BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found t...BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.展开更多
Cancer is the leading cause of death all over the world.Among the chemotherapy drugs,taxanes play an important role in cancer treatment.6258-70 is a new semi-synthetic taxane which has a broad spectrum of antitumor ac...Cancer is the leading cause of death all over the world.Among the chemotherapy drugs,taxanes play an important role in cancer treatment.6258-70 is a new semi-synthetic taxane which has a broad spectrum of antitumor activity.A fast and reliable high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS） method was developed for quantification of 6258-70 in rat plasma and tissues in this paper.After extraction by liquid-liquid extraction method with methyl tert-butyl ether,the samples were separated on a Kinetex C_（18） column（50 mm × 2.1 mm,2.6 μm,Phenomenex,USA） within3 min.The method was fully validated with the matrix effect between 87.7%and 99.5%and the recovery ranging from 80.3%to 90.1%.The intra- and inter-day precisions were less than 9.5%and the accuracy ranged from-3.8%to 6.5%.The reliable method was successfully applied to the pharmacokinetics and tissue distribution studies of 6258-70 after intravenous administration in rats.The pharmacokinetic results indicated that the pharmacokinetic behavior of 6258-70 in rats was in accordance with linear features within tested dosage of 1 to 4 mg/kg,and there was no significant difference between the two genders.The tissue distribution study showed that 6258-70 had an effective penetration,spread widely and rapidly and could cross blood-brain barrier.The results of pharmacokinetics and tissue distribution may provide a guide for future study.展开更多
AM: To produce high-quality potyclonal antibody to lysosomeassociated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellul...AM: To produce high-quality potyclonal antibody to lysosomeassociated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC). METHODS: The 297 bp 5' end of LAPTM4BcDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the recombinant pGEX-KG-N_(1-99) was transformed into E. coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharose^(TM) 4B agarose. The purified GST-LAPTM4B-N_(1-99) was characterized by SDSPAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B-35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N_(1-99) was isolated from 1 L of bacteria. The GST-LAPTM4B-N_(1-99) produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4BN_(1-99)-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.展开更多
Objective： To investigate the effects of Biejia Ruangan Tablet （复方鳖甲软肝片, BRT）- containing serum on the expression of matrix metalloproteinase （MMP-9） and tissue inhibitor of metalloproteinase （TIMP-1） in...Objective： To investigate the effects of Biejia Ruangan Tablet （复方鳖甲软肝片, BRT）- containing serum on the expression of matrix metalloproteinase （MMP-9） and tissue inhibitor of metalloproteinase （TIMP-1） in cultured renal interstitial fibroblasts. Methods： Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose （7 g/kg）, mid-dose （3.5 g/kg）, and low-dose （1.75 g/kg） BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-β1 （recombined human TGF-β 1）, and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1. Results： The high dose BRT-containing serum could decrease the type Ⅰ and Ⅲ procollagen gene expression which boosted by TGF- 13 1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF- β1 （P〈0.05）. Treatment of cells with recombined human TGF-β 1 had no significant effect on MMP-9 expression and BRT- containing serum also had no effect on MMP-9 expression. Conclusions： High dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type Ⅰ and Ⅲ procollagen and TIMP-1 expression.展开更多
Background It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways ( ) epigallocatechin 3 gallate (EGCG) is the major effective component in green tea and can offer...Background It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways ( ) epigallocatechin 3 gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet induced damage In this study, we investigated the protective mechanism of EGCG on human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro Methods Transcription factor Jun protein levels were measured by Western blot Matrix metalloproteinase 1 (MMP 1) and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA were studied by reverse transcription polymerase chain reaction (RT PCR) analysis in conjunction with computer assisted image analysis MMP 1 and TIMP 1 proteins were quantified by enzyme linked immunosorbent assay (ELISA) Results EGCG decreased transcription activity of Jun protein after induction by UVA Both the mRNA and protein levels of MMP 1 were increased by UVA irradiation, while no significant changes were observed in TIMP 1 levels The ratio of MMP 1 to TIMP 1 showed statistically significant differences compared with the control EGCG decreased the ratio of MMP 1 to TIMP 1 by inhibiting UVA induced MMP 1 expression ( P <0 05) Conclusion EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP 1 The ratio of MMP 1 to TIMP 1, rather than the levels of MMP 1 or TIMP 1 alone, may play a significant role in human skin photodamage展开更多
Meniscus is a wedge-shaped fibrocartilaginous tissue,playing important roles in maintaining joint stability and function.Meniscus injuries are difficult to heal and frequently progress into structural breakdown,which ...Meniscus is a wedge-shaped fibrocartilaginous tissue,playing important roles in maintaining joint stability and function.Meniscus injuries are difficult to heal and frequently progress into structural breakdown,which then leads to osteoarthritis.Regeneration of heterogeneous tissue engineering meniscus(TEM)continues to be a scientific and translational challenge.The morphology,tissue architecture,mechanical strength,and functional applications of the cultivated TEMs have not been able to meet clinical needs,which may due to the negligent attention on the importance of microenvironment in vitro and in vivo.Herein,we combined the 3D(three-dimensional)-printed gradient porous scaffolds,spatiotemporal partition release of growth factors,and anti-inflammatory and anti-oxidant microenvironment regulation of Ac2-26 peptide to prepare a versatile meniscus composite scaffold with heterogeneous bionic structures,excellent biomechanical properties and anti-inflammatory and anti-oxidant effects.By observing the results of cell activity and differentiation,and biomechanics under anti-inflammatory and anti-oxidant microenvironments in vitro,we explored the effects of anti-inflammatory and anti-oxidant microenvironments on construction of regional and functional heterogeneous TEM via the growth process regulation,with a view to cultivating a high-quality of TEM from bench to bedside.展开更多
Spindle is a specific substructure in cell during meiosis and gamete maturity division and its structure and function directly influence the development, differentiation, growth and reproduction of individuals. Recent...Spindle is a specific substructure in cell during meiosis and gamete maturity division and its structure and function directly influence the development, differentiation, growth and reproduction of individuals. Recent researches also show that mouse Mos/MAPK pathway is tightly related to spindiin phosphorylation. We report that (i) a novel gene encoding a putative human spindlin (SPIN) has been cloned, which is highly homologous to mouse Spin; (ii) tissue expression pattern of SPIN shows that SPIN is highly expressed as a 4.8 kb transcript in ovary, testis, heart, brain, kidney, pancreas, placenta and spleen, also with a specific 2.0 kb transcript in testis, but there is no hybridization band in thymus and small intestine, and (iii) SPIN has been mapped to human chromosome 9q22.1-22.3 by Radiation Hybrid Mapping. This novel gene was registered in GenBank as SPIN with accession number AF087864.展开更多
Dysregulation of microRNAs(miRNAs)in adipocytes plays a critical role in the pathogenesis of obesity.However,the signaling mechanisms regulating miRNAs production in adipose tissue remain largely unclear.Here,we show ...Dysregulation of microRNAs(miRNAs)in adipocytes plays a critical role in the pathogenesis of obesity.However,the signaling mechanisms regulating miRNAs production in adipose tissue remain largely unclear.Here,we show that adipose tissue-specific knockout of Ras homolog enriched in brain(Rheb),a direct upstream activator of mTOR,increases miR-182-5p level in mouse subcutaneous white adipose tissues.Interestingly,the inhibition of mTOR signaling by rapamycin has no effect on miR-182-5p level in primary subcutaneous white adipocytes,suggesting the presence of a mTOR-independent mechanism regulating Rheb-mediated miR-182-5p expression.Consistent with this view,Rheb-ablation activates the cAMP/PPARγsignaling pathway.In addition,treatment of white adipocytes with pioglitazone,a PPARγagonist,dramatically upregulates miR-182-5p levels.Our study reveals a unique mechanism by which Rheb regulates miR-182-5p in adipocytes.Given that increasing miR-182-5p in adipose tissue promotes beige fat development,our study also suggests a unique mechanism by which Rheb promotes thermogenesis and energy expenditure.展开更多
文摘BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.
文摘Cancer is the leading cause of death all over the world.Among the chemotherapy drugs,taxanes play an important role in cancer treatment.6258-70 is a new semi-synthetic taxane which has a broad spectrum of antitumor activity.A fast and reliable high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS） method was developed for quantification of 6258-70 in rat plasma and tissues in this paper.After extraction by liquid-liquid extraction method with methyl tert-butyl ether,the samples were separated on a Kinetex C_（18） column（50 mm × 2.1 mm,2.6 μm,Phenomenex,USA） within3 min.The method was fully validated with the matrix effect between 87.7%and 99.5%and the recovery ranging from 80.3%to 90.1%.The intra- and inter-day precisions were less than 9.5%and the accuracy ranged from-3.8%to 6.5%.The reliable method was successfully applied to the pharmacokinetics and tissue distribution studies of 6258-70 after intravenous administration in rats.The pharmacokinetic results indicated that the pharmacokinetic behavior of 6258-70 in rats was in accordance with linear features within tested dosage of 1 to 4 mg/kg,and there was no significant difference between the two genders.The tissue distribution study showed that 6258-70 had an effective penetration,spread widely and rapidly and could cross blood-brain barrier.The results of pharmacokinetics and tissue distribution may provide a guide for future study.
基金Supported by the 248 Major R&D Program of Beijing, No. H020220020310, and Special Fund for Promotion of Education, Ministry of Education, China
文摘AM: To produce high-quality potyclonal antibody to lysosomeassociated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC). METHODS: The 297 bp 5' end of LAPTM4BcDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the recombinant pGEX-KG-N_(1-99) was transformed into E. coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharose^(TM) 4B agarose. The purified GST-LAPTM4B-N_(1-99) was characterized by SDSPAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B-35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N_(1-99) was isolated from 1 L of bacteria. The GST-LAPTM4B-N_(1-99) produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4BN_(1-99)-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.
基金Supported by the National Science Foundation of China(No.30130220 and No.30873345)National Natural Science Found for Innovative Research Groups Science Foundation of China(No.30121005)
文摘Objective： To investigate the effects of Biejia Ruangan Tablet （复方鳖甲软肝片, BRT）- containing serum on the expression of matrix metalloproteinase （MMP-9） and tissue inhibitor of metalloproteinase （TIMP-1） in cultured renal interstitial fibroblasts. Methods： Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose （7 g/kg）, mid-dose （3.5 g/kg）, and low-dose （1.75 g/kg） BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-β1 （recombined human TGF-β 1）, and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1. Results： The high dose BRT-containing serum could decrease the type Ⅰ and Ⅲ procollagen gene expression which boosted by TGF- 13 1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF- β1 （P〈0.05）. Treatment of cells with recombined human TGF-β 1 had no significant effect on MMP-9 expression and BRT- containing serum also had no effect on MMP-9 expression. Conclusions： High dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type Ⅰ and Ⅲ procollagen and TIMP-1 expression.
文摘Background It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways ( ) epigallocatechin 3 gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet induced damage In this study, we investigated the protective mechanism of EGCG on human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro Methods Transcription factor Jun protein levels were measured by Western blot Matrix metalloproteinase 1 (MMP 1) and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA were studied by reverse transcription polymerase chain reaction (RT PCR) analysis in conjunction with computer assisted image analysis MMP 1 and TIMP 1 proteins were quantified by enzyme linked immunosorbent assay (ELISA) Results EGCG decreased transcription activity of Jun protein after induction by UVA Both the mRNA and protein levels of MMP 1 were increased by UVA irradiation, while no significant changes were observed in TIMP 1 levels The ratio of MMP 1 to TIMP 1 showed statistically significant differences compared with the control EGCG decreased the ratio of MMP 1 to TIMP 1 by inhibiting UVA induced MMP 1 expression ( P <0 05) Conclusion EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP 1 The ratio of MMP 1 to TIMP 1, rather than the levels of MMP 1 or TIMP 1 alone, may play a significant role in human skin photodamage
基金the National Natural Science Foundation of China(NSFC,82002298,51920105006,51973226)the China Postdoctoral Science Foundation(2020M670066)+1 种基金the National Key Research and Development Program of China(2016YFC1100704)the Youth Innovation Promotion Association CAS(2019031).
文摘Meniscus is a wedge-shaped fibrocartilaginous tissue,playing important roles in maintaining joint stability and function.Meniscus injuries are difficult to heal and frequently progress into structural breakdown,which then leads to osteoarthritis.Regeneration of heterogeneous tissue engineering meniscus(TEM)continues to be a scientific and translational challenge.The morphology,tissue architecture,mechanical strength,and functional applications of the cultivated TEMs have not been able to meet clinical needs,which may due to the negligent attention on the importance of microenvironment in vitro and in vivo.Herein,we combined the 3D(three-dimensional)-printed gradient porous scaffolds,spatiotemporal partition release of growth factors,and anti-inflammatory and anti-oxidant microenvironment regulation of Ac2-26 peptide to prepare a versatile meniscus composite scaffold with heterogeneous bionic structures,excellent biomechanical properties and anti-inflammatory and anti-oxidant effects.By observing the results of cell activity and differentiation,and biomechanics under anti-inflammatory and anti-oxidant microenvironments in vitro,we explored the effects of anti-inflammatory and anti-oxidant microenvironments on construction of regional and functional heterogeneous TEM via the growth process regulation,with a view to cultivating a high-quality of TEM from bench to bedside.
文摘Spindle is a specific substructure in cell during meiosis and gamete maturity division and its structure and function directly influence the development, differentiation, growth and reproduction of individuals. Recent researches also show that mouse Mos/MAPK pathway is tightly related to spindiin phosphorylation. We report that (i) a novel gene encoding a putative human spindlin (SPIN) has been cloned, which is highly homologous to mouse Spin; (ii) tissue expression pattern of SPIN shows that SPIN is highly expressed as a 4.8 kb transcript in ovary, testis, heart, brain, kidney, pancreas, placenta and spleen, also with a specific 2.0 kb transcript in testis, but there is no hybridization band in thymus and small intestine, and (iii) SPIN has been mapped to human chromosome 9q22.1-22.3 by Radiation Hybrid Mapping. This novel gene was registered in GenBank as SPIN with accession number AF087864.
基金supported by grants 82170886 and 81800758 from the National Nature Science Foundation of Chinapartially supported by the Fundamental Research Funds for the Central Universities of Central South University(2021zzts1048)。
文摘Dysregulation of microRNAs(miRNAs)in adipocytes plays a critical role in the pathogenesis of obesity.However,the signaling mechanisms regulating miRNAs production in adipose tissue remain largely unclear.Here,we show that adipose tissue-specific knockout of Ras homolog enriched in brain(Rheb),a direct upstream activator of mTOR,increases miR-182-5p level in mouse subcutaneous white adipose tissues.Interestingly,the inhibition of mTOR signaling by rapamycin has no effect on miR-182-5p level in primary subcutaneous white adipocytes,suggesting the presence of a mTOR-independent mechanism regulating Rheb-mediated miR-182-5p expression.Consistent with this view,Rheb-ablation activates the cAMP/PPARγsignaling pathway.In addition,treatment of white adipocytes with pioglitazone,a PPARγagonist,dramatically upregulates miR-182-5p levels.Our study reveals a unique mechanism by which Rheb regulates miR-182-5p in adipocytes.Given that increasing miR-182-5p in adipose tissue promotes beige fat development,our study also suggests a unique mechanism by which Rheb promotes thermogenesis and energy expenditure.
