G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K d = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.
With the recently solved crystal structure of the murine mu opioid receptor, the elucidation of the structure function relationships of the human mu receptor becomes feasible. In this study, we analyzed the available structural information along with ligand binding and G protein activation of human mu receptor. Affinity determinations were performed in a HEK293 cell line stably transfected with the human mu opioid receptor for 6 different agonists (morphine, DMAGO, and herkinorn) and antagonists (naloxone, beta-Funaltrexamine, and Norbinaltorphimine) based on the method. G protein activation was investigated in membrane preparations containing human mu receptors treated with the agonist, partial agonist, or antagonist compounds. 4DKL.pdb was utilized for structural analysis and docking calculations for 28 mu receptor ligands. The predicted affinities from docking were compared with those experimentally determined. While all known ligands bind to the receptor through the same binding site that is large enough to accommodate molecules of various sizes, interaction with D147 (D149 in human mu receptor) is essential for binding. No distinguishable interaction pattern in the binding site for agonist, partial agonist, or antagonist to predict pharmacological activities was found. The failure to reconcile the predicted affinities from docking with experimental values indicates that the receptor might undergo significant conformational changes from one state to the other states upon different ligand binding. A simplified model to understand the complicated system is proposed and further study on these multiple conformations using high resolution structural approaches is suggested.
Sedation induces changes in electroencephalography (EEG) dynamics. However, the distinct EEG dynamic characteristics at comparable sedation levels have not been well studied, resulting in potential interpretation errors in EEG monitoring during sedation. We aimed to analyze the EEG dynamics of dexmedetomidine and propofol at comparable sedation levels and to explore EEG changes with increased sedation levels for each agent. We measured the Bispectral Index (BIS) and 20-channel EEG under dexmedetomidine and propofol sedation from wakefulness, moderate sedation, and deep sedation to recovery in healthy volunteers (n = 10) in a randomized, 2-day, crossover study. Observer’s Assessment of Alertness and Sedation (OAA/S) score was used to assess sedation levels. Despite similar changes in increased delta oscillations, multiple differences in the EEG spatiotemporal dynamics were observed between these two agents. During moderate sedation, both dexmedetomidine and propofol induced increased spindle power; however, dexmedetomidine decreased the global alpha/beta/gamma power, whereas propofol decreased the alpha power in the occipital area and increased the global spindle/beta/gamma power. During deep sedation, dexmedetomidine was associated with increased fronto-central spindle power and decreased global alpha/beta/gamma power, but propofol was associated with increased theta/alpha/spindle/beta power, which was maximized in the frontal area. The transition of topographic alpha/spindle/beta power distribution from moderate sedation to deep sedation completely differed between these two agents. Our study demonstrated that there was a distinct hierarchy of EEG changes with increased sedation depths by propofol and dexmedetomidine. Differences in EEG dynamics at the same sedation level might account for differences in the BIS value and reflect the different sedation mechanisms. EEG-based clinical sedation monitoring should consider the effect of drug types on EEG dynamics.
Ca/calmodulin-dependent protein kinase II (CaMKII) activation through autophosphorylation at threonine 286 was involved in the modulation of neuronal excitability and neurotransmission. Both propofol and ketamine may affect the intracellular Ca levels through N-methyl-D-aspartate receptors or voltage-dependent Ca channels, but they have different mechanisms in general anesthesia. The purpose of this study was to investigate the effects of propofol and ketamine on CaMKII total protein and phosphorylation (p-CaMKII) levels in the brain of rats. We found that both propofol and ketamine could induce a decrease of p-CaMKII, not CaMKII total protein, in an anesthetic depth-dependent manner, whereas only ketamine caused a dose (50, 100, and 150 mg/kg)-dependent depression of p-CaMKII in hippocampus and frontal cortex of rats after intraperitoneal injections for 30 minutes. The significant depressions of p-CaMKII started at 5 minutes both in hippocampus and frontal cortex of rats after 100 mg/kg propofol treatment, whereas 100 mg/kg ketamine-induced significant depression of p-CaMKII initiated at 30 minutes in hippocampus and 5 minutes (no reduction observed at 15 min) in frontal cortex of rats. The maximum reduction of p-CaMKII with both drugs was at 60 minutes, and then restored to control level at 240 minutes. In addition, we confirmed the depression of p-CaMKII in hippocampus and frontal cortex of rats after 100 mg/kg of propofol or ketamine treatment for 60 minutes by using immunostaining. These results suggested that decreased p-CaMKII levels correlate with anesthetic depths achieved by propofol and ketamine, which may be related to the effects of propofol and ketamine on central nervous system function and their clinical effect.
Objectives Although the flexible laryngeal mask airway (FLMA) provides considerable advantages in head and neck procedures, little is known about its safety and efficacy in functional endoscopic sinus surgery (FESS). We conducted a retrospective study to evaluate the success rate of FLMA and relevant airway complications in FESS under general anaesthesia. Methods A retrospective review of consecutive patients who underwent FESS for chronic rhinosinusitis was performed from 2015 to 2019. All patients scheduled for FLMA ventilation were identified. Patient characteristics, length of the surgery, FLMA size, failed FLMA cases requiring endotracheal intubation, immediate adverse airway events and delayed airway injuries were recorded. The primary outcomes included the FLMA success rate, which was defined as primary success after induction and final success after the whole surgical procedure. The secondary outcomes were specific clinical factors associated with FLMA failure and airway complications related to FLMA usage. Results Of the 6661 patients included in our study, primary success was achieved in 6572 (98.7%), and final success was achieved in 6512 (97.8%). Failure occurred in 89 patients (1.3%) during induction, in 14 (0.2%) during surgical preparation and in 46 (0.7%) during the intraoperative procedure. All patients with failed FLMA ventilation were successfully switched to endotracheal intubation. Male sex, advanced age, higher American Society of Anesthesiologists grade (ASA) and higher body mass index (BMI) were independent risk factors associated with failed FLMA. Immediate adverse respiratory events were observed in 0.85% of the patients, and delayed airway injuries associated with use of FLMA were observed in 0.07%. Conclusion This retrospective study demonstrates a high success rate for FLMA (97.8% in 6661 patients undergoing FESS). Adverse airway events and injuries associated with FLMA are rare, but clinicians should remain vigilant so that early diagnosis and prompt treatment can be provided.
Background Although enhanced recovery after surgery (ERAS) protocols have been widely applied during perioperative periods for different diseases, there are few reports of ERAS in patients undergoing endoscopic sinus surgery (ESS). This study therefore aimed to evaluate the benefits of ERAS protocol compared to traditional care following ESS. Methods A total of 55 patients with chronic rhinosinusitis undergoing ESS were prospectively assigned to 1 of 5 treatment groups; ERAS groups with postoperative intravenous Flubiprofen Axetil or analgesia pump, traditional care with Flubiprofen Axetil or analgesia pump (NERAS groups), or traditional care without postoperative intravenous analgesia group (control). All patients completed the Kolcaba General Comfort Questionnaire, Medical Outcomes Study Sleep Scale, and Self-rating Anxiety Scale at admission and before discharge. Pain scores were recorded at 2, 6, 24, and 48 hours postsurgery and adverse reactions to analgesics were noted. Results Patients in ERAS group demonstrated significantly higher general comfort scores and lower self-rating anxiety scores compared to patients in NERAS and control groups. Compared to control patients, patients in ERAS group reported significantly lower pain scores at 6, 24, and 48 hours. Moreover, pain alleviated from 6 hours postsurgery in ERAS group compared to 48 hours in NERAS group. Patients using opioids experienced more adverse nausea events than patients using only nonsteroidal anti-inflammatory drugs (NSAIDs). Conclusions The use of patient-tailored ERAS programs following ESS may help to attain higher general comfort and to alleviate perioperative anxiety compared with traditional perioperative care. Adequate postoperative analgesia with NSAIDs in ERAS protocol may alleviate pain earlier with fewer adverse reactions.
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