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Nuclear reprogramming: the zygotic transcription program is established through an“erase-and-rebuild” strategy

Cell Research 2007年第2期17卷 117-134页

作者： Feng Sun~(1,*) Haiyan Fang~(2,*) Ruizhen Li~(2,*) Tianlong Gao~1 Junke Zheng~2 Xuejin Chen~2 Wenqin Ying~2 Hui Z Sheng~2 ~1Program for Graduation Studies,shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,shanghai 200031,china ~2center for developmental biology,xinhua hospital,school of medicine,shanghai jiao tong university,1665 long jiang road,shanghai 200092,china Program for Graduation Studies Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China center for developmental biology Xinhua Hospital School of Medicine Shanghai Jiao Tong University Shanghai China

Oocytes display a maternal-specific gene expression profile,which is switched to a zygotic profile when a haploidset of chromatin is passed on to the fertilized egg that develops into an *** mechanism underlying this tran-scription reprogramming is currently *** we demonstrate that by the time when transcription is shut downin germinal vesicle oocytes,a range of general transcription factors and transcriptional regulators are dissociated fromthe *** global dissociation of chromatin factors(CFs)disrupts physical contacts between the chromatin andCFs and leads to erasure of the maternal transcription program at the functional *** transcription factors andregulators remain separated from chromatin for a prolonged period,and become re-associated with chromatin shortlyafter pronuclear *** is followed temporally by the re-establishment of nuclear functions such as DNA rep-lication and *** propose that the maternal transcription program is erased during oogenesis to generatea relatively naive chromatin and the zygotic transcription program is rebuilt de novo after *** process istermed as the"erase-and-rebuild"process,which is used to reset the transcription program,and most likely other nuclearprocesses as well,from a maternal one to that of the *** further show in the accompanying paper(Gao T,et al.,Cell Res 2007:17:135-150.)that the same strategy is also employed to reprogram transcriptional profiles in somatic cellnuclear transfer and parthenogenesis,suggesting that this model is universally applicable to all forms of transcriptionalreprogramming during early *** of CFs from chromatin also offers an explanation for thephenomenon of transcription silence during the maternal to zygotic transition.

关键词： nuclear reprogramming transcription transcription silence chromatin factors embryogenesis fertilization oogenesis

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Regulated expression of TATA-binding protein-related factor 3(TRF3)during early embryogenesis

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Cell Research 2006年第7期16卷 8+611-621页

作者： Ye Yang~1 Jian Cao~1 Lu Huang~1 Hai Yan Fang~2 Hui Zhen Sheng~(1,2) ~1Laboratory of Stem Cell biology,Institute of Biochemistry and Cell biology,shanghai Institutes.for Biological Sciences,Chinese Academy of Sciences,shanghai 200031 china ~2center for developmental biology,xinhua hospital,school of medicine,shanghai jiao tong university,1665 Kong jiang road,shanghai 200092,china Laboratory of Stem Cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China center for developmental biology Xinhua Hospital School of Medicine Shanghai Jiao Tong University Shanghai China

RNA polymerase（Pol）Ⅱ transcription persists in TATA-box-binding protein（TBP）-/-mutant mouse embryos,indicat-ing TBP-independent mechanisms for Pol Ⅱ transcription in early ***-related factor3（TRF3）has beenproposed to substitute for TBP in TBP-/-mouse *** examined the expression of TRF3 in maturing oocytes andearly embryos and found that TRF3 was co-expressed with TBP in the meiotic oocytes and early embryos from the lateone-cell stage *** amounts of TBP and TRF3 changed dynamically and correlated well with *** immunoprecipitation（CHIP）assay revealed that different gene promoters in mouse embryonic stem（ES）cells recruited TRF3 and TBP *** analyses of TRF3 and TBP during cell cycle showed thatboth factors proceeded through cell cycle in a similar pace,except that TRF3 was slightly delayed than TBP in enteringthe nucleus when cells were exiting the *** from expression and biochemical analyses therefore support thehypothesis that TRF3 plays a role in early mouse *** addition,results from co-localization study suggestthat TRF3 may be also involved in Pol I transcription.

关键词： TBP TRF3 oocyte embryo ES cell NIH3T3 cell

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Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis

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Cell Research 2007年第2期17卷 135-150页

作者： Tianlong Gao~(1,*) Junke Zheng~(2,*) Fengying Xing~(2,*) Haiyan Fang~2 Feng Sun~1 Ayong Yan~2 Xun Gong~2 Hui Ding~3 Fan Tang~2 Hui Z Sheng~2 ~1Program for Graduation Studies,shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,shanghai 200031,china ~2center for developmental biology,xinhua hospital,shanghai jiao tong university,school of medicine,1665 Kong jiang road,shanghai 200092,china ~3school of Animal Science and Technology,Yangzhou university,Yangzhou 225009,china Program for Graduation Studies Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China center for developmental biology Xinhua Hospital Shanghai Jiao Tong University School of Medicine Shanghai China school of Animal Science and Technology Yangzhou University Yangzhou China

Somatic cell nuclear transfer(SCNT)and parthenogenesis are alternative forms of reproduction and development,building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin,*** thepreceding paper(Sun F,et al.,Cell Res 2007;17:117-134.),we showed that an"erase-and-rebuild"strategy is used innormal development to transform the maternal gene expression profile to a zygotic ***,we investigate if the samestrategy also applies to SCNT and *** relationship between chromatin and chromatin factors(CFs)during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein *** fromthese studies indicated that soon after nuclear transfer,a majority of CFs dissociated from somatic nuclei and were re-distributed to the cytoplasm of the *** erasure process in oogenesis is recaptured during the initial phase in *** CFs entered pseudo-pronuclei shortly after their *** parthenogenesis,all parthenogenotes underwentnormal oogenesis,and thus had removed most CFs from chromosomes before the initiation of *** CFswere subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized *** these data,we conclude that the"erase-and-rebuild"process observed in normal development also occurs in SCNTand in parthenogenesis,albeit in altered *** process is responsible for transcription reprogramming in ***"erase"process in SCNT is compressed and the efficiency is compromised,which likely contribute tothe developmental defects often observed in nuclear transfer(nt)***,results from this study indicatedthat the cytoplasm of an egg contains most,if not all,essential components for assembling the zygotic program and canassemble them onto appropriate diploid chromatin of distinct origins.

关键词： nuclear reprogramming SCNT parthenogenesis chromatin factors chromatin embryogenesis

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Skeletal myogenesis by human embryonic stem cells

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Cell Research 2006年第8期16卷 713-722页

作者： Jun Ke Zheng~1 Yi Wang~1 Aditi Karandikar~1 Qian Wang~1 Hui Gai~1 Ai Lian Liu~1 Chao Peng~1 Hui Zhen Sheng~(1,2) ~1center for developmental biology,xinhua hospital,school of medicine,shanghai jiao tong university,1665 Kong jiang road,shanghai 200092,china:~2Laboratory of Stem Cell biology,Institute of Biochemisny and Cell biology,shanghai Institutes of Biological Sciences,Chinese Academy of Sciences,shanghai 200037,china center for developmental biology Xinhua Hospital School of Medicine Shanghai Jiao Tong University Shanghai China Laboratory of Stem Cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes of Biological Sciences Chinese Academy of Sciences Shanghai China

We have examined the myogenic potential of human embryonic stem(hES)cells in a xeno-transplantation *** we show that precursors differentiated from hES cells can undergo myogenesis in an adult environmentand give rise to a range of cell types in the myogenic *** study provides direct evidences that hES cells canregenerate both muscle and satellite cells in viro and are another promising cell type for treating muscle degenerativedisorders in addition to other myogenic cell types.

关键词： Skeletal myogenesis embryonic stem cell differentiation

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Potential therapeutic role of spermine via Rac1 in osteoporosis:Insights from zebrafish and mice

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Zoological Research 2024年第2期45卷 367-380页

作者： Rui-Xue jiang Nan Hu Yu-Wei Deng long-Wei Hu Hao Gu Nan Luo Jin Wen Xin-Quan jiang Department of Prosthodontics Shanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghai 200011China College of Stomatology Shanghai Jiao Tong UniversityNational Center for StomatologyNational Clinical Research Center for Oral DiseasesShanghai Key Laboratory of StomatologyShanghai Research Institute of StomatologyShanghai Engineering Research Center of Advanced Dental Technology and MaterialsShanghai 200125China Department of Endodontics Shanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghai Jiao Tong UniversityShanghai 200011China Department of Oral and Maxillofacial-Head and Neck Oncology Shanghai Ninth People’s HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai 200011China Department of Preventive Dentistry Shanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghai Jiao Tong UniversityShanghai 200011China

Osteoporosis(OP)is a prevalent metabolic bone *** drug therapy is essential to prevent bone loss in osteoporotic patients,current treatments are limited by side effects and high costs,necessitating the development of more effective and safer targeted *** a zebrafish(Danio rerio)larval model of osteoporosis,we explored the influence of the metabolite spermine on bone *** showed that spermine exhibited dual activity in osteoporotic zebrafish larvae by increasing bone formation and decreasing bone *** not only demonstrated excellent biosafety but also mitigated prednisolone-induced embryonic neurotoxicity and ***,spermine showcased protective attributes in the nervous systems of both zebrafish embryos and *** the molecular level,Rac1 was identified as playing a pivotal role in mediating the antiosteoporotic effects of spermine,with P53 potentially acting downstream of *** findings were confirmed using mouse(Mus musculus)models,in which spermine not only ameliorated osteoporosis but also promoted bone formation and mineralization under healthy conditions,suggesting strong potential as a bonestrengthening *** study underscores the beneficial role of spermine in osteoporotic bone homeostasis and skeletal system development,highlighting pivotal molecular *** their efficacy and safety,human endogenous metabolites like spermine are promising candidates for new anti-osteoporotic drug development and daily bone-fortifying agents.

关键词： Osteoporosis Spermine Rac1 Zebrafish Mice

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Wrinkled petals and stamens 1,is required for the morphogenesis of petals and stamens in Lotus japonicus

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Cell Research 2006年第5期16卷 499-506页

作者： jiang Hua Chen~1 Ji Liang Pang~2 Li Lin Wang~2 Yong Hai Luo~1 Xin Li~3 Xiang Ling Cao~1 Kui Lin~3 Wei Ma~3 Xiao He Hu~1 Da Luo~(1,3) ~1National Laboratory of Plant Molecular Genetics,Institute of Plant Physiology and Ecology,shanghai Institutes for Biological Sciences,and Graduate school of the Chinese Academy of Sciences,Chinese Academy of Sciences,300 Fenglin road,shanghai 200032,china ~2school of Life Science,Hangzhou Normal College,16 Xuelin road,Hangzhou 310018,china ~3school of Life Science and Biotechnology,shanghai jiao tong university,1954 Huashan road,shanghai 200030,china National Laboratory of Plant Molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences and Graduate School of the Chinese Academy of Sciences Chinese Academy of Sciences Shanghai China school of Life Science Hangzhou Normal College Hangzhou China school of Life Science and Biotechnology Shanghai Jiao Tong University Shanghai China

Although much progress has been made in understanding how floral organ identity is determined during the floraldevelopment,less is known about how floral organ is elaborated in the late floral developmental *** we de-scribe a novel floral mutant,wrinkledt petals and stamensl (wps1),which shows defects in the development of petalsand *** analysis indicates that wps1 mutant is corresponding to a single recessive locus at the long arm ofchromosome *** early development of floral organs in wps1 mutant is similar to that in wild type,and the malfunctionof the mutant commences in late developmental stages,displaying a defect on the appearance of petals and *** mature flower,petals and stamen filaments in the mutant are wrinkled or folded,and the cellular morphology underL1 layer of petals and stamen filaments is *** is found that the expression patterns of floral organ identity genesare not affected in wps1 mutants compared with that of wild type,consistent with the unaltered development of allfloral ***,the identities of epidermal cells in different type of petals are *** histologicalanalysis shows that in wps1 flowers all petals are irregularly folded,and there are knotted structures in the petals,whilethe shape and arrangement of inner cells are malformed and *** on these results,we propose that Wps1acts downstream to the class B floral organ identity genes,and functions to modulate the cellular differentiation duringthe late flower developmental stages.

关键词： Lotus japonicus wps1 mutant ABC model floral organ differentiation

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Acquired immune thrombotic thrombocytopenic purpura(TTP)associated with inactivated COVID-19 vaccine CoronaVac

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Frontiers of medicine 2024年第4期18卷 744-751页

作者： Zhangbiao long Suyu jiang Honglei Xin Lu Zhang Ruinan Lu Fengqi Liu Yong Xu Linv Wang Jun Wang Xuezhong Zhang Hui Liao Jinning Shi Xue Yan Xiang Zhu Ruonan Shao Zijian Li Yilin Zhu Han Yan jiao Wu Chao Fang Xiaodong Xi Xiaofeng Shi Department of Hematology The First Affiliated Hospital of Anhui Medical UniversityHefei230022China Department of Hematology The Second Affiliated Hospital of Nanjing Medical UniversityNanjing210003China Department of Hematology The First Affiliated Hospital of Nanjing Medical UniversityJiangsu Province HospitalNanjing210029China Department of Hematology Nanjing Drum Tower HospitalThe Affiliated Hospital of Nanjing University Medical SchoolNanjing210008China Department of Hematology The Affiliated Hospital of Nanjing University of Chinese MedicineThe First Clinical Medical College of Nanjing University of Chinese MedicineNanjing210009China Department of Hematology Zhongda Hospital of Southeast UniversityNanjing210044China Department of Hematology Nanjing First HospitalNanjing Medical UniversityNanjing210006China Department of Hematology The Air Force Hospital of Eastern Theater commandNanjing210016China Department of Hematology Jiangning Hospital Affiliated of Nanjing Medical UniversityNanjing211199China Department of Hematology Nanjing Hospital of Chinese MedicineNanjing210006China Department of Pharmacology School of Basic MedicineTongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious DiseasesHuazhong University of Science and TechnologyThe Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei ProvinceWuhan430030China shanghai Institute of Hematology State Key Laboratory of Medical GenomicsRuijin Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghai200025China

Corona virus disease 2019(COVID-19)due to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection has affected the whole *** thrombotic thrombocytopenic purpura(TTP)has been reported after administration of mRNA-or adenoviral vector-based COVID-19 vaccines,including ***2-S,BNT162b2,mRNA-1273,and ChAdOx1 ***,whether inactivated vaccines,such as CoronaVac,could cause TTP and whether the symptoms in TTPs caused by inactivated vaccines are different from previously reported cases are *** this study,two cases were *** cases developed TTP after the second CoronaVac vaccination shot,but not the *** demonstrated symptoms of fever,neurological abnormalities,renal dysfunction,thrombocytopenia,and *** patients achieved complete remission through several sessions of plasma exchanges and immune *** incidence of TTP in Nanjing area was *** number of patients with TTP was 12 in 2019,6 in 2020,16 in 2021,and 19 in *** the authors’knowledge,this report is the first report of TTP associated with inactivated COVID-19 vaccine(CoronaVac).The rarity and delayed onset may be due to the relatively milder immune response caused by the inactivated vaccines than mRNA-based *** plasma exchange is a vital treatment for CoronaVac-related TTP,similar to activated vaccine-related TTP.

关键词： thrombotic thrombocytopenia purpura(TTP) corona virus disease 2019(COVID-19) CoronaVac acute respiratory syndrome coronavirus 2(SARS-CoV-2) vaccine

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IMpower210:A phase Ⅲ study of second-line atezolizumab vs. docetaxel in East Asian patients with non-small cell lung cancer

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Chinese Journal of Cancer Research 2024年第2期36卷 103-113页

作者： Yi-long Wu Shun Lu Gongyan Chen Jianxing He Jifeng Feng Yiping Zhang Liyan jiang Hongming Pan Jianhua Chang Jian Fang Amy Cai Lilian Bu Jane Shi Jinjing Xia Guangdong Lung Cancer Institute Guangdong Provincial Key Laboratory of Translational Medicine in Lung CancerGuangdong Provincial People's Hospital&Guangdong Academy of Medical SciencesSouthern Medical UniversityGuangzhou 510080China shanghai Lung Cancer center Shanghai Chest HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai 200025China Department of Respiration Harbin Cancer HospitalHarbin Medical UniversityHarbin 150081China Department of Thoracic Oncology and Surgery the First Affliated Hospital of Guangzhou Medical UniversityGuangzhou 510120China Department of Medical Oncology Jiangsu Cancer HospitalNanjing 210009China Department of Medical Oncology Zhejiang Cancer HospitalHangzhou 330022China Department of Pulmonary medicine Shanghai Chest HospitalShanghai Jiao Tong UniversityShanghai 200030China Department of Medical Oncology Sir Run Run Shaw HospitalZhejiang University School of MedicineHangzhou 310016China Department of Medical Oncology Shenzhen HospitalCancer Hospital of Chinese Academy of Medical SciencesShenzhen 518116China Department of Thoracic Oncology Beijing Cancer HospitalBeijing 100142China Product Development Shanghai Roche Pharmaceutical LtdShanghai 201203China

Objective: IMpower210(NCT02813785) explored the efficacy and safety of single-agent atezolizumab *** as second-line treatment for advanced non-small cell lung cancer(NSCLC) in East Asian ***: Key eligibility criteria for this phase Ⅲ, open-label, randomized study included age ≥18 years;histologically documented advanced NSCLC per the Union for International Cancer Control/American Joint Committee on Cancer staging system(7th edition);Eastern Cooperative Oncology Group performance status of 0 or 1;and disease progression following platinum-based chemotherapy for advanced or metastatic NSCLC. Patients were randomized 2:1 to receive either atezolizumab(1,200 mg) or docetaxel(75 mg/m^(2)). The primary study endpoint was overall survival(OS) in the intention-to-treat(ITT) population with wild-type epidermal growth factor receptor expression(ITT EGFR-WT) and in the overall ITT ***: Median OS in the ITT EGFR-WT population(n=467) was 12.3 [95% confidence interval(95% CI),10.3-13.8] months in the atezolizumab arm(n=312) and 9.9(95% CI, 7.8-13.9) months in the docetaxel arm[n=155;stratified hazard ratio(HR), 0.82;95% CI, 0.66-1.03]. Median OS in the overall ITT population was 12.5(95% CI, 10.8-13.8) months with atezolizumab treatment and 11.1(95% CI, 8.4-14.2) months(n=377) with docetaxel treatment(n=188;stratified HR, 0.87;95% CI, 0.71-1.08). Grade 3/4 treatment-related adverse events(TRAEs) occurred in 18.4% of patients in the atezolizumab arm and 50.0% of patients in the docetaxel ***: IMpower210 did not meet its primary efficacy endpoint of OS in the ITT EGFR-WT or overall ITT populations. Atezolizumab was comparatively more tolerable than docetaxel, with a lower incidence of grade3/4 TRAEs.

关键词： Atezolizumab East Asia non-small cell lung cancer programmed death-ligand 1 inhibitors monoclonal antibody

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A Transgenic Zebrafish Model of Macrophage Migration and Chemotaxis

A Transgenic Zebrafish Model of Macrophage Migration and Che...

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中国遗传学会“发育、遗传和疾病”研讨会

作者： Yong Zhang~1 Xue-Tao Bai~1 Yi Jin~(1,2) Min Deng~2 Yi Chen~(1,2) Huang-Ying Le~(1,2) Yah-Fang Fu~2 Xiao-Jian Sun~(1,2) jiang Zhu~1 Zhu Chen~(1,4) Sai-Juan Chen~1 Ting Xi Liu~(1,2,3) 1.State Key Laboratory for Medical Genomics and shanghai Institute of Hematology,RuiJin hospital affiliated to shanghai jiao tong university school of medicine,shanghai 200025,china 2.Laboratory of Development and Diseases,Institute of Health Sciences,shanghai Institutes for Biological Sciences,Chinese Academy of Sciences & shanghai jiao tong university school of medicine,shanghai 20002,china 3.Model Organism Division,E-Institutes of shanghai Universities,shanghai 200025,china 4.shanghai center for Systems Biomedicine,shanghai jiao tong university,shanghai 200240,china

We have established an in vivo model for visualizing the developmental and chemotactic movement of primitive macrophages,by generating a stable transgenic line expressing EGFP under the zebrafish lysozyme C promoter [TG(zlyz:EGFP)].We show that the earliest macrophages originate from the head mesenchyme nearby the bilateral plate mesoderm at～22 hpf,then migrate either within the head or toward the yolk sac close to the circulation *** macrophages subsequently colonize the posterior intermediate cell mass(ICM)through ***,the resident macrophages,but not the circulating macrophages,migrate rapidly to the site of inflammation induced by tail transection,with their number peaking at approximately 5-6 hours after *** hours post-injury,these recruited macrophages initiate retrograde migration from the inflamed tail back to the ICM mesenchyme,suggesting a resolution of the ***,an anti-inflammatory glucocorticoid hormone (dexamethasone)suppresses both the chemotactic and retrograde migration of macrophages during the onset and resolution of ***,the TG(zlyz:EGFP)zebrafish provides an invaluable tool not only for elucidating the pleiotropic roles of macrophages in innate immunity,tumorigenesis and organogenesis during embryogenesis,but for identifying the therapeutic small molecules against human disorders associated with deregulated migration and chemotaxis of macrophage in vivo.

关键词： A Transgenic Zebrafish Model of Macrophage Migration and Chemotaxis Model

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Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries:a new approach to autologous stem cell therapy

Parthenogenetic embryonic stem cells derived from cryopreser...

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第九届中国实验动物科学年会（2010新疆）

作者： Fengying Xing,Ph.D.,a Zhenfu Fang,Ph.D.,a Bolin Qin,M.S.,a Yao Li,Ph.D.,a Jian Hou,Ph.D.,b and Xuejin Chen,Ph.D.a a center for developmental biology,xinhua hospital,school of medicine,shanghai jiao tong university, shanghai,china and b College of Biological Sciences,china Agricultural university,Beijing,People’s Republic of china

Objective It has been suggested that somatic cell nuclear transfer techniques can produce embryonic stem (ES) cells for stem cell therapy without the risk of immune ***,somatic cell nuclear transfer requires complex techniques and culture conditions and is accompanied by numerous ethical *** contrast,parthenogenesis involves embryos derived directly from unfertilized *** work demonstrated that parthenogenetic embryos always arrest in the early limb bud stage(1) and that they do not produce live ***,parthenogenetic ES cells used in stem cell therapy avoid ethical disputes(2).A parthenogenetic ES cell line from nonhuman primates was first established using ovulated oocytes(3).Recently,Lee et al.(2) reported that autologous ES cells can be obtained using mouse preantral follicles cultured in vitro and oocyte *** has been shown that primordial ovarian follicles survive the cryopreservation process for a long time and maintain their fertility(4-7).Therefore,we chose to use cryopreserved newborn mouse ovaries as a source of oocytes for establishing parthenogenetic ES *** procedures included cryopreservation of newborn mouse ovaries,thawing them,grafting the ovaries under the kidney capsule of immunodeficient mice,promotion of follicular maturation using pregnant mare serum gonadotropin(PMSG),and in vitro parthenogenetic activation of oocytes matured in *** this new approach,large numbers of oocytes were harvested from newborn mouse ovaries and activated parthenogenetically to derive parthenogenetic mouse ES(pmES) cell lines for use in autologous cell *** pm ES cell lines generated by these procedures have not yet been reported.

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