Nitrogen-Centered Lactate Oxidase Nanozyme for Tumor Lactate Modulation and Microenvironment Remodeling.

Designing nanozymes that match natural enzymes have always been an attractive and challenging goal. In general, researchers mainly focus on the construction of metal centers and the control of non-metallic ligands of nanozyme to regulate their activities. However, this is not applicable to lactate oxidase, i.e., flavoenzymes with flavin mononucleotide (FMN)-dependent pathways. Herein, we propose a coordination strategy to mimic lactate oxidase based on engineering the electronic properties at the N center by modulating the Co number near N in the Cox-N nanocomposite. Benefitting from the manipulated coordination fields and electronic structure around the electron-rich N sites, Co4N/C possesses a precise recognition site for lactate and intermediate organization and optimizes the absorption energies for intermediates, leading to superior oxidation of the lactate α-C-sp(3)-H bond toward ketone. The optimized nanozyme delivers much improved anticancer efficacy by reversing the high lactate and the immunosuppressive state of the tumor microenvironment, subsequently achieving excellent tumor growth and distant metastasis inhibition. The developed Co4N/C NEs open a new window for building a bridge between chemical catalysis and biocatalysis.

Novel variants of engineered water soluble mu opioid receptors with extensive mutations and removal of cysteines.

We have shown that water-soluble variants of the human mu opioid receptor (wsMOR) containing a reduced number of hydrophobic residues at the lipid-facing residues of the transmembrane (TM) helices can be expressed in E. coli. In this study, we tested the consequences of increasing the number of mutations on the surface of the transmembrane domain on the receptor's aqueous solubility and ligand binding properties, along with mutation of 11 cysteine residues regardless of their solvent exposure value and location in the protein. We computationally engineered 10 different variants of MOR, and tested four of them for expression in E. coli. We found that all four variants were successfully expressed and could be purified in high quantities. The variants have alpha helical structural content similar to that of the native MOR, and they also display binding affinities for the MOR antagonist (naltrexone) similar to the wsMOR variants we engineered previously that contained many fewer mutations. Furthermore, for these full-length variants, the helical content remains unchanged over a wide range of pH values (pH 6 ~ 9). This study demonstrates the flexibility and robustness of the water-soluble MOR variants with respect to additional designed mutations in the TM domain and changes in pH, whereupon the protein's structural integrity and its ligand binding affinity are maintained. These variants of the full-length MOR with less hydrophobic surface residues and less cysteines can be obtained in large amounts from expression in E. coli and can serve as novel tools to investigate structure-function relationships of the receptor.

The C-terminus of the mu opioid receptor is critical in G-protein interaction as demonstrated by a novel graphene biosensor.

Several water-soluble variants of the human mu opioid receptor (wsMORs) have been designed and expressed, which enables the detection of opioids in the nM to pM range using biosensing platforms. The tools previously developed allowed us to investigate MOR and G-protein interactions in a lipid free system to demonstrate that the lipid bilayer might not be essential for the G-protein recognition and binding. In this study, we are able to investigate G-protein interactions with MOR by using graphene enabled technology, in a lipid free system, with a high sensitivity in a real time manner. A new wsMOR with the native C-terminus was designed, expressed and then immobilized on the surfaces of scalable graphene field effect transistor (GFET)-based biosensors, enabling the recording of wsMOR/G-protein interaction with an electronic readout. G-protein only interacts with the wsMOR in the presence of the native MOR C-terminus with a KA of 32.3±11.1 pM. The electronic readout of such interaction is highly reproducible with little variance across 50 devices in one biosensor array. For devices with receptors that do not have the native C-terminus, no significant electronic response was observed in the presence of G-protein, indicating an absence of interaction. These findings reveal that lipid environment is not essential for the G-protein interaction with MOR, however, the C-terminus of MOR is essential for G-protein recognition and high affinity binding. A system to detect MOR-G protein interaction is developed. wsMOR-G2_Cter provides a novel tool to investigate the role of C terminus in the signaling pathway.

A Multi-Dimensional Goal Aircraft Guidance Approach Based on Reinforcement Learning with a Reward Shaping Algorithm.

Guiding an aircraft to 4D waypoints at a certain heading is a multi-dimensional goal aircraft guidance problem. [d=Zu]In order to improve the performance and solve this problem, this paper proposes a multi-layer RL approach.To enhance the performance, in the present study, a multi-layer RL approach to solve the multi-dimensional goal aircraft guidance problem is proposed. The approach [d=Zu]enablesassists the autopilot in an ATC simulator to guide an aircraft to 4D waypoints at certain latitude, longitude, altitude, heading, and arrival time, respectively. To be specific, a multi-layer RL [d=Zu]approach is proposedmethod to simplify the neural network structure and reduce the state dimensions. A shaped reward function that involves the potential function and Dubins path method is applied. [d=Zu]Experimental and simulation results show that the proposed approachExperiments are conducted and the simulation results reveal that the proposed method can significantly improve the convergence efficiency and trajectory performance. [d=Zu]FurthermoreFurther, the results indicate possible application prospects in team aircraft guidance tasks, since the aircraft can directly approach a goal without waiting in a specific pattern, thereby overcoming the problem of current ATC simulators.

Impact of the COVID-19 Epidemic on Stroke Care and Potential Solutions.

BACKGROUND AND PURPOSE: When the coronavirus disease 2019 (COVID-19) outbreak became paramount, medical care for other devastating diseases was negatively impacted. In this study, we investigated the impact of the COVID-19 outbreak on stroke care across China. METHODS: Data from the Big Data Observatory Platform for Stroke of China consisting of 280 hospitals across China demonstrated a significant drop in the number of cases of thrombolysis and thrombectomy. We designed a survey to investigate the major changes during the COVID-19 outbreak and potential causes of these changes. The survey was distributed to the leaders of stroke centers in these 280 hospitals. RESULTS: From the data of Big Data Observatory Platform for Stroke of China, the total number of thrombolysis and thrombectomy cases dropped 26.7% (P<0.0001) and 25.3% (P<0.0001), respectively, in February 2020 as compared with February 2019. We retrieved 227 valid complete datasets from the 280 stroke centers. Nearly 50% of these hospitals were designated hospitals for COVID-19. The capacity for stroke care was reduced in the majority of the hospitals. Most of the stroke centers stopped or reduced their efforts in stroke education for the public. Hospital admissions related to stroke dropped ≈40%; thrombolysis and thrombectomy cases dropped ≈25%, which is similar to the results from the Big Data Observatory Platform for Stroke of China as compared with the same period in 2019. Many factors contributed to the reduced admissions and prehospital delays; lack of stroke knowledge and proper transportation were significant limiting factors. Patients not coming to the hospital for fear of virus infection was also a likely key factor. CONCLUSIONS: The COVID-19 outbreak impacted stroke care significantly in China, including prehospital and in-hospital care, resulting in a significant drop in admissions, thrombolysis, and thrombectomy. Although many factors contributed, patients not coming to the hospital was probably the major limiting factor. Recommendations based on the data are provided.

Molecular Interaction Between Butorphanol and κ-Opioid Receptor.

BACKGROUND: The misuse of opioids stems, in part, from inadequate knowledge of molecular interactions between opioids and opioid receptors. It is still unclear why some opioids are far more addictive than others. The κ-opioid receptor (KOR) plays a critical role in modulating pain, addiction, and many other physiological and pathological processes. Butorphanol, an opioid analgesic, is a less addictive opioid with unique pharmacological profiles. In this study, we investigated the interaction between butorphanol and KOR to obtain insights into the safe usage of this medication. METHODS: We determined the binding affinity of butorphanol to KOR with a naltrexone competition study. Recombinant KORs expressed in mammalian cell membranes (Chem-1) were used for G-protein activation studies, and a human embryonic kidney-293 (HEK-293) cell line stably transfected with the human KOR was used for ß-arrestin study as previously described in the literature. The effects of butorphanol on KOR internalization were investigated using mouse neuroblastoma Neuro2A cells stably transfected with mKOR-tdTomato fusion protein (N2A-mKOR-tdT) cells overexpressing KOR. The active-state KOR crystal structure was used for docking calculation of butorphanol to characterize the ligand binding site. Salvinorin A, a full KOR agonist, was used as a control for comparison. RESULTS: The affinity of KOR for butorphanol is characterized by Kd of 0.1 ± 0.02 nM, about 20-fold higher compared with that of the µ-opioid receptor (MOR; 2.4 ± 1.2 nM). Our data indicate that butorphanol is more potent on KOR than on MOR. In addition, butorphanol acts as a partial agonist of KOR in the G-protein activation pathway and is a full agonist on the ß-arrestin recruitment pathway, similar to that of salvinorin A. The activation of the ß-arrestin pathway is further confirmed by KOR internalization. The in silico docking model indicates that both salvinorin A and butorphanol share the same binding cavity with the KOR full agonist MP1104. This cavity plays an important role in determining either agonist or antagonist effects of the ligand. CONCLUSIONS: In conclusion, butorphanol is a partial KOR agonist in the G-protein activation pathway and a potent KOR full agonist in the ß-arrestin recruitment pathway. The structure analysis offers insights into the molecular mechanism of KOR interaction and activation by butorphanol.

Dezocine Alleviates Morphine-Induced Dependence in Rats.

BACKGROUND: Opioid dependence is a major public health issue without optimal therapeutics. This study investigates the potential therapeutic effect of dezocine, a nonaddictive opioid, in opioid dependence in rat models. METHODS: Dezocine was administered intraperitoneally to a morphine-dependent rat model to investigate its effect on withdrawal and conditioned place preference (CPP). Effect of dezocine on morphine withdrawal syndrome and CPP was analyzed using 2-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Buprenorphine and vehicle solution containing 20% (v/v) dimethyl sulfoxide were used for positive and negative control, respectively. The astrocytes activation in nucleus accumbens was assessed by immunofluorescence assay of glial fibrillary acidic protein. Effect of dezocine and buprenorphine on the internalization of κ opioid receptor (KOR) was investigated using Neuro2A expressing KOR fused to red fluorescent protein tdTomato (KOR-tdT). Buprenorphine and dezocine were screened against 44 G-protein-coupled receptors, ion channels, and transporter proteins using radioligand-binding assay to compare the molecular targets. RESULTS: The mean withdrawal score was reduced in rats treated with 1.25 mg·kg dezocine compared to vehicle-treated control animals starting from the day 1 (mean difference: 7.8; 95% confidence interval [CI], 6.35-9.25; P < .0001 by 2-way ANOVA). Significance was observed at all treatment days, including day 7 (mean difference: 2.13; 95% CI, 0.68-3.58; P < .001 by 2-way ANOVA). Furthermore, dezocine inhibited the reinstatement of morphine-induced CPP (mean difference: 314; 95% CI, 197.9-430.1; P < .0001 by 2-way ANOVA) compared to the control group. Chronic morphine administration induced astrocytes activation in nucleus accumbens, which was attenuated by dezocine. Dezocine blocked the agonist-induced KOR internalization in vitro, 1 of the mechanisms involved in the downstream signaling and development of opioid dependence. Dezocine had affinity to norepinephrine and serotonin transporters and sigma-1 receptor, whereas buprenorphine showed no activity against these targets. CONCLUSIONS: Dezocine could potentially be used to alleviate opioid dependence. Due to the unique molecular target profile different from buprenorphine, it might have important value in studying the mechanisms of morphine dependence and developing novel therapeutic approaches.

Significant Stroke Knowledge Deficiencies in Community Physician Improved with Stroke 120.

BACKGROUND AND PURPOSE: In the existing model of community health service in China, community general practitioners play important roles in health promotion as well as prehospital stroke recognition and management. We recently engineered Stroke 120 based on FAST for China. This investigation aimed to investigate its acceptance in community physicians and promote their stroke related knowledge. METHODS: We conducted an stroke education session to community physicians or family doctors (total of 435 participants), teaching both FAST and Stroke 120. Online survey was distributed to the participants before and after the education session to evaluate the awareness of stroke and the acceptance of the stroke recognition tool. RESULTS: Significant stroke knowledge deficiencies were found in community physicians. After the education session, percent of the participants knew that the thrombolytic therapeutic window (<4.5 hours) was improved from 54.0% to 91.6% (P < .001). A total of 88.5% of them would send their patients who had stroke to the nearest hospital with stroke center by emergency medical service, compared to baseline (64.4%, P < .001). In total, 95.2% of them would recommend thrombolytic therapy in the treatment of acute ischemic stroke compared to 82.7% (baseline P < .001). Although majority mastered both FAST (95.5%) and Stroke 120 (98.0%) through our education session, 96.3% of them believe that Stroke 120 is the most suitable for Chinese in stroke education. CONCLUSIONS: Stroke 120 strategy was well accepted by the community physicians in China and in the meantime improved knowledge regarding stroke was observed.

Stroke 112: A Universal Stroke Awareness Program to Reduce Language and Response Barriers.

BACKGROUND AND PURPOSE: To improve stroke awareness and reduce life-threatening prehospital delays worldwide, a universal stroke educational program is needed. To meet this unmet need, we developed a universal program without language barriers and tested its acceptance in Taiwan, where Chinese is the native language. METHODS: Stroke 112 was developed using the universal emergency phone number, 112. The numbers imply an emergency and correspond to the 3 stroke recognition signs used in FAST (Face, Arm, Speech, and Time): 1 uneven face (crooked mouth); 1 weak arm (arm weakness); 2 incoherent lips (slurred speech). An online survey was used to determine the acceptance of the Stroke 112 program compared with that of FAST in Chinese. The surveys were delivered using SurveyMonkey (http://www.surveymonkey.com) on 2 separate occasions in Taiwan; in August 2017 for an initial estimation of the acceptance of Stroke 112 and in March 2018, 2 weeks after the official release of Stroke 112 in Taiwan, including a special introductory lecture for neurologists hosted by the STARS-Taiwan (Stroke Treatment and Research Society-Taiwan). RESULTS: The initial survey with 465 survey responders, 54.6% thought that Stroke 112 was easier to remember for people in Taiwan compared with FAST (41.2%). After Stroke 112's official release in Taiwan, 610 individuals completed the survey, and the majority (66.4%) thought that Stroke 112 was easier to remember, a significant increase compared with the initial survey (P=0.0001). Among the 130 neurologists who attended the Stroke 112 introductory lecture, 55 completed the online survey. A greater acceptance of Stroke 112 (74.6%) compared with FAST (16.4%) was observed among these 55 neurologists (P=0.0001). CONCLUSIONS: Stroke 112, a universal stroke educational program without language barriers was developed. It could potentially be implemented worldwide, especially where 112 is used as an emergency phone number.

Salvinorin A ameliorates cerebral vasospasm through activation of endothelial nitric oxide synthase in a rat model of subarachnoid hemorrhage.

OBJECTIVE: This study aimed to demonstrate the potential of salvinorin A (SA) for cerebral vasospasm after subarachnoid hemorrhage (SAH) and investigate mechanisms of therapeutic effect using rat SAH model. METHODS: Salvinorin A was injected intraperitoneally, and the neurobehavioral changes were observed at 12 hours, 24 hours, 48 hours, and 72 hours after SAH. Basilar artery was observed by magnetic resonance imaging (MRI). The inner diameter and thickness of basilar artery were measured. The morphological changes and the apoptosis in CA1 area of hippocampus were detected. Endothelin-1 (ET-1) and nitric oxide (NO) levels were detected by ELISA kit. The protein expression of endothelial NO synthase (eNOS) and aquaporin-4 (AQP-4) was determined by Western blot for potential mechanism exploration. RESULTS: Salvinorin A administration could relieve neurological deficits, decrease the neuronal apoptosis, and alleviate the morphological changes in CA1 area of hippocampus. SA alleviated CVS by increasing diameter and decreasing thickness of basilar artery, and such changes were accompanied by the decreased concentration of ET-1 and increased level of NO. Meanwhile, SA increased the expression of eNOS and decreased the expression of AQP-4 protein in the basilar artery and hippocampus. CONCLUSIONS: Salvinorin A attenuated CVS and alleviated brain injury after SAH via increasing expression of eNOS and NO content, and decreasing ET-1 concentration and AQP-4 protein expression.

Chronic Opioid Usage in Surgical Patients in a Large Academic Center.

OBJECTIVE: The objective of this study is to investigate the prevalence and disparity of chronic opioid usage in surgical patients and the potential risk factors associated with chronic opioid usage. BACKGROUND: Chronic opioid usage is common in surgical patients; however, the characteristics of opioid usage in surgical patients is unclear. In this study, we hypothesize that the prevalence of chronic opioid usage in surgical patients is high, and that significant disparities may exist among different surgical populations. METHODS: Data of opioid usage in outpatients among different surgical services were extracted from the electronic medical record database. Patient demographics, clinical characteristics of sex, age, race, body mass index (BMI), specialty visited, duration of opioid use, and opioid type were collected. Chronic opioid users were defined as patients who had been recorded as taking opioids for at least 90 days determined by the first and last visit dates under opioid usage during the investigation. RESULTS: There were 79,123 patients included in this study. The average prevalence is 9.2%, ranging from 4.4% to 23.8% among various specialties. The prevalence in orthopedics (23.8%), neurosurgery (18.7%), and gastrointestinal surgery (14.4%) ranked in the top three subspecialties. Major factors influencing chronic opioid use include age, Ethnicitiy, Subspecialtiy, and multiple specialty visits. Approximately 75% of chronic users took opioids that belong to the category II Drug Enforcement Administration classification. CONCLUSIONS: Overall prevalence of chronic opioid usage in surgical patients is high with widespread disparity among different sex, age, ethnicity, BMI, and subspecialty groups. Information obtained from this study provides clues to reduce chronic opioid usage in surgical patients.

Hypoxia Induces Internalization of κ-Opioid Receptor.

BACKGROUND: It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation. METHODS: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor-tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. RESULTS: Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization. CONCLUSIONS: Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.

The Role of κ Opioid Receptor in Brain Ischemia.

OBJECTIVES: Our previous studies indicated that highly selective κ opioid receptor agonists could protect the brain, indicating an important role of κ opioid receptor agonist in brain ischemia. In this study, we investigated the role and related mechanisms of κ opioid receptor agonists in brain ischemia in a middle cerebral artery occlusion mouse model. DESIGN: Animal model. SETTING: Laboratory. SUBJECTS: The middle cerebral artery occlusion model was established by 120 minutes of ischemia followed by 24-hour reperfusion in male adult mice. INTERVENTIONS: Various doses of salvinorin A, a highly selective and potent κ opioid receptor agonist, were administered intranasally 10 minutes after initiation of reperfusion. Norbinaltorphimine (2.5 mg/kg, IP) as a κ opioid receptor antagonist was administered in one group before administration of salvinorin A (50µg/kg) to investigate the specific role of κ opioid receptor. MEASUREMENTS AND MAIN RESULTS: Infarct volume, κ opioid receptor expression, and Evans blue extravasation in the brain, and neurobehavioral outcome were determined. Immunohistochemistry and western blot were performed to detect the activated caspase-3, interleukin-10, and tumor necrosis factor-α levels to investigate the role of apoptosis and inflammation. κ opioid receptor expression was elevated significantly in the ischemic penumbral area compared with that in the nonischemic area. Salvinorin A reduced infarct volume and improved neurologic deficits dose-dependently. Salvinorin A at the dose of 50 µg/kg reduced Evans blue extravasation, suggesting reduced impairment of the blood-brain barrier and decreased the expression of cleaved caspase-3, interleukin-10, and tumor necrosis factor-α in the penumbral areas. All these changes were blocked or alleviated by norbinaltorphimine. CONCLUSIONS: κ opioid receptors were up-regulated and played a critical role in brain ischemia and reperfusion. κ opioid receptor activation could potentially protect the brain and improve neurologic outcome via blood-brain barrier protection, apoptosis reduction, and inflammation inhibition.

Scalable production of highly sensitive nanosensors based on graphene functionalized with a designed G protein-coupled receptor.

We have developed a novel, all-electronic biosensor for opioids that consists of an engineered µ-opioid receptor protein, with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor to read out ligand binding. A variant of the receptor protein that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. A shadow mask process was developed to fabricate arrays of hundreds of graphene transistors with average mobility of ∼1500 cm(2) V(-1) s(-1) and yield exceeding 98%. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, an opioid receptor antagonist, with a detection limit of 10 pg/mL.

Novel molecular targets of dezocine and their clinical implications.

BACKGROUND: Although dezocine is a partial µ-opioid receptor agonist, it is not a controlled substance. Thus, the characterization of the molecular targets of dezocine is critical for scientific and clinical implications. The goal of this study is to characterize molecular targets for dezocine and determine their implications. METHODS: A binding screen for dezocine was performed on 44 available receptors and transporter proteins. Functional assays for the novel targets were performed along with computation calculations to locate the binding site. A G protein activation study was performed for the human κ opioid receptor to determine whether dezocine is a κ-antagonist. Data are presented as mean ± standard error. RESULTS: The affinities for dezocine were 3.7 ± 0.7 nM for the µ receptor, 527 ± 70 nM for the δ-receptor, and 31.9 ± 1.9 nM for the κ-receptor. Dezocine failed to induce G protein activation with κ-opioid receptor and concentration dependently inhibited κ-agonist (salvinorin A and nalbuphine)-induced receptor activation, indicating that dezocine is a κ-antagonist. Two novel molecular targets (norepinephrine transporter and serotonin transporter) were identified. Dezocine concentration-dependently inhibited norepinephrine and serotonin reuptake in vitro. The half maximal inhibitory concentrations (expressed as pIC50) were 5.68 ± 0.11 for norepinephrine transporter and 5.86 ± 0.17 for serotonin transporter. Dezocine occupied the binding site for known norepinephrine transporter and serotonin transporter inhibitors. CONCLUSIONS: The unique molecular pharmacological profile of dezocine as a partial µ-receptor agonist, a κ-receptor antagonist, and a norepinephrine and serotonin reuptake inhibitor (via norepinephrine transporter and serotonin transporter) was revealed. These discoveries reveal potentially important novel clinical implications and drug interactions of dezocine.

Characterization of a computationally designed water-soluble human µ-opioid receptor variant using available structural information.

BACKGROUND: The recent X-ray crystal structure of the murine µ-opioid receptor (MUR) allowed the authors to reengineer a previously designed water-soluble variant of the transmembrane portion of the human MUR (wsMUR-TM). METHODS: The new variant of water-soluble MUR (wsMUR-TM_v2) was engineered based on the murine MUR crystal structure. This novel variant was expressed in Escherichia coli and purified. The properties of the receptor were characterized and compared with those of wsMUR-TM. RESULTS: Seven residues originally included for mutation in the design of the wsMUR-TM were reverted to their native identities. wsMUR-TM_v2 contains 16% mutations of the total sequence. It was overexpressed and purified with high yield. Although dimers and higher oligomers were observed to form over time, the wsMUR-TM_v2 stayed predominantly monomeric at concentrations as high as 7.5 mg/ml in buffer within a 2-month period. Its secondary structure was predominantly helical and comparable with those of both the original wsMUR-TM variant and the native MUR. The binding affinity of wsMUR-TM_v2 for naltrexone (K(d) approximately 70 nM) was in close agreement with that for wsMUR-TM. The helical content of wsMUR-TM_v2 decreased cooperatively with increasing temperature, and the introduction of sucrose was able to stabilize the protein. CONCLUSIONS: A novel functional wsMUR-TM_v2 with only 16% mutations was successfully engineered, expressed in E. coli, and purified based on information from the crystal structure of murine MUR. This not only provides a novel alternative tool for MUR studies in solution conditions but also offers valuable information for protein engineering and structure-function relations.

Non-scheduled short-acting opioid to taper off opioids?

This commentary discusses the issues related to the current pharmacotherapy using super long-acting opioids (for the potential convenience for both patients and medical providers) for opioid addiction and argues for the potential to use a non-scheduled short-acting opioid to taper off opioids to reduce total number of patients on opioids and ultimately reduce opioid-related death. This article also proposes to develop short-acting opioids for addiction management instead of the current long-acting regimen. The authors further suggest that dezocine, a previously FDA approved medication for perioperative pain management and a non-scheduled opioid, be brought back to clinical practice in the US as a potential alternative addiction management medication, especially for those who are highly motivated to quit opioids completely using a taper off strategy.