1.Effects of fractionated low-dose ionizing radiation on differentially expressed genes in ferroptosis of human bronchial epithelial cells
Min ZHANG ; Lingyu ZHANG ; Yashi CAI ; Huixian LI ; Yanting CHEN ; Guanyou CHEN ; Xin LAN ; Changyong WEN ; Weixu HUANG ; Jianming ZOU ; Huifeng CHEN
Chinese Journal of Radiological Health 2025;34(3):310-317
		                        		
		                        			
		                        			Objective To investigate the effects of fractionated low-dose ionizing radiation (LDIR) on the ferroptosis in human bronchial epithelial (HBE) cells as well as the associated differentially expressed genes (DEGs), biological processes, and signaling pathways. Methods HBE cells were exposed to different single doses of X-ray irradiation (0, 25, 50, 75, and 100 mGy) for 24, 48, and 72 h, respectively. The change in cell viability was detected by MTT assay. Cells were irradiated with 0, 25, 50, and 100 mGy X-rays 5 times, with 48 h between each irradiation and a dose rate of 50 mGy/min. Cells were harvested 24 h after irradiation for the measurement of the expression of ferroptosis-related genes SLC7A11 and GPX4 at the mRNA and protein levels, cellular iron content, and the expression of FTH1 and FTL mRNAs. High-throughput sequencing was used to screen for the DEGs in each dose group, followed by Gene Ontology-Biological Process (GO-BP) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA). Results Compared with the control group, single-dose LDIR significantly increased cell proliferation at 75 mGy after 24 h (P < 0.05), at 50, 75, and 100 mGy after 48 h (P < 0.05), and at 75 and 100 mGy after 72 h (P < 0.05). Compared with the control group, at the end of the fifth fractionated LDIR, SLC7A11 and GPX4 mRNAs decreased at all doses (P < 0.05), SLC7A11 protein decreased at all doses, GPX4 protein decreased at 25 and 100 mGy, iron content increased at all doses, and FTH1 and FTL mRNAs decreased at all doses (P< 0.05). Sequencing analysis identified 248, 30, and 291 DEGs and 10, 2, and 9 ferroptosis-associated genes at the three doses compared to the control. Gene Ontology-Biological Process analysis showed that DEGs were mainly enriched in biological processes such as response to lipids, cell death, and response to unfolded proteins. Kyoto Encyclopedia of Genes and Genomes analysis showed that DEGs were mainly enriched in the JAK-STAT signaling pathway, lipids and atherosclerosis, ferroptosis, protein processing in the endoplasmic reticulum, and FoxO signaling pathway. Gene set enrichment analysis showed that DEGs were mainly enriched in ferroptosis, fatty acid degradation, and glutathione metabolism. Conclusion Fractionated low-dose radiation induced ferroptosis in HBE cells, and DEGs were predominantly enriched in biological processes and signaling pathways related to inflammation, ferroptosis, and endoplasmic reticulum stress.
		                        		
		                        		
		                        		
		                        	
2.Effects of imperatorin on malignant biological behavior of gastric cancer cells by regulating ThPOK expression
Lan CHEN ; Lingli XIA ; Ying CHEN ; Gang ZHANG ; Feng WEN
China Pharmacy 2025;36(2):191-196
		                        		
		                        			
		                        			OBJECTIVE To investigate the effects of imperatorin (IMP-SD) on malignant biological behavior of gastric cancer (GC) cells by regulating zinc finger and BTB domain 7B (ThPOK). METHODS Human GC cells MKN-7 were used as the research object and then divided into control group (no treatment), IMP-SD low-, medium- and high-concentration groups (40, 80 and 160 μmol/L IMP-SD), si-ThPOK and si-NC group [treated with 160 μmol/L IMP-SD and then transfected with ThPOK small interfering RNA (si-ThPOK) or its negative control (si-NC)]. After treatment, cell clone formation, migration and invasion abilities and apoptosis of MKN-7 cells were detected; the killing activity of NK cells, T cells classification, the protein expressions of ThPOK, programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) were all determined. RESULTS Compared with the control group, the number of cell clones, migration number, invasion number, and the protein expressions of PD-1 and PD-L1 were decreased or down-regulated significantly in IMP-SD groups, while the cell apoptotic rate, NK cell killing activity, CD4+ T proportion, the ratio of CD4+ T proportion and CD8+ T proportion (CD4+ T/CD8+ T), and the protein expression of ThPOK were increased or up-regulated significantly, in a concentration-dependent manner (P<0.05). Compared with IMP-SD high-concentration group and si-NC group, the number of cell clones, migration number, invasion number, and the protein expressions of PD-1 and PD-L1 were increased or up-regulated significantly in si-ThPOK group, while the cell apoptotic rate, NK cell killing activity, CD4+ T proportion, CD4+ T/CD8+ T, and the protein expression of ThPOK were decreased or down-regulated significantly (P<0.05). CONCLUSIONS IMP-SD may reduce the clonal formation, migration and invasion abilities of GC cells, promote their apoptosis and inhibit their immune escape by promoting ThPOK expression.
		                        		
		                        		
		                        		
		                        	
3.Effects of imperatorin on malignant biological behavior of gastric cancer cells by regulating ThPOK expression
Lan CHEN ; Lingli XIA ; Ying CHEN ; Gang ZHANG ; Feng WEN
China Pharmacy 2025;36(2):191-196
		                        		
		                        			
		                        			OBJECTIVE To investigate the effects of imperatorin (IMP-SD) on malignant biological behavior of gastric cancer (GC) cells by regulating zinc finger and BTB domain 7B (ThPOK). METHODS Human GC cells MKN-7 were used as the research object and then divided into control group (no treatment), IMP-SD low-, medium- and high-concentration groups (40, 80 and 160 μmol/L IMP-SD), si-ThPOK and si-NC group [treated with 160 μmol/L IMP-SD and then transfected with ThPOK small interfering RNA (si-ThPOK) or its negative control (si-NC)]. After treatment, cell clone formation, migration and invasion abilities and apoptosis of MKN-7 cells were detected; the killing activity of NK cells, T cells classification, the protein expressions of ThPOK, programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) were all determined. RESULTS Compared with the control group, the number of cell clones, migration number, invasion number, and the protein expressions of PD-1 and PD-L1 were decreased or down-regulated significantly in IMP-SD groups, while the cell apoptotic rate, NK cell killing activity, CD4+ T proportion, the ratio of CD4+ T proportion and CD8+ T proportion (CD4+ T/CD8+ T), and the protein expression of ThPOK were increased or up-regulated significantly, in a concentration-dependent manner (P<0.05). Compared with IMP-SD high-concentration group and si-NC group, the number of cell clones, migration number, invasion number, and the protein expressions of PD-1 and PD-L1 were increased or up-regulated significantly in si-ThPOK group, while the cell apoptotic rate, NK cell killing activity, CD4+ T proportion, CD4+ T/CD8+ T, and the protein expression of ThPOK were decreased or down-regulated significantly (P<0.05). CONCLUSIONS IMP-SD may reduce the clonal formation, migration and invasion abilities of GC cells, promote their apoptosis and inhibit their immune escape by promoting ThPOK expression.
		                        		
		                        		
		                        		
		                        	
4.Clinical Observation on Qigui Tongluo Oral Liquid Combined with Moxibustion for the Treatment of Chronic Fatigue Syndrome of Qi Deficiency and Blood Stasis Type Based on Qi-Collateral Theory
Yi ZHANG ; Shao-Lan CHEN ; Mei-Ling WANG ; Hai-Wen HUANG ; Min GAO
Journal of Guangzhou University of Traditional Chinese Medicine 2024;41(1):41-47
		                        		
		                        			
		                        			Objective To observe the clinical efficacy of Qigui Tongluo Oral Liquid(a hospital preparation developed by Guangdong Second Traditional Chinese Medicine Hospital and being composed of Astragali Radix,Angelicae Sinensis Radix,Notoginseng Radix et Rhizoma,Paeoniae Radix Rubra,Achyranthis Bidentatae Radix,Spatholobi Caulis,Salviae Miltiorrhizae Radix et Rhizoma,Pheretima,etc.)combined with moxibustion for the treatment of chronic fatigue syndrome(CFS)of qi deficiency and blood stasis type on the basis of qi-collateral theory.Methods A retrospective study was conducted in 60 CFS patients with qi deficiency and blood stasis type.The patients were divided into an observation group and a control group,with 30 patients in each group according to the therapy.The control group was treated with mild moxibustion on Shenque(CV8)point with moxa sticks,and the observation group was treated with Qigui Tongluo Oral Liquid on the basis of treatment for the control group.The course of treatment lasted for 4 weeks.The changes of traditional Chinese medicine(TCM)syndrome scores,Fatigue Scale-14(FS-14)scores,serum immunoglobulin IgA,IgM,IgG levels,and cortisol(COR)level in the two groups were observed before and after the treatment.After treatment,the clinical efficacy and safety of the two groups were evaluated.Results(1)After 4 weeks of treatment,the total effective rate of the observation group was 96.67%(29/30),and that of the control group was 80.00%(24/30).The intergroup comparison showed that the clinical efficacy of the observation group was significantly superior to that of the control group(P<0.05).(2)After treatment,the TCM syndrome scores and FS-14 scores of patients in the two groups were significantly decreased compared with those before treatment(P<0.01),and the effect on decreasing TCM syndrome scores and FS-14 scores in the observation group was significantly superior to that in the control group(P<0.01).(3)After treatment,the serum IgA and IgG levels of the two groups as well as the serum IgM and COR levels of the observation group were significantly increased compared with those before treatment(P<0.01),and the effect of the observation group on increasing serum IgA,IgM,IgG,and COR levels was significantly superior to that of the control group(P<0.05 or P<0.01).(4)During the treatment,there were no significant adverse reactions occurring in the two groups.Conclusion Healthy-qi deficiency and collateral obstruction contribute to the core pathogenesis of CFS.Based on the TCM qi-collateral theory and following the therapeutic principle of replenishing deficiency and unblocking collaterals,Qigui Tongluo Oral Liquid combined with moxibustion for the treatment of CFS patients with qi deficiency and blood stasis type can achieve certain efficacy.The combined therapy could significantly alleviate the clinical symptoms,improve the immunity level,and regulate the neuro-endocrine-immune(NEI)network of the patients.
		                        		
		                        		
		                        		
		                        	
5.Effects of Electroacupuncture on Skeletal Muscle Mitochondrial Dynamics in SAMP8 Mice
Ruolan WEN ; Wanqing GUO ; Weiguo DONG ; Limin CHEN ; Jinquan LI ; Chenguang WEN ; Lan LIN ; Feng WANG
Chinese Journal of Information on Traditional Chinese Medicine 2024;31(2):104-109
		                        		
		                        			
		                        			Objective To observe the effects of electroacupuncture on the motor function and mitochondrial dynamics of skeletal muscle of SAMP8 mice;To explore the mechanism of electroacupuncture in improving the motor dysfunction of Alzheimer disease(AD)from the perspective of mitochondrial dynamics.Methods Totally 18 SAMP8 mice were divided into model group and electroacupuncture group,with 9 mice in each group,and the SAMR1 mice with the same age were set as control group."Baihui","Dazhui"and"Shenshu"were selected in the electroacupuncture group,and electroacupuncture was performed daily for 20 min,8 d as a course of treatment.Each course of treatment was separated by 2 d,for a total of 3 courses of treatment.The model group and the control group were not intervened.The motor function of mice was tested by grip strength test,suspension test,hind limb extension test and Morris water maze experiment.The morphology and structure of gastrocnemius were observed by HE staining,ATP content in gastrocnemius was determined by colorimetry,the mRNA expression of optic atrophy 1(OPA1),mitofusin 2(MFN2)and dynamin-related protein 1(DRP1)in gastrocnemius were detected by real-time quantitative PCR,the expressions of OPA1,MFN2 and DRP1 in gastrocnemius were detected by Western blot.Results Compared with the control group,the grip strength,the score in suspension test,and the average speed and maximum speed of Morris water maze experiment of mice in model group significantly decreased(P<0.01);the arrangement of fibers in the gastrocnemius muscle tissue was disordered,the gaps become wider,and the distribution of nuclei was uneven;the ATP content in the gastrocnemius muscle tissue was significantly decreased(P<0.01),the mRNA and protein expressions of OPA1 and MFN2 were significantly decreased(P<0.01),and the expression of DRP1 mRNA and protein significantly increased(P<0.01).Compared with the model group,the grip strength,the score in suspension test,and the average speed and maximum speed of Morris water maze experiment in electroacupuncture group significantly increased(P<0.01);the arrangement of gastrocnemius muscle tissue was relatively neat,the gaps become narrower,and the distribution of nuclei was more uniform;the ATP content in gastrocnemius muscle tissue significantly increased(P<0.01),while the mRNA and protein expressions of OPA1 and MFN2 significantly increased(P<0.05,P<0.01),the expression of DRP1 mRNA and protein significantly decreased(P<0.01).Conclusion Electroacupuncture can improve the skeletal muscle morphological structure and motor dysfunction of SAMP8 mice,and the mechanism may be related to the correction of skeletal muscle mitochondrial dynamic imbalance and the increase of skeletal muscle ATP content.
		                        		
		                        		
		                        		
		                        	
6.A Case Report of Multidisciplinary Diagnosis and Treatment of a Patient with Tuberous Sclerosis Complex and Multi-Organ Involvement
Hua ZHENG ; Yunfei ZHI ; Lujing YING ; Lan ZHU ; Mingliang JI ; Ze LIANG ; Jiangshan WANG ; Haifeng SHI ; Weihong ZHANG ; Mengsu XIAO ; Yushi ZHANG ; Kaifeng XU ; Zhaohui LU ; Yaping LIU ; Ruiyi XU ; Huijuan ZHU ; Li WEN ; Yan ZHANG ; Gang CHEN ; Limeng CHEN
JOURNAL OF RARE DISEASES 2024;3(1):79-86
Tuberous sclerosis complex(TSC)is a rare genetic disease that can lead to benign dysplasia in multiple organs such as the skin, brain, eyes, oral cavity, heart, lungs, kidneys, liver, and bones. Its main symptoms include epilepsy, intellectual disabilities, skin depigmentation, and facial angiofibromas, whilst incidence is approximately 1 in 10 000 to 1 in 6000 newborns. This case presents a middle-aged woman who initially manifested with epilepsy and nodular depigmentation. Later, she developed a lower abdominal mass, elevated creatinine, and severe anemia. Based on clinical features and whole exome sequencing, the primary diagnosis was confirmed as TSC. Laboratory and imaging examinations revealed that the lower abdominal mass originated from the uterus. CT-guided biopsy pathology and surgical pathology suggested a combination of leiomyoma and abscess. With the involvement of multiple organs and various complications beyond the main diagnosis, the diagnostic and therapeutic process for this patient highlights the importance of rigorous clinical thinking and multidisciplinary collaboration in the diagnosis and treatment of rare and challenging diseases.
7.Cost-effectiveness analysis of chemical treatment with drones for Oncomelania hupensis control in marshland and lake areas
Yong CHEN ; Xiaojuan XU ; Daolong WEN ; Bo DAI ; Lan GAO ; Rong ZHANG ; Qingqing HUANG ; Linlin LI ; Fan ZHA ; Liang FANG ; Ping ZHANG ; Shiqing ZHANG ; Chunli CAO
Chinese Journal of Schistosomiasis Control 2024;36(5):502-506
		                        		
		                        			
		                        			 Objective To evaluate the molluscicidal effect and cost of spraying molluscicides with drones against Oncomelania hupensis snails in marshland and lake areas, so as to provide new insights into field snail control in China. Methods A marshland and lake setting measuring approximately 12 000 m2 was selected in Wanzhi District, Wuhu City on June 2023 as the test field, and assigned to four groups, of 3 000 m2 in each group. Environmental cleaning was not conducted in groups A or B, which were given 5% niclosamide ethanolamine salt granules sprayed with knapsack-type sprayers and drones at a dose of 40 g/m2, and environmental cleaning was conducted in groups C and D, which were given 5% niclosamide ethanolamine salt granules sprayed with drones and knapsack-type sprayers at a dose of 40 g/m2, respectively. O. hupensis snails were surveyed before chemical treatment and 1, 3, 5, 7, 14 days post-treatment. The uniformity of chemicals was determined on the day of treatment, and the snail mortality, corrected snail mortality and density of living snails were calculated and compared among groups. The cost of molluscicides, labor fees of environmental cleaning and chemical treatment and cost of equipment were calculated, and the cost for a 1% reduction in the mean density of living snails was calculated 14 days post-treatment. Results The mean densities of living snails and mortality rates of snails were 1.82 to 2.85 snails/0.1 m2 and 1.41% to 2.94% in groups A, B, C and D before chemical treatment, and the mortality and corrected mortality of snails were 55.75%, 49.32%, 85.94% and 87.50%, and 55.00%, 48.47%, 85.70% and 87.29% in groups A, B, C and D 14 days post-treatment. There was a significant difference in the mortality of snails among the four groups 14 days post-treatment (χ2 = 38.735, P < 0.005), and there was a higher snail mortality in Group D than in Group A (χ2 = 16.876, P < 0.005), and higher in Group C than in Group B (χ2 = 20.508, P < 0.005). The density of living snails reduced by 55.00%, 43.94%, 90.43% and 87.14% 14 days post-treatment relative to pre-treatment in groups A, B, C and D, respectively. The test for uniformity of chemicals showed that the mean dose of molluscicides were 57.34, 55.21, 40.19 g/m2 and 32.37 g/m2 in groups A, B, C and D, respectively, and the minimal standard deviation (7.07) and coefficient of variation (0.18) of mean doses were seen in Group C. The costs for chemical treatment were 0.33 Yuan in groups A and B and 1.53 Yuan in groups C and D, respectively. The costs for a 1% reduction in the mean density of living snails were 17.82, 22.47, 50.73 Yuan and 52.56 Yuan in groups A, B, C, and D 14 days post-treatment, respectively. Conclusions The molluscicidal effect and cost of spraying 5% niclosamide ethanolamine salt granules with drones are comparable to manual spraying, and chemical treatment with drones are high in uniformity of molluscicides, time- and labor-saving, and feasible for applications in complex environments, which deserves widespread applications in the field of snail control. 
		                        		
		                        		
		                        		
		                        	
8.Iodine Nutrition,Thyroid-stimulating Hormone,and Related Factors of Postpartum Women from three Different Areas in China:A Cross-sectional Survey
Yun Xiao SHAN ; Yan ZOU ; Chun Li HUANG ; Shan JIANG ; Wen Wei ZHOU ; Lan Qiu QIN ; Qing Chang LIU ; Yan Xiao LUO ; Xi Jia LU ; Qian De MAO ; Min LI ; Yu Zhen YANG ; Chen Li YANG
Biomedical and Environmental Sciences 2024;37(3):254-265
		                        		
		                        			
		                        			Objective Studies on the relationship between iodine,vitamin A(VA),and vitamin D(VD)and thyroid function are limited.This study aimed to analyze iodine and thyroid-stimulating hormone(TSH)status and their possible relationships with VA,VD,and other factors in postpartum women. Methods A total of 1,311 mothers(896 lactating and 415 non-lactating)from Hebei,Zhejiang,and Guangxi provinces were included in this study.The urinary iodine concentration(UIC),TSH,VA,and VD were measured. Results The median UIC of total and lactating participants were 142.00 μg/L and 139.95 μg/L,respectively.The median TSH,VA,and VD levels in all the participants were 1.89 mIU/L,0.44 μg/mL,and 24.04 ng/mL,respectively.No differences in the UIC were found between lactating and non-lactating mothers.UIC and TSH levels were significantly different among the three provinces.The rural UIC was higher than the urban UIC.Obese mothers had a higher UIC and a higher prevalence of excessive TSH.Higher UICs and TSHs levels were observed in both the VD deficiency and insufficiency groups than in the VD-sufficient group.After adjustment,no linear correlation was observed between UIC and VA/VD.No interaction was found between vitamins A/D and UIC on TSH levels. Conclusion The mothers in the present study had no iodine deficiency.Region,area type,BMI,and VD may be related to the iodine status or TSH levels.
		                        		
		                        		
		                        		
		                        	
9.Clinical Observation on the Electroacupuncture at Shuigou Point Combined with Warming-Needle Moxibustion in the Treatment of Acute Cerebral Infarction
Xin CHEN ; Ti MO ; Shu-Wen LIU ; Lan-Zhu LI ; Jian-Hao LIU
Journal of Guangzhou University of Traditional Chinese Medicine 2024;41(6):1498-1503
		                        		
		                        			
		                        			Objective To observe the clinical efficacy of electroacupuncture at Shuigou(DU26)combined with warming-needle moxibustion in the treatment of acute cerebral infarction.Methods Nighty-six patients with acute cerebral infarction were randomly divided into observation group and control group,48 cases in each group.Both groups were given routine basic treatment.The control group was treated with warming-needle moxibustion.The observation group was treated with electroacupuncture at Shuigou point on the basis of the control group.The treatment was performed 5 days a week for a total of 3 weeks.After 3 weeks of treatment,the clinical efficacy of the two groups was evaluated,and the changes of National Institutes of Health Stroke Scale(NIHSS)score,Barthel Index(BI)score and Fugl-Meyer motor function assessment scale score of the two groups were observed before and after treatment.The changes of interleukin-6(IL-6),high-sensitivity C-reactive protein(hs-CRP)and homocysteine(Hey)levels were compared before and after treatment between the two groups.The safety and adverse reactions of the two groups were evaluated.Results(1)The total effective rate of the observation group was 95.83%(46/48),and that of the control group was 81.25%(39/48).The efficacy of the observation group was superior to that of the control group,and the difference was statistically significant(P<0.05).(2)After treatment,the NIHSS scores,BI scores,and FMA scores of the patients in the two groups were significantly improved(P<0.05),and the observation group was significantly superior to the control group in improving the NIHSS scores,BI scores,and FMA scores,with a statistically significant difference(P<0.05).(3)After treatment,the serum IL-6,hs-CRP,and Hey levels of patients in the two groups were significantly improved(P<0.05),and the observation group was significantly superior to the control group in improving the serum inflammatory factors of IL-6,hs-CRP,and Hey levels,with statistically significant differences(P<0.05).(4)Comparing the incidence of adverse reactions between the observation group and the control group,the difference was not statistically significant(P>0.05).Conclusion Electroacupuncture at Shuigou point combined with warming-needle moxibustion in the treatment of acute cerebral infarction can significantly promote the recovery of patients'neurological function,thus improving their motor and living ability,effectively reducing the levels of inflammatory factors and homocysteine,and the clinical efficacy is remarkable.
		                        		
		                        		
		                        		
		                        	
10.Inhibition of sigma-1 receptor reduces DRG cell apoptosis of rats with neuropathic pain
Dongling YU ; Shaoe MO ; Wen FU ; Shi CHEN ; Chouqin XIE ; Yuyan LAN
Basic & Clinical Medicine 2024;44(8):1101-1106
		                        		
		                        			
		                        			Objective To investigate the effect of sigma-1 receptor(sig-1R)inhibition on apoptosis in the dorsal root ganglion(DRG)of rats with sciatic nerve chronic constriction injury(CCI)-mediated neuropathic pain.Methods Rats undergoing intrathecal intubation were randomly divided into three groups with 12 in each:sham group,model group(CCI modeling one week after intrathecal intubation)and sig-1R inhibitor group(BD1047 group)that was injected intrathecally on the fourth,fifth,and sixth days after the CCI operation.The mechanical withdrawal threshold(MWT)of rats was detected on the day before surgery and then first,third,fifth and sev-enth days after surgery.The expression of sig-1R,Bcl-2 and Bax was detected by Western blot and immunofluo-rescence in DRG cell;TUNEL staining microscopy was used to observe the apoptosis of DRG cells.The changes of endoplasmic reticulum and mitochondria were observed by transmission electron microscopy of DRG cells.Results Compared with the sham group,the model group showed a decreased MWT at all time points after surgery,up-regulation of DRG cell apoptosis,up-regulation of sig-1R and Bax,down-regulation of Bcl-2,swelling of en-doplasmic reticulum,disruption of mitochondrial membrane and reduction of mitochondrial cristae in the DRG cell endoplasmic reticulum after surgery(P<0.05).BD1047 group showed elevated MWT at the fifth and seventh postoperative days,down-regulation of DRG cell apoptosis,down-regulated expression of sig-1R and Box,up-regulated expression of Bcl-2 and slightly damaged endoplasmic reticulum as well as mitochondria of DRG cells compared with model group(P<0.05).Conclusions Inhibition of sig-1R up-regulates mechanical withdrawal threshold and reduces DRG cell apoptosis in CCI rats.
		                        		
		                        		
		                        		
		                        	
            
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