Structural and functional basis of Endothelin-1 type A receptor (ETAR) activation

Endothelin-1 type A (ETAR) and B receptors (ETBR) belong to the GPCR subfamily A and mediate the actions of their peptide agonist Endothelin-1 (ET-1). ET-1 is one of the most potent vasoconstrictors in the human organism and regulates blood pressure and local and systemic homeostasis. ETAR can be activated not only by ET-1 but also by agonistic antibodies. As recently demonstrated, extracellular binding of agonistic autoantibodies targeting ETAR (ETAR-IgG) promotes downstream signaling through allosteric activation of the receptor, independent of ET-1. The resultant signaling induced severe renovascular disease, exemplified on systemic sclerosis related renal crisis (SSc). Despite their clinical relevance, the potential differences and similarities to natural ligand-mediated activation have not been studied yet. To investigate relevant extracellular loops required for ET-1 and ETAR-IgG binding and downstream signaling responses, each of the three extracellular loops (ECL) was individually mutated. Functional effects were assessed with two different assays. The first GPCR activation assay relied on the MMY yeast model, in which a single human GPCR controls yeast growth to assess the effects of the mutations on ETAR-IgG-mediated ETAR activation. In the second assay, luciferase reporter plasmids enabled monitoring of G-proteins binding to ETAR in response to ET-1 and ETAR-IgG. Change of ECL3 structure resulted in the constitutive activation of ETAR both in MMY yeast model and in luciferase reporter assays. Besides, mutating ECL1 to Alanine or replacing ECL2 demonstrated no effect on ETAR-IgG -induced ETAR activation in yeast GPCR activation assay. In addition, these mutations did not influence ETAR-IgG -mediated Gq/11 and G12/13 binding to ETAR as revealed by luciferase reporter assays. On the contrary, results showed that intact structure of ECL1 and ECL2 is necessary for G12/13 activation upon ET-1 stimulation. This work demonstrates that unlike similar antibodies against other GPCRs, antibodies directed against Endothelin-1 type A receptor do not bind to the extracellular loops of the receptor; thereby eliciting intracellular signaling differences from the natural ligand. Elucidation of these epitopes is a prerequisite for rational design of new potent and more precise pharmacological compounds to replace old, relatively inefficient ones.Endothelin-1 Typ A (ETAR) und B Rezeptoren (ETBR) gehören zur Unterfamilie A der GPCR und vermitteln die Signale ihres Peptidagonisten Endothelin-1 (ET-1). ET-1 ist einer der stärksten Vasokonstriktoren im menschlichen Organismus und reguliert Blutdruck und lokale und systemische Homöostase. ETAR können nicht nur durch ET-1 aktiviert werden, sondern auch durch agonistische Antikörper. Wie vor kurzem gezeigt, induziert die extrazelluläre Bindung agonistischer Autoantikörper am ETAR (ETAR-IgG) downstream Signaling durch allosterische Rezeptoraktivierung unabhängig von ET-1. Dadurch können schwere renovaskuläre Erkrankungen ausgelöst werden, wie sich am Beispiel der renalen Krise bei Systemischer Sklerose (SSc) zeigt. Trotz ihrer klinischen Bedeutung, wurden die möglichen Unterschiede und Ähnlichkeiten zur Rezeptoraktivierung durch den natürlichen Liganden bisher noch nicht erforscht. Um die für die Bindung von ET-1 und der ETAR-IgG entscheidenden extrazellulären Schleifen und intrazelluläre Signalantworten untersuchen zu können, wurden jede der drei extrazellulären Schleifen (ECL) einzeln mutiert. Funktionelle Effekte wurden mit zwei verschiedenen Assays überprüft. Der erste GPCR Aktivierungsassay beruhte auf einem MMY Hefemodell, bei dem ein einzelner humaner GPCR das Hefewachstum bestimmt. Beim zweiten Assay ermöglichte ein Luziferase-Reporterplasmidassay, die Bindung von G-Proteinen an den ETAR nach Stimulation mit ET-1 und ETAR-IgG zu monitoren. Veränderungen an der Struktur der 3. ECL führten zur konstitutiven Aktivierung des ETAR, sowohl im MMY Hefemodell, als auch im Luziferase-Reporterassay. Dagegen blieben die Mutation zu Alanin an der 1. ECL oder der Ersatz der 2. ECL ohne Wirkung auf die ETAR-IgG -induzierte ETAR Aktivierung im Hefe GPCR Aktivierungsassay. Darüberhinaus beeinflussten diese Mutation die ETAR-IgG vermittelte Bindung von Gq/11 und G12/13 an den ETAR nicht, wie in Luziferase-Reporterassays gezeigt wurde. Im Gegensatz dazu zeigten die Ergebnisse, dass die intakten Strukturen der 1. ECL und 2. ECL für die G12/13 Aktivierung durch ET-1 notwendig sind. Diese Arbeiten zeigen, dass anders als bei ähnlichen GPCR-Antikörpern, die gegen den Endothelin-1 type A Rezeptor gerichtete Antiköper nicht an die extrazellulären Rezeptorschleifen binden. Dadurch ergeben sich Unterschiede zum intrazellulären Signaling des natürlichen Liganden. Die Aufklärung der Bindungsepitope ist die Voraussetzung für die Entwicklung neuer, präziser und wirksamerer Medikamente, um die alten, relativ ineffizienten Arzneien zu ersetzen

Quality and Flavor Analysis of Six Commercially Available Stewed Pork Balls

In order to clarify the quality differences of existing commercially available stewed pork ball products, this study comprehensively analyzed the quality traits of commercially available stewed pork ball of six different brands (A1-Mouth Edge, A2-Wutting Bridge, A3-Kijin, A4-Lao Yangcheng, A5-Sanzhenzhai, and A6-Huangjue) on the physicochemical indices of organoleptic, colour and lustre, textural characteristics, water loss rate and fat oxidation, combined with the free fatty acids and volatile flavour compounds to characterize. The results showed that the sensory score of the A1 stewed pork ball was as high as 34 points, and the redness value of A4 was as high as 3.46±0.08 among the six types of commercially available stewed pork balls, while the fat oxidation value of A1 was the lowest at 0.44 mg MDA/kg, and the masticatory of A5 and A6 was lower at 0.21±0.02 and 0.32±0.11 mJ, respectively. The free fatty acids composition of the six commercially available stewed pork balls, which were mainly composed of oleic acid and secondly composed of saturated fatty acids such as palmitic acid and stearic acid. The polyunsaturated fatty acids in stewed pork balls were decomposed into aldehydes and other substances by a large number of oxidation reactions, while the content of saturated fatty acids was significantly increased. γ-Pinene, hexanal, linalool, nonanal, etc. were the common volatile flavor substances, in addition, the main key volatile flavor substances of A1 were 2-pentyl furan, the main volatile flavor compounds of A2 were Valencia orange alkene, methyl heptane, isovaleraldehyde, while A4 was ethyl caprylate, A5 was 4-terpineol, undecyl alcohol, methyl mercaptan, furfuryl alcohol, furfural, methyl stearate, anethole, and A6 was undecyl alcohol, phenylpropanoid. Thus, hydrocarbons and aldehydes, along with alcohols and ketones, might be the major factors affecting the flavor quality of various commercially available stewed pork ball products. The results of this study may provide a theoretical reference for the construction of the product quality evaluation system for commercially available stewed pork ball products, as well as flavor unification and product development and production

Research on recognition algorithm for gesture page turning based on wireless sensing

When a human body moves within the coverage range of Wi-Fi signals, the reflected Wi-Fi signals by the various parts of the human body change the propagation path, so analysis of the channel state data can achieve the perception of the human motion. By extracting the Channel State Information (CSI) related to human motion from the Wi-Fi signals and analyzing it with the introduced machine learning classification algorithm, the human motion in the spatial environment can be perceived. On the basis of this theory, this paper proposed an algorithm of human behavior recognition based on CSI wireless sensing to realize deviceless and over-the-air slide turning. This algorithm collects the environmental information containing upward or downward wave in a conference room scene, uses the local outlier factor detection algorithm to segment the actions, and then the time domain features are extracted to train Support Vector Machine (SVM) and eXtreme Gradient Boosting (XGBoost) classification modules. The experimental results show that the average accuracy of the XGBoost module sensing slide flipping can reach 94%, and the SVM module can reach 89%, so the module could be extended to the field of smart classroom and significantly improve speech efficiency

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1RAbs- induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong