1.Relationship between long non-coding RNA and osteoarthritis
Shanbin ZHENG ; Tianwei XIA ; Jiahao SUN ; Zhiyuan CHEN ; Xun CAO ; Chao ZHANG ; Jirong SHEN
Chinese Journal of Tissue Engineering Research 2025;29(11):2357-2367
BACKGROUND:As a common disease in middle-aged and elderly,osteoarthritis is difficult to cure,and the pathogenesis is not clear.Long non-coding RNA participates in the pathogenesis of osteoarthritis through many ways,such as regulating translation,promoting or inhibiting mRNA,and adsorbing miRNAs. OBJECTIVE:To review the types of common long non-coding RNA in osteoarthritis,and the influence of multiple long non-coding RNAs on the pathological factors related to osteoarthritis,to analyze the future application of long non-coding RNAs in osteoarthritis. METHODS:Literature retrieval was conducted in CNKI,WanFang Data,VIP database,PubMed,Web of Science and Sciencedirect databases,using the search terms of"osteoarthritis,degenerative joint disease,degenerative arthritis,OA,LncRNA,long non-coding RNA,long noncoding RNA,long intergenic non-coding RNA"in Chinese and English.All relevant literature published from 1976 and May 2024 was retrieved.After literature screening,induction,analysis and summary,93 articles were finally included for review. RESULTS AND CONCLUSION:This review collected 25 long non-coding RNAs that are well studied with osteoarthritis.Long non-coding RNAs,as a molecular sponge for miRNA,are competing endogenous RNAs to competitively adsorb miRNAs and then affect downstream targets.Long non-coding RNAs can regulate physiopathological processes such as chondrocyte apoptosis and proliferation,cartilage extracellular matrix degradation,and inflammatory responses.Long non-coding RNAs are expected to become a biomarker and potential therapeutic target for the clinical diagnosis and therapeutic prognosis of osteoarthritis,and it may become a new strategy for the clinical treatment of osteoarthritis in the future.
2.Dynamic Monitoring and Correlation Analysis of General Body Indicators, Blood Glucose, and Blood Lipid in Obese Cynomolgus Monkeys
Yanye WEI ; Guo SHEN ; Pengfei ZHANG ; Songping SHI ; Jiahao HU ; Xuzhe ZHANG ; Huiyuan HUA ; Guanyang HUA ; Hongzheng LU ; Yong ZENG ; Feng JI ; Zhumei WEI
Laboratory Animal and Comparative Medicine 2025;45(1):30-36
ObjectiveThis study aims to investigate the dynamic changes in general body parameters, blood glucose, and blood lipid profiles in obese cynomolgus monkeys, exploring the correlations among these parameters and providing a reference for research on the obese cynomolgus monkey model. Methods30 normal male cynomolgus monkeys aged 5 - 17 years old (with body mass index < 35 kg/m² and glycated hemoglobin content < 4.50%) and 99 spontaneously obese male cynomolgus monkeys (with body mass index ≥35 kg/m² and glycated hemoglobin content < 4.50%) were selected. Over a period of three years, their abdominal circumference, skinfold thickness, body weight, body mass index, fasting blood glucose, glycated hemoglobin, and four blood lipid indicators were monitored. The correlations between each indicator were analyzed using repeated measurement ANOVA, simple linear regression, and multiple linear regression correlation analysis method. Results Compared to the control group, the obese group exhibited significantly higher levels of abdominal circumference, skinfold thickness, body weight, body mass index, and triglyceride (P<0.05). In the control group, skinfold thickness increased annually, while other indicators remained stable. Compared with the first year, the obese group showed significantly increased abdominal circumference, skinfold thickness, body weight, body mass index, triglyceride, and fasting blood glucose in the second year(P<0.05), with this increasing trend persisting in the third year (P<0.05). In the control group, the obesity incidence rates in the second and third years were 16.67% and 23.33%, respectively, while the prevalence of diabetes remained at 16.67%. In the obese group, the diabetes incidence rates were 29.29% and 44.44% in years 2 and 3, respectively. Among the 11-13 year age group, the incidence rates were 36.36% and 44.68%, while for the group older than 13 years, the rates were 28.13% and 51.35%. Correlation analysis revealed significant associations (P<0.05) between fasting blood glucose and age, abdominal circumference, skinfold thickness, body weight, and triglyceride in the diabetic monkeys. Conclusion Long-term obesity can lead to the increases in general physical indicators and fasting blood glucose levels in cynomolgus monkeys, and an increase in the incidence of diabetes. In diabetic cynomolgus monkeys caused by obesity, there is a high correlation between their fasting blood glucose and age, weight, abdominal circumference, skinfold thickness, and triglyceride levels, which is of some significance for predicting the occurrence of spontaneous diabetes.
3.Effectiveness of Traditional Chinese Medicine Chronic Disease Management Programme after Colorectal Polyp Surgery:A Retrospective Cohort Study
Jiahao MO ; Yi CHENG ; Chang LIU ; Peixin HU ; Cailing ZHONG ; Beiping ZHANG
Journal of Traditional Chinese Medicine 2025;66(12):1241-1248
ObjectiveTo explore the effectiveness of traditional Chinese medicine (TCM) chronic disease management programme in preventing postoperative recurrence of colorectal polyps. MethodsThe clinical data of 447 postoperative colorectal polyp patients were retrospectively collected, and the patients were divided into an exposure group and a control group taking the acceptance of TCM chronic disease management programme as exposure factor, and the polyp recurrence rate as the main outcome indicator, comparing the differences in baseline characteristics, outcome events, and safety assessment between the two groups, and conducting correlation analysis between the length of medication and polyp recurrence. Multifactorial logistic regression was used to analyse the effects of receiving the TCM chronic disease management programme (TCM treatment and life management for spleen deficiency and dampness stasis syndrome, dampness and stasis obstruction in collaterals syndrome, and intestinal dampness and heat syndrome), gender, age, co-morbidities, TCM syndrome, and dietary and exercise factors on the outcome events. ResultsAmong 257 postoperative patients with colorectal polyps, there were 172 in the exposure group and 85 in control group. The recurrence rate of polyps in exposure group was 22.7% (39/172), while the recurrence rate in control group was 57.6% (49/85), and the difference between groups was statistically significant (P<0.01). The diameter of recurrent polyps in exposure group (median= 4.0 mm) was smaller than that in control group (median= 5.0 mm, P<0.01). The correlation analysis between the duration of medication taking and the recurrence of polyps in the spleen deficiency and dampness stasis syndrome group showed Phi value as -0.345 (P<0.001); the correlation analysis within the group of dampness and stasis obstruction in collaterals syndrome showed Phi value as -0.361 (P<0.05), indicating a negative correlation between the duration of medication taking and polyp recurrence. The results of multivariate logistic regression analysis indicated that the positive effect of accepting TCM chronic disease management programme on preventing polyp recurrence is statistically significant (OR=0.224, P<0.01). ConclusionAccepting TCM chronic disease management programme for colorectal polyps can help reducing the recurrence rate after polyp surgery, which is a protective factor for patients to the outcome event.
4.Danhong Injection Regulates Mitochondrial Dynamics in Rat Model of Chronic Heart Failure via AMPK/Drp1 Pathway
Jiahao YE ; Zizheng WU ; Yao ZHANG ; Lichong MENG ; Zhixi HU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):126-135
ObjectiveTo investigate the effects of Danhong injection on mitochondrial dynamics, morphology, and function in the rat model of chronic heart failure by mediating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/dynamin-related protein 1 (Drp1) pathway. MethodsFrom 75 SD rats, 15 rats were randomly selected as the sham group, and the remaining 60 rats were used to prepare a rat model of chronic heart failure by abdominal aortic constriction (AAC). The modeled rats were randomly allocated into model, Danhong Injection (6 mL·kg-1), and captopril (8.8 mg·kg-1) groups and administrated with corresponding agents for 15 consecutive days. The levels of N-terminal pro-brain natriuretic peptide (NT-pro BNP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ were determined by enzyme-linked immunosorbent assay. The changes in cardiac function were detected by echocardiography. The ultrastructural changes of myocardial mitochondria were observed by transmission electron microscopy. Western blot was employed to assess the protein levels of AMPK, p-AMPK, Drp1, p-Drp1, optic atrophy 1 (Opa1), mitofusin (Mfn2), and fission l (Fis1) in the myocardial tissue. Real-time PCR was performed to determine the mRNA levels of Opa1, Mfn2, and Fis1, and immunohistochemistry to detect the expression of p-AMPK. ResultsCompared with the sham group, the model group showed elevated levels of NT-pro BNP, ADP, TNF-α, IL-6, and IL-1β (P<0.01), declined ATP level (P<0.01), weakened activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ (P<0.01), decreased left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) (P<0.01), and increased left ventricular internal diameter at end-diastole (LVDd) and leaf ventricular internal diameter at end-systole (LVIDs) (P<0.01). Electron microscopy results showed that the model group presented heavily abnormal myocardial structure, with large areas of myofilament structure destroyed and dissolved, significantly enlarged residual structural gaps, and fragmented mitochondria. Western blot results showed that the model group demonstrated down-regulated protein levels of p-AMPK, Mfn2, and Opa1 (P<0.01) and up-regulated protein levels of p-Drp1 and Fis1 (P<0.01) in the myocardial tissue. Real-time PCR results showed that the model group presented up-regulated mRNA level of Fis1 (P<0.01) and down-regulated mRNA levels of Mfn2 and Opa1 (P<0.01). Immunohistochemistry results showed reduced expression of p-AMPK in the model group compared with sham group (P<0.01). Compared with the model group, Danhong injection lowered the levels of NT-pro BNP, ADP, TNF-α, IL-6, and IL-1β (P<0.01), raised the level of ATP (P<0.01), increased the activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ (P<0.05, P<0.01), increased the LVEF and LVFS (P<0.01), decreased the LVDd and LVIDs (P<0.05, P<0.01), alleviated mitochondrial damage, up-regulated the protein levels of p-AMPK, Mfn2, and Opa1 (P<0.05, P<0.01), down-regulated the protein levels of p-Drp1 and Fis1 (P<0.01), reduced the mRNA level of Fis1 (P<0.01), elevated the mRNA levels of Mfn2 and Opa1 (P<0.05, P<0.01), and promoted the expression of p-AMPK (P<0.05). ConclusionDanhong injection repairs the imbalance of mitochondrial dynamics, restores the mitochondrial function, improves the myocardial energy metabolism, and reduces the inflammatory response by regulating the AMPK/Drp1 pathway, thus improving the cardiac function.
5.Research Progress in Tong Du Tiao Shen of Mental Disorders
Jiahao ZHANG ; Cheng CHI ; Mengyue FAN ; Lin YAN ; Feixue WANG ; Meng ZHANG ; Yongjun CHEN
World Science and Technology-Modernization of Traditional Chinese Medicine 2024;26(1):61-68
Mental disorders are characterized by disturbances in behavior,volition,emotion,and cognition and are considered emotional diseases in traditional Chinese medicine.Acupuncture is one of the most widely used complementary alternative therapies for the treatment of mental disorders.Recently,there has been growing interest in the use of the Tong Du Tiao Shen(Dredging Du meridian to regulate the spirit)as a primary treatment.However,a comprehensive summary of the establishment and related acupuncture methods of Tong Du Tiao Shen is lacking.This paper aims to address this gap by exploring the origin and development of Tong Du Tiao Shen,its application in treating mental disorders,and the modern biological mechanisms involved.Ultimately,this paper seeks to expand the clinical application of Tong Du Tiao Shen acupuncture and provide a scientific basis for future research in this field.
6.The pathological progression of steroid-induced osteonecrosis of the femoral head caused by oxidative stress-induced osteoblast ferroptosis
Jiahao ZHANG ; Yuhao LIU ; Chi ZHOU ; Liang MO ; Hanjun FANG ; Zhenqiu CHEN
Chinese Journal of Tissue Engineering Research 2024;28(20):3202-3208
BACKGROUND:Studies have shown that imbalance of bone metabolism during glucocorticoid-induced osteonecrosis of the femoral head necrosis is closely related to oxidative stress. OBJECTIVE:To investigate the pathological mechanism by which oxidative stress-induced ferroptosis promote apoptosis in osteoblasts involved in steroid-induced osteonecrosis of the femoral head. METHODS:General data and serum specimens were collected from 47 patients with steroid-induced osteonecrosis of the femoral head.In addition,six femoral head specimens were collected from these patients.According to the Association Research Circulation Osseous(ARCO)staging system,serum specimens were grouped into ARCO Ⅱ,Ⅲ,and IV,while femoral head specimens were classified into ARCO Ⅲ and IV.Serum levels of malondialdehyde and superoxide dismutase 1 were measured.The protein expression of superoxide dismutase 1,glutathione peroxidase 4,Bcl-2 in the femoral head was detected and verified by Data independent acquisition(DIA)for quantitative sequencing,western blot and alkaline phosphate detection. RESULTS AND CONCLUSION:The ARCO stage of patients with steroid-induced osteonecrosis of the femoral head was independent of age,sex and necrotic side.The serum levels of malondialdehyde and superoxide dismutase 1 were higher in patients with ARCO stage Ⅲ compared with those with ARCO stage Ⅱ and IV.The results of DIA protein quantification showed that the function of differential proteins was mainly related to redox.The levels of superoxide dismutase 1,glutathione peroxidase 4,and Bcl-2 in the necrotic region were lower than in the normal region,as well as lower in ARCO stage IV than in ARCO stage Ⅲ.Western blot verified the results of DIA protein quantification.The alkaline phosphatase activity was lower in the necrotic region than in the normal region,as well as lower in ARCO stage IV than in ARCO stage Ⅲ.In the necrotic and sclerotic regions,the function of differential proteins was also related to redox,and superoxide dismutase 1,glutathione peroxidase 4,Bcl-2 protein expression and alkaline phosphatase activity were lower in the necrotic area than in the sclerotic region,as well as lower in ARCO stage IV than in ARCO stage Ⅲ.To conclude,glucocorticoids can influence the progression of steroid-induced osteonecrosis of the femoral head by upregulating oxidative stress levels,inducing osteoblast ferroptosis,and inhibiting osteogenic function.
7.Value of mean platelet volume combined with thromboelastography in predicting thrombosis after total knee arthroplasty
Jiahao CHEN ; Shuo FENG ; Hu WANG ; Qiang ZHANG ; Xiangyang CHEN
Chinese Journal of Tissue Engineering Research 2024;28(21):3334-3338
BACKGROUND:Thromboelastography plays an important role in identifying the hypercoagulable state of blood and thrombosis in humans.Recent studies have shown a correlation between an increase in mean platelet volume and thrombosis.We can therefore ask whether the combined diagnosis of thromboelastography and mean platelet volume is a more accurate predictor of thrombosis. OBJECTIVE:To predict the status of blood and the occurrence of thrombosis after total knee arthroplasty by means of mean platelet volume combined with thromboelastography. METHODS:One hundred and twenty patients who underwent unilateral total knee arthroplasty between May 2015 and March 2022 were collected.Patients were divided into 60 patients in the thrombosis group and 60 patients in the control group based on ultrasound findings on postoperative day 7.Whole blood cell and thromboelastography were performed 1 day before,1 and 7 days after surgery,respectively.Multifactorial analysis was used to investigate independent predictors of thrombosis after total knee arthroplasty.The receiver operating characteristic curve and area under the curve were measured in the subjects. RESULTS AND CONCLUSION:Mean platelet volume correlated most strongly with maximum amplitude,followed by coagulation angle.Mean platelet volume and coagulation angle on postoperative day 1 were independent predictors of thrombosis.Mean platelet volume tended to rise and then fall in patients with thrombosis.The best critical value for mean platelet volume to predict thrombosis was 10.73 fL.The area under the receiver operating characteristic curve of subjects was 0.665(95%CI:0.568-0.762,P<0.05];whereas the area under the receiver operating characteristic curve for subjects using mean platelet volume combined with coagulation angle was 0.815(95%CI:0.750-0.879,P<0.05).In addition,the maximum amplitude,coagulation angle,coagulation index and mean platelet volume were significantly higher in the thrombosis group than in the control group postoperatively(P<0.05).The results suggest that the mean platelet volume can reflect the hypercoagulable state of blood after surgery,and the combination of mean platelet volume and coagulation angle on day 1 after total knee arthroplasty can improve the prediction of thrombosis.
8.Effect of varus and valgus angles of tibial prosthesis on short-term outcome of fixed bearing unicompartmental knee arthroplasty
Hu WANG ; Shuo FENG ; Qiang ZHANG ; Jiahao CHEN ; Xiangyang CHEN
Chinese Journal of Tissue Engineering Research 2024;28(24):3827-3832
BACKGROUND:Previous studies on the effects of valgus and varus angles of tibial component on short-term postoperative outcomes after mobile bearing unicompartmental knee arthroplasty have been reported in and outside China.However,there are few reports on the effect of the valgus and varus angles of tibial component on short-term postoperative outcomes after fixed bearing unicompartmental knee arthroplasty. OBJECTIVE:To investigate the effect of valgus and varus angles of tibial component on short-term clinical outcomes in patients with medial knee osteoarthritis undergoing fixed bearing unicompartmental knee arthroplasty. METHODS:120 patients(122 knees)who underwent fixed bearing unicompartmental knee arthroplasty for medial knee osteoarthritis in Department of Orthopedic Surgery,Affiliated Hospital of Xuzhou Medical University from August 2020 to January 2023 were selected as the study subjects.Two physicians measured the varus angle of femoral prosthesis,valgus and varus angles of tibial prosthesis,flexion and extension angles of femoral prosthesis,and posterior inclination angle of tibial prosthesis after unicompartmental knee arthroplasty based on postoperative X-ray.After excluding the influence of the other three angles,the measurement results of the tibial component varus angle were divided into three groups:<-2°,-2° to 2°,>2°,which were denoted as groups 1,2,and 3,respectively.The range of knee motion,the hospital for special surgery knee score,the American knee society score,and forgotten joint score were recorded and compared before and after the operation. RESULTS AND CONCLUSION:(1)A total of 120 patients(122 knees)were enrolled in this study.They were divided into three groups according to the size of the valgus and varus angles of the tibial prosthesis after operation:37 patients in the first group,60 patients in the second group,and 23 patients in the third group.There was no significant difference between the three groups in terms of baseline information such as age,gender,and side of surgery(P>0.05).(2)Patients were followed up for 3-30 months after arthroplasty.(3)The hospital for special surgery knee score of the second group was higher than that of the first group(P=0.015)and the third group(P=0.012).The American knee society score of the second group was significantly higher than that of the first group(P=0.014)and the third group(P<0.001).The forgotten joint score of the second group was higher than that of the first group(P=0.033)and the third group(P=0.016).(4)After fixed bearing unicompartmental knee arthroplasty,when the valgus angle of tibial prosthesis was-2° to 2°,which can achieve better short-term clinical results,the degree of prosthesis self-realization is higher.
9.Predictors of a forgotten joint after medial open wedge high tibial osteotomy
Yiwei HUANG ; Bo PENG ; Chen ZHANG ; Hao GE ; Jiahao LI ; Yijin LI ; Jinlun CHEN ; Wenjun FENG ; Yirong ZENG
Chinese Journal of Tissue Engineering Research 2024;28(24):3903-3909
BACKGROUND:Medial open wedge high tibial osteotomy is an effective procedure for preserving the knee joint in patients with medial compartmental osteoarthritis.Previous studies have demonstrated that the forgotten joint score provides a lower ceiling effect and consistency of medial open wedge high tibial osteotomy outcomes compared to traditional assessment tools. OBJECTIVE:To identify predictive factors associated with the occurrence of a forgotten joint after medial open wedge high tibial osteotomy. METHODS:117 patients with medial open wedge high tibial osteotomy who were treated at First Affiliated Hospital of Guangzhou University of Chinese Medicine were selected,including 35 males and 82 females,with an average age of 61 years.They were followed up for at least 2 years.Patients were divided into a forgotten joint group(n=28)and a non-forgotten joint group(n=89)by evaluating whether they achieved forgotten joint after surgery.Univariate and multivariate logistic regression analyses were performed with preoperative patient characteristics and surgery-related factors as potential predictors. RESULTS AND CONCLUSION:(1)There were significant differences in the proximal medial tibial angle between the two groups before surgery(P<0.05).There were significant differences in the forgotten joint score,Knee Injury and Osteoarthritis Outcome Score,knee society knee score,function score,and patients joint perception between the two groups after surgery(P<0.05).There was a significant difference between the hip-knee-ankle angle and the medial proximal tibial angle after operation(P<0.05).(2)Univariate Logistic regression analysis showed that the medial proximal tibial angle had a significant influence on the forgotten joint before operation[OR=0.755,95%CI(0.635-0.897),P<0.001].There were significant effects on the forgotten joint of hip-knee-ankle angle and medial proximal tibial angle[OR=1.546,95%CI(1.242-1.924),P<0.001;OR=0.815,95%CI(0.713-0.931),P=0.003].(3)Multivariate logistic regression analysis showed that preoperative K-L grade 1 was a favorable factor for obtaining forgotten joints.Preoperative medial proximal tibial angle and postoperative hip-knee-ankle angle were independent predictors of forgetting joints,and they had a curvilinear relationship with the probability of achieving forgetting joints.When preoperative medial proximal tibial angle increased by 1°,the probability of achieving a forgotten joint decreased by 27.7%[OR=0.723,95%CI(0.593-0.882),P<0.001].Conversely,when postoperative hip-knee-ankle angle increased by 1°,the probability of achieving a forgotten joint increased by 46.4%[OR=1.464,95%CI(1.153-1.860),P=0.002].(4)The results showed that patients with preoperative knee osteoarthritis K-L grade 1,small medial proximal tibial angle(<85.5°),and large postoperative hip-knee-ankle angle(>176.0°)were predictors of forgotten joint.
10.Effectiveness of exercise interventions for fibromyalgia syndrome:a Meta-analysis
Jinpu ZHANG ; Junli WANG ; Siqi ZHANG ; Jiahao CHEN ; Qiushi YANG
Chinese Journal of Tissue Engineering Research 2024;28(32):5210-5216
OBJECTIVE:Exercise intervention is one of the main treatments for fibromyalgia,but there is no consistent conclusion on the choice of different exercise modalities.In this article,a network Meta-analysis was used to comprehensively and quantitatively evaluate the effects of different exercise modalities on fibromyalgia syndrome. METHODS:PubMed,EMbase,Scoups,The Cochrane Library,Web of Science,CNKI,WanFang Database,and China Biomedical Literature Database were searched for relevant literature,with a search timeframe from the establishment of each database to June 2023.The outcome indicators included five continuous variables,including fibromyalgia impact questionnaire-revised(FIQ)scores,visual analogue scale(VAS)scores,quality of life,quality of sleep,and depression.The Cochrane Risk of Bias Assessment Tool was used to evaluate the quality of the included literature.RevMan 5.4 software was used to perform effect sizes,subgroup analyses,and sensitivity analyses of the data.Stata 17 software was used to perform reticulation and network Meta-analysis of the data. RESULTS:A total of 13 articles with 14 randomized controlled trials were finally included.The overall methodological quality of the literature was high.The results of traditional Meta-analysis showed that,compared with the control group,exercise therapy significantly improved the FIQ score[standardized mean difference(SMD)=-0.67,95%confidence interval(CI):-0.83 to-0.50,P<0.01],VAS score(SMD=-0.72,95%CI:-0.90 to-0.54,P<0.01),quality of life(SMD=1.03,95%CI:0.45 to 1.61,P=0.000 5),sleep quality(SMD=-0.62,95%CI:-0.98 to-0.25,P=0.001),and depression(SMD=-0.63,95%CI:-1.09 to-0.18,P=0.007).Network Meta-analysis showed that the probability of optimal intervention effect of exercise modalities on FIQ scores was ranked as:mind-body exercise(86.5)>resistance exercise(70.5)>aerobic exercise(41.7);the probability of optimal intervention effect of exercise modalities on VAS scores was ranked as:resistance exercise(85.3)>mind-body exercise(74.3)>aerobic exercise(34.5). CONCLUSION:Exercise therapy significantly improves FIQ scores,VAS scores,quality of life,sleep quality,and depression in patients with fibromyalgia syndrome.Mind-body exercise and resistance exercise are the most effective exercise modalities to reduce FIQ scores and VAS scores in patients with fibromyalgia syndrome.

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