Case report: Safety and efficacy of synergistic treatment using selinexor and azacitidine in patients with atypical chronic myeloid leukemia with resistance to decitabine.

Background: Atypical chronic myeloid leukemia (aCML) is a BCR::ABL1 negative myelodysplastic/myeloproliferative neoplasm with poor overall survival. Some patients can be treated by allogeneic hematopoietic stem cell transplantation (allo-HSCT) from suitable donors. The effectiveness of decitabine or azacitidine (AZA) has recently been reported; however, their combined efficacy with selinexor has not yet been reported. Case description: In this study, we report the case of a patient with aCML who was successfully treated with selinexor combined with AZA. A 67-year-old man with a history of gastric mucosa-associated lymphoid tissue (MALT) lymphoma was admitted to the hospital with fatigue and emaciation. He was diagnosed with aCML and no longer responded to decitabine treatment after undergoing seven cycles. The patient was subsequently administered hydroxyurea (HU), selinexor, and AZA. After four courses of combination therapy, his blood cell counts improved; he no longer required transfusions and was able to discontinue HU. The patient continued receiving selinexor and AZA without severe complications. This case is the first to show that combinatorial selinexor and AZA therapy can effectively treat aCML. Conclusion: Our case sheds light on the importance of selinexor and AZA combined therapy in the exploration of new treatment strategies for aCML. Moreover, this treatment approach offers the possibility of bridging with allo-HSCT.

3D density imaging using gravity and gravity gradient in the wavenumber domain and its application in the Decorah.

Density imaging is a method that uses the inversion of the gravity and gravity gradient spectra in the wavenumber domain to create accurate 3D reconstructions of subsurface density distributions. This approach offers computational efficiency and rapid calculations. This research used preliminary inversions to examine the spectral characteristics of gravity and gravity gradient anomalies, as well as the resulting models, were scrutinized through preliminary inversions. 3D density imaging of gravity and gravity gradient was performed in the wavenumber domain using depth weighting on both noise-added and theoretical data, producing a density model that was consistent with the theoretical one. The technique was then used in the Decorah region of the United States, where 3D density imaging was performed and an examination of the properties of gravity and gravity gradient anomalies was conducted. The results showed where high-density Decorah complexes, low-density siliceous intrusive rocks, and high-density intrusive rock masses, were the distributed within the surrounding rock. Each of these provided comprehensive insights into the intrusive pathways to the rock mass. Thus, the appropriateness and effectiveness of the density imaging method were confirmed, supporting a deeper understanding of the structural division and geological evolution in the region.

Causal relationship between gut microbiota and immune thrombocytopenia: a Mendelian randomization study of two samples.

Background: Some observational studies have shown that immune thrombocytopenia (ITP) is highly associated with the alteration-composition of gut microbiota. However, the causality of gut microbiota on ITP has not yet been determined. Methods: Based on accessible summary statistics of the genome-wide union, the latent connection between ITP and gut microbiota was estimated using bi-directional Mendelian randomization (MR) and multivariable MR (MVMR) analyses. Inverse variance weighted (IVW), weighted median analyses, and MR-Egger regression methods were performed to examine the causal correlation between ITP and the gut microbiota. Several sensitivity analyses verified the MR results. The strength of causal relationships was evaluated using the MR-Steiger test. MVMR analysis was undertaken to test the independent causal effect. MR analyses of reverse direction were made to exclude the potential of reverse correlations. Finally, GO enrichment analyses were carried out to explore the biological functions. Results: After FDR adjustment, two microbial taxa were identified to be causally associated with ITP (PFDR < 0.10), namely Alcaligenaceae (PFDR = 7.31 × 10-2) and Methanobacteriaceae (PFDR = 7.31 × 10-2). In addition, eight microbial taxa were considered as potentially causal features under the nominal significance (P < 0.05): Actinobacteria, Lachnospiraceae, Methanobacteria, Bacillales, Methanobacteriales, Coprococcus2, Gordonibacter, and Veillonella. According to the reverse-direction MR study findings, the gut microbiota was not significantly affected by ITP. There was no discernible horizontal pleiotropy or instrument heterogeneity. Finally, GO enrichment analyses showed how the identified microbial taxa participate in ITP through their underlying biological mechanisms. Conclusion: Several microbial taxa were discovered to be causally linked to ITP in this MR investigation. The findings improve our understanding of the gut microbiome in the risk of ITP.

[Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification].

Antibodies play an essential role in cancer diagnosis and treatment because of the specificity for target biomolecules and reduction of side effects. However, antibodies separation and purification still face some challenges. Antibody elution from columns using a low-pH aqueous solution leads to aggregation or loss of activity of the antibody drugs. In this paper, a block copolymer-based temperature-responsive affinity chromatography (TRAC) stationary phase, SiO2-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopropylacrylamide-b-4-vinylpyridine) (P[NIPAM-b-4VP]) as the space arms and 4-mercaptoethyl pyridine (MEP) as the ligand was prepared for antibody separation. The TRAC column was tested using bovine serum albumin (BSA) and γ-globulin as model proteins, and the effects of salt concentration in the mobile phase and temperature on their separation were studied in detail. At 40 ℃, the TRAC stationary phase only selectively retained γ-globulin due to the specific affinity interaction between antibodies and the ligand MEP. At 5 ℃, γ-globulin can be eluted from the column with a mass recovery of 92.7% using a Tris-HCl buffer (pH 8.0) solution containing 0.6 mol/L NaCl. The adsorption capacity of γ-globulin on this stationary phase was (71.5 ±2.1) mg/g (n=3), which was twice that of a traditional temperature-sensitive affinity chromatography stationary phase SiO2-PNIPAM-MEP. The stationary phase was also used to separate and purify immunoglobulin (IgG) in human serum in one step by altering the temperature and ion strength of the mobile phase, resulting in a purity of 97.4%±0.7%. Thus, this new technology has specific selectivity for antibodies, as well as mild and green elution conditions, ultimately resolving the problem of traditional affinity chromatography using acid elution, which can lead to the antibodies aggregation/inactivation. This technology has great application potential for the industrial production of antibody drugs.

Anti-Oomycete Effect and Mechanism of Salicylic Acid on Phytophthora infestans.

Pathogenic oomycetes infect a wide variety of organisms, including plants, animals, and humans, and cause massive economic losses in global agriculture, aquaculture, and human health. Salicylic acid (SA), an endogenous phytohormone, is regarded as an inducer of plant immunity. Here, the potato late blight pathogen Phytophthora infestans was used as a model system to uncover the inhibitory mechanisms of SA on pathogenic oomycetes. In this research, SA significantly inhibited the mycelial growth, sporulation, sporangium germination, and virulence of P. infestans. Inhibition was closely related to enhanced autophagy, suppression of translation initiation, and ribosomal biogenesis in P. infestans, as shown by multiomics analysis (transcriptomics, proteomics, and phosphorylated proteomics). Monodansylcadaverine (MDC) staining and Western blotting analysis showed that SA promoted autophagy in P. infestans by probably targeting the TOR signaling pathway. These observations suggest that SA has the potential to control late blight caused by P. infestans.

Decision-making experiences related to mastectomy: A descriptive qualitative study.

AIM: To obtain an in-depth understanding of women's decision-making experiences related to mastectomy. DESIGN: A descriptive qualitative interview study. METHODS: Individual semi-structured interviews were conducted face-to-face with 27 Chinese women with breast cancer who underwent mastectomy at two tertiary hospitals in mainland China between September 2020 and December 2021 after obtaining the appropriate ethical approvals. Interviews were conducted in Mandarin. Data were analysed using inductive content analysis. RESULTS: Mean age of participants was 48 years (range 31-70). Most participants had low education, low monthly family income, had a partner and health insurance, had been diagnosed with early breast cancer, and had not undergone reconstructive surgery. Six categories related to decision-making experiences emerged: (1) Emotions affecting decision-making, (2) Information seeking for decision-making, (3) Beliefs about mastectomy and the breast, (4) Participation in decision-making, (5) People who influence decision-making, and (6) Post-decision reflection. Participants did not mention the role of nurses in their decision-making process for mastectomy. CONCLUSIONS: This study adds valuable insights into the limited evidence on women's experience with decision-making about mastectomy from a Chinese perspective, which is important given the continuing high prevalence of mastectomy in many regions. Future studies from other countries and ethnic groups are recommended to gain diverse knowledge. IMPACT: The findings of this study are useful for nurses and other healthcare professionals in the multidisciplinary team to better support women with breast cancer in their decision-making process regarding mastectomy. The findings could inform future interventions to support treatment decision-making and may be relevant to women living in similar socio-medical contexts to those in mainland China. REPORTING METHOD: The study was reported following the Standards for Reporting Qualitative Research checklist. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution.

Preparation and Characterization Evaluation of Poly(L-Glutamic Acid)-g-Methoxy Poly(Ethylene Glycol)/Combretastatin A4/BLZ945 Nanoparticles for Cervical Cancer Therapy.

Purpose: Cervical cancer (CC) is a highly vascularized tumor with abundant abnormal blood vessel, which could be targeted by therapeutic strategies. Poly(L-glutamic acid)-g-methoxy poly(ethylene glycol)/combretastatin A4 (CA4)/BLZ945 nanoparticles (CB-NPs) have shown great potential as nano vascular disrupting agents (VDAs) in the realm of synergistic cancer therapy. Methods: In this study, we investigated the nanocharacteristics of CB-NPs, focusing on active pharmaceutical ingredients (API), as well as lyophilized samples combining API with protective agents (PAs). The in vivo efficacy of final sample (API + PAs) was evaluated. Results: The assembled sphere of API with complex core and thin-shell structure was confirmed. PAs were found to significantly influence in vivo efficacy. Collaborative efforts between API and PAs, namely mannitol and lactose, resulted in the most promising lyophilized sample, ie, the final sample (FS2) for CC therapy. Impressively, FS2 demonstrated an exceptional 100% cure rate on the CC U14-bearing mice model. Conclusion: FS2 has provided significant insights for cervical cancer therapy. It is also crucial to develop a comprehensive evaluation strategy for the formulation of nanomedicine, which has the potential to serve as a guideline for future clinical trials.

Comparison of tandospirone and escitalopram as a symptomatic treatment in Multiple System Atrophy-cerebellar ataxia: An open-label, non-controlled, 4 weeks observational study.

BACKGROUND: Multiple system atrophy (MSA) is a neurodegenerative disorder characterized by autonomic failure and motor dysfunction in parkinsonism and/or cerebellar ataxia. Patients with MSA usually present with depression and anxiety symptoms. This observational study of patients with MSA-cerebellar subtype (MSA-C) with subthreshold depression/anxiety symptoms aimed to compare the efficacy of escitalopram oxalate (an antidepressant drug) and tandospirone citrate (an anxiolytic drug). METHODS: Fifty-six MSA-C patients were included, with 28 patients in each treatment group. One group received escitalopram oxalate 10 mg/day and the other group received tandospirone citrate 30 mg/day. The patients were evaluated at baseline and after 4 weeks. Several psychiatric and neurological tests were performed, including the Hamilton Anxiety Rating Scale (HAMA), Hamilton Depression Rating Scale (HAMD), Scale for the Assessment and Rating of Ataxia (SARA), and the Scale for Outcomes in Parkinson's Disease for Autonomic Symptoms (SCOPA-AUT). Furthermore, post-void residual urine volume (PVR) and blood pressure were measured. RESULTS: There was a more substantial reduction in the HAMA/HAMD, scores of stance, finger tracking, and finger nose test in the SARA, and PVR in the tandospirone group. There was a more substantial reduction in scores of dysuria, light-headed when standing up, syncope and hyperhidrosis in the SCOPA-AUT in the escitalopram group (p's < 0.05). CONCLUSIONS: Tandospirone citrate was more effective in improving depression/anxiety and some cerebellar ataxia symptoms, whereas escitalopram was more effective in improving some autonomic symptoms in MSA-C patients over a short-term period in an open-label observational study without a control group. Further research is needed to evaluate the long-term effects of tandospirone and escitalopram in MSA-C in long-term placebo controlled trials.

Hormone effects of eighteen bisphenol analogues and their effects on cellular homeostasis and the typical signal pathways.

Through the transfer chain of surroundings from feed to the farmed-animals and ultimately the corresponding livestock and poultry products, people are exposed to large amounts of bisphenol analogues (BPs), such as rational emissions from manufacturing plants, feed packaging bags and food packaging contact. Some BPs have been reported to show certain toxicological effects, especially, estrogen and endocrine disrupting effect. With the increasing application of BPs, the problem is becoming more and more serious. We systematically studied the hormonal effects of 18 BPs and their effects on cell homeostasis and classical signaling pathways by using classical E-SCREEN assay, fluorescent probes and western blotting. The results confirmed the estrogen-like effect of 13 BPs and 6 BPs obtained high docking scores (Scores < -9.0) for the three receptors simultaneously with the main interactions of hydrophobic, hydrogen and π-stacking of T-type bonds. BPAP regulates cells via apoptosis and steroid signaling pathway by intracellular ROS and mitochondrial followed the caspase pathway. BPE and BPS were involved in the classical NF-κB and Hippo signaling pathways. All data provides scientific basis for the safety risk assessment of endocrine disrupting and cellular homeostasis evaluation of BPs as chronic environmental pollution.

TMM: A comprehensive CAD system for hepatic fibrosis 5-grade METAVIR staging based on liver MRI.

BACKGROUND: Precise staging of hepatic fibrosis with MRI is necessary as it can assist precision medicine. Computer aided diagnosis (CAD) system with distinguishing radiomics features and radiologists domain knowledge is expected to obtain 5-grade meta-analysis of histological data in viral hepatitis (METAVIR) staging. PURPOSE: This study aims to obtain the precise staging of hepatic fibrosis based on MRI as it predicts the risk of future liver-related morbidity and the need for treatment, monitoring and surveillance. Based on METAVIR score, fibrosis can be divided into five stages. Most previous researches focus on binary classification, such as cirrhosis versus non-cirrhosis, early versus advanced fibrosis, and substantial fibrosis or not. In this paper, a comprehensive CAD system TMM is proposed to precisely class hepatic fibrosis into five stages for precision medicine instead of the common binary classification. METHODS: We propose a novel hepatic fibrosis staging CAD system TMM which includes three modules, Two-level Image Statistical Radiomics Feature (TISRF), Monotonic Error Correcting Output Codes (MECOC) and Monotone Multiclassification with Deep Forest (MMDF). TISRF extracts radiomics features for distinguishing different hepatic fibrosis stages. MECOC is proposed to encode monotonic multiclass by making full use of the progressive severity of hepatic fibrosis and increase the fault tolerance and error correction ability. MMDF combines multiple Deep Forest network to ensure the final five-class classification, which can achieve more precise classification than the common binary classification. The performance of the proposed hepatic fibrosis CAD system is tested on the hepatic data collected from our rabbits models of fibrosis. RESULTS: A total of 140 regions of interest (ROI) are selected from MRI T1W of liver fibrosis models in 35 rabbits with F0(n = 16), F1(n = 28), F2(n = 29), F3(n = 44) and F4(n = 23). The performance is evaluated by five-fold cross-validation. TMM can achieve the highest total accuracy of 72.14% for five fibrosis stages compared with other popular classifications. To make a comprehensive comparison, a binary classification experiment have been carried out. CONCLUSIONS: T1WI can obtain precise staging of hepatic fibrosis with the help of comprehensive CAD including radiomics features extraction inspired by radiologists, monotonic multiclass according to the severity of hepatic fibrosis, and deep learning classification.

Decisional Conflict, Patient Involvement, and the Associated Psychological Factors Relating to Mastectomy Decisions Among Women With Breast Cancer: A Cross-sectional Study.

BACKGROUND: Most women with breast cancer in China have received a mastectomy despite emerging breast-conserving alternatives. Their decision-making relating to mastectomy is unclear. OBJECTIVE: To investigate decisional conflict, women's involvement, and psychological factors relating to mastectomy decisions. METHODS: Women with breast cancer 18 years and older who had a mastectomy were recruited from 2 hospitals in China. A conceptual framework adapted from the Ottawa Decisional Support Framework was used to guide this study. Data were collected using the 16-item Decisional Conflict Scale, the 9-item Shared Decision-Making Questionnaire, and a 19-item psychological factor list. RESULTS: A total of 304 women participated. Overall, they reported a low-level conflict and high-level involvement. "Cancer not return" was rated as the most important psychological factor influencing mastectomy decisions. Lower decisional conflict was predicted by higher involvement. Higher involvement was predicted by younger age and increased family income. CONCLUSIONS: This study is the first to demonstrate decisional conflict, involvement, and the associated factors specifically in Chinese women undergoing mastectomy. Results determined the importance of several psychological factors influencing mastectomy decisions. Future qualitative studies are required to deepen understanding of women's decision-making experiences regarding this surgery. IMPLICATIONS FOR PRACTICE: Nurses need to provide support to Chinese women in making treatment decisions, especially for older women, and those who are economically disadvantaged. Measures are needed to promote their involvement and improve their understanding of breast cancer and its treatments, which may help reduce decisional conflict, and potentially improve their satisfaction with treatment and quality of life.

Depth distribution of Moho model and tectonic patterns in South China Sea and adjacent areas.

This study aims to investigate the depth distribution of Mohorovicic discontinuity (Moho) and its relationship with the tectonic pattern of the South China Sea and adjacent areas. To achieve this, the spatial characteristics of the full tensor gravity gradient data are analyzed to identify 17 large and deep faults and to divide the study area into 9 tectonic units with distinct geological structures. Using a three-dimensional (3D) interface inversion method, the Moho depth is determined, constrained by the Moho depth information obtained from sonar-buoy detection and submarine seismograph detection profiles. By analyzing the relationship between the distribution characteristics of Moho and tectonic units, the study summarizes the trend, relief, gradient of Moho, and crustal properties in the study area. Additionally, the seismically constrained Moho undulation combined with the gravity data, gravity gradient anomalies and unconstrained 3D correlation imaging are employed to study the crustal structure of the South China Sea, investigate the vertical and horizontal changes of the crustal structure, and reveal the large-scale crustal and regional structure of the South China Sea. Through the coupling analysis of shallow and deep structures, the study reveals that the gravity gradient anomalies and 3D correlation imaging are consistent with the variations of the Moho depth, indicating the presence of a trench-island arc-back arc basin system and the distribution of continental crust, oceanic crust, and transitional crust in the South China Sea.

Application of role reversal and standardized patient simulation (SPS) in the training of new nurses.

PURPOSE: To explore the effect of role reversal and standardized patient simulation on the training of new nurses. METHOD: This study was conducted in a territory hospital in China between August 2021 and August 2022. The selected staff were all newly recruited and trained nurses, with a total of 58 cases. This study is a randomised controlled trial. The selected nurses were randomly divided into two groups. One group of 29 nurses (the control group) received routine training and assessment; the other group (the experimental group) was given role reversal combined with a standardized vertebral patient training examination. The implementation effects of different training and assessment methods were compared and analysed. RESULTS: Before the training, the core competence scores of nurses in the two groups were lower, and there was no significant data difference (P > 0.05). After training, the core competence scores of nurses were improved, and the score of nurses in the experimental group was 165.49 ± 22.34. The difference was statistically significant when compared with the score of nurses in the control group (P < 0.05), indicating that nurses in the experimental group had better abilities. At the same time, the satisfaction of the two groups of nurses with the training was 96.55% (experimental group) and 75.86% (control group), and the difference in data was significant (P < 0.05). The satisfaction of the experimental group of nurses was higher, and the training effect was better. CONCLUSION: In the training of new nurses, the combined application of role interchange and standardized patient training and assessment methods has significant effects, which can improve the core competency of nurses and improve the training satisfaction of nurses, which is significant.

Biomimetic Synthesis of Organic Molecular-Doped Fluorescent Guanine Crystals with Pearlescence.

Biomimetic synthesis of guanine crystals has been focused on in the last years. However, multi-functional guanine crystals occluded with fluorescent molecules have not been realized in the laboratory. Here, the controllable synthesis of guanine crystal microplatelets with fluorescence and pearlescence was achieved for the first time by incorporating Nile red (NR) or fluorescein isothiocyanate (FITC) molecules into guanine crystals. The synthesized fluorescent guanine crystals are pure ß-phase anhydrous guanine single crystals. Aqueous suspensions with NR- and FITC-doped guanine crystals exhibit distinct pearlescence. The fluorescence intensities of NR and FITC were greatly enhanced after being doped into guanine crystals due to the inhibition of aggregation-caused quenching. Moreover, films composed of fluorescent guanine microplatelets exhibit high diffuse reflection intensity (70 %). This work provides a strategy to synthesize multifunctional materials composed of organic crystals occluded with dyes.

Mutagenicity of PM2.5 and Ethnic Susceptibility in Chengdu-Chongqing Economic Circle, China.

PM2.5 seriously endangers human health, and its mutagenicity is considered an important pathogenic mechanism. However, the mutagenicity of PM2.5 is mainly determined by traditional bioassays, which are limited in the large-scale identification of mutation sites. Single nucleoside polymorphisms (SNPs) can be used for the large-scale analysis of DNA mutation sites but have not yet been used on the mutagenicity of PM2.5. The Chengdu-Chongqing Economic Circle is one of China's four major economic circles and five major urban agglomerations, and the relationship between the mutagenicity of PM2.5 and ethnic susceptibility in this circle remains unclear. In this study, the representative samples are PM2.5 from Chengdu in summer (CDSUM), Chengdu in winter (CDWIN), Chongqing in summer (CQSUM) and Chongqing in winter (CQWIN) respectively. PM2.5 from CDWIN, CDSUM and CQSUM induce the highest levels of mutation in the regions of exon/5'Utr, upstream/splice site and downstream/3'Utr respectively. PM2.5 from CQWIN, CDWIN and CDSUM induce the highest ratio of missense, nonsense and synonymous mutation respectively. PM2.5 from CQWIN and CDWIN induce the highest transition and transversion mutations respectively. The ability of PM2.5 from the four groups to induce disruptive mutation effects is similar. For ethnic susceptibility, PM2.5 in this economic circle is more likely to induce DNA mutation in Chinese Dai from Xishuangbanna among Chinese ethnic groups. PM2.5 from CDSUM, CDWIN, CQSUM and CQWIN may particularly tend to induce Southern Han Chinese, Dai in Xishuangbanna, Dai in Xishuangbanna and Southern Han Chinese respectively. These findings may assist in the development of a new method for analyzing the mutagenicity of PM2.5. Moreover, this study not only promotes attention to ethnic susceptibility to PM2.5, but also introduces public protection policies for the susceptible population.

Root and root canal morphology of mandibular second permanent molars in the Gansu province population: A CBCT study.

To study the anatomical characteristics of the root and root canal system of the mandibular second molars in the population of Gansu province, and to provide theoretical and clinical references for improving the success rate of root canal therapy (RCT) of mandibular second molars. The number of roots and root canals, root canal type and pulp chamber height of mandibular second molars were determined by observing cone-beam computed tomography (CBCT) images of people living in Gansu. The most common type of mandibular second molars in the Gansu province population was a double root with three root canals (47.55%), followed by a C-shaped root (35.56%). There were more females than males with a C-shaped root (p < 0.05). The most common root canal subtype of the C-type root was C3 (13.91%). Most of the population (77.11%) had bilateral mandibular second molars with symmetrical root canal morphology. With an increase in age, the height of the pulp chamber decreased significantly. The incidence of root canal variation of the mandibular second molars is relatively high in the population of Gansu province. Preoperative examination with CBCT is essential for mandibular second molars that need RCT to avoid root canal treatment failure and decrease the occurrence of postoperative pain as much as possible.

Analgesic tolerance and cross-tolerance to the bifunctional opioid/neuropeptide FF receptors agonist EN-9 and µ-opioid receptor ligands at the supraspinal level in mice.

The chimeric peptide EN-9 was reported as a κ-opioid/neuropeptide FF receptors bifunctional agonist that modulated chronic pain with no tolerance. Many lines of evidence have shown that the effect of the κ-opioid receptor is mediated by not only its specific activation but also downstream events participation, especially interaction with the µ-opioid receptor pathway in antinociception and tolerance on most occasions. The present study investigated the acute and chronic cross-tolerance of EN-9 with µ-opioid receptor agonist EM-2, DAMGO, and morphine after intracerebroventricularly (i.c.v) injection in the mouse tail-flick test. In the acute tolerance test, EN-9 showed symmetrical acute cross-tolerance to DAMGO but no cross-tolerance to EM2. In the chronic tolerance test, EN-9 had no tolerance after eight days of repeated administration. However, EN-9 illustrated complete cross-tolerance to morphine and symmetrical cross-tolerance to EM2. In addition, inhibition of NPFF receptor could induce the tolerance development of EN-9. These findings indicated that supraspinal EN-9-induced antinociception contains additional components, which are mediated by the downstream µ-opioid receptor pathway both in acute and chronic treatment, whereas the subtypes of µ-opioid receptor or NPFF system pathway involved in antinociceptive effects induced by EN-9 are complex. Identifying the receptor mechanism could help design preferable bifunctional opioid compounds.