Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys220 residue, Gln207, Gly211, Arg213, Gly218, Ile222, Phe224, Leu225, and Pro228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance

The atomistic level structure for the activated human κ-opioid receptor bound to the full Gi protein and the MP1104 agonist

The kappa opioid receptor (κOR) is an important target for pain therapeutics to reduce depression and other harmful side effects of existing medications. The analgesic activity is mediated by κOR signaling through the adenylyl cyclase-inhibitory family of Gi protein. Here, we report the three-dimensional (3D) structure for the active state of human κOR complexed with both heterotrimeric Gi protein and MP1104 agonist. This structure resulted from long molecular dynamics (MD) and metadynamics (metaMD) simulations starting from the 3.1-Å X-ray structure of κOR–MP1104 after replacing the nanobody with the activated Gi protein and from the 3.5-Å cryo-EM structure of μOR–Gi complex after replacing the 168 missing residues. Using MD and metaMD we discovered interactions to the Gi protein with strong anchors to two intracellular loops and transmembrane helix 6 of the κOR. These anchors strengthen the binding, contributing to a contraction in the binding pocket but an expansion in the cytoplasmic region of κOR to accommodate G protein. These remarkable changes in κOR structure reveal that the anchors are essential for activation

IL-4 Inhibits IL-1β-Induced Depressive-Like Behavior and Central Neurotransmitter Alterations

It has been known that activation of the central innate immune system or exposure to stress can disrupt balance of anti-/proinflammatory cytokines. The aim of the present study was to investigate the role of pro- and anti-inflammatory cytokines in the modulation of depressive-like behaviors, the hormonal and neurotransmitter systems in rats. We investigated whether centrally administered IL-1β is associated with activation of CNS inflammatory pathways and behavioral changes and whether treatment with IL-4 could modulate IL-1β-induced depressive-like behaviors and central neurotransmitter systems. Infusion of IL-4 significantly decreased IL-1β-induced anhedonic responses and increased social exploration and total activity. Treatment with IL-4 markedly blocked IL-1β-induced increase in PGE2 and CORT levels. Also, IL-4 reduced IL-1β-induced 5-HT levels by inhibiting tryptophan hydroxylase (TPH) mRNA and activating serotonin transporter (SERT) in the hippocampus, and levels of NE were increased by activating tyrosine hydroxylase (TH) mRNA expression. These results demonstrate that IL-4 may locally contribute to the regulation of noradrenergic and serotonergic neurotransmission and may inhibit IL-1β-induced behavioral and immunological changes. The present results suggest that IL-4 modulates IL-1β-induced depressive behavior by inhibiting IL-1β-induced central glial activation and neurotransmitter alterations. IL-4 reduced central and systemic mediatory inflammatory activation, as well as reversing the IL-1β-induced alterations in neurotransmitter levels. The present findings contribute a biochemical pathway regulated by IL-4 that may have therapeutic utility for treatment of IL-1β-induced depressive behavior and neuroinflammation which warrants further study

Work-Related Perceptions, Injuries, and Musculoskeletal Symptoms: Comparison Between U.S.-Educated and Foreign-Educated Nurses.

Immigrants account for a significant proportion of the nursing workforce in the United States (U.S.). Although different cultural backgrounds may affect immigrant nurses' perceptions of work and occupational health risks, little research has been conducted. Defining immigrant nurses as those who received initial nursing education in foreign countries, this study examined the differences in work-related perceptions and experiences of musculoskeletal (MS) symptoms and injuries between U.S.-educated and foreign-educated nurses. We analyzed data from a cross-sectional study using a statewide random sample of 419 California registered nurses. Foreign-educated nurses reported a more positive safety climate (p = .017) and perceived their jobs as less demanding (p = .008) than did U.S.-educated nurses. The prevalence of work-related MS symptoms was significantly lower in foreign-educated nurses than in U.S.-educated nurses (p = .044), but the difference was not significant in the multivariable analyses. Positive safety climate was significantly associated with a decreased risk of work-related MS symptoms and injuries, and this relationship was greater among U.S.-educated nurses than among foreign-educated nurses. Our findings suggest that immigrant nurses may have different perceptions about safety climate and job demand, which may modify their occupational health risks

Genome-wide analysis to predict protein sequence variations that change phosphorylation sites or their corresponding kinases

We define phosphovariants as genetic variations that change phosphorylation sites or their interacting kinases. Considering the essential role of phosphorylation in protein functions, it is highly likely that phosphovariants change protein functions and may constitute a proportion of the mechanisms by which genetic variations cause individual differences or diseases. We categorized phosphovariants into three subtypes and developed a system that predicts them. Our method can be used to screen important polymorphisms and help to identify the mechanisms of genetic diseases

A Compact Optical Six-Axis Force/Torque Sensor for Legged Robots Using a Polymorphic Calibration Method

This paper presents a novel design for a compact, lightweight 6-axis force/torque sensor intended for use in legged robots. The design promotes easy manufacturing and cost reduction, while introducing innovative calibration methods that simplify the calibration process and minimize effort. The sensor's advantages are achieved by streamlining the structure for durability, implementing noncontact sensors, and providing a wider sensing range compared to commercial sensors. To maintain a simple structure, the paper proposes a force sensing scheme using photocouplers where the sensing elements are aligned in-plane. This strategy enables all sensing elements to be fabricated on a single printed circuit board, eliminating manual labor tasks such as bonding and coating the sensing elements. The prototype sensor contains only four parts, costs less than $250, and exhibits high response frequency and performance. Traditional calibration methods present challenges, such as the need for specialized equipment and extensive labor. To facilitate easy calibration without the need for specialized equipment, a new method using optimal control is proposed. To verify the feasibility of these ideas, a prototype six-axis F/T sensor was manufactured. Its performance was evaluated and compared to a reference F/T sensor and previous calibration methods.Comment: 12 pages, 13 figures, 9 table