WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

POU 抄写因素 OCT4 不仅在维持 pluripotent 和房间而且幕作为通过基因剂量的一个房间命运决定因素完成的胚胎的茎(ES ) 的自我更新的状态起一个必要作用。然而，控制细胞内部的 OCT4 蛋白质水平的分子的机制留下逃犯。这里，我们报导那人的 WWP2， E3 ubiquitin (Ub ) 蛋白质 ligase，通过它的 WW 领域明确地与 OCT4 交往并且在 vitro 并且在 vivo 提高 OCT4 的 Ub 修正。我们首先证明在人的 ES 房间的内长的 OCT4 能被 Ub post-translationally 修改。而且，我们发现 WWP2 以一种剂量依赖者方式，和 WWP2 的活跃地点半胱氨酸残余通过 26S proteasome 支持了 OCT4 的降级在 OCT4 上为它的酶的活动和解朊的效果被要求。显著地，我们当 WWP2 表示是由特定的 RNA 干扰(RNAi ) 的 downregulated 时，内长的 OCT4 蛋白质水平显著地被提高的数据表演，建议那 WWP2 是为在人的 ES 房间维持合适的 OCT4 蛋白质水平的一个重要管理者。而且，北污点分析证明 WWP2 抄本在多样的人的织物 / 器官是广泛地在场的并且高度在无差别的人的 ES 房间表示了。然而，它的表示水平快速在区分的人的 ES 房间以后被减少，显示 WWP2 表示力量发展地被调整。我们的调查结果证明 WWP2 是在人的 ES 房间的 OCT4 蛋白质水平的一个重要管理者。

Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts

胚胎的茎(ES ) 细胞从胚囊阶段胚胎被导出。他们自强和 pluripotency 的唯一的性质为治疗为基本研究和一个潜在的房间资源使他们成为一个吸引人的工具。老鼠和人的 ES 房间成功地在大量研究被产生了并且适用。然而，没有真 ES 房间线迄今为止从老鼠被获得了。在这研究，我们对 pluripotent 干细胞的标记用特定的抗体在早老鼠胚胎识别了 pluripotent 房间。由为老鼠胚囊修改文化媒介，随后，我们导出 pluripotent 老鼠象 ES 一样房间线，它表示了 pluripotency 标记并且在 vitro 形成了胚胎植物或动物身体(EB ) 。重要地，这些老鼠象 ES 一样房间能生产 teratomas。EB 和 teratomas 从所有三胚胎的细菌层包含了纸巾。从老鼠象 ES 一样房间，另外，我们导出一根老鼠原语内胚叶(PrE ) 房间线。而且，我们进行了老鼠象 ES 一样房间介绍的 transcriptional 并且识别了老鼠 pluripotent 干细胞的唯一的分子的签名。我们的分析表明那多重发信号小径包括 BMP， Activin 和 mTOR 小径，可以涉及处于一个无差别的状态把老鼠作为象 ES 一样房间。在这研究获得的房间线和信息将加速我们位于 pluripotency 下面的分子的规定的理解并且从特别的种在 ES 房间的适当操作指导我们。

Mesenchymal stem cells： a new strategy for immunosuppression and tissue repair

间充质的干细胞(MSC ) 为治疗各种各样的疾病有大潜力，特别那些与织物有关损坏包含有免疫力的反应。各种各样的研究证明了 MSC 在 vitro 并且在 vivo 是强烈抑制免疫力的。我们的最近的研究证明了那未刺激的 MSC 确实不能免疫力的抑制；他们与 TNF- 伪， IL-1 伪或 IL-1 尾与激活的淋巴细胞的上层清液，或与 IFN- 纬的联合在刺激之上变得 potently 抑制免疫力。这观察表明在某些情形下面，煽动性的 cytokines 能实际上变得抑制免疫力。我们证明在调停 MSC 的免疫力的抑制的机制有一个种类变化：由告知 cytokine 的老鼠 MSC 的免疫力的抑制被氮的氧化物调停(没有) 而由告知 cytokine 的人的 MSC 的免疫力的抑制通过 indoleamine 被执行 2， 3-dioxygenase (伊多语) 。在有煽动性的 cytokines 的刺激之上，另外，老鼠和人的 MSC 分泌几白血球 chemokines 显然服务与 MSC 吸引有免疫力的房间进最近，在哪儿不或伊多语被预言很活跃。因此，由煽动性的刺激 cytokine 的 MSC 的免疫力的抑制经由 chemokines 的一致行动发生并且免疫者禁止没有或伊多语由 MSC 生产了。因此，我们的结果在对待有免疫力的回答导致的织物损害关于调停 MSC 的免疫力的抑制并且为 MSC 的更好的申请的机制提供新奇信息。

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

基本 helix-loop-helix (bHLH ) 的成员基因家庭在脊椎动物神经发生起重要作用。在这研究，共焦的基于显微镜学的荧光回声精力转移(烦恼) 被用来在各种各样的生理的条件下面监视 bHLH 蛋白质蛋白质相互作用。织物特定的 bHLH 使活跃之物， NeuroD1， Mash1， Neurogenin1 (Ngn1 ) ， Neurogenin2 (Ngn2 ) ，和无所不在的表示 E47 蛋白质与提高的黄荧光蛋白质(EYFP ) 被标注并且分别地提高了青色荧光蛋白质(ECFP ) 。潜水艇 bHLH 熔化蛋白质的细胞的本地化和活动性在 HEK293 房间被检验。由短暂 transfection 并且在 ovo electroporation，四织物特定的 bHLH 使活跃之物和 E47 蛋白质在 HEK293 房间和发展中的小鸡胚胎是过去表示的神经试管。与漂白的领受人相片方法，烦恼能在 transfected 的原子核在这些 bHLH 蛋白质对之间被检测房间和发展中的神经试管。Mash1/E47 和 Ngn2/E47 烦恼对更高出现烦恼效率在中间并且小鸡胚胎的侧面的一半神经试管分别地。它建议这些 bHLH 蛋白质对在特定的区域与他们的下游的目标基因的规章的元素形成了功能的 DNA 蛋白质建筑群。这个工作将帮助一个在 vivo 理解 bHLH 因素的行为。

Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts

一个单个哺乳动物的抄本通常编码一蛋白质，但是 GNAS (G 蛋白质伪 - 子单元) 的抄本包含二个读物框架并且生产二在结构上无关的蛋白质， XL 伪 s 和 ALEX。没有其它证实象 GNAS 一样双编码的抄本迄今为止被报导了，尽管许多如此的候选人基因被生物信息学分析预言了。在这研究，我们构造了一系列向量测试二个蛋白质产品怎么在 vitro 从一个单个抄本被翻译。ORF (开的读框架) 的长度，第一 8 月的位置和 Kozak 主题被发现是重要因素。这些因素，以及 55-bp NMD (调停胡说八道的 mRNA 腐烂) 规则，为候选人在生物信息学搜索被使用双编码的抄本。分别地， 1307， 750 和 474 个 two-ORF-containing 抄本的一个总数分别地， 170， 89 和 70 哪个被发现是潜在的双编码的抄本在人，老鼠和老鼠被发现。大多数抄本在种类之中显示出低保存。有趣地，双编码的抄本显著地从锌手指蛋白质家庭为抄本被充实，它通常是涉及抄写过程的规定的 DNA 有约束力的蛋白质。

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

囊包含域的蛋白质属于 phosphoinositide 磷酸酶(PIPPase 家庭) 的一个最新识别的家庭。尽管有描绘得好的酶的活动，这个哺乳动物的囊领域 PIPPase 家庭的生物功能仍然保持大部分未知。我们识别了新奇的囊包含域的蛋白质，老鼠 Sac3 (rSac3 ) 它是广泛地在各种各样的纸巾表示了并且对 endoplasmic 蜂窝胃局部性， Golgi 复杂、再循环的内涵体。rSac3 在 vitro 作为底层与 PI (3 ) P， PI (4 ) P 和 PI (3,5 ) P (2 ) 显示 PIPPase 活动，并且在囊域的催化核心的一个变化废除它的酶的活动。rSac3 的表示起来在刺激的神经生长因素(NGF ) 期间调整了 PC12 细胞 neuronal 区别，并且在这的表示上，蛋白质在 PC12 细胞支持神经突长出。相反地，由反感觉 oligonucleotides 的 rSac3 表示的抑制减少刺激 NGF 的 PC12 房间的神经突长出，并且 rSac3 的活跃地点变化消除它的 neurite-outgrowth-promoting 活动。这些结果显示 rSac3 在通过它的 PIPPase 活动区分神经原支持神经突长出，建议囊领域 PIPPase 蛋白质可以参予从到血浆膜的 endoplasmic 蜂窝胃和 Golgi 建筑群的前面的膜交通国王，并且可以作为 neuronal 房间生长和区别的这个关键过程的管理者工作。

Identification of karyopherin-alpha 2 as an Oct4 associated protein

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem(ES) cells.To further explore the functional network of Oct4,the yeast two-hybrid system was used to search for Oct4 interacting proteins.PH domain(containing POU domain and homeodomain) of human OCT4 was used as a bait.From the human testis cDNA library,we identi-fied a strong interaction between OCT4 and karyopherin-alpha 2(KPNA-2).KPNA2 is involved in active nuclear import of proteins.This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays.The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins.In addition,we studied nuclear localization signal(NLS) of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization.Thus,our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

Cell–cell contact with proinflammatory macrophages enhances the immunotherapeutic effect of mesenchymal stem cells in two abortion models

Mesenchymal stem cells(MSCs),which are pluripotent cells with immunomodulatory properties,have been considered good candidates for the therapy of several immune disorders,such as inflammatory bowel diseases,concanavalin A-induced liver injury,and graft-versus-host disease.The embryo is a natural allograft to the maternal immune system.A successful pregnancy depends on the timely extinction of the inflammatory response induced by embryo implantation,followed by the switch to a tolerant immune microenvironment in both the uterus and the system.Excessive infiltration of immune cells and serious inflammatory responses are triggers for embryo rejection,which results in miscarriage.Here,we demonstrated that adoptive transfer of MSCs could prevent fetal loss in a lipopolysaccharide(LPS)-induced abortion model and immune response-mediated spontaneous abortion model.The immunosuppressive MSCs alleviated excessive inflammation by inhibiting CD4+T cell proliferation and promoting the decidual macrophage switch to M2 in a tumor necrosis factor-stimulated gene-6(TSG-6)-dependent manner.Cell-tocell contact with proinflammatory macrophages increased the TSG-6 production by the MSCs,thereby enhancing the suppressive regulation of T cells and macrophages.Moreover,proinflammatory macrophages in contact with the MSCs upregulated the expression of CD200 on the stem cells and facilitated the reprogramming of macrophages towards an anti-inflammatory skew through the interaction of CD200 with CD200R on proinflammatory macrophages.Therefore,the results demonstrate that a TSG-6-mediated paracrine effect,reinforced by cell-to-cell contact between MSCs and proinflammatory macrophages,is involved in the mechanism of MSC-mediated abortion relief through the induction of immune tolerance.Our study also indicates the potential application of MSCs in clinical recurrent miscarriages.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells

Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

Overexpression of mechanical sensitive miR-337-3p alleviates ectopic ossification in rat tendinopathy model via targeting IRS1 and Nox4 of tendon-derived stem cells

Tendinopathy,which is characterized by the ectopic ossification of tendon,is a common disease occurring in certain population,such as athletes that suffer from repetitive tendon strains.However,the molecular mechanism underlying the pathogenesis of tendinopathy caused by the overuse of tendon is still lacking.Here,we found that the mechanosensitive miRNA,miR-337-3p,had lower expression under uniaxial cyclical mechanical loading in tendon-derived stem cells(TDSCs)and negatively controlled chondro-osteogenic differentiation of TDSCs.Importantly,downregulation of miR-337-3p expression was also observed in both rat and human calcified tendons,and overexpressing miR-337-3p in patellar tendons of rat tendinopathy model displayed a robust therapeutic efficiency.Mechanistically,we found that the proinflammatory cytokine interleukin-1^was the upstream factor of miR-337-3p that bridges the mechanical loading with its downregulation.Furthermore,the target genes of miR-337-3p,NADPH oxidase 4,and insulin receptor substrate 1,activated chondro-osteogenic differentiation of TDSCs through JNK and ERK signaling,respectively.Thus,these findings not only provide novel insight into the molecular mechanisms underlying ectopic ossification in tendinopathy but also highlight the significance of miR-337-3p as a putative therapeutic target for clinic treatment of tendinopathy.

Wwp2 mediates Oct4 ubiquitination and its own auto-ubiquitination in a dosage-dependent manner

抄写因素 Oct4 在维持 pluripotency 并且控制胚胎的干细胞(转换字符) 的系承诺起关键作用。我们的以前的学习显示那 Wwp2，鼠标 HECT 类型 E3 ubiquitin ligase， ubiquitinates Oct4 并且在一个 heterologous 系统支持它的降级。然而，在象 Wwp2 的功能的分子的特征一样调整内长的 Oct4 蛋白质层次的 Wwp2 的角色一直不是坚定的。这里，我们报导 Wwp2 在 embryonal 癌房间(ECC ) 的区别期间在 Oct4 ubiquitination 和降级起一个重要作用，尽管它不看起来在无差别的 ECC 和转换字符影响 Oct4 蛋白质层次。重要地，由特定的 RNA 干扰的 Wwp2 表示的抑制提高 Oct4 蛋白质水平，导致在 retinoid 的变细区别相关的标记基因的导致酸的激活。机械学地， Wwp2 经由离氨酸催化 Oct4 poly-ubiquitination 以一种剂量依赖者方式的 63 连接。有趣地， Wwp2 也以一种类似的方式调整它的自己的 ligase 活动。而且， Wwp2 的 auto-ubiquitination 通过 intra 分子的机制发生。一起拿，这些结果在在 ECC 的区别过程期间控制内长的 Oct4 蛋白质层次表明 Wwp2 的一个关键角色并且为调整 E3 ubiquitin ligase 的催化活动建议有趣的剂量依赖者机制， Wwp2。

The histone H3 lysine-27 demethylase Jmjd3 plays a critical role in specific regulation of Th17 cell differentiation

Interleukin(IL)17-producing T helper(Th17)cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases,such as multiple sclerosis,psoriasis,and ulcerative colitis.Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently,the participation of epigenetic modifications in the differentiation process has yet to be elucidated.We demonstrated here that histone H3 lysine-27(H3K27)demethylation,predominantly mediated by the H3K27 demethylase Jmjd3,crucially regulated Th17 cell differentiation.Activation of naı¨ve CD41 T cells immediately induced high expression of Jmjd3.Genetic depletion of Jmjd3 in CD41 T cells specifically impaired Th17 cell differentiation both in vitro and in vivo.Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficientCD41 T cells.Importantly,Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis(EAE).Furthermore,inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro.At the molecular level,Jmjd3 directly bound to and reduced the level of H3K27 trimethylation(me3)at the genomic sites ofRorc,which encodes the masterTh17 transcription factorRorgt,and Th17 cytokine genes such as Il17,Il17f,and Il22.Therefore,our studies established acritical role of Jmjd3-mediatedH3K27demethylation inTh17 cell differentiation andsuggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses.

Generation and application of human iPS cells

Human embryonic stem (ES) cells are capable of unlimited proliferation and maintenance of pluripo-tency in vitro;these properties may lead to potential applications in regenerative medicine. However,immune rejection hampers the allogenic application of human ES cells. Over-expression of several specific transcription factors has been used to reprogram human adult cells into induced pluripotent stem (iPS) cells,which are similar to hESCs in many aspects. The iPS technique makes it possible to produce patient-specific pluripotent stem cells for transplantation therapy without immune rejection. However,some challenges remain,including viral vector integration into the genome,the existence of exogenous oncogenic factors,and low induction efficiency. Here,we review recent advances in human iPS methodology,as well as remaining challenges and its potential applications.

GLUT3 induced by AMPK/CREB1 axis is key for withstanding energy stress and augments the efficacy of current colorectal cancer therapies

Cancer cells are usually characterized by hyperactive glucose metabolism,which can often lead to glucose scarcity;thus,alternative pathways to rewire cancer metabolism are required.Here,we demonstrated that GLUT3 was highly expressed in colorectal cancer(CRC)and negatively linked to CRC patient outcomes,whereas GLUT1 was not associated with CRC prognosis.Under glucoselimiting conditions,GLUT3 expedited CRC cell growth by accelerating glucose input and fuelling nucleotide synthesis.Notably,GLUT3 had a greater impact on cell growth than GLUT1 under glucose-limiting stress.Mechanistically,low-glucose stress dramatically upregulated GLUT3 via the AMPK/CREB1 pathway.Furthermore,high GLUT3 expression remarkably increased the sensitivity of CRC cells to treatment with vitamin C and vitamin C-containing regimens.Together,the results of this study highlight the importance of the AMPK/CREB1/GLUT3 pathway for CRC cells to withstand glucose-limiting stress and underscore the therapeutic potential of vitamin C in CRC with high GLUT3 expression.

Expression regulation and function of NLRC5

The NOD like receptors(NLRs),a class of intracellular receptors that respond to pathogen attack or cellular stress,have gained increasing attention.NLRC5,the largest member of the NLR protein family,has recently been identified as a critical regulator of immune responses.While NLRC5 is constitutively and widely expressed,it can be dramatically induced by interferons during pathogen infections.Both in vitro and in vivo studies have demonstrated that NLRC5 is a specifi c and master regulator of major mistocompatibility complex(MHC)class I genes as well as related genes involved in MHC class I antigen presentation.The expression of MHC class I genes is regulated by NLRC5 in coordination with the RFX components through an enhanceosome-dependent manner.And the involvement of NLRC5 in MHC class I mediated CD8^(+)T cell activation,proliferation and cytotoxicity is proved to be critical for host defense against intracellular bacterial infections.Nevertheless,the role of NLRC5 in innate immunity remains to be further explored.Here,we review the research advances on the structure,expression regulation and function of NLRC5.