Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/β-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knock-down.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/β-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alle-viated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) is a serious and irreversible injury. Bone marrow-derived mesenchymal stem cells (MSCs) is considered to be a potential therapy for I/R injury due to the paracrine effects. High-mobility group box 1 (HMGB1) is a novel mediator in MSC and regulates the response of inflammation injury. Signal Transduction and Transcription Activator 3 (STAT3) is a critical transcription factor and important for release of paracrine factors. However, the relationship between HMGB1 and STAT3 in paracrine effect of MSC remains unknown. METHODS: In vitro, hypoxia/reoxygenation injury model was established by AnaeroPack System and examined by Annexin V flow cytometry, CCK8 assay and morphology observation. Detection of apoptotic proteins and protein expression of HMGB1 and STAT3 by Western blot. RESULTS: The conditioned medium of MSCs with or without LPS pretreatment was cocultured with H9C2 cells for 24 h before hypoxia treatment and MSC showed obvious cardiomyocytes protect role, as evidence by decreased apoptosis rate and improved cells viability, and LPS pretreated MSC exhibited better protect role than untreated MSC. However, such effect was abolished in HMGB1 deficiency group, silencing HMGB1 decreased the secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin growth factor (IGF), cell viability, and the expression of STAT3. Furthermore, STAT3 silence attenuated the protective effect of LPS in MSC. CONCLUSIONS These findings suggested that LPS improved MSC-mediated cardiomyocytes protection by HMGB1/STAT3 signaling.

Objective: To establish a predictive model for survival benefit of patients with intrahepatic cholangiocarcinoma (ICC) who received adjuvant chemotherapy after radical resection. Methods: The clinical and pathological data of 249 patients with ICC who underwent radical resection and adjuvant chemotherapy at 8 hospitals in China from January 2010 to December 2018 were retrospectively collected. There were 121 males and 128 females,with 88 cases>60 years old and 161 cases≤60 years old. Feature selection was performed by univariate and multivariate Cox regression analysis. Overall survival time and survival status were used as outcome indicators,then target clinical features were selected. Patients were stratified into high-risk group and low-risk group,survival differences between the two groups were analyzed. Using the selected clinical features, the traditional CoxPH model and deep learning DeepSurv survival prediction model were constructed, and the performance of the models were evaluated according to concordance index(C-index). Results: Portal vein invasion, carcinoembryonic antigen>5 μg/L,abnormal lymphocyte count, low grade tumor pathological differentiation and positive lymph nodes>0 were independent adverse prognostic factors for overall survival in 249 patients with adjuvant chemotherapy after radical resection (all P<0.05). The survival benefit of adjuvant chemotherapy in the high-risk group was significantly lower than that in the low-risk group (P<0.05). Using the above five features, the traditional CoxPH model and the deep learning DeepSurv survival prediction model were constructed. The C-index values of the training set were 0.687 and 0.770, and the C-index values of the test set were 0.606 and 0.763,respectively. Conclusion: Compared with the traditional Cox model, the DeepSurv model can more accurately predict the survival probability of patients with ICC undergoing adjuvant chemotherapy at a certain time point, and more accurately judge the survival benefit of adjuvant chemotherapy.

Objectives: To construct a nomogram for prediction of intrahepatic cholangiocarcinoma (ICC) lymph node metastasis based on inflammation-related markers,and to conduct its clinical verification. Methods: Clinical and pathological data of 858 ICC patients who underwent radical resection were retrospectively collected at 10 domestic tertiary hospitals in China from January 2010 to December 2018. Among the 508 patients who underwent lymph node dissection,207 cases had complete variable clinical data for constructing the nomogram,including 84 males,123 females,109 patients≥60 years old,98 patients<60 years old and 69 patients were pathologically diagnosed with positive lymph nodes after surgery. Receiver operating characteristic curve was drawn to calculate the accuracy of preoperative imaging examinations to determine lymph node status,and the difference in overall survival time was compared by Log-rank test. Partial regression squares and statistically significant preoperative variables were screened by backward stepwise regression analysis. R software was applied to construct a nomogram,clinical decision curve and clinical influence curve,and Bootstrap method was used for internal verification. Moreover,retrospectively collecting clinical information of 107 ICC patients with intraoperative lymph node dissection admitted to 9 tertiary hospitals in China from January 2019 to June 2021 was for external verification to verify the accuracy of the nomogram. 80 patients with complete clinical data but without lymph node dissection were divided into lymph node metastasis high-risk group and low-risk group according to the score of the nomogram among the 858 patients. Log-rank test was used to compare the overall survival of patients with or without lymph node metastasis diagnosed by pathology. Results: The area under the curve of preoperative imaging examinations for lymph node status assessment of 440 patients was 0.615,with a false negative rate of 62.8% (113/180) and a false positive rate of 14.2% (37/260). The median survival time of 207 patients used to construct a nomogram with positive or negative postoperative pathological lymph node metastases was 18.5 months and 27.1 months,respectively (P<0.05). Five variables related to lymph node metastasis were screened out by backward stepwise regression analysis,which were combined calculi,neutrophil/lymphocyte ratio,albumin,liver capsule invasion and systemic immune inflammation index,according to which a nomogram was constructed with concordance index(C-index) of 0.737 (95%CI: 0.667 to 0.806). The C-index of external verification was 0.674 (95%CI:0.569 to 0.779). The calibration prediction curve was in good agreement with the reference curve. The results of the clinical decision curve showed that when the risk threshold of high lymph node metastasis in the nomogram was set to about 0.32,the maximum net benefit could be obtained by 0.11,and the cost/benefit ratio was 1∶2. The results of clinical influence curve showed that when the risk threshold of high lymph node metastasis in the nomogram was set to about 0.6,the probability of correctly predicting lymph node metastasis could reach more than 90%. There was no significant difference in overall survival time between patients with high/low risk of lymph node metastasis assessed by the nomogram and those with pathologically confirmed lymph node metastasis or without lymph node metastasis (Log-rank test:P=0.082 and 0.510,respectively). Conclusion: The prediction accuracy of preoperative nomogram for ICC lymph node metastasis based on inflammation-related markers is satisfactory,which can be used as a supplementary method for preoperative diagnosis of lymph node metastasis and is helpful for clinicians to make personalized decision of lymph node dissection for patients with ICC.

By referring to the materials of ancient materia medica， prescriptions and medical books， combining with modern literature， this paper made textual research on the name， origin， producing area， quality evaluation， harvesting and processing methods of Ligustici Rhizoma et Radix in famous classical formulas， so as to provide a basis for the selection and use of Ligustici Rhizoma et Radix in the development of famous classical formulas. Through textual research， it can be seen that Ligustici Rhizoma et Radix is the correct name in all dynasties， but it still has many aliases. The main strain of Ligustici Rhizoma et Radix was Ligusticum sinense and L. jeholense， in addition， there are also L. tenuissimum， Sinodielsia yunnanensis， and Conioselinum vaginatum， L. sinense var. hupehense used in different regions. Since modern times， the textual examination of its scientific name is more complicated. Different foreign scholars have given many scientific names from their local observation， but most of them do not match the actual situation of Ligustici Rhizoma et Radix in ancient China. It is probably because they did not collect the original plants of Chinese Ligustici Rhizoma et Radix and conduct accurate identification. After the founding of the People's Republic of China， the L. sinense and L. jeholense was identified as the original plants through systematic investigation and arrangement of Ligustici Rhizoma et Radix. Since modern times， L. sinense is distributed in the upper and middle reaches of the Yellow River and the south of the region， a small amount of production in Sichuan， Hubei， Hunan and Jiangxi. L. jeholense is distributed in the middle and lower reaches of the Yellow River and the north of the region， mostly produced in Liaoning and Hebei. The ancient harvesting period of Ligustici Rhizoma et Radix was concentrated in January and February according to the lunar calendar with thick roots， dry， fragrant， heavy quality as the best. But now the collection of roots and rhizome more than concentrated in spring before seedling emergence and autumn when leaves were gone， and take the large， dry， yellow， strong， fragrant， less roots， residual short as the best. In the past dynasties， raw products were the main processing methods， and there were also roasting， grinding and so on. Based on the research results， it is suggested that the raw roots and rhizomes of L. sinense and L. jeholense should be used in the development of famous classical formulas.

Objective To summarize clinical characteristics and investigate possible pathogenic gene of Klippel-Feil syndrome(KFS)by the self-designed multigene panel sequencing,so as to decipher the molecular basis for early diagnosis and targeted therapy.Methods From January 2015 to December 2018,we consecutively recruited 25 patients who were diagnosed with KFS in Peking Union Medical College Hospital.The demographic information,clinical manifestations,physical examination and radiological assessments were analyzed.Multigene panel sequencing was performed after DNA extraction from peripheral blood.The possible pathogenic mutations of KFS were explored on the basis of bioinformatics analysis.Results The KFS cohort consisted of 25 patients,including 15 males and 10 females,with a mean age of(12.9±7.3)years.Limited cervical range of motion was the most common clinical feature(12 cases,48%).Based on the Samartzis classification,the proportion of patients suffered from short neck(P=0.031)and limited cervical range of motion(P=0.026)in type Ⅲ KFS was significantly higher than that in type Ⅱ and type Ⅰ KFS.Panel sequencing detected a total of 11 pathogenic missense mutations in eight patients,including COL6A1,COL6A2,CDAN1,GLI3,FLNB,CHRNG,MYH3,POR,and TNXB.There was no pathogenic mutation found in five reported pathogenic genes(GDF6,MEOX1,GDF3,MYO18B and RIPPLY2)associated with KFS.Conclusions Our study has shown that patients with multiple contiguous cervical fusions are more likely to manifest short neck,limited cervical range of motion,and clinical triad.Therefore,these patients need additional attention and follow-up.Our analysis highlights novel KFS-related genetic variants,such as COL6A and CDAN1,extending the spectrum of known mutations contributing to this syndrome and providing a basis for elucidating the pathogenesis of KFS.
Cervical Vertebrae ; Child ; Cohort Studies ; Female ; Glycoproteins ; Humans ; Klippel-Feil Syndrome/genetics* ; Male ; Mutation ; Nuclear Proteins ; Radiography ; Transcription Factors/genetics*

In recent years， the incidence of neurological diseases has been increasing year by year. To give full play to the advantages of traditional Chinese medicine （TCM） in the treatment of neurological disorders， identify the breakthrough point of integrating TCM with western medicine， and further standardize the clinical diagnosis and treatment of TCM， the China Association of Chinese Medicine organized neurologists in TCM and western medicine to carry out in-depth discussion on the neurological diseases responding specifically to TCM and integrated TCM and western medicine， such as stroke， headache， vertigo， multiple sclerosis， and epilepsy， aiming to formulate a well-recognized and integrated treatment protocol for TCM and western medicine and improve the efficacy of neurological disorders. Furthermore， the treatment suggestions of the corresponding diseases in TCM and western medicine were proposed to provide references for clinical practice and scientific research.

Objective: To study and create the algorithm for the diversity (AD) of extensible markup language (XML) tree map, and provide a new tool for the identification of Artemisiae Annuae Herba. Method: According to the literature research, the key information of Artemisiae Annuae Herba was selected from the macroscopic, mesoscopic and microscopic information, etc. Based on the key information, the relevant upper standards of domestic and foreign professional fields were cited to assign the unique identification independent of language for each data element, and the coding rules of relevant data elements were established. The digital coding technology was applied to the flexible structure editor, and the tree map was created, which could be returned as the 5^th version of hypertext markup language (HTML5) or XML format. Based on the diversity related algorithms, the authors innovatively developed the AD of XML tree map of Artemisiae Annuae Herba, which took into account both of topology and semantics, and the expression model of related mathematical functions of Artemisiae Annuae Herba was established. By comparing the calculation results with the reality, the algorithm model was debugged continuously until the convergence of the core-culvert algorithm model. Result: Through the research on AD, the diversity between two XML tree maps could be calculated, and the discrimination or identification model of Artemisiae Annuae Herba also could be finally optimized and established. After calculation and analysis of the tested tree maps, the effective rate of the model was 100%. Conclusion: In this study, the establishment of the AD of XML tree map can effectively assist in the identification of Artemisiae Annuae Herba, which provides certain technical support and theoretical guidance for the research on intelligent application of traditional Chinese medicine.

【Objective】To investigate the effects of Tc17 cells on disease severity and elimination of HBV-DNA in patients with HBV related acute-on-chronic liver failure（HBV-ACLF）.【Methods】Forty-three patients with HBV- ACLF were enrolled. Twenty patients with chronic hepatitis B（CHB）and 12 healthy subjects（NC）were enrolled as controls. Flow cytometry was used to detect the expression of peripheral Tc17 cells. HBV-DNA loads were measured，and the MELD，MELD-Na，CLIF-C ACLF and AARC scores were used to evaluate the disease severity. The effects of Tc17 cells on disease severity and decline of HBV-DNA were analyzed.【Results】Compared with NC group and CHB group， the expression of Tc17 cells in HBV-ACLF patients was significantly increased（P < 0.001 and P = 0.017）. Correlation analysis showed that Tc17 cells were positively correlated with AARC score，MELD score and MELD-Na score（r = 0.504，P = 0.001；r = 0.417，P = 0.005 and r = 0.382，P = 0.012），and a correlated trend was found with CLIF-C ACLF score（r = 0.294，P = 0.055）. And as the disease progressed，the expression of Tc17 cells gradually increased.In addition，Tc17 cells were positively correlated with HBV-DNA levels（r = 0.339，P = 0.026）at baseline，and the HBV-DNA levels were significantly decreased in patients with higher expression of Tc17 cells than in lower group after 4 weeks of treatment（P < 0.001）. Furthermore，it was found that baseline Tc17 cells and AST levels were associated with the HBV-DNA decline by multivariate linear regression analysis.【Conclusion】Tc17 cells-mediated inflammatory response helps clear the HBV-DNA，but excessive inflammatory response may aggravate the disease severity.

Acute Disease ; Dendritic Cells ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; Skin Neoplasms