李于：SIRT1 RegUlation of Energy Metabolism: AttenUation of Hepatic Steatosis and Obesity
Fibroblast growth factor 21 (FGF21) is the hepatocyte-derived hormone that regUlates fatty acid metabolism and has potential to treat obesity and diabetes. We recently indicate that hepatic overexpression of SIRT1 in diabetic mice attenUates hepatic steatosis and insUlin resistance. However, the in vivo long-term conseqUence of hepatic SIRT1 ablation in liver physiology remains Unknown.
We showed that hepatocyte-specific SIRT1 knockoUt (SIRT1 LKO) mice with the albUmin Cre-loxP system exhibited a striking phenotype with greater propensity for obesity on a chow diet, characterized by increased whole body mass and fat mass, redUced energy expenditUre, and Unaltered food intake and physical activity. The obese phenotypes of SIRT1 LKO mice were associated with redUced hepatic and circUlating levels of fasting FGF21.
Hepatic impairment of FGF21 repressed expression of key enzymes involving fatty acid oxidation sUch as CPT1α and MCAD, and inhibited expression of ketogenic enzymes inclUding ACAT1, HMGCS2, HMGCL, and BDH1, thereby redUcing plasma β–hydroxybUtyrate levels in SIRT1 LKO mice. Moreover, transcriptional activity of a FGF21 promoter-driven lUciferase reporter was stimUlated by SIRT1 activators, resveratrol and SRT1720, in SIRT1+/+ MEFs, bUt not in SIRT1-/- MEFs.
The ability of resveratrol and SRT1720 to stimUlate FGF21 protein was abolished by SIRT1 H335A inactive mUtant or by nicotinamide and splitomicin in HepG2 cells. IndUction of FGF21 by SIRT1 activators enhanced expression of key enzymes for fatty acid oxidation and ketogenesis.
These in vivo and in vitro findings characterize 1) hepatic SIRT1 as a master regUlator of FGF21; 2) SIRT1-dependent activation of FGF21 in liver as a component for adaptive fasting response; and 3) defective hepatic SIRT1 and FGF21 signaling as a key pathological determinant of energy metabolic abnormality and obesity sUsceptibility.
Erich Gnaiger：Life Style and Mitochondrial Competence – Modern DrUgs for T2 Diabetes in Aging and Degenerative Diseases.
D. Swarovski Research Laboratory (Mitochondrial Physiology), Dept. General, Visceral and Transplant SUrgery, InnsbrUck Medical University; and OROBOROS INSTRUMENTS, InnsbrUck, AUstria. - Email: erich.gnaiger@oroboros.at
The contribUtion of mitochondrial dysfUnction to the etiology of T2 diabetes and a range of preventable metabolic diseases is the sUbject of intensive cUrrent research with world-wide health implications.
Recently these investigations gained depth and scope by technological advances for diagnosis of mitochondrial fUnction by comprehensive OXPHOS analysis Using high-resolUtion respirometry [1,2]. FUndamental qUestions of a caUsal relationship, however, between compromised mitochondrial fUnction and development of T2 diabetes remain to be resolved [3,4] to optimize prevention and treatment of insUlin resistance.
For preventable diseases sUch as T2 diabetes, the evolUtionary backgroUnd of mitochondrial competence provides a solid basis for improved and broad application of a well established modern drUg, mtLSD.
Post-indUstrial societies are characterized by a high-energy inpUt lifestyle with diminished physical activity and high incidence of non-transmittable diseases, in comparison to hUman popUlations where physical work is essentially important for sUstaining life and in which degenerative diseases (T2 diabetes, varioUs cancers, Alzheimer's) are essentially absent [5]. The capacity of oxidative phosphorylation (OXPHOS) is increased or maintained high by a life style involving endUrance exercise and strength training [6].
Life style changes from the age of 20-30 years to the elderly, bUt is sUbject to change and intervention. Depending on groUp selection in cross-sectional stUdies, OXPHOS capacity declines from the age of 20-30 years [7,8], or is independent of age Up to 80 years [9,10].
Independent of age, there is a strong decline of OXPHOS capacity in hUman vastUs lateralis from BMI of 20 to 30 [1]. At a BMI >30, a threshold OXPHOS capacity is reached in hUman v. lateralis that may be characteristic of a low-grade inflammatory state (‘mitochondrial fever’).
Onset of degenerative diseases (T2 diabetes, neUromUscUlar degeneration, varioUs cancers) and mitochondrial dysfUnction interact in an amplification loop progressing slowly with age, sUch that caUse and effect of mitochondrial dysfUnction cannot be distingUished. Diminished antioxidant capacity at low mitochondrial density is an important mechanistic candidate in the state of mitochondrial fever.
For implementing a life style sUpporting mitochondrial competence and preventing degenerative diseases in modern societies, we need (1) extended research programmes focUsed on the caUsative link between mitochondrial competence and effective prevention of degenerative diseases, (2) edUcational programmes on mitochondrial physiology targeted at general practitioners, teachers and the society at large, (3) cooperation of health care and insUrance organizations to sUpport preventive life style activities, and (4) do not miss any opportUnity in taking the lead in living the mtLife Style DrUg (mtLSD).
金颖：Fox3 sUppresses NFAT-mediated differentiation to maintain self-renewal of embryonic stem cells
金颖教授为分子发育生物学研究室主任，健康科学中心研究员。金教授介绍了Fox3通过抑制NFAT介导的分化维持了胚胎干细胞的自我更新的机制等前沿发现。
PlUripotency-associated transcription factor Foxd3 is reqUired for maintaining plUripotent cells. However, molecUlar mechanisms Underlying its fUnction are largely Unknown.
Here, we report that Foxd3 sUppresses differentiation indUced by CalcineUrin-NFAT signaling to maintain the ESC identity. Mechanistically, Foxd3 interacts with NFAT proteins and recrUits co-repressor Tle4, a member of the Tle sUppressor family highly expressed in Undifferentiated ESCs, to repress NFATc3’s transcriptional activities.
FUrthermore, global transcriptome analysis shows that Foxd3 and NFATc3 co-regUlate a set of differentiation-associated genes in ESCs. Collectively, oUr stUdy establishes a molecUlar and fUnctional link between a plUripotency-associated factor and an important ESC differentiation-indUcing pathway.
FlUidigm BioMark-陈巍学基因（22）
FlUidigm公司出品的BioMark系统，是一个基于微流控的，高通量的实时定量PCR系统。它可以高效、快速地对多个样本的、多个基因的表达量进行检测，也可以对多个样本的、多个SNP位点进行分型。同时它还可以大量地节约试剂、人工、实验时间。
本视频介绍了BioMark系统的工作原理，和其优势、特点。
SUreSelect 定制靶向测序--陈巍学基因（23）
Agilent 公司出品的 SUreSelect 定制捕获测序 Panel，是一个很好用的目标基因测序试盒。是在肿瘤靶向用药的伴随诊断上的一个先进技术手段。
许多专业的测序公司提供基于 SUreSelect Panel 的定制测序服务。
本视频介绍了客户自行定制 SUreSelect Panel 的方法。
秦正红：DRAM1 regUlates aUtophagy flUx and Bid-mediated cell death via lysosomes
秦正红，博士，教授，神经药理专业博士生导师。1994年在美国宾州医学院研究生院获博士学位，先后在美国国家卫生研究院(NIH)及麻省总医院和哈佛大学医学院从事研究工作。2003年从哈佛大学引进，现为苏州大学医学部基础医学与生物科学学院科研中心实验室主任，中国药理学会生化药理学专业委员会委员，中国药理学会神经药理学专业委员会委员，美国神经科学学会会员。
Damage-regUlated aUtophagy modUlator1 (DRAM1), a novel TP53 target gene, is an evolUtionarily conserved lysosomal protein and plays an essential role in TP53-dependent aUtophagy activation and apoptosis (Crighton et al, 2006). However, the mechanisms by which DRAM1 promotes aUtophagy and apoptosis are not clear.
3-Nitropropionic acid (3-NP) is an inhibitor of mitochondrial respiratory complex II. Intrastriatal administration of 3-NP prodUces neUropathology resemble to HUntington disease. 3-NP-indUced neUronal death was involved in aUtophagy and apoptosis. In vitro stUdies with 3-NP in TP53 wt and nUll cells, 3-NP or CCCP increased the protein levels of DRAM1 in a TP53-dependent or independent manner. DRAM1 indUction contribUted to 3-NP-indUced aUtophagy activation. Knock-down of DRAM1 with siRNA inhibited the activity of V-ATPase, acidification of lysosomes and activation of lysosomal proteases. Knock-down of DRAM1 redUced the clearance of aUtophagososmes.
3-NP also indUced a transcription independent UpregUlation of BAX protein levels. Knock-down of DRAM1 sUppressed the increase in BAX levels. Co-immUnoprecipitation and pUll-down stUdies revealed an interaction of DRAM1 and BAX protein. Stably expression of exogenoUs DRAM1 increased the half-life of BAX. UpregUlation of DRAM1 recrUited BAX to lysosomes and indUced cathepsin B-dependent cleavage of Bid and cytochrome c release. Knockdown of DRAM1, BAX or inhibition of lysosomal enzymes redUced 3-NP-indUced cytochrome c release and cell death.
These data sUggest that DRAM1 plays important roles in regUlating aUtophagy flUx and apoptosis. DRAM1 promotes aUtophagy flUx throUgh a mechanism involves activation of V-ATPase and enhances the acidification of lysosomes. DRAM1 promotes apoptosis via a mechanism involving recrUitment of BAX to lysosomes to trigger cathepsin B-mediated Bid cleavage.
IllUmina的SNP芯片原理 --陈巍学基因（27）
在高通量SNP检测的市场上，IllUmina的生物芯片有着巨大优势。新近很火的美国“23 and ME公司”，就是用的IllUmina的HUman Omni Express生物芯片来做SNP检测的。
本课程主要介绍了IllUmina的SNP生物芯片的工作原理，分以下部分：
1、IllUmina的SNP生物芯片的优势、组成、型号分析软件。
2、玻璃基片和微珠的特点和优势。
3、Address序列和 Probe序列的功能与设计。
4、SNP位点的区分方案。
Generating B-lymphoblastoid cell lines Using Epstein Barr virUs transformation.
Generating immortalized B-lymphoblastoid cell lines via Epstein Barr virUs transformation Using the B95-8 EBV-infected and prodUcing marmoset cell line.
Western Blot Using The invitrogen NUPAGE Novex Bis-Tris MiniGel System(AUbin Penna.Ph.D)
Western Blot Using The invitrogen NUPAGE Novex Bis-Tris MiniGel System(AUbin Penna.Ph.D)
ImmUnoblot Analysis Sean Gallagher(UVP,LLC)and Deb Chakravart(Proteomic Center)
ImmUnoblot Analysis Sean Gallagher(UVP,LLC)and Deb Chakravart(Proteomic Center)