1.Effect of salvianolic acid B on inflammatory responses of vascular smooth muscle cells in septic mice: role of circACTA2
Manli ZHANG ; Jingru ZHAO ; Hua YIN ; Manna ZHANG ; Xuan SONG ; Fei TONG
Chinese Journal of Anesthesiology 2024;44(2):225-231
Objective:To evaluate the effect of Salvianolic acid B (Sal B) on the inflammatory responses of vascular smooth muscle cells (VSMCs) in septic mice and the role of circACTA2.Methods:In vivo experiment Eighty-one healthy male C57BL/6 mice, aged 6-8 weeks, were divided into 3 groups ( n=27 each) by a random number table method: sham operation group, sepsis group and Sal B group. Sepsis model was developed by cecal ligation and puncture. After sucessful preparation of the model, Sal B 7 mg/kg/d was intraperitoneally injected once a day for 2 consecutive days in Sal B group. Twenty mice in each group were randomly selected to measure systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and whole blood lactic acid (Lac) and to record the survival within 7 days after developing the model. Seven mice in each group were randomly selected at 48 h after developing the model, and the arterial vascular tissues were collected for determination of the expression of interleukin-1beta (IL-1β) (by immunofluorescence staining), expression of IL-1β, tumor necrosis factor-alpha (TNF-α) and IL-6 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction, respectively), and expression of circACTA2 (by quantitative real-time polymerase chain reaction). Cell experiment Mouse VSMCs were cultured and divided into 6 groups ( n=3 each) by a random number table method: control group (C group), lipopolysaccharide (LPS) group, Sal B group, si-circACTA2+ C group, si-circACTA2+ LPS group, and si-circACTA2+ Sal B group. The cells were incubated for 24 h with LPS (final concentration 1 μg/ml) in LPS group and with LPS (final concentration 1 μg/ml) and Sal B (final concentration 5 μmol/L) in Sal B group. VSMCs were transfected with si-circACTA2 only in si-circACTA2+ C group. At 24 h after transfection of si-circACTA2 into VSMCs, the cells were incubated with LPS (final concentration 1 μg/ml) in si-circACTA2+ LPS group and with LPS (final concentration 1 μg/ml) and Sal B (final concentration 5 μmol/L) for 24 h in si-circACTA2+ Sal B group. The expression of IL-1β, TNF-α and IL-6 protein and mRNA was detected using Western blot and quantitative real-time polymerase chain reaction, and the expression of circACTA2 was determined by the quantitative real-time polymerase chain reaction. Results:In vivo experiment Compared with sham operation group, SBP, DBP and MAP were significantly decreased, the concentrations of whole blood Lac were increased, 7-day survival rate was decreased, the expression of IL-1β, TNF-α and IL-6 protein and mRNA in arterial vascular tissues was up-regulated, circACTA2 expression was down-regulated ( P<0.05), and the fluorescence of IL-1β was enhanced in sepsis group. Compared with sepsis group, SBP, DBP and MAP were significantly increased, whole blood Lac concentrations were decreased, 7-day survival rate was increased, the expression of IL-1β, TNF-α and IL-6 protein and mRNA in arterial vascular tissues was down-regulated, the expression of circACTA2 was up-regulated ( P<0.05), and the fluorescence of IL-1β was weakened in Sal B group. Cell experiment Compared with group C, the expression of IL-1β, TNF-α and IL-6 protein and mRNA was significantly up-regulated, and the expression of circACTA2 was down-regulated in LPS group ( P<0.05). Compared with LPS group, the expression of IL-1β, TNF-α and IL-6 protein and mRNA was significantly down-regulated, and the expression of circACTA2 was up-regulated in Sal B group ( P<0.05). Compared with si-circACTA2+ C group, the expression of IL-1β, TNF-α and IL-6 protein and mRNA was significantly up-regulated in si-circACTA2+ LPS group ( P<0.05). There were no significant differences in the expression of IL-1β, TNF-α and IL-6 protein and mRNA between si-circACTA2+ LPS group and si-circACTA2+ Sal B group ( P>0.05). Conclusions:Sal B can reduce the inflammatory responses of VSMCs, and the mechanism may be related to promoting the expression of circACTA2 in septic mice.
2.Meta-synthesis of qualitative studies on the authentic experience of moral dilemmas of ICU nurses
Baosheng ZHAO ; Juan WANG ; Manli WANG ; Junjie SHI ; Jun DU ; Fengxia LI
Chinese Journal of Nursing 2024;59(10):1263-1269
Objective To systematically evaluate the psychological feelings and real experience of ICU nurses'moral dilemma,and the aim is to provide references for the formulation of targeted psychological intervention programs.Methods Pubmed,CINAHL,Embase,Web of Science,Cochrane Library,PsycNET,CNKI,Wanfang database,VIP database,and China Biomedical Literature Database were searched by computer to retrieve qualitative studies on psychological perceptions and real experiences of moral dilemmas of ICU nurses,and the search time was from the establishment of databases to March 2023.The quality of literature was evaluated using the 2016 version of the Australian JBI Centre for Evidence-Based Health Care Quality Evaluation Criteria for qualitative studies,and the results were integrated and analyzed by the Meta integration method.Results A total of 10 pieces of literature were included,including 3 phenomenological studies and 7 descriptive studies.The results of 27 studies were summarized into 8 new categories,and 3 integrated results were obtained:①The internal and external causes of moral dilemmas,including conflicting concepts of life and death,differences of the idea of the superior death,conflict of clinical decision-making,and insufficient organizational support;②The progressive effects of moral dilemmas,including aggravating negative emotional burden and breeding professional identity bias;③ Coping strategies for moral dilemmas,including active self-debugging and seeking support from colleagues.Conclusion It is urgent to popularize the scientific concept of life and death of patients and families,deepen the concept of superior death of ICU nurses,and alleviate the conflict of nursing and patient decision-making.It is important to pay attention to the negative emotional state of ICU nurses,improve their professional identity,improve the self-adjustment ways of ICU nurses,and to provide multi-organization support for ICU nurses.
3.Carrier screening for 223 monogenic diseases in Chinese population:a multi-center study in 33 104 individuals
Wei HOU ; Xiaolin FU ; Xiaoxiao XIE ; Chunyan ZHANG ; Jiaxin BIAN ; Xiao MAO ; Juan WEN ; Chunyu LUO ; Hua JIN ; Qian ZHU ; Qingwei QI ; Yeqing QIAN ; Jing YUAN ; Yanyan ZHAO ; Ailan YIN ; Shutie LI ; Yulin JIANG ; Manli ZHANG ; Rui XIAO ; Yanping LU
Journal of Southern Medical University 2024;44(6):1015-1023
Objective To investigate the epidemiological characteristics and mutation spectrum of monogenic diseases in Chinese population through a large-scale,multicenter carrier screening.Methods This study was conducted among a total of 33 104 participants(16 610 females)from 12 clinical centers across China.Carrier status for 223 genes was analyzed using high-throughput sequencing and different PCR methods.Results The overall combined carrier frequency was 55.58%for 197 autosomal genes and 1.84%for 26 X-linked genes in these participants.Among the 16 669 families,874 at-risk couples(5.24%)were identified.Specifically,584 couples(3.50%)were at risk for autosomal genes,306(1.84%)for X-linked genes,and 16 for both autosomal and X-linked genes.The most frequently detected autosomal at-risk genes included GJB2(autosomal recessive deafness type 1A,393 couples),HBA1/HBA2(α-thalassemia,36 couples),PAH(phenylketonuria,14 couples),and SMN1(spinal muscular atrophy,14 couples).The most frequently detected X-linked at-risk genes were G6PD(G6PD deficiency,236 couples),DMD(Duchenne muscular dystrophy,23 couples),and FMR1(fragile X syndrome,17 couples).After excluding GJB2 c.109G>A,the detection rate of at-risk couples was 3.91%(651/16 669),which was lowered to 1.72%(287/16 669)after further excluding G6PD.The theoretical incidence rate of severe monogenic birth defects was approximately 4.35‰(72.5/16 669).Screening for a battery of the top 22 most frequent genes in the at-risk couples could detect over 95%of at-risk couples,while screening for the top 54 genes further increased the detection rate to over 99%.Conclusion This study reveals the carrier frequencies of 223 monogenic genetic disorders in the Chinese population and provides evidence for carrier screening strategy development and panel design tailored to the Chinese population.In carrier testing,genetic counseling for specific genes or gene variants can be challenging,and the couples need to be informed of these difficulties before testing and provided with options for not screening these genes or gene variants.
4.Carrier screening for 223 monogenic diseases in Chinese population:a multi-center study in 33 104 individuals
Wei HOU ; Xiaolin FU ; Xiaoxiao XIE ; Chunyan ZHANG ; Jiaxin BIAN ; Xiao MAO ; Juan WEN ; Chunyu LUO ; Hua JIN ; Qian ZHU ; Qingwei QI ; Yeqing QIAN ; Jing YUAN ; Yanyan ZHAO ; Ailan YIN ; Shutie LI ; Yulin JIANG ; Manli ZHANG ; Rui XIAO ; Yanping LU
Journal of Southern Medical University 2024;44(6):1015-1023
Objective To investigate the epidemiological characteristics and mutation spectrum of monogenic diseases in Chinese population through a large-scale,multicenter carrier screening.Methods This study was conducted among a total of 33 104 participants(16 610 females)from 12 clinical centers across China.Carrier status for 223 genes was analyzed using high-throughput sequencing and different PCR methods.Results The overall combined carrier frequency was 55.58%for 197 autosomal genes and 1.84%for 26 X-linked genes in these participants.Among the 16 669 families,874 at-risk couples(5.24%)were identified.Specifically,584 couples(3.50%)were at risk for autosomal genes,306(1.84%)for X-linked genes,and 16 for both autosomal and X-linked genes.The most frequently detected autosomal at-risk genes included GJB2(autosomal recessive deafness type 1A,393 couples),HBA1/HBA2(α-thalassemia,36 couples),PAH(phenylketonuria,14 couples),and SMN1(spinal muscular atrophy,14 couples).The most frequently detected X-linked at-risk genes were G6PD(G6PD deficiency,236 couples),DMD(Duchenne muscular dystrophy,23 couples),and FMR1(fragile X syndrome,17 couples).After excluding GJB2 c.109G>A,the detection rate of at-risk couples was 3.91%(651/16 669),which was lowered to 1.72%(287/16 669)after further excluding G6PD.The theoretical incidence rate of severe monogenic birth defects was approximately 4.35‰(72.5/16 669).Screening for a battery of the top 22 most frequent genes in the at-risk couples could detect over 95%of at-risk couples,while screening for the top 54 genes further increased the detection rate to over 99%.Conclusion This study reveals the carrier frequencies of 223 monogenic genetic disorders in the Chinese population and provides evidence for carrier screening strategy development and panel design tailored to the Chinese population.In carrier testing,genetic counseling for specific genes or gene variants can be challenging,and the couples need to be informed of these difficulties before testing and provided with options for not screening these genes or gene variants.
5.Chinese Medicine Intervention in Diabetic Cardiomyopathy Based on Autophagy:A Review
Yunfeng YU ; Gang HU ; Manli ZHOU ; Xiahui ZHOU ; Yanzhen ZHAO ; Weixiong JIAN
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(22):276-282
Autophagy is a lysosome-dependent intracellular degradation process,and it is a key mechanism of diabetic cardiomyopathy (DCM). Autophagy has dual regulatory effects on DCM. Under physiological conditions,normal autophagy can promote the decomposition of damaged cardiomyocytes and metabolites,so as to reduce the damage of harmful substances to the body and provide energy for cardiomyocytes. Under pathological conditions,the inhibited autophagy of cardiomyocytes will cause the accumulation of damaged cells and metabolites,which will cause damage to cardiomyocytes and eventually aggravate cardiac dysfunction in the patients with DCM. However,the over autophagy of cardiomyocytes will lead to autophagic death of a large number of cardiomyocytes and result in pathological myocardial remodeling and cardiac dysfunction,thus promoting the progression of DCM. Therefore,the restoration of a normal autophagy level is the key means to protect cardiomyocytes and improve the prognosis of DCM. Chinese medicine can regulate autophagy to treat DCM. Specifically,it can promote autophagy (making up for deficiency) or inhibit autophagy (removing excess) to restore the balance of autophagy,thereby alleviating DCM.
6.Chinese expert consensus on emergency surgery for severe trauma and infection prevention during corona virus disease 2019 epidemic (version 2023)
Yang LI ; Yuchang WANG ; Haiwen PENG ; Xijie DONG ; Guodong LIU ; Wei WANG ; Hong YAN ; Fan YANG ; Ding LIU ; Huidan JING ; Yu XIE ; Manli TANG ; Xian CHEN ; Wei GAO ; Qingshan GUO ; Zhaohui TANG ; Hao TANG ; Bingling HE ; Qingxiang MAO ; Zhen WANG ; Xiangjun BAI ; Daqing CHEN ; Haiming CHEN ; Min DAO ; Dingyuan DU ; Haoyu FENG ; Ke FENG ; Xiang GAO ; Wubing HE ; Peiyang HU ; Xi HU ; Gang HUANG ; Guangbin HUANG ; Wei JIANG ; Hongxu JIN ; Laifa KONG ; He LI ; Lianxin LI ; Xiangmin LI ; Xinzhi LI ; Yifei LI ; Zilong LI ; Huimin LIU ; Changjian LIU ; Xiaogang MA ; Chunqiu PAN ; Xiaohua PAN ; Lei PENG ; Jifu QU ; Qiangui REN ; Xiguang SANG ; Biao SHAO ; Yin SHEN ; Mingwei SUN ; Fang WANG ; Juan WANG ; Jun WANG ; Wenlou WANG ; Zhihua WANG ; Xu WU ; Renju XIAO ; Yang XIE ; Feng XU ; Xinwen YANG ; Yuetao YANG ; Yongkun YAO ; Changlin YIN ; Yigang YU ; Ke ZHANG ; Xingwen ZHANG ; Guixi ZHANG ; Gang ZHAO ; Xiaogang ZHAO ; Xiaosong ZHU ; Yan′an ZHU ; Changju ZHU ; Zhanfei LI ; Lianyang ZHANG
Chinese Journal of Trauma 2023;39(2):97-106
During coronavirus disease 2019 epidemic, the treatment of severe trauma has been impacted. The Consensus on emergency surgery and infection prevention and control for severe trauma patients with 2019 novel corona virus pneumonia was published online on February 12, 2020, providing a strong guidance for the emergency treatment of severe trauma and the self-protection of medical staffs in the early stage of the epidemic. With the Joint Prevention and Control Mechanism of the State Council renaming "novel coronavirus pneumonia" to "novel coronavirus infection" and the infection being managed with measures against class B infectious diseases since January 8, 2023, the consensus published in 2020 is no longer applicable to the emergency treatment of severe trauma in the new stage of epidemic prevention and control. In this context, led by the Chinese Traumatology Association, Chinese Trauma Surgeon Association, Trauma Medicine Branch of Chinese International Exchange and Promotive Association for Medical and Health Care, and Editorial Board of Chinese Journal of Traumatology, the Chinese expert consensus on emergency surgery for severe trauma and infection prevention during coronavirus disease 2019 epidemic ( version 2023) is formulated to ensure the effectiveness and safety in the treatment of severe trauma in the new stage. Based on the policy of the Joint Prevention and Control Mechanism of the State Council and by using evidence-based medical evidence as well as Delphi expert consultation and voting, 16 recommendations are put forward from the four aspects of the related definitions, infection prevention, preoperative assessment and preparation, emergency operation and postoperative management, hoping to provide a reference for severe trauma care in the new stage of the epidemic prevention and control.
7.Inflammatory Mechanism of Coronary Heart Disease and Intervention Strategies of Chinese Medicine Based on ''Phlegm,Stasis,Toxin'' Theory
Pei LIU ; Yunfeng YU ; Xinyu YANG ; Manli ZHOU ; Yanzhen ZHAO ; Weixiong JIAN
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(12):185-191
More and more evidence shows that there is a close relationship between the inflammatory state and coronary heart disease. Inflammatory state triggers the damage of vascular endothelium in the early stage of coronary heart disease and ultimately mediates the formation of atherosclerotic plaque. The mechanism of occurrence and development of heart disease is of great significance. Phlegm is a pathological product formed by the subtle imbalance of the spleen and stomach in the transportation and transformation of water and grain. It is the general summary of a series of abnormally accumulated inflammatory substances, such as low density lipoprotein, inflammatory cells, and inflammatory factors. The nature of Phlegm determines the invasiveness and turbidity of Phlegm. Phlegm invades the meridians, causing damage to the meridians and gradually accumulating, which eventually causes the local meridian damage to aggravate. This process is similar to the persistent damage of the vascular endothelium caused by inflammation. Phlegm blocks the meridians, affects the operation of Qi and blood, causes Qi stagnation and blood stasis, and finally forms the outcome of heart and blood stasis. This process is similar to the mechanism of atherosclerotic plaques formed by continuous inflammatory damage. Heart blood stasis, depression and heat, heat toxin endogenous, forming the syndrome of heat toxin stasis, which is similar to the process of atherosclerotic plaque rupture and thrombosis causing acute cardiovascular events.The formation of Phlegm is rooted in the deficiency of spleen. Based on the ''phlegm,stasis,toxin'' theory, spleen deficiency is the intrinsic pathogenesis of the inflammatory state of coronary heart disease, and the invasion of phlegm, blood stasis of heart, heat and blood stasis are the evolution of inflammatory damage of coronary heart disease. Traditional Chinese medicine differentiation and treatment is based on strengthening the spleen and nourishing Qi to treat the root and removing phlegm and blood stasis, and clearing heat and detoxifying to treat symptoms. The related Chinese medicine compounds, Chinese patent medicines, and single Chinese medicines can reduce the inflammatory indicators of coronary heart disease, thereby improving the prognosis of coronary heart disease.
8.Strategies of Traditional Chinese Medicine Standardized Treatment Based on Syndrome Differentiation of Bipolar Disorder
Yunfeng YU ; Manli ZHOU ; Xiaoxin LUO ; Yanzhen ZHAO ; Weixiong JIAN
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(21):180-187
Bipolar disorder (BD) is a serious chronic emotional disorder with a high suicide rate and a common psychiatric disease. Traditional Chinese medicine (TCM) treatment based on syndrome differentiation of BD has unique advantages and good safety, which is expected to become a breakthrough in the treatment. Based on Expert Consensus on TCM Syndrome Differentiation Criteria for Bipolar Disorder by Professor Yin Dongqing and Professor Jia Hongxiao, this study collated the treatment protocols of BD with various syndrome types according to Meta-analysis of the existing literature in the database and evaluated the evidence level according to the evidence evaluation standard issued by the US Agency for Healthcare Research and Quality (AHRQ). (1) Depression attack. ① Liver depression and spleen deficiency syndrome: Xiaoyaosan pills or Shugan Jieyu capsules, ② Phlegm dampness and spleen stagnation syndrome: Wendantang modified with Tianwang Buxindan, ③ Heart and spleen deficiency syndrome: Jiuwei Zhenxin Granules or DANG's Ganmai Dazhaotang, ④ Fire heat and internal depression syndrome: Danzhi Xiaoyaosan Granules or Chaihu Longgu Mulitang, ⑤ Liver and kidney deficiency syndrome: JIANG's Buganshen Decoction. (2) Mania episode. ① Heart and liver fire hyperactivity syndrome: Zhengan Ningshen Formula, ② Phlegm heat harassing spirit syndrome: Huatan Xiehuo Dingshen decoction, Lianzhi Tongqiao Anshen decoction, Qingshen Dingkuang decoction or Qingshen Xingnao decoction, ③ Liver and gallbladder dampness-heat syndrome: Longdan Xiegantang. (3) Other syndrome types. ① Liver qi stagnation syndrome: modified Tongqiao Huoxue decoction, Shengyang Yiwei Acupuncture, ② Deficiency of kidney yang syndrome: Jingui Shenqitang, ③ Phlegm accumulation and blood stasis syndrome: modified Tongqiao Huoxue decoction, ④ Qi and Yin deficiency, stagnation of blood stasis syndrome: Xinnaoxin pills, ⑤ Syndrome of blood deficiency generating wind and fire heat harassing spirit: Fangji Dihuangtang.
9.Application of holographic image in transperineal prostate targeted biopsy
Lei WANG ; Zichen ZHAO ; Hongfeng GUO ; Manli NA ; Mengshen LI ; Yi WANG ; Ningchen LI ; Yanqun NA
Chinese Journal of Urology 2022;43(2):111-115
Objective:To investigate the feasibility and accuracy of transperineal prostate targeted biopsy guided by holographic image.Methods:Clinical data of 10 patients with transperineal prostate targeted biopsy guided by holographic image in Peking University Shougang Hospital between May and September 2020 were analyzed retrospectively. The average age was (70.9±10.3) years old, the median PSA was 15.1(6.02-1110.14) ng/ml, prostate MRI were performed before biopsy and the PI-RADS scores were all ≥ 3, and the number of suspicious target lesions was 1.4±0.5. CT examinations of urinary system were performed on the premise of mild lithotomy position and positioning stickers pasted on the skin of perineum and lower abdomen. The original data of CT and MRI were obtained, holographic image models were firstly made separately and then fused into a complete model, and the puncture paths were planned for the target lesions. At the time of puncture, the patient took the same body position as in CT scan, the operator wore a mixed reality head mounted display (HoloLens glasses), and the skin positioning stickers were used for visual registration between the holographic model and the real human body. Then under the guidance of the virtual puncture path, the puncture biopsy gun was placed, fired after reaching the predetermined depth, a transrectal ultrasound probe was placed to clarify the position of the puncture needle, and the objective accuracy of puncture was judged by comparison of ultrasound and MRI images. If the first shot was judged to be inaccurate, it was allowed to make a supplementary shot after adjusting the angle. After holographic guided biopsies, cognitive fusion targeted biopsies and 12-needle systematic biopsies were performed routinely, and the proportion of positive needles of the three different biopsy methods were calculated respectively.Results:All the 10 cases were successfully completed, including 16 holographic image guided shots, 28 cognitive fusion targeted shots and 116 systematic shots. The objective accuracy of holographic image guided biopsy after first shot judgments was 68.8% (11/16), while it raised to 87.5% (14/16) after supplementary shots. The proportion of positive needles in the three puncture methods were 56.3% (9/16), 42.9% (12/28) and 19.8% (23/116), respectively ( P=0.002). The results of subjective questionnaire showed that holographic model was helpful to improve the spatial understanding of lesions. The satisfaction of intraoperative holographic registration and guided puncture were 90% and 60%, respectively. No puncture related complication occurred in this group. Conclusion:The study preliminarily confirmed the feasibility of holographic image-guided prostate targeted biopsy. This new puncture method has better objective accuracy, and the proportion of positive needles is significantly better than systematic biopsy.
10.Chinese Medicine Regulates Atherosclerosis-related Signaling Pathway: A Review
Manli ZHOU ; Yanzhen ZHAO ; Yunfeng YU ; Yifan ZHANG ; Yilei HU ; Weixiong JIAN
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(15):232-239
Atherosclerotic cardiovascular disease is a common disease with high incidence rate and mortality worldwide. Atherosclerosis is an important pathological basis for the formation of ischemic cardiovascular diseases such as cardiovascular disease, which is related to inflammation, oxidative stress, apoptosis, vascular endothelial damage, foam cell formation, platelet activation, and so on, involving mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK), nuclear factor-kappa B (NF-κB), and other signaling pathways. In the past few decades, high-intensity statins were mainly used to treat atherosclerosis by reducing blood lipid levels, which usually caused obvious side effects. Therefore, the development of safer and more effective drugs and treatment modes is the focus of research at this stage. In recent years, Chinese medicine has been playing an increasingly important role in the prevention and treatment of cardiovascular diseases in China. There are many studies on the mechanism of Chinese medicine in the prevention and treatment of atherosclerosis, and it is found that a variety of single Chinese medicine regulate the formation process of atherosclerosis by regulating targeted signal molecules. This paper reviewed the research results of related signaling pathways involved in the pathological formation of atherosclerosis and the mechanism of Chinese medicine in the prevention and treatment of cardiovascular diseases, thereby providing references for the clinical treatment of cardiovascular diseases.

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