THE EFFECTS OF PROCESSING FLUENCY ON RESTORATIVE ENVIRONMENTS

Attention Restoration Theory (ART) maintains that directed attention, a type of attention that requires effort, is a resource that is susceptible to fatigue after prolonged use. This directed attention fatigue, commonly known as mental fatigue, is a factor linked to performance and safety decline in the workplace. Many studies have found that contact with nature promotes restoration from directed attention fatigue; however, there is little research on how nature has this effect. The aim of the present study was to explore whether natural environments have restorative potential given their higher degree of processing fluency. College students (N=78) were mentally fatigued by performing a sustained attention task. Then they viewed a natural photograph or urban photograph presented in various degrees of fluency (manipulated via visual clarity) and performed the sustained attention task again. Mood ratings were also examined. In line with ART, participants who viewed the natural environment photograph showed a larger improvement in the sustained attention task compared to those who saw the urban environment photograph; however, this was not influenced by fluency levels. Mood ratings were also not influenced by the type of environment or fluency levels. The research promotes the exploration of specific mechanisms underlying restorative environments which in turn would provide landscape designers, spatial planners, and employers a basis for green designs and interventions

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination

Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease

The PMIP4 contribution to CMIP6 – Part 2: two interglacials, scientific objective and experimental design for Holocene and last interglacial simulations

Two interglacial epochs are included in the suite of Paleoclimate Modeling Intercomparison Project (PMIP4) simulations in the Coupled Model Intercomparison Project (CMIP6). The experimental protocols for Tier 1 simulations of the mid-Holocene (midHolocene, 6000 years before present) and the Last Interglacial (lig127k, 127,000 years before present) are described here. These equilibrium simulations are designed to examine the impact of changes in orbital forcing at times when atmospheric greenhouse gas levels were similar to those of the preindustrial period and the continental configurations were almost identical to modern. These simulations test our understanding of the interplay between radiative forcing and atmospheric circulation, and the connections among large-scale and regional climate changes giving rise to phenomena such as land-sea contrast and high-latitude amplification in temperature changes, and responses of the monsoons, as compared to today. They also provide an opportunity, through carefully designed additional CMIP6 Tier 2 and Tier 3 sensitivity experiments of PMIP4, to quantify the strength of atmosphere, ocean, cryosphere, and land-surface feedbacks. Sensitivity experiments are proposed to investigate the role of freshwater forcing in triggering abrupt climate changes within interglacial epochs. These feedback experiments naturally lead to a focus on climate evolution during interglacial periods, which will be examined through transient experiments. Analyses of the sensitivity simulations will also focus on interactions between extratropical and tropical circulation, and the relationship between changes in mean climate state and climate variability on annual to multi-decadal timescales. The comparative abundance of paleoenvironmental data and of quantitative climate reconstructions for the Holocene and Last Interglacial make these two epochs ideal candidates for systematic evaluation of model performance, and such comparisons will shed new light on the importance of external feedbacks (e.g., vegetation, dust) and the ability of state-of-the-art models to simulate climate changes realistically

Two interglacials: scientific objectives and experimental designs for CMIP6 and PMIP4 Holocene and last interglacial simulations

Two interglacial epochs are included in the suite of paleoclimate simulations in the present phase of the Coupled Model Intercomparison Project (CMIP6). Equilibrium simulations of the mid-50 Holocene (midHolocene, 6000 years before present) and the Last Interglacial (lig127k, 127,000 years before present) are designed to examine the impact of changes in orbital forcing at times when atmospheric greenhouse gas levels were similar to those of the preindustrial period and the continental configurations were almost identical to modern. These simulations test our understanding of the interplay between radiative forcing and atmospheric circulation, and the connections among large-scale and regional climate changes giving rise to phenomena such as land-sea contrast and high-latitude amplification in temperature changes, and responses of the monsoons, as compared to today. They also provide an opportunity, through carefully designed additional sensitivity experiments as part of the Paleoclimate Modeling Intercomparison Project (PMIP4), to quantify the strength of atmosphere, ocean, cryosphere, and land-surface feedbacks. Sensitivity experiments are proposed to investigate the role of freshwater forcing in triggering abrupt climate changes within interglacial epochs. These feedback experiments naturally lead to a focus on climate evolution during interglacial periods, which will be examined through transient experiments. Analyses of the sensitivity simulations will also focus on interactions between extratropical and tropical circulation, and the relationship between changes in mean climate state and climate variability on annual to multi-decadal timescales. The comparative abundance of paleoenvironmental data and of quantitative climate reconstructions for the Holocene and Last Interglacial make these two epochs ideal candidates for systematic evaluation of model performance, and such comparisons will shed new light on the importance of external feedbacks (e.g., vegetation, dust) and the ability of state-of-the-art models to simulate climate changes realistically

Analysis of β-adrenergic receptor activation

Die Funktionalität β1- und β2-adrenerger Rezeptoren wird durch Polymorphismen in ihrer kodierenden Region moduliert. Wir haben uns die Technik des Fluoreszenz-Resonanz- Energie-Transfers (FRET) zu Nutze gemacht, um den Einfluss der am häufigsten vorkommenden Polymorphismen (Ser49Gly und Gly389Arg im β1AR, Arg16Gly und Gln27Glu im β2AR) auf die Rezeptorkonformation nach Aktivierung zu untersuchen. Dafür wurden FRET-Sensoren für die beiden βAR-Subtypen mit einem gelb-fluoreszierenden Protein (YFP) sowie einem cyan-fluoreszierenden Protein (CFP oder Cerulean) in der dritten intrazellulären Schleife bzw. am C-Terminus verwendet. Nach Stimulierung der βARSensoren konnte die Aktivierung der polymorphen Rezeptorvarianten in lebenden Zellen in Echtzeit untersucht werden. Dabei behielten die FRET-Sensoren sowohl die Bindungsaffinitäten der nativen Rezeptoren als auch eine intakte Funktionalität hinsichtlich der Bildung von sekundären Botenstoffen. Der Vergleich der Aktivierungskinetiken der verschieden polymorphen Varianten des β1AR und β2AR ergab keine signifikanten Unterschiede nach einer einmaligen Stimulation. Es zeigte sich jedoch, dass Rezeptorpolymorphismen die Aktivierungskinetik vorstimulierter βAR erheblich beeinflussen. So konnten wir im Vergleich zur ersten Aktivierung eine schnellere Aktivierung der Gly16-Varianten des β2AR sowie des Gly49Arg389-β1AR feststellen, während die Arg16-β2AR-Variante und der Ser49Gly389-β1AR dagegen bei einer wiederholten Stimulation langsamer aktiviert wurden. Diese Ergebnisse lassen auf ein "Rezeptorgedächtnis" schließen, das spezifisch für bestimmte polymorphe Rezeptorvarianten ist und eine βAR-Subtyp-spezische Ausprägung zeigt. Die Ausbildung der unterschiedlichen Aktivierungskinetiken hing von der Interaktion des Rezeptors mit löslichen intrazellulären Faktoren ab und bedurfte einer Phosphorylierung intrazellulärer Serin- und Threonin-Reste durch G-Protein-gekoppelte Rezeptorkinasen. Die Interaktion mit löslichen intrazellulären Faktoren scheint für den β1AR weniger stark ausgeprägt zu sein als für den β2AR. Die cAMP-Produktion war für die schneller werdenden, “hyperfunktionellen” Gly16-β2ARVarianten signifikant um mehr als 50% höher im Vergleich zur “hypofunktionellen” Arg16- Variante. Die unterschiedliche Funktionalität spiegelte sich im Therapieausgang bei Tokoysepatientinnen wider, dessen Erfolg mit dem Arg16Gly Polymorphismus verknüpft war. Die Daten implizieren eine intrinsische, polymorphismusabhängige Eigenschaft der βAR, die die Aktivierungskinetik der Rezeptoren bei wiederholten Stimulationen determiniert. Diese könnte auch für die zwischen Individuen variierende Ansprechbarkeit auf β-Agonisten und β-Blocker mitverantwortlich sein.Signaling through G protein-coupled receptors is known to be influenced by receptor polymorphisms, yet the molecular basis for the functional differences is unclear. To investigate the impact of the most frequent polymorphic sites of the β1- and the β2– adrenergic receptor (Ser49Gly and Gly389Arg for β1AR, Arg16Gly and Gln27Glu for β2AR) on receptor conformation we used a fluorescence resonance energy transfer (FRET) based approach. We made use of βAR-FRET sensors with a yellow fluorescent protein (YFP) inserted into the third intracellular loop and a cyan fluorescent protein (CFP or Cerulean) fused to the C-terminal tail of the βAR. These sensors retained key pharmacological and functional characteristics of the native receptors. Upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells and found that βAR respond to repeated activation with a change of their activation kinetics during subsequent stimulations. This phenomenon differed between polymorphic variants of the βAR. The “hyperfunctional” Gly16-β2AR variants as well as the Gly49Arg389-β1AR became faster in their activation kinetics, while the “hypofunctional” Arg16-β2AR and the Ser49Gly389-β1AR became slower compared to their initial activation. These differences depended on the interaction with soluble cytosolic factors that occurred after the initial activation, and on the phosphorylation of agonist-bound receptors through G protein-coupled receptor kinases. The “memory“ of previous activation is formed already after a first stimulation of only five seconds, whereas the β1AR memory necessitates prestimulation for five minutes and seems to be based on a less stable interaction with intracellular proteins compared to the β2AR. Assuming short-lived and repetitive receptor-ligand interaction under native conditions, we hypothesized that faster activation during single ligand-receptor interaction represents the basis for more effective signaling to downstream effectors. Indeed, the extent of cAMP formation was enhanced by 50% upon stimulation of the Gly16-β2AR compared to the Arg16 variant. The different functionality reflected the outcome of tocolysis treatment with the β2-agonist fenoterol whose success correlated with the Arg16Gly genotype of the patients. Our findings suggest an intrinsic, polymorphism-specific property of the βAR that alters activation kinetics upon continued stimulation and that might account for individual drug responses

Two serines in the distal C-terminus of the human ß₁-adrenoceptor determine ß-arrestin2 recruitment

<div><p>G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β₁-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.</p></div

Phosphorylation at the distal C-terminus of the ADRB1 determines receptor internalization, but does not alter stimulation-dependent MAP kinase activation.

<p>(<b>A</b>) Representative Western blots using antibodies directed against activated MAPK1/3 (pMAPK1/3), MAPK1/3 and ADRB1. HSP90 was used as a loading control. Lysates were prepared from HEK293 cells stably expressing the indicated ADRB1 mutants and stimulated with NE (100 μM for 5 min) or control. (<b>B</b>) Quantification of MAPK1/3 activity from (A). Data are means+SEM, n = 3–9. (<b>C</b>) Internalization of ADRB1 mutants expressed in HEK293 cells (together with ARRB2) as determined by loss of cell surface receptor labeling with ³H-CGP-12177. Time points indicate duration of treatment with NE prior to labeling with ³H-CGP-12177. Data are means+SEM of n = 5 independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 as determined by two-way ANOVA.</p

Phosphorylation at Ser461/Ser462 in the distal C-terminus determines arrestin binding.

<p>(<b>A</b>) Schematic of the human ADRB1 highlighting the phosphosites mutated in the distal C-terminus: ADRB1(Ala459/461/462) (light blue), ADRB1(Ala461/462) (orange) and ADRB1(Ala473/475) (dark grey). (<b>B</b>) Averaged FRET tracings comparing ADRB1(Ala459/461/462), ADRB1(Ala461/462) and ADRB1 (Ala473/475). Data are means±SEM of n ≥ 6 representative tracings. (<b>C</b>) Quantification of NE-induced changes of receptor-arrestin FRET obtained for the ADRB1 variants depicted in (B). Data are means+SEM of n ≥ 9 cells. *** p ≤ 0.001 vs. wild-type and n.s. = not significant. (<b>D</b>) Conservation of serine 459 (light blue), serine 461 and serine 462 (orange) among twelve species.</p

Phosphorylation sites in the C-terminus are crucial for β-arrestin2 recruitment.

<p>(<b>A</b>) Schematic of the FRET assay for β-arrestin2 recruitment (left) and confocal microscopy of representative cells expressing Cerulean-tagged ADRB1 and YFP-tagged β-arrestin2 (right, scale = 10 μm). (<b>B</b>) Representative tracings (left) and their ratiometric representation (right) of simultaneous recording of emissions from YFP (535 nm) and Cerulean (480 nm) upon stimulation of cells with norepinephrine (NE, 10 μM). (<b>C, D</b>) Schematics of the human ADRB1 highlighting the phosphosites mutated in the third intracellular loop and the C-terminus. (<b>E</b>) Averaged FRET tracings comparing wild-type ADRB1, ADRB1Δ3rdloop^a (= ADRB1(Ala260), light green) and a mutant devoid of all phosphosites in the third intracellular loop and C-terminus (ADRB1Δphos). (<b>F</b>) Quantification of NE-induced changes of receptor-arrestin FRET obtained for the ADRB1 variants depicted in (C). (<b>G, H</b>) Analogous experiments as in (E, F) for C-terminal mutants. Data are means±SEM from ≥10 tracings (E, G) and ≥ 30 cells (F, H). Kruskal-Wallis-Test with Dunn‘s post test. *** p ≤ 0.001 vs. wild-type and n.s. = not significant.</p

The β₁-adrenoceptor is phosphorylated at multiple sites in the third intracellular loop and at the C-terminus.

<p>(<b>A</b>) Phosphorylation of the ADRB1 with and without stimulation with NE (100 μM) for 5 min. Cells stably expressing the human ADRB1 were cultured in medium containing 1% FCS and ³²P. After cell lysis and IP of the ADRB1, the amount of phosphorylated ADRB1 (pADRB1) was quantified by phosphor imager analysis. Total ADRB1 was subsequently visualized by western blotting. (<b>B</b>) Quantification of ADRB1 phosphorylation from (A), n = 3. (<b>C</b>) Preparation of samples for mass spectrometry. (<b>D</b>) Western blot after crosslink IP of the ADRB1. HEK: untransfected HEK293 cells (negative control); ADRB1: HEK293 cells stably expressing the β₁-adrenoceptor. (<b>E</b>) Silver staining of SDS gel showing the ADRB1 after IP. (<b>F</b>) Phosphorylation pattern of the ADRB1. Amino acids marked in light grey and black indicate phosphorylation under basal conditions and after stimulation with 100 μM NE for 5 min, respectively. Pool of five experiments. Annotated spectra of the detected phosphosites are presented in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176450#pone.0176450.s001" target="_blank">S1 Fig</a>.</p