1.Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells
Liangliang MA ; Ke ZHANG ; Jiangning LU ; Lixin SUN ; Long YU ; Yuliang RAN ; Lichao SUN
Chinese Journal of Oncology 2024;46(9):878-888
Objectives:To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells.Methods:The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation.Results:The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group ( P<0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group ( P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group ( P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P<0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group ( P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group ( P<0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group ( P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group ( P<0.001). Conclusion:Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.
2.Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells
Liangliang MA ; Ke ZHANG ; Jiangning LU ; Lixin SUN ; Long YU ; Yuliang RAN ; Lichao SUN
Chinese Journal of Oncology 2024;46(9):878-888
Objectives:To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells.Methods:The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation.Results:The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group ( P<0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group ( P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group ( P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P<0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group ( P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group ( P<0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group ( P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group ( P<0.001). Conclusion:Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.
3.Trend in the epidemic characteristics of human brucellosis in Hangzhou City from 2004 to 2021
Sujuan ZHU ; Xingyi JIN ; Liangliang HUO ; Zhou SUN ; Tingting ZHAO ; Weimin XU
Chinese Journal of Endemiology 2024;43(5):370-375
Objective:To analyze the changing trends of the epidemic characteristics of human brucellosis in Hangzhou City, and to provide a basis for further formulating prevention and control measures.Methods:The epidemic data of brucellosis in Hangzhou City from 2004 to 2021 were collected from the "China Disease Control and Prevention Information System" and the annual reports, compilations and summaries of prevention and control work of brucellosis in Hangzhou City, and the epidemiological characteristics and trends of brucellosis cases in Hangzhou City from 2004 to 2009, 2010 to 2015, and 2016 to 2021 were compared and analyzed.Results:A total of 186 cases of brucellosis were diagnosed in Hangzhou City from 2004 to 2021, with an average annual incidence rate of 0.151/100 000, ranging from 0.016/100 000 to 0.286/100 000. The incidence rate showed a stage-by-stage upward trend from 2004 to 2009, 2010 to 2015, and 2016 to 2021 ( Z = 3.15, P = 0.002). The age of onset of brucellosis cases was predominantly 18 - 60 years old, accounting for 78.49% (146/186). The male-to-female ratio was 2.10∶1.00 (126∶60). The occupational and non-occupational populations accounted for 62.90% (117/186) and 37.10% (69/186), respectively, with the proportion of non-occupational cases in the three stages being 28.57%, 23.29% and 51.76%, showing an increasing trend (χ 2trend = 9.85, P = 0.002). The regional distribution was dominated by Yuhang District, accounting for 55.91% (104/186), followed by Jianggan District (9.14%, 17/186) and Fuyang District (7.53%, 14/186). The number of brucellosis epidemic counties (cities, districts) increased from 6 to 9. The seasonal analysis showed that the proportion of cases from January to June decreased from 84.93% (23/28) from 2004 to 2009 to 54.12% (46/85) from 2016 to 2021. The proportion of infection from outside the province in the three stages was 0, 10.96% and 27.38%, respectively, showing an upward trend (χ 2trend = 18.16, P < 0.001), the main infected areas were Henan Province, Heilongjiang Province, and Inner Mongolia Autonomous Region. In clinical symptoms and signs, the incidence of muscle and joint pain and low back pain increased from 60.71% (17/28) and 10.71% (3/28) from 2004 to 2009, respectively, to 83.53% (71/85) and 25.88% (22/85) from 2016 to 2021, both showing an upward trend (χ 2trend = 9.92, 4.67, P = 0.002, 0.031). Conclusions:The incidence of human brucellosis in Hangzhou City is on the rise, and the number of cases of non-occupational population has increased significantly in recent years. The sheep breeding places have gradually become the high incidence areas of brucellosis in Hangzhou City. It is recommended to strengthen the promotion and education of knowledge on brucellosis prevention among the general public, and focus on actively monitoring human brucellosis in high-risk areas.
4.Epidemiological characteristics and spatio-temporal aggregation of hemorrhagic fever with renal syndrome in Hangzhou City from 2010 to 2022
Zhe WANG ; Renjie HUANG ; Lei ZHU ; Shuang FENG ; Zhaokai HE ; Liangliang HUO ; Zhou SUN
Chinese Journal of Endemiology 2024;43(7):586-592
Objective:To study the epidemiological and spatio-temporal distribution characteristics of hemorrhagic fever with renal syndrome (HFRS) in Hangzhou City, providing a scientific basis for prevention and control of HFRS.Methods:Data of HFRS cases reported in Hangzhou City from January 1, 2010 to December 31, 2022 were collected through the Infectious Disease Surveillance and Reporting Information System of China Disease Prevention and Control Information System. Descriptive epidemiological methods were used to analyze the prevalence and three-distribution characteristics of HFRS in Hangzhou City. Joinpoint regression was used to analyze the trend of HFRS incidence in Hangzhou City from 2010 to 2022. Global and local spatial autocorrelation were used to analyze the spatial distribution pattern of HFRS and the hotspots of incidence in Hangzhou City. And spatio-temporal scanning was used to analyze the spatio-temporal aggregation areas of HFRS in Hangzhou City.Results:From 2010 to 2022, a total of 224 HFRS cases were reported in Hangzhou City, with an average annual incidence of 0.18/100 000. The distribution of cases showed obvious seasonality, with peak incidence in spring (March to May) and autumn (September to November), accounting for 30.80% (69/224) and 26.34% (59/224), respectively. HFRS cases were reported in all districts (counties, cities) of the city, among which Xiaoshan District (66 cases, 29.46%), Chun'an County (41 cases, 18.30%) and Jiande City (25 cases, 11.16%) ranked the top three. The majority of the cases were individuals aged 31 to 60 (65.18%, 146/224), males (74.55%, 167/224), and farmers (46.43%, 104/224). Joinpoint regression analysis indicated that the overall incidence of HFRS in Hangzhou City was in downward trend from 2010 to 2022 [average annual percent change (AAPC) = - 5.01%, 95% confidence intervals ( CI): - 9.46% to - 0.34%, t = - 2.10, P = 0.036]. Global spatial autocorrelation analysis showed that there was a positive spatial correlation in the incidence of HFRS among various streets (townships) in Hangzhou City from 2011 to 2014, 2018, and 2020 (Moran's I > 0, Z > 1.96, P < 0.05). Local spatial autocorrelation analysis showed that from 2010 to 2022, the number of streets (towns) in hot areas (high-high) in Hangzhou City was 0, 2, 3, 3, 3, 3, 0, 0, 4, 0, 1, 0, and 1, respectively, and was relatively fixed in the southwest districts (counties, cities). Spatio-temporal scan analysis identified three clusters: Cluster I was from August 2011 to January 2015, centered on Fenkou Town in Chun'an County, involving 5 townships in Chun'an County; Cluster Ⅱ-1 was from August 2012 to March 2016, centered on Puyang Town in Xiaoshan District, involving 5 townships in Xiaoshan District; Cluster Ⅱ-2 was from June 2019 to June 2020, centered on Xiaya Town in Jiande City, not involving other streets (townships). Conclusions:From 2010 to 2022, the majority of HFRS cases in Hangzhou City are middle-aged male farmers. The overall trend of HFRS epidemic is decreasing, mainly concentrated in the southwest districts (counties, cities) of Hangzhou City. In the future, precise prevention and control measures should be implemented in key areas and among key populations.
5.Investigation and analysis of indoor radon concentration of urban residents in Shiyan, China
Pei LI ; Wenshan ZHOU ; Fang WANG ; Liangliang GUO ; Shaohua YANG ; Tongqiang ZHAO ; Jiabing WU ; Rongbin SUN
Chinese Journal of Radiological Health 2023;32(1):26-29
Objective To monitor the indoor radon concentration of urban residents in Shiyan, China, and to analyze the related influencing factors. Methods From April to July, 2019, RSKS standard detectors were used to measure the indoor radon concentration of 125 households in Shiyan, and the results were analyzed. Results The indoor radon concentration of residents in Shiyan showed a skewed distribution, ranging from 13.8 to 145 Bq/m3, and M (P25,P75) was 38.3 (29.0,62.0) Bq/m3. The estimated annual effective dose of radon and radon daughters from inhalation was 0.52-5.50 mSv, and M (P25,P75) was 1.45 (1.10, 2.36) mSv, which was consistent with literature. Building structure (H = 14.10, P < 0.001), floor (H = 24.41, P < 0.001), and geographical region (H = 8.963, P < 0.05) were influencing factors of indoor radon concentration, and the differences were significant. Conclusion The indoor radon concentration of urban residents in Shiyan is lower than the national standard limit. However, in daily life, it is still necessary to take appropriate measures to reduce the concentration of indoor radon as much as possible.
6.Clinical analysis of streptococcal toxic shock syndrome caused by Group A Streptococcus infection in children
Qing MENG ; Yuhui WU ; Yanlan YANG ; Yanxia HE ; Lintao ZHOU ; Huabao CHEN ; Hui XIE ; Liangliang KANG ; Nannan HE ; Lifang SUN
Chinese Journal of Applied Clinical Pediatrics 2022;37(21):1665-1668
Objective:To analyze the clinical characteristics of Group A Streptococcal(GAS) toxic shock syndrome (STSS) in children. Methods:The clinical data of 10 STSS children hospitalized in Shenzhen Children′s Hospital from January 2015 to March 2022 were downloaded from the electronic medical record system.The clinical manifestations were analyzed and treatment experience was summarized respectively.Results:There were 5 males and 5 females, with an average age of (5.29±2.87) years.All the patients were healthy in the past.The diagnoses were confirmed by blood culture in 2 cases, pus culture in 5 cases, and blood metagenomics next generation sequencing in 3 cases.The rapid detection of GAS antigen was positive in 7 cases.All cases had fever, and 9 cases of them developed fever after viral infection, including pneumonia in 7 cases, skin and soft tissue infections in 6 cases, necrotizing fasciitis in 3 cases, and purulent meningitis in 1 case.All cases also presented with shock.Six cases had liver function injury, and 4 cases suffered from acute kindey injury.Four cases had infection-related encephalopathy, and 7 cases were afflicted with disseminated intravascular coagulation.Two cases had respiratory failure, and 2 cases had rhabdomyolysis.There were 3 cases with a decreased white blood cell (WBC) count and 7 cases with an increased WBC count on admission.Seven cases were found to have thrombocytopenia, but their platelet levels were all elevated after recovery.C-reactive protein and procalcitonin and the proportion of neutrophils were markedly increased in all cases.All cases suffered from hypoalbuminemia, hyponatremia and hypocalcemia.All the 10 positive strains were sensitive to Penicillin, Ceftriaxone/Cefotaxime and Vancomycin.Eight cases were treated with combined antibiotics after admission.Eight patients received intravenous immunoglobulin.All cases were cured and discharged.Conclusions:The STSS progresses rapidly in children, so pediatricians should pay great attention to the disease.Early identification, diagnosis of infection sources, infusion of antibiotics and surgical treatment are the keys to disease management.
7.Path analysis of the influence of professional values and self-concept of nursing professional on academic burnout of nursing students
Chen CHEN ; Hui CAO ; Fanjuan ZENG ; Lili CHEN ; Liangliang SUN ; Shouli WANG
Chinese Journal of Practical Nursing 2022;38(31):2456-2461
Objective:To investigate the academic burnout level of nursing interns, and to analyze the influence path of nursing professional self-concept and professional values on the academic burnout of nursing interns.Methods:From September to December 2021, 204 nursing students who practiced for 6 months in the First Affiliated Hospital of Anhui Medical University were surveyed using the College Student Learning Burnout Scale, the Nursing Professional Self-Concept Scale and the Nursing Professional Values Scale. Structural equation modeling using AMOS 23.0.Results:The total score of academic burnout was (52.37 ± 11.60), which was in the middle level. The results of correlation analysis showed that the nurses' professional values were negatively correlated with depression, misbehavior, and low sense of achievement ( r=-0.253, -0.256, -0.406), and the differences were statistically significant ( P<0.01). Nursing professional self-concept was negatively correlated with depression, misbehavior, and low sense of achievement ( r=-0.466, -0.414, -0.525), and the differences were statistically significant ( P<0.01). Intern nursing students' nursing occupational self-concept had a partial mediating effect on their professional values and academic burnout, and the mediating effect accounted for 48.57% of the total effect. Conclusions:Professional values of nursing students can directly affect academic burnout, and also indirectly affect them through nursing professional self-concept.
8.Investigation on knowledge and practice of iodine deficiency disorders prevention and control among main caregivers of pregnant women in trimester
Liangliang HUO ; Zhaoying QIAN ; Bing ZHU ; Zhou SUN ; Tingting ZHAO ; Shuchang CHEN ; Qingxin KONG
Chinese Journal of Endemiology 2022;41(7):561-565
Objective:To learn about the knowledge and practice of iodine deficiency disorders (IDD) prevention and control among main caregivers of pregnant women in trimester and its related influencing factors, so as to provide a scientific basis for effectively improving the iodine nutrition level of pregnant women.Methods:In April 2020, using the stratified sampling method, 2 districts and counties were selected in the urban, suburban and rural areas of Hangzhou City, respectively, and 5 maternal and child health care institutions were selected as the survey sites in each district and county according to the five directions of East, West, South, North and Middle. A face-to-face questionnaire investigation was used to investigate the knowledge and practice of IDD prevention and control among main caregivers of pregnant women.Results:Totally 601 caregivers of pregnant women were investigated. Among them, 68.9% (414/601) knew that eating iodized salt was the best way to prevent IDD. However, 15.0% (90/601) thought that eating seafood was the best way to prevent IDD. And 41.9% (252/601) caregivers of pregnant women would actively purchase iodine-rich food for cooking, and 63.6% (382/601) would choose iodized salt. There were statistically significant differences in the knowledge and practice of IDD prevention and control among caregivers of pregnant women in urban, suburban and rural areas ( P < 0.05). And 50.3% (87/173) male caregivers would actively purchase iodine-rich food for cooking, which was higher than that of female caregivers [38.6% (165/428)], the difference was statistically significant (χ 2 = 6.97, P = 0.008). There was a statistically significant difference between the caregivers of pregnant women of different ages in actively purchasing iodine-rich food for cooking (χ 2 = 12.61, P = 0.013). And there were statistically significant differences between the caregivers of pregnant women with different educational levels in actively purchasing iodine-rich food for cooking and choosing iodized salt (χ 2 = 29.34, 39.42, P < 0.001). Conclusions:The awareness of knowledge about IDD prevention and control and the formation of prevention and control behavior among caregivers of pregnant women in Hangzhou City are poor. Active education of caregivers of pregnant women should be strengthened to promote maternal and infant health.
9.LncRNA DACH1 protects against pulmonary fibrosis by binding to SRSF1 to suppress CTNNB1 accumulation.
Jian SUN ; Tongzhu JIN ; Zhihui NIU ; Jiayu GUO ; Yingying GUO ; Ruoxuan YANG ; Qianqian WANG ; Huiying GAO ; Yuhan ZHANG ; Tianyu LI ; Wenxin HE ; Zhixin LI ; Wenchao MA ; Wei SU ; Liangliang LI ; Xingxing FAN ; Hongli SHAN ; Haihai LIANG
Acta Pharmaceutica Sinica B 2022;12(9):3602-3617
Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unknown etiology and limited therapeutic options. Activation of fibroblasts is a prominent feature of pulmonary fibrosis. Here we report that lncRNA DACH1 (dachshund homolog 1) is downregulated in the lungs of IPF patients and in an experimental mouse model of lung fibrosis. LncDACH1 knockout mice develop spontaneous pulmonary fibrosis, whereas overexpression of LncDACH1 attenuated TGF-β1-induced aberrant activation, collagen deposition and differentiation of mouse lung fibroblasts. Similarly, forced expression of LncDACH1 not only prevented bleomycin (BLM)-induced lung fibrosis, but also reversed established lung fibrosis in a BLM model. Mechanistically, LncDACH1 binding to the serine/arginine-rich splicing factor 1 (SRSF1) protein decreases its activity and inhibits the accumulation of Ctnnb1. Enhanced expression of SRSF1 blocked the anti-fibrotic effect of LncDACH1 in lung fibroblasts. Furthermore, loss of LncDACH1 promoted proliferation, differentiation, and extracellular matrix (ECM) deposition in mouse lung fibroblasts, whereas such effects were abolished by silencing of Ctnnb1. In addition, a conserved fragment of LncDACH1 alleviated hyperproliferation, ECM deposition and differentiation of MRC-5 cells driven by TGF-β1. Collectively, LncDACH1 inhibits lung fibrosis by interacting with SRSF1 to suppress CTNNB1 accumulation, suggesting that LncDACH1 might be a potential therapeutic target for pulmonary fibrosis.
10.Epidemiological characteristics of malaria in Hangzhou City from 2004 to 2021
Sujuan ZHU ; Xingyi JIN ; Liangliang HUO ; Weimin XU ; Zhou SUN ; Qingxin KONG ; Junfang CHEN
Journal of Preventive Medicine 2022;34(10):1026-1031
Objective:
To investigate the epidemiological characteristics of malaria in Hangzhou City from 2004 to 2021, so as to provide the evidence for formulating the post-elimination control strategy for malaria in Hangzhou City.
Methods:
The epidemic situation of malaria in Hangzhou City from 2004 to 2021 were collected from the National Information System for Disease Control and Prevention in China, and the temporal, spatial and human distributions of malaria cases and the source of malaria infections were analyzed in Hangzhou City during the pre-elimination stage (2004 to 2009), the elimination stage (2010 to 2015) and the post-elimination stage (2016 to 2021).
Results:
Totally 602 malaria cases were reported in Hangzhou City from 2004 to 2021,and the annual mean incidence of malaria was 0.22/105, 0.20/105 and 0.18/105 during the pre-elimination, elimination and post-elimination stages, appearing a tendency towards a decline. Men accounted for 63.96%, 85.07% and 93.75% of all malaria cases and there were 67.86%, 82.84% and 80.00% of cases at ages of 18 to 50 years during the pre-elimination, elimination and post-elimination stages, both appearing a tendency towards a decline (χ2trend=56.748, P<0.001; χ2trend=39.971, P<0.001). The predominant occupation of malaria cases shifted from farmers or migrant workers to multiple occupations, and the proportion of commercial servants increased from 4.87% during the pre-elimination stage to 24.38% during the post-elimination stage (χ2trend=73.308, P<0.001). The proportion of Plasmodium vivax malaria cases reduced from 96.43% during the pre-elimination stage to 7.50% during the post-elimination stage, and the proportion of P. falciparum malaria cases increased from 3.57% to 71.25%, while P. ovale, P. malariae and mixed infections were identified since 2010. There was a significant season-specific incidence of P. vivax malaria during the pre-elimination stage, and the period between May and October was an epidemic season; however, there was no season-specific incidence of P. vivax malaria during the elimination and post-elimination stages. The regional distribution of malaria cases presented a tendency towards a shift from suburb and rural areas to urban areas (χ2trend=74.229, P<0.001). No local cases were detected in Hangzhou City since 2010, and 94.22% of malaria cases were overseas imported cases after malaria elimination, including 90.61% from Africa.
Conclusions
oung and middle-aged men were high-risk populations for malaria in Hangzhou City from 2004 to 2021, and overseas commercial servants gradually became the predominant source of malaria infections, with malaria parasite species tending to be diverse. Improving the management of overseas imported cases and timely identification and treatment of cases are major interventions to consolidate malaria elimination achievements in Hangzhou City.


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