INTRODUCTIONThe treatment of human epithelial malignancies islimited by drug resistance and toxic and side effects,which results in the failure in the treatment ofmajority of advanced cancer victims.To seek for anew,a...INTRODUCTIONThe treatment of human epithelial malignancies islimited by drug resistance and toxic and side effects,which results in the failure in the treatment ofmajority of advanced cancer victims.To seek for anew,and specific antineoplastic therapy willprovide hope for tumor treatment.Althoughdisordered intermediary metabolism in cancer cellshas been known for many years,much of the展开更多
Objective:To determine fatty acid synthase(FAS)expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma.Methods:FAS expression was determined by immunohistochemistry,re...Objective:To determine fatty acid synthase(FAS)expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma.Methods:FAS expression was determined by immunohistochemistry,re- verse-transcription polymerase chain reaction(RT-PCR)and immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma(MM patients)and peripheral blood mononuclear cells(PBMCs)obtained from 12 healthy donors. In parallel,additional analyses were performed on 2 human multiple myeloma cell lines,U266 and RPMI8226.U266 cells were treated with cerulenin at various concentrations(5 to 320μg/ml)for 24 h,and metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays.Apoptosis was evaluated by dual Annexin V/PI (propidium iodide)labeling and flow cytometry(FCM)in U266 cells treated with 20μg/ml cerulenin for 12 h or 24 h.Results:By immunohistochemistry,we found that 19 of 27 bone marrow samples obtained from MM patients expressed significantly high levels of FAS.Similarly,by RT-PCR,22 of 27 bone marrow samples obtained from MM patients,U266 and RPMI8226 showed FAS expression,whereas PBMC samples from 12 healthy donors did not express detectable level of FAS.FAS protein expression was confirmed by immunoblot analysis in 16 of 27 bone marrow samples obtained from MM patients,U266 and RPMI8226 cell lines,and no FAS protein expression was detected in PBMC samples from 12 healthy donors.U266 cells were highly sensitive to cerulenin treatment,with a dosage-related effect on metabolic activity,as a measure for cell proliferation.U266 cells treated with 20μg/ml cerulenin for 12 and 24 h also showed early sign of apoptosis with 56.9%and 69.3%Annexin V+/PI ?cells,and late apoptotic and necrotic cells with 3.2%and 17.6%Annexin V+/PI +cells.Conclusion:Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines.Cerulenin greatly inhibited metabolic activity/cell proliferation of U266 cells and induced apoptosis,suggesting that FAS is an effective target for pharmacological therapy in human multiple myeloma.展开更多
Objective:Our aim was to test the hypothesis that fatty acid synthase(FASN)expression contributes to radioresistance of nasopharyngeal carcinoma(NPC)cells and that inhibiting FASN enhances radiosensitivity.Methods:Tar...Objective:Our aim was to test the hypothesis that fatty acid synthase(FASN)expression contributes to radioresistance of nasopharyngeal carcinoma(NPC)cells and that inhibiting FASN enhances radiosensitivity.Methods:Targeting FASN using epigallocatechin gallate(EGCG)or RNA interference in NPC cell lines that overexpress endogenous FASN was performed to determine their effects on cellular response to radiationin vitro using MTT and colony formation assays,andin vivo using xenograft animal models.Western blot,immunohistochemistry,real-time PCR arrays,and real-time RT-PCR were used to determine the relationship between FASN and frizzled class receptor 10(FZD10)expression.FZD10 knockdown and overexpression were used to determine its role in mediating FASN function in cellular response to radiation.Immunohistochemical staining was used to determine FASN and FZD10 expressions in human NPC tissues,followed by analysis of their association with the overall survival of patients.Results:FASN knockdown or inhibition significantly enhanced radiosensitivity of NPC cells,bothin vitro andin vivo.There was a positive association between FASN and FZD10 expression in NPC cell lines grown as monolayers or xenografts,as well as human tissues.FASN knockdown reduced FZD10 expression,and rescue of FZD10 expression abolished FASN knockdown-induced enhancement of radiosensitivity.FASN and FZD10 were both negatively associated with overall survival of NPC patients.Conclusions:FASN contributes to radioresistance,possiblyvia FZD10 in NPC cells.Both FZD10 and FASN expressions were associated with poor outcomes of NPC patients.EGCG may sensitize radioresistance by inhibiting FASN and may possibly be developed as a radiosensitizer for better treatment of NPCs.展开更多
Objective To explore the effects of zinc-α2-glycoprotein(ZAG) on body weight and body fat in high-fat-diet(HFD)-induced obesity in mice and the possible mechanism.Methods Thirty-six male mice were fed with standard f...Objective To explore the effects of zinc-α2-glycoprotein(ZAG) on body weight and body fat in high-fat-diet(HFD)-induced obesity in mice and the possible mechanism.Methods Thirty-six male mice were fed with standard food(SF)(n=9) and HFD(n=27),respectively.Five weeks later,9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method,and another 9 mice to negative control plasmid transfection.Two weeks later,serum ZAG level in the mice was assayed by Western blot,and the effects of ZAG over-expression on body weight,body fat,serum biochemical indexes,and adipose tissue of obese mice were evaluated.The mRNA expressions of fatty acid synthase(FAS) and hormone-sensitive lipase(HSL) in liver tissue were determined by reverse transcription-polymerase chain reaction.Results Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice(0.51±0.10 AU vs.0.75±0.07 AU,P<0.01).Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight(r =-0.56,P<0.001),epididymal fat mass(r=-0.67,P<0.001),percentage of epididymal fat(r=-0.65,P<0.001),and increased weight(r=-0.57,P<0.001) in simple SF-and HFD-fed mice.ZAG over-expression in obese mice reduced body weight and the percentage of epididymal fat.Furthermore,FAS mRNA expression decreased(P<0.01) and HSL mRNA expression increased(P<0.001) in the liver in ZAG over-expressing mice.Conclusions ZAG is closely related to obesity.Serum ZAG level is inversely correlated with body weight and percentage of body fat.The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.展开更多
Background: The liver is the corner stone in lipid metabolism, free fatty acid uptake, synthesizing, storing and exporting lipids;non-alcoholic fatty liver disease (NAFLD) develops if there is any interruption or dera...Background: The liver is the corner stone in lipid metabolism, free fatty acid uptake, synthesizing, storing and exporting lipids;non-alcoholic fatty liver disease (NAFLD) develops if there is any interruption or derangements in lipid metabolim. Fatty acid synthase (FAS) is the major enzyme in lipogenesis, and its circulating level is a bi-omarker of metabolically demanding human diseases. Aim of the Work: To evaluate the level of circulating FAS in NAFLD patients and to correlate it to serum lipid pa-rameters. Materials and Methods: The study included forty NAFLD patients and forty age and sex-matched healthy subjects as controls. Results: FAS levels were signifi-cantly higher in NAFLD patients compared to their level in the controls (P < 0.05). Ad-ditionally, a positive correlation was found between the levels of FAS and BMI (r = 0.57), and between FAS levels and triglycerides and low density lipoprotein cholesterol levels in NAFLD patients (r = 0.79 & 0.53, respectively). Conclusion: Elevated levels of circulating FAS can be considered as a biomarker of fatty liver disease.展开更多
Fatty acid composition of four Sargassum species from Qingdao and Shidao,Shandong Provincewas investigated.16:0（palmitic acid）was the major saturated fatty acid.C<sub>18</sub> and C<sub>20</sub&...Fatty acid composition of four Sargassum species from Qingdao and Shidao,Shandong Provincewas investigated.16:0（palmitic acid）was the major saturated fatty acid.C<sub>18</sub> and C<sub>20</sub>were the mainpolyunsaturated fatty acids（PUFAs）.Arachidonic acid and eicosapentaenoic acid predominated amongpolyenoic acids in all the algal species examined.except for Sargassum sp.which had low concentrationof eicosapentaenoic acid.展开更多
The feeding trial was conducted to determine the effects of flaxseed on layer＇s performance and egg quality, and the effects of the dietary ratio of ω-6 fatty acid to ω-3 fatty acid on fatty acid deposition in yolk...The feeding trial was conducted to determine the effects of flaxseed on layer＇s performance and egg quality, and the effects of the dietary ratio of ω-6 fatty acid to ω-3 fatty acid on fatty acid deposition in yolk using flaxseed as resource of ω-3 fatty acid and sunflower seed as resource of ω-6 fatty acid. 192 20-wk-old Newroman commercial hens were randomly divided into 4 treatment groups with 6 replicates containing 8 birds. The rations corresponding with the four treatments were formulated with 4% flaxseed ＋ 9% sunflower seed, 8% flaxseed ＋ 18% sunflower seed, 15% flaxseed, and soybean meal, respectively. Sunflower seed was unshelled and flaxseed was whole. Ratios of ω-6 fatty acid to ω-3 fatty acid in 4 dietary treatments were 5.67, 5.21, 1.07, and 11.83, respectively. It can be concluded that long-term feeding 15% flaxseed had no significant （P〉 0.05） influence on egg production, egg weight, eggshell quality, yolk weight, and percentage of yolk weight. Dietary ratio of ω-6 fatty acid to ω-3 fatty acid had impact on the 0-3 fatty acid levels in yolk, nevertheless yolk fatty acid profiles were different from those of dietary fatty acid compositions.展开更多
Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in ins...Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in insects they are important in both cuticular hydrocarbon (CHC) formation and reproduction. Here we characterized the sequence structure and function of an FAS from the small brown planthopper (SBPH), Laodelphax striatellus. The full-length open reading frame (ORF) sequence of LsFAS1 was 7122 bp, encoding a predicted protein of 2373 amino acid residues. There were 7 functional domains in the LsFAS1 protein sequence. Gene expression screening by real-time quantitative polymerase chain reaction (RT-qPCR) showed that LsFAS1 was expressed in all developmental stages. Relative expression was highest at the 4th-instar and female adult stages. Among different tissues, the expression level of LsFAS1 in the ovary was the highest. Phylogenetic analysis showed that LsFAS1 clustered in a clade with 2 FASs from Nilaparvata lugens. Furthermore, these 3 FASs are related to cockroach BgFAS and locust LmFAS. After RNA interference-mediated knock-down, most treated insects died at eclosion. In addition, the lifespan of dsFAS1-treated female adults was shorter than that of the dsGFP-injected control, and offspring production decreased. Also, the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes decreased. Virgin females dissected at days 2 and 4 post-eclosion showed many matured oocytes in planthoppers treated with dsGFP but not with dsFAS1. These data highlight the importance of LsFAS1 in SBPH, including a role in reproduction.展开更多
Fatty acids(FAs) provide energy and also can be used to trace trophic relationships among organisms.Sea cucumber Apostichopus japonicus goes into a state of aestivation during warm summer months.We examined fatty acid...Fatty acids(FAs) provide energy and also can be used to trace trophic relationships among organisms.Sea cucumber Apostichopus japonicus goes into a state of aestivation during warm summer months.We examined fatty acid profiles in aestivated and non-aestivated A.japonicus using multivariate analyses(PERMANOVA,MDS,ANOSIM,and SIMPER).The results indicate that the fatty acid profiles of aestivated and non-aestivated sea cucumbers differed significantly.The FAs that were produced by bacteria and brown kelp contributed the most to the differences in the fatty acid composition of aestivated and nonaestivated sea cucumbers.Aestivated sea cucumbers may synthesize FAs from heterotrophic bacteria during early aestivation,and long chain FAs such as eicosapentaenoic(EPA) and docosahexaenoic acid(DHA) that produced from intestinal degradation,are digested during deep aestivation.Specific changes in the fatty acid composition of A.japonicus during aestivation needs more detailed study in the future.展开更多
基金the Applied Science Foundation of Jiangsu Province,China,No.BJ97071
文摘INTRODUCTIONThe treatment of human epithelial malignancies islimited by drug resistance and toxic and side effects,which results in the failure in the treatment ofmajority of advanced cancer victims.To seek for anew,and specific antineoplastic therapy willprovide hope for tumor treatment.Althoughdisordered intermediary metabolism in cancer cellshas been known for many years,much of the
基金Project supported by the Medicine and Health Research Fund of Zhejiang Province(No.2007B091)the Office of Education of Zhejiang Province,China(No.20070104)
文摘Objective:To determine fatty acid synthase(FAS)expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma.Methods:FAS expression was determined by immunohistochemistry,re- verse-transcription polymerase chain reaction(RT-PCR)and immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma(MM patients)and peripheral blood mononuclear cells(PBMCs)obtained from 12 healthy donors. In parallel,additional analyses were performed on 2 human multiple myeloma cell lines,U266 and RPMI8226.U266 cells were treated with cerulenin at various concentrations(5 to 320μg/ml)for 24 h,and metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays.Apoptosis was evaluated by dual Annexin V/PI (propidium iodide)labeling and flow cytometry(FCM)in U266 cells treated with 20μg/ml cerulenin for 12 h or 24 h.Results:By immunohistochemistry,we found that 19 of 27 bone marrow samples obtained from MM patients expressed significantly high levels of FAS.Similarly,by RT-PCR,22 of 27 bone marrow samples obtained from MM patients,U266 and RPMI8226 showed FAS expression,whereas PBMC samples from 12 healthy donors did not express detectable level of FAS.FAS protein expression was confirmed by immunoblot analysis in 16 of 27 bone marrow samples obtained from MM patients,U266 and RPMI8226 cell lines,and no FAS protein expression was detected in PBMC samples from 12 healthy donors.U266 cells were highly sensitive to cerulenin treatment,with a dosage-related effect on metabolic activity,as a measure for cell proliferation.U266 cells treated with 20μg/ml cerulenin for 12 and 24 h also showed early sign of apoptosis with 56.9%and 69.3%Annexin V+/PI ?cells,and late apoptotic and necrotic cells with 3.2%and 17.6%Annexin V+/PI +cells.Conclusion:Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines.Cerulenin greatly inhibited metabolic activity/cell proliferation of U266 cells and induced apoptosis,suggesting that FAS is an effective target for pharmacological therapy in human multiple myeloma.
基金This work was supported by grants from the National Natural Science Foundation of China(Grant Nos.81572588,81872147)Shantou University Medical College Clinical Trial Uplift Program(Grant No.201423)+4 种基金the Medical Scientific Research Foundation of Guangdong Province,China(Grant No.B2018222)the Traditional Chinese Medicine Research Project from Traditional Chinese Medicine Bureau of Guangdong Province(Grant No.20191182)the Youth Research Grant from Shantou University Medical College Cancer Hospital(Grant No.2018A001,2018A008)the key Project of Science and Technology of Shantou[Grant No.(2018)37]and the Natural Science Foundation of Guangdong Province of China(Grant No.2020A1515010094).
文摘Objective:Our aim was to test the hypothesis that fatty acid synthase(FASN)expression contributes to radioresistance of nasopharyngeal carcinoma(NPC)cells and that inhibiting FASN enhances radiosensitivity.Methods:Targeting FASN using epigallocatechin gallate(EGCG)or RNA interference in NPC cell lines that overexpress endogenous FASN was performed to determine their effects on cellular response to radiationin vitro using MTT and colony formation assays,andin vivo using xenograft animal models.Western blot,immunohistochemistry,real-time PCR arrays,and real-time RT-PCR were used to determine the relationship between FASN and frizzled class receptor 10(FZD10)expression.FZD10 knockdown and overexpression were used to determine its role in mediating FASN function in cellular response to radiation.Immunohistochemical staining was used to determine FASN and FZD10 expressions in human NPC tissues,followed by analysis of their association with the overall survival of patients.Results:FASN knockdown or inhibition significantly enhanced radiosensitivity of NPC cells,bothin vitro andin vivo.There was a positive association between FASN and FZD10 expression in NPC cell lines grown as monolayers or xenografts,as well as human tissues.FASN knockdown reduced FZD10 expression,and rescue of FZD10 expression abolished FASN knockdown-induced enhancement of radiosensitivity.FASN and FZD10 were both negatively associated with overall survival of NPC patients.Conclusions:FASN contributes to radioresistance,possiblyvia FZD10 in NPC cells.Both FZD10 and FASN expressions were associated with poor outcomes of NPC patients.EGCG may sensitize radioresistance by inhibiting FASN and may possibly be developed as a radiosensitizer for better treatment of NPCs.
基金Supported by the National Natural Science Foundation of China (30771026)Beijing Natural Science Foundation (7082079)
文摘Objective To explore the effects of zinc-α2-glycoprotein(ZAG) on body weight and body fat in high-fat-diet(HFD)-induced obesity in mice and the possible mechanism.Methods Thirty-six male mice were fed with standard food(SF)(n=9) and HFD(n=27),respectively.Five weeks later,9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method,and another 9 mice to negative control plasmid transfection.Two weeks later,serum ZAG level in the mice was assayed by Western blot,and the effects of ZAG over-expression on body weight,body fat,serum biochemical indexes,and adipose tissue of obese mice were evaluated.The mRNA expressions of fatty acid synthase(FAS) and hormone-sensitive lipase(HSL) in liver tissue were determined by reverse transcription-polymerase chain reaction.Results Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice(0.51±0.10 AU vs.0.75±0.07 AU,P<0.01).Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight(r =-0.56,P<0.001),epididymal fat mass(r=-0.67,P<0.001),percentage of epididymal fat(r=-0.65,P<0.001),and increased weight(r=-0.57,P<0.001) in simple SF-and HFD-fed mice.ZAG over-expression in obese mice reduced body weight and the percentage of epididymal fat.Furthermore,FAS mRNA expression decreased(P<0.01) and HSL mRNA expression increased(P<0.001) in the liver in ZAG over-expressing mice.Conclusions ZAG is closely related to obesity.Serum ZAG level is inversely correlated with body weight and percentage of body fat.The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.
文摘Background: The liver is the corner stone in lipid metabolism, free fatty acid uptake, synthesizing, storing and exporting lipids;non-alcoholic fatty liver disease (NAFLD) develops if there is any interruption or derangements in lipid metabolim. Fatty acid synthase (FAS) is the major enzyme in lipogenesis, and its circulating level is a bi-omarker of metabolically demanding human diseases. Aim of the Work: To evaluate the level of circulating FAS in NAFLD patients and to correlate it to serum lipid pa-rameters. Materials and Methods: The study included forty NAFLD patients and forty age and sex-matched healthy subjects as controls. Results: FAS levels were signifi-cantly higher in NAFLD patients compared to their level in the controls (P < 0.05). Ad-ditionally, a positive correlation was found between the levels of FAS and BMI (r = 0.57), and between FAS levels and triglycerides and low density lipoprotein cholesterol levels in NAFLD patients (r = 0.79 & 0.53, respectively). Conclusion: Elevated levels of circulating FAS can be considered as a biomarker of fatty liver disease.
文摘Fatty acid composition of four Sargassum species from Qingdao and Shidao,Shandong Provincewas investigated.16:0（palmitic acid）was the major saturated fatty acid.C<sub>18</sub> and C<sub>20</sub>were the mainpolyunsaturated fatty acids（PUFAs）.Arachidonic acid and eicosapentaenoic acid predominated amongpolyenoic acids in all the algal species examined.except for Sargassum sp.which had low concentrationof eicosapentaenoic acid.
基金supported by the Heilongjiang Province Government Foundation,China(HL-98)
文摘The feeding trial was conducted to determine the effects of flaxseed on layer＇s performance and egg quality, and the effects of the dietary ratio of ω-6 fatty acid to ω-3 fatty acid on fatty acid deposition in yolk using flaxseed as resource of ω-3 fatty acid and sunflower seed as resource of ω-6 fatty acid. 192 20-wk-old Newroman commercial hens were randomly divided into 4 treatment groups with 6 replicates containing 8 birds. The rations corresponding with the four treatments were formulated with 4% flaxseed ＋ 9% sunflower seed, 8% flaxseed ＋ 18% sunflower seed, 15% flaxseed, and soybean meal, respectively. Sunflower seed was unshelled and flaxseed was whole. Ratios of ω-6 fatty acid to ω-3 fatty acid in 4 dietary treatments were 5.67, 5.21, 1.07, and 11.83, respectively. It can be concluded that long-term feeding 15% flaxseed had no significant （P〉 0.05） influence on egg production, egg weight, eggshell quality, yolk weight, and percentage of yolk weight. Dietary ratio of ω-6 fatty acid to ω-3 fatty acid had impact on the 0-3 fatty acid levels in yolk, nevertheless yolk fatty acid profiles were different from those of dietary fatty acid compositions.
基金the Jiangsu Agriculture Science and Technology Innovation Fund(grant no.CX(20)1004)the Earmarked Fund for China Agriculture Research System(grant no.CARS-01).
文摘Fatty acid synthase (FAS) is a multifunctional enzyme that plays an important role in the formation of fatty acids. The fatty acids take part in many processes, such as cell signaling and energy metabolism, and in insects they are important in both cuticular hydrocarbon (CHC) formation and reproduction. Here we characterized the sequence structure and function of an FAS from the small brown planthopper (SBPH), Laodelphax striatellus. The full-length open reading frame (ORF) sequence of LsFAS1 was 7122 bp, encoding a predicted protein of 2373 amino acid residues. There were 7 functional domains in the LsFAS1 protein sequence. Gene expression screening by real-time quantitative polymerase chain reaction (RT-qPCR) showed that LsFAS1 was expressed in all developmental stages. Relative expression was highest at the 4th-instar and female adult stages. Among different tissues, the expression level of LsFAS1 in the ovary was the highest. Phylogenetic analysis showed that LsFAS1 clustered in a clade with 2 FASs from Nilaparvata lugens. Furthermore, these 3 FASs are related to cockroach BgFAS and locust LmFAS. After RNA interference-mediated knock-down, most treated insects died at eclosion. In addition, the lifespan of dsFAS1-treated female adults was shorter than that of the dsGFP-injected control, and offspring production decreased. Also, the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes decreased. Virgin females dissected at days 2 and 4 post-eclosion showed many matured oocytes in planthoppers treated with dsGFP but not with dsFAS1. These data highlight the importance of LsFAS1 in SBPH, including a role in reproduction.
基金Supported by the National Marine Public Welfare Research Project(No.201305043)the National Natural Science Foundation of China(No.41106134)the National Key Technology R&D Program of China(Nos.2011BAD13B02,2010BAC68B01)
文摘Fatty acids(FAs) provide energy and also can be used to trace trophic relationships among organisms.Sea cucumber Apostichopus japonicus goes into a state of aestivation during warm summer months.We examined fatty acid profiles in aestivated and non-aestivated A.japonicus using multivariate analyses(PERMANOVA,MDS,ANOSIM,and SIMPER).The results indicate that the fatty acid profiles of aestivated and non-aestivated sea cucumbers differed significantly.The FAs that were produced by bacteria and brown kelp contributed the most to the differences in the fatty acid composition of aestivated and nonaestivated sea cucumbers.Aestivated sea cucumbers may synthesize FAs from heterotrophic bacteria during early aestivation,and long chain FAs such as eicosapentaenoic(EPA) and docosahexaenoic acid(DHA) that produced from intestinal degradation,are digested during deep aestivation.Specific changes in the fatty acid composition of A.japonicus during aestivation needs more detailed study in the future.
