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Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.
Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h−1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).
Clostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.
Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals [1–3]. C. thermocellum forms specialized biofilms for growth on cellulose  and produces mobile  and cell-bound enzyme complexes termed cellulosomes  that mediate deconstruction of lignocellulosic substrates to short, β1,4-linked glucose oligosaccharides for fermentation to ethanol and organic acids. Wild type C. thermocellum fermentation products include acetic acid, lactic acid, formic acid, H2, ethanol, and amino acids such as valine , with additional products being made at high substrate loadings as a result of overflow metabolism .
Organic acid production decreases biofuel yields and acidifies culture medium, each of which are undesirable for an industrial process. Therefore, metabolic engineering efforts have focused on modifying carbon metabolism to decrease production of acidic co-products and increase ethanol production [9–17]. In the highest yielding C. thermocellum strain published to date, strain LL1210, pathways for the synthesis of lactic acid, acetic acid, formic acid, and most H2 production were eliminated , followed by adaptive laboratory evolution to improve growth rate and ethanol titer . This allowed the strain to produce 22 g/L ethanol from 60 g/L cellulose, with a maximum theoretical yield for ethanol of 75% although further advances are required for the bacterium to be industry-ready. Ethanol yields and titers of up to 80% and 38 g/L, respectively, have also been achieved with an engineered C. thermocellum strain in co-culture with an engineered Thermoanaerobacterium saccharolyticum strain . While these strains synthesize essentially no organic acids as end products, fermentation of sugars to reduced compounds such as ethanol or butanol results in the production of a more oxidized compound such as bicarbonate, which will also result in acidification of the medium.
Organisms must be tolerant to the fermentation conditions and the products formed during growth, especially at the high substrate levels required for industrial fermentations. The response to ethanol stress and ethanol-tolerant strains of C. thermocellum have been characterized [19, 20]. C. thermocellum inhibition by pentose sugars  and compounds generated within switchgrass fermentations  has also been characterized. However, deep understanding of the C. thermocellum response to low pH remains underexplored.
While the anaerobic cellulolytic bacterium Fibrobacter succinogenes has been evolved for steady-state growth at pH 5.75 , known anaerobic cellulolytic bacteria typically do not grow at pH values lower than pH 6.0 . High-yielding- and highly productive ethanologens such as Saccharomyces cerevisiae and Zymomonas mobilis, which are not natively cellulolytic, have broad pH ranges (pH 2–6.5  and pH 3.5–7.5 , respectively). C. thermocellum is typically cultured in medium maintained at neutral pH and it maintains its intercellular pH between ~ 7.3–8.5 [24, 25]. For different C. thermocellum strains, growth has been reported between pH 5.9 and 8.1 , as low as pH 6.0 , and between pH 6.2 and 7.7 . The lower pH limit of the only C. thermocellum strain that is currently genetically tractable, strain DSM1313, has not been reported.
A major mechanism of low pH toxicity is dissipation of the proton gradient across the cytoplasmic membrane. Weak acids such as acetic acid become protonated at the low pH in the supernatant, resulting in an uncharged molecule that can cross the cytoplasmic membrane. The cytoplasm is more alkaline, so the weak acid becomes deprotonated, effectively transporting a proton from outside the cell to the inside, collapsing the ∆pH and acidifying the cytoplasm. Cytoplasmic pH can be maintained by a variety of mechanisms that include proton pumps, antiporters, production of ammonia via urease and arginine deiminase pathways, and organic acid decarboxylation reactions that cleave a carboxylic acid side chain (pKa ~ 4.7) to bicarbonate (pKa = 6.4 and more volatile); tolerance to lower pH can be further enhanced by macromolecule repair or protection, lipid changes and biofilm formation [29–31]. Understanding and enhancing the mechanisms by which C. thermocellum responds to decreased pH will be critical for the industrial application of C. thermocellum for lignocellulosic biofuel production, but those mechanisms are not currently known. Therefore, to better understand the physiological response of C. thermocellum to acidic conditions, we cultured strain LL1210 at different pH values in chemostats and applied systems biology tools.
Clostridium thermocellum LL1210 was cultured in duplicate chemostats fed with defined medium (MTC) containing 3 g/L cellobiose for 1058 h (Fig. 1, Additional file 1). After initial batch fermentation, steady-state chemostat growth was achieved for pH 7.0 at a dilution rate of 0.1 h−1. The culture pH was then reduced to pH 6.5 at hour 189.5 and stead-state growth was maintained. Culture pH was checked using a regularly calibrated external probe and reported in Additional file 1. A pH adjustment to pH 6.10 resulted in substantial decreases in cell densities (~ 308 h, Fig. 1), along with lower ethanol concentrations and concomitant increases in cellobiose concentrations (Additional file 1), which suggests the specific growth rate of strain LL1210 was lower than the dilution rate of 0.1 h−1. Medium flow was temporarily halted for 37.5 h and the culture pH set to 6.25, which restored cellobiose consumption, ethanol production and cell density and permitted medium flow to be resumed at dilution rate of 0.1 h−1. After the equivalent of three fermentor vessel volumes, the chemostats were adjusted from pH 6.25 to 6.15, which coincided with lower culture densities. The mean culture turbidity measurements nearest to the omics sampling points, as measured by optical density (OD600 nm) and their standard deviations were 0.655 (0.02), 0.651 (0.04), 0.533 (0.06), and 0.143 (0.05) for pH values pH 7, 6.5, 6.25 and 6.1, respectively. A change in dilution rate to 0.05 h−1 permitted growth at pH 6.10 and 6.00. At pH 5.90, culture turbidity steadily declined; the dilution rate was reduced to 0.01 h−1 and the OD continued to decline for 144 h in one chemostat while the other oscillated, indicative of stress, and eventually recovered. A single chemostat was able to maintain growth at pH 5.90 at a dilution rate of 0.01 h−1, although culture turbidity showed a steady decline at pH 5.80 over a 133 h period and was unable to recover when the flow was stopped for 84 h. Thus, growth-limiting pH for this system is approximately pH 6.25 using strain LL1210 in a chemostat with a dilution rate of 0.1 h−1 and slower dilution rates are required for steady-state growth below this pH.
Samples at dilution rate of 0.1 h−1 were withdrawn from each bioreactor at pH 7 (actual pH = 6.98), pH 6.5 (actual pH = 6.48), pH 6.25 (actual pH = 6.24), and at apparent washout pH 6.15 (actual pH = 6.12) for analysis by transcriptomics and metabolomics (Fig. 1). Additional metabolomics samples were taken at pH 5.80 at a lower dilution rate (0.05 h−1) to help elucidate the physiological changes.
A greater number of differentially expressed genes were observed as the pH diverged further from pH 6.98. Using pH 6.98 as a reference, there were 80, 469, and 536 differentially expressed genes, respectively, for cells growing at pH 6.48, 6.24, and pH 6.12 (Additional file 2). To more easily categorize gene expression responses to pH changes, differentially expressed genes were clustered (Fig. 2a), and then gene clusters were checked for enrichment of gene ontology (GO) terms in comparison to the frequency of GO terms for all genes in the C. thermocellum genome by Fisher’s exact test (Fig. 2b). Cluster 1 contained only one gene (a 2-isopropylmalate synthase; Clo1313_0857) and cluster 3 only contained tRNA genes and were omitted from enriched function tests. Cluster 2 contained genes for rRNA, tRNA, tRNA modification, purine metabolism, and polyamine transport but no enrichment of GO terms was found. Flagella biosynthesis and chemotaxis, sulfate transport and reduction, nitrate transport and nitrogen fixation, glutamate dehydrogenase (GLDH, Clo1313_1847), and fatty acid biosynthesis (clusters 4, 5, 6) were downregulated, while F0F1-ATP synthase genes (cluster 7) were upregulated at pH 6.24. Diguanylate cyclase genes Clo1313_1339, Clo1313_1404 (with TRP repeats), Clo1313_1813 (with putative polar amino acid sensor domains and a phosphodiesterase domain), and Clo1313_2478 (putative response regulator) were all found in cluster 4. Genes for MotAB (Clo1313_0056-7) were part of cluster 6. MotAB system has been show to generate flagellar torque using proton-motive force . Upon being shocked with pH 6.12, cells increased expression of genes for translational machinery (cluster 8), while gene expression decreased for carbohydrate binding and polysaccharide catabolic processing enzymes (cluster 9) including those for cellulosomal proteins CipA, OlpB, Orf2p, OlpA, CelD, CelH, CelR, CelV, CprA, CtManF, LecA, XynA, and XynD (Additional file 2). Cluster 9 also contained a malate shunt gene, phosphoenolpyruvate carboxykinase (PEPCK, Clo1313_0415) . All but one of the glutamate synthase (GOGAT) genes (Clo1313_2032-3, Clo1313_2035-6) were also in cluster 9.
Previous studies have identified genes commonly expressed during acid stress by Gram-positive neutrophilic bacteria , which includes genes for amino acid decarboxylases/antiporter systems, DnaK, GroEL, ClpP protease, urease, F1F0-ATPase, arginine deiminase (ADI), agmatine deiminase (AgDi), and cyclopropane fatty acid (CFA) synthase. Most of these gene systems were not differentially expressed in C. thermocellum and genes for ADI, AgDi, or CFA are not annotated in the genome. Rather than increasing in expression at low pH, several decarboxylases were downregulated at pH 6.24 and 6.12, including aspartate 1-decarboxylase (Clo1313_1318), diaminopimelate decarboxylase (Clo1313_1540), orotidine 5′-phosphate decarboxylase (Clo1313_1266), putative oxaloacetate decarboxylase (Clo1313_1523), and a putative sodium pump decarboxylase γ-subunit (Clo1313_1525). The only decarboxylase that was upregulated at any pH was the S-adenosylmethionine decarboxylase proenzyme (Clo1313_1509), which produces the aminopropyl substrate need for synthesis of the polyamines spermidine and spermine. However, expression of the agmatinase and spermidine synthase (Clo1313_1529-1530) that synthesize the spermidine and spermine were either not differentially expressed or showed decreased expression at lower pH. Then again, genes for a putative spermidine/putrescine ABC transporter (Clo1313_1471-5) were upregulated at pH 6.24 and 6.12 (Additional file 2). Because a polyamine transporter and biosynthetic gene were upregulated, and polyamines can decrease membrane permeability and may protect cells in acid environments , their potential to augment C. thermocellum LL1210 growth at lower pH values was evaluated. Medium was supplemented with 100 μM of the polyamines putrescine, spermidine, spermine, or metabolic precursor arginine and the terminal pH was determined after 144 h. C. thermocellum LL1210 fermentations with and without amendments initiated at pH 7 and 6.75 had similar growth profiles (Additional file 3).
Downregulation of the GLDH (Clo1313_1847) and GOGAT (Clo1313_2032-3, Clo1313_2035-6) genes were observed at pH 6.12, but no significant expression changes were observed for the Type I glutamine synthetase (GS, Clo1313_2031), two Type III glutamine synthetases (Clo1313_1357 and Clo1313_2038), or the glutamate synthetase (Clo1313_1849) (Additional file 2). The type III glutamine synthase (Clo1313_2303) was significantly upregulated at pH 6.24 (Additional file 2). To examine the role of nitrogen metabolism in relation to pH and physiology, deletion strains for GLDH, GOGAT, GS, GS–GOGAT, and an annotated NifH (Clo1313_2339) were characterized using unbuffered (MOPS free) carbon-replete medium (10 g/L) (Additional file 4). Consistent with an earlier study , the parental strain had a higher specific growth rate compared to strain LL1210 (Additional file 4a). The final pH for the GS-mutant- and LL1210 cultures were about pH 6.3 and pH 5.9, respectively, whereas the range for the other strains was ~ pH 5.6–5.8 (Additional file 4b). In the unbuffered system, the GS mutant achieved the highest culture turbidity and produced the second highest titer of ethanol after strain LL1210 (2.0 and 2.7 g/L, respectively) (Additional file 4c). The higher final ethanol titers and pH values for strains GS and LL1210 coincided lower levels of residual substrate.
Genes for two orphan histidine kinases previously predicted to be part of the sporulation cascade in C. thermocellum , a pro-σE processing protease and BofA, inhibitor of the stage IV pro-σK processing protease SpoIVFB, were upregulated in C. thermocellum LL1210 at pH 6.24 compared to 6.98 (Additional file 5). To better understand the role of sporulation in the pH response of C. thermcellum, growth and sporulation of strains LL1210, asporogenous strains M1726 (DSM1313 ∆hpt ∆spo0A1) and M1725 (DSM1313 ∆hpt ∆spo0A1 ∆ldh ∆pta), and the DSM1313 ∆hpt parental strain were examined. C. thermocellum sporulation was inefficient, consistent with an earlier study  and sporulation-deficient mutant strains had neither a growth advantage or disadvantage at lower pH values for the conditions used in this study (Additional file 5).
The molecular chaperone GrpE (Clo1313_0932) and heat shock protein Hsp 20 (Clo1313_0678) were upregulated (2.0- and 2.2-fold) at pH 6.12, which were previously found to be upregulated after furfural shock and heat shock in C. thermocellum ATCC 27405 . Hsp33 (Clo1313_2544) and MutS (Clo1313_1982) were upregulated at pH 6.24 (2.2- and 2.0-fold, respectively). Hsp33 was previously found to be more highly expressed in cellulose-adhered C. thermocellum ATCC 27405 compared to planktonic cells . At pH 6.12, downregulated genes included one of the annotated Clp protease genes (Clo1313_1116) (0.48-fold), an annotated DNA repair proteins RadC (Clo1313_1465) (0.36-fold), and UvrD (Clo1313_2004) (0.31-fold) and a small subunit of an exodeoxyribonuclease VII (Clo1313_1389) (0.50-fold).
To further investigate the physiological changes that occur at lower pH values, metabolomics was performed to compare intercellular concentrations of metabolites when cells were exposed to pH 6.98, 6.48, 6.24, 6.12, and an additional time point at pH 5.80 (at a lower dilution rate 0.05 h−1). Peak areas for 91 metabolites identified by GS–MS were calculated for comparison (Additional file 6) and 39 of these were significantly higher at one or more of the pH values compared to pH 6.98 (Table 1). Most significantly, higher metabolites in cells at pH 6.24 were nitrogen (N)-containing metabolites that included glutamate, lysine, glycine, and five unknown N-metabolites (Table 1). There were also two unknown phosphate (P)-containing metabolites. Glutamate showed the largest differences in average fold change across all samples tested at lower pH values (Table 1). At pH 6.24, cells contained 267 times more glutamate than cells at pH 6.98 (significant α = 0.05). Intracellular concentrations of glutamate ranged from 0.004 μmol/g pellet at pH 6.98 to 1.798 μmol/g pellet at pH 5.80 (Additional file 6). Concentrations of intercellular valine, alanine, and threonine were also quantified on a μmol/g of pellet basis for comparison with glutamate concentrations. While glutamate and alanine peaked at pH 5.80 (2.660 μmol/g of pellet for alanine), valine and threonine peaked at 6.24 with concentrations of 5.312 and 0.267 μmol/g of pellet, respectively. Long-chain fatty acids, their iso-counterparts and glycerol conjugates accumulated at growth-limiting pH values, pH 6.12 and 5.80 (Table 1). Citramalate accumulated at pH 6.24, 6.12, and 5.80 (Table 1) and an accumulation of glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose at pH values 6.12 and 5.80 was consistent with downregulation of genes for major downstream pathways, malate shunt (Clo1313_0415) and the TCA cycle (Clo1313_0708-9) (Additional file 2).
At pH 6.24, only valine, alanine, and threonine extracellular concentrations were significantly higher than when pH was 6.98 (α = 0.05) (Additional file 7). Asparagine was significantly lower at pH 6.24 compared to 6.98 (α = 0.05). Extracellular glutamate was not significantly different at pH 6.24 compared to 6.98, although it was significantly lower at pH 6.48 compared to 6.24 (α = 0.05). When cells were shocked with pH 6.12 compared to when they were growing at pH 6.98, there were significantly lower concentrations of all detectable extracellular amino acids except methionine (α = 0.05). The amino acids that had the highest average extracellular concentrations in chemostats were valine (1.5 mM at pH 6.24), asparagine (147 μM at pH 6.98), and alanine (104 μM at pH 6.24). The highest average concentration of glutamate was 32 μM (near the limit of detection) at pH 6.24 (Additional file 7). An ABC-type branch-chain amino acid transporter gene (Clo1313_0822) was upregulated at pH 6.24 and 6.12 and ABC-type polar amino acid transport genes (Clo1313_0794-5) were upregulated at pH 6.24 (Additional file 2).
To gain a global perspective on metabolic inhibition at growth-limiting pH values, at and below pH 6.24, key mechanisms and responses for pH homeostasis were summarized (Fig. 3). A key feature of the physiological response at lower pH values was changes in expression for use and conservation of ATP. At pH 6.24, LL1210 increased expression of its F1F0-ATPase (Fig. 2). The F1F0-ATPase likely pumps protons to maintain pH homeostasis by ATP hydrolysis as is the case of many other neutrophiles . Competing uses for ATP included valine transport, chaperones protecting cytoplasmic macromolecules from acid damage, and assimilation of ammonium by GS, GOGAT, and GLDH (Table 1, Additional file 2). ATP is also consumed by fatty acid biosynthesis (Table 1, Fig. 2), but there are many metabolites and downregulated genes for this aspect of metabolism. Therefore, it was omitted from Fig. 3, with the exception of biosynthesis pathways for valine and leucine, which are precursors for biosynthesis of iso-fatty acids. The accumulation of fatty acids and iso- derivatives may improve the capacity cytoplasmic membrane to repel protons, but adjustments to membrane lipid composition in response to an increase in proton concentrations are diverse among bacteria and not well understood . Though fatty acid adjustments may have reduced membrane permeability to protons, downregulation of fatty acid biosynthesis genes suggests that ATP conservation was more important. Other gene expression changes that decrease ATP consumption include downregulation of sulfate and nitrate ABC transporter and reduction genes and 80 putative flagella biosynthesis and motility genes (listed in Additional file 2). Reduction of proton influx may also have been achieved through downregulation of gene expression for proton-channeling motility proteins MotAB (Clo1313_0056-7) at pH 6.24 (Fig. 2) . The putative proton-pumping PPi-ase (Clo1313_0823) was constitutively and highly expressed at all pH values. Accumulation of intercellular phosphate at pH 5.80 (Table 1) may have been due to PPi-ase activity, ATP hydrolysis or other cellular activities. Another possibility is that collapse of the membrane gradient would have made PPi hydrolysis to free phosphate much more thermodynamically favorable via the membrane-bound PPi-ase. Both reduction of proton influx through MotAB and proton-pumping through the putative PPi-ase would allow more ATP conservation.
Clostridium thermocellum strain LL1210 has deletions in key genes for metabolic pathways to produce acetate, lactate, formate, and H2, and it has among the highest reported ethanol yields and titers for this bacterium. LL1210 grew at pH 6.24 at a dilution rate of 0.1 h−1. At pH values at or below 6.24, we observed gene expression patterns and metabolite levels consistent with five pH homeostasis mechanisms used by C. thermocellum LL1210, namely (i) proton pumping by F1F0-ATPase, (ii) macromolecule protection by chaperones, and (iii) prevention of proton influx through MotAB were mechanisms supported by changes in gene expression (Fig. 2, Additional file 2); (iv) proton pumping by PPi-ases was supported by metabolomics (Table 1); and (v) changes in membrane fatty acid composition was supported by both techniques (Fig. 2, Additional file 2, Table 1).
Differential expression analysis at pH 6.24 revealed that the F1F0-ATPase genes were upregulated (Fig. 2), consistent with a model that the bacterium uses F1F0-ATPases to pump-protons at the expense of ATP (Fig. 3). Increased expression and activity of F1F0-ATPases are associated with pH tolerance in several other bacteria challenged with sublethal pH conditions [29, 30, 38]. In addition to the F1F0-ATPase system, proton-pumping may be possible via a constitutively and highly expressed putative proton-pumping PPi-ase (Clo1313_0823) (top 9% of genes) [39, 40]. Rhodospirillum rubrum and plant proton-pumping PPi-ases are reversible  and the C. thermocellum proton-pumping PPi-ase has been proposed to be important in energy metabolism in this organism ; further studies are required to experimentally test the function in C. thermocellum. Many ATP-utilizing enzymes and pathways were downregulated at lower pH, which included most of the flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes (Fig. 2, Additional file 2). Genes for sulfate transport/reduction and for nitrogen metabolism have shown differential expression in response to stresses [36, 52] and their roles in maintaining homeostasis and for channeling electrons to augment biofuels productively could better understood through modeling, biochemistry and genetic studies. In this study, clustered and enriched gene ontologies (GO) analyses indicate genes for enzymes using ATP had inverse differential expression patterns compared to F1F0-ATPase genes, which suggests metabolism is redirected to conserve energy (Fig. 2). Indeed, maintaining ∆pH and protecting macromolecules takes precedence over many reactions since critical enzymes for growth cannot function if they are damaged. In addition to a direct burden on ATP, flagella motility may have an indirect burden since protons channeled into the cell through MotAB would need to be pumped back out by to maintain pH homeostasis. Therefore, motAB downregulation at pH 6.24 would support pH homeostasis (Additional file 2, Fig. 3).
Genes involved in polyamine transport and biosynthesis were differentially expressed at lower pH, suggesting a potential functional role for these compounds in pH tolerance. Specifically, the transporter (Clo1313_1471-5) and SAM decarboxylase gene (Clo1313_1509) were upregulated (Additional file 2). Medium supplementation with polyamines or arginine (a polyamine precursor) did not significantly improve growth or enable C. thermocellum to ferment to a lower terminal pH value compared to unsupplemented fermentations (Additional file 3). Instead, a functional role for stabilization of tRNAs during translation  matches well with polyamine biosynthesis and transport gene expression profiles clustering with expression profiles of genes for tRNAs, tRNA modification proteins, rRNA, ribosomal proteins, ribosomal maturation factors, and translational initiation factors (Fig. 2, Additional file 2). Upregulation of translational machinery, tRNAs, and the polyamines that stabilize them, is likely a regulatory response to the abundance of available carbon at pH-limited growth.
Genes involved in sporulation were upregulated at pH 6.24 in chemostat culture. Interestingly, strain LL1210 has a mutation in one of the two copies of the spo0A gene that results in an amino acid change ; Spo0A is generally the master regulator of sporulation, and the role of this mutation has not been established. However, examination of sporulation in different genetic backgrounds at acidic pH fermentation demonstrated that C. thermocellum is inefficient at sporulation, and that LL1210 is similar to the wild type strain. Further, a growth advantage for sporulation mutants in acidic, MOPS-free, carbon-replete medium was not observed (Additional file 5), suggesting that a programmed metabolic shutdown via sporulation is not a major driver of sensitivity to low pH.
Nitrogen metabolism was strongly effected at lower pH values, resulting in increased secretion of amino acids including valine and alanine, which has been seen in other studies [8, 18], and also intracellular accumulation of glutamate with minimal secretion (Table 1, Additional files 5, 7). Some neutrophilic bacteria express glutamate decarboxylase in response to pH stress to convert glutamate into γ-aminobutyric acid; the decarboxylation reaction consumes protons from the cytoplasm which reduces intercellular pH . Heterologous expression of a glutamate decarboxylase in C. thermocellum LL1210 could make use of the accumulating intracellular glutamate, possibly enabling steady-state growth at pH values less than 6.25 with a dilution rate of 0.1 h−1. Glutamate decarboxylases with pH and temperature ranges suitable for C. thermocellum have already been characterized [44, 45], and heterologous expression of glutamate decarboxylase genes with enhanced production of γ-aminobutyric acid in distantly related organisms has been demonstrated . Urease is another target for improvement of pH tolerance in C. thermocellum. Urease activity in neutrophilic bacteria produces ammonia which is protonated intercellularly or exported and protonated to form ammonium thereby raising intracellular or extracellular pH . The urease genes had low expression in LL1210 at all pH values sampled (Additional file 2) in urea-containing growth medium. Previous work has showed that the parental strain (DSM1313 Δhpt) also had low urease expression in urea-containing fermentation medium, but that deletion of the GS gene caused significant upregulation of urease genes . Consistent with higher expression of the urease genes, higher terminal pH was also previously observed for the GS-mutant fermentations compared to the parental strain fermentations . It remains to be determined if higher urease expression increases C. thermocellum’s intracellular pH and enables steady-state growth at pH values below 6.25 with a dilution rate of 0.1 h−1. The GS mutant had less residual cellobiose and glucose in unbuffered medium than LL1210 and other nitrogen assimilation mutant strains tested (Additional file 4). Therefore, deletion of the GS in LL1210 might also improve substrate utilization and reduce or eliminate the need for buffering fermentation media.
Clostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for related C. thermocellum strains. In nature, C. thermocellum may be exposed to acidic pH values, and if it is, metabolism will slow or the bacterium could sporulate, which are not aligned with industrial processes. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.
Clostridium thermocellum strains DSM1313 ∆hpt , LL1210 , M1726 (LL376) , M1725 (LL375) , and AG1329  were used in this study. The GLDH (Clo1313_1847), GOGAT (Clo1313_2032-6), GS–GOGAT (Clo1313_2031-6), and a nifH (Clo1313_2339) strain were deleted in a C. thermocellum DSM1313 ∆hpt background to create AG1327, AG1328, AG2068, and AG2078, respectively. Mutants were constructed using the procedures described in detail [49, 50]. Strains are available upon request. Primers used to construct strain AG2078 and other strains used in this study as well as SNPs identified from genome sequencing are provided in Additional file 8. Strains were stored frozen at − 80 °C in a modified version of DSM 122 medium, referred to as CTFUD , that contains 50 mM of MOPS (morpholinepropanesulfonic acid) and 10 mM of sodium citrate. Actively growing cultures were transferred twice from freezer stocks prior to batch fermentation experiments and inocula were 2% of the final volume. Cells were routinely cultured at 55 °C in either 50 ml (160 ml serum bottles) or 10 ml (26 ml Balch tubes) of MTC medium as described earlier , except that MOPS was omitted in this study. The LL1210 strain was also cultured in 1 ml volumes in a 48-well plate reader (Biotek Eon, Winooski, VT, USA) inside a Coy anaerobic chamber kept at 55 °C with growth data collected at OD600 nm every 15 min. Growth in MTC-containing 100 μM of spermine (AC132750010, Fisher Scientific, Hampton, NH, USA), spermidine (S0266, Sigma-Aldrich, St. Louis, MO, USA), putrescine (AC112120250, Fisher Scientific) or arginine (A5006, Sigma-Aldrich) was assessed in plate reader assays.
Strain LL1210 was grown at 55 °C in duplicate 750 ml (total vessel capacity 1.3 L) chemostat cultures using water-jacketed BioFlo110 bioreactors (New Brunswick Scientific, Edison, NJ, USA), essentially as described previously in another C. thermocellum chemostat study , except that the medium contained 3 g/L of cellobiose as the carbon source. Briefly, the MTC medium was MOPS free and fed at a dilution rate of 0.1 h−1, except where noted, for 1058 h. Temperature, pH, and agitation speed were monitored and controlled during fermentation using the BioCommand software (version 2.62). Gel-reference pH electrodes Mettler-Toledo (Woburn, MA) calibration, accuracy checks and pH control were maintained as described earlier , except that 1 N HCl and 3 N KOH were used for acid and base additions, respectively. Recalibration of the bioreactor probes was performed at 546 h after pH drifted by 0.08 and 0.12. Samples for omics were taken in 50 ml volumes; then harvested and stored as described in . For transcriptomics, two samples were taken from different chemostats at pH 6.48 and 6.12, and for pH values 6.98 and 6.24 biological duplicates and two technical replicates separated by at least one change of vessel medium were collected. For intracellular metabolomics, samples were taken immediately after transcriptomic samples, and additional biological duplicate samples were taken at pH 5.8 for metabolomics only. Culture turbidity was measured taking optical density readings at 600 nm using a Genesys 20 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Cells were enumerated using a modified version of the direct cell counting procedure described in . Briefly, 1 ml of culture samples were preserved for up to 1 week in 0.025 mg/ml (final concentration) paraformaldehyde (Sigma-Aldrich,). Cells were diluted 1:1000 with 0.9% (m/v) saline solution and stained with 25 nM 4′,6-diamidino-2-phenylindole (DAPI) (final concentration), immobilized to a 1.5 cm diameter section of a black MilliQ filter, and then imaged with a Zeiss Axio Imager.M1 with a Plan Apochromat 63×/1.4 Oil Immersion objective and X-Cite Series 120 Exfo fluorescence power source (Carl Zeiss, Germany). 8-bit images were imported into ImageJ (http://imagej.nih.gov/ij/) for manual or automated counting. Manual counting was performed with the cell counter plugin, and automated counting was performed with JAVA scripts (Additional file 1) which assumed cells were visible at a % Area (saturation threshold) of 1% (± 0.1%) with a particle size greater than 5 pixels after binary images had been processed with Watershed. Spherical morphologies were assumed to have a particle size range of 20–200 pixels and circularity of 0.85–1.00 for conservative estimates, and a particle size range of 50–100 pixels and circularity of 0.95–1.00 for liberal estimates. Automated parameters were validated with manual counts of 24 images. Stepwise calculations of standard deviation of progressively more sample counts were performed to ensure the count variance plateaued. Highly refractive mature spores were also observed by light microscopy after heat fixing to 20 μl of stationary cells to glass slides.
Extracellular fermentation product samples for high-performance liquid chromatography (HPLC) were collected and processed as described in . HPLC data were generated using a Shimadzu Prominence LC-20A Series system (Shimadzu Scientific Instruments, Columbia, MD) fitted with a refractive index detector (model RID-20A) and an Aminex HPX 87H HPLC column (300 × 7.8 mm) (Bio-Rad, Hercules, Dallas, TX, USA) against known standards for cellobiose, glucose, ethanol, acetate, lactate, and formate. Intracellular metabolite fold changes and select metabolite concentrations were determined using a 5975C inert XL gas chromatography–mass spectrometer (Agilent, Santa Clara, CA) . Extracellular amino acid concentrations were quantified by an Aracus Amino Acid Analyzer (membraPure, Berlin, Germany) . Two-tailed paired T tests were performed to determine if amino acid concentrations were significantly different. Homoscedasticity was confirmed by performing Breusch–Pagan and Abridged White’s tests prior to performing T tests.
Total RNA was isolated from 50 ml of chemostat sample using the procedures described in , and quantity and quality were determined using a Nanodrop instrument (ThermoFisher Scientific Waltham, MA). Depletion, library preparation and sequencing were completed at the Joint Genome Institute (JGI, Walnut Creek, CA). In short, 100 ng of total RNA was depleted of ribosomal material using a Ribo-Zero (TM) rRNA Removal Kit (Epicentre). The rRNA depleted RNA was fragmented and reversed transcribed using random hexamers and SSII (Invitrogen) followed by second strand synthesis. Stranded cDNA libraries were generated using the Illumina Truseq Stranded RNA LT kit, following the manufactures protocols, using ten cycles of PCR. qPCR was used to determine the concentration of the libraries. Libraries were pooled and sequenced on an Illumina HiSeq 2500 platform with version 2 chemistry in a 2 × 151 bp configuration (JGI, Walnut Creek, CA; Project ID 503106). Fastq files were verified for integrity by checksum and read quality checked using FastQC . RNAseq reads were trimmed using Trimmomatic MAXINFO method with target length and strictness parameters set to 40 and 0.8, respectively . Reads were mapped to C. thermocellum DSM1313 (NC_017304, last modified 2015/07/30) using Bowtie2 with the same parameters as the very-sensitive preset option expect the number of mismatches was set to 1 [57, 58], and reads were counted with HTSeq . Principle component analysis (PCA) was used to check for sample variation using JMP Genomics 8 (SAS Institute, Cary, NC, USA). Differential expression analysis was performed with DESeq 2 . Genes were considered significantly differential expressed when their adjusted p values were less than or equal to 0.05 and log2-fold change values were ≥ 1 or ≤ 1. Raw RNA-seq data have been deposited in NCBI SRA under project number PRJNA395926. PCA plots, raw counts, RPKM normalized gene counts, and differential expression are provided in Additional file 2. Genes found to be differentially expressed at pH 6.48, 6.23, and pH 6.12 compared to pH 6.98 were clustered by their expression patterns using the K-means cluster procedure in JMP Genomics 8. Fisher’s exact test was then performed on 7 of the 9 K-means clusters to identify enriched GO terms. Prior to this analysis, GO terms were identified for the entire C. thermocellum DSM1313 genome using the standard workflow in Blast2GO 4.1.5 .
JMW designed and conducted experiments, analyzed data and wrote the manuscript. J-WM contributed to chemostat cultivation and sampling. MRJr contributed to chemostat cultivation and sampling, and HPLC analysis. NLE generated metabolomics data. DMK extracted RNA. TR generated and analyzed extracellular amino acid data and constructed the nifH mutant. MMA contributed nifH mutant construction. TJT analyzed metabolomics data. AMG oversaw nifH mutant construction, assisted with data interpretation and manuscript preparation. SDB designed experiments, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
This work is supported by the BioEnergy Science Center (BESC), which is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. ORNL is managed by UT-Battelle, LLC, Oak Ridge, TN, USA, for the DOE under contract DE-AC05-00OR22725. RNA-Seq data were generated by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231.
The manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the US Department of Energy. The funders had no role in study design, data collection and interpretation, preparation of the manuscript, or the decision to submit the work for publication. The publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).
This work is supported by the BioEnergy Science Center (BESC), which is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. ORNL is managed by UT-Battelle, LLC, Oak Ridge, TN, USA, for the DOE under contract DE-AC05-00OR22725.
13068_2018_1095_MOESM1_ESM.xlsx Additional file 1. Data logs, substrate and product concentrations, internal and external pH readings, and media formulation for chemostat reactor cultures of C. thermocellum LL1210.
13068_2018_1095_MOESM2_ESM.xlsx Additional file 2. Raw and processed read counts, alignment statistics, log2-fold changes in the gene expression, and K-means clusters and GO enrichment of differentially expressed genes from samples taken from C. thermocellum LL1210 cultured in chemostats at pH values 6.98, 6.48, pH 6.24, and pH 6.12 (washout conditions). Gene expression at pH 6.98 was used as a reference for differential expression at lower pH values.
13068_2018_1095_MOESM3_ESM.docx Additional file 3: Figure S1. Average optical density at wave-length 600nm (A and C) and average terminal pH (B and D) of C. thermocellum LL1210 cultured in 48-well plates. OD600nm readings were taken automatically every 15 min in a microplate spectrophotometer (Biotek Eon, Winooski, VT) kept in an anaerobic chamber. Only 3-h time points are shown. Nine hundred microliters of inoculated medium was mixed with 100 μl of uninoculated medium supplemented with spermine, spermidine, or putrescine (polyamines), or arginine (polyamine precursor) so that the final concentration was 100 μM. Initial culture pH was 7.00 (A and B) or 6.75 (C and D). Averages were calculated from at least three biological replicates. Error bars indicate standard deviation and are colored the same as the amendments in the legend. Table S1. Average and standard deviation of maximum and terminal optical densities (600nm) and specific growth rate of C. thermocellum LL1210 cultured in media with and without amendments and having initial pHs of 7.00 and 6.75.
13068_2018_1095_MOESM4_ESM.docx Additional file 4: Figure S2. Average growth (A), terminal pH (B), and remaining substrates and products at the end of C. thermocellum-mutant strain fermentations of cellobiose in MOPS-free carbon-replete medium (C). Averages were computed with data from four biological replicates. Error bars in each graph indicate standard deviation. Some error bars are too small to see. Deletion mutants are designated as LL1210 (hydrogenase maturation protein, lactate dehydrogenase, pyruvate formate lyase, phosphotransacetylase and acetate kinase), GLDH (glutamate dehydrogenase), GS (glutamine synthetase), GOGAT (glutamate synthase), GS-GOGAT (both), and NifH (nitrogenase iron protein). The parental strain designated DSM1313 has a deletion in the hypoxanthine phosphoribosyltransferase.
13068_2018_1095_MOESM5_ESM.docx Additional file 5: Figure S3. Differential expression of genes found in Clostridia sporulation cascades. pro-σE processing protease is a stage III sporulation factor. BofA is an inhibitor of the stage IV pro-σK processing protease SpoIVFB. Table S2. Percentage of spherical morphologies 144 and 216 h after inoculation. Figure S4. Substrates and products (A) and the pH (B) after 144 and 216 h of C. thermocellum-mutant fermentations on MOPS-free carbon-replete medium starting with an initial pH of 6.75. Significant differences at α = 0.001 for comparisons with DSM1313 (∆hpt) are indicated with a “*” and comparisons with DSM1313 (∆hpt) and LL1210 are indicated with “**”. Averages were calculated with six biological replicates. Error bars indicate standard deviation.
13068_2018_1095_MOESM6_ESM.xlsx Additional file 6: Table S3. Fold changes of intercellular metabolite there were significantly higher or lower in concentration at growth-limiting pHs.
13068_2018_1095_MOESM7_ESM.xlsx Additional file 7. Extracellular amino acid concentrations in media from C. thermocellum LL1210 chemostats that were sampled when pH values were pH 6.48, pH 6.24, pH 6.12, and below. Demonstrations of data homoscedasticity for T tests.
13068_2018_1095_MOESM8_ESM.docx Additional file 8: Table S4. Strains and primers used in this study.
Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010. http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Accessed 5 Feb 2016.

industry_corpus2: Biopharmaceuticals


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Ultra modern design, while still keeping comfort in mind. Featuring spacious seating and 3 adjustable backrests.
Quality, steel mechanism with several different postions.
The Newport is on display in our showroom. You are welcome to pop in and try it out for comfort, and build quality anytime during our business hours. We also have all of the fabric and leather samples available, and if you require, we can give you a cutting to take home.

industry_corpus2: Manufacturing


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Available for Citroen Xsara 2001 onwards in h7-twin-pack fitting, the Ring H7 Long Life Headlight Bulbs lasts up to 3 times longer than standard H7 bulbs, and are the cost effective solution for high mileage drivers seeking ultimate bulb reliability.
H7 bulbs are single filament, compact in size and high in power, this means normal H7 bulbs can be more susceptible to early failure.
The Ring Long Life H7 is a standard Halogen 55w H7 fitting but benefits from stronger construction methods to bear out the manufacturers claim of lasting 3 times longer than standard bulbs.
These bulbs are sold in a pack of 2.
As with all our products, Care4car Guarantees this item for 12 months from the date of order.

industry_corpus2: Automobiles


text: Hi there! Welcome to another video tutorial brought to you by iSenseLabs in cooperation with OpenCart. This time you’re gonna learn one of the most basic parts of running an OpenCart store. Installing modules. It’s really easy. After you download your module either from the OpenCart marketplace or a third-party vendor, you usually have a ZIP with all the extension files inside. The one you need is the OCMOD dot zip file. This is the module you need to upload to your store. So, let’s show you how! Go to your OpenCart administration and click on Extensions and then on Installer. This is where you manage your module uploads. You have your Install history where you can see all the modules you’ve uploaded, with an option to delete the files using this button. Let’s upload an extension now. We’ve downloaded NitroPack and we’re gonna install it in our store now. Click the Upload button and select the ocmod file. Upload it and wait for the progress bar to finish loading. When it’s done, it means your module files are now uploaded in your OpenCart store. The next step is to go to your Modifications. In OpenCart 3, not all modules come with modifications, due to the new Event system. Still, there are still some extensions that use modifications, so if you’ve installed one of them, you need to hit the Refresh button here. Okay, now your module is ready to be configured. Go to extensions. The dropdown here is used to direct you to the type of extension you just installed. For example, it can be a new shipping or payment method. In our case, it’s a module, so we click on modules. Find the extension you just uploaded and hit the Install button here. After that, hit Edit and enable your new extension. Now you just need to enter your license to receive support and extension updates. You’re now ready to configure the settings and start using your new OpenCart module! So, that’s basically how to install extensions in your store. A helpful tip before we conclude - be careful with the modules you install, especially the free and cheapest ones that you can find in the OpenCart marketplace. Do your research on the developer and make sure that you’re using quality extensions. That’s it for today! Give us a like if this was helpful and subscribe to our channel for more OpenCart 3 tutorials! Thanks for watching!

industry_corpus2: Computing & Telecommunications


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Max White, BCom, writes that he’s now working full time in music. He plays in the underground Montreal rock bands Light Bulb Alley and the Count Ferrara. He also co-founded the psychedelic blues punk duo, the White Brothers Band, with his brother Jackson. They made their debut at the Horseshoe Tavern in Toronto in July 2018. Max says he’s working on a number of new projects including the White Brothers Band’s debut album, and Light Bulb Alley’s third LP. In addition to these records, he contributed sounds for the soon-to-be released Netflix series V-Wars.
Mark Steenhof, MBA, is director of structural engineering at Steenhof Building Services Group in Orillia, Ont. The firm provides residential and commercial engineering services, specializing in modular building engineering.
Steve Camacho, EMBAA, was appointed chief information officer for the Toronto Catholic District School Board in March. Prior to this, Steve was director of technology services for the Institute of Technology and Advanced Learning at Humber College in Toronto.
Laird Choi, EMBAA, and Lulu Fan, EMBAA, have launched Allosentry, an app to help reunite parents and caregivers with their lost children, using crowdsearch methods.
Denisse Cieza, MIB, is working in Calgary as a Trade Marketing Account Representative for Imperial Tobacco Canada. She manages business accounts in Calgary, working as a strategic partner on product knowledge, pricing and distribution. Prior to this, Denisse completed a summer internship at Imperial Tobacco head office in Montreal in the area of trade marketing optimization.
Emmanuel Fombu, EMBAA, was asked to serve as an external advisory board member on the Massachusetts Institute of Technology’s MIT.nano project. He just published a rationale and design paper on the first-ever study using remote devices to measure physical activity and sleep in heart-failure patients. He was also selected as a finalist in the New York City Health Business Leaders 2017 Boldest Digital Health Influencer Awards.

industry_corpus2: Film & Entertainment


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Red morocco leather, high heels.
These women's high-heel shoes in a refined style are made of morocco leather. In the 18th century, women's shoes for dressing up were often made of the same gorgeous silk fabric as their robes. These, however, are an example of high quality morocco (tanned goat leather), used for luxury shoes, gloves, bookbindings, and furniture coverings.

industry_corpus2: Others


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Until the early 1990s, handcuffs linked both wrists by a short metal chain, but apart from restricting arm movements, they offered little else in terms of restraint, and if only one wrist were attached to them, the handcuffs could quickly become a flail-like weapon. Rigid handcuffs, such as Kwik Cuffs, were first trialed in 1993 and have since become standard issue in the United Kingdom and the United States. In Australia, there is a mixed use of chain-link and fixed-link handcuffs.
Although the ratchet mechanism is the same as with the older cuffs, the fixed joint between the cuffs gives several distinct advantages. Holding the fixed joint allows easy application because simple pressure against the wrist enables the single bar to release over the wrist and engage the ratchet. The ratchet can be locked to prevent further tightening but can also only be released with the key, which requires the detainee to cooperate by keeping still. If the cuffs are not locked, then progressive tightening can occur. Correctly tightened cuffs should just have enough space for an additional finger between the applied cuff and wrist. The hands are usually cuffed behind the back one above the other, because handcuffing to the front may provide opportunities to resist detention.
Even with only one wrist in the cuffs, control by the officer can be gained by essentially using the free cuff and rigid link as a lever to apply local painful pressure to the restrained wrist. Techniques allow a detainee to be brought to the ground in a controlled manner or the other wrist to be put within the cuffs.
A gentle application, such as may be experienced by the forensic physician in a personal trial, will demonstrate that it is clearly an effective way of gaining control of most individuals. This may not be the case in those who are intoxicated, have mental health issues, or are violent. Cuffs should fit firmly but not tightly at the narrowest part of the wrist just distal to the radial and ulna sty-loid processes.

industry_corpus2: Law & Justice


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Join us for Playdough Palooza at the Pleasant Hill Library. We will provide the materials for a fun and creative time at this family event, no matter your age!
No registration is required. Call the library at 937.676.2731 for more information.
The library is located at 12 North Main Street, Pleasant Hill, OH.

industry_corpus2: Subject Education


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How did you swing Jabong your way against formidable suitors for Jabong like Snapdeal and Future Group?
I believe that across Myntra and Jabong, between two lifestyle and fashion destinations, we can really shape the market. In some way it makes a lot of logical sense and then, Flipkart as a group, in any case dominate fashion. In all deals the real question is where is the value creation? In my mind the value creation is the ability to shape the fashion and lifestyle market and to see how we get our 15 million monthly customers across Myntra and Jabong really shop differently for fashion.
What changed at the last moment as others were also in the fray?
I cannot really comment and all I would say that both Kinnevik, others saw value with Myntra. I think it is a good deal. I think we gain, they gain and it is always the best about any deal. (In) Any good deal, both sides gain. That is what really changed in my mind and that is fair deal for all of us.
There are some sceptical voices and they think Jabong as a brand is going to get killed with this acquisition.
You have some strengths and Jabong has some. What are the strengths that Jabong brings to the table?
Are there territories where you are strong and some where Jabong is?
You have been in and out of talks to acquire Jabong and others were considered front runners until recently.
Look these are conversations and they evolve and all of us saw real value in the deal. Therefore we decided to do the deal. I would call it the real 800 pound gorilla of fashion and lifestyle in the making and that is what we are excited as a group.
What does it mean for the competition, be it Amazon or upcoming Alibaba or Rakuten?
Across the group we are fairly dominant in the online fashion space among Flipkart, Myntra and Jabong. I don’t think of competition and we think of consumers. What we can offer to consumers better through this. Now we can offer more choice, more variety and continued fabulous consumer experience.
Will it go through any competition commission or any other regulatory hurdle?
I don’t see any competition hurdle here. I think fashion and lifestyle is a very, very, fragmented market. So I don’t think there is a competition commission issue here. We have to shape the market as we still are a very small percent of the overall pie. If you look globally, no particular fashion destination actually dominates anywhere because the tastes among people are dramatically different. So each brand offers something unique and different.


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the gardiner's dirt: Oh Birmingham!
On our travels through the deep south we hit up Birmingham next. We had only intended on breezing through. We had a quick bite at a Jimmy Johns then headed over to Vulcan Park.
The girls enjoyed cruising through the museum, pressing pennies, and climbing to the top of Vulcan Tower. Well sort of, we learned that Blondes is deathly afraid of heights and had quite the panic attack at the top. MaeMae and Roo on the other hand were running around it giggling and chasing each other as if their feet were still flat on the ground.
On our way back to the car, Roo followed right behind her sisters walking on a 3ft rock wall when BAM! She went down. Easy had Rosie and I bolted to my screaming 2 toddler, scooped her up, and assessed the damage. A mouth full of blood and a gash in her lip too deep for my comfort. I pressed her dress on it hard as we thought of what to do. Easy insisted that we "just get it fixed right," and so we all took off down the hill. Luckily the Children's Hospital was right around the corner. I held her in my lap and she insisted that her dress was kept pressed to her lip.
I really didn't think I'd be back in a hospital this quick!! Poor girl was so patient and good though. We waited and waited. Easy in the car with the 2 olders + a screaming Rosie and I with Roo.
We switched off so I could feed the baby, we fought with the hospital security about parking, gassed up the car, and the girls watched movies while waiting.
Roo was alert through the whole thing and watched like a deer in the headlights as the tiny needle pulled the string through her lip. Unfortunately the resident that stitched it was a newbie and it had to be done twice. I was slightly irritated. They told me to try to keep it dry since the stitches would dissolve.
So we got a hotel and crashed in Birmingham.
With our 2 little stitches.
And this time with 2 beds in the room.
Our other trip to get stitched here.
Ah! That is kind of a trauma and reminds me of when Mae crashed and burned at the community center. Your ladies have a thing for split lips.

industry_corpus2: Travel & Geography


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Description : Clarke and Finn are captured by Anya, who forces them to save the life of a victim of the explosion. Meanwhile, Raven deals with her split from Finn, and Kane fights to save the Ark and it's survivors.
The Calm (Episode 11) is a Drama, Mystery, Sci-Fi. movie released in .
The Calm (Episode 11) Season 01 download in HINDI in our website moviesyug.net easly with NOT AVAILABLE subtitles.
The Calm (Episode 11) is avilable for download in two part of mp4 formate and full hd format with smaple files for instant quality check.


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Colour Consultations focus on choosing the correct colour palette for your home, including accents, features and finishes. From this meeting you’ll receive a full Colour Schedule to provide to your painter for implementation.
Only available local to Aurora or Collingwood, Ontario.

industry_corpus2: Real Estate & Construction


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Opinion: Celebrating National Good Neighbor Day | Tenleytown, D.C.
It may not be a major holiday or even appear on your calendar, but to me September 28, National Good Neighbor Day is one of the most important holidays to celebrate. The day is a reminder of the importance of communities, large and small, and the simple, but generous act of being kind to those who live around you. Today’s world moves at lightning speed, with people rushing past each other on their way to work, meetings, children’s activities, or a myriad other pressing events. Our connected lives means more often than not we’re staring at a screen rather than the person who might be right next to us. It takes a concerted effort to slow down, look up, and engage with our communities.
In his 1978 proclamation establishing Good Neighbor Day, President Jimmy Carter stated: “Understanding, love and respect build cohesive families and communities. The same bonds cement our Nation and the nations of the world. For most of us, this sense of community is nurtured and expressed in our neighborhoods where we give each other an opportunity to share and feel part of a larger family.” At the root of the founding of this website two years ago, was a desire to connect more with Tenleytown and learn more about the people and places at the heart of the neighborhood. In the process of writing Tenleytown, D.C. I have had the pleasure of meeting residents who are making great contributions to the community, whether as members of community groups, local government representatives, business owners, leaders of local events or simply individual citizens offering assistance and or a friendly word each day.
Just on my block alone I have frequent reminders of what it means to be a good neighbor. From the retirees who live next door and, no matter how early I go out on a snowy day, have already helped clear my walk, to the couple across the street who keeps an eye out for my son whenever he’s playing outside. From the neighbors on the block who have welcomed us to open houses and organized block parties, to my own husband who helped the woman up the way when she accidentally locked herself in an upstairs room the day she moved into her home. In ways big and small, these neighbors make me feel part of an extended family.
The one event, though, that has crystallized the true meaning of community for me was the outpouring of support for Wilson High School when it was targeted by a vile hate group in June 2014. To see hundreds gather in front of the school with messages of love and solidarity was to witness the very essence of what it means to love thy neighbor.
Amy Carden Suardi, author of the blog Frugal Mama and a member of our greater Tenleytown community, writes eloquently about why we need strong, nurturing communities, drawing upon her own experiences building relationships at home, in her neighborhood, and even overseas. “Children prosper if the context where they grow up — their family, school, peer group, and larger community — have relationships of trust and cooperation. Neighborhoods with high levels of social capital are safer, cleaner, and friendlier. And the democracy that our country was built upon depends on our participation, trust, and solidarity.” If you haven’t read Amy’s blog before, you should definitely do so. While many of her posts are about making the most out of life while also living frugally, to me her blog is really about living authentically. And being a good neighbor is part of her creed.
when I really am pressed for time, giving a hearty wave, a smile, and a promise to catch up later.
None of these are earth-shattering ideas, just simple ways to say hello and that you care. It doesn’t matter so much what you do. It’s the intent behind it – to connect with those who live around us.
I love this post and how you show that a neighborhood can become an extension of one’s family and home: nurturing, friendly, and safe. I loved hearing more about the Wilson counter-protest. Thank you for putting the focus on communities, and thank you for writing this blog! Knowing more about our community is a big piece toward feeling one with it.


text: For those who’ve attended any occasions sponsored by the Art Deco Society of California, you recognize that dressing up in deco type is a part of the fun, and a preservation activity in its personal right. Get digicam-prepared for Style Week 2017. NYC shall be stuffed with a stampede of posh editors, bloggers, stylists and shutterbugs. (They will be invading your favourite espresso retailers and cafes. You’ve got been warned). Tickets to a few of the main runway exhibits aren’t accessible to most people, but newsflash: you do not have to be a part of the elite style world to really feel like an insider. Possibly you do not have a front-row seat to the exhibits or a spot reserved next to Anna Wintour, however do not fret—we’ve got you lined. From free New York Trend Week occasions you possibly can really attend to tips on how one can get observed by avenue type photographers, we’ll ensure to maintain you within the loop. Ensure to e book an appointment at top-of-the-line hair salons NYC has to supply and hit the perfect clothing stores and plan out your most fashionable outfits for the week—you by no means know who is likely to be taking your image.


text: At Riverbank Motor Company we aim to bring you the widest choice of used cars in Greenock, Renfrewshire and at great prices. We have some fantastic deals on used BMW cars, which are always changing. Browse our used cars below and contact us for more information on any of our second hand cars.


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Coasters are available in bulk within a cost-effective wholesale price for people and companies. With wholesale coasters can help to promote business products & items economically. Are you looking for bulk coasters? Discount Designing & Printing certainly the best choice for those who want to promote their valuable products and save their bucks. By making use of prestigious graphic design tools, we create the best designs for your wholesale custom coasters in a stylish way. If you require any sort of modification, then you do not need to bother because we create coasters for your fulfillment. Our customized wholesale coasters look very suitable and dynamic to satisfy your specific business requirements. You could promote as many products & accessories by way of our personalized wholesale coasters as you want. We are pleased to provide cheap wholesale coasters along with free design service and free lamination (gloss & matte finish) for cardboard wholesale coasters.
Discount Designing & Printing believes in absolute professionalism, honesty, dedication and commitment.
We would certainly like to fulfill the desires of our valued customers by giving them discounted coasters along with free delivery at your door-step.
Remember that we provide unique, original, topnotch quality and full color retail and wholesale coasters for our clients in USA and Canada (Now offering our services in Australia).


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GvHD is a possible complication of a bone marrow or stem cell transplant from another person.
A bone marrow or stem cell transplant from another person is called a donor or an allogeneic transplant.
Bone marrow and stem cell transplants are treatments that use very high doses of chemotherapy, sometimes with radiotherapy. The high dose treatment can kill cancer cells but it also kills off the stem cells in your bone marrow that produce your blood cells.
So after the high dose treatment you have stem cells or bone marrow from your donor through a drip. The donor stem cells make their way into your bone marrow and start to produce blood cells again.
GvHD means the graft reacts against the host. The graft is the donated marrow or stem cells. The host is the person receiving the transplant. GvHD can cause diarrhoea, skin rashes and liver damage.
GvHD happens when particular types of white blood cell (T cells) in the donated bone marrow or stem cells attack your own body cells. This happens because the donated cells (the graft) see your body cells (the host) as foreign and attack them.
Some people have a very mild form which doesn’t last long. For others, GvHD can be severe. It may even be life threatening in a few cases. Some people may have GvHD over many months, or even years.
GvHD happens because the transplant affects your immune system.
The donor's bone marrow or stem cells will contain some T cells. T cells are a type of white blood cell that help us fight infections. T cells attack and destroy cells they see as foreign, and potentially harmful such as viruses.
Normally T cells don’t attack our own body cells, because they recognise proteins on the cells called HLA (human leukocyte antigens). We inherit our HLA from our parents. Apart from identical twins, HLA is unique to each person.
Before a bone marrow or stem cell transplant, you and your donor have blood tests to check how closely your HLA matches. This test is called tissue typing. If you and your donor have very similar HLA this lowers the chance of GvHD. The more differences there are between your HLA and your donor's, the more likely you are to get GvHD.
After a transplant your bone marrow starts making new blood cells from the donor stem cells. These new blood cells have the donor's HLA pattern. They recognise the HLA pattern on your body cells as different (foreign) and may begin to attack some of them.
The GvHD may affect different areas of your body. Most commonly it affects the skin, digestive system (including the bowel and stomach) or the liver.
Acute GvHD generally starts within 100 days of your transplant but it can sometimes happen after this time. It usually happens about 2 to 3 weeks after your transplant when your new bone marrow begins to make blood cells. This is called engraftment.
Chronic GvHD starts more than 100 days after your transplant. You are more likely to get it if you have had acute GvHD but it can happen even if you haven't. It can be mild or severe, and for some people can go on for several months or even years.
They are both more likely to happen after mini transplants (reduced intensity conditioning). Nowadays, doctors use mini transplants more often.
It is difficult to say who will develop GvHD after a transplant. We don’t know exactly, but somewhere between 1 and 4 out of every 5 people (20 to 80%) having a donor transplant will develop some degree of GvHD.
The symptoms of GvHD can be difficult to cope with. In some cases GvHD is a serious, even life threatening, problem.
But having mild GvHD could be a good thing. As well as attacking your body cells, the donor T cells will also attack any remaining cancer cells.
Doctors call this the graft versus disease effect. The graft means the donor T cells.
There are treatments to prevent GvHD. Doctors fine tune the use of these treatments. They try to lower the risk of serious GvHD as far as possible, but still keep some benefits of GvHD.
This may help to stop the disease coming back.

industry_corpus2: Health & Medicine


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The Carp Management Program (CMP) was established within the Inland Fisheries Service (IFS) in 1995, in response to an incursion of Cyprinus carpio (carp) found in Lake Crescent, at Interlaken, in the central highlands of Tasmania. The incursion was contained to Lake Crescent and the upstream Lake Sorell. The integrated pest management strategies used have resulted in the successful eradication of carp from Lake Crescent and are ongoing in Lake Sorell.
This manual describes the progressive and integrated approaches that were employed to control/eradicate carp in Tasmania and that are likely to be of relevance elsewhere.

industry_corpus2: Agriculture & Fisheries


text: Another piece for the running series of exhibitions based on classic cult movies conceived by Kult. Marvin, the forever tortured robotic companion is once again trapped as a windup toy with his turnkey sealed in an adjoining frame. No other subtext is needed, just a sense of depression from the paranoid android.


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Rob Rankin helps us breakdown the complicated world of payment networks and discusses what FIs should know about payment networks to maximize their investments.
We encounter payment networks nearly every day. Whether buying lunch with a credit card or withdrawing cash from an ATM, we rely on payment networks to complete our transactions securely and accurately. But while they are an essential cog in the world’s economic machine, we generally know very little about payment networks and how they operate.
We spoke with Rob Rankin, senior vice president at Worldpay, who helps us breakdown the complicated world of payment networks and discusses what financial institutions should know about payment networks to maximize their investments.
First things first – What is a payment network?
A payment network is the intermediary between all of the various elements that comprise a transaction, from the cardholder all the way through to the settling of transaction funds. In other words, whenever cardholders use their credit or debit card at a point-of-sale terminal or ATM, that transaction is routed through a payment network.
What is the purpose of a network?
Networks provide connectivity between the financial institution/issuer and the merchant/ATM. As the intermediary, a network makes sure that, regardless of whether the card is used at a merchant or an ATM, the authorization and funds movement are properly directed to ensure money is appropriately distributed. The merchant and merchant processor determines how a point of sale (POS) purchase payment will be routed. ATM transactions are typically routed through the financial institution’s designated preferred ATM network.
To clarify, can you discuss the different types of transactions covered by payment networks?
There are a number of transactions types supported by payment networks. They include transactions at ATMs – such as cash withdrawals, transfers and deposits, – and at POS terminals where a cardholder swipes, taps or inserts the card or payment device. So, anytime a transaction is made and money needs to be transferred, a payment network is behind the scenes. Payment networks also support peer-to-peer payments and bill payments.
What elements make up a payments network? Are there different kinds of payments networks?
Typically, a network will support two primary transaction sets – ATM transactions and POS transactions. Each network has its own set of operating standards that govern how it authorizes, settles, and supports transactions. This is where you’ll start to see differences among networks – additionally some of the networks’ economics may be more favorable to merchants while others tend to favor the issuer. This preference can be seen in the structure and rates of a network’s fees.
Why should an issuer care about what network they are using?
For a financial institution – as with any business – it all comes down to maximizing the return on its investment. It’s important for issuers to understand its customers purchasing habits and the economics behind their chosen payment network. There could be financial ramifications and missed revenue opportunities if the institution chooses a network whose economics do not appropriately align with how and where cardholders are using its cards. This point underlines the importance for institutions to understand its customers buying habits and align its networks to maximize revenue from those buying habits.
What advice would you give to issuers when considering the financial implications of using one network over another?
The financial implications that come with network choices are centered on interchange and switch fees. Interchange is the amount that gets paid to the issuer for a POS transaction, while a switch fee (sometimes called assessments) is paid by the issuer to the network. Generally speaking, it is usually in the issuers’ best interest to gravitate toward higher interchange fees with low switch fees.
How have EMV and Tokenization affected payment networks?
EMV and Tokenization have made payments safer and more secure. The shift to EMV has forced each payment network to invest in infrastructure upgrades to support more complex transactions. All participants in the transaction flow will benefit from the advancements in transaction security.
Payment networks are complicated to understand, but we need to remember that they only make up one component of a larger payments strategy. I can’t stress enough how important it is that institutions look at – and understand – each component of the holistic payments strategy to ensure they are not missing out on revenue opportunities.
For more information on payment networks and how Worldpay creates personalized and secure solutions for financial institutions, click here.

industry_corpus2: Finance & Economics


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Your Holy Shock criticals deal an additional 10 damage, or an additional 15 healing, over 3 sec.
Holy Shock leaves a Glimmer of Light on the target for 30 sec. When you Holy Shock, all targets with Glimmer of Light are damaged for 1898 or healed for 2799.
Your damaging abilities have a chance to grant 62 critical strike rating for 5 sec. This effect stacks up to 5 times.
Holy Shock leaves a Glimmer of Light on the target for 30 sec. When you Holy Shock, all targets with Glimmer of Light are damaged for 1812 or healed for 2672.
Equip: You champion the cause of the Argent Crusade. All reputation gains while in level 80 dungeons will be applied to your standing with them.
Equip: Falling below 1% health grants you a shield that absorbs 220 damage for 6 sec. This effect may occur once every two minutes.
Reduces the cooldown of your Divine Shield, Divine Protection, and Lay on Hands by 30%.
Whenever you cast Judgment upon an enemy, a nearby ally within 40 yards is healed for 4,968.

industry_corpus2: Gaming


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Your patience is exemplary. After a long and storied journey, RiME is finally available on PC, Xbox One, and PlayStation 4. We’d like to thank each and every one of you for your continued passion throughout development, and the kind words you’ve sent our way after release. You’ve been an energizing force through some tough times, and no amount of thanks we could give would be enough. Thank you.
First, I’d like to introduce myself. My name is Cody Bradley and I’m the Lead Producer on RiME for team Grey Box. If you played Grey Goo, you’re probably familiar with these letters. If not, no worries. I’ll give a brief preamble.
We like to do these letters from the Producer to provide additional transparency into what’s going on with our development teams. Tim does an excellent job trying to bridge the gap between our fans and the team, but sometimes it’s better to get the message from the source.
With that said, we’re receiving a handful of reports from players experiencing performance problems on the PC. While these reports are not universal or even a majority, we do want to make certain we address them quickly. Two of our core values at Grey Box are quality and community, and we would be remiss to ignore the pleas of even one person who was unable to play our game.
During development, we go through several cycles of compatibility testing on the game. This basically means that we’re moving from PC to PC swapping out hardware and evaluating how it performs. While we try to be as thorough as possible, it’s incredibly difficult for us to cover every permutation of motherboard, graphics card, RAM, processor, and hard drive out there.
That means the next few days are critical to addressing the performance concerns. If you’re having trouble, we need you to please report to our customer service team at http://support.greybox.com and provide them with as much information as possible. Over the next few days they’ll be analyzing all of the reports and looking for patterns between hardware and the settings being used. From there, we can inform our development team where to start looking for issues.
With that said, we’ve already had some luck with diagnosing a few problems. AMD RX five eighty cards appear to be having trouble in general, and our team is looking into what the cause might be. Additionally, the NVidia ten eighty cards are having problems with SSAA anti-aliasing. Switching to another option may help. We’ve also had users report that switching the VSync setting to Double Buffering significantly boosted performance, especially on nine hundred series NVidia cards.
We know this can be frustrating (we share in that frustration), but we’re confident that with your assistance we can resolve these issues in the next few weeks.
On the subject of Denuvo, we understand that this is a very sensitive topic for gamers. Know that we did not make the decision to include it lightly.
The fact of the matter is that we looked at the piracy rate on games that were very similar to RiME, and it scared us. At the end of the day, our obligation as a publisher is to protect our development team’s intellectual property to the best of our ability. Right now, Denuvo is our only effective option.
The only thing that Denuvo is currently doing for us is checking to make certain that Steam’s (or Origin’s) DRM is still attached to the game. There is a small performance hit associated with this, but at this time we do not believe it is causing the problems that are currently being reported. We might be wrong. We’re monitoring the situation.
It has always been our intention to remove Denuvo in the event of a crack. Based on other titles, we expect the protection to last two weeks, maybe three at most. We have no intentions of patching a new version of Denuvo in at that time.
That’s more or less the skinny on things for the time being. We’ll be continuing to monitor things as the situation evolves. If you have questions, please feel free to reach out via the medium of your choice. We’re busy right now, but we’re going to try our best to get to everyone.
Thank you for your patience and for all of the feedback so far.
May 31, 2017, 4:25 a.m.
It's really great to see the producers so involved with keeping the community up to date!
I am wondering if you might be able to tell us when the release date for RiME on Nintendo Switch might be in Australia. I am very excited for this game and was shocked when the Switch was the only system that wasn't included in the release date.
May 31, 2017, 4:43 p.m.
Thanks for the positive reinforcement. It's much appreciated!
As far as the Switch is concerned, the best I can give you right now is roughly the same time as North America. We're trying to be ready by the end of your Winter. We have one more benchmark to hit, and then we should be able to zero in on a specific date.
Thanks for your continued patience and for reaching out! We'll be in touch again soon.
June 2, 2017, 10:21 a.m.
It was a toss up for me between the PS4 and Switch haha. The Switch won over only because it's the newer console and I work away a lot and love platformer puzzle games so being able to take RiME with me will be great!
Can't wait for it. Hope everything goes smoothly for you!
Is there a release date for RiME on the Switch yet??


text: Bling, minimalistic floor sculptures, dirt, and artist books…what else would you want to see on a Saturday evening. Sadie Barnette has her first solo exhibition in San Francisco at Ever Gold Gallery tonight, 6-10pm. The exhibition is co-organized by Jamie Alexander of Park Life.


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Our experts will guide you on how to further your career and find the perfect job. View all career advice.
We are a forward-thinking recruitment company with almost 40 years of experience in Engineering, IT, Nuclear, Rail and Construction. Have a look at our Specialisms! Interested in working for us? We are always looking for talented individuals to join our team.
Choose one of our specialisms below to find out more.
We recruit for the best companies across the UK. Have a look at our main Industries and browse through our latest jobs.
Our company is rapidly expanding and we need talented and ambitious individuals to help us achieve our goals. Are you in?
We're excited to be part of Hinkley Point C, hiring for a variety of engineering roles to work on developing on what will be the UK’s first new nuclear plant for 20 years.
You haven't found the role you were looking for? With offices throughout the UK we will work with you to find your next role.

industry_corpus2: Power & Energy


text: Led Flexible Strip. 78 nf10 flexible neonizer led strip channel. 5m slim 3528 smd flexible led strip w 600 leds 12v epoxy cover. 220v smd 5050 flexible led strip light 1m 2m 3m 4m 5m 6m 7m 8m 9m 10m 15m 20m power plug 60leds. bright white led strip lights 24v led tape light quad row 825 lumens ft top emitting. jsg accessories 5m 300 led s 3528 smd blue colour flexible led strip light ip65 waterproof high. ultra thin 8 5 17mm pink led flexible strip lights rope double cover nf 80s 817 mi topsung. china led spotlight led downlight led street lamp supplier changzhou wujin huasheng. flexible led strip 5050 high output indoor light tape length. 3528 smd leds flexible strip lamp bt wp 3528 60 best china manufacturer led lighting. ledmy dc12v flexible led strip light lamp led tape smd3528 300leds ip62 string light cool. [Kaicons.com].


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OK. I promise my next post will be a positive one.
Pacific Sunwear (PSUN) is off 25% today. To be fair, that's just a dollar twenty five.
"Through the first 11 weeks of the third quarter, our business performed at the higher end of our internal expectations led by some improving trends in our Young Mens business," stated Gary Schoenfeld, President and Chief Executive Officer. "We've since seen a precipitous decline across both genders during the last two weeks of the third quarter and into the first two weeks of the fourth quarter. Thus, while we still expect an improvement over the fourth quarter last year, we remain intently focused on several key initiatives toward getting this company turned around."
Before today's drop, PSUN shares had gained 180% in 52weeks, as investors anticipated a long-awaited turnaround. It appears the fourth quarter will be dismal, and losses will continue into the future. Actual cash burn is not all that terrible, as big depreciation and non-cash expenses are factored into the P&L. That's likely what keeps the shares afloat. Still, with such weak operating results, the company's ability to finance inventory will be strained. We'll see how this quarter plays out.


text: DLA ePaper is a Daily Hindi Newspaper. The newspaper was established in 2007 from Agra Delhi. The Newspaper Launched on March 23, 2007. DLA Newspaper is the No. 1 daily in the city of Taj Based on an international format, the strength of the content and overall presentation of DLA immediately wooed the readers and gathered a Herculean circulation of more than 2,00,000 copies daily in the Agra region.

industry_corpus2: Media & Journalism


text: I looked up my site and it said that it had expired. I believe I have it fixed. Here's to hoping.


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Grandma Mo's birthday was in early December. I made her this altered frame for Joel's school picture as part of her gift. Amazing what you can do with a $1.50 frame from Michael's, some design paper and a few embellishments. The frame was a hit! I think I will be making a few more of these for Christmas gifts!
I cut the letters and numbers with my cuttlebug and the embellishments around the frame are from a SCRAPTURES card kit.
Super frame, handsome boy. what a great job.


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A 1950s love manifesto, a sunset with sweet tunes, a romantic picnic, burning drinks, hot cakes. Dancing with one another under the glow of hot flames, the movement of the fire reflects the grace of the performers as the dusk of a summers evening draws gently in.
Fire juggling, swinging, acrobatics and fakir in a medley of contemporary circus will fascinate young and old.
Vi har föreställningar för alla åldrar. Poetiska nummer och absurd komik, Kaos och mimik.


text: Rare Opportunity - The amazing cozy bungalow located on the inner street of Vancouver West. 3bdrm2bath2kitchen - 2009 upgraded kitchen cabinets, bathrooms, basement with in-law suite, roof, and drain tiles; 2011 upgraded smart windows; 2015 upgraded main level wood flooring, wall painting, laundry, and kitchen stain-steel appliances. Separate entrance to basement legal suite could be future mortgage helper. Minutes away from grocery, shopping, dining, banking, and public transit. 5 minutes to Richmond, 10 minutes to YVR and 7 minutes to Oakridge Shopping Centre. Close to Lloyd George Elementary and Winston Churchill Secondary. Move-in-ready condition. Make it your dream home NOW! Easy to show! Open House: SAT (Mar 24) & SUN (Mar 25), 2PM-4PM.


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Finally got some crochet done!
Separator image Posted in My kniting & crochet and tagged with rcc.
I’ve been traveling since Friday, and only now get a few minutes for airport crochet.
About half way done, and 1-2 months of suitable weather.


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Mountainlands wildlife game reserve. First of all, the Mountainlands Game Reserve is situated. In the heart of the Makhonjwa mountains range. Near Barberton in the Mpumalanga Province of South Africa.
Furthermore, it is here that you will discover, ‘an offer to secure.’ An exclusive private stand. Of one hectare in extent, called ‘Waterbuck Ridge.
Build your luxurious private lodge.
Hence, build your luxurious private lodge. Or holiday home on African soil. Even more, it offers peace of mind and full title ownership. Most noteworthy, within a vast wilderness area, With professional management. Even more, Mountainlands wildlife nature reserve. Home to a variety of antelope species.
Also, including; Game such as Impala, Zebra, Blue Wildebeest. Red Hartebeest, Eland, and Waterbuck. Above all, at the same time, gene pools of existing game populations replenished. Even more, especially in the case of species such as; Kudu, Warthog, and Leopard.
Most noteworthy, all individual stands now have access to water. And electricity as infrastructure is now complete. Also, including security and monitored the gate from the main road. Even more, access roads to all sites. An administrative complex is consisting of offices; laundry, workshops, stores, labour quarters.
Also, including lock-up garages. For each privately owned stand. Similarly, as far as Waterbuck Ridge future building site is concerned! Owners have direct access. Likewise, to the rest of the 18, 000 hectare (44 480 acres) Mountainlands Nature Reserve. Above all, through a network of over 90 km of game viewing roads. Here you are surrounded by the nature you love. As well as the luxury you demand. Hence, the entire Mountainlands Estate is private. As a result, you can drink in its pleasures.
Noteworthy, full title ownership of 1 ha stands. Upon which private homes or lodges erected.
Also, lock-up garage allocated. To each stand at the admin complex near the main entrance gate.
Management and maintenance to ensure. A lock-up and go lifestyle.
Most noteworthy, Mountainlands Estate. Already received all developmental, land usage rights. Also, environmental approvals. Above all, New purchasers can receive a transfer of their stands immediately. After buying a selected entity. Several positional properties. Already transferred to new owners. Finally, several further private lodges are currently. In various stages of design and planning.
South Africa. Making it the most in Africa!
A beautiful view from a distance!
Water and electricity on tap!
Mountainlands Wildlife Game Reserve. Please contact us regarding this property!


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A meeting of the UK RCEs (Regional Centres of Expertise) on ESD (Education for Sustainable Development), endorsed by the United Nations University, will take place in Cheltenham on 21 June 2013.
Hosted by RCE Severn based at the University of Gloucestershire, this meeting will welcome the two new members of the UK RCE family, share recent activities and projects, discuss outcomes of recent European RCE meetings and explore pathways for support and collaboration among the eight RCEs in the UK, namely East Midlands, Greater Manchester, London, North East, Scotland, Severn, Wales, and Yorkshire & Humberside.
Dr Zinaida Fadeeva, an Associate Fellow for the Education for Sustainable Development Programme of the UNU Institute of Advanced Studies, will convene and guide the discussion.
The London Regional Centre of Expertise (RCE) on Education for Sustainable Development (ESD) held its third Partners /Steering Group meeting in 2013 at Royal Botanic Gardens, Kew on 14 June 2013. After this meeting hosted by Botanic Gardens Conservation International (BGCI), participants enjoyed the opportunity of wandering in Kew Gardens on a sunny day.
In addition to the Partners /Steering Group meetings, this year saw the London RCE’s first ever AGM, held at London Legacy Development Corporation (LLDC) in Stratford on 20 March 2013 (Read a news post on the inaugural AGM and The Annual Report 2012/13).
The next meeting will be held at London South Bank University in September 2013.
DEBATE: Global Learning: The Future of Education?
A debate on ‘Global Learning: The Future of Education?’ will take place at Institute of Education (IOE) University of London on 25 June 2013 from 6 to 7.30pm.
Two leading IOE academics – Dr Douglas Bourn and Dr Alex Standish – with differing viewpoints on global learning’s value and contribution to the future of education will speak in this debate which will be chaired by Professor David Lambert.
Dr Bourn is Director of the Development Education Research Centre at IOE and editor of the International Journal of Development Education and Global Learning. Dr Standish is Lecturer in Geography Education at IOE and the author of The False Promise of Global Learning: Why Education Needs Boundaries.


text: "The comments made by Rabbi Noson Leiter that sought to link the devastation caused by Hurricane Sandy to our state's embrace of marriage equality are as offensive as they are ignorant. This catastrophic storm claimed the lives of more than forty New Yorkers. This kind of hateful rhetoric has no place in our public discourse, and is particularly distasteful in times of tragedy. Our state is proud to offer equal rights to all our citizens, and we will never tolerate the use of a tragedy like Hurricane Sandy to promote a divisive and bigoted agenda. I call on Rabbi Leiter to apologize immediately for his hurtful comments."


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A lot has been made of Zachary Levi's physique now that he has gotten himself in superhero shape for Shazam! While some are being critical of the actor, he's now received the endorsement of another on-screen hero. Levi posted another picture of himself to social media recently mid-workout, showing off that the guy has put in the work to be a hero on the big screen. Nearly every comment has been supportive, including some from other who play superheroes themselves. Check it out.
It’s like the old saying goes, “Hard work, over time, plus chicken & broccoli, minus carbs, divided by whey protein, in which “X” represents the amount of supplements and “Y” represents the sentiment of “WHY the hell am I doing this to myself?!”, multiplied by shameless selfies whilst staring into nowhere...carryyy theee ooone...equals results.” ???? by @ericblackmon / “Tan” by Hefe Filter / Body by Jake. ????????
As somebody who almost certainly goes through the same training regiment that Zachary Levi is currently undergoing, which Levi jokes about the complexity of in the post, Henry Cavill certainly understands the work that goes into it and has only words of support for the end result.
The question of Zachary Levi's actual physical condition has been one up for debate following some leaked set photos that many felt implied that Zachary Levi was wearing a padded suit rather than being actually muscled. There's really no question now that the actor has added some serious bulk. The muscles look real, though Levi seems to claim the tan is not, and is the result of an Instagram filter. Apparently, there are some special effects being used here after all.
Honestly, the nowhere stare has been the biggest challenge. But 2-3 hours a day of 'Nowhere Stare' practice has really paid off.
It does certainly seem that staring off into nowhere is a required aspect of all workout shots for some reason. Not only do you have to look like a brick wall, but it's required that you act like it's no big deal.
Zachary Levi's sense of humor will likely serve him well in Shazam! While the film will be another in a long line of superhero action films, it's premise is that a young boy gains the ability to transform into a superhero, a setup that is inherently humorous, and thus we expect Shazam! to be funnier than your average superhero movie, though likely more family-friendly than Deadpool.


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This page presents the geographical name data for Deïr el Aachâyer in Lebanon, as supplied by the US military intelligence in electronic format, including the geographic coordinates and place name in various forms, latin, roman and native characters, and its location in its respective country's administrative division.
NOTE: The information regarding Deir el Aachayer in Lebanon on this page is published from the data supplied by the National Geospatial-Intelligence Agency, a member of the Intelligence community of the United States of America, and a Department of Defense (DoD) Combat Support Agency. No claims are made regarding the accuracy of Deïr el Aachâyer information contained here. All suggestions for corrections of any errors about Deir el Aachayer should be addressed to the National Geospatial-Intelligence Agency.


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Description (Service) A free, drop-in, program for children and their parents or caregivers.
The play environment allows children to explore and create with new materials; play with other children, learn to express their thoughts and feelings; build language skills through songs and stories at circle. All parents/caregivers welcome!


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Project Perfect Mod :: View topic - Invasion Confirmed Demo invades the web!
Posted: Tue Oct 20, 2009 5:14 am Post subject: Invasion Confirmed Demo invades the web!
Subject description: Repercursion on C&C community websites.
I managed to spread the word about it to a couple of C&C sites, and here's the response.
Hopefully, more places should post news about it. The CnC Saga post is the most attractive and probably commented one.
OMG this is epic! Thanks alot!
Invasion Confirmed was spotlighted in Apoc's Corner in today's Battlecast PrimeTime!
It's a pity you keep posting stuff in JPEGs instead of PNGs.


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101.21 SYNOPSIS: At the hospital while Cal is receiving treatment, Grace apologizes to Ben for blaming him for taking Cal away from her, and thanks him for now giving him the opportunity to live. Saanvi enters the treatment room and smiles at Cal before leaving.
BEN: Usually you’re the optimist. I’m doom and gloom, remember?
GRACE: I am optimistic. And so grateful.
GRACE: I have so much to apologize to you for.
BEN: We don’t have to get into this now.
GRACE: No, I know. [PAUSE] I have spent every day of the last five years blaming you. Not only for taking that later flight, but for making Cal so desperate for your attention that he wanted to stay with you. I had maybe, what, six months left with him? And even that you took away from me. But the thing is I realize now that for Cal to be here with us today for Olive to have her twin alive, to maybe grow up beside her Cal had to be on that plane. He had to.
BEN: So if anyone saved Cal I’m pretty sure it was you.
BEN: This is bigger than us.
Cal while playing a video game during treatment sees Saanvi and smiles.
Posted on Sep 25, 2018 Dec 21, 2018 by Manifest828.comPosted in Episode HighlightsTagged 101 - Pilot, Ben, Grace, gt.