Dysfunction of protein quality control contributes to the cellular pathology of polyglutamine (polyQ) expansion diseases and other neurodegenerative disorders associated with aggregate deposition. Here we analyzed how polyQ aggregation interferes with the clearance of misfolded proteins by the ubiquitin-proteasome system (UPS). We show in a yeast model that polyQ-expanded proteins inhibit the UPS-mediated degradation of misfolded cytosolic carboxypeptidase Y(∗) fused to green fluorescent protein (GFP) (CG(∗)) without blocking ubiquitylation or proteasome function. Quantitative proteomic analysis reveals that the polyQ aggregates sequester the low-abundant and essential Hsp40 chaperone Sis1p. Overexpression of Sis1p restores CG(∗) degradation. Surprisingly, we find that Sis1p, and its homolog DnaJB1 in mammalian cells, mediates the delivery of misfolded proteins into the nucleus for proteasomal degradation. Sis1p shuttles between cytosol and nucleus, and its cellular level limits the capacity of this quality control pathway. Upon depletion of Sis1p by polyQ aggregation, misfolded proteins are barred from entering the nucleus and form cytoplasmic inclusions.

Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.

The low efficiency of reprogramming and genomic integration of virus vectors obscure the potential application of induced pluripotent stem (iPS) cells; therefore, identification of chemicals and cooperative factors that may improve the generation of iPS cells will be of great value. Moreover, the cellular mechanisms that limit the reprogramming efficiency need to be investigated. Through screening a chemical library, we found that two chemicals reported to upregulate E-cadherin considerably increase the reprogramming efficiency. Further study of the process indicated that E-cadherin is upregulated during reprogramming and the established iPS cells possess Ecadherin-mediated cell-cell contact, morphologically indistinguishable from embryonic stem (ES) cells. Our experiments also demonstrate that overexpression of E-cadherin significantly enhances reprogramming efficiency, whereas knockdown of endogenous E-cadherin reduces the efficiency. Consistently, abrogation of cell-cell contact by the inhibitory peptide or the neutralizing antibody against the extracellular domain of E-cadherin compromises iPS cell generation. Further mechanistic study reveals that adhesive binding activity of E-cadherin is required. Our results highlight the critical role of E-cadherin-mediated cell-cell contact in reprogramming and suggest new routes for more efficient iPS cell generation. STEM CELLS

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Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.

SummaryReprogramming of somatic cells to a pluripotent state was first accomplished using retroviral vectors for transient expression of pluripotency-associated transcription factors. This seminal work was followed by numerous studies reporting alternative (noninsertional) reprogramming methods and various conditions to improve the efficiency of reprogramming. These studies have contributed little to an understanding of global mechanisms underlying reprogramming efficiency. Here we report that inhibition of the mammalian target of rapamycin (mTOR) pathway by rapamycin or PP242 enhances the efficiency of reprogramming to induced pluripotent stem cells (iPSCs). Inhibition of the insulin ⁄ IGF-1 signaling pathway, which like mTOR is involved in control of longevity, also enhances reprogramming efficiency. In addition, the small molecules used to inhibit these pathways also significantly improved longevity in Drosophila melanogaster. We further tested the potential effects of six other longevity-promoting compounds on iPSC induction, including two sirtuin activators (resveratrol and fisetin), an autophagy inducer (spermidine), a PI3K (phosphoinositide 3-kinase) inhibitor (LY294002), an antioxidant (curcumin), and an activating adenosine monophosphate-activated protein kinase activator (metformin). With the exception of metformin, all of these chemicals promoted somatic cell reprogramming, though to different extents. Our results show that the controllers of somatic cell reprogramming and organismal lifespan share some common regulatory pathways, which suggests a new approach for studying aging and longevity based on the regulation of cellular reprogramming.

Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types of the human body and provide potential applications in regenerative medicine [1]. However, the concerns of immune rejection hamper transplantation therapies using human ES cells. To avoid the complications of immune rejection, diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and fusion of somatic cells with human ES cells [2], have been attempted to produce patient-specific pluripotent stem cells. Most of these approaches have resulted in little success. The generation of human iPS cells (induced pluripotent stem cells) from somatic cells with defined transcription factors makes it possible to produce patientspecific ES-like stem cells for therapeutic purposes [3,4]. Two sets of four-factors, OCT4, SOX2, C-MYC, KLF4 reported by Yamanaka's laboratory and OCT4, SOX2, NANOG, LIN28 reported by Thomson's laboratory, have been shown to reprogram human somatic cells to pluripotency with similar efficiency (10-20 iPS cell colonies from 0.1 million initial fibroblasts) [3,4]. We speculated that C-MYC and KLF4 might synergize with Thomson's 4 factors (OCT4, SOX2, NANOG, LIN28) to reprogram the human somatic cells. Our present study shows that a combination of 6 transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, significantly increases the efficiency of generating iPS cells from human somatic cells.The human genes encoding the transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, were cloned into a lentiviral vector to produce lentivirus.Half million human newborn foreskin fibroblasts were transduced with the lentivirus carrying GFP (serving as negative control), or a cocktail of lentivirus carrying 4 factors (OCT4, NANOG, SOX2 and LIN28) or 6 factors (OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4). Twenty-four hours later, the cells were dissociated with trypsin, transferred to five flasks (0.1 million initial cells per flask) coated with murine embryonic fibroblast (MEF) feeder, and cultured in human embryonic stem cell media. Colonies with a human ES cell-like morphology (iPS cell colonies) first became visible 12 days after transduction with 4 factors, whereas iPS cell colonies became visible 7 days after transduction with 6 factors. In order to quantify the efficiency of the reprogramming, the cells in one flask for each combination of factors were fixed on day 17 to analyze the alkaline phosphatase expression. A total of 16±3 colonies from 0.1 million initial fibroblast cells transduced with 4 factors were alkaline phosphatase-positive, 166±6 colonies from 0.1 million initial fibroblast cells transduced with 6 factors were alkaline phosphatase-positive, and no colonies were observed in GFP-lentivirus transduced controls ( Figure 1A). The efficiency of 6 factors is 10.4-fold more than that of 4 factors. The iPS colonies generated by transduction of 4 factors were picked on day 26. As reported by Yu et al. [4], these cells expressed alkaline phosphatase and undifferentia...

npg Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their overexpression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cellcycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.