients than B implant

Orexin neurons track temporal features of blood glucose in behaving mice

Does the brain track how fast our blood glucose is changing? Knowing such a rate of change would enable the prediction of an upcoming state and a timelier response to this new state. Hypothalamic arousal-orchestrating hypocretin/orexin neurons (HONs) have been proposed to be glucose sensors, yet whether they track glucose concentration (proportional tracking) or rate of change (derivative tracking) is unknown. Using simultaneous recordings of HONs and blood glucose in behaving male mice, we found that maximal HON responses occur in considerable temporal anticipation (minutes) of glucose peaks due to derivative tracking. Analysis of >900 individual HONs revealed glucose tracking in most HONs (98%), with derivative and proportional trackers working in parallel, and many (65%) HONs multiplexed glucose and locomotion information. Finally, we found that HON activity is important for glucose-evoked locomotor suppression. These findings reveal a temporal dimension of brain glucose sensing and link neurobiological and algorithmic views of blood glucose perception in the brain's arousal orchestrators

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization