1.Effects of LINC00626 on proliferation,apoptosis and drug resistance of colorectal cancer SW480 cells
Liang LI ; Hao QIANG ; Shui-ri WANG ; Fu-long YU ; Song WANG ; Hui YUAN ; Ya-ru YANG ; Zhi-ning LIU
Chinese Pharmacological Bulletin 2025;41(10):1900-1905
Aim To investigate the high expression of LINC00626 in colorectal cancer,and explore the effects of LINC00626 on the proliferation,apoptosis,and drug sensitivity of colorectal cancer SW480 cells,as well as its underlying mechanisms.Methods Flu-orescence in situ hybridization(FISH)was used to de-tect the expression levels of LINC00626 in 38 colorec-tal cancer tissues and their corresponding adjacent nor-mal tissues.The JASPAR database was utilized to pre-dict co-expressed genes and their possible binding sites.Cell transfection technology was employed to knockdown LINC00626.Western blot and qRT-PCR techniques were used to verify the transfection efficien-cy.CCK-8 assay,cell apoptosis and necrosis staining,and Western blot were used to detect the changes in the proliferation,apoptosis,drug sensitivity,and ap-optotic proteins of SW480 cells,respectively.Results The FISH results indicated that LINC00626 was highly expressed in colorectal cancer tissues(P<0.05).The expression of LINC00626 was not associat-ed with the age or gender of patients,but was related to the TNM stage and the presence of lymph node me-tastasis($ P<0.05 $).The results of CCK-8 assay and cell apoptosis and necrosis staining showed that af-ter knockdown of LINC00626,the proliferation ability of SW480 cells decreased,the apoptosis level in-creased,and the drug resistance decreased(P<0.05).Western blot results showed that with the de-crease in the expression level of LINC00626,the ex-pression of caspase-3 protein decreased,the expression of cleaved caspase-3 protein increased,and the expres-sion of Bcl-2 protein decreased(P<0.05).Conclu-sions LINC00626 is highly expressed in colorectal cancer and is associated with the TNM stage and the presence of lymph node metastasis.LINC00626 can af-fect the proliferation,apoptosis,and drug sensitivity of SW480 cells and alter the expression of apoptotic pro-teins.
2.Effects of Aqueous Extract of Corn Silk Combined with Training on Exercise Function and Glycolipid Metabolism in Mice with Metabolic Syndrome
Yi-lin LIU ; Zi-ling SONG ; Ting ZHOU ; Ji-ping CHEN ; Zi-han LIN ; Yu-xuan ZHANG ; Ji-qiang ZENG ; Shan-rong ZHANG ; Zhi-peng WU ; Chen LU ; Ying ZHU
Progress in Modern Biomedicine 2025;25(15):2411-2420
Objective:To investigate the effects of combination therapy with aqueous extract of corn silk(CS)and training on exercise capacity and glycolipid metabolism in mice with metabolic syndrome(MS).Methods:In this study,db/db mice were used as the animal model of MS.The mice were administered aqueous extract of CS via gavage and subjected to different intensities of training for 12 weeks(3 months).The specific experimental design was as follows:24 db/db mice were randomly divided into four groups on average:negative control group(NC),aqueous extract of CS group(CS),aqueous extract of CS+moderate-intensity training group(CS+MT),and CS aqueous extract of CS+high-intensity training group(CS+HT).The maximum running speed,forelimb grip strength,body weight and fasting blood glucose of mice were measured before and after treatment.After the intervention,oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)were conducted to assess glucose metabolism,while serum triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)levels were measured to evaluate lipid metabolism.Results:After 3 months of intervention,there were significant differences in the maximum running speed and forelimb grip strength among the four groups(P<0.05).The maximum running speed and forelimb grip strength of CS group,CS+MT group and CS+HT group were higher than those of NC group(P<0.05).The CS+MT group exhibited higher forelimb grip strength,and the CS+HT group showed higher maximum running speed and forelimb grip strength compared to the CS group(P<0.05),while no significant difference was found between the CS+MT and CS+HT groups(P>0.05).Significant differences in body weight were observed among the four groups after 3 months of intervention(P<0.05).Specifically,the CS+MT and CS+HT groups exhibited significantly lower body weight compared to both the NC and CS groups(P<0.05),with the CS+MT group having the lowest body weight(P<0.05).Fasting blood glucose levels also differed significantly among the groups after 2 and 3 months of intervention(P<0.05).The CS,CS+MT,and CS+HT groups had lower fasting blood glucose levels compared to the NC group(P<0.05),with the CS+MT and CS+HT groups showing the lowest levels(P<0.05).No significant difference was found between the CS+MT and CS+HT groups(P>0.05).After 3 months of intervention,significant differences in the area under the curve(AUC)of OGTT and ITT were observed among the four groups(P<0.05).The AUC of OGTT and ITT were significantly lower in the CS,CS+MT,and CS+HT groups compared to the NC group(P<0.05).The CS+MT and CS+HT groups exhibited the lowest AUC values for both OGTT and ITT(P<0.05),with the CS+MT group showing the lowest AUC for OGTT(P<0.05).Significant differences in serum lipid levels were observed among the four groups after 3 months of intervention(P<0.05).TG,TC,and LDL-C levels were significantly lower,while HDL-C levels were higher in the CS,CS+MT,and CS+HT groups compared to the NC group(P<0.05).The CS+MT group had the lowest TG levels and the highest HDL-C levels compared to the CS+HT group(P<0.05),with no significant differences in TC and LDL-C levels between these two groups(P>0.05).Conclusion:Aqueous extract of CS combined with different intensity training can significantly improve the exercise capacity and glycolipid metabolism of MS mice and reduce body weight,especially CS combined with MT treatment is more effective in improving lipid metabolism.In addition,when combined with HT,aqueous extract of CS can also play an auxiliary role in reducing the side effects of high-intensity exercise and improving the therapeutic effect.
3.Effect of CYFIP1 on proliferation and apoptosis of colorectal cancer cell HT29
Fu-long YU ; Liang LI ; Hao QIANG ; Hui YUAN ; Song WANG ; Xiao-hu CHENG ; Run-ben JIANG ; Ya-ru YANG ; Zhi-ning LIU
Chinese Pharmacological Bulletin 2025;41(1):116-121
Aim To investigate the expression levels of cytoplasmic FMR1-interacting protein-1(CYFIP1)in colorectal cancer and assess the impact of CYFIP1 interaction on the proliferation and apoptosis of colorec-tal cancer cell HT29,along with its potential mecha-nisms.Methods Immunohistochemistry was em-ployed to assess CYFIP1 expression in 32 colorectal cancer tissues and adjacent tissues.Coexpressed genes were identified using the GEPIA2 website to predict potential correlations and binding sites.Following the construction of a siRNA-CYFIP1,alterations in cell proliferation,apoptosis,and levels of apoptosis-related proteins were evaluated through CCK-8 assay,Hoechst 33342/PI double staining assay,and Western blot a-nalysis,respectively.Results The immunohisto-chemical findings revealed a significantly elevated level of CYFIP1 expression in colorectal cancer tissues com-pared to paracancer tissues(P<0.05).The expres-sion of CYFIP1 did not show any correlation with age and gender,but exhibited associations with TNM stage and lymph node metastasis(P<0.05).A conserved TP53 binding site was predicted in the 3kbps DNA re-gion upstream of the CYFIP1 gene using GEPIA2,JASPAR databases,and rVista 2.0 promoter prediction software.Following transfection of HT29 cells with siRNA-CYFIP1,the clonogenesis and proliferation of cells significantly decreased(P<0.05).Additional-ly,the levels of cleaved caspase-3 were elevated,while the expression levels of caspase-3 and Bcl-2 were reduced after transfection with siRNA-CYFIP1(P<0.05),which might be related to the interaction be-tween CYFIP1 and TP53.Conclusions The upregu-lation of CYFIP1 in colorectal cancer is associated with TNM stage and lymph node metastasis.Upon silen-cing,CYFIP1 demonstrates the ability to suppress pro-liferation in HT29 cells and modulate the expression of apoptotic proteins.
4.Effects of LINC00626 on proliferation,apoptosis and drug resistance of colorectal cancer SW480 cells
Liang LI ; Hao QIANG ; Shui-ri WANG ; Fu-long YU ; Song WANG ; Hui YUAN ; Ya-ru YANG ; Zhi-ning LIU
Chinese Pharmacological Bulletin 2025;41(10):1900-1905
Aim To investigate the high expression of LINC00626 in colorectal cancer,and explore the effects of LINC00626 on the proliferation,apoptosis,and drug sensitivity of colorectal cancer SW480 cells,as well as its underlying mechanisms.Methods Flu-orescence in situ hybridization(FISH)was used to de-tect the expression levels of LINC00626 in 38 colorec-tal cancer tissues and their corresponding adjacent nor-mal tissues.The JASPAR database was utilized to pre-dict co-expressed genes and their possible binding sites.Cell transfection technology was employed to knockdown LINC00626.Western blot and qRT-PCR techniques were used to verify the transfection efficien-cy.CCK-8 assay,cell apoptosis and necrosis staining,and Western blot were used to detect the changes in the proliferation,apoptosis,drug sensitivity,and ap-optotic proteins of SW480 cells,respectively.Results The FISH results indicated that LINC00626 was highly expressed in colorectal cancer tissues(P<0.05).The expression of LINC00626 was not associat-ed with the age or gender of patients,but was related to the TNM stage and the presence of lymph node me-tastasis($ P<0.05 $).The results of CCK-8 assay and cell apoptosis and necrosis staining showed that af-ter knockdown of LINC00626,the proliferation ability of SW480 cells decreased,the apoptosis level in-creased,and the drug resistance decreased(P<0.05).Western blot results showed that with the de-crease in the expression level of LINC00626,the ex-pression of caspase-3 protein decreased,the expression of cleaved caspase-3 protein increased,and the expres-sion of Bcl-2 protein decreased(P<0.05).Conclu-sions LINC00626 is highly expressed in colorectal cancer and is associated with the TNM stage and the presence of lymph node metastasis.LINC00626 can af-fect the proliferation,apoptosis,and drug sensitivity of SW480 cells and alter the expression of apoptotic pro-teins.
5.Effects of Aqueous Extract of Corn Silk Combined with Training on Exercise Function and Glycolipid Metabolism in Mice with Metabolic Syndrome
Yi-lin LIU ; Zi-ling SONG ; Ting ZHOU ; Ji-ping CHEN ; Zi-han LIN ; Yu-xuan ZHANG ; Ji-qiang ZENG ; Shan-rong ZHANG ; Zhi-peng WU ; Chen LU ; Ying ZHU
Progress in Modern Biomedicine 2025;25(15):2411-2420
Objective:To investigate the effects of combination therapy with aqueous extract of corn silk(CS)and training on exercise capacity and glycolipid metabolism in mice with metabolic syndrome(MS).Methods:In this study,db/db mice were used as the animal model of MS.The mice were administered aqueous extract of CS via gavage and subjected to different intensities of training for 12 weeks(3 months).The specific experimental design was as follows:24 db/db mice were randomly divided into four groups on average:negative control group(NC),aqueous extract of CS group(CS),aqueous extract of CS+moderate-intensity training group(CS+MT),and CS aqueous extract of CS+high-intensity training group(CS+HT).The maximum running speed,forelimb grip strength,body weight and fasting blood glucose of mice were measured before and after treatment.After the intervention,oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)were conducted to assess glucose metabolism,while serum triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)levels were measured to evaluate lipid metabolism.Results:After 3 months of intervention,there were significant differences in the maximum running speed and forelimb grip strength among the four groups(P<0.05).The maximum running speed and forelimb grip strength of CS group,CS+MT group and CS+HT group were higher than those of NC group(P<0.05).The CS+MT group exhibited higher forelimb grip strength,and the CS+HT group showed higher maximum running speed and forelimb grip strength compared to the CS group(P<0.05),while no significant difference was found between the CS+MT and CS+HT groups(P>0.05).Significant differences in body weight were observed among the four groups after 3 months of intervention(P<0.05).Specifically,the CS+MT and CS+HT groups exhibited significantly lower body weight compared to both the NC and CS groups(P<0.05),with the CS+MT group having the lowest body weight(P<0.05).Fasting blood glucose levels also differed significantly among the groups after 2 and 3 months of intervention(P<0.05).The CS,CS+MT,and CS+HT groups had lower fasting blood glucose levels compared to the NC group(P<0.05),with the CS+MT and CS+HT groups showing the lowest levels(P<0.05).No significant difference was found between the CS+MT and CS+HT groups(P>0.05).After 3 months of intervention,significant differences in the area under the curve(AUC)of OGTT and ITT were observed among the four groups(P<0.05).The AUC of OGTT and ITT were significantly lower in the CS,CS+MT,and CS+HT groups compared to the NC group(P<0.05).The CS+MT and CS+HT groups exhibited the lowest AUC values for both OGTT and ITT(P<0.05),with the CS+MT group showing the lowest AUC for OGTT(P<0.05).Significant differences in serum lipid levels were observed among the four groups after 3 months of intervention(P<0.05).TG,TC,and LDL-C levels were significantly lower,while HDL-C levels were higher in the CS,CS+MT,and CS+HT groups compared to the NC group(P<0.05).The CS+MT group had the lowest TG levels and the highest HDL-C levels compared to the CS+HT group(P<0.05),with no significant differences in TC and LDL-C levels between these two groups(P>0.05).Conclusion:Aqueous extract of CS combined with different intensity training can significantly improve the exercise capacity and glycolipid metabolism of MS mice and reduce body weight,especially CS combined with MT treatment is more effective in improving lipid metabolism.In addition,when combined with HT,aqueous extract of CS can also play an auxiliary role in reducing the side effects of high-intensity exercise and improving the therapeutic effect.
6.Integrated molecular characterization of sarcomatoid hepatocellular carcinoma
Rong-Qi SUN ; Yu-Hang YE ; Ye XU ; Bo WANG ; Si-Yuan PAN ; Ning LI ; Long CHEN ; Jing-Yue PAN ; Zhi-Qiang HU ; Jia FAN ; Zheng-Jun ZHOU ; Jian ZHOU ; Cheng-Li SONG ; Shao-Lai ZHOU
Clinical and Molecular Hepatology 2025;31(2):426-444
Background:
s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated.
Methods:
In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs.
Results:
Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment.
Conclusions
We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.
7.Progress on antisense oligonucleotide in the field of antibacterial therapy
Jia LI ; Xiao-lu HAN ; Shi-yu SONG ; Jin-tao LIN ; Zhi-qiang TANG ; Zeng-ming WANG ; Liang XU ; Ai-ping ZHENG
Acta Pharmaceutica Sinica 2025;60(2):337-347
With the widespread use of antibiotics, drug-resistant bacterial infections have become a significant threat to human health. Finding new antibacterial strategies that can effectively control drug-resistant bacterial infections has become an urgent task. Unlike small molecule drugs that target bacterial proteins, antisense oligonucleotide (ASO) can target genes related to bacterial resistance, pathogenesis, growth, reproduction and biofilm formation. By regulating the expression of these genes, ASO can inhibit or kill bacteria, providing a novel approach for the development of antibacterial drugs. To overcome the challenge of delivering antisense oligonucleotide into bacterial cells, various drug delivery systems have been applied in this field, including cell-penetrating peptides, lipid nanoparticles and inorganic nanoparticles, which have injected new momentum into the development of antisense oligonucleotide in the antibacterial realm. This review summarizes the current development of small nucleic acid drugs, the antibacterial mechanisms, targets, sequences and delivery vectors of antisense oligonucleotide, providing a reference for the research and development of antisense oligonucleotide in the treatment of bacterial infections.
8.Current status and suggestions on regulation of traditional Chinese medicine raw materials and preparations under regulatory system of drugs.
Li-Ping QU ; Yong-Dan XU ; Wei-Jing HE ; Ding-Kun ZHANG ; Nan YANG ; Min-Xian SONG ; Zhi-Qiang MIN ; Ting-Mo ZHANG
China Journal of Chinese Materia Medica 2025;50(3):824-832
At present, the cause of traditional Chinese medicine(TCM) in China has entered a new period of high-quality development. How to strengthen the foundation for the TCM industry from the source is an important issue that deserves the attention of the authorities, industry, and academia. This study systematically analyzed the regulatory system of TCM raw materials and preparations. The study took the TCM industry chain and the product life cycle as a clue and focused on the dimensions of TCM resource protection and plant cultivation(farming), production and quality supervision of TCM raw materials and preparations, and their market access and distribution. It analyzed the current situation of the regulation of TCM raw materials and preparations under the regulatory system of drugs, discussed the main problems, and put forward corresponding suggestions. The results can provide an important reference value for the subsequent improvement of the regulatory system of drugs and the construction of a prominent regulatory system of drugs in accordance with TCM characteristics.
Drugs, Chinese Herbal/economics*
;
Medicine, Chinese Traditional/standards*
;
China
;
Quality Control
;
Humans
;
Plants, Medicinal/chemistry*
9.Multicenter randomized controlled trial of Yiqi Huoxue formula() for the treatment of ruptured lumbar disc herniation.
Yu ZHU ; Zhi-Qiang WANG ; Shun LIN ; Ying-Ying YAO ; Xue-Qiang SHEN ; Xiao-Chun LI ; Feng YU ; Xiao-Yang XIONG ; Yi SONG ; Meng-Fei CHEN ; Peng-Fei YU ; Hong JIANG ; Jin-Tao LIU
China Journal of Orthopaedics and Traumatology 2025;38(11):1112-1118
OBJECTIVE:
To observe the clinical symptoms and MRI outcomes of patients with ruptured lumbar disc herniation(LDH) through a multicenter randomized controlled study, and to evaluate the clinical efficacy and safety of Yiqi Huoxue formula() in the treatment of this disease.
METHODS:
A total of 160 outpatients and inpatients with ruptured LDH admitted to 4 medical centers from January 2023 to June 2023 were selected and randomly divided into the Yiqi Huoxue formula group and the control group, with 80 patients in each group. In the Yiqi Huoxue formula group, there were 43 males and 37 females, with an age of (41.03±9.56) years and a disease duration of (10.45±25.37) days, and the patients were treated with Yiqi Huoxue formula. In the control group, there were 34 males and 46 females, with an age of (42.14±8.73) years and a disease duration of (11.31±21.14) days;during the acute phase, patients in this group could take celecoxib capsules orally, and methylcobalamin orally at the same time. The Japanese Orthopaedic Association (JOA) score, Oswestry disability index (ODI), changes in the volume of herniated disc tissue on MRI, herniation rate, and absorption rate were recorded at the time of enrollment and during follow-ups at the 3rd, 6th, and 12th month after treatment.
RESULTS:
A total of 156 patients completed the clinical follow-up, and 4 patients withdrew midway. The clinical symptoms of all patients who completed the study were relieved to varying degrees, and reabsorption of herniated disc tissue was observed in all patients in the Yiqi Huoxue formula group after treatment. For the JOA score:in the Yiqi Huoxue formula group, it was (10.73±2.76) points before treatment and (24.65±2.19) points at the 12th month after treatment;in the control group, it was (11.01±1.20) points before treatment and (17.07±3.26) points at the 12th month after treatment. For the ODI score:in the Yiqi Huoxue formula group, it was (26.21±3.55) points before treatment and (5.65±2.19) points at the 12th month after treatment;in the control group, it was (27.92±2.51) points before treatment and (9.09±2.15) points at the 12th month after treatment. At the 12th month after treatment, the JOA and ODI scores of both groups were better than those before treatment, and the scores of the Yiqi Huoxue formula group were better than those of the control group, with statistically significant differences (P<0.05). In terms of the herniated disc volume and herniation rate on MRI, the Yiqi Huoxue formula group was superior to the control group, with statistically significant differences(P<0.05). Reabsorption occurred in 56.96%(45/79) of patients in the Yiqi Huoxue formula group, which was significantly higher than the 37.66%(29/77) in the control group.
CONCLUSION
After treatment with Yiqi Huoxue formula, patients with ruptured LDH show significant improvement in clinical symptoms and a marked reduction in the volume of herniated discs. During the follow-up period, no obvious adverse drug reactions are observed in patients, and no recurrence of symptoms is found at the last follow-up, indicating that the formula has safe and reliable efficacy.
Humans
;
Male
;
Female
;
Intervertebral Disc Displacement/drug therapy*
;
Adult
;
Drugs, Chinese Herbal/adverse effects*
;
Middle Aged
;
Lumbar Vertebrae
10.Effectiveness of Lianhua Qingwen Granule and Jingyin Gubiao Prescription in Omicron BA.2 Infection and Hospitalization: A Real-World Study of 56,244 Cases in Shanghai, China.
Yu-Jie ZHANG ; Guo-Jian LIU ; Han ZHANG ; Chen LIU ; Zhi-Qiang CHEN ; Ji-Shu XIAN ; Da-Li SONG ; Zhi LIU ; Xue YANG ; Ju WANG ; Zhe ZHANG ; Lu-Ying ZHANG ; Hua FENG ; Yan-Qi ZHANG ; Liang TAN
Chinese journal of integrative medicine 2025;31(1):11-18
OBJECTIVE:
To examine the effectiveness of Chinese medicine (CM) Lianhua Qingwen Granule (LHQW) and Jingyin Gubiao Prescription (JYGB) in asymptomatic or mild patients with Omicron infection in the shelter hospital.
METHODS:
This single-center retrospective cohort study was conducted in the largest shelter hospital in Shanghai, China, from April 10, 2022 to May 30, 2022. A total of 56,244 asymptomatic and mild Omicron cases were included and divided into 4 groups, i.e., non-administration group (23,702 cases), LHQW group (11,576 cases), JYGB group (12,112 cases), and dual combination of LHQW and JYGB group (8,854 cases). The length of stay (LOS) in the hospital was used to assess the effectiveness of LHQW and JYGB treatment on Omicron infection.
RESULTS:
Patients aged 41-60 years, with nadir threshold cycle (CT) value of N gene <25, or those fully vaccinated preferred to receive CM therapy. Before or after propensity score matching (PSM), the multiple linear regression showed that LHQW and JYGB treatment were independent influence factors of LOS (both P<0.001). After PSM, there were significant differences in LOS between the LHQW/JYGB combination and the other groups (P<0.01). The results of factorial design ANOVA proved that the LHQW/JYGB combination therapy synergistically shortened LOS (P=0.032).
CONCLUSIONS
Patients with a nadir CT value <25 were more likely to accept CM. The LHQW/JYGB combination therapy could shorten the LOS of Omicron-infected individuals in an isolated environment.
Humans
;
Drugs, Chinese Herbal/therapeutic use*
;
Male
;
Female
;
Middle Aged
;
Adult
;
China/epidemiology*
;
Hospitalization
;
COVID-19 Drug Treatment
;
COVID-19/epidemiology*
;
SARS-CoV-2
;
Retrospective Studies
;
Treatment Outcome
;
Length of Stay
;
Young Adult
;
Aged

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