text:
During evolution, enzyme‐coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC‐1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis‐regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino‐acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB‐regulated metabolic network includes enzyme‐coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co‐evolution in which TrmB integrates the expression of metabolic enzyme‐coding genes of diverse origins.
Visual Overview
Several lateral gene transfers and homologous gene replacement events are speculated to have an important function in evolution of archaeal metabolic networks (Galperin and Koonin, 1999; Siebers and Schonheit, 2005). If so, then this raises important questions regarding the evolution and the architecture of gene regulatory networks (GRNs) that integrate and coordinate these enzymes in the face of unique environmental challenges. Metabolism of sugars in Halobacterium salinarum NRC‐1 represents one such central process in which several enzymes and entire segments seem to have been acquired through lateral gene transfers. Taking a systems approach, we have characterized global regulation of these core processes by a single regulator TrmB. Specifically, we integrated data from classical physiology and genetics experiments with orthogonal sources of genome‐wide evidence, including (i) protein–DNA interactions measured globally with ChIP‐chip; (ii) transcriptional responses of genetically and environmentally perturbed strains using microarray analysis; (iii) genome‐wide distribution of a conserved TF‐binding motif signature); (iv) a reconstructed metabolic network (Figure 6).
Deletion of this regulator resulted in severe growth defects in diverse environments including nutrient replete or limitation, metal excess or limitation, and oxidative stress conditions. This generalized growth phenotype was likely because of a reduced NAD+/NADH ratio relative to the parent strain and readily complemented by glucose or glycerol. Global transcription analysis of the ΔtrmB strain revealed perturbed regulation of 182 genes (16 down‐ and 166 upregulated) with significant overrepresentation of carbohydrate metabolism genes. Surprisingly, the deletion of TrmB also resulted in defective regulation of amino acid, cofactor, vitamin, and purine biosynthesis genes. These observations were further refined through ChIP‐chip experiments, which showed that TrmB was physically associated with the promoters of many of these genes in a glucose‐ or glycerol‐dependent manner (P=5.5 × 10−11). We have verified the requirement of a conserved cis‐regulatory motif within many of these promoters to be essential for TrmB function. Together these results support the hypothesis that, depending on the carbon source (glucose or glycerol), TrmB acts as both a transcriptional activator and a repressor to directly coordinate enzymes of central metabolism with associated pathways.
Upon integration of the gene regulatory network with a reconstructed metabolic network, we observed several instances of direct TrmB‐mediated transcriptional control of metabolic enzymes with the biosynthesis of their cognate cofactors. For example, TrmB directly controls over 10 enzymes that require adenosine phosphates (AXP; Figure 6, e.g. reactions 3, 7, 10, 11, 27, 33, 63) and six genes that encode the biosynthesis of these cofactors (Figure 6, reactions 43, 44, 45, 46, 47, 52). Our data suggest that TrmB represses the semi‐phosphorylative Entner–Doudoroff (E–D) glycolytic pathway (Danson et al, 2007; Kanai et al, 2007; van der Oost and Siebers, 2007; Pfeiffer et al, 2008) (e.g. gap, pykA, VNG0442G) (Figure 6), and induces gluconeogenesis (e.g. ppsA; Figure 6). Thus, a deletion in trmB would lead to an inability to generate energy through gluconeogenesis in the absence of glucose. Furthermore, ΔtrmB cultures grown in the absence of glucose upregulate enzymes that reduce NAD(P)+ to NAD(P)H, potentially forcing the cell toward an oxidized state (e.g. Figure 6, reactions 19, 31). If so, then this would lead to a shortage of reducing equivalents. The hypersensitivity to oxidative stress and reduced NAD+/H ratio observed in ΔtrmB mutant cells are consistent with this hypothesis. We conclude that TrmB acts to maintain redox and energy balance in response to nutrient availability in H. salinarum.
The TrmB‐specified regulatory network coordinates the transcription of enzymes of mixed evolutionary lineage (Figure 6). For example, in the shikimate biosynthesis pathway, only one gene encoding shikimate kinase is of archaeal origin, whereas all other genes are conserved throughout evolution (orange shaded area, Figure 6). Strikingly, all genes of this pathway except shikimate kinase are direct TrmB targets (Figure 6), suggesting that shikimate kinase may have been acquired by homologous gene replacement (Galperin and Koonin, 1999). In this regard, the TrmB regulatory network might be a specific example of an active evolutionary process, because several lateral gene transfer or homologous gene replacement events are thought to have occurred in the evolutionary compilation of metabolic networks (Galperin and Koonin, 1999). In summary, this study provides insight into how the architecture of a large metabolic network and an associated GRN may have co‐evolved using components of diverse origins, and how this assembly may be conserved across the archaeal lineage.
We have discovered an evolutionarily conserved gene regulatory network (GRN) specified by a single transcription regulator TrmB that coordinates over 100 enzymes of diverse ancestry.
Depending on the carbon source, TrmB functions either as an activator or repressor to coordinate enzymes of core metabolism with pathways for synthesis of their co‐factors.
Given that many TrmB targets are NAD(P)+‐dependent enzymes, disruption of its activity alters the redox and energy balance to result in a generalized growth defect under diverse environmental conditions.
This study provides insight into the co‐evolution of a GRN and a large metabolic network that has assembled from components of diverse origins.
Archaeal genomes encode unusual metabolic enzymes with homologs in either eukarya or bacteria (Siebers and Schonheit, 2005). Several homologous gene replacement events are speculated to have an important function in evolution to integrate these enzymes into archaeal metabolic networks that are otherwise comprised of enzymes conserved across two or more domains of life (Galperin and Koonin, 1999; Siebers and Schonheit, 2005). If so, then this raises important questions regarding the evolution and the architecture(s) of gene regulatory networks (GRNs) that integrate and coordinate enzyme‐coding genes within archaeal metabolic networks in the face of unique environmental challenges.
GRNs evolve by internalizing environmental factor changes to coordinate the efficient uptake and usage of limited nutritional resources (Tagkopoulos et al, 2008). Not surprisingly, the activity of many transcription factors (TFs) in these GRNs reflects cellular adaptations to environmental niches. For example, greater than half of all TFs in bacteria are thought to bind small molecules to monitor changes in environmental and cellular status (Madan Babu and Teichmann, 2003). Likewise, at least 50 eukaryotic TFs coordinate central metabolic pathways in multiple cellular compartments (Herrgard et al, 2006; Reece et al, 2006). Although limited information exists on archaeal GRNs, it is known that the pre‐initiation complex (PIC) is made up of orthologs of the eukaryotic general transcription factors (GTFs): transcription factor II B (TFB), a TATA‐binding protein (TBP), and a eukaryotic RNA‐Pol II‐like polymerase (Geiduschek and Ouhammouch, 2005). In contrast, many of their sequence‐specific repressors and activators of transcription share ancestry with bacterial transcription regulators (Bell, 2005). However, only 10 of these regulators have been characterized to date (Bell, 2005), and even fewer have a known function in vivo (Lie et al, 2005; Muller and DasSarma, 2005; Lee et al, 2008). Those that have a known function in vivo include transcription regulators for glycolytic/gluconeogenic, nitrogen, and lysine usage pathways (Brinkman et al, 2002; Lie et al, 2005; Kanai et al, 2007). For example, the TrmB transcription factor (thermococcus regulator of maltose binding) acts as a repressor for genes encoding glycolytic enzymes and as activator for genes encoding gluconeogenic enzymes (Kanai et al, 2007). In these systems, TrmB also binds to glucose, maltose, trehalose, maltodexterins, and sucrose molecules to differentially regulate the genes encoding corresponding sugar uptake systems in a sequence‐specific manner (van de Werken et al, 2006; Lee et al, 2008). However, the regulation of all other metabolic pathways in archaea is currently unknown.
The limited information on regulation of metabolism in archaea is a significant handicap in comparative analysis for understanding evolutionary similarities and differences in the architecture(s) of GRNs. Here we have characterized the TrmB regulatory network in the halophilic archaeon Halobacterium salinarum NRC‐1 by integrating three disparate sources of evidence (protein–DNA interactions measured globally with ChIP‐chip, transcriptional responses of genetically and environmentally perturbed strains using microarray analysis, and genome‐wide distribution of a conserved TF‐binding motif signature) with a metabolic reconstruction. These results demonstrate that the haloarchaeal TrmB ortholog (VNG1451C) coordinates the transcription of more than 100 central metabolic enzyme‐coding genes with genes involved in de novo synthesis of their cognate cofactors. We hypothesize that this balanced regulation allows the cell to modulate redox and energy status. More importantly, we show that the TrmB‐dependent metabolic network integrates the transcription of enzyme‐coding genes that are uniquely archaeal with those that are conserved across all three domains of life. In sum, this study provides insight into how the architecture of a large metabolic network and an associated GRN may have co‐evolved using components of diverse origins, and how this assembly may be conserved across the archaeal lineage.
We used a combination of classical genetics, genome‐wide experimental, and computational approaches to identify the TrmB ortholog and characterize the architecture of the network it specifies to control central metabolism in the archaeon H. salinarum. These approaches included (i) sequence analysis to identify a putative TrmB homolog; (ii) phenotypic characterization of a ΔtrmB deletion strain; (iii) transcriptomic analysis of the ΔtrmB strain under defined growth conditions associated with the defective phenotypes; (iv) ChIP‐chip (genome‐wide in vivo localization of TrmB binding); (v) genome‐wide distribution of a conserved motif signature discovered de novo within experimentally mapped TrmB‐binding sites; (vi) promoter: reporter fusion assays to validate TrmB targets identified by the high‐throughput methods; (vii) computational integration of the results of these experiments and data from earlier studies to construct transcriptional and metabolic networks governed by TrmB. We conclude from the results of these experiments that TrmB is a bifunctional regulator that governs the transcription of genes in central metabolic pathways of diverse ancestry to manage cellular redox and energy status. Results of these experiments are described in detail below.
Sequence analysis suggests that VNG1451C encodes a putative sugar‐binding transcription regulator
Given the central nature of sugar metabolism in cellular physiology, we searched for putative TFs that may control this process. At least seven proteins in the H. salinarum NRC‐1 proteome (http://baliga.systemsbiology.net) have significant matches to protein family signatures and sequences of known sugar metabolism regulators. Among these candidate regulators, the VNG1451C amino‐acid sequence (Figure 1A) significantly matches (e‐value=2 × 10−8) the 50aa TrmB family signature (PF01978, http://pfam.sanger.ac.uk/) with 21% identity to the consensus sequence (Figure 1B). According to ClustalW analysis, VNG1451C possesses at least three active site residues known to be critical for sugar binding in the characterized TrmB orthologs (Krug et al, 2006; Kanai et al, 2007; Lee et al, 2008). Interestingly, although the TrmB signature is conserved across 175 bacterial and archaeal species, no TrmB orthologs have been identified to date in bacteria (Lee et al, 2008). TFs of the TrmB family have been implicated in the regulation of maltose and glucose usage in thermophilic archaea (van de Werken et al, 2006; Kanai et al, 2007; Lee et al, 2008). In these archaea, the genetic loci encoding TrmB also harbor genes coding for the maltose and/or trehalose ABC transporters. Notably, these genes are absent in chromosomal vicinity of VNG1451C in the H. salinarum genome (Figure 1A). This combined evidence suggests that VNG1451C encodes a widely conserved regulator with a putative function related to sugar metabolism.
Phenotypic analysis suggests that TrmB is involved in sugar metabolism and maintenance of redox balance
We investigated the phenotypic consequence of deleting trmB in diverse environments. This revealed a severe growth defect in the mutant under nearly every condition tested, including standard growth in rich media, nutrient starvation in defined media, metal depletion and excess, and oxidative stress (Figure 2A; Supplementary Tables 1 and 2; Materials and methods). In addition, the NAD+/NADH ratio in mid‐logarithmic phase ΔtrmB cultures was, on average, significantly lower than in the parent strain in the absence of glucose (Figure 2B). Wild‐type growth rates were recovered by functional complementation in trans with a plasmid‐borne copy of trmB in the ΔtrmB background, ruling out polar effects of the gene deletion on surrounding genes (Supplementary Table 2). The addition of glucose to the growth media also complemented both the growth defect and NAD+/NADH ratio imbalance (Figures 2A and B). Partial complementation of the mutant growth phenotype was observed in the presence of glycerol. However, the inability of sucrose, galactose, raffinose, maltose, and pyruvate to remedy these defects (P<0.001) indicated some nutrient specificity in the function of this regulator (Figure 2A; Supplementary Table 2). Together these observations suggest that the function of TrmB is associated with glucose or glycerol metabolism and potentially linked to the maintenance of cellular redox balance.
Transcriptome analysis reveals that TrmB might regulate functions in diverse metabolic pathways
To characterize the TrmB regulon, genome‐wide transcriptional changes were analyzed in the ΔtrmB deletion background during growth in the presence and absence of glucose (Figure 2C; Supplementary Table 3). On culturing in the absence of glucose, the transcription of 182 genes showed perturbed expression in the ΔtrmB background compared with the isogenic parent (16 down and 166 upregulated; Figure 2C; Supplementary Table 3). These genes were grouped into 11 functional categories according to GO and KEGG databases (Ashburner et al, 2000; Kanehisa and Goto, 2000). As expected from the aforementioned experiments, genes whose products function in carbohydrate metabolism were significantly overrepresented in these categories (P∼5 × 10−6; e.g. ppsA, PEP synthase; pykA, pyruvate kinase; Figure 2C; Supplementary Table 3). Surprisingly, genes encoding additional metabolic pathways were also significantly perturbed in the mutant; including amino acid, cofactor, vitamin, and purine biosynthetic pathways (Figure 2C). Consistent with the phenotypic data, growth phase‐specific regulation of these genes was restored in ΔtrmB on the addition of glucose (Figure 2C). Two possible molecular mechanisms could lead to this result: (i) direct TrmB binding to affected promoters, including those of other TFs; and (ii) an indirect consequence of perturbing sugar metabolism.
TrmB binds target promoters that function in diverse metabolic pathways in the absence of glucose or glycerol
TF‐binding location analysis with ChIP‐chip.
To differentiate between direct and indirect regulatory influences of TrmB, its transcription‐factor‐binding sites (TFBS) were localized throughout the genome using ChIP‐chip. This procedure localizes DNA fragments within transcription factor complexes enriched with chromatin immunoprecipitation (ChIP) using whole genome tiling arrays (chip). We mapped TFBSs in the presence and absence of varying concentrations of glucose or glycerol (Materials and methods). TrmB bound to 113 sites throughout the chromosome in the absence of glucose or glycerol (Figure 3A). Interestingly, no TrmB binding was observed across the genome in the presence of high glucose or glycerol (Figures 3B and C; Supplementary Figures 1 and 2). This finding is consistent with the observation that both glucose and glycerol can complement the physiological consequences of deleting TrmB (Figure 2) and with earlier reports on TrmB function in other systems (Lee et al, 2008).
The 113 binding sites were ranked according to statistical confidence (Table I). The top seven high‐confidence (P<10−6) hits reside in intergenic regions upstream of genes encoding functions in glycolysis and gluconeogenesis (functional enrichment P=1.11E−02) (Table I; Figures 3A–C). These genes also exhibited the most significant enrichment in immunoprecipitated TrmB–DNA complexes (gpm, ppsA, pgk, pykA, VNG0683C; Figure 3A). In addition, one gene, ppsA, remained bound under low glucose concentrations, suggesting a high‐affinity interaction with TrmB at this site (Figure 3B).
Other high‐confidence targets in the list (10−3>P>10−6) also showed a strong transcriptional perturbation in the ΔtrmB background (Table I; Supplementary Table 4). Consistent with the transcriptome data, genes coding for biosynthesis of purine nucleotides, cobalamin, thiamin, and amino acids were significantly overrepresented across all 113 targets (Table I; Supplementary Table 4). We also observed binding to the intergenic region upstream of five TFs, including VNG0156C, VNG0247C, VNG0878G, putative regulators; VNG1179C, a regulator of copper homeostasis (Kaur et al, 2006); and TrmB itself (Supplementary Table 4). This could explain the differential regulation of a large number of genes whose promoters are not directly bound by TrmB. Together these data suggest that TrmB directly controls the expression of genes functioning in diverse metabolic pathways.
Although TF binding in intergenic regions is generally considered as evidence for direct regulation of downstream genes, we found that ∼40% (45 of 113) of TrmB‐binding sites were inside coding sequences. However, given that the H. salinarum genome is ∼85% coding, our sample of binding sites is actually somewhat enriched in intergenic regions (P=0.18), with 60% of these binding sites falling within 250 bp of an experimentally determined transcription start site (Koide et al, 2009). ChIP‐chip studies for other bacterial TFs have found up to 70% of targets in intergenic regions (Shimada et al, 2008). Combined, these results suggest that these binding events might be functional. However, further investigation is required to elucidate the physiological function of these unusual TrmB targets, at least two of which are near loci that encode newly discovered putative noncoding RNAs (Koide et al, 2009).
Identification of a cis‐regulatory sequence motif.
To further define the TrmB‐binding site, we searched for a conserved cis‐regulatory sequence motif within 250 bp of its genomic binding locations identified by ChIP‐chip. Locations of transcription start sites (Koide et al, 2009), translation start sites (http://baliga.systemsbiology.net)(Ng et al, 2000), and putative GTF‐binding sites (Facciotti et al, 2007) were used to constrain the sequence search space (Materials and methods). We identified a conserved cis‐element [TACT‐N(7‐8)‐GAGTA (P<2 × 10−5)] (Figure 4A) within 250 bp of 115 (P=8.7 × 10−50) of all genes nearby TrmB‐binding sites identified by ChIP‐chip (Figure 4B). Matches to this signature were also detected in the vicinity of other sites, albeit farther from the ChIP‐chip location (>250 bp) (Supplementary Table 4). This motif is divergent from other characterized TrmB‐binding sites (e.g. Thermococcales glycolytic motif (TGM), TATCAC‐N5‐GTGATA) (van de Werken et al, 2006). However, consensus sequences for different HTH‐domain containing TFs can be quite divergent (Rigali et al, 2004). A genome‐wide pattern searching algorithm identified a total of 317 matches to this motif signature (Supplementary Table 5; Materials and methods) which are nearby 396 genes in operons, suggesting that TrmB may bind to additional loci across the genome. This motif signature is enriched in intergenic regions (P∼0.002). However, given that functional promoter binding is a product of combinatorial interactions of TFs, GTFs, cofactors, and RNA polymerase; further studies will determine which of these additional putative TrmB‐binding sites are indeed functional.
In vivo validation of key TrmB‐binding sites using promoter: reporter fusion assays.
To ensure that the TrmB‐binding motif represents a physiologically relevant regulatory region, the ppsA promoter was fused to the GFP reporter and assayed in the wild type and ΔtrmB backgrounds (Figure 4C). As expected from the microarray and ChIP‐chip data, FACS assays validated that transcription from the ppsA promoter is activated three‐fold in the absence of glucose in the parent background, whereas ΔtrmB cells are impaired for induction (Figure 4C). Strikingly, when the distal TrmB‐binding site is removed, the glucose responsiveness is reduced in the parent strain (Figures 4C and D). Activity from this shorter promoter remains at background levels in the ΔtrmB mutant regardless of condition (Figure 4C). Together these data verify that the cis‐regulatory TrmB‐binding site identified by high‐throughput methods is physiologically relevant and requires TrmB for regulation in the absence of glucose. Further, both the promoter proximal and distal binding sites contribute to TrmB‐mediated response to glucose, with both binding sites required for full activation, suggesting binding site synergy (Figure 4D).
TrmB governs an integrated transcriptional and metabolic network to balance the expression of evolutionarily diverse cofactor and enzyme‐coding genes
TrmB is a bifunctional regulator that activates some targets and represses others.
To construct the H. salinarum TrmB‐dependent transcriptional network, we calculated the significance of the overlap between the integrated ChIP‐chip, transcriptome, and motif location data generated here. This was further integrated with genome‐wide transcription start site data (Koide et al, 2009) and ChIP‐chip data for seven GTFs (Facciotti et al, 2007). This enabled identification of a significant group of 37 genes (organized among 20 operons) in the overlap between integrated system‐wide datasets (Figures 4A and B). Functional annotations (Materials and methods) revealed that these 37 genes encode enzymes of (i) glycolytic and gluconeogenic pathways, (ii) purine biosynthesis, (iii) cobalamin biosynthesis, (iv) TCA cycle, and (v) glutamate dehydrogenase (Figure 4A).
The mechanism by which TrmB activates and/or represses these genes was investigated further by analyzing the locations of its binding sites relative to those of seven GTFs (Facciotti et al, 2007) (Figure 5A). TrmB‐binding sites were located upstream of GTF‐binding sites in promoters of genes it activates (e.g. ppsA; Figure 5B; P<0.03). In contrast, no GTF‐binding locations were detected within promoters of genes that are repressed by TrmB (e.g. gap; Figures 5A and C). Alternatively, GTF‐binding sites were located upstream of repressed genes (e.g. VNG0303G, VNG0382G, VNG1128G, Figure 5A). Thus, our system‐wide ChIP‐chip data support a model in which TrmB binds to its motif downstream of the PIC to occlude transcription. In contrast, TrmB‐binding upstream of the PIC facilitates transcription at weak promoters (Figure 5D). Although this model was suggested earlier (Kanai et al, 2007; Lee et al, 2008), this study presents the first in vivo experimental evidence of these interactions and places them in a global context. Similar analysis of the remaining TrmB‐bound promoters revealed a more complex distribution of GTF and TrmB‐binding sites, which may indicate the involvement of additional mechanisms in the regulation of those genes (Facciotti et al, 2007). Nevertheless, this integrated systems analysis has provided both a global perspective and valuable mechanistic insight into combinatorial regulation by GTFs and a sequence‐specific transcription regulator (Bell et al, 1999; Kanai et al, 2007).
The combined evidence presented thus far strongly suggests that TrmB acts as both a transcriptional activator and a repressor in response to carbon source availability. Further, these results suggest that TrmB directly and coordinately controls genes significantly overrepresented for functions in central metabolism and its associated pathways in the metabolic network.
Metabolic network reconstruction analysis suggests that TrmB coordinates the expression of evolutionarily diverse enzymes.
To gain a systems‐scale perspective on the role of TrmB in metabolism, we reconstructed the metabolic network of H. salinarum in the context of the TrmB transcription regulatory network and known archaeal reactions reported in the literature (Figure 6; Supplementary Table 6). Many TrmB‐regulated enzymes catalyze reactions at critical regulatory branch points but not the end stages of several metabolic pathways, including amino acid, purine, thiamine, cobalamin biosynthesis. (Figure 6; Falb et al, 2008; Gonzalez et al, 2008). This is illustrated by TrmB‐mediated transcriptional control of genes encoding branch points between purine and thiamine metabolism (purK, purE, and purM; reactions 46 and 47, Figure 6), carbon and nitrogen metabolism (gdhB, korAB, and gdhA1; reactions 31, 32, and 39, respectively, Figure 6)
We also observed several instances of direct TrmB‐mediated transcriptional control of metabolic enzymes with the biosynthesis of their cognate cofactors. Three examples support this assertion. (i) TrmB directly controls over 10 enzymes that require adenosine phosphates (AXP; Figure 6, e.g. reactions 3, 7, 10, 11, 27, 33, 63) and six genes that encode the biosynthesis of these cofactors (Figure 6, reactions 43, 44, 45, 46, 47, 52). (ii) TrmB target genes encoding functions such as 5‐phosphoribosyl‐N‐formylglycinamidine (FGAM) synthase (purL2; Figure 6, reaction 45) and asparagine synthase (asnA, reaction 63) use glutamate or glutamine as co‐reactants and ATP as a cofactor. Concomitantly, TrmB regulates GOGAT pathway genes (reactions 31, 39). (iii) TrmB coordinately controls thiamin (TPP) synthesis (thiC) with thiamine‐requiring enzymes (porAB and menD, reactions 12 and 25, respectively) (Rodionov et al, 2003).
In addition, among direct TrmB targets were genes encoding enzymes of uniquely archaeal lineage (Figure 6; Supplementary Table 6). Of the enzyme‐coding genes under TrmB control included in the metabolic pathways shown in Figure 6, 13 are unique to archaea (Supplementary Table 6) and 35 are conserved across species from all three domains of life. Integrated analysis of the metabolic and gene regulatory network architecture reveals two opposing scenarios. (i) In the shikimate biosynthesis pathway, only one gene encoding shikimate kinase (VNG1245C, reaction 20) is of archaeal origin, whereas all other genes are conserved throughout evolution (orange shaded area, Figure 6; Supplementary Table 6). Strikingly, all genes of this pathway except shikimate kinase are direct TrmB targets (Figure 6), suggesting that shikimate kinase may have been acquired by homologous gene replacement (Galperin and Koonin, 1999). This finding is especially surprising given that enzymes in bacterial metabolic pathways without branch points tend to be co‐regulated (Seshasayee et al, 2008). (ii) The latter half of the TCA cycle from 2‐oxoglutarate to fumarate is directly TrmB‐dependent (purple shaded area, Figure 6), including the genes encoding 2‐oxoglutarate oxidoreductase, the only uniquely archaeal enzyme in the pathway (Supplementary Table 6). These findings suggest that TrmB governs the transcription of a metabolic network with hybrid evolutionary origin.
TrmB coordinately regulates metabolic enzyme‐coding genes with cofactor genes
From the evidence presented in this study, we conclude that TrmB governs a sugar‐responsive global metabolic regulatory network to coordinate the expression of genes with diverse evolutionary ancestry. Remarkably, TrmB coordinates the transcription of enzyme‐coding genes involved in the synthesis of cofactors required for the function of these metabolic enzymes. We hypothesize that this TrmB‐directed coordination may enable redox and energy balance. Specifically, our data suggest that TrmB represses the semi‐phosphorylative Entner–Doudoroff (E–D) glycolytic pathway (Kanai et al, 2007; Danson et al, 2007; van der Oost and Siebers, 2007; Pfeiffer et al, 2008) (e.g. gap, pykA, VNG0442G) (Figures 5 and 6), and induces gluconeogenesis (e.g. ppsA; Figure 2). Thus, a deletion in trmB would lead to an inability to generate energy through gluconeogenesis in the absence of glucose. Secondly, ΔtrmB cultures grown in the absence of glucose overexpress genes coding for enzymes that convert NAD(P)+ to NAD(P)H, this may force the cell toward an oxidized state (e.g. Figure 6, reactions 19, 31). If so, then this would lead to a shortage of reducing equivalents. The hypersensitivity to oxidative stress and reduced NAD/H ratio observed in ΔtrmB mutant cells are consistent with this hypothesis (Supplementary Table 2; Figure 2B). We conclude that TrmB acts to maintain redox and energy balance in response to nutrient availability in H. salinarum.
Interestingly, our evidence points to additional regulatory mechanisms that may cooperate with the TrmB transcription network. First, TrmB does not seem to regulate the end stages of several pathways (e.g. amino acid, purine, thiamine, cobalamin biosynthesis). Second, TrmB is not only autoregulated, but also seems to directly control four other TFs (Supplementary Table 4). Third, the transcription of some genes with a direct TrmB–promoter interaction remains unchanged in the ΔtrmB strain (e.g. amino‐acid biosynthesis gene asnA). Finally, in several instances TrmB regulates an entire pathway except for one gene (e.g. enolase, shikimate kinase). Together, this evidence suggests the cooperation of other regulatory mechanisms with the TrmB transcription network. This interpretation is in line with earlier metabolic regulatory network analyses in Escherichia coli, which found that the majority of central metabolic pathways are controlled by multiple TFs (Seshasayee et al, 2008).
Evolutionary context for the TrmB transcription‐metabolism network
Among known global regulators of central metabolic genes in prokaryotes, no single transcription factor has been shown to directly control both metabolic enzyme and cognate cofactor biosynthesis genes (Grainger et al, 2005; Supplementary Table 4). For example, CRP, a global regulator of carbon and nitrogen metabolic pathways in enteric bacteria, is required for the condition‐specific transcriptional induction of cobalamin biosynthesis genes (cob). However, a direct CRP–cob promoter interaction has not been established (Ailion et al, 1993; Grainger et al, 2005). Instead, the cob‐specific transcription factor PocR may be a more likely candidate for direct regulation (Ailion et al, 1993). Similarly, in Bacillus subtilis, CcpA controls global targets in carbon metabolism (Sonenshein, 2007), whereas the PurR repressor is specific for purine biosynthetic genes (Saxild et al, 2001).
In contrast, in other archaeal species, it is possible that TrmB or TrmB‐type global transcriptional control of metabolism is operative. For example, in species in which TrmB is known to control glycolysis and gluconeogenic pathways (e.g. Pyrococcus furiosus), an unknown transcription factor upregulates genes in other metabolic pathways such as the TCA cycle and chorismate synthesis in response to maltose (Schut et al, 2003). In addition, divergent TrmB‐binding sites can be detected in the vicinity of the transcription start site for some of these genes in this and other thermophilic archaeal species, although they have not been experimentally validated (van de Werken et al, 2006). It will be interesting to confirm these preliminary findings, because it suggests that the network motif of integrated control of cofactor and enzyme genes could be widespread in archaea. Therefore, the metabolic network model presented here will be useful as a structural framework for other archaeal systems and a starting point for evolutionary comparisons with other understudied representatives in other domains of life.
In light of this evolutionary context, it was striking to observe that genes encoding enzymes of uniquely archaeal lineage were included among the direct TrmB targets in H. salinarum (Figure 6; Supplementary Table 6). Combined with the observation that the TrmB regulon genes encode enzymes of diverse ancestry, these results are consistent with the hypothesis that several lateral gene transfer or homologous gene replacement events occurred in the evolutionary compilation of the TrmB network (Galperin and Koonin, 1999). However, additional information is required to determine whether the TrmB regulatory network architecture (e.g. promoter elements, nutrient responsiveness) was in place before or after the acquisition of these new genes, because our data support both scenarios (e.g. compare the opposing shikimate pathway and TCA cycle examples. See shaded areas in Figure 6). Nevertheless, the TrmB regulatory network may represent a unique window into an active evolutionary process.
In summary, this study reveals that TrmB regulatory control is restricted primarily to central metabolism and branch points, suggesting combinatorial control between TrmB and pathway‐specific regulatory mechanisms. In addition, TrmB seems to balance the expression of genes coding for metabolic enzymes with those of their cognate cofactors in a sequence‐specific manner. Finally, the TrmB metabolic regulatory network is an evolutionary mosaic, controlling genes coding for uniquely archaeal enzymes with those that are more widely distributed, even within the same metabolic pathway.
Materials and methods
Strains, media, plasmids, and growth curve assays
H. salinarum NRC‐1 (ATCC700922) was grown routinely in complex medium (CM; 250 NaCl, 20 g/l MgSO4 7H2O, 3 g/l sodium citrate, 2 g/l KCl, 10 g/l peptone) or a complete defined medium (CDM) containing 19 amino acids (Supplementary Table 1). For growth in CDM, starter cultures were grown in CM to mid‐logarithmic phase and washed three times in basal salts buffer (CM lacking peptone) and resuspended in CDM at OD600 ∼0.1. Subsequent growth was conducted for all media conditions at 37°C with 225 r.p.m. shaking in the presence of low light intensity (24.6 μmol photons/m2 s from fluorescent lamps).
For routine culturing and growth assays of the Δura3 parent and ΔVNG1451C mutant strains, CM or CDM was supplemented with 0.05 mg/ml uracil. For growth curve assays, NAD+/H assays and growth complementation experiments in the Δura3 parent and ΔVNG1451C mutant strains, CM or CDM was supplemented with various sugars (Figure 2) at 7% w/v except for glycerol at 0.08% v/v. Samples were grown in 200 μl cultures for 6 days under continuous ∼225 r.p.m. shaking in a Bioscreen C (Growth Curves USA, Piscataway, NJ), set to measure optical density (OD) at 600 nm automatically every 30 min for 200 culture samples simultaneously. The average and standard deviation of the doubling time for Δura3 and Δura3ΔVNG1451C during the logarithmic phase is shown in Figure 2, which represents at least 9 and at most 20 biological replicate samples for each strain under each condition. Two sets of non‐parametric paired t‐tests were performed comparing (i) wild type versus mutant growth (asterisks in figure represent this set of tests); and (ii) mutant growth without carbon source versus mutant growth in each of the carbon sources shown in Figure 2. By the latter measure, mutant growth rates in the absence of carbon source were significantly different from growth in glycerol and glucose but not for any other source tested. For TFBS location array experiments with this strain, glucose was either omitted or added at 0.01% or 7% w/v or glycerol at 0.08% v/v where indicated in the text. Strain constructions and NAD+/H assays are described in detail in Supplementary information.
Gene expression arrays
10 ml of H. salinarum NRC‐1 Δura3ΔVNG1451C and Δura3 parent strain sample cultures grown in CM or CDM in the presence or absence of glucose were collected at three time points throughout the growth curve (OD600∼0.2, 0.6, and 1.2). Cells were immediately pelleted by room temperature centrifugation at 8820 g for 8 min at 4°C and snap‐frozen on a dry‐ice ethanol bath. Sample pellets were stored overnight at −80°C, followed by RNA preparation using the Absolutely‐RNA kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. RNA quality was checked using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and freedom from DNA contamination was ensured by PCR amplification of 200 ng of RNA sample. 5 μg of each quality‐checked RNA sample was hybridized against the H. salinarum NRC‐1 reference RNA prepared under standard conditions (mid‐logarithmic phase batch cultures grown at 37°C in CM). This common reference RNA has been used across all ∼950 microarray experiments in the H. salinarum NRC‐1 microarray data repository (Bonneau et al, 2007). Samples were hybridized to a 70‐mer oligonucleotide array containing the 2400 nonredundant open reading frames (ORFs) of the H. salinarum NRC‐1 genome as described in Baliga et al (2004). Each ORF was spotted on each array in quadruplicate and dye flipping was conducted (to rule out bias in dye incorporation) for all samples, yielding eight technical replicates per gene per time point. At least two independent biological replicates exist for all experimental conditions for a total of 16 replicates per gene per condition. Direct RNA or DNA (TFBS location arrays, see below) labeling, slide hybridization, and washing protocols were performed as described earlier (Facciotti et al, 2007; Schmid et al, 2007). Raw intensity signals from each slide were processed by the SBEAMS‐microarray pipeline (Marzolf et al, 2006) (www.SBEAMS.org/microarray), in which resultant data were median normalized and subjected to significant analysis of microarrays (SAM) and variability and error estimates (VERA) analysis. Each data point was assigned a significance statistic, λ, using maximum likelihood (Ideker et al, 2000).
Microarray data were analyzed using the TM4 MultiExperiment Viewer (MeV) application (http://www.tm4.org/) within the Gaggle data analysis environment (Shannon et al, 2006). Specifically, all 2400 genes across mutant and wild‐type microarray experiments, described above, were subjected to three independent analyses: significance analysis of microarrays (SAM), KMEANS clustering, and hierarchical clustering. Resultant clusters of genes in the union of all three analyses that displayed significant differential transcription between the parent and mutant strains in the presence and/or absence of glucose were considered to be VNG1451C dependent. Biological replicates were considered independently to ensure statistical rigor.
TFBS location array analysis
ChIP of VNG1451C‐cmyc‐tagged constructs was performed as described (Ren et al, 2000; Facciotti et al, 2007) in cultures grown in the presence or absence of glucose or glycerol. Resultant TFBS location data were analyzed for statistically significant enrichment of features in the ChIP‐chip sample versus the unenriched sample using MeDiChI, a regression‐based deconvolution algorithm (Reiss et al, 2008). Enrichment lists from each of the five independent MeDiChI runs were combined into a density algorithm (Koide et al, 2009) to find TFBS locations overrepresented in the data. To be considered as part of the final TFBS enrichment list (Supplementary Table 4), we required that each enrichment peak from the density output be composed of at least two biological replicate peaks with a combined MeDiChI P‐value <0.001 (product of replicate P‐values). A peak was considered to be 'intergenic' if it fell within 250 bp of a transcription start site or termination site (a conservative estimate of the resolution of the data from MeDiChI‐derived binding sites; (Reiss et al, 2008)). Subsequently, the resultant binding sites from the combined dataset were compared with orthogonal datasets (i.e. genome‐wide mRNA expression and binding motif searches, details below).
To analyze the TrmB TFBS location data in the context of the GTF TFBS data (Facciotti et al, 2007), the distance of the genomic position for each high‐resolution GTF‐binding site (Reiss et al, 2008) to that of TrmB (GTF coordinate—TrmB coordinate=relative position) at each target promoter was calculated. The Pearson correlation between this distance and the mRNA expression data in ΔtrmB for the gene of interest was then calculated. The P‐value for these correlations reported in the text were calculated based on 100 000‐fold resamplings of the data.
DNA‐binding motif searching
To find the consensus binding motif for VNG1451C, the sequence search space was limited for each putative promoter region enriched in the TFBS location data through several constraints: (i) sequence ±250 bp from the center of each MeDiChI‐based peak; (ii) sequence ±20 bp from the putative transcription start site (Koide et al, 2009); (iii) annotated translation start site location (Ng et al, 2000). Resultant sequences were used as input for three independent motif‐finding algorithms: (i) Bioprospector (http://ai.stanford.edu/~xsliu/BioProspector/), which finds gapped motifs in query sequences (Liu et al, 2001); (ii) MEME/MAST (http://meme.sdsc.edu/meme/) (Bailey and Gribskov, 1998; Bailey et al, 2006); and (iii) RSAT pattern finding (http://rsat.ulb.ac.be/rsat/). Only motifs represented in the intersection of all the three algorithm outputs were considered in further analysis. To generate the P‐value for each motif, the three algorithms were re‐run on randomized query sequences. Results were compared with algorithm outputs from original sequences using the Wilcoxon test (Frith et al, 2008). One motif had a statistically significant P‐value (P=2.0 × 10−5), and the resultant consensus motif described in the text was generated using weblogo.berkeley.edu/logo.cgi. To scan the remainder of the genome for the resultant motif, we used the pattern‐finding program at rsat.ulb.ac.be/rsat/ with the parameters of (i) no more than 1 bp away from the consensus motif, (ii) unbiased for genomic position (i.e. coding and noncoding sequences were searched); (iii) containing a 7 or 8 bp gap within the motif; (iv) located on the chromosome (as no TrmB hits were found on either of pNRC100 or pNRC200).
GFP promoter: reporter fusion validation experiments
The ppsA p1+p2 construct contains both putative TrmB‐binding sites in a 115‐bp fragment upstream of the translation start site of the ppsA (VNG0330G) gene (Figure 4D). The ppsA p2 construct is an 88‐bp truncated version of the p1+p2 construct and lacks the promoter‐distal TrmB‐binding site (Figure 4D). These promoter fragments were fused to a red‐shifted GFP variant optimized to function in haloarchaea, which was adapted from (Reuter and Maupin‐Furlow, 2004). See Supplementary information for details on strain construction. Constructs were transformed into the Δura3 parent strain and the ΔtrmB deletion mutant. Resultant constructs were grown in CM media in the presence or absence of 7% glucose to mid‐logarithmic phase (OD600∼0.4–0.8). Cultures were diluted to OD600=0.2 and fixed in 0.25% formaldehyde dissolved in basal salts (CM lacking peptone) for 10 min at 4°C and subsequently washed in basal salt to remove fixative. Fluorescence of fixed cells was measured by flow cytometry on a FACS‐Calibur instrument (Becton Dickinson, San Jose, CA) in the presence of 1 μM fluorescent beads (Polysciences, Warrington, PA) spiked in at a concentration of 4 × 108 beads/ml. A negative control strain carrying the empty GFP vector with no promoter insert was treated identically to gauge background fluorescence levels (black bar in Figure 4C). Resultant data were analyzed using Flow‐Jo software (Tree Star, Inc., Ashland, OR). The average absolute fluorescent cell counts normalized to bead counts from three biological replicate experiments±s.d. are shown in the graph (Figure 4C).
Data integration analysis
To assess the extent of agreement between the three system‐wide datasets presented in this study (gene expression, ChIP‐chip, and motif search data), the hypergeometric distribution P‐values were calculated, which reflect the likelihood that the intersection of any two of these three datasets are due to chance. Specifically, we calculated the significance of (i) the number of genes within 250 bp of both the ChIP‐chip hits and binding motif sequences (one extra bp in motif degeneracy was allowed for a few of the genes in Figure 4A, which were nearby ChIP‐chip hits, which showed a highly significant change in the microarray data;Figure 4A); (ii) the number of genes whose expression changed in the trmB mutant, which were also within 250 bp of a ChIP‐chip hit; (iii) the number of genes changing in the transcriptome data, which contained a motif within 250 bp of their transcription start site (Figure 4B).
Detailed annotation analysis of the 37 genes in the intersection of the three high‐throughput datasets (Figure 4B) was conducted using protein functional data from online databases (http://baliga.systemsbiology.net/halobacterium; KEGG, GO, STRING, HaloLex) (Bonneau et al, 2004; Bare et al, 2007; Pfeiffer et al, 2008; Jensen et al, 2009). To build the metabolic network governed by TrmB, a four‐step bioinformatic process was conducted according to the flowchart shown in Supplementary Figure 3.
Accession numbers
All ChIP‐chip and gene expression array data presented in this study are available at the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) under the accessions GSE13531, GSE13529, and GSE13498.
We are indebted to Ludmila Chistoserdova and Monica Orellana for their critical reading of the paper, Kenia Whitehead for useful discussions, Christopher Bare for software support, and Lee Pang and Noel Blake for assistance with the FACS analysis. This work was supported by grants from NIH (P50GM076547 and 1R01GM077398‐01A2), DoE (MAGGIE: DE‐FG02‐07ER64327), NSF (DBI‐0640950) to NSB, and from NIH (5F32GM078980‐02) to AKS.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary materials
This file contains supplementary results, methods, and figures S1–3 [msb200940-sup-0001.pdf]
Supplementary Table 1
Supplementary Table 1. Halobacterium salniarum NRC‐1 complete defined synthetic medium (CDM) [msb200940-sup-0002.xls]
Supplementary Table 2
Supplementary Table 2. High throughput growth data for VNG1451C (trmB) deletion mutant compared to parent strain(s) [msb200940-sup-0003.xls]
Supplementary Table 3
Supplementary Table 3. Microarray gene expression data. [msb200940-sup-0004.xls]
Supplementary Table 4
Supplementary Table 4. Direct targets of TrmB: all 113 ChIP‐chip hits. [msb200940-sup-0005.xls]
Supplementary Table 5
Supplementary Table 5. TrmB binding motifs located throughout the H. salinarum NRC‐1 genome. [msb200940-sup-0006.xls]
Supplementary Table 6
Supplementary Table 6. Details regarding metabolic reactions depicted in Figure 5. [msb200940-sup-0007.xls]
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
- Copyright © 2009 EMBO and Nature Publishing Group


text:
True Highlands Blog
Wild Swimming in the Highlands & Islands of Scotland
Posted by True Highlands in Kit and Caboodle | 4 comments
There is something truly special about going for a wild swim. Whether it's to escape the midges, to cool down after a long day hillwalking or to explore some remote or curious locations then the Highlands are blessed with some of the finest locations for a quick dip, an epic swim or a wee paddle. Part of the attraction is not just the act of swimming, it is the places that this can take you and the things you can see. Here is our selection of rivers, waterfalls, lochs, pools and bays that can demonstrate why the highlands is the most exciting swimming destination in the world. Sculptures, castles, wildlife, and mythology, all you have to do is get wet.
Your #HighlandHighlights w/e 7 August 2015
Posted by True Highlands in Kit and Caboodle | 0 comments
We admit the summer hasn't been up to scratch so far. However, no matter what the weather brings, there is no doubting that the Highlands & Islands are spectacular at any time! Here are our pick of 10 from this weeks Facebook and Twitter submissions.

subset: commoncrawl


text:
Zanetti to finish career at Chelsea? All the latest
Rumours have started suggesting that Zanetti could play for just 1 last year for Chelsea and finish his career before becoming a sport director. All the latest are from Twitter:
Exclusive: #Mourinho wants Javier #Zanetti to Chelsea. 1 year as player, then sport director by 2015. Zanetti's priority is for Inter #CFC
— Fabrizio Romano (@FabrizioRomano) February 20, 2014
Inter's Zanetti will meet with Thohir. But there is a "Special"offer from Chelsea: the details http://t.co/JwK1Hzk9cv
— David Amoyal (@DavidAmoyal) February 20, 2014
What's your take on this? An old guard for Chelsea?Will it work out?
Feedback is always appreciated
Follow me- @TheRealBluearmy


text:
Or connect with:
All site tags
Categories: journals, books
The effective use of digital games for empowerment and social inclusion (DGEI) of people and communities at risk of exclusion will be shaped by, and may influence the development of a range of sectors that supply products, services, technology and research. The principal industries that would appear to be implicated are the 'videogames' industry, and an emerging 'serious games' industry. The videogames industry is an ecosystem of developers, publishers and other service providers drawn f...


text:
ERIC Number: ED175577
Record Type: RIE
Publication Date: 1978-Oct
Pages: 337
Abstractor: N/A
Reference Count: 0
Lasting Effects After Preschool. A Report of the Consortium for Longitudinal Studies.
Lazar, Irving; And Others
This second general technical report of the Consortium for Longitudinal Studies summarizes the findings of current analyses of longitudinal studies of low income children who participated in experimental preschool programs initiated in the 1960s and includes additional data and further analyses of the 1976-77 follow-up study. The pooled data bank of the 12 research groups making up the Consortium provided information on approximately 3,000 low income children who either participated in early intervention programs or served as controls. The common information across projects includes various pre-enrollment measures of the children's home background, a pretest IQ score, and at least one IQ test score collected immediately after the preschool experience. Each investigator also collected a variety of cognitive and behavioral measures which are not common across all projects. In 1976-77, members of the Consortium collaborated in a common follow-up data collection effort. These data include both child and parent interviews, children's IQ and achievement test scores, and information on whether the children had ever failed a grade or been assigned to special education classes. The children were aged 9 through 19 years old at the 1976-77 follow-up. The findings showed that high quality early education programs for low income children had lasting effects in five areas: (1) reducing the number of children assigned to special education classes, (2) reducing the number of children retained in grade, (3) increasing children's math achievement scores at fourth grade, (4) increasing IQ scores at least up to age 13, and (5) influencing aspects of children's and mothers' achievement orientation. (Author/JMB)
Descriptors: Academic Achievement, Achievement Need, Achievement Rating, Disadvantaged Youth, Early Childhood Education, Followup Studies, Grade Repetition, Intelligence Quotient, Intervention, Longitudinal Studies, Low Income Groups, Preschool Education, Program Effectiveness, Special Education
Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 (Stock Number 017-090-00047-0)
Publication Type: Reports - Evaluative
Education Level: N/A
Audience: N/A
Language: English
Sponsor: New Jersey State Dept. of Higher Education, Trenton.; Administration for Children, Youth, and Families (DHEW), Washington, DC.; William and Flora Hewlett Foundation, Palo Alto, CA.
Authoring Institution: Education Commission of the States, Denver, CO.
Note: For summary report, see PS 010 386; For related documents, see ED 148 471-72


text:
In 2010-2011 a good friend of mine visited Japan and taught Australian Studies at Todai (Tokyo University). I was overjoyed because it had been some time since I had last seen him and also because in addition to being highly astute he is a great conversationalist. Finally, someone with whom I could share my own experiences of living in Japan (coming up on close to ten years altogether). What was so refreshing about the limited conversations we had was his total lack of bitterness prevalent among gaijin. As I have written elsewhere there is a general persistent malaise infecting Anglosphere gaijin in Japan, a malaise that might correctly be diagnosed as a failure to recognise the limits of their cultural universality and the resultant bitterness that manifests as they are trapped within a hyper-speed game of conceptual Pong in which they are always on the losing team.
An admission: I have had moments of bitterness. While I take full responsibility for them, I am also able to locate, on reflection their origins: the miasma of self-perpetuating "opinion" offered up as knowledge across what seems to be the entire English-language internet relating to Japan. In other words, going online for help resulted in self-fulfilling, paranoid and unreconstructed retreads of other people's stories. What is worse, is that a number of supposedly authoritative online voices from high profile activists such as Arudo Debito seem to relish in and gleefully spread cynical screeds against issues that with a little more thought and a tad more contextual awareness would not be as difficult or terrible as they make out.
Debito is an odd bird. On emigrating to Japan he eventually attained naturalised Japanese citizenship, quite a rare feat for a Westerner. Indeed, his precedent, along with his civic-rights-oriented activist bent caused him to rise to some degree of fame. His fifteen minutes came when he won a lawsuit against a Hokkaido hot springs owner who had denied him and one of his daughters entry on grounds of race. But it seems that Debito's hunger for fame or at least admiration as a vanguard activist eclipsed his capacity for considered reflection and contextual awareness. A casual read of his blog reveals a number of interesting and high profile stories to do with illegal discrimination and everyday life in Japan. The blog also seems regularly frequented by a number of people with similar interests. However, with sustained, continuous engagement it begins to be possible to locate an underlying bitterness and personal dissatisfaction that quite clearly colours his writings. What had started out as spotlighting and acting on injustice has turned into smug, chauvinistic critique.
In his latest Japan Times column "These are a few of my favourite things in Japan" in the semi-regular opinion series "Just Be Cause," Debito employs a spot of silver tonguing in an attempt to both "be positive" and offer salient critique. Indeed, the former idea is a rather antiquated internet discussion rhetorical device frequently employed by those inclined to plead "but you never say anything good" or "If it's so bad, just go home". As for the latter, observation has always been Debito's strong suit, critique, less so. Both combined make for a dire reading experience in which each compliment appears poisoned by an invisible yet omnipresent sarcastic smirk. One further issue is his flippant disregard of Japanese satire and the role of irony/sarcasm in Japanese society.
While correct in asserting a lack of emphasis on sarcasm as a legitimate means of communicating humour or social analysis, he fails to tell us just why it matters. After all, he points out how Anglosphere cultures have a rich tradition of sarcasm and stinging social critique, but fails to let us know that a lot of this critique has its origins in class politics as a way of shoring up class hierarchies and dismissing lower class cultural practice. In fact I would be more interested in his take on social critique from a Japanese perspective in relation to Anglosphere critique and ironic modes of humour. But then again, whenever Debito writes, it is always from the location of a particular kind of gaijin crystallised in time and place, a kind of outsider who knows more than his green-behind-the-ears fan base but seems incapable of getting inside a local, cultural perspective. While clearly proficient in Japanese to a certain level, Debito appears to be culturally illiterate when it comes to examining similar phenomena from different cultural perspectives.
Obviously, his latest column is supposed to be an exercise in humour and appeal to the gaijin readership of the Japan Times. In such a genre, he hardly has the time or space to address in detail any of the critiques I have raised here. It would be nice, however, to see writing from such a prominent gaijin who attained permanent residency in 1996 and citizenship in 2000. That is a long time to hold onto such limited outsider concepts at the expense of digging deep into the local mind beyond adversarial escapades in so-called activism. Why not start writing in Japanese? Why not start engaging with actual local community beyond the gaijin Anglosphere, in their own language. So in regards to the shots fired at Japan from an American distance away, I say, "Stop it, Debito, just stop it!" If you want to have any relevance at all in terms of Japan's multicultural future, you may need to redraft your terms of war. In fact, leave the war behind and make peace. Dude.


text:
Blog Archives
Character Kids 2014
It's a new year and a new opportunity to dream again! This year I'm excited about adding a youth council to my character education program. I look forward to seeing what great things will become of this merging of young and mature minds. I think that it is so important to allow youth to have a voice. I've learned so many things from my own children, like how to keep things simple when I want to make a "mountain out of a molehill," or that I really don't understand "everything" that they are experiencing these days in school, even though I've "been there, done that". Sure, I agree, for the most part, that there is nothing new under the sun, but surely 21st century youth are experiencing some issues that those of us over 35 never imagined we'd have to deal with during our youth.
Take for example the terms, cyberbullying, road rage, knock-out, Internet, cell phones, website, tweets, Twitter, Facebook and any other 21st century term you can think of. They were unheard of when I was growing up over 35 years ago. We may have seen a form of road rage, and yes, cell phones and computers have been around for much longer than someone younger than 20 may realize, but most of these terms, issues and products did not exist, at least in their present form, 30 years ago. Youth are dealing with the age of technology, knowledge and major life issues that require consistent adult guidance, advocacy and wisdom as well as their own voice to express how these issues affect them. After all, they will be leading our world in a few years, so it is imperative for adults to listen to what they have to say and gain some insight into their perspective of life.
My hope is that the youth council will not only represent the Maximizing Character program by exhibiting good character in their respective homes and communities, but I hope that they will become global leaders. One way that I believe that adults can give youth an opportunity to express themselves and develop into productive citizens is through role play, service learning, mentoring opportunities, designing and implementing projects and through cultural arts. Through these modes of expression, youth can develop their communication skills, increase their capacity to lead others and develop social responsibility. 21st century youth also have an opportunity to make sense of their current environments, the world and establish a worldview.
I'm rooting for our youth and expecting great things from my group of character leaders as well as those children and young adults that we hope to impact as we promote excellence in character. I look forward to shining our light as we travel from school to school, community event to community event and even as we connect to children and families around the world in order to support an important cause or create positive change in areas affected by poverty, disease or injustices. Keep checking in with us this year to see what the Maximizing Character youth group is up to. Here's to 2014! Elevation, Manifestation and Multiplication!
Posted in Character Building, Community, Life Skills
Tags: Behavior, Bullying, Character education, Community, Education, Facebook, Family, Parenting, School, Twitter, Youth
Got Character?
Developing character can be a challenging process. It is not always easy to do the right thing when everyone else is "doing it". As I mentioned last month, since I have started the Maximizing Character youth program, my own integrity has been challenged. I have been confronted relationally, professionally, spiritually, financially and physically to consistently do the right thing. Don't worry. It's a good thing. I'm up for the challenge.
There are times when we all want to fit in and not necessarily stand up or stand out. In most cases, we want to blend in with the crowd at the sporting event by wearing casual apparel as opposed to a tuxedo or ball gown. Ok, that wasn't a good example, but hopefully you get the point. Kids, for example, want to fit in by wearing the latest designer clothes and shoes or they don't want to be the snitch that tells the school administrator what they know about the latest school crime. For me, starting a program focusing on people's character has been a challenge in and of itself. I'm challenging myself and others to do a self-evaluation of the behaviors and motives that cause us to do what we do. This is not always a popular topic in society. People actually want to hear about the latest "dirt", scandal, celebrity gossip or breakup. Stories that involve a lack of character seem to hold the spotlight more than the good news that peers through the rubbish.
As I watch the news and observe the lack of character in politics, business and entertainment as well as the increase in violent acts—increased bullying, children killing their peers and adults, acts of suicide as a result of being bullied, youth acting out dangerous and even life-threatening scenes from the latest video games or movies, I have to ask myself, what can I do to help reduce the occurrence of these events? Obviously I can't help everyone, but I can create positive change in my sphere of influence. In the past few months I've learned that it is fulfilling to be able to take a stand for what I believe in. I believe it takes a resolute mind and steadfast determination to do what is right, walking in integrity.
As I encourage myself, I also encourage my readers to continue to do the right thing. Don't quit. Don't forfeit the intrinsic benefits that come with possessing good character. Commit to developing a better you, not only the external you that we as a society tend to focus on, but also the internal you, the essence of who you are and want to be in order to make a positive impact in the world. It starts with one. Got character?
Posted in Character Building, Community, Health & Wellness, Life Skills
Tags: Bullying, Clothing, Cyberbullying, Maximizing Character, Sport, Video game, Youth, Youth program
Bullying: Our Experience
October is National Bullying Prevention Month. During the course of this month I hope that readers will take this opportunity to participate in the discussion about this nationwide concern. All across the nation schools, organizations and the media are tackling this topic in hopes of making people aware of its prevalence and to provide strategies to reduce the occurrence of bullying behaviors. Although the issue of bullying has become a national issue in recent years, bullying is not a new issue. It has been going on since the beginning of time. However, in this day and age, bullying has evolved into a form of violence that not only plagues youth and young adults, but it occurs among adults in the workplace and other arenas. Additionally, with the evolution of technological advances, cyber bullying has emerged, causing a whirlwind of problems such as suicide, low self-esteem among youth and other forms of violence.
The issue of bullying is one that I know all too well. Not only have I known individuals and groups who have been affected by this issue, but I was a victim of bullying many years ago. My twin sister and I dealt with bullying during the 70's and 80's. Although times have changed and we did not have to experience cyber bullying, the effects were still the same. Hurt, fear, lack of confidence and low self-esteem are just some of the feelings that come to mind when I think about being bullied.
My sister and I did not deserve the pushing, shoving, name-calling, lies and rumors that took place in an effort to hurt us or provoke fights. I'll never understand what went on in the minds of the elementary and middle school aged students who bullied us. I remember asking the loving adults in my life why this was happening and what had we done to deserve the hateful behaviors and comments. We simply wanted to make friends and go to and from school safely.
I'm sure that there are children (and adults) out there today who shared some of the same feelings I did thirty years ago. Last year one of my sons experienced bullying for the first time as he entered middle school. He recalled being fearful of going to the bus stop and to ride the bus. There were all sorts of "initiations" for the new comers and it scared him. Initially I had no idea that there was a problem. The school year got off to a good start. However as we entered the third or fourth week of school, he began to have asthma attacks and spent several days home from school. For the next month or so he would ask to stay home, citing that his stomach hurt or that he was not feeling well. One day it occurred to me that something was wrong. I had gone through a similar situation with my oldest son and it wasn't until I opened up the lines of communication and asked questions that I was able to find out what was really going on.
My son revealed that kids were hitting others at the bus stop and on the bus. He, and the kids his age, were being told to switch seats and were often being "removed" if they did not get up. My son shared that one of his biggest fears was coming home after school, especially on Fridays, deemed "Freshman Fridays". He shared that the older kids would chase them off of the bus and hit or beat them up as part of the "initiations". I was horrified and it broke my heart to know that my son was experiencing this.
My son's account of what was going on, mainly in my neighborhood at the bus stop, brought back the memories of my own experiences with bullying. I remembered that my mother advocated for my sister and me when we were bullied. She went so far as to go to the police regarding one incident. I truly believe that because of her actions, support, encouragement and positive affirmations, my sister and I are women who are now thriving in life. We have been able to move on, learn from the experiences and help others. That being said, I went straight to the school. We were blessed to receive the prompt support and response of my son's school administration. When my son experienced another potential incident, this time during school, his sixth grade assistant principle stepped in again to squash the situation.
I hope that the bullying awareness and prevention goes beyond the month of October. In the meantime, I urge you to support the various activities, events and discussions that may take place in your community or online. Most of all, I encourage you to stand up for those who are experiencing bullying or may be potentially targeted.


text:
Fireplace Shelving Units How To Style Bookcase Bookshelf Decorating Ideas Do
Wooden bedroom furniture designs mid century modern cupboard ideas new room design with wood furnished catalogue image of box for bedrooms indian homes. Magnificent brown polished bedroom furniture set plus master and dresser also wooden vanity drawer with new room design wood furnished modern images decor designs for home.
Modern Bedroom Cupboard Designs Full Size Of Bedroomsweet Interior Idea In For
- Category : Interior Design
- Added by: Mertise Billie Terilynn, 2017-11-01 23:01:14
- Tags : wooden furniture designs for home | wardrobe designs for bedroom indian | master bedroom wardrobe designs | solid wood bedroom furniture manufacturers | modern wooden furniture images | indian wooden bed designs catalogue pdf | wooden box bed designs catalogue
Wooden Box Bed Designs Catalogue Furniture Design For Living Room Cupboard Bedrooms Indian Homes Photos Best Low Beds Ideas On Pinterest Frame Platform
Woodwork designs for bedroom cupboards stunning home living room furnishing design inspiration establish wooden new with wood furnished colors furniture eo bedrooms light. Wooden bedroom furniture home decorating designs interior design paint colors that go with oak modern cupboard master wardrobe new room wood furnished box catalogue simple. Modern bedroom with wooden floor fully furnished model max light wood furniture decorating ideas latest wardrobe designs for woodwork cupboards new room design photos. Interior design wooden bedroom woodwork designs for cupboards modern furniture to furnish your house ideas new room with wood furnished cheap living wardrobe catalogue box.
Wardrobe designs for bedroom indian modern wooden cheap furniture house pinterest bedrooms diy new room design with wood furnished box image cupboard small interior latest. Elegant interior furniture for small bedroom design ideas with execellent decor apartment brown teak wooden frame cozy studio london room wardrobe catalogue master designs. Indian wooden designs catalogue pdf unique traditional carving king size headboard double mirror cupboard dresser to decorate brown bedroom antique furnishing paint color.<< new bed room design with wood furnished wooden designs pictures interior


text:
Marc Smith, Chief Social Scientist at Connected Action Consulting Group, to Give Lecture on Using Software to Navigate the Sea of Social Networking
We now live in a sea of tweets, posts, blogs, and updates coming from a significant fraction of the people in the connected world. Marc Smith knows that social media can be a bewildering stream of comments, a daunting fire hose of content. However, with better tools and a few key concepts from the social sciences, the social media swarm of favorites, comments, tags, likes, ratings, updates and links can be brought into clearer focus to reveal key people, topics and sub-communities.
WHEN: Tuesday, Nov 15th at 3:30 PM
Abstract> Charting Collections of Connections in Social Media: Creating Maps and Measures with NodeXL
Networks are a data structure commonly found in any social media service that allows populations to author collections of connections. The Social Media Research Foundation's NodeXL project makes analysis of social media networks accessible to most users of the Excel spreadsheet application. With NodeXL, network charts become as easy to create as pie charts. Recent research created by applying the tool to a range of social media networks has already revealed the variations in network structures present in online social spaces. A review of the tool and images of Twitter, flickr, YouTube, Facebook and email networks will be presented.
Bio: Marc Smith is a sociologist specializing in the social organization of online communities and computer mediated interaction. Smith leads the Connected Action consulting group and lives and works in Silicon Valley, California. Smith co-founded and directs the Social Media Research Foundation (http://www.smrfoundation.org/), a non-profit devoted to open tools, data, and scholarship related to social media research.
Smith is the co-editor with Peter Kollock of Communities in Cyberspace (Routledge), a collection of essays exploring the ways identity; interaction and social order develop in online groups. Along with Derek Hansen and Ben Shneiderman, he is the co-author and editor of Analyzing Social Media Networks with NodeXL: Insights from a connected world, from Morgan-Kaufmann which is a guide to mapping connections created through computer-mediated interactions.
Smith's research focuses on computer-mediated collective action: the ways group dynamics change when they take place in and through social cyberspaces. Many "groups" in cyberspace produce public goods and organize themselves in the form of a commons (for related papers see: http://www.connectedaction.net/marc-smith/). Smith's goal is to visualize these social cyberspaces, mapping and measuring their structure, dynamics and life cycles. While at Microsoft Research, he founded the Community Technologies Group and led the development of the "Netscan" web application and data mining engine that allowed researchers studying Usenet newsgroups and related repositories of threaded conversations to get reports on the rates of posting, posters, crossposting, thread length and frequency distributions of activity. He contributes to the open and free NodeXL project (http://nodexl.codeplex.com/) that adds social network analysis features to the familiar Excel spreadsheet. NodeXL enables social network analysis of email, Twitter, Flickr, WWW, Facebook and other network data sets.
The Connected Action consulting group (http://www.connectedaction.net) applies social science methods in general and social network analysis techniques in particular to enterprise and internet social media usage. SNA analysis of data from message boards, blogs, wikis, friend networks, and shared file systems can reveal insights into organizations and processes. Community managers can gain actionable insights into the volumes of community content created in their social media repositories. Mobile social software applications can visualize patterns of association that are otherwise invisible.
Smith received a B.S. in International Area Studies from Drexel University in Philadelphia in 1988, an M.Phil. in social theory from Cambridge University in 1990, and a Ph.D. in Sociology from UCLA in 2001. He is an adjunct lecturer at the College of Information Studies at the University of Maryland. Smith is also a Distinguished Visiting Scholar at the Media-X Program at Stanford University.


text:
Wednesday, April 27, 2016
Tuck Everlasting
This is a sad state of affair when I, who can't be accused of anglophilia, keep wishing that Brits would handle all family shows. The Brits seem to be repelled by sugary sentimentality, and the new Tuck Everlasting could have used a touch of dakrness. My review's here.
No comments:
Subscribe to: Post Comments (Atom)


text:
Poetry Friday: Tumbling Thunder
May 21st, 2010
This week's poem comes from Mark Overmeyer's recent book, What Student Writing Teaches Us: Formative Assessment in the Writing Workshop. In this chapter on feedback as formative assessment, Mark discusses the use of rubrics in poetry study and uses student examples from a sixth-grade class. Here is Renee's poem about "Tumbling Thunder:"
Tumbling Thunder
by Renee
thunder can be strong
a warning
a sign
tumbling around all hope
inviting a great flash
of astounding light
it starts a melody of echo
all around
splitting trees
cry out in fear
of the fury that
will soon strike
when the entire world is calm
and all lamenting stops
you will know
that thunder has shuffled on
the thunder's ramble will
always cease
but the trance
will carry on
Entry Filed under: Poetry Friday
2 Comments Add your own
1. laurasalas | May 21st, 2010 at 1:38 pm
Love this poem, especially
you will know
that thunder has shuffled on
2. Laura Evans | May 21st, 2010 at 3:27 pm
Wow, Renee, is a poet in the making! Nice poem!
Laura Evans
all things poetry
Leave a Comment
Some HTML allowed:
  <acronym title="">
      <q cite=""> <s>
Trackback this post | Subscribe to the comments via RSS Feed


text:
Fullness Factor
Struggling to manage that empty-stomach feeling dieting gives you? Has over-eating ruined your previous diet attempts? Our in-house doctors have developed our Fullness Factor appetite suppressant to assist your weight loss plan more effectively by satiating your hunger.
Fullness Factor is designed to help you manage your hunger between meals so you don't fall off your diet plan due to hunger. These hunger suppressant pills combine soluble fiber to replicate the sensation of fullness with a patented vegetable protein that signals to your brain that you are full.
The Benefits of Fullness Factor:
- Eat less and still feel satisfied sooner and longer
- Helps in controlling portion size
- Reduced hip to waist ratio
- Stimulant and caffeine free
- Can aid with constipation
- Can reduce cholesterol
- Can reduce blood sugar
- Slows the absorption of carbohydrates stabilizing blood sugar
How Does Fullness Factor Work?
Fullness Factor's hunger suppressant pills are intended to be taken about an hour before a meal. As the soluble fiber glucomannan expands and creates a mechanical sensation of fullness in the stomach, the active ingredient, a patented vegetable protein, increases gastric signaling to the brain by promoting CCK, so you'll begin to feel less hungry leading up to your meal. Before and during your meal, Fullness Factor will increase the signals from your stomach to your brain that tell your brain that you're full.
The result? While no two patients experience the exact same effect, you may eat less during meals, feel fuller longer, and lose weight without suffering through the anguish of constant food cravings. Long-term Diet Doc patients often notice that Fullness Factor helps make a reduced calorie diet much more bearable.
Each ingredient in this appetite suppressant helps you boost your weight loss results and achieve your goals safely and consistently. Our doctors designed Fullness Factor by incorporating proven ingredients that work together seamlessly to keep you on track. In addition to appetite suppression, Diet Doc patients taking Fullness Factor have noticed other improvements such as reduced constipation, reduced cholesterol, and lower blood sugar.
Fullness Factor is a patented Slendesta product. Slendesta is a safe and effective weight loss supplement that contains a unique protein called Proteinase inhibitor II (P12). This protein stimulates the natural release of cholecystokinin (CCK), which signals to the brain that the stomach is full. Slendesta is also rich in natural potato protein extract, and studies show that this extract significantly aids with appetite suppression. Fullness Factor also combines Slendesta with a second ingredient: the natural, water-soluble fiber, glucomannan which expands in the digestive tract and creates a mechanical sensation of fullness inside of the stomach. This one-two punch allows dieters to feel less hungry throughout the day, thus eliminating sabotaging cravings and hunger pangs.
The Fullness Factor appetite suppressant is safe for long-term weight loss plans. It contains no harmful ingredients, stimulants, or caffeine, and can be combined with any of Diet Doc's other weight loss aids. And there's no need to worry about purity or quality with Diet Doc products – all of our prescription medication is formulated by a FDA-approved compounding pharmacy in the U.S.
How Do I Get Started with Fullness Factor?
Fullness Factor is only available from Diet Doc - click here to shop it today. Have questions about this supplement or ready to really get moving with your weight loss plans? Give us a ring today, and we'll schedule a complimentary consultation with one of our expert doctors. During your consultation, you'll be evaluated for all underlying factors that have caused your weight to increase or have been making weight loss difficult for you. Call us toll free or send us a message to schedule a free consultation with a leading weight loss doctor today!
Join the 55,000 Diet Doc patients who met their weight loss goals by signing up for your free consultation today.


text:
|| Home | Free Articles for Your Site | Submit an Article | Advertise | Link to Us | Search | Contact Us |
Custom Search
Article Surfing Archive
Easy Distance Off The Tee - Articles Surfing
How can I generate more distance off the tee? Recreational players, it seems, are always looking for golf tips on how to generate more distance off the tee. While proper technique is the true secret to generating distance off the tee, other isolated factors also affect distance. Taking advantage of these factors, as I urge players who take my golf lessons, is an easy way to get more distance without swinging harder or faster.
Loft is one of those isolated factors. Unfortunately, some players who take my golf lessons or read my golf tips don't truly understand the significance of loft. Some players also have a psychological barrier against loft, which is natural given what happens on the course when you hit a tee shot.
Conventional Wisdom versus Reality
Reality in golf is always what it seems. Conventional wisdom suggests that higher-lofted drivers produce shots that fly high and stop dead when they land. Lower-lofted drivers produce shots that fly low but run more when they land, generating more distance off the tee. That theory makes sense, or so it would seem.
The key to maximizing power at any swing speed, however, is launching the ball on the proper trajectory with the right amount of spin to take advantage of the golf ball's aerodynamic properties. That launch angle is generally 10 to 15 degrees of loft (slower swingers can get more distance with a higher loft angle). Ideal spin varies, but it's about 2000 to 3000 revolutions per minute, again a little higher with slower swing speeds.
Loft, it would seem, is a key factor when hitting a driver. In fact, adding loft is the single most important thing I've found in my years of providing golf instruction that helps people hit their drivers better. For some golfers adding loft to their drivers'without making any other adjustments'generates an average of at least 10 yards to their drives. For others, it generates even more.
Determining the Right Loft
So how do you determine what loft is right for you? Pick a driver that gives you as high a trajectory as you can get without sacrificing accuracy. When you feel you're uncomfortably high, take the loft one step down to see if you still are consistently long most of the time. When in doubt, take more loft rather than less.
Once you've determined the right loft, try the following tips for generating more distance of the tee. They'll help you hit the ball farther without changing your swing speed or adding more muscle power.
1. Widen stance at address for greater stability
2. Shift more weight on right side (for a right-hander)
3. Turn our your left foot slightly
4. Take a full 90-degree turn.
5. Hover club head above ground
6. Hit hard with right hand through impact
7. Sense right forearm crossing over left
8. Finish high with your swing
Some of these tips focus on changes in your address position. Hovering the club head above the ground is a technique both Greg Norman and Jack Nicklaus use. It takes a little practice to learn how to do it, but it produces dividends when executed correctly. And finishing correctly is a must on every swing. When combined with the right loft, these tips will add more distance to your drives.
Two Drills for Generating More Distance
Two proven drills for increasing driving distance are the lead hand drill and the weighted-club drill. I often suggest them to players who take my golf lessons or in written golf tips in my newsletter.
1. The lead hand drill strengthens the 'pulling' muscles of your left arm. (Right fore- arm for left-handers.) With this drill, practice hitting a golf ball with just your left hand. Start with a pitching wedge. Try it without a ball first. Swing easily. When you're ready, tee the ball up and swing away. Take about 25 swings. When you can hit the ball off the tee consistently, drop down to an 8 iron. Repeat the process.
2. The weighted-club drill stretches the back muscles, leading to a fuller turn. Use a heavyweight practice club, a practice 'donut,' or a weighted cover for your club. Take about 50 practice swings a day. Swing easily and smoothly. After a month or two, you will find the arc of your swing is larger than before, without losing control of the swing.
Adding more loft to your driver won't make you a Jack Nicklaus or Greg Norman over night; but it will improve your game without involving hours and hours of practice. More importantly, adding loft will help you hit the kinds of drives that can help lower your golf handicap.
Copyright © 1995 - Photius Coutsoukis (All Rights Reserved).
Arts and Crafts
Auto and Trucks
Business and Finance
Cancer Survival
Computers and Internet
Computers and Technology
Education #2
Food and Drink
Food and Drink B
Gadgets and Gizmos
Home Improvement
Home Management
Kids and Teens
Learning Languages
Legal B
Marketing B
Medical Business
Medicines and Remedies
Music and Movies
Online Business
Parenting B
Pets and Animals
Politics and Government
Real Estate
Recreation and Sports
Self Help
Self Improvement
Short Stories
Site Promotion
Travel and Leisure
Travel Part B
Web Development
Wellness, Fitness and Diet
World Affairs
Writing B


text:
Used a heavy duty paint stripper and got down to bare metal, of course some remnants remain after over 25 years sitting on there.
This is the bare material with which I have to work. The condition of the metal is fortunately very excellent, as crisp as was cast. The main body and the arm with the axe are assembled, comprised of a few pieces. These were pinned with epoxy and thick steel paper clips.
Detail is quite nice on the sculpt.
Usually I'm enthusiastic about the build, but in this case I'm really more enthusiastic about the painting involved in such a large piece. Incentive to get it done.


text:
Coming home from holiday was like coming back to Earth with a bump. With a mountain of laundry to do, reorganising our house after the decorators had been and generally getting back to the routines of day to day life it all had me feeling a bit gloomy. Let's face it, not matter what job you do, coming back after a break is often not the most exciting prospect, and I guess being a stay at home mum isn't that different. I'm not even sure how to put it into words other than that I was feeling glum.
But one thing I have learnt as a stay at home mum is that life must go on, even if you are feeling low. The jobs around the house still need doing (though at least they can be delayed) but most importantly your little one still needs entertaining and to be cared for. What's the phrase, no rest for the wicked? No rest for the mummy more like So I plodded my way through the week, doing what I could do plaster a smile on my face. I resumed the morning walks with my lovely neighbour, and that definitely helped. I really find that when I am feeling down, getting out of the house is more important than ever. We all know the feeling of wanting to just curl up at home, but with a little one that isn't really an option and in my opinion, isn't very fair on the monkey, and actually never makes me feel any better.
So when a couple of my other mummy friends suggested taking the kiddies to a local farm and play centre on Friday I ignored my initial reservations and jumped at the chance. Friday came and it was the gloomiest morning of all but I sucked it up and went anyway because I knew I would enjoy it when I was there. And you know what, I really did. I was so glad I went and was able to catch up with my friends. Monkey is still afraid of sheep unfortunately and so we had a very clingy start to the visit, but once we moved into play areas (and after he had some food) he loved it too.
We came home after a few hours and Monkey had a lovely long nap, and after a gloomy few days I felt much happier and much more like myself again. Just being able to chat with my friends really helped and honestly I think it does us all good. Talking to other mummy friends just reminds you that you are not alone and that we all have good days and less good days. We empathise, commiserate and laugh with each other and feel much more refreshed afterwards. I think it helps that we also tire the kiddies out to the point where they have a lovely long sleep when we get home :).
So yes, thank goodness for my other mummy friends


text:
Medulloblastoma is a type of brain tumor. A brain tumor begins when healthy cells in the brain change and grow out of control, forming a mass. A tumor can be cancerous or benign. A cancerous tumor is malignant, meaning it can grow and spread to other parts of the body. A benign tumor means the tumor can grow but will not spread.
The brain and spinal column make up the central nervous system (CNS), where all vital functions are controlled, including thought, speech, and body strength. Medulloblastoma begins in different cells in the cerebellum, which is the back of the brain. The cerebellum controls body movement and coordination.
Relevant Videos
Medulloblastoma Trials
Abstracts and Publications
PLOS Phase 1 Published Paper
A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/ Refractory Neuroblastoma. Download PDF.
Oncotarget LIN28/Let 7 Published Paper
Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma. Download PDF.
ANR 2016 Abstract Book
DFMO maintains remission and increases overall survival in high risk neuroblastoma: results of a phase II prevention trial. PDF.


text: Six Pakistanis among 21 hostages freed by US Navy Seals Posted by: Editor in Top News March 17, 2014 0 958 Views S Navy Seals conducted an operation and freed 21 crew members, six Pakistanis among them, held hostage on a North Korea-flagged vessel in international Mediterranean waters. Pentagon officials said the US commandos raid the vessel, Morning Glory, on the request of Cyprus and Libya and got freed 21 crew members, including six Pakistanis. There were that a North Korea-flagged oil freighter has been seized by some armed Libyan rebels in sea territory of Republic of Cyprus, an island country in the Eastern Mediterranean Sea. Officials said three of the anti-government Libyan rebels were also arrested. They said the Morning Glory will soon head to Libya under the control of a team of US forces. Media reports said the freighter was a North Korean-flagged vessel loaded with 200,000 barrels of oil. There were no further details available about the raid. 0.00 avg. rating (0% score) - 0 votes featured national news pakistan news top news 2014-03-17 Editor tweet