Acetylation Blocks cGAS Activity and Inhibits Self-DNA-Induced Autoimmunity.

The presence of DNA in the cytoplasm is normally a sign of microbial infections and is quickly detected by cyclic GMP-AMP synthase (cGAS) to elicit anti-infection immune responses. However, chronic activation of cGAS by self-DNA leads to severe autoimmune diseases for which no effective treatment is available yet. Here we report that acetylation inhibits cGAS activation and that the enforced acetylation of cGAS by aspirin robustly suppresses self-DNA-induced autoimmunity. We find that cGAS acetylation on either Lys384, Lys394, or Lys414 contributes to keeping cGAS inactive. cGAS is deacetylated in response to DNA challenges. Importantly, we show that aspirin can directly acetylate cGAS and efficiently inhibit cGAS-mediated immune responses. Finally, we demonstrate that aspirin can effectively suppress self-DNA-induced autoimmunity in Aicardi-Goutières syndrome (AGS) patient cells and in an AGS mouse model. Thus, our study reveals that acetylation contributes to cGAS activity regulation and provides a potential therapy for treating DNA-mediated autoimmune diseases.

The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA.

Cyclic GMP-AMP synthase (cGAS) functions as a key sensor for microbial invasion and cellular damage by detecting emerging cytosolic DNA. Here, we report that GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) primes cGAS for its prompt activation by engaging cGAS in a primary liquid-phase condensation state. Using high-resolution microscopy, we show that in resting cells, cGAS exhibits particle-like morphological characteristics, which are markedly weakened when G3BP1 is deleted. Upon DNA challenge, the pre-condensed cGAS undergoes liquid-liquid phase separation (LLPS) more efficiently. Importantly, G3BP1 deficiency or its inhibition dramatically diminishes DNA-induced LLPS and the subsequent activation of cGAS. Interestingly, RNA, previously reported to form condensates with cGAS, does not activate cGAS. Accordingly, we find that DNA - but not RNA - treatment leads to the dissociation of G3BP1 from cGAS. Taken together, our study shows that the primary condensation state of cGAS is critical for its rapid response to DNA.

NADPHnet: a novel strategy to predict compounds for regulation of NADPH metabolism via network-based methods.

Identification of compounds to modulate NADPH metabolism is crucial for understanding complex diseases and developing effective therapies. However, the complex nature of NADPH metabolism poses challenges in achieving this goal. In this study, we proposed a novel strategy named NADPHnet to predict key proteins and drug-target interactions related to NADPH metabolism via network-based methods. Different from traditional approaches only focusing on one single protein, NADPHnet could screen compounds to modulate NADPH metabolism from a comprehensive view. Specifically, NADPHnet identified key proteins involved in regulation of NADPH metabolism using network-based methods, and characterized the impact of natural products on NADPH metabolism using a combined score, NADPH-Score. NADPHnet demonstrated a broader applicability domain and improved accuracy in the external validation set. This approach was further employed along with molecular docking to identify 27 compounds from a natural product library, 6 of which exhibited concentration-dependent changes of cellular NADPH level within 100 µM, with Oxyberberine showing promising effects even at 10 µM. Mechanistic and pathological analyses of Oxyberberine suggest potential novel mechanisms to affect diabetes and cancer. Overall, NADPHnet offers a promising method for prediction of NADPH metabolism modulation and advances drug discovery for complex diseases.

Characteristics of white matter alterations along fibres in patients with bulimia nervosa: A combined voxelwise and tractography study.

Accumulating evidence supports the hypothesis that white matter (WM) abnormalities are involved in the pathophysiology of bulimia nervosa (BN); however, findings from in vivo neuroimaging studies have been inconsistent. We aimed to investigate the possible brain WM alterations, including WM volume and microstructure, in patients with BN. We recruited 43 BN patients and 31 healthy controls (HCs). All participants underwent structural and diffusion tensor imaging. Differences in WM volume and microstructure were evaluated using voxel-based morphometry, tract-based spatial statistics, and automated fibre quantification analysis. Compared with HCs, BN patients showed significantly decreased fractional anisotropy in the middle part of the corpus callosum (nodes 31-32) and increased mean diffusivity in the right cranial nerve V (CN V) (nodes 27-33 and nodes 55-88) and vertical occipital fasciculus (VOF) (nodes 58-85). Moreover, we found decreased axial diffusivity in the right inferior fronto-occipital fasciculus (node 67) and increased radial diffusivity in the CN V (nodes 22-34 and nodes 52-89) and left VOF (nodes 60-66 and nodes 81-85). Meanwhile, WM microstructural changes were correlated with patients' clinical manifestations. We did not find any significant differences in WM volume and the main WM fibre bundle properties between BN patients and HCs. Taken together, these findings provide that BN shows significant brain WM reorganization, but primarily in microstructure (part of WM fibre bundle), which is not sufficient to cause changes in WM volume. The automated fibre quantification analysis could be more sensitive to detect the subtle pathological changes in a point or segment of the WM fibre bundle.

Rosmarinic acid ameliorates autoimmune responses through suppression of intracellular nucleic acid-mediated type I interferon expression.

Recognition of intracellular nucleic acids is a vital step for host to mount prompt immune responses against microbial pathogens. However, inappropriate response to self-nucleic acids leads to sustained type I interferon (IFN) production, which is implicated in the development of several autoimmune diseases, such as Aicardi-Goutières syndrome (AGS). Therefore, effective confinement of intracellular nucleic acid-induced IFN expression is a potential strategy for the treatment of such autoimmune diseases. In this study, we found that rosmarinic acid (RA), a natural compound isolated from rosemary, inhibits intracellular nucleic acid-stimulated IFN expression. Mechanistic investigation revealed that RA binds to both G3BP1 and cGAS, and impairs cGAS activation through disrupting the binding of DNA with cGAS. More importantly, we showed that RA could effectively attenuate the expression of IFN-stimulated genes (ISGs) in the well-established cell models for AGS. Thus, our study provides a promising compound for the treatment of autoimmune responses induced by aberrant nucleic acid-sensing.

G3BP1 Inhibition Alleviates Intracellular Nucleic Acid-Induced Autoimmune Responses.

The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing.

The contradictory roles of tightly bound and loosely bound extracellular polymeric substances of activated sludge in trimethoprim adsorption process.

Extracellular polymeric substances (EPS) of activated sludge are a mixture of high molecular weight polymers secreted by microorganisms, which have the double structure of tightly-bound EPS (TB-EPS) in inner layer and loosely-bound EPS (LB-EPS) in outer layer. The characteristic of LB- and TB-EPS were different, which would affect their adsorption of antibiotics. However, the adsorption process of antibiotics on LB- and TB-EPS was still unclear yet. Therefore, in this work, the roles of LB-EPS and TB-EPS in adsorption of a typical antibiotic-trimethoprim (TMP) at environmentally relevant concentration (25.0 µg/L) were investigated. The results showed the content of TB-EPS was higher than that of LB-EPS, which was 17.08 and 10.36 mg/g VSS, respectively. The adsorption capacity of raw, LB-EPS extracted and both LB- and TB-EPS extracted activated sludges for TMP were 5.31, 4.65 and 9.51 µg/g VSS, respectively, which indicated LB-EPS had positive effect on TMP removal, while TB-EPS had negative effect. The adsorption process can be well described by a pseudo-second-order kinetic model (R2 > 0.980). The ratio of different functional groups was calculated and the CO and C-O bond might be responsible for the adsorption capacity difference between LB- and TB-EPS. The fluorescence quenching results indicated that tryptophan protein-like substances in LB-EPS provided more binding sites (n = 0.36) than that of tryptophan amino acid in TB-EPS (n = 0.1). Furthermore, the extend DLVO results also demonstrated that LB-EPS promoted the adsorption of TMP, while TB-EPS inhibited the process. We hope the results of this study were helpful for understanding the fate of antibiotics in wastewater treatment systems.

Uncovering neural pathways underlying bulimia nervosa: resting-state neural connectivity disruptions correlate with maladaptive eating behaviors.

PURPOSE: Bulimia nervosa (BN) is characterized by recurrent binge-eating episodes and inappropriate compensatory behaviors. This study investigated alterations in resting-state surface-based neural activity in BN patients and explored correlations between brain activity and eating behavior. METHODS: A total of 26 BN patients and 28 healthy controls were enrolled. Indirect measurement of cerebral cortical activity and functional connectivity (FC) analyses were performed in Surfstat. A principal component analysis (PCA) model was used to capture the commonalities within the behavioral questionnaires from the BN group. RESULTS: Compared with the healthy control group, the BN group showed decreased surface-based two-dimensional regional homogeneity in the right superior parietal lobule (SPL). Additionally, the BN group showed decreased FC between the right SPL and the bilateral lingual gyrus and increased FC between the right SPL and the left caudate nucleus and right putamen. In the FC-behavior association analysis, the second principal component (PC2) was negatively correlated with FC between the right SPL and the left caudate nucleus. The third principal component (PC3) was negatively correlated with FC between the right SPL and the left lingual gyrus and positively correlated with FC between the right SPL and the right lingual gyrus. CONCLUSION: We revealed that the right SPL undergoes reorganization with respect to specific brain regions at the whole-brain level in BN. In addition, our results suggest a correlation between brain reorganization and maladaptive eating behavior. These findings may provide useful information to better understand the neural mechanisms of BN. LEVEL OF EVIDENCE: V, descriptive study.

May Measurement Month 2019: an analysis of blood pressure screening results from China.

To report the blood pressure (BP) data obtained in the May Measurement Month (MMM) 2019 in China. Study participants were recruited if ≥18 years of age and had ideally not had their BP measured for ≥1 year. BP was measured three times consecutively with a 1-min interval in the sitting position, using a validated electronic BP monitor. Trained volunteer investigators administered a questionnaire to collect information on lifestyle, medical history, and use of medications. The measurement was performed in 238 387 participants in 250 sites across 31 China provinces. The majority of screening took place in hospitals or clinics (78.7%), with 17.1% in outdoor public areas and 4.2% in other settings. The study participants included 127 853 women (53.6%) and had a mean (±SD) age of 48.9 ± 16.2 years. The mean (of readings two and three) systolic/diastolic BP was 121.8/73.8 mmHg. In all hypertensive patients (n = 66 181, 27.8%), the awareness, treatment, and control rates of hypertension were 51.5%, 48.4%, and 29.1%, respectively. Linear regression models showed differences in systolic and diastolic BP according to sex and age and several other major characteristics, such as previous stroke, myocardial infarction, and diabetes mellitus, antihypertensive medication use and known hypertension, previous hypertension in pregnancy and current pregnancy, alcohol intake and current smoking, and body mass index. The MMM 2019 campaign has been successful in measuring BP in a large member of participants in China.

Spatial Heterojunction in Nanostructured TiO2 and Its Cascade Effect for Efficient Photocatalysis.

A highly efficient photoenergy conversion is strongly dependent on the cumulative cascade efficiency of the photogenerated carriers. Spatial heterojunctions are critical to directed charge transfer and, thus, attractive but still a challenge. Here, a spatially ternary titanium-defected TiO2@carbon quantum dots@reduced graphene oxide (denoted as VTi@CQDs@rGO) in one system is shown to demonstrate a cascade effect of charges and significant performances regarding the photocurrent, the apparent quantum yield, and photocatalysis such as H2 production from water splitting and CO2 reduction. A key aspect in the construction is the technologically irrational junction of Ti-vacancies and nanocarbons for the spatially inside-out heterojunction. The new "spatial heterojunctions" concept, characteristics, mechanism, and extension are proposed at an atomic-/nanoscale to clarify the generation of rational heterojunctions as well as the cascade electron transfer.

Transcriptomic analysis reveals recovery strategies in strawberry roots after using a soil amendment in continuous cropping soil.

BACKGROUND: In strawberry cultivation, continuous cropping (CC) obstacles seriously threaten production. A patented soil amendment (SA) can effectively relieve the CC obstacles to strawberry cultivation, but knowledge of the recovery mechanisms underlying this phenomenon is limited. RESULTS: In this study, transcriptomic profiling of strawberry roots in soil with and without the SA was conducted using RNA-Seq technology to reveal gene expression changes in response to SA treatment. In total, 188 differentially expressed genes (DEGs), including 144 upregulated and 44 downregulated DEGs, were identified. SA treatment resulted in genotype-dependent responses, and the response pattern, including an overall increase in the expression of nutrient transport genes and a decrease in the expression of defense response genes, may be a possible mechanism underlying recovery strategies in strawberry roots after the application of the SA to CC soil. We also found that 9 Hsp genes involved in plant defense pathways were all downregulated in the SA-treated roots. CONCLUSIONS: This research indicated that strawberry plants reallocated defense resources to development when SA treatment alleviated the stress caused by a CC soil environment. The present study provides an opportunity to reveal the fundamental mechanisms of the tradeoff between growth and defense in strawberry.

Deactivation of the kinase IKK by CUEDC2 through recruitment of the phosphatase PP1.

Despite rapid progress in elucidating the molecular mechanisms of activation of the kinase IKK, the processes that regulate IKK deactivation are still unknown. Here we demonstrate that CUE domain-containing 2 (CUEDC2) interacted with IKKalpha and IKKbeta and repressed activation of the transcription factor NF-kappaB by decreasing phosphorylation and activation of IKK. Notably, CUEDC2 also interacted with GADD34, a regulatory subunit of protein phosphatase 1 (PP1). We found that IKK, CUEDC2 and PP1 existed in a complex and that IKK was released from the complex in response to inflammatory stimuli such as tumor necrosis factor. CUEDC2 deactivated IKK by recruiting PP1 to the complex. Therefore, CUEDC2 acts as an adaptor protein to target IKK for dephosphorylation and inactivation by recruiting PP1.

Simultaneous determination and determination of sildenafil and its active metabolite in human plasma using LC-MS/MS method.

A sensitive and selective high-performance liquid chromatography-tandam mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma. Sildenafil-d8 was used as an internal standard. The analytes were extracted by precipitation extraction and chromatographed on a C18 column using mobile phase A of water (containing 0.1% formic acid) and mobile phase B of acetonitrile (containing 0.1% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 475.4 → m/z 283.3 for sildenafil, m/z 461.4 → m/z 283.2 for N-desmethyl sildenafil and m/z 483.3 → m/z 108.1 for IS in positive ionization mode. The calibration curve was established over the range of 2.00-1,000 ng/ml and the correlation coefficient was >0.99. The intra-day and inter-day relative standard deviations were <6.5% for sildenafil and 6.3% for N-desmethyl sildenafil respectively. Accuracy determinaed at four concentrations was 86.50-105.67% for sildenafil and 96.83-114.40% for N-desmethyl sildenafil. This method was successfully applied to a pharmacokinetic description of sildenafil and the effect of food intake on the pharmacokinetics of sildenafil was also demonstrated in healthy Chinese volunteers.

Distinctive Signatures of the Spin- and Momentum-Forbidden Dark Exciton States in the Photoluminescence of Strained WSe2 Monolayers under Thermalization.

With both spin and valley degrees of freedom, the low-lying excitonic spectra of photoexcited transition-metal dichalcogenide monolayers (TMDC-MLs) are featured by rich fine structures, comprising the intravalley bright exciton states as well as various intra- and intervalley dark ones. The latter states can be classified as those of the spin- and momentum-forbidden dark excitons according to the violated optical selection rules. Because of their optical invisibility, these two types of the dark states are in principle hardly observed and even distinguished in conventional spectroscopies although their impacts on the optical and dynamical properties of TMDC-MLs have been well noticed. In this Letter, we present a theoretical and computational investigation of the exciton fine structures and the temperature-dependent photoluminescence spectra of strained tungsten diselenide monolayers (WSe2-MLs) where the intravalley spin-forbidden dark exciton lies in the lowest exciton states and other momentum-forbidden states are in the higher energies that are tunable by external stress. The numerical computations are carried out by solving the Bethe-Salpeter equation for an exciton in a WSe2-ML under the stress-control in the tight-binding scheme established from the first principle computation in the density functional theory. According to the numerical computation and supportive model analysis, we reveal the distinctive signatures of the spin- and momentum-forbidden exciton states of strained WSe2-MLs in the temperature-dependent photoluminescences and present the guiding principle to infer the relative energetic locations of the two types of dark excitons.

A pilot-scale three-dimensional electrochemical reactor combined with anaerobic-anoxic-oxic system for advanced treatment of coking wastewater.

Coking wastewater is highly concentrated and extremely toxic, greatly challenging the treatment technologies. Conventional biological technology such as anaerobic-anoxic-oxic (A2O) system is inefficient, since various biological reactions are inhibited by toxicants in coking wastewater. In this work, a pilot-scale three-dimensional electrochemical reactor (3DER) is integrated into the A2O system as a pretreatment unit to improve the treatment efficiency of coking wastewater. The results indicate that 3DER pretreatment increased the biodegradability of coking wastewater, promoting the degradation of coking wastewater in A2O system. The integrated 3DER-A2O system can remove 94.4% of COD and 76.2% of TN from coking wastewater, and the energy consumption was only 0.22 kWh/kg COD and 4.69 kWh/kg TN. The components of coking wastewater were significantly simplified and the acute toxicity was reduced from 99% to 12% after the treatment. The integrated 3DER-A2O system provides a new solution for coking wastewater treatment, showing a promising application potential.

May Measurement Month 2017: an analysis of blood pressure screening results in China-East Asia.

Elevated blood pressure (BP) is a growing burden worldwide, leading to over 10 million deaths each year. May Measurement Month (MMM) is a global initiative aimed at raising awareness of high BP and to act as a temporary solution to the lack of screening programmes worldwide. In China, several hypertension screening programmes are undertaken in the elderly in the community and in youths at university entrance and graduation. However, most people, especially the middle-aged working population, do not often have their BP measured. The current awareness (46.9%), treatment (40.7%), and control rates (15.3%) of hypertension remain low, while the proportion of screenees with hypertension is high in adult Chinese (23.2%). An opportunistic cross-sectional survey of volunteers aged ≥18 years was carried out in May 2017. Blood pressure measurement, the definition of hypertension and statistical analysis followed the standard MMM protocol. About 125 236 individuals were screened. After multiple imputation, with 124 623 as denominator, 32 089 (25.7%) had hypertension. Of the 103 981 individuals not on antihypertensive medication, 11 447 (11.0%) were hypertensive. Of the 20 547 individuals on antihypertensive medication, 7392 (36.0%) had uncontrolled BP (≥140/90 mmHg). An opportunistic screening may effectively identify those with high BP regardless of the use of antihypertensive medication and shows similar information on BP as a survey in a randomly selected population sample.

An improved LC-MS/MS method for determination of cinacalcet in human plasma and its application to the evaluation of food intake effect on the pharmacokinetics of cinacalcet in healthy volunteers.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cinacalcet in human plasma was developed and validated. This assay was based on liquid-liquid extraction and cinacalcet-d4 was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase A of water (containing 0.1% formic acid) and the mobile phase B of acetonitrile-water (95:5, v/v) (containing 0.2% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 358.2 → m/z 155.2 for cinacalcet and m/z 362.3 → m/z 155.0 for IS at positive ionization mode. The calibration curve was established over the range 0.05-20.0 ng/mL and the correlation coefficient was >0.99. The intra- and inter-day relative standard deviations were <5.8%. Accuracy determined at four concentrations ranged between 96.0 and 106.0%. This method was successfully applied to a pharmacokinetic description of oral dose of cinacalcet and the significant effect of food intake on the pharmacokinetics of cinacalcet was first demonstrated in Chinese healthy volunteers.

Signaling protein signature predicts clinical outcome of non-small-cell lung cancer.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is characterized by abnormalities of numerous signaling proteins that play pivotal roles in cancer development and progression. Many of these proteins have been reported to be correlated with clinical outcomes of NSCLC. However, none of them could provide adequate accuracy of prognosis prediction in clinical application. METHODS: A total of 384 resected NSCLC specimens from two hospitals in Beijing (BJ) and Chongqing (CQ) were collected. Using immunohistochemistry (IHC) staining on stored formalin-fixed paraffin-embedded (FFPE) surgical samples, we examined the expression levels of 75 critical proteins on BJ samples. Random forest algorithm (RFA) and support vector machines (SVM) computation were applied to identify protein signatures on 2/3 randomly assigned BJ samples. The identified signatures were tested on the remaining BJ samples, and were further validated with CQ independent cohort. RESULTS: A 6-protein signature for adenocarcinoma (ADC) and a 5-protein signature for squamous cell carcinoma (SCC) were identified from training sets and tested in testing sets. In independent validation with CQ cohort, patients can also be divided into high- and low-risk groups with significantly different median overall survivals by Kaplan-Meier analysis, both in ADC (31 months vs. 87 months, HR 2.81; P <  0.001) and SCC patients (27 months vs. not reached, HR 9.97; P <  0.001). Cox regression analysis showed that both signatures are independent prognostic indicators and outperformed TNM staging (ADC: adjusted HR 3.07 vs. 2.43, SCC: adjusted HR 7.84 vs. 2.24). Particularly, we found that only the ADC patients in high-risk group significantly benefited from adjuvant chemotherapy (P = 0.018). CONCLUSIONS: Both ADC and SCC protein signatures could effectively stratify the prognosis of NSCLC patients, and may support patient selection for adjuvant chemotherapy.

Computational insights into the G-protein-biased activation and inactivation mechanisms of the µ opioid receptor.

The µ opioid receptor (OR), a member of the class A subfamily of G-protein coupled receptors (GPCRs), is a major target for the treatment of pain. G-protein biased µ-OR agonists promise to be developed as analgesics. Thus, TRV130, the first representative µ-OR ligand with G-protein bias, has entered into phase III clinical trials. To identify the detailed G-protein-biased activation and inactivation mechanisms of the µ-OR, we constructed five µ-OR systems that were in complexes with the G-protein-biased agonists TRV130 and BU72, the antagonists ß-FNA and naltrexone, as well as the free receptor. We performed a series of conventional molecular dynamics simulations and analyses of G-protein-biased activation and inactivation mechanisms of µ-OR. Our results, together with previously reported mutation results, revealed the operating mode of the activation switch composed of residues W6.48 and Y7.43 (Ballesteros/Weinstein numbering), the activity of which was responsible for down- and up-regulation, respectively, of the ß-arrestin signaling, which in turn affected G-protein-biased activation of µ-OR. TRV130 was found to stabilize W6.48 by interacting with Y7.43. In addition, we obtained useful information regarding µ-OR-biased activation, such as strong stabilization of W7.35 through a hydrophobic ring interaction in the TRV130 system. These findings may facilitate understanding of µ-OR biased activation and the design of new biased ligands for GPCRs.

Computational insights into the subtype selectivity and "message-address-efficacy" mechanisms of opioid receptors through JDTic binding and unbinding.

In drug design and discovery, binding affinity and selectivity are two basic properties of a drug candidate. Opioid receptors (ORs) are the main targets of strong analgesics. Like some other class A members of G-protein-coupled receptors (GPCRs), ORs exhibit complex selectivity on their ligands. The diversity of binding activity and selectivity among opioids has deeply attracted researchers for a long time. To investigate the subtype selectivity of µ, δ and κ ORs in detail, using the κ-selective antagonist JDTic as a probe, we performed a series of computational simulations, including molecular dynamics and metadynamics, on JDTic-µ/δ/κ-OR complexes. From the simulations, we found that the decisive factor of JDTic selectivity on the µ-subtype was the 2.63 position, which affected the efficacy of JDTic through changing the dynamics of the Q2.60 residue. In addition to the 2.63-position residue, the 7.35 position was the other crucial aspect of JDTic selectivity for the δ-subtype. Based on the results, we suggest a new concept, the "message-address-efficacy" hypothesis, to explain the relationships among the affinity, selectivity and function between ORs and opioids. Thus, all the detailed dynamics of JDTic-bound ORs might be helpful to deeply understand the subtype selectivity and binding mechanisms of other GPCRs.