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The pluripotent transcriptome
Marica Grskovic,
Miguel Ramalho-Santos,
Departments of Ob/Gyn and Pathology, Institute for Regeneration Medicine and Diabetes Center, University of California San Francisco, San Francisco, CA 94143, USA
Pluripotent stem cells, such as Embryonic Stem cells, have the extraordinary ability to give rise to all cell types of the body, and hold great potential for regenerative medicine. Pluripotent stem cells are derived from in vitro culture of early embryos or by over-expression of specific transcription factors in somatic cells. In recent years great progress has been made towards understanding the molecular basis of pluripotency. The transcriptomes of various pluripotent stem cell lines have been examined in order to identify molecular mechanisms that control self-renewal and pluripotency. These studies have begun to reveal the components and architecture of the transcriptional regulatory networks of pluripotency, and suggest that transcription factors maintain the pluripotent state in the backdrop of a loose, accessible chromatin. Such a chromatin state may allow for increased global transcription, resulting in the expression of many novel and as yet uncharacterized transcripts. Fluctuations in the transcriptional networks may alternate pluripotent stem cells between states with distinct probabilities for self-renewal or differentiation. In this chapter, we review studies of the transcriptome of pluripotent stem cells, highlight intriguing transcriptional similarities between pluripotent stem cells, cancer cells and the germline, and discuss future avenues of research.
Embryonic Stem (ES) cells are the prototypical pluripotent stem cell: they are able to both self-renew and differentiate into all cell types. Because of these properties, ES cells are an excellent system to study cellular differentiation in both normal and diseased states. In addition, cells derived from ES cells have great potential for therapeutic applications, provided they can be efficiently expanded and differentiated in vitro. An important step towards therapeutic applications of ES cells is to identify the molecules and pathways governing ES cell proliferation and differentiation.
ES cells are derived from in vitro culture of the inner cell mass (ICM) of the blastocyst, prior to implantation. Another pluripotent cell type, Embryonic Germ (EG) cells, can be derived from in vitro culture of the embryonic germline, Primordial Germ Cells (PGCs). Recently, induced Pluripotent Stem (iPS) cells have been derived from non-embryonic somatic cells by the introduction of specific key transcription factors (Aoi et al., 2008; Blelloch et al., 2007; Hanna et al., 2008; Lowry et al., 2008; Maherali et al., 2007; Meissner et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). Both embryo-derived (ES and EG) and non-embryo-derived (iPS) pluripotent cells share highly similar transcriptional profiles (Sharova et al., 2007; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007).
In recent years there has been great progress towards understanding the molecular basis of pluripotency. The transcriptomes of various pluripotent cell lines have been extensively examined in order to identify molecular factors and mechanisms that control self-renewal and differentiation. Genome-wide expression profiling using expressed sequence tag (EST) analysis (Brandenberger et al., 2004), serial analysis of gene expression (SAGE; Richards et al., 2004), massively parallel signature sequencing (MPSS; Brandenberger et al., 2004; Wei et al., 2005) and DNA microarrays (Abeyta et al., 2004; Bhattacharya et al., 2004; Ivanova et al., 2002; Ramalho-Santos et al., 2002; Sato et al., 2003; Sperger et al., 2003; see Figure 1) have uncovered gene networks and signaling pathways believed to be essential for maintenance of the pluripotent state. Here, we review studies of the transcriptome of pluripotent stem cells, and highlight intriguing similarities between pluripotent stem cells, cancer cells and the germline.
Gene expression profiling techniques used in studies of pluripotent cells.
Genome-wide expression profiling using expressed sequence tag (EST) analysis, serial analysis of gene expression (SAGE), DNA microarrays and massively parallel signature sequencing (MPSS) have all been used to study the transcriptome of ES cells. ESTs are generated by inserting cDNA into cloning vectors to produce a cDNA library. The inserts are sequenced and the resulting EST sequences are analyzed. SAGE involves the generation of gene-specific tags, typically 10–14 basepairs in length. These tags are ligated to form di-tags, amplified by PCR, ligated again to form concatamers and sequenced. Expression microarrays are arrays of synthesized oligonucleotides or spotted cDNAs in a predetermined spatial orientation. Total RNA is reverse transcribed, fluorescently labeled and hybridized to the microarray. Specific hybridization signals are detected by a fluorescent scanner, and gene expression is deduced. Tiling arrays are modified microarrays where all non-repetitive genomic DNA is represented at various sequence resolutions. Unlike conventional microarrays, tiling arrays enable the discovery of novel transcribed sequences and regulatory elements through the unbiased examination of the genome. MPSS involves the cloning of a cDNA library on beads and the acquisition of 17–20-nucleotide tags from these cDNAs using an unconventional sequencing method that allows for large-scale sequencing. Recently developed deep sequencing approaches using 454 and Solexa platforms promise to provide a comprehensive description of the pluripotent transcriptome.
2. Molecular signature of pluripotent stem cells
To define the molecular signature of pluripotent stem cells, several groups compared the expression profiles of ES cells to those of differentiated cells (Bhattacharya et al., 2004; Brandenberger et al., 2004; Liu et al., 2006; Miura et al., 2004; Zhou et al., 2007), to other stages of the developing embryo (Falco et al., 2007; Sharov et al., 2003), to other types of pluripotent and adult stem cells (Abeyta et al., 2004; Ivanova et al., 2002; Ramalho-Santos et al., 2002; Sharova et al., 2007; Zeng et al., 2004), or to ES cells of other species (Sato et al., 2003; Sun et al., 2007; Wei et al., 2005). An emerging picture from these studies is that some genes are predominantly or exclusively expressed in pluripotent cells, such as Oct4, Nanog, Rex1 and Utf1. These studies also point to the activity of particular signaling pathways in ES cells, such as Jak/Stat, Bmp, Wnt and FGF. Many of these studies highlight an over-representation of transcription, chromatin remodeling and DNA repair factors in the pluripotent transcriptome, suggesting that these play an essential role in pluripotency. Upon in vitro differentiation of ES cells, pluripotency-associated genes are consistently down-regulated regardless of the method of differentiation, while the induced genes depend on the method of differentiation (Walker et al., 2007).
Predominant or exclusive expression of a gene in ES cells provides insufficient evidence for a role of that gene in self-renewal or pluripotency. Some genes predominantly expressed in ES cells, such as Oct4 or Sox2, had been shown to be essential regulators of pluripotency before transcriptional profiling became widely used (Avilion et al., 2003; Nichols et al., 1998). By combining expression profiling with RNA interference (RNAi), Ivanova and colleagues identified several novel regulators of ES cell self-renewal, such as Esrrb, Tbx3 and Tcl1 (Ivanova et al., 2006). In addition, gene expression profiling of ES cells in which expression of a critical transcription factor has been perturbed can provide insight into the transcription regulatory networks of pluripotency (Ivanova et al., 2006; Matoba et al., 2006; Walker et al., 2007). For example, genes that are affected by downregulation of Oct4 are similarly affected by downregulation of Nanog or Sox2, indicating that Oct4, Sox2 and Nanog cooperate to control transcription of target genes (Ivanova et al., 2006; Walker et al., 2007). Downregulation of Esrrb, Tbx3 and Tcl1 affected the expression of a different set of genes, suggesting that they regulate a distinct ES cell pathway (Ivanova et al., 2006). Thus, transcriptional profiling, in particular in the setting of gene-manipulated ES cells, will likely continue to provide important insights into the regulation of the pluripotent transcriptome.
3. Co-expression to co-regulation: regulatory pathways
Genes co-expressed in pluripotent cells are likely to be (at least in part) co-regulated by the same transcription factors. Several key transcription factors have been identified as expressed in ES cells and involved in the maintenance of pluripotency. Among these are factors known as "core regulators of pluripotency": Oct4, Sox2 and Nanog (see Figure 2).
The transcriptome of pluripotent cells.
The "core" regulators of pluripotency Oct4, Sox2 and Nanog, act in concert with other transcription and chromatin modifying factors to establish and maintain the transcriptional program of pluripotency. The accessible chromatin of ES cells may allow for increased global transcription resulting in expression of many genes, their splicing isoforms, intergenic spliced mRNAs, non-coding intergenic transcripts and antisense transcripts. The function of these novel transcripts in ES cells is not yet understood. Antisense transcripts are thought to regulate the expression of their sense counterparts, possibly by sense-anti-sense pairing and production of siRNAs. Endogenous siRNAs have been recently detected in oocytes (Tam et al., 2008; Watanabe et al., 2008), but so far they have not been described in ES cells. miRNAs however, have been shown to be expressed in ES cells and are required for their proliferation and differentiation (reviewed in (Bibikova et al., 2008)).
The POU-family transcription factor Oct4 is predominantly expressed in pluripotent cells of the developing mouse embryo (ICM, PGCs) and their in vitro counterparts (ES and EG cells, respectively; Pesce and Scholer, 2001). Embryos lacking Oct4 die at the blastocyst stage, and ES cells with perturbed levels of Oct4 expression exit self-renewal and differentiate (Nichols et al., 1998; Niwa et al., 2000). The Sry-related transcription factor Sox2 can act synergistically with Oct4 to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct4 and Sox2 itself (Chew et al., 2005; Kuroda et al., 2005; Nishimoto et al., 2005; Okumura-Nakanishi et al., 2005; Rodda et al., 2005; Tomioka et al., 2002). A recent report indicates that the essential function of Sox2 may be to stabilize ES cells in a pluripotent state by maintaining the appropriate level of Oct4 expression (Masui et al., 2007).
Similarly to Oct4, the homeobox transcription factor Nanog is predominantly expressed in ICM, PGCs, ES and EG cells. Nanog has been shown to sustain pluripotency in ICM and ES cells, and to support self-renewal of mouse ES cells in the absence of LIF, a cytokine essential for mouse ES cells (Chambers et al., 2003; Mitsui et al., 2003). Recent genome-wide studies using chromatin immunoprecipitation combined with microarrays (ChIP-chip) or paired-end ditag sequencing (ChIP-PET) revealed Nanog as part of a transcriptional network that regulates the activity of numerous genes in collaboration with Oct4 and Sox2 (Boyer et al., 2005; Loh et al., 2006). Since each of these three genes contains binding sites for each of the three transcription factors, it was suggested that they may act as a self-organizing feed-forward loop that sustains the expression levels needed to maintain pluripotency. However, Chambers et al recently showed that it is possible to derive ES cells that lack Nanog but still retain the expression of pluripotency markers, including Oct4 and Sox2 (Chambers et al., 2007). Although they are prone to differentiate, these ES cells are capable of self-renewal in the absence of Nanog, and they can contribute to all three germ layers in chimeras. Therefore, it appears that, rather than being essential for establishing pluripotency, Nanog may function to reinforce the stability of the pluripotent state.
To delineate regulatory relationships between Oct4, Sox2 and Nanog and their targets, Walker and colleagues combined available ChIP-chip data (Boyer et al., 2005; Boyer et al., 2006; Loh et al., 2006) with time-course gene expression profiling of differentiating ES cells, as well as ES cells deficient for Oct4 or Sox2 (Walker et al., 2007). They found that polycomb group genes Eed and Phc1are both bound by Oct4 and Nanog, are downregulated upon downregulation of Oct4 and Nanog, and all four factors target the promoters of the same set of genes, implicating them as partners in repressing genes associated with development. While gene expression or transcription factor binding studies alone are not sufficient to identify regulatory transcription factor-target interactions, this integrative approach allowed for prediction of novel regulators and regulatory networks controlling ES cell fate, some of which were verified using RNAi combined with gene expression analysis.
To further expand the core transcriptional regulatory network of pluripotency, Kim et al. used a modified ChIP-chip to examine the function of six additional transcription factors (Klf4, c-Myc, Nac1, Zfp281, Dax1 and Rex1) in mouse ES cells (Kim et al., 2008). They showed that regulation of target genes correlates with the extent of promoter occupancy by multiple factors: promoters bound by few factors tend to be repressed, while promoters bound by more than four factors are largely active in the pluripotent state and become repressed upon differentiation. Interestingly, while the majority of the nine factors tested share similar targets, c-Myc and Rex1 occupy promoters of a distinct subset. In addition, c-Myc target genes are associated with the active histone mark H3K4me3 and are more frequently expressed than targets of other factors in ES cells. These results are in agreement with the proposed function of c-Myc in the widespread maintenance of active chromatin (Knoepfler et al., 2006) and suggest a distinct function of c-Myc in regulation of chromatin accessibility in pluripotent cells. In a similar study, Chen et al. used ChIP-seq to determine binding sites of 13 sequence-specific transcription factors (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2F1 and CTCF) and 2 transcriptional regulators (p300 and Suz12; Chen et al., 2008). They found that the majority of genes upregulated in ES cells are bound by either Nanog, Oct4, Sox2, Smad1 and STAT3, or c-Myc and n-Myc. Smad1 and STAT3 share many target sites with Nanog, Oct4 and Sox2, integrating BMP and LIF signaling pathways, respectively, with the core ES cell transcription regulatory network. These two studies demonstrate that gene clustering based on combinatorial transcription factor occupancies has the potential to predict ES-cell specific gene expression.
ChIP-chip, ChIP-PET and/or RNAi in combination with gene expression profiling have proven to be valuable methods to dissect transcriptional regulatory networks. Nevertheless, they all require prior knowledge of the relevant transcription factors. With computational approaches, however, it is possible to identify novel transcription factors using only gene expression profiling data (GuhaThakurta et al., 2002; Liu et al., 2001; Roth et al., 1998; Zhu et al., 2002). Genes with co-regulated expression often share cis-regulatory motifs, corresponding to transcription factor binding sites, in their promoter and enhancer regions (MacIsaac and Fraenkel, 2006; Pennacchio and Rubin, 2001). Using a computational approach, we recently identified DNA motifs that are over-represented in the cis******-acting regions of genes highly expressed in pluripotent cells (Grskovic et al., 2007). These motifs include the binding sites for transcription factors Oct4, Sox2 and Myc, as well as several novel motifs. The analysis of one of the novel motifs identified transcription factor Nfy as a regulator of gene expression in ES cells that is required for their proliferation (Grskovic et al., 2007). Nfy had not previously been implicated in regulation of pluripotency. In addition, gene expression profiling failed to detect Nfy as enriched in pluripotent cells, possibly because one of the Nfy subunits, Nfya, has two splicing isoforms. During ES cell differentiation, the expression of the two isoforms changes in opposite directions (Grskovic et al., 2007), which would be overlooked if expression levels of only common exons were analyzed, as is normally the case with standard microarrays. Therefore, it is important to use unbiased gene profiling approaches to obtain a more comprehensive description of the pluripotent transcriptome.
4. Novel genes and splice variants
A limitation of gene expression studies using standard microarrays is that novel non-annotated genes may not be probed for in the array platform used. A further limitation is that standard microarrays do not generally distinguish between differentially spliced isoforms (see above). Studies of the ES cell transcriptome that did not rely on microarrays revealed that ES cells express numerous uncharacterized or novel transcripts (Araki et al., 2006; Sharov et al., 2003). A large number of ESTs and SAGE tags with no match to a known gene were found to be abundantly and specifically expressed in ES cells (Brandenberger et al., 2004; Richards et al., 2004). In addition, the analysis of gene trap screens revealed thousands of novel exons and genes, many of which are found in gene trapping hotspots associated with loci significantly expressed in ES cells (Roma et al., 2007). Further studies identified naturally occurring antisense transcripts for several pluripotency genes, including Oct4 and Nanog, suggesting that sense-antisense pairing may have a role in regulation of pluripotency (Richards et al., 2006). It should be noted that antisense transcription is not specific for ES cells: recent analysis of global transcription in mouse revealed that a large proportion of the genome produces transcripts from both strands (Katayama et al., 2005).
A next step will be to characterize novel genes expressed in ES cells and evaluate their role in the regulation of pluripotent cells. To this end, Kunarso et al. have analyzed several novel transcripts predominantly expressed in ES cells and found that they represent novel protein-coding genes or intergenic splicing isoforms (Kunarso et al., 2008). Using combined computational and experimental approaches, Pritsker et al. have identified alternative splicing of numerous genes in ES cells, with high levels of alternative splicing in components of signaling pathways that are functional in stem cells (Pritsker et al., 2005). While the identification of novel transcripts and alternative splicing isoforms increases the complexity of the pluripotent transcriptome, future work should reveal what role, if any, they play in maintaining the pluripotent state of ES cells.
5. Is everything transcribed? In every ES cell?
The increase in the number of expressed genes as well as a high number of novel transcripts in ES cells may be due to two possibilities: 1) the transcriptome of ES cells only appears to be more complex than that of other cell types, due to the extensive investigation of ES cells; or 2) ES cells do express a higher number of genes compared to differentiated cells. To investigate these two possibilities, Efroni et al. used tiling microarrays to compare genome-wide transcription profiles of ES cells and neuronal progenitor cells (Efroni et al., 2008). Their analysis revealed genome-wide hypertranscription specifically in ES cells, including expression of normally silent, noncoding regions. In addition, several tissue-specific genes are expressed at low levels in ES cells, suggesting their expression might occur stochastically within the population (Efroni et al., 2008). Increased global transcription in ES cells is in agreement with specific features of the ES cell chromatin, such as a "loose" chromatin structure and the presence of bivalent chromatin marks (Azuara et al., 2006; Bernstein et al., 2006; Meshorer et al., 2006; Niwa, 2007). In addition, ES cells contain higher levels of acetylated H3K9 and methylated H3K4, markers of open chromatin (Azuara et al., 2006). Therefore, it seems that the increase in global transcription in undifferentiated ES cells is an inherent characteristic of ES cells (see Figure 2). However, it is still not clear whether this hypertranscription is simply a consequence of the chromatin properties of ES cells, or whether it has a major role in regulation of pluripotency and self-renewal of ES cells. Furthermore, it is possible that some of the hypertranscription detected in ES cells is derived from heterogeneity in the cell population.
Genome-wide transcriptional profiling typically requires a high number of cells and has therefore been performed using populations of ES cells. However, recent reports have demonstrated cell-to-cell differences in the expression patterns of several genes among undifferentiated ES cells, suggesting that ES cells are not a homogeneous population (Carter et al., 2008; Chambers et al., 2007; Toyooka et al., 2008). Indeed, Toyooka et al. have identified two populations within ES cells that differ in Rex1 expression level and have distinct differentiation potential (Toyooka et al., 2008). Similarly, Nanog is undetectable in a subpopulation of undifferentiated ES cells (Chambers et al., 2007). This subpopulation of cells is predisposed to differentiate but may remain undifferentiated and can subsequently re-express Nanog. These results show that ES cells are a heterogeneous cell population consisting of different types or states of cells. It has been suggested that ES cells may exist in a metastable state which fluctuates between a Nanog-Oct4-Sox2 stabilized state, where differentiation-associated transcription is maintained below threshold levels, and a Nanog-deficient state, where an increase in specific signals, such as phosphorylated-Erk, may trigger differentiation-associated transcriptional networks (Silva and Smith, 2008). Future work should reveal if heterogeneity of undifferentiated cells is limited to the cells tested, or if it is an intrinsic property of all ES cells.
The differential expression of genes in subpopulations of ES cells may be a product of stochastic gene expression, or it may be regulated by an as yet unidentified mechanism (for example, linked to the cell cycle stage). It is also possible that only a subset of undifferentiated ES cells in culture retain full pluripotency. Future work focusing on single-cell transcriptome analysis should clarify the profile and potential of different subpopulations of ES cells.
6. Differences between mouse and human ES cells
Pluripotent mouse and human ES cells by definition share the fundamental properties of self-renewal and the ability to give rise to cells of all three germ layers. Therefore, it is not surprising that they both express components of the core transcriptional network of pluripotency, such as Oct4, Sox2 and Nanog. Nevertheless, there are important differences in the targets of Oct4, Sox2 and Nanog between mouse and human ES cells (Boyer et al., 2005; Loh et al., 2006). These differences are reflected in the divergence between mouse and human transcriptomes, and correlate with differential cytokine and growth factor requirements, growth rates and marker expression between the ES cells of the two species (Wei et al., 2005). For example, human ES cells lack transcripts of the LIF signaling pathway, while this signaling pathway is expressed, active and required for mouse ES cells. Similarly, several components of the FGF pathway are expressed in human ES cells while being absent from mouse ES cells. Cell surface markers SSEA1, SSEA3 and SSEA4 are also differently expressed in mouse and human ES cells (Thomson et al., 1998).
The differences between mouse and human ES cells may represent species-specific transcriptional programs. Alternatively, mouse and human ES cells may represent different stages of early development. Consistent with the latter hypothesis, pluripotent epiblast cells (EpiSCs) can be derived from post-implantation mouse embryos (Brons et al., 2007; Tesar et al., 2007). Unlike mouse ES cells, but similarly to human ES cells, mouse EpiSCs require Activin/Nodal and FGF signaling for their derivation and maintenance (reviewed in (Yu and Thomson, 2008)), and are able to differentiate into trophectoderm. In addition, there is a greater overlap between genes bound by Oct4 in human ES cells and mouse EpiSCs than between human and mouse ES cells (Tesar et al., 2007). Therefore, it is possible that mouse and human ES cells correspond to different stages of embryonic development. Future studies of EpiSCs may reveal the extent of species- and stage-specific differences between the mouse and human pluripotent transcriptome.
In summary, there is substantial evidence that both conserved and divergent pathways act to regulate self-renewal and pluripotency in mouse and human ES cells. Even though mouse ES cells are an excellent model to study developmental processes and diseases, care must be taken when extrapolating results obtained using mouse ES cells to human ES cells.
7. Expression of the pluripotent transcriptome in cancer
Cancer cells are capable of self-renewal and possess high phenotypic plasticity, which are the major features of stem cells. In fact, it is thought that some cancers may arise from aberrant stem or progenitor cells, or that cancer cells develop by progressive de-differentiation of their normal counterparts. Such de-differentiation might occur if a particular combination of stem cell-associated factors is expressed in cancer cells, perhaps in a manner analogous to the generation of iPS cells. Indeed, ectopic expression of Oct4 inhibits progenitor cell differentiation and causes dysplasia in epithelial tissues (Hochedlinger et al., 2005). It should be noted, however, that somatic cancers have not been conclusively shown to express Oct4: the expression reported by several groups (Atlasi et al., 2007; Ezeh et al., 2005; Gibbs et al., 2005; Iki and Pour, 2006; Ponti et al., 2005; Tai et al., 2005) was recently attributed to the expression of an Oct4 pseudogene or of Oct4B, an isoform of Oct4 that is not associated with pluripotency (Cantz et al., 2008). Recently, two groups have analyzed the expression of particular ES cell-associated gene sets in various tumors (Ben-Porath et al., 2008; Wong et al., 2008). Interestingly, they found that the ES cell-like transcriptional program is active in several human tumors and is correlated with metastasis and mortality. This ES cell-like transcriptional program includes genes with roles in cell cycle, signaling, transcription, DNA repair, stress response and differentiation. The targets of Oct4/Sox2/Nanog are found to be expressed in some cancers despite the lack of Oct4 and Nanog, indicating that the pluripotency-associated transcriptional program may be activated by alternative mechanisms. Furthermore, Wong et al. have shown that c-Myc can activate the ES cell-like transcriptional program in adult epithelia and in an in vivo model of human epithelial cancer, leading to an increase in the number of tumor-initiating cells. In particular, expression of c-Myc led to induction of Sox2 and repression of differentiation regulators, such as Hox genes. These findings are in agreement with the role of c-Myc as one of the key transcription factors involved in inducing pluripotency (Lowry et al., 2008; Maherali et al., 2007; Meissner et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007) and may relate to the function of c-Myc in the widespread maintenance of active chromatin (Knoepfler et al., 2006). It should be noted, however, that pluripotent cells can be derived from somatic cells in the absence of c-Myc, albeit with a lower efficiency (Nakagawa et al., 2008; Wernig et al., 2008).
Some germ cell tumors, which are thought to arise from transformation of PGCs (Stevens, 1967), have transcriptional profiles similar to ES cells, including the expression of Oct4, Sox2 and Nanog (Santagata et al., 2007; Skotheim et al., 2005; Sperger et al., 2003). It is important to note that the expression of Oct4, Sox2 and Nanog is not sufficient to cause tumors: normal PGCs express all three factors at levels similar to those in ES cells. To identify genes with possible role in germ cell tumor formation, we compared the expression profiles of PGCs to pluripotent stem cell types (ES cells, EG cells and ICM) and to publicly available expression data from germ cell tumors (Wei et al., submitted). We found that the genes absent from PGCs but highly expressed in pluripotent cell types are up-regulated in some germ cell tumors. Among these is Klf4, a transcription factor that functions with c-Myc to induce pluripotency in somatic cells. It is possible that Klf4, similarly to c-Myc, activates the ES cell-like transcriptional program and induces tumor-initiating cells. In agreement with this model, expression of Klf4 in basal keratinocytes leads to squamous epithelial dysplasia (Foster et al., 2005). These results suggest that similar mechanisms may govern the conversion of PGCs to the pluripotent stem cell state (EGs) and germ cell tumorigenesis. Further functional studies should address the role of regulators of pluripotency in tumorigenesis. Detailed characterization of the ES cell-like transcriptional networks in cancer cells may reveal novel diagnostic markers and potential therapeutic targets (see Figure 3).
Future avenues in research of pluripotent cells.
The development of high-throughput genome-wide expression analyses has led to the identification of the ES cell transcriptome. There are still several issues that need to be addressed, such as permissive transcription, stochastic gene expression and heterogeneity in the population of undifferentiated ES cells (see text). Insights gained by analyzing the transcriptome of pluripotent cells will feed into, and feed back from, research focusing on: a) the role of pluripotency in vivo, in particular through the study of the function of regulators of pluripotency in PGCs, b) the role of ES cell-like gene expression in cancer, which may lead to development of novel diagnostic and prognostic markers as well as new therapies; and c) the regulation of ES cell self-renewal and differentiation and of somatic cell reprogramming to the pluripotent state.
8. In vivo transcriptomics – a germline connection?
The majority of the transcriptional profiling studies aimed at understanding pluripotency were performed using pluripotent stem cells grown in vitro, mostly because this type of analysis requires large numbers of cells that are difficult to obtain in vivo. However, understanding the transcriptome of pluripotent cells in vivo may provide novel insights into the significance and regulation of pluripotency. Recently, we performed a comparative study of the gene expression profiles of mouse pluripotent stem cells and the cells in the embryo from which they are derived (Wei et al, submitted). We found that PGCs express the global transcriptional program for pluripotency at levels similar to ES cells. Furthermore, PGCs express high levels of pluripotency regulators such as Oct4, Sox2, Nanog, Sall4, N-Myc, Utf1 and Rex1, indicating that some of the factors and mechanisms that maintain pluripotency in early embryos and ES cells may also be operating in the embryonic germline.
The evolution of the molecular mechanisms that regulate pluripotency has not been studied in detail. The sequence and function of known regulators of pluripotency, such as Oct4 and Nanog, do not appear to be well conserved outside of mammals. Putative orthologs of Oct4 or Nanog have been identified in Xenopus and chicken (Lavial et al., 2007; Morrison and Brickman, 2006), but they are more broadly expressed than in the mouse embryo. XenopusOct4 is required for maintaining the numbers of uncommitted cells during gastrulation, suggesting an ancestral role for Oct4-like proteins in preserving the undifferentiated state in early embryos (Morrison and Brickman, 2006). Oct4 and Nanog promote self-renewal in chicken ES cells, but their function in the chick embryo has yet to be determined (Lavial et al., 2007). It is interesting to note that chicken Oct4 and Nanog are expressed in PGCs, and that the axolotl also contains an Oct4 gene expressed in gastrulating embryos and oocytes (Bachvarova et al., 2004). It is possible that the program for pluripotency evolved in mammals, from molecular components already present in low vertebrates, to inhibit somatic differentiation and preserve germline competence until the germline is induced and fully developed. If that is the case, regulators of pluripotency are expected to have essential roles in PGC development. Interestingly, Nanog-deficient PGCs fail to mature into germ cells (Chambers et al., 2007). As the timing of the defect corresponds to the timing when PGCs undergo extensive epigenetic changes (imprint erasure, genome-wide demethylation and X chromosome inactivation in female cells), it was suggested that Nanog functions by maintaining PGCs in a state of minimal epigenetic control, similar to the ES cell state (Chambers et al., 2007). In addition, inactivation of Oct4 specifically in PGCs leads to a loss of germ cells (Kehler et al., 2004). Further studies of gene expression and dissection of the transcriptional regulatory networks in PGCs should shed light on the role of pluripotency in the germline (see Figure 3).
9. Concluding remarks
It is becoming apparent that a detailed understanding of regulation of gene expression in pluripotent cells will continue to make important contributions to developmental, stem cell and cancer biology. The emerging picture is one where specific transcription factors, operating through self-enforcing mechanisms, maintain the pluripotent state in the backdrop of a loose, non-restricted chromatin. Increased global transcription in ES cells may be a by-product of loose chromatin. Alternatively, increased transcription may be required to maintain the chromatin in accessible, pluripotent state. Fluctuations in the transcriptional regulatory network may cause ES cells to oscillate between different states with distinct propensities for self-renewal or differentiation. It will be of interest to investigate whether probabilistic behavior is central to ES cell pluripotency, and if so how the probabilities of self-renewal or differentiation can be modulated (Ramalho-Santos, 2004).
Accessible chromatin and fluctuating transcriptional networks could allow for stochastic gene expression that escapes deterministic transcriptional regulation. Therefore, it will be necessary to annotate and characterize novel transcripts found in ES cells and determine whether they have a role in regulating pluripotency. The development of new, more sensitive methods, such as deep sequencing, should allow for unbiased identification of low-abundance transcripts, as well as newly emerging small RNA species. As even more sensitive large-scale analytical techniques to assess gene expression are developed, it will be important to analyze gene expression in single ES cells, and ideally to correlate individual transcriptomes to developmental potential. The results of transcriptome analysis will have to be complemented by proteome and RNAome analysis to account for the role that post-transcriptional regulation may play in pluripotent cells. Several miRNAs were found to be predominantly expressed in ES cells, and their analysis revealed that miRNAs play crucial roles in ES cell self-renewal and differentiation (reviewed in (Blakaj and Lin, 2008). Future work will determine the role that miRNAs may have on modulating the transcriptional regulatory network of pluripotency (Marson et al., 2008). Finally, it will be crucial to investigate the role(s) of pluripotency in vivo in the germline and in tumor development.
Elucidation of mechanisms governing pluripotency should deepen our fundamental understanding of early embryogenesis and germline development. It will also inform our efforts to generate disease- or patient-specific pluripotent stem cells and to target differentiation towards cell types of therapeutic interest (see Figure 3). Finally, insights gained into the regulation of pluripotency may facilitate the identification of new cancer markers and therapeutic targets. Clearly, the pluripotent transcriptome is "poised" for significant impact in a variety of fields in the years ahead.
We would like to thank Robert Blelloch, Kevin Corbit, Erica Jackson, Collin Melton, Yuki Ohi, Jeremy Reiter and Yangming Wang for helpful comments on the manuscript. M.G. was supported by CIRM, and M.R.S. by CIRM, JDRF and IRM.
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Copyright: © 2008 Marica Grskovic and Miguel Ramalho-Santos.
To whom correspondence should be addressed. E-mail: mrsantos@diabetes.ucsf.edu
*Edited by Bradley E. Bernstein. Last revised September 5, 2008. Published October 10, 2008. This chapter should be cited as: Grskovic, M. and Ramalho-Santos, M., The pluripotent transcriptome (October 10, 2008), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.24.1, https://www.stembook.org.
2.Molecular signature of pluripotent stem cells
3.Co-expression to co-regulation: regulatory pathways
4.Novel genes and splice variants
5.Is everything transcribed? In every ES cell?
6.Differences between mouse and human ES cells
7.Expression of the pluripotent transcriptome in cancer
8.In vivo transcriptomics – a germline connection?
9.Concluding remarks


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We won't sit here and argue that San Francisco does pizza better than a city like New York or Chicago (but honestly: apples and oranges), but we will argue that San Francisco can make a darn good slice of pie. There are many, many options in this town. But here are the top ten.
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There's something so special about dough so fluffy and billowy, with blistered bubbles that are perfectly charred. The high ceilings, communal tables, Edison bulbs and friendly staff make the pizza taste even better. Don't forget to order a cocktail at the bar if there's a wait. The drink is definitely worth it.
Deep dish is the specialty at this Chicago-inspired spot, but it's hard to just focus on the pie when the menu is chalk full of delicious options. Start with a seasonal cocktail like the new trick with bourbon and fernet branca (hey, got to have a digestif for all the pizza you're about to consume). Then order a Farmer's Pie, which is loaded with four cheeses, sausage, bacon, wild mushrooms, eggs, and arugula. If there's two of you, there will definitely be leftovers.
Get ready to indulge in the best Grandma's pie-style pizza you've ever tasted. Golden Boy has been serving up slices to-go wrapped in red and white gingham paper since 1978. Your options are limited to sausage, cheese, veggie, pepperoni or clam and pesto, but you'll be satisfied with any one of those. It's also open late, though there's a line of hungry night owls to go with it.
Delfina has been king of the pizza crown ever since it burst onto the scene in 2005. Their Neapolitan-style pizzas are piled high with farm-fresh ingredients like homemade fennel sausage and bell peppers or cherrystone clams and tomatoes and oregano. The Mission location definitely has an old-school takeout pizza spot feel, but the newer one in the Fillmore is a bit more upscale. The pizza is just as delicious either way.
There's always a wait, but you can always snag a fireside martini at Original Joe's while you wait. Once you get in, you'll be drooling over the thin crust pizza covered with artichokes and provolone or soppressata and red hot pepper oil. You might have to sit outside, so make sure you bundle up!
Little Star has developed a bit of a cult following in San Francisco. But who could blame the sheep? This crusty cornmeal edges with an inside oozing with marinara sauce, mozzarella, and extras like zucchini and mushrooms (that's in the vegetarian) or chicken, bell peppers and artichoke hearts (that's the Mediterranean Chicken).
When the dough can be fluffy, chewy, and crispy at the same time, you've reached pizza heaven. The Rocket Man, a doughy combo of garlic, arugula, egg, lemon, and mozzarella is a crowd favorite. Chalkboard walls and multi-colored string lights give the shop a casual feel so the focus is on the food.
Not too thin or too thick, Zero Zero's pizza is just right. The hen of the woods mushrooms are perfectly paired with leeks and pecorino, making a rich combo in the Fillmore but then there's also the slow-roasted tomatoes, eggplants, and ricotta on the Hayes. There are too many decisions, so don't make any. Invite all your friends.


The CARD therapeutic riding program is offered through three sessions each year: Fall, Winter and Spring.
The Fall, Winter & Spring sessions are 9-12 weeks long.
The Summer Program is 6 weeks long.
There is a weight restriction of 160 lbs for riders with no physical impairments. Riders with balance or motor control challenges will be assessed on a case-by-case basis for participation in mounted programs.


The Coleshill Uncovered Project has now ended.
Please Note: The Coleshill House site and grounds are owned by the National Trust. The woods and grounds of the house are all strictly private and access is limited to set days a year. See our events page for their official open days. Attempting to access the site outside of these times is not only trespassing but could damage the future of our work and relationship with the Trust and villagers.
The Coleshill grounds had never been investigated so CART set up Coleshill Uncovered. It's objective, to learn more about the site through historical desktop research and archaeological study. An archaeological site proposal was submitted to the National Trust. This was approved and the first survey was conducted on January 22nd and 23rd 2011.
The second stage of work was conducted in July 2011.
The overall project is managed by CART. John Winterburn and Anna Gow lead the archaeological work.
CART's Coleshill Uncovered project undertook its first work over the weekend of 22nd – 23rd of January 2011 with about 30 enthusiastic volunteers.
On Saturday morning a project outline was given by Tom Sykes (CART), and the archaeological strategy and methodology was given by John Winterburn and Anna Gow, who handled these aspects throughout the weekend with an easy-going yet professional touch.
2/ general survey (including metal detection) employing a line of people visually examining the ground of Sector 1, and subsequently Sector 2, woodland east of the ornamental main gates.
3/ close visual examination of the Ha-Ha; a 17th century ornamental wall and ditch that follows just beyond the outline of Coleshill house and the woodland adjacent to the north wall around the grounds.
Team one worked to find and clear the concrete bases of the Nissen huts and discovered one large base, perhaps previously unknown.
Team two in that area discovered several bullet cases and further east some light aluminium work perhaps originating from road vehicles or even an aircraft (auxiliers report both at Coleshill for demolitions/explosives training).
Team three, looked for the Ha-Ha on the partially open grass areas south of Coleshill house itself. No trace was found, but the surface remains of a water or sewage cistern was investigated just south of a large Cedar tree (detailed report forthcoming). The approximate line of the Ha-Ha was followed around the house and garden to the east and searched for through the adjacent woodland (Sector 1), again without trace. Further east clear remains of the Ha-ha became visible and examination of the wall began in earnest.
Because the known underground OB (operational base) has its escape tunnel exiting in the Ha-ha wall, team 3 were looking for disturbance/changes to the stonework; particularly deep gaps between stones were examined with long probes and a torch.
As so much of the wall was similar the team chose to examine the several hundred metres of the Ha-ha more than once. This methodology proved worthwhile when Peter Antill found one or two slightly newer looking stones and, upon gentle removal, discovered a small concealed chamber containing a complete British Army bayonet.
Saturday afternoon, team one continued clearing the concrete bases in Sector 1, while team 2 (metal detectorists) moved into Sector 2, woodland east of the ornamental gates.
Team three examined the Ha-Ha close to the known OB locating ceramic pipe fragments (a possible breather for the OB's chemical toilet), along with metal hinges and hooks (in likely outfill from the collapsed escape tunnel - previously excavated). Cement filling was found at the escape tunnel exit between the Ha-Ha's stonework; an application presumably designed to prevent access when the main entrance was also sealed to put the OB beyond use. Also found, a sizeable fragment of concrete, including chicken wire and wooden frame impressions; the latter pair being structural materials described by auxiliers as commonly constituting an original main hatch; and so this find was almost certainly part of the concrete seal over the OB's everyday entrance.
Subsequently team three examined the very dilapidated remains of the Ha-Ha east of the OB, and searched for telephone lines (postulated to run between the OB and the possible observation post near by on the north side of the Coleshill to Faringdon road). By evening, the Ha-Ha and the telephone line search had yielded no finds except (via metal detector) the team identified a live WW2 British 2 inch mortar round.
After bomb disposal experts confirmed and blew up the mortar round the teams continued. Generally, at the same locations (as Saturday) but now pursuing new clues towards further finds.
Large anomalies were identified to the east of the known OB using deep field metal detection; the Ha-Ha's entire length was re-examined (twice) for any other areas of concrete infill around the stonework, but without result. The various teams recorded finds locations with GPS.
Later, team three focused on testing CART's special recording sheet for OBs. These are designed to help lone researchers (like our CIOs - County Information Officers) when working in the field. One observation made concerned the possible original construction technique of the escape tunnel.
The tunnel was partially made through solid rock, some two to three feet below the woodland surface, and in this are a number of channels (approx 1 to 2 inches wide) on both side walls. It was posited that these were perhaps drilled down from the surface and subsequently filled with high explosive in order to blast the rock, allowing easier digging of the tunnel.
As the light began to fade in the afternoon, the various teams packed up their equipment and headed back to the courtyard. And as we all departed we agreed on the completion of a very exciting and satisfying stage of the Coleshill Uncovered project.
With the help and advice of the former Royal Armouries curator at the Museum of London one of the Coleshill Uncovered team, Guy Taylor, has successfully managed to remove the bayonet from the scabbard.
The corrosion on the blade appears to be relatively superficial for the most part; the worst appears to be on the cross-guard, but even this is not too bad overall. It is clear that the edges of the blade have not been sharpened, so the fighting-knife theory appears not to hold up. Such is archaeology!
Guy has used a fine round scalpel blade to remove a small amount of the corrosion from the ricasso, to see if it is possible to find any markings stamped on the blade. There do not appear to be any deep, clearly defined, impressions alas, but there is possibly a rather light letter R (VR?). As far as further treatment is concerned, the separation of scabbard and bayonet will make the conservation process much easier. When Guy spoke initially to the senior MoL conservator, she said that if he could separate them, the scabbard could be treated with a proprietary leather treatment. As the leather is already drying and stiffening, Guy will go ahead with this. There is some corrosion on the scabbard metalwork but it is not too far gone and after the loose material has been removed it will probably be sufficient to keep it in a relatively dry atmosphere; a small silica gel bag will be adequate until it goes on display.
As far as the bayonet itself is concerned, Guy suspects that the blade would benefit from some form of cleaning and/or chemical inhibition. The wood grips probably need only an application of some commercially available wood preservative; treatment with PEG and freeze-drying is thankfully not called for.
See finds images from Stage One here and here.
See working photos here and here.
1. Complete the clearance work started during the January 2011 evaluation weekend on the concrete slabs in Sector 1. This work will also now include exploratory trenches that will hopefully provide both further information about the construction of the structures and also regarding the original use of the structures.
2. Undertake clearance work and excavate two trenches at the MT ramp.
3. Work carried out so far at Structure 2, previously known as the Generator Base, has not really yielded any useful information about it's use. We therefore propose excavating two trenches at this feature.
4. The metal detecting that was carried out over the evaluation weekend provided both a substantial number of artefacts and a number of strong signals that should be investigated further. Subject to number of detectorists on site, we believe that a larger systematic gridded metal detecting survey of these areas (Sectors 1 & 2) will provide us with even more evidence of the training landscape.
5. Follow-up all strong MD & geophysics signals recorded during the January 2011 evaluation weekend with some excavation to see if any further structural remains (including another possible OB) can be identified.
6. Depending on manpower available we will also look to conduct an initial walking survey of Sectors 3 and 4.
All of this is obviously dependant on how many people we have during the week.
See finds images here, here, here and here.
Stage One - 1st April 2011: The official report and findings for work carried out in January can be downloaded here. Images from the report can be seen here.
All our blog posts on this project can be seen here.
The press can be seen here under the respective catagories.


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85. Kerames to the sea Walk and Cycle in Crete .
Twelve kilometres of road, track and path. This route includes long sandy deserted beaches. We almost missed the track back up as we were walking along in the sea with all our clothes in our rucksacks. Cyclists would find the shore section very difficult because of the soft sand.
On the western edge of Kerames a good tarmac road leads down to the sea through the olive groves. Cyclists should beware that some of the hairpins are very sharp.
45 min. The right fork is a gravel track down through olive groves to a small bay with a mixture of sand and boulders. There is a single taverna that may open only in the high season. A steep scrub covered hillside makes walking west from here difficult, but a track runs from above the scrub down towards Palm beach and Preveli. The shore can be followed east, but access is easier from the left fork of the road down from Kerames to the shore one kilometre further on at Agia Fotini.
At this fork in the road turn left for Agia Fotini and follow it down until the road ends at the sea.
1 hour Here large boulders sit in a crystal clear sea and there is one small taverna that may open in high season.
1 hr 10 min. then continue along the shore where the sand sweeps in dunes up the cliffs.
2 hours Keep a good lookout for the next track which drops down from the village of Agalianos. Beyond this inland is a very broken landscape.
2 hr 20 min. One kilometre up this track a concrete tank intercepts the flow from a line of springs and a chorus of frogs call their appreciation of the moisture.
North of the tank turn left and west on a track that winds its way up through carob scrub and small olive groves for a further two kilometres to rejoin the narrow tarmac road at Agalianos.
3 hr 20 min back to Kerames.


With winter just beginning, TravelInsurance.com reminds travelers to stay protected as Pacific weather phenomenon weighs on the season ahead with warm weather in the northeast and flash flooding on the west coast.
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Home Columns The New Mamie Van Doren Exhibit
The New Mamie Van Doren Exhibit
By Denise Ortuno Neil
On Saturday October 25th, the Palm Springs Historical Society will be unveiling the Mamie Van Doren exhibit honoring the famous blonde bombshell. The opening of the exhibit will be followed by a decadent cocktail party at the historic Andreas Hotel and Spa, which was Van Doren's favorite place to stay in Palm Springs.
Mamie Van Doren is one of old Hollywood's most coveted blonde bombshells from the 1950's and 60's era. Van Doren first came to Hollywood with her parents when she was only 13. She came into the limelight at a young age…and as time ensued, joined iconic platinum blondes such as Marilyn Monroe and Jane Mansfield. She was discovered by Howard Hughes, and he cast her in his epic film, Jet Pilot in 1957. But before the Hughes film, Van Doren gained the title of Miss Palm Springs…making the city near and dear to her heart.
Van Doren was known for her sexy allure and went on to be featured in many films and had many love interests including Nicky Hilton, Steve McQueen and Jack Dempsey just to name a few. Her 1987 biography, Playing the Field, Sex, Stardom, Love and Life in Hollywood, chronicled her famous and infamous life.
The Andreas Hotel and Spa is situated in downtown Palm Springs and was built in the 1930's. It was a hot spot for Hollywood stars and is a very popular hotel today. The hotel features the Mamie Van Doren suite, where the star used to stay. The suite has since been redecorated, but has the essence of the star all around it…very vintage Hollywood….the room sparkles. The suite will be available for viewing during the cocktail party.
The exhibit opens at the Palm Springs Historical Society at 10am to 4pm on Saturday October 25th. The following cocktail party will take place from 5:30pm to 7:30pm and honoring Van Doren and will also serve as the 2014/2015 season kick-off for the Palm Springs Historical Society. The party will offer hors d'oeuvres provided by LULU California Bistro, dessert by Manhattan of the Desert and a no host bar will be present. The highlight will be an appearance by Mamie Van Doren in all of her glamorous glory. Tickets are $55 for members and $65 for non-members of the Palm Springs Historical Society (all proceeds benefit the Palm Springs Historical Society). Reservations are required and vintage cocktail party attire is encouraged. It is sure to be a night filled with old school glitz and glamour…Palm Springs style!
For more information visit www.pshistoricalsociety.org or call 760-323-8297
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Home » Rain Drop Splash
Rain Drop Splash
Doesn't make much of a splash.
This seems to be the story of a flood but done in like a cutesy tone, so it's kind of poetic. It talks about how the rain is dripping and dropping, and how it "Fell from a daisy's face", things like that. "There were so many raindrops they made a puddle." And then it grows larger and becomes a pond, and suddenly, poof, there are fish in it. What? This is all one rainstorm, but fish kind of spontaneously generate in the pond. And the pond gets bigger. It spills over into a brook. The brook runs down the mountain, a chipmunk runs away because it's scared, some deer drink from it, and it overflows into a lake. I guess the lake was already there? But the lake gets bigger and floods a meadow and a farm, and everything gets muddy. Then it covers an entire road and the children "had to go to school in a boat". Then it goes into a river, and the river goes through the town, and then it goes all the way out through the town and the city to the ocean. Then the sun comes out and the rain stops.
Is all of this supposed to be happening in a single day? It takes place more or less in the modern era, as it mentions things like "ocean liners". I might just be out of my depth, having been born and raised in southern California, where rain is pretty scarce, but going from the creation of a puddle to the flooding of a lake seems like it would take more than one rainstorm. At least more than one day. I guess there's nothing specific that signals that it takes place over one day, but the time period seems either very short, or vaguely long with the (fully adult) fish appearing in the new pond. A whole ecosystem doesn't appear in that short of a time period, but the length of time over which the book's events occur is very nebulous. How much of it is literal? Metaphorical?
The art is dated looking and somewhat strange, in a blocky style with crisp lines at the ends of everything. My guess would be watercolor paintings. It's not bad; it's pretty typical for the time period. And the book isn't bad either, though not great, and mostly just odd.
Water flows from rain and down the mountains, and eventually ends up in the ocean.
Alvin Tresselt
Leonard Weisgard


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How the Dyson Bladeless Fan Works
by Jonathan Strickland & Nathan Chandler
The 10-inch Air Multiplier. Take a look inside gadgets with essential gadgets pictures.
Image courtesy Dyson
In October 2009, James Dyson's consumer electronics company, famous for its line of vacuum cleaners, introduced a new device to the market called the Dyson Air Multiplier. The Air Multiplier is a fan with an unusual characteristic: It doesn't have any visible blades. It appears to be a circular tube mounted on a pedestal. The shallow tube is only a few inches deep.
Looking at the device, you wouldn't expect to feel a breeze coming from the mounted circle. There are no moving parts in sight. But if the fan is switched on, you'll feel air blowing through the tube. How does it work? How can an open circle push air into a breeze without fan blades?
As you might imagine, there are a few scientific principles at play here. There's also an electronic element. While the tube doesn't have any blades inside it, the pedestal of the fan contains a brushless electric motor that takes in air and feeds it into the circular tube. Air flows along the inside of the device until it reaches a slit inside the tube. This provides the basic airflow that creates the breeze you'd feel if you stood in front of the fan.
According to Dyson, the breeze generated by the Air Multiplier is more consistent and steady than one from a standard fan with blades. Since there are no rotating blades, the breeze from the fan doesn't buffet you with short gusts of air.
What's the secret behind the technology?
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Risdall announces the addition of Dave Folkens as vice president of Risdall Public Relations.
Folkens specializes in providing strategic counseling to clients. With a broad range of communications experience, he will leverage Risdall's resources to set the course for clients' success.
Folkens has worked in health care communications for two decades, starting with corporate work at Orphan Medical and Medtronic Inc. He then moved to the agency side of public relations when he joined Fleishman-Hillard. To further his experience, he entered the nonprofit world with the Minnesota AIDS Project. Folkens returned to the agency setting at PadillaCRT where he led several health care accounts before joining Risdall.
Folkens graduated from Moorhead State University in Minnesota with a degree in mass communication with an emphasis in public relations. He is an active member of the Minnesota chapter of the Public Relations Society of America and serves on the board for Children's Cancer Research Fund.


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HIPP, HIPP, Hooray! 04 July 2019
High integrity pressure protection systems (HIPPS) have become increasingly a solution of choice in managing the risks of ever more demanding environments – and none more so than offshore gas installations. How exactly do they work?
High integrity pressure protection systems (HIPPS) are regarded as a 'must have' for many offshore gas installations, and it isn't hard to see why. Crucially, a HIPP serves as the last line of defence in the event of an over-pressurisation incident, automatically shutting off and isolating the source of the high pressure before the design pressure of the system can be exceeded. What such systems do is prevent an uncontrolled loss of containment – in effect, creating a barrier between a high-pressure and low-pressure section of pipe, ensuring people, plant and equipment downstream are protected.
Unlike an emergency shut down (ESD) system, which provides a safe and orderly cessation of a process, HIPPS is very much an emergency response to a pressure build-up by rapidly closing the pipeline. Without such systems in place, a catastrophic outcome to plant, equipment and personnel might well be the result.
While overpressure events have been resolved historically using mechanical pressure release systems, the downside is that these solutions generally need a sizeable footprint to function effectively, and may not be suitable where space is limited, or if the fluid is flammable or toxic, as is the case in offshore applications. Enter the fray, therefore, safety instrumented systems, and most particularly HIPPS, which have become increasingly a solution of choice in managing the risks of ever more demanding environments – and none more so than offshore gas installations.
CATASTROPHE IN WAITING
"As high temperature/high pressure (HT/HP) applications become more commonplace, the consequences of a significant increase in upstream pressure can be catastrophic, not only to people and equipment, but also to the environment," says Tony Brayford (pictured, above right), director for oil and gas at Schneider Electric UK & Ireland.
Trigger points, he identifies, for HIPPS being called into action are where: an application involves high pressures or flow rates; the environmental risks are great; the economics of the process need to be improved; and the risk level is too high.
"There are also commercial incentives," adds Brayford. "One context where a HIPPS could deliver value to an offshore asset would be when an existing mature well, which operates at a certain pressure, is having another new well brought online. The new well may exceed the pressures that the existing infrastructure can handle. The introduction of a HIPPS system would enable the facility to exploit new wells safely, without needing to upgrade existing infrastructure."
There are several stages involved in a HIPPS operation. Critically, block valves are used, because they have high-reliability and low operating torque requirements. "Three pressure sensors are installed to detect upstream pressure," he says. "To prevent any spurious trips, they use a 'two out of three' voting logic. This means that, if all three sensors don't agree, the two that align are taken as correct and the system responds accordingly." (See also nuclear seismic sensor article, Operations Engineer April 2019, or visit www.is.gd/apodev).
On top of that there is the logic solver. "This reacts to the conditions presented by the pressure sensors and determines whether the block valves should close," he explains. "In an overpressure situation, at least two of the three pressure sensors report pressure equal to, or greater than, the high pressure set point."
What are the downsides of not having HIPPS installed, in his view? "The high pressures associated with deep water drilling can cause oil wells to burst. A HIPPS can isolate the well that loses control, resolving the crisis before it becomes a catastrophe."
With so much at stake, it is essential that the right levels of professional instruction on the safe operation of the system are delivered, once the provider has completed the handover. Brayford adds: "Functional safety expertise in the design of the system, and both SIF and SIL requirements from international SIS standards such as ANSI/ISA 84 and IEC 61511, are equally crucial."
CLEAR DISADVANTAGES
Where traditional systems might be seen as having a disadvantage (when set alongside HIPPS) is that over-pressure is dealt with through relief valves, leading to venting systems. "These systems have obvious disadvantages, such as release of (flammable and toxic) process fluids in the environment, and often a large footprint of the installation," says Niels Huttenhuis, account manager at Safety Solutions (Yokogawa Europe Solutions BV). "With the increasing environmental awareness, relief systems are no longer an acceptable solution."
How do HIPPS actually function when determining an emergency shutdown needs to be implemented? Essentially, they are constructed as a complete functional loop, consisting of the initiators that detect the unacceptable levels of high pressure that are occurring. "These initiators are typically electronic pressure transmitters and they measure the actual pressure in the pipeline," he points out. "Meanwhile, a safety logic solver processes the input from the initiators to determine the need to close the final elements – these elements physically performing the corrective actions in the field by, for example, closing the valves and bringing the process to a safe state. The final element consists of a valve, plus actuator and solenoids."
HIPPS can be used for a variety of reasons, he continues: "To avoid human injuries, due to chemical release, fire and explosion; to avoid financial loss, such as damage to the asset, unnecessary insurance costs, reduction of pressure relief capacity, piping classes (weight, cost); or to avoid damage to the environment, by spilling or CO2 emission."
An example, he cites, of where HIPPS could have prevented a major incident was the Deepwater Horizon event in 2010. On 20 April, the Macondo well – located about 50 miles from New Orleans – blew out, costing the lives of 11 men and spilling more than four million barrels of crude oil into the Gulf of Mexico. The root causes, identified by the President's Oil Spill Commission, were associated with zonal isolation during cementing and the failure to create a competent barrier to uncontrolled flow. Other risk factors were associated with well monitoring equipment on the Deepwater Horizon, including data displays. The environmental damage is enormous and irreversible, Huttenhuis points out.
According to a US Department of Energy 'Offshore Safety and Spill Prevention' review (www.is.gd/zerejo), in the wake of the Deepwater Horizon catastrophe, increased interest in HIPPs is an important trend. "This system can be helpful in pressure protection of existing low-pressure pipelines, when new high-pressure pipelines are connected to the systems," it states.
Huttenhuis also emphasises the imperative for the highest levels of training and instruction to ensure that safety is the priority when operating in such environments. "The applicable safety standard IEC-61511 demands that everybody working in one part of the safety lifecycle (design, installation, operation, maintenance and decommissioning) shall be proven competent, but also working under a functional safety management system. Competency basically consists of knowledge and experience. This can be cultivated by hands-on training and kept relevant by frequent practice. The functional safety management system is part of the HSE system, and consists of a strict set of work instructions and operating procedures necessary to reduce human and systematic failures."
Graphic: Yokogawa Electric Corporation/Phil Holmes


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Video: Michigan State's Fake Punt ('Mouse Trap') From Northwestern Game
By Sean Yuille Oct 27, 2010, 8:11am EDT
Share All sharing options for: Video: Michigan State's Fake Punt ('Mouse Trap') From Northwestern Game
Last Saturday against Northwestern, Michigan State ran another fake involving punter Aaron Bates. Earlier in the year, Bates became well known for more than simply being MSU's punter when he threw the game-winning touchdown pass in overtime against Notre Dame on a fake field goal. The play became known as "Little Giants" and made the Notre Dame game an instant classic.
Against Northwestern, Michigan State was playing from behind and needed a game-changing play to pull off the comeback. That play turned out to be a fake punt called "Mouse Trap." Dantonio said in the post-game interview that it was called "Mouse Trap" because Northwestern took the cheese, which in this case was a delay of game penalty to sell the punt. Despite taking the penalty, MSU still ran a fake, and although Northwestern was actually in a punt safe formation, it still worked for the Spartans.


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Webinar: How Coal Plant Retirements Will Drive Midstream Investment in the Midwest and Northeast
06/11/2013 at 1:18pm EST by Sara Wilmer
Levitan & Associates is partnering with B2BWebinars.net to present the "How Coal Plant Retirements Will Drive Midstream Investment in the Midwest and Northeast" webinar. This 90-minute online business intelligence session will answer the following questions:
How will coal-fired generation retirements affect gas generation builds throughout the Midwest, New England, and the remainder of the Northeast?
When and where is new generation likely to come on line?
What are the current infrastructure constraints in the said regions?
What projects are planned to alleviate these constraints?
What are the potential market innovations that could promote fuel security objectives?
Richard Levitan, President & Principal, LEVITAN & ASSOCIATES
Eric Thoms, Technical Lead, Transmission Access Planning, MIDWEST ISO (MISO)
The webinar will be held on Thursday, June 20, 2013 from 1:00 – 2:30 PM (Eastern)
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