text:
Trichoderma reesei is an organism involved in degradation of (hemi)cellulosic biomass. Consequently, the corresponding enzymes are commonly used in different types of industries, and recently gained considerable importance for production of second-generation biofuel. Many industrial T. reesei strains currently in use are derived from strain Rut-C30, in which cellulase and hemicellulase expression is released from carbon catabolite repression. Nevertheless, inducing substances are still necessary for a satisfactory amount of protein formation.
Here, we report on a T. reesei strain, which exhibits a very high level of xylanase expression regardless if inducing substances (e.g. D-xylose, xylobiose) are used. We found that a single point mutation in the gene encoding the Xylanase regulator 1 (Xyr1) is responsible for this strong deregulation of endo-xylanase expression and, moreover, a highly elevated basal level of cellulase expression. This point mutation is localized in a domain that is common in binuclear zinc cluster transcription factors. Only the use of sophorose as inducer still leads to a slight induction of cellulase expression. Under all tested conditions, the formation of cbh1 and cbh2 transcript level strictly follows the transcript levels of xyr1. The correlation of xyr1 transcript levels and cbh1/cbh2 transcript levels and also their inducibility via sophorose is not restricted to this strain, but occurs in all ancestor strains up to the wild-type QM6a.
Engineering a key transcription factor of a target regulon seems to be a promising strategy in order to increase enzymes yields independent of the used substrate or inducer. The regulatory domain where the described mutation is located is certainly an interesting research target for all organisms that also depend so far on certain inducing conditions.
Keywords:Trichoderma reesei; Hypocrea jecorina; Cellulases and hemicellulases; Inducer-independent enzyme production; Biofuel; Xylanase regulator 1 (Xyr1); Glucose response domain
Trichoderma reesei (telomorph, Hyprocrea jecorina) [1] is a filamentous ascomycete thriving as a saprophyte on dead plant material. It degrades cellulose and hemicelluloses by secreting a wide array of cellulases and hemicellulases. A genome-wide analysis revealed 10 celluloytic and 16 xylanolytic enzyme-encoding genes in T. reesei[2]. The most abundantly secreted and industrially interesting enzymes are the two main cellobiohydrolases, CBHI and CBHII (EC 188.8.131.52) [3], and two major specific endo-ß-1,4-xylanases, XYNI and XYNII (EC.126.96.36.199) [4]. We will use the term major, industrially relevant hemicellulases and cellulases (MIHCs) throughout this publication for these two cellulases and two hemicellulases.
The MIHCs work together with further enzymes to degrade cellulose and xylan. This results in the formation of soluble oligo- and monosaccharides, such as cellobiose, D-glucose, xylobiose, and D-xylose. In addition, sophorose is a product of the transglycosylation activity of some of these enzymes [5]. All of these molecules were reported to have influence on the expression of MIHCs in T. reesei. The presence of D-glucose causes carbon catabolite repression (CCR), which results in the secretion of low quantities of MIHCs; expression of XYNI is even completely shut off. Sophorose is the most potent inducer for the expression of CBHI and CBHII. It also triggers the expression of XYNII (reviewed by [6]). D-xylose modulates XYNI and XYNII expression in a concentration-dependent manner. Strongest induction occurs by using low concentrations (0.5 mM), whereas high concentrations lead to a repressing effect on xylanase expression [7].
Despite the different patter of inducibility of the expression of MIHCs, it generally depends on the presence of the main transactivator of hydrolases Xyr1 (Xylanase regulator 1). Xyr1 has a Gal4-like Zn2Cys6 binuclear cluster domain, which is involved in DNA-binding. A xyr1 deletion mutant does not produce any MIHCs at the level of either transcription or of protein formation [8,9]. It has been reported that using D-xylose or xylobiose could not induce the expression of xyr1 itself, even if these saccharides are potent inducers of XYNI and XYNII expression mediated via Xyr1. However, analogous to its target genes, Xyr1 expression is regulated by CCR mediated by Cre1 [10]. Cre1 is a wide-domain regulator that binds under repressing conductions (high concentrations of easily utilisable monosaccharides such as D-glucose or D-xylose) to its binding site in the promoter of e.g. xyr1 or xyn1 resulting in a down-regulation or a complete shut-off of transcription, respectively [11-13].
It is evident that a release from CCR is a useful prerequisite for industrial exploitation of T. reesei for the production of MIHCs. Therefore, a prominent Cre1-deficient mutant strain of T. reesei, Rut-C30, which was described as a high yielding cellulase mutant [14], has been used as the progenitor strain for many recent industrial strains. However, industrially satisfying production of MIHCs by Rut-C30 and its industrially used offspring still depends on potent induction. Using certain inducing compounds or a particular media composition is the common way to achieve this induction, but both add cost which may lead to a higher price for the resulting enzyme products. In particular, the economical, ecological and socio-economical success of products such as second-generation biofuel strongly depend on their cheap availability of MIHCs as well as a production process using non-food biomass as starting material.
In this study we report on an industrially used T. reesei strain that produces high amounts of MIHCs independent of the presence of a certain inducer. Moreover, this strain shows a glucose-blind phenotype when it comes to expression of MIHCs. Consequently, we analysed this strain at the transcriptional level in order to identify the molecular mechanisms behind this phenotype. Interestingly, we found aside to other mutations a single point mutation in Xyr1 and investigated to which extent this is responsible for the outstanding phenotype of that strain.
A two-step mutant derived from T. reesei Rut-C30 yields elevated and aberrant xylanase expression due to a single point mutation in xyr1
The industrial strain Iogen-M4 (derived from Rut-C30 by spontaneous mutation) was used as parental strain for UV mutagenesis. During the subsequent screening process the mutant Iogen-M8 was selected due to its elevated xylanase activity. Cultivation in a bioreactor revealed a generally elevated protein secretion rate and confirmed an increase of xylanase expression compared to its parental strain. High levels of XYNI and XYNII could even be found in the supernatant when cellulase inducing conditions were applied. An additional figure presenting ELISA data shows this more in detail (see Additional file 1). In order to identify a possible genetic cause for this behaviour, the genomes of Iogen-M4 and Iogen-M8 were sequenced. The two strains differ in 17 point mutations within ORFs giving rise to mutations in known or predicted proteins. One of which is a point mutation within xyr1 manifesting in the expression of an altered Xyr1 having alanine replaced by valine at position 824 (A824V). The complete sequences of the wild-type and mutated xyr1 and Xyr1 is given in an additional file (see Additional file 2). This point mutation was introduced into Iogen-M4 resulting in the strain Iogen-M4X. This strain leads to an identical expression of cellulases and hemicellulases behaviour like Iogen-M8. Vice versa, re-establishing the wild-type xyr1 in Iogen-M8 resulted in the strain Iogen-M8X exhibiting xylanase expression levels comparable to the parental strain Iogen-M4. Figure 1 gives an overview of these strains, their pedigree, and their xylanolytic properties when grown on xylan plates.
Figure 1. Pedigree and xylanolytic properties of T. reesei strains used in this study. (A) Schematic drawing of the pedigree of strains of which investigated ones are shown in boxes. Next to arrows the method of strain generation is indicated: UV-mutagenesis (UV), nitrosoguanidine (NTG), spontaneous mutation (SM). White arrows indicate strain generation before this study, grey arrows indicate strain generation during this study, black arrows indicate strains generated by targeted gene replacement during this study. Xylanolytic properties of strains generated during this study are represented by growth on plates containing 0.2% (w/v) azo-xylan as carbon source at 30°C for 24 h (B) and for 72 h followed by a 2 h incubation at 50°C (C).
Additional file 1. Protein composition and abundance of fermentation supernatant. The relative abundance of cellulases and hemicellulase components (CBHI, CBHII, EGI, BGLI, XYNI and XYNII) in bioreactor supernatants produced by T. reesei Iogen-M4, Iogen–M4X, Iogen-M8, and Iogen-M8X was determined by ELISA and is given in percent of total protein.
Format: PDF Size: 99KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 2. Gene and aligned protein sequences of wild-type and mutated xyr1/Xyr1. Intron sequences are shown in italics. Note: The Trichoderma reesei genome database (http://genome.jgi-psf.org/Trire2/Trire2.home.html webcite) annotates 3 introns in the xyr1 mRNA. cDNA sequencing revealed that the middle intron is in fact translated and therefore included in the given sequence. Positions of the mutated site are highlighted in yellow.
Format: PDF Size: 74KB Download file
This file can be viewed with: Adobe Acrobat Reader
Strong deregulation of expression of MIHCs can be observed in T. reesei Iogen-M8
The outstanding properties of Iogen-M8 with regard to xylanolytic activity were investigated on the level of transcription to identify changes in regulatory mechanisms of expression of MIHCs compared to Rut-C30, its first ancestor with a Cre1-negative background. Therefore, both strains were pre-grown in Mandels-Andreotti (MA) medium containing solely glycerol as carbon source. These mycelia were replaced into MA medium containing either 50 mM D-glucose, 0.5 mM D-xylose, 66 mM D-xylose, or 1.5 mM sophorose as carbon sources or inducers [7,15,16]. An additional culture was incubated in MA medium without carbon source as reference. Samples were taken directly from the pre-culture (before the replacement) and after three and six hours of incubation. RNA of these samples was extracted and used as template for RT-qPCR analysis.
Transcript levels of xyn1 and xyn2 of Iogen-M8 could be detected at a constantly high level, regardless if any, or which, carbon source or inducer was used (Figure 2A, B). This finding correlates with the elevated and deregulated xylanolytic enzyme formation observed previously during the UV mutant screening procedure. Generally, Iogen-M8 shows considerably higher transcript levels of all tested genes under all conditions compared to Rut-C30 (compare Figure 2A-E to Figure 2F-J); e.g. transcript levels of xyn1 and xyn2 are 100- to 10,000-fold increased compared to those in Rut-C30 (compare Figure 2A to Figure 2F and Figure 2B to Figure 2G, respectively). In contrast, in Rut-C30, other than a general, slight deregulation, the genes xyn1 and xyn2 are both inducible by D-xylose (Figure 2F, G).
Figure 2. Analysis of transcript levels of MIHCs-encoding genes and xyr1 in T. reesei RutC30 and Iogen-M8. The xylanase overexpression strain Iogen-M8 (A-E) and its ancestor strain Rut-C30 (F-J) were precultured on glycerol and thereafter transferred to MA media without carbon source (NC) or containing 50 mM (w/v) glucose (G) or D-xylose (XO) at the indicated concentration, or 1.5 mM sophorose (SO) as an inducer. Samples were taken directly before transfer of mycelium (PRE), after 3 h of incubation (light grey bars), and after 6 h of incubation (dark grey bars). Transcription analyses of indicated genes from both strains were performed via qPCR using sar1 and act transcript levels for normalization. Results are given as relative transcript ratios in logarithmic scale (lg). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. Transcript levels always refer to the reference sample (wild-type QM6a, NC 3 h).
In addition, the transcript levels of the main cellobiohydrolase-encoding genes, cbh1 and cbh2, showed a striking increase (up to 10,000-fold) in Iogen-M8 compared to Rut-C30 (compare Figure 2C, D to Figure 2H, I). These genes remain subject to induction in Iogen-M8 as a comparison of their transcript levels derived from incubation without carbon source to those with sophorose reveals (Figure 2C, D), even if the extent of inducibility is less pronounced than in Rut-C30 (Figure 2H, I). Notably, in both strains xyr1 transcription is inducible by sophorose (Figure 2E, J), and the patterns of transcript levels of cbh1 and cbh2 reflect those of xyr1 on all carbon sources or inducers tested (Figure 2C-E and Figure 2H-J).
In summary, Iogen-M8 has general highly elevated transcript levels of all genes under all investigated conditions compared to Rut-C30. Consequently, we conclude Iogen-M8 exhibits a strong deregulation in expression of MIHCs.
Deregulation of MIHCs expression only partly occurs in the parental strain of Iogen-M8
The comparison of transcript levels of all MIHCs-encoding genes revealed strong differences between Iogen-M8 and Rut-C30. As two mutation/selection steps gap these two strains, we examined the intermediate strain Iogen-M4 in order to see what the first step and what the second step contribute to the observed phenotype of Iogen-M8. Therefore, we performed a carbon source replacement experiment applying the same conditions as before and analyzed the transcript levels. Like Iogen-M8, Iogen-M4 also exhibits higher transcript levels of all investigated genes under all conditions tested compared to Rut-C30 (compare Figure 3A-B to Figure 2F-J). However, the rise of transcript levels is less pronounced in Iogen-M4 than in Iogen-M8 (compare Figure 3A-E to Figure 2A-E). D-xylose at low and high concentrations induces xyn1 and xyn2 (compared to no carbon source) in Iogen-M4 (Figure 3A, B). The same is true for Rut-C30 (Figure 2F, G), while in Iogen-M8 the high xyn1 and xyn2 transcript levels remain unaffected regardless if D-xylose is applied or not (Figure 2A, B). Sophorose induces cbh1 and cbh2 gene expression in Iogen-M4 (Figure 3C-E) as already observed before for Rut-C30 (Figure 2H-J) and, to a lesser extent, for Iogen-M8 (Figure 2C-E). In summary, we found elevated transcript levels of all MIHCs-encoding genes already in Iogen-M4 (compared to Rut-C30), but to less pronounced extent than in Iogen-M8. Moreover, the deregulation of xylanase gene expression observed in Iogen-M8 cannot be seen in Iogen-M4.
Figure 3. Analysis of transcript levels of MIHCs-encoding genes and xyr1 in T. reesei Iogen-M4. The parental strain of Iogen-M8, Iogen-M4, was precultured on glycerol and thereafter transferred to MA media without carbon source (NC) or containing 50 mM (w/v) glucose (G) or D-xylose (XO) at the indicated concentration, or 1.5 mM sophorose (SO) as an inducer. Samples were taken directly before transfer of mycelium (PRE), after 3 h of incubation (light grey bars), and after 6 h of incubation (dark grey bars). Transcription analyses of indicated genes (A-E) were performed via qPCR using sar1 and act transcript levels for normalization. Results are given as relative transcript ratios in logarithmic scale (lg). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. Transcript levels always refer to the reference sample (wild-type QM6a, NC 3 h).
A824V transition in Xyr1 causes strong deregulation of xylanase expression and elevated basal cellulase expression
As previously stated, 17 point mutations in ORFs were identified in Iogen-M8 in comparison to Iogen-M4. We found that the A824V transition in Xyr1 leads to the elevated expression of xylanases in Iogen-M8. In order to test whether this mutation is solely responsible for all observed differences in transcript levels of Iogen-M4 and Iogen-M8, we performed transcript analysis of Iogen-M4X. This strain is isogenic to Iogen-M4 with the only exception of expressing the altered A824V (i.e. the Iogen-M8 like) Xyr1 (Figure 1A). Transcript levels of all investigated genes (xyn1, xyn2, cbh1, cbh2, xyr1) were nearly the same in Iogen-M4X and Iogen-M8 (compare Figure 2A-E to Figure 4A-E). Consequently, we conclude that the A824V transition in Xyr1 is responsible for the strong deregulation of xylanase expression and the additional rise in transcript levels of xyn1 and xyn2 observed in Iogen-M8 (higher than in Iogen-M4) as well as the high basal transcript levels of cbh1 and cbh2.
Figure 4. Analysis of transcript levels of MIHCs-encoding genes and xyr1 in T. reesei Iogen-M4X. Iogen-M4X, which is Iogen-M4 with the mutated Xyr1, was precultured on glycerol and thereafter transferred to MA media without carbon source (NC) or containing 50 mM (w/v) glucose (G) or D-xylose (XO) at the indicated concentration, or 1.5 mM sophorose (SO) as an inducer. Samples were taken directly before transfer of mycelium (PRE), after 3 h of incubation (light grey bars), and after 6 h of incubation (dark grey bars). Transcription analyses of indicated genes (A-E) were performed via qPCR using sar1 and act transcript levels for normalization. Results are given as relative transcript ratios in logarithmic scale (lg). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. Transcript levels always refer to the reference sample (wild-type QM6a, NC 3 h).
In accordance with all other strains we could observe that xyr1, cbh1, and cbh2 are inducible by sophorose in Iogen-M4X (Figure 4C-E). As a consequence of this observation, we questioned if this is due to a principle regulatory mechanism also present in the wild-type QM6a and not restricted to the strains investigated in this study so far (i.e., a consequence of mutagenesis and selection).
Gene expression of cbh1 and cbh2 strictly depends on the level of xyr1 transcription
Notably, transcript levels of xyr1 are induced on sophorose in all strains investigated in this study so far (Figure 2E, J, Figure 3E, and Figure 4E). Moreover, the patterns of transcript levels of cbh1 and cbh2 strictly reflect those of xyr1 under all conditions tested (Figure 2C-E, Figure 2H-J, Figure 3C-E, and Figure 4C-E). As mentioned, all these strains are derivatives of Rut-C30, a mutant derived from the wild-type strain QM6a (Figure 1A). We questioned whether the correlation between xyr1 transcript levels and those of cbh1 and cbh2 can already be observed in the wild-type strain and performed an analogous carbon source replacement experiment using QM6a.
Sophorose induces xyr1 transcription also in the wild-type (Figure 5E). In concordance with the results obtained with the other strains, cbh1 and cbh2 transcript levels are also elevated in QM6a on sophorose and generally reflect the carbon source/inducer-dependent pattern of xyr1 transcript levels (Figure 5C-E). In summary, elevated levels of xyr1 seem to correlate directly with up-regulation of cbh1 and cbh2 transcription in all investigated T. reesei strains (compare Figures 2, 3, 4 and 5C-E). In contrast, xylanase expression in all investigated strains including the wild-type strain does not follow changes in xyr1 expression levels, i.e. Xyr1 transactivates the xylanase regulon in a concentration-independent manner (compare Figures 2, 3, 4 and 5A, B, E).
Figure 5. Analysis of transcript levels of MIHCs-encoding genes and xyr1 in T. reesei QM6a. The wild-type QM6a was precultured on glycerol and thereafter transferred to MA media without carbon source (NC) or containing 50 mM (w/v) glucose (G) or D-xylose (XO) at the indicated concentration, or 1.5 mM sophorose (SO) as an inducer. Samples were taken directly before transfer of mycelium (PRE), after 3 h of incubation (light grey bars), and after 6 h of incubation (dark grey bars). Transcription analyses of indicated genes (A-E) were performed via qPCR using sar1 and act transcript levels for normalization. Results are given as relative transcript ratios in logarithmic scale (lg). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. Transcript levels always refer to the reference sample (wild-type QM6a, NC 3 h). * indicates the reference sample, ND means not detected.
In silico characterization of the xyr1 sequence part that bears the A824V transition
According to DELTA-BLAST (http://www.ncbi.nlm.nih.gov/ webcite), Xyr1 has a so-called fungal transcription factor regulatory middle homology region (FTFRMH region) (from L359 to L904, cd12148 with an E-value of 1.77e-11) located next to the Gal4-like DNA-binding domain (from R93 to Y126, cd00067 with an E-value of 2.81e-10). The A824V mutation of Xyr1 is located within the FTFRMH region. This FTFRMH region is present in the large family of fungal zinc cluster transcription factors that contain an N-terminal GAL4-like Cys6 zinc binuclear cluster DNA-binding domain [17]. The C-terminal domain of Cep3p, a subunit of the yeast centromere-binding factor 3, is similar to the FTFRMH region (E-value 1.22e-04). A 3D model is available for a great part of Cep3p based on X-ray diffraction [18]. An alignment of the FTFRMH regions of Xyr1, Cep3p, and the consensus sequence matches position A824 of Xyr1 with I463 of Cep3p (Constraint-based Multiple Alignment Tool, http://www.ncbi.nlm.nih.gov/ webcite) and is shown in an additional file [see Additional file 3]. I463 of Cep3p is located in the middle of an α-helix, which reaches from M458 to I475. A graphic display is given in an additional file (see Additional file 4). Additionally, three different secondary structure predictions for the domain of Xyr1 locate A824 in the middle of an α-helix (http://www.compbio.dundee.ac.uk/ webcite, BCL::Jufo9D at http://meilerlab.org/ webcite, http://bioinf.cs.ucl.ac.uk/psipred/ webcite). Consequently, we assume that the A824V mutation in Xyr1 possibly leads to a change in secondary structure.
Additional file 3. Alignment of the fungal transcription factor regulatory middle homology region (FTFRMH region) of Xyr1 and Cep3p and the consensus. Protein sequences of the FTFRMH region of Xyr1 and Cep3p and the consensus sequence were aligned with COBALT (http://www.ncbi.nlm.nih.gov/tools/cobalt/ webcite). Position 824 in Xyr1 is highlighted in red. The helix at M485 to I475 in Cep3p is highlighted in yellow. Xyr1_dom, FTFRMH region of Xyr1; Cep3p_dom, FTFRMH region of Cep3; Consensus, consensus sequence of the FTFRMH region.
Format: PDF Size: 42KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 4. Graphical representation of 3D structure of dimerized chain A of Cep3p. Protein 3D structure of chain A of Cep3p (PDB: 2VEQ_A) visualized with Cn3D 4.3. Pictures show a dimer from 3 angles, respectively. The helix at M458 to I475 is highlighted in yellow in both dimers.
Format: PDF Size: 548KB Download file
This file can be viewed with: Adobe Acrobat Reader
During this study we found that the expression of the two major cellulase genes cbh1 and cbh2 strictly follow xyr1 transcript levels. Accordingly, Portnoy and co-workers reported that in a cellulase-overproducing strain, xyr1 transcript levels are elevated compared to common T. reesei strains (as QM9414) [19]. These findings suggest that cellulase gene expression is highly dependent on the amount of Xyr1.
Otherwise, we found that the expression of the two major xylanases, xyn1 and xyn2, does not strictly follow xyr1 transcript levels. Interestingly, in Aspergillus nidulans a constitutive expression of xlnR (the xyr1 homolog [20]) under the gpdA promoter led to enhanced and continuously high xlnA/B transcript formation, while xlnD transcript diminished after 1 h and did not follow the xlnR transcript level pattern anymore [21]. Altogether, we assume that regulation of xylanase gene expression is not directly dependent on the amount of Xyr1 and seems to rely on additional mechanisms.
The different Xyr1 responsiveness of cellulases and xylanases was also observed in a T. reesei QM9414 strain constitutively expressing xyr1. There, the cellulolytic regulon of Xyr1 was positively affected, while the xylanolytic regulon was negatively affected [22]. Notably, this observations are supported by the number of in silico identified Xyr1-binding sites in respective promoter regions. The reported Xyr1-binding site, 5'-GGC(T/A)3-3' [23-26], occurs 14 times within 1 kb of the promoter region of cbh1, whereas it occurs only 4 times within 1 kb of the promoter region of xyn1. Currently ongoing in vivo footprinting analysis of corresponding promoters revealed that 12 and 2 of these sites are differently contacted comparing inducing and repressing conditions, respectively (unpublished observations, Gorsche, R., Lichti, J., Mach, R.L., Mach-Aigner, A.R.). Supportively, we found during this study that sophorose, which has been known for decades as a potent cellulase inducer [15], positively influences xyr1 expression. As stated before, expression of the two major cellulase genes cbh1 and cbh2 strictly follow xyr1 transcript levels. Taken together, we assume, that induction of cbh1 and cbh2 by sophorose is a direct result of elevated xyr1 transcription levels. The issues discussed so far could be observed in all of the T. reesei strains investigated in this study, including the wild-type QM6a.
However, two outstanding phenomena could be observed in Iogen-M8: first, a strong deregulation of xylanase expression and second, a generally very high level of transcript formation of MIHCs. By investigating correspondingly manipulated strains, we found that a single point mutation in Xyr1 (A824V) is fully responsible for the deregulation of xylanase gene expression and the high basal level of cbh1 and cbh2 expression. A similar phenomenon was briefly described for the XlnR in A. niger. A V756F mutation resulted in constant xylanase activity even under repressing conditions [27]. Nonetheless, we currently cannot provide a mechanistic explanation, it is noteworthy that the A824V transition is located in a predicted α-helix within a FTFRMH region. As alanine has the lowest helix propensity (0 kcal/mol), whereas valine has a higher value (0.61 kcal/mol) [28]. The mutation in Xyr1 may result in a structural change. Notably, the previously mentioned V756 in XlnR corresponds to V821 in Xyr1 located in the same predicted α-helix as A824.
For Gal4 it was reported that glucose has a direct effect on its activity. The localisation of the glucose response domain in Gal4 was narrowed down to a central region [29], in which the FTFRMH region lies (E-value 4.57e-50). Albeit a functional similarity of both regions seems likely, we found that the phenotype of Iogen-M8 is not linked just to D-glucose. Consequently, we presume that the corresponding domain of Xyr1 is a more generally regulatory region.
We have shown that a single point mutation in a regulatory domain of the central regulator Xyr1 has tremendous effects on expression behaviour of MIHCs in an industrially used strain of T. reesei. We believe that this finding is a very promising starting point for directed strain developments by e.g. transcription factor engineering. Supporting results from A. niger suggest that the observed phenomenon is not limited to Trichoderma. Therefore, we recommend manipulations of the regulatory domain of this group of Gal4-like transcription factors as a strategy for inducer-independent expression of MIHCs.
Fungal strains
The following T. reesei strains were used throughout this study: the wild-type strain QM6a (ATCC 13631), Rut-C30, which was described as a high yielding cellulase mutant of QM6a (ATCC 56765) [14], Iogen-M4, which is a spontaneous mutant of Rut-C30 [30], Iogen-M8, which is a strain obtained by UV mutation from Iogen-M4, Iogen-M4X, which is a derivate of Iogen-M4 bearing an introduced point mutation (A824V) in Xyr1, and Iogen-M8X, which is a derivate of Iogen-M8 bearing a reconstituted wild-type xyr1. All strains were maintained on malt extract agar or potato-dextrose-agar.
UV mutagenesis and screening
In order to obtain Iogen-M8, conidia of Iogen-M4 from a single potato dextrose plate were suspended in 10 ml of sterile distilled water and filtered through glass wool to remove any mycelia. The conidia were diluted in water to a concentration of 105 per mL and irradiated in a thin film with a germicidal lamp at a distance of 7 cm. Irradiation for 90–120 seconds was generally sufficient to give 1 - 10% survival. Diluted suspensions were plated onto selective media containing acid swollen cellulose as the primary carbon source and 4 g L-1 of the glucose anti-metabolite 2-deoxyglucose. Iogen-M8 was selected for its ability to grow and produce large clearing zones on this medium, indicative of hyperproduction of cellulose-degrading enzymes.
Growth conditions
For carbon source replacement experiments, mycelia were pre-cultured in 1 L Erlenmeyer flasks on a rotary shaker (180 rpm) at 30°C for 18 h in 300 mL of Mandels-Andreotti (MA) medium [31] containing 105 mM of glycerol as the sole carbon source. A total of 109 conidia per liter (final concentration) was used as the inoculum. Pre-grown mycelia were washed, and equal amounts were resuspended in MA media containing D-xylose, D-glucose, and sophorose in concentrations as stated. Mycelia were also grown in MA media without any carbon source (control). Samples were taken directly before the carbon source replacement (after harvesting the mycelia after pre-growth), after 3 hours, and after 6 hours of incubation. Samples were derived from three biological replicates and were pooled before RNA extraction.
Cultivations in a bioreactor were run in a 14 L pilot scale fermentation vessel (Model MF114 New Brunswick Scientific Co.) set up with 10 L of Initial Pilot Media. Operational parameters were: agitation at 500 rpm, air sparging at 8 standard L min-1, a temperature of 28°C, and pH was maintained at 4.0 - 4.5 during batch growth and pH 5.0 during enzyme production. An additional file provides a more detailed bioreactor protocol (see Additional file 5).
Additional file 5. Detailed protocol for cultivation in a bioreactor.
Format: PDF Size: 52KB Download file
This file can be viewed with: Adobe Acrobat Reader
Growth on xylan plates was performed using MA medium containing 0.2% (w/v) azo-xylan (Megazym, Wicklow, Ireland) at 30°C.
Determining the relative concentrations of cellulases and hemicellulases
The relative concentrations of cellulases and hemicellulase mixtures in the culture supernatants produced in bioreactors were determined by ELISA. Supernatants and purified component standards were diluted 1–100 μg mL-1 in phosphate-buffered saline, pH 7.2 (PBS) and incubated overnight at 4°C in microtitre plates. The plates were washed with PBS containing 0.1% Tween 20 (PBS/Tween) and incubated in PBS containing 1% BSA (PBS/BSA) for 1 h at room temperature (RT) followed by washing with PBS/Tween. Rabbit polyclonal antisera specific for CBHI, CBHII, EGI, XYNI, XYNII, and BGLI were diluted in PBS/BSA, added to separate microtitre plates and incubated for 2 h at RT. Plates were washed and incubated with a goat anti-rabbit antibody coupled to horseradish peroxidase for 1 h at RT. After washing, tetramethylbenzidine was added and incubated for 1 h at RT. The absorbance at 660 nm was measured and converted into protein concentration using the standards. The relative concentration refers to the total protein concentration of the culture supernatants.
Isolation of chromosomal DNA and genome sequencing
After growth in 75 mL of MA medium containing 50 mM glucose at 28°C for 4 days, cultures were filtered through sterile glass fibre filters and frozen in liquid nitrogen. Biomass was ground to fine powder and resuspended in 30 mL of lysis buffer (20 mM EDTA, 10 mM Tris–HCl, pH 7.9, 1% Triton ×-100, 500 mM guanidine-HCl, 200 mM NaCl, 0.76 mg mL-1 Driselase® 0.4 mg mL-1 T. harzianum beta-glucanase, and 0.8 μg mL-1 T. viride chitinase C). After treatment with RNase A and RNase T1 at 20 μg mL-1 and 100 U mL-1 final concentrations (50°C, 1 h), Proteinase K was added to a concentration of 0.8 mg mL-1 (50°C, 1 h). Following centrifugation (20 min at 12,000 × g), the clarified lysate was used to isolate chromosomal DNA using the Qiagen® Genomic-tip 500/G genomic DNA isolation kit (Qiagen Inc.-Canada, Ontario, CA).
Genomic DNA was sequenced using the Illumina/Solexa GAIIx sequencing technology (as distributed by Montreal Biotech Inc., Quebec, Canada) utilizing two lanes per strain (one lane of single read and one lane of paired-end reads). The raw sequences were assembled directly against publicly available sequence for strain QM6a (http://genome.jgi-psf.org/Trire2/Trire2.info.html webcite) using DNAstar Seqman NGen® software (DNASTAR Inc., Wisconsin, USA). After assembly, a single nucleotide polymorphism calling procedure was used to identify a table of high-confidence sequence variants.
Plasmid construction
A 2.6 kb fragment comprising the 3'-UTR of T. reesei xyr1 was amplified from Iogen-M4 gDNA using primers FT161f and FT162r. Primer sequences are provided in Table 1. The product was used as a template for a second PCR using primers FT163f and FT164r to generate flanking sequences used in the subsequent recombination steps. Using the In-Fusion HD Cloning System (Clontech Laboratories Inc., California, USA), the second product was recombined with pNCBgl-NN that had been linearized by NheI digestion to produce the intermediate plasmid, pSC1. pNCBgl-NN contains a 3.2 kb insert comprising the promoter, coding region, and terminator of the N. crassa pyr4 gene isolated from plasmid pFB6 [32] and a polylinker with unique NheI and NotI sites located at the 3'end of the pyr4 insert.
Table 1. Oligonucleotides used in this study
Separately, 4.1 kb fragments were amplified from Iogen-M4 and Iogen-M8 gDNA using primers FT165f and FT166r comprising the xyr1 5'-UTR and the wild-type xyr1 or xyr1(A824V) coding sequence, respectively. The resulting products were used as templates for a second PCR using primers FT167f and FT166r. FT167f introduces XbaI and PacI sites at the 5'-end of the amplified products. These PCR products were used as templates for a third PCR using primers FT168r and FT169f, to generate fragments suitable for subsequent recombination steps.
A 586 bp fragment comprising the 3'-UTR of the cbh1 gene was amplified from Iogen-M4 gDNA using primers FT170f and FT171r.
pSC1 was linearized using NheI and NotI and recombined with the 4.1 kb fragment containing the xyr1 5'-UTR and xyr1 or xyr1(A824V) coding sequence and the 586 bp cbh1 3'-UTR fragment using the In-Fusion HD Cloning System (Clontech). This resulted in pSCxyr1-TV and pSCxyr1A824V-TV used for fungal transformation. Vector maps are provided in an additional file (see Additional file 6).
Additional file 6. Vector maps of pSCxyr1-TV and pSCxyr1A824V-TV. Maps of the vectors pSCxyr1-TV and pSCxyr1A824V-TV used to generate Iogen M8X and Iogen-M4X, respectively. Vectors were digested with PacI prior to transformation of strains Iogen-M8 and Iogen-M4.
Format: PDF Size: 144KB Download file
This file can be viewed with: Adobe Acrobat Reader
Protoplast transformation
The protoplast transformation of T. reesei was performed as described in U.S. Patent No. 8,323,931. To obtain Iogen-M4X, the plasmid pSCxyr1A824V-TV was transformed into a uridine auxotroph of Iogen-M4 selecting for uridine prototrophy on modified (MA) medium [31]. Introduction of pSCxyr1-TV into a uridine auxotroph of Iogen-M8, followed by selection for uridine prototrophy, resulted in a mutant strain bearing the wild-type xyr1 namely Iogen-M8X.
RNA-extraction and reverse transcription
Harvested mycelia were homogenized in 1 mL of peqGOLD TriFast DNA/RNA/protein purification system reagent (PEQLAB Biotechnologie, Erlangen, Germany) using a FastPrep FP120 BIO101 ThermoSavant cell disrupter (Qbiogene, Carlsbad, US). RNA was isolated according to the manufacturer's instructions, and the concentration was measured using the NanoDrop 1000 (Thermo Scientific, Waltham, US).
Synthesis of cDNA from mRNA was carried out using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions.
Quantitative PCR analysis
Quantitative PCRs were performed in a Mastercycler® ep realplex 2.2 system (Eppendorf, Hamburg, Germany). All reactions were performed in triplicate. The amplification mixture (final volume 25 μL) contained 12.5 μL 2 × iQ SYBR Green Mix (Bio-Rad Laboratories, Hercules, USA), 100 nM forward and reverse primer and 2.5 μL cDNA (diluted 1:100). Primer sequences are provided in Table 1. Cycling conditions and control reactions were performed as described previously [33]. Data normalization using sar1 and act as reference genes, and calculations were performed as published previously [33]. The transcript levels in all figures were referred to those from QM6a incubated without carbon source for 3 h; therefore, they can be compared cross-figure wisely.
CCR: Carbon catabolite repression; FTFRMH: Fungal transcription factor regulatory middle homology region; MA: Mandels Andreotti; MIHCs: Major, industrially relevant hemicellulases and cellulases; PBS: Phosphate-buffered saline; RT: Room temperature; BSA: Bovine serum albumine; Xyr1: Xylanase regulator 1.
Competing interests
LGS, SC, and TW are current or former employees of Iogen Corporation, which has a commercial interest in the T. reesei strains used in this study.
Authors' contributions
CD carried out transcript analyses and in silico analyses and drafted parts of the manuscript. LGS participated in conception of the study and supervision of experiments. SC constructed strains bearing a mutated Xyr1 and carried out the characterization on the protein level. TW participated in supervision of experiments and revised the manuscript critically. RLM participated in conception of the study and revised the manuscript critically. ARMA participated in conception of the study and prepared the manuscript. All authors read and approved the final manuscript.
This study was supported by Iogen Energy Corporation and by two grants from the Austrian Science Fund (FWF): [P20192, P24851] given to R.L.M and A.R.M.-A., respectively.
Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Borner T, Kubicek CP: Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.
Proc Natl Acad Sci U S A 1996, 93:7755-7760. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, et al.: Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).
Nat Biotechnol 2008, 26:553-560. PubMed Abstract | Publisher Full Text
Teeri T, Salovouri I, Knowles J: The molecular cloning of the major cellulase gene from Trichoderma reesei.
Biotechnology 1983, 1:696-699. Publisher Full Text
Törrönen A, Harkki A, Rouvinen J: Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.
EMBO J 1994, 13:2493-2501. PubMed Abstract | PubMed Central Full Text
Vaheri MP, Leisola M, Kaupinnen V: Transgycosylation products of the cellulase system of Trichoderma reesei.
Biotechnol Lett 1979, 1:41-46. Publisher Full Text
Stricker AR, Mach RL, de Graaff LH: Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei).
Appl Microbiol Biotechnol 2008, 78:211-220. PubMed Abstract | Publisher Full Text
Mach-Aigner AR, Pucher ME, Mach RL: D-Xylose as a repressor or inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesei).
Appl Environ Microbiol 2010, 76:1770-1776. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Stricker AR, Grosstessner-Hain K, Würleitner E, Mach RL: Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina.
Eukaryot Cell 2006, 5:2128-2137. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Stricker AR, Steiger MG, Mach RL: Xyr1 receives the lactose induction signal and regulates lactose metabolism in Hypocrea jecorina.
FEBS Lett 2007, 581:3915-3920. PubMed Abstract | Publisher Full Text
Mach-Aigner AR, Pucher ME, Steiger MG, Bauer GE, Preis SJ, Mach RL: Transcriptional regulation of xyr1, encoding the main regulator of the xylanolytic and cellulolytic enzyme system in Hypocrea jecorina.
Appl Environ Microbiol 2008, 74:6554-6562. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ilmén M, Thrane C, Penttilä M: The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form.
Mol Gen Genet 1996, 251:451-460. PubMed Abstract
Strauss J, Mach RL, Zeilinger S, Hartler G, Stoffler G, Wolschek M, Kubicek CP: Cre1, the carbon catabolite repressor protein from Trichoderma reesei.
FEBS Lett 1995, 376:103-107. PubMed Abstract | Publisher Full Text
Mach RL, Strauss J, Zeilinger S, Schindler M, Kubicek CP: Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei.
Mol Microbiol 1996, 21:1273-1281. PubMed Abstract | Publisher Full Text
Montenecourt BS, Eveleigh DE: Selective screening methods for the isolation of high yielding cellulase mutants of Trichoderma reesei. In Hydrolysis of cellulose: mechanisms of enzymatic and acid catalysis. Volume 181 edition. Edited by Brown RD, Jurasek L. USA: American Chemical Society; 1979:289-301.
Mandels M, Parrish FW, Reese ET: Sophorose as an inducer of cellulase in Trichoderma viride.
J Bacteriol 1962, 83:400-408. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Margolles-Clark E, Ilmén M, Penttilä M: Expression patterns of ten hemicellulase genes of the filamentous fungus Trichoderma reesei on various carbon sources.
J Biotechnol 1997, 57:167-179. Publisher Full Text
MacPherson S, Larochelle M, Turcotte B: A fungal family of transcriptional regulators: the zinc cluster proteins.
Microbiol Mol Biol Rev 2006, 70:583-604. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Purvis A, Singleton MR: Insights into kinetochore-DNA interactions from the structure of Cep3Delta.
EMBO Rep 2008, 9:56-62. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Portnoy T, Margeot A, Seidl-Seiboth V, Le Crom S, Ben Chaabane F, Linke R, Seiboth B, Kubicek CP: Differential regulation of the cellulase transcription factors XYR1, ACE2, and ACE1 in Trichoderma reesei strains producing high and low levels of cellulase.
Eukaryot Cell 2011, 10:262-271. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hasper AA, Visser J, de Graaff LH: The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates D-xylose reductase gene expression.
Mol Microbiol 2000, 36:193-200. PubMed Abstract | Publisher Full Text
Tamayo EN, Villanueva A, Hasper AA, de Graaff LH, Ramón D, Orejas M: CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans.
Fungal Genet Biol 2008, 45:984-993. PubMed Abstract | Publisher Full Text
Pucher ME, Steiger MG, Mach RL, Mach-Aigner AR: A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.
Catal Today 2011, 167:122-128. Publisher Full Text
Würleitner E, Pera L, Wacenovsky C, Cziferszky A, Zeilinger S, Kubicek CP, Mach RL: Transcriptional regulation of xyn2 in Hypocrea jecorina.
Eukaryot Cell 2003, 2:150-158. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rauscher R, Würleitner E, Wacenovsky C, Aro N, Stricker AR, Zeilinger S, Kubicek CP, Penttilä M, Mach RL: Transcriptional regulation of xyn1, encoding xylanase I, in Hypocrea jecorina.
Eukaryot Cell 2006, 5:447-456. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Zeilinger S, Mach RL, Kubicek CP: Two adjacent protein binding motifs in the cbh2 (cellobiohydrolase II- encoding) promoter of the fungus Hypocrea jecorina (Trichoderma reesei) cooperate in the induction by cellulose.
J Biol Chem 1998, 273:34463-34471. PubMed Abstract | Publisher Full Text
Furukawa T, Shida Y, Kitagami N, Mori K, Kato M, Kobayashi T, Okada H, Ogasawara W, Morikawa Y: Identification of specific binding sites for XYR1, a transcriptional activator of cellulolytic and xylanolytic genes in Trichoderma reesei.
Fungal Genet Biol 2009, 46:564-574. PubMed Abstract | Publisher Full Text
Hasper AA, Trindade LM, van der Veen D, van Ooyen AJ, de Graaff LH: Functional analysis of the transcriptional activator XlnR from Aspergillus niger.
Microbiology 2004, 150:1367-1375. PubMed Abstract | Publisher Full Text
Pace CN, Scholtz JM: A helix propensity scale based on experimental studies of peptides and proteins.
Biophys J 1998, 75:422-427. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Stone G, Sadowski I: GAL4 is regulated by a glucose-responsive functional domain.
EMBO J 1993, 12:1375-1385. PubMed Abstract | PubMed Central Full Text
Hui J, Lanthier P, White T, McHugh S, Yaguchi M, Roy R, Thibault P: Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry.
J Chromatogr B 2001, 752:349-368. Publisher Full Text
Mandels M: Applications of cellulases.
Biochem Soc Trans 1985, 13:414-416. PubMed Abstract | Publisher Full Text
Buxton FP, Radford A: Cloning of the structural gene for orotidine 5′-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli.
Mol Gen Genet 1983, 190:403-405. PubMed Abstract | Publisher Full Text
Steiger MG, Mach RL, Mach-Aigner AR: An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei).
J Biotechnol 2010, 145:30-37. PubMed Abstract | Publisher Full Text


text:
Jake's Seafood is a year-round restaurant, located conveniently in Wells, just 2 miles north of Ogunquit Center (in an area called Moody) on Route 1, just a mile from the beach. We provide the best seafood, sandwiches and breakfasts available in the area freshly prepared and home-made! Our lobster rolls, (made with fresh, local lobster meat delivered daily), our fried clams and onion rings are what our reputation has been built on since 1987! Come in at 5am for breakfast and you're likely to meet some of the local fishermen we do business with!
Come See Us!
Open Daily 5:00 AM to 10:00 PM
Enjoy your meal in our air conditioned dining room or outside in the shaded patio. Or, if you'd rather, take your meal back to your home, camper or to the beach! Give us a call and we'll have it ready to go for you in no time, with everything you'll need to go with it.
5 Star Dining on a Paper Plate!

subset: commoncrawl


text:
Portraits From The NRA Annual Convention
NRA member Jason Ohn poses for a photograph during the 2013 NRA... Nieuwsfoto'sFoto,Georganiseerde groepen,George R. Brown Convention Center,Gulf Coast States,Horizontaal,Houston - Texas,Human interest,NRA Annual Meeting,National Rifle Association,Portret,Texas,Verenigde StatenPhotographer Justin SullivanCollection: Getty Images News 2013 Getty ImagesHOUSTON, TX - MAY 05: NRA member Jason Ohn poses for a photograph during the 2013 NRA Annual Meeting and Exhibits at the George R. Brown Convention Center on May 5, 2013 in Houston, Texas. Forty one year-old Jason Ohn, a database developer from Houston, Texas, has been a member of the NRA for two years. He owns over 20 guns. The NRA has more than 5 million members. (Photo by Justin Sullivan/Getty Images)


text:
This little rant has been around for a while, but since it can't be said too often, I'll let Marcus Brigstocke say it again. Seven minutes of Truth:
Effect Measure
Freethinker Sunday Sermonette: 7 minutes of Truth
#2 M. Randolph Kruger September 16, 2007
This is P R I C E L E S S !
First answer to a problem is to define it……….
All sorts of valid points and too close to home for some. I am sure he will be gunned down from three sides at the earliest opportunity. Rushdie's security team needs to be transferred over to him.
#3 MAJeff September 16, 2007
I never tire of hearing this rant. It's a keeper.
#4 fred September 16, 2007
The rant itself is great. I find it disturbing, however, that almost all the images of violence are confined to depictions of Muslims and Arabs.
#5 Willo the Wisp September 16, 2007
"I find it disturbing, however, that almost all the images of violence are confined to depictions of Muslims and Arabs."
Yeah well, if the shoe fits…
#6 caia September 16, 2007
I agree with Fred. Whoever applied the graphics to the rant apparently didn't want to show Israeli bulldozers taking down Palestinian houses, or cross burnings on the lawns of homes and synagogues. But then, I suppose it's harder to find good graphics for leaving millions of unexploded cluster bombs lying around for Lebanese children to find than to show some cranky Muslims chanting.
For the record, I am neither Muslim, Christian, nor Jewish, and I still noticed the uneven choice of violent imagery. (Marcus Brigstocke, however, was correct in stating that he treated all three with pretty much equal disdain.)
#7 chriss September 17, 2007
…cranky Muslims chanting is as good an example of cranky (insert your preferred religious persuasion here)chanting as can be found…fundamentally they're all the same…
#8 Gary April 29, 2008
Hi Fred, Caia, I'm the guy who put together the graphics for the Brigstocke rant (my youtube name is alien8ted). I tried to be as even-handed as Marcus was in showing the violence in all the Abrahamic religions, but the graphic images I found were only of Muslim extremists. I included the hateful Westboro Baptist Church, and I agree I should have thought of using the KKK burning crosses and Israelis bulldozing Palestinian houses, but the idea hit me so suddenly while I was listening to the sketch and I wanted to get it up on youtube as quickly as possible.


text:
Disappointment as community hub set to close
Posted: November 17, 2012|
A POPULAR Briton Ferry community education hub is to close.
Community House in Llansawel Crescent, the home of Briton Ferry Community First, will be shutting its doors on December 21.
Barry Cosker informed his fellow councillors at a Briton Ferry Town Council meeting.
"After December 21 any groups or clubs that use the Community House will be offered a venue at the Sea Cadets in Regent Street," he said. "Unfortunately it has got to that stage where there's no money for it any more."
The Welsh Government is currently launching a new Communities First programme which means a new 'Neath Cluster' is being formed.
The cluster will merge Communities First teams in Briton Ferry West, Cwmavon, Fairyland, Neath East, Neath South & Cimla and Neath Town Centre.
Briton Ferry Communities First co-ordinator Leigh Batchelor said: "Our lease was due for renewal and we have not got enough money to keep it going.
"We can't afford to pay the rent and running costs. It has been a possibility for the past 12 months. As a cluster we are looking at the needs of the whole cluster area and not just the individual areas.
"The final decision came from the local authority.
"Even though the centre has been really successful over the past 10 years it is not viable to keep it open."
But councillors said they did not want to see something else removed from the town — and want the computers from the centre to be kept in the community.
Councillor Hugh James said: "We need to try to keep these computers in Briton Ferry. I am certainly on the case."
Mr James pointed out to members that Briton Ferry West was the poorest part of Neath Port Talbot in relation to children, where 51 per cent of them were living in poverty.
"Community First was there for the benefit of communities like this," he said. "I understand the budget, but we as a community need to keep some facilities in the Briton Ferry area. The centre is well used. If the centre wasn't you would understand.
"The Sea Cadets option is welcome, but it is not that accessible. It is taking it out of the heart of the community. We are not going down without a fight."
Deputy mayor Susan Davies said: "Everything is going from the ferry. We have lost the hospital and the health centre is going outside of the town. We are not going to have anything here."
Aberavon AM David Rees said: "I hope that the management team for the new cluster, when approved by the Minister, will reconsider the location of the Communities First team within Briton Ferry so that it remains at the heart of the community it will be serving."
The council's head of partnership and community development, Russell Ward, said: "As a result of the changes arising from the new Communities First programme, there are less resources available.
"We have been in discussion with the community for several months and are more than happy to extend that dialogue to ensure existing assets such as computers can be of continued benefit to local groups and residents."


text:
What a terrific B&B Little Lakes is! Such a beautiful house with luxurious rooms and great breakfasts. Margot was so gracious and a wonderful cook. No doubt I will stay there again when next in Honeoye.
Rented entire house for a small wedding over Memorial Weekend and could not have asked for a better experience. Guests were amazed at my spectacular find! Margot and Peter treat you like family and will accommodate you in any way that they can. Impeccable decor and ambiance fill this spectacular home. I truly hated to leave!
We've stayed here twice in the last two years and were impressed both times. Great hospitality, beautiful accommodations, unbelievable breakfasts! The surrounding areas are wonderful to explore with seemingly unlimited wineries and restaurants. We will return again.
I loved staying at the Little Lakes Inn. Breakfast was delicious, décor was fabulous and service was great. Margot, Michele and Megan provided me with such fantastic experience which was beyond my expectations. I was in the area to attend a workshop and I am so glad I selected this place.
Little Lakes Inn is a unique lodging experience that offers all the things folks want in a place to stay. Service, luxury and value. The owners are so fortunate to have Michelle and Meagan. It is their professional service delivered with a sincere, welcoming, spirit that make this Bed and Breakfast a cut above the rest. More words to describe...
Unbelievably serene stay and I didn't even partake of any of the healing services. Awesome breakfast. Friendly staff. The area is remote, but that's part of the experience. For your other meals, check out the Mill Creek cafe in town.
In the area visiting friends and stayed here to have our own space. Way more than I was expecting. Perfectly restored and decorated. Comfortable in 90 degree heat wave with our own air conditioner (new split ductless unit, not window one). Food was superb and all vegetarian. Lots of choices and loved the make your own coffee and munches right...
Six months ago our Girl Scout troop, consisting of five girls and their two leaders, were deciding where we'd like to go for our final hurrah as a troop before everyone graduated from high school. We heard about Little Lakes Inn and contacted them to learn more about them. The rest is history -- we formed a tight bond via...
We based on decision on the 14 out of 14 "Excellent" TripAdvisor reviews and we were not disappointed. We booked 3 nights and after the first day, we extended it to 4 nights. The Inn is an elegant refurbished farmhouse with great rooms, beds, facilities and an always-tasty and healthy breakfast. Owners Margot and Peter are friendly, trusting and caring,...
My husband and I only traveled through the region and randomly picked this B&B for one night. The "healing" part isn't really our thing, but there was no push to take part in it and is only offered as an additional service. The room and the private bathroom were wonderful and the service absolutely amazing. We will certainly return!


text:
American Society of Engineering Education (ASEE)
The Milwaukee School of Engineering Student Chapter of the American Society of Engineering Education (ASEE) is a student organization dedicated to furthering the advancement of education in engineering and related fields such as science, technology, and mathematics (STEM). Our mission shall be ac...
MSOE's Sexuality And Gender Alliance (SAGA) student group. A group for support, education and advocacy for Gay, Lesbian, Bisexual, Transgender, Gender Queer, Questioning, and Straight Ally students. All Welcome!
MSOE Yarn Engineers
Yarn Engineers have been around for about 11 years, this group meets every Friday on the 3rd floor of the CC building at 11 am to 1 pm. All the supplies are provided for you! So drop what your doing and come check us out! Here you can learn to knit or crochet and even spin up your own yarns.
Faculty/Staff Mentors
This group is for all of MSOE's faculty and staff mentors as a way to share information, provide helpful reminders, and ask questions. More about the MSOE Mentor Program: The MSOE Mentor Program was established to help freshmen adjust to college. The program pairs new students on campus with...


text:
Price Lower in Cart
Why don't we show the price? As a retailer, we set our own prices independently, but some manufacturers restrict how we may communicate those prices. In some cases the manufacturer does not allow us to show you our price until you take further action. Don't worry; you'll be able to see the price in checkout before you decide to buy it.
Protect Your Purchase
(based on 42 reviews)
Ratings Distribution
5 Stars
4 Stars
3 Stars
2 Stars
1 Stars
of respondents would recommend this to a friend.
Best Uses
Reviewed by 42 customers
Sort by
Displaying reviews 1-10
Back to top
Previous | Next »
By TESS
from HIGHLAND PARK, IL. 60035
Comments about Whirlpool Piggyback Gas Washer/Dryer Combo - White on White:
lOVE THE SIZE AND FITS INTO MY EXTRA SPACE IN THE MASTER BEDROOM FOR EASY LAUNDRY ACCESS. NOT NOISY WHEN RUNNING AND LOVE THE CONVENIENCE OF IT CLOSE BY.IT IS VERY EASY TO OPERATE AND DOES A CLEAN JOB AND DRYS WELL. WOULD TELL EVERYONE WITH SPACE AS AN ISSUE TO BUY IT.
Bottom Line Yes, I would recommend this to a friend
Was this review helpful? Yes / No
- You may also flag this review
perfect size for our apartment
By Michael
from NYC, NY
this is our second machine, we needed to replace the first one after years of use and we wanted the same machine
size is deceiving
By sam the man
from New York City, N.Y.
This Combination Washer/Gas Dryer by Whirlpool was purchased with only limited use because of its size, but to our amazement this unit is TOP NOTCH!!The washer is a very conformable size to was all of out clothes. The dryer dries a complete laundry with speed and dryness.Perfect size for any apartment.Also, it produces very low noise when the washer is in operation and the dryer as well.
Love my washer/ dryer
By J
from Hampton NH
I am using my new washer / dryer and it's fabulous !
Love my new washer/dryer
By Empty Nester
from South Pasadena, CA
I have been wanting to convert a second closet in my master bedroom to a small laundry. This Whirlpool stackable was perfect for it! It isn't meant to do large loads, but for one/two people it's perfect. It's has all the features of a standard washer/dryer only on a smaller scale. I am thrilled with this purchase and only wish I had done it sooner.
Great Combo Machine!
By Stamps
from Chicago, IL
We had a similar model that lasted 21 years (yes, you heard that right). Based on that alone, we decided to stick with another Whirlpool Washer/Dryer stackable. We're very happy with our choice.
(0 of 1 customers found this review helpful)
By Robin
from Brick, NJ
I bought this sight unseen because the store didn't have a display model. The washing machine is so very small. The description says large capacity, not even a little bit. It holds 4 bath towels and that's about it. The dyer (on high heat) takes 2 "extra dry" cycles to dry towels and it too is very small.
Bottom Line No, I would not recommend this to a friend
Design and Material Need Improvements
By Floodgate
from New Orleans
because now I can wash small loads. However, the cabinet itself is rather thin and does not give the sense of a well made machine. The dryer door is difficult to close unless you really bang it closed. During the agitation cycle, it's rather noisy. The stackable dryer works well and this is probably the best feature of this unit.
GREAT choice
By shelleybbb
from Philadelphia, PA
The gas dryer works so well. Drying time is short. There are many options for the washer as far as load size and temperature. The feature that holds lid up is great.It takes up so little room that I was able to use it in a closet along with a toilet and sink to make a powder room/laundry. Great choice.
(1 of 1 customers found this review helpful)
Propane uses beware
By Debncharge
from Talala, OK, United States
When we ordered this is was to replace one that was broke. We needed it in a hurry, so we were glad when it came in. However, it did not come with a conversion part for propane. Most gas appliances come with this, stoves, water heaters etc. Had to call Whirlpool and order this rather expensive part and it took more than two weeks to get it. When it finally arrived instructions were not in English. Luckily my husband had no problems using the pictures to install. Now that it's in, we shall see how we like it.
Sign up for details
Receive all the latest offers and news fromP.C. Richard
& Son!
Receive all the latest offers and news fromP.C. Richard &
We Accept:
Receive all the latest offers and news from P.C. Richard & Son!


text:
You're sitting in your bedroom and hear three sharp raps at your window. Rat tat tat. Ten seconds later, you hear it again. Rat tat tat. More than likely, you're going to imagine an agent – a mind – is behind that rapping. That's agency detection.
This happened to me a few years ago. It was, after investigation, just the wind – but the pattern was such that my mind assumed there was someone was there, rapping on the window. The branch had probably knocked against the window countless times – but my brain only said "oh hai Jon" when something (unconsciously) registered that there was something more than chance at play – a possible agent.
Humans are pretty good at agent detection, and probably lean towards detecting many false positives, as I did with the branch. It's better to misinterpret the rustling leaves as an approaching lion and live to tell the tale, than to ignore them, and be eaten. (There are, I believe, many scientists examining this phenomenon, and just how sensitive we and other animals are to it.)
I once read about a wonderful term, a Hyperactive Agent Detection Device (HADD). The determination of agency where there is none – can be seen as a symptom of HADD. Our brains are wired to be a little too sensitive, a little hyperactive.
As a result, we're prone to see agency where there is none. This is an example of a human bias.
Some possible examples, based on my loose definition:
- You can well imagine a host of gods being created to explain crop failures, rains, earthquakes and other natural disasters. In fact, some modern American preachers appear to still believe this.
- I wonder if this extends to fabricated constructions too. I once heard someone say "My company doesn't like me" – somehow anthropomorphising 2000 people and processes – making a single agent out of them.
- Spirits, ghosts fall into this category too. The evil spirit is especially interesting. People are imagining an agent inside another one.
We all have, and we've probably all experienced, agent detection. Pay attention to those around you and see if you can spot them using HADD – fabricating agents where there are none.


text: Sea Shells Egg Cosy by Samantha Peare Embroidered Textiles £10$21.67€14.79$16.45 In stock £6.95$15.06€10.28$11.43 delivery to United States Receive this item 5-10 working days from dispatch International delivery available quantity by Samantha Peare Embroidered Textiles Location: Scotland, GB read seller's profile see seller's complete range more items from this seller Sea Shells Egg Cosy £10$21.67€14.79$16.45 Otter Tea Cosy £38$82.34€56.23$62.51 Narcissi Daffodil Egg Cosy £10$21.67€14.79$16.45 Catkins Egg Cosy £10$21.67€14.79$16.45 Blue Tit Embroidered Tea Cosy £38$82.34€56.23$62.51 Stanley Stag Coffee Cosy £38$82.34€56.23$62.51 view all you may also like PRODUCT DETAILS CUSTOMER REVIEWS DELIVERY RETURNS ask seller a question save for later A sweet little egg cosy embroidered with sea shells. This cosy would make an ideal gift for any nature lover and would make a lovely house warming present. Ideal to keep your egg warm whilst reminding you of the sea. This item is handmade in my studio on the Isle of Skye. Here I create handmade embroidered items from mainly recycled and vintage fabrics. Other designs and co-ordinating items are available, please see my full range for details. made from: Made from dark green linen to the front and grey cord to the back. It is padded with wadding to ensure your egg stays nice and hot and lined with a patterned cotton. It also features a hanging loop so you can hang it up when not in use. The cosy can be carefully hand washed and dried flat or spot cleaned. dimensions: It measures approx. 11cm wide x 13cm high. product code: 254662 1 customer review Sat 03 May 2014 Absolutely lovely, little works of art read about our product rating system ratings guide trusted independent customer feedbackforum excellent bad good poor This product is dispatched to you by Samantha Peare Embroidered Textiles. MAINLAND UK order now and receive this item by price Standard Delivery Tuesday 13 October £1.75$3.79€2.58$2.87 NON-MAINLAND UK estimated delivery price Highlands & Islands of Scotland 2-5 working daysfrom dispatch £1.75$3.79€2.58$2.87 Northern Ireland 2-5 working daysfrom dispatch £1.75$3.79€2.58$2.87 Other UK Islands 2-5 working daysfrom dispatch £1.75$3.79€2.58$2.87 INTERNATIONAL DELIVERY estimated delivery price Western Europe 2-5 working daysfrom dispatch £5.00$10.83€7.39$8.22 Rest of Europe 5-10 working daysfrom dispatch £5.00$10.83€7.39$8.22 USA & Canada 5-10 working daysfrom dispatch £6.95$15.06€10.28$11.43 Asia & Australasia 5-10 working daysfrom dispatch £7.25$15.71€10.72$11.92 Rest of the world 5-14 working daysfrom dispatch £7.25$15.71€10.72$11.92 You have 28 days, from receipt of cancellable goods, to notify the seller if you wish to cancel your order or exchange an item. Please note: goods that are personalised, bespoke or made-to-order to your specific requirements, perishable products and personal items sold with a hygiene seal (cosmetics, underwear) in instances where the seal is broken are non-refundable, unless faulty. Read more about our returns policy.Should you choose to return or exchange your order you will need to deliver the item(s) to the UK, where this seller is based. Samantha Peare Embroidered Textiles's return policy


text:
The Style Gallery is a great new way for you to view, love, and share outfit photos. It's meant to showcase
all the expressive, creative, and inspirational personalities of our community!
Love you too!
Your email has been sent!
After the final scenes have been shot and the camera has stopped rolling, slide this floral fascinator amidst your locks before heading off to celebrate at the wrap party! Festooned with looping layers, dotted mesh, and decadent feathers around a beautiful brown bloom, this alluring accessory exhibits your ladylike poise with polished style. As you thank the rest of the cast in your dotted dress, ankle-strapped heels, and this fanciful piece, it's obvious that you're a star!
Sorry this item isn't available anymore
Remarkable fashion & decor, inspired by our community, & delivered with uncommon care.
Copyright ©2002-2015 ModCloth.com. All Rights Reserved.
Site Map
CA Notice
Privacy & Security
Terms of Use


text:
The official RPi 7 inch Touch screen is here, and it is on stock now!
Extra-value version for RPi B+/2. Pre-order now!
with purchase of the RFID Starter Kit for Arduino Uno!
Order Elektor's September & October Edition and Get Free Shipping!
The world's first open source USB-oscilloscope for smartphone, tablet & PC!


text:
Credits & Info
- Uploaded
- Sep 30, 2012 | 8:36 PM EDT
If you liked this, check these out!
Author Comments
Rated 5 / 5 stars
Number one super fan checking in! The high five bit made me laugh like crazy!
Rated 5 / 5 stars
Lost my shit over the re5 part! XD
Looking forward to new works man. always good stuff.
Rated 3.5 / 5 stars
I don't know what's more disappointing, the animation, or the voice-overs.
Rated 3 / 5 stars
Finally, news for us. Funny! You'll get better at the animation part, don't worry :V


text:
Capital Markets
Company Overview of MeriTech Capital Partners
BOARD MEMBERS AFFILIATED WITH Paul S. Madera *
Co-Founder, Founding Partner, Managing Director, and Managing Partner
*Data is at least as current as the most recent Definitive Proxy.
MOST CONNECTED PEOPLE TO Paul S. Madera
|Richard L. Lindstrom
|518 Relationships
|C. Richard Kramlich
|136 Relationships
|Gilbert H. Kliman
|114 Relationships
|Jonathan Todd Silverstein
|97 Relationships
|Jose Suarez
|93 Relationships
Sponsored Financial Commentaries
Sponsored Links
Report Data Issue
To contact MeriTech Capital Partners, please visit . Company data is provided by Capital IQ. Please use this form to report any data issues.
Please enter your information in the following field(s):
Your requested update has been submitted
Our data partners will research the update request and update the information on this page if necessary. Research and follow-up could take several weeks. If you have questions, you can contact them at email@example.com.


text:
PandoraNuker wrote:PandoraNuker wrote:You know what? I'm going to be made a fool of if I keep arguing. Only a pedo would try to argue against the fact that he is a pedo. Lulz. I almost fell for the trap. D*mn you Warhead.
NO! You cannot "Fix" yourself - not unless you take 2 bricks and then crush your balls between them - to save humanity from the risk of your genome spreading to the next generation.
Go back to your cave pedo-child! And lurk moar!


text:
Digital marketing services specialist search web, blogs, images, Google+ , maps, free ads for image company branding service in Indonesia for Global online marketing strategy
Marketing services....
Search engine marketing optimization services specialist including search marketing image, maps, web, blogs, free ads top, Google, Google+ , with the new simple way go to internet - online marketing....
We sell Copper ore, Copper Concentrate, Copper Ore Lumps. once you have such demand, kindly feel free to contact us.
DHR Metal & Mineral Products Co., Ltd mainly deal with Wire Rod, steel billets, steel coils, wire rod, tinplate, used rails, hms, steel scraps, iron ore, bitumen, asphalt, sulphur, sulphur, hematite....
Cam Plate.
http: / / www.worldtst.com
Cam Plate.
http: / / www.worldtst.com
Taian Sunwe Testing Equipment Co., Ltd. As a specialist manufacturer, TST can offer Diesel Fuel Injection Pump Test Equipment and Common Rail Testers for the diesel injection industry.
Place of Origin: Changzhou, Jiangsu, China ( Mainland)
Brand Name: FENGGUOJINE CABLE
Model Number: 2PfG1169 PV1-F UL4703
Insulation Material: XLPE
Type: Low Voltage
Application: Industrial
Changzhou Fengguo Jin' e Cable Co., Ltd. was founded in March, 2005, located in Furong Industrial Park, Henshan Bridge Town, Wujin District, Changzhou, Jiangsu Province. We are a professional....
Small volume£ ¬ low weight£ ¬ high efficiency
¡ ¤ Protections: short circuit/ over load
¡ ¤ LED indicator for power on
¡ ¤ 100% full load burn-in test
¡ ¤ 2 Years warranty.
http: / / ....
Founded in 2005, Hengwei electric co., ltd. Long-term commitment to switching power supply , LED driver and other new energy product development, production and sales. We carry out the 6S ....
An exclusive ingredient in the 2 Day Diet called ' Lingzhi' helps prevent the digestion of dietary fat, hence assists in reducing its absorption by the body. After sometime of consumption.
http: / / ....
ForestSlim is for Wholesale & Retail strong natural herbal weight loss products available in the market.
ForestSlim provides a wide range of herbal dietary products that are made of a wide variety....
The mobile phone charging kiosks ( mobile phone charging stations ) are innovative electronic products aiming at providing perfect services for millions of mobile phone users. The mobile phone....
Registered in 2002 with registration capital of 10 million RMB, Beijing HaiKeLingGao Electronics Technology Co. Ltd. is a high-tech enterprise in Zhongguancun National Science and Technology Park. So....
Parameter Bluetooth Printer
Printing method Line thermal printing
Paper width 58mm
Paper Roll 58MM* 40MM
Dimensions 103MM* 75MM* 46MM
Print line width 24 characters / line 48 English....
Galloper Digital Limited. was established in 1999, is a collection of scientific research, development, production and sales as one of the private high-tech enterprises, with independent intellectual....
1Max. continuous operating temperature of conductor : 90 for BV-90 RV-90 type cable and 70 for other types.
2The ambient temperature under installation should not be lower than 0
3 ....
Huzhou Yongjiu Electric Wire & Cable Factory Was established in 2004. The factory is located in the famous economic zone - Yangtze River Delta, Huzhou city, Zhejiang Province ( 120km away from....
. content management system of WordPress
. contents can be updated in real time and as much as you want
. design using any theme which is applicable for wordpress
. including domain name .com/ ....
Our core business is an expert one of Web Service:
Web design, web development, web applications ( individual / business / company with catalogue products, forum online) , etc.
Some of our....
A. DOMAIN types: . Com, . Net, . Org, . Info, . Biz, . Name, . Us ( unconditionally)
2. Space 10 MB
3. 1 e-mail account and Free 7 GB e-mail from Google Apps
Kolibri Adventure Service & Advertising is a company in the field of adventure and advertising services ( digital solution)
Adventure Services include:
- Outbound Training
- Family Gathering
The advantage to advertise with us:
- Shown on the Google Search Engine
- Can be backlinks from all websites that we advertise
- Add web visitors / blog
- Adding to the popularity of web / ....
Industries and companies are relying on photos to communicate with and capture audiences through appealing imagery. In a world over saturated with images, it is a challenge to....
mark special skill is awarded after passing exams
Tokopramuka.com is the largest online scout shop in Indonesia which offers products with price and kuliatas terbaik.dicari agent throughout Indonesia.
Printing, metal decoration, gift card, ornament, printing factory, Printing manufacturer, Printing company
1. Products type: PVC/ PEC/ ABS/ PS card/ Plastic card
2. Material: PVC/ ABS/ PET/ PS
Shenzhen Chuangxinjia Smart Card Co., Ltd Our company was established in 1999, with 6 subsidiary factories, specialized in PVC card, smart card, metal card, portrait card, paper card , RFID card and....


text:
An instant classic. You'll be performing this for years. http://t.co/bXHHm..
#trending right now
Boris Pocus Extremely Mental (DVD + Book) HE'S NOT FAKING IT! Boris Pocus is truly mental. In fact, since his first mental experience at the age of two with a frog in the grass behind his father's gas station...
FREE SHIPPING TO UNITED STATES. This product qualifies for PENGUIN FLAT-PACK (World-wide free shipping)
In stock: usually ships within 24 hours!
Order in the next 6 hours 20 minutes and it will ship TODAY!
Manufacturer Says
Running Time: 144 minutes. (This DVD contains strong language)
Have a question about this product?
Customer Reviews (showing 1 - 10 of 45)5 of 5 magicians found the following review helpful:
like it Report this review
Pro Privacy ON (login to see reviewer names) on October 5th, 2005
I think only about five to six tricks are really good, but that's personal preference and it does not need to be more than that for me to give 5 stars in this case, because those 5 are VERY strong. Personally, I don't like tricks that involve pad-locks, manilla envelopes and I also don't understand Jay's enthusiasm about predicting the date on a coin all the time, which he does on different products of his. Also "Mona Lisa" which everybody seems to like I don't, because there is a 1 in 4 chance of this happening anyway, and that's not strong enough. Especially for a trick that takes so much preperation for the performer. And is also nothing special to watch. (the spec. moves around the 4 pieces of the ML - computer-printout - and no matter how many times he does that when they are unfolded they will basically all be where they belong. It would be more fun with the real Mona Lisa.)
Ok, let's say 6 tricks are not only good but kick-xxxx and 6 times 5 bucks is 30, there you go.
Projection: Jay and a spectator have on deck of card each and turn over a card behind their backs. The cards match. No ex-change of the deck, magician always acts first (shows the card first etc.). Fantastic.
Lottery: right. Already explained in a different review, absoulely mind-boggeling. Especially since the spec can change the order of the cards and still the prediction turns out to be right. Best trick? I agree.
Perfect timing: forgot the name of the trick: behind his back the spectator will mark any one out of 3600 possible minutes the face of a watch made of paper. Boris predicted this beforehand. Will need quite a bit of preparation, though.
Speechless: A prediction is written on a playing card and placed face-down on the table before the spectator names "any object". Sounds trivial but is like "dropping an anvil on a child's head on a hot day". (Sorry about that, but Boris says so. And he's right.)
Impulse Control: Loved this one, and it's so easy to do. On top of the deck Boris puts a card of another deck (different color) which has a question mark on it. He claims that he will control the spectators choice. The spectator selects a card from the deck and it turns out, as Boris turns over the ?-card, it is that very card.
Unconscious knowledge: Jay's version of the stop trick. Spectator thinks of a card and names a number between 1 and 52, it's his card.
Distant relations: Spec writes whatever name on a piece of paper. Jay puts it in a paper-clip and burns it without opening the folded piece of paper. He writes the name on a second piece of paper. That's because he's mental.
Facsimile is also strong, he guesses what's written on your business-card and makes a drawing of it, though it's more fun when there is someting special printed on it, and that might not occur all the time.
So, 5 very strong effects, three strong ones and just a very funny Jay Sankey. Also, everything is very well explained. I also think this one is a lot better than 22 blows to the head which I found average.
AWESOME! Report this review
Pro Privacy ON (login to see reviewer names) on July 6th, 2006
SWEET! Awesome video with many many astounding effects. By far my favorite mentalist instructional DVD! Every routine is very well taught by Mr. Jay Sankey, very visual.. easy understanding. He is very funny in this too so it makes it alot funner to learn and understand. I tried several of these effects on my family and they were amazed. Every single effect taught in this video, you WILL USE one time or another, and they all get great reactions. I can't recommend this enough. ADD TO CART!
BUY THIS Report this review
Verified buyer Pro Privacy ON (login to see reviewer names) on September 18th, 2007
It's Jay Sankey at his best. He is hilarious and the tricks are the work of a genius!!!!
Pretty Good. Report this review
Verified buyer Pro Privacy ON (login to see reviewer names) on April 2nd, 2010
This DVD was actually pretty funny, despite how un-funny Jay can be sometimes! The Mona Lisa trick alone was worth the price of the DVD.
Very Weird Report this review
Verified buyer Pro Privacy ON (login to see reviewer names) on April 3rd, 2010
Jay did it again. He went out of his way to create an entertaining, but high quality product.
There is soooo much material on this DVD to watch, but it's just hilarious to watch as well.
This is good if you want to add some mentalism to your act without needing to carry around a bunch of crazy props.
good & funny Report this review
Pro Privacy ON (login to see reviewer names) on May 31st, 2006
this DVD is a must have it funny and its good mental magic easy to do but hit ppl hard
Better than 22 Blows to the Head Report this review
Pro Privacy ON (login to see reviewer names) on October 13th, 2004
I saw Sankey Lecture in Cincinnati, and picked up this DVD. It exceeded my expectations. The effects are very strong, and so simple. The entertainment value is alone worth the price. I plan on using three or four effects from the DVD. They are very strong and practical.
Note: I do not like how people review products when they don't even own them. It clutters up the review pages. How can someone knock Sankey's new DVD when they don't even own it? Just my 2 cents... Jason
AWESOME Report this review
Pro Privacy ON (login to see reviewer names) on October 11th, 2004
FUNNIEST MAGIC DVD EVER Report this review
Pro Privacy ON (login to see reviewer names) on October 11th, 2004
The funniest mentalism dvd out there.
reply luciano Report this review
Pro Privacy ON (login to see reviewer names) on October 18th, 2004
Look, magic is for entertainment purposes and the point of entertainment is to capture your audiences attention and to keep it within your grasp. With magic, you capture it by performing feats which people would never think possible. That is what you are missing.
As for the DVD, I think that it is a very good DVD with some amazing effects in there. If there is one thing you will buy, GET IT!
More Featured Magic Tricks
0 0 : 0 0 : 0 0 remaining
Mind Stunts 2 by Patrik Kuffs
Every minute the price goes down. Only 1 available.
Updated hourly
0 0 : 0 0 : 0 0 remaining
Tarbell 39: Fundamental Card Sleights
See the next offer NOW with the CoolBox app
Act Builder beta
Get To Know Us
Shipping and Store Policies
Need Help?


text:
See all St. Gallen hostels
Hostel | Trogenerstrasse, St. Gallen, Switzerland 9042
87.6% Overall Rating
12 out of 12 customers would recommend this property
Recent review (23/09/2009)
"Good service , clean. Kept kitchen open later for us on arrival .Quiet , yet on tram line.Beautiful area."Read all Appenzellerhof reviews
- Credit cards accepted
- Car Parking
- Currency Exchange
- Secure lockers
- Lounge area
- Internet / Wi-Fi
- Restaurant
- Safety deposit
- 24 hour hot showers
- Laundry service
- Reception (limited hours)
- Wake-up calls
- Common area / room
- Internet access in public areas
- Pets allowed
What's Included
- Luggage Room
- Breakfast
- Linen
- Towel
Cancellation Policy
Customers can cancel their reservation free of charge up to 2 days before arrival (local time). Payments made to HostelBookers are non-refundable.
Check-In/Out Details
Earliest check-in: 15:00
Latest Check-out: 12:00
Group booking?
Group of 15 or more traveling to St. Gallen?Contact us directly
Speicher is 10 min form St.Gallen away und whether as a restaurant, holidays or hotel guest, you will be always spoiled by us. Read more
Enter dates to check prices
About Appenzellerhof
Whether as a restaurant, holidays or hotel guest, you will be always spoiled by us with well-balanced, natural and recover to culinary specialities. If always possibly we use products of farmers from the region and from biology production.Otherwise, we pay attention to fresh, healthy and tasty ingredients from biological cultivation.
See all St. Gallen hostels
Other languages
- Deutsch: Appenzellerhof
- Español: Appenzellerhof
- Français: Appenzellerhof
- Italiano: Appenzellerhof
- Português: Appenzellerhof


text:
Pryor Mountain Wild Horse Range
Public comment sought on Pryor wild horse population control programs
Many folks who comment on our website say that the BLM does not really listen to public comments. Unfortunately the only way we can be heard is by persistently communicating. This is directed especially to the biologists and other equine professionals out there – YOUR dialog due to your professional knowledge and scientific understanding is key. Statements which are ...Full Article
Fence must come down, say wild horse advocates
Word is spreading – the fence planned for by the US Forest Service and the Bureau of Land Management is NOT in the best interest of the wild horses ...Full Article
Pryor Mountain's Wild Mustangs
Jerry recently did a post about an article on the wild horses of the Pryor Mountain Mustang Center. It was due to Ginger Kathrens and the Cloud Foundation that ...Full Article
Wild Horse Management Suit Bucked by Judge
39,000 acres….120 horses equals not enough land??? ~ HfH From: Courthouse News Service By: Annie Youderian (CN) – The government's decision to rein in the wild horse population on ...Full Article


text:
Filipino bishops will return vehicles donated by government office
Catholic World News - July 08, 2011
Catholic bishops in the Philippines have announced that they will return luxury vehicles they received as gifts from the government's Charity Sweepstakes Office.
Political leaders have scolded the bishops for accepting gifts from the Sweepstakes Office, charging that the hierarchy was too close to the government of former President Gloria Magapagal-Arroyo. The charges of corruption have clearly been designed to weaken the bishops' opposition to proposed family-planning legislation.
The Filipino bishops' conference issued a statement this week saying that the gifts were used for legitimate charitable purposes. While denying any wrongdoing, the bishops nevertheless said that they would accept any legal consequences of receiving the gifts.
Additional sources for this story
Some links will take you to other sites, in a new window.
An appeal from our founder, Dr. Jeffrey Mirus:
Dear reader: If you found the information on this page helpful in your pursuit of a better Catholic life, please support our work with a donation. Your donation will help us reach seven million Truth-seeking readers worldwide this year. Thank you!
Our Fall Campaign
Progress toward our year-end goal ($162,782 to go):
$200,000.00 $37,217.72
81% 19%
All comments are moderated. To lighten our editing burden, only current donors are allowed to Sound Off. If you are a donor, log in to see the comment form; otherwise please support our work, and Sound Off!


text:
- Home
- » Eminem
- Back to Search Results
See below for Eminem concert tickets. Check 2015 tour dates, gigs and concerts information. Buy Eminem tickets in confidence with Live Nation.
Back to Search Results
We have no Eminem events at this time
Add this Artist to your Favourites to get the latest News and Updates


text:
Capital Markets
Company Overview of AP Asset Management (UK) Limited
BOARD MEMBERS AFFILIATED WITH Kwok Tai Tsang *
Chairman and Managing Partner
*Data is at least as current as the most recent Definitive Proxy.
MOST CONNECTED PEOPLE TO Kwok Tai Tsang
|James E. Rogers
|462 Relationships
|Margaret C. Whitman
|171 Relationships
|Thomas J. Tierney
|171 Relationships
|Steven A. Denning
|170 Relationships
|Muneer A. Satter
|166 Relationships
Sponsored Financial Commentaries
Sponsored Links
Report Data Issue
To contact AP Asset Management (UK) Limited, please visit . Company data is provided by Capital IQ. Please use this form to report any data issues.
Please enter your information in the following field(s):
Your requested update has been submitted
Our data partners will research the update request and update the information on this page if necessary. Research and follow-up could take several weeks. If you have questions, you can contact them at email@example.com.


text: Andreas Dippel | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Andreas Dippel Producer ( b. circa 1867 GERMANY - d. May 19, 1932 Hollywood, California, USA ) Male Productions ProductionsDate of ProductionsThe Love Mill[Musical, Comedy, Original] Produced by Andreas Dippel Feb 07, 1918 - Mar 23, 1918The Lilac Domino[Musical, Operetta, Original] Produced by Andreas Dippel Oct 28, 1914 - Jan 30, 1915 Find out where Andreas Dippel and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


text:
Chemical Resistance Charts
Product Announcement from GRI Pumps (A Gorman-Rupp Company)
The Chemical Resistance Charts provide ratings to reveal which chemicals work best with specific materials. These ratings have been developed from technical publications, material suppliers and laboratory tests, and are presented for your evaluation and not as a guarantee. Our staff will help determine material selections for testing.
Part Saved
You have successfully added from to your part list.
Save Part
For more information:
|Get More Info on Supplier's Site
|Request A Quote
|Email Supplier


text:
To embed, copy and paste the code into your website or blog:
In a ruling that may restrict the future viability of false advertising class actions, the Ninth Circuit recently overturned a lower court decision certifying nationwide class status to individuals who purchased or leased an Acura vehicle equipped with an optional "collision mitigation braking system" between August 2005 and December 2008. See Mazza v. American Honda Motor Company – opinion here. According to plaintiffs, advertisements for the $4,000 system were misleading in that they failed to disclose certain important facts, including that the system did not work in inclement weather (when collisions have been known to occur).
As certified, the class consisted of individuals who purchased their Acura vehicle in one of 44 states. Plaintiffs filed suit in federal court in California and sought false advertising relief solely under California's consumer protection law. Plaintiffs argued that California law should apply to all class members' claims, regardless of the point of actual purchase, because the defendant is headquartered in California and because the consumer protection law of that state is not materially different from the consumer protection laws of the other 43 states in which class members purchased the collision mitigation system.
Please see full article below for more information.
Firefox recommends the PDF Plugin for Mac OS X for viewing PDF documents in your browser.
We can also show you Legal Updates using the Google Viewer; however, you will need to be logged into Google Docs to view them.
Please choose one of the above to proceed!
LOADING PDF: If there are any problems, click here to download the file.
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
© Foley Hoag LLP - Trademark, Copyright & Unfair Competition
Your first step to building a free, personalized, morning email brief covering pertinent authors and topics on JD Supra:
Sign up to create your digest using LinkedIn*
Back to Top
Copyright ©


text:
Outdoors brushing her svelte, naked body against the soft flowers Caramel walks towards you through the long grass, her pert breasts bobbing as she walks.
Sliding off her bag and laying on a blanket Caramel lets her hands explore her tanned body. Moving over her smooth skin and towards her shaved vulva she bends over exposing her round bum. Exploring further she touches the sides of her beautiful pink labia...
Real people. HQ Leidsestraat 32D, 1017PB Amsterdam. +31 (0)20 622 7418.
♥ General enquiries: firstname.lastname@example.com;
♥ Owner: email@example.com.


text:
An overview of Different Software development models with a deeper look at RUP Development model
Posted by kalali on April 20, 2006 at 3:27 PM PDT
Here is an Presentation that i made last year . it is about different lifecycle models like spiral , waterfall and iterative models.
this presentation contain following contents :
- Part I
- Basics and Explanations
- Terms and definitions
- Well known Life Cycle models / Methodologies
- Waterfall
- Spiral
- Iterative
- Basics and Explanations
- Part II
- RUP as a SDP Life Cycle Model
- Phases and Milestones
- RUP and System Evolution
- EUP (Explanation)
- RUP as a SDP Life Cycle Model
you can get OO.org ODP from Here
Hoping you find it useful.
Related Topics >>
Blog Links >>
- Login or register to post comments
- Printer-friendly version
- kalali's blog
- 2055 reads


text:
| WDFW Home |
Favorites |
You must submit a hunter report even if you did not hunt. Failing to report by the deadline date will result in a hunter report fee.
Last Updated27th of March, 2015
Print this page
Email this page
Post a comment
Subscribe me
Add to favorites
Remove Highlighting
Edit this Question
Quick Edit
Export to PDF
Thank you for rating this answer.


text:
Building your own electric guitar was never easier! Here is your chance to express yourself by creating a one-of-a-kind masterpiece. Saga Electric Guitar Kit: LC-Style - The LC-10 is a single cut-away, arch-top machine that will easily accommodate any of today's music styles, design being limited only by your imagination! Hack it and make it your own!
Note: Drop Ships Via Manufacturer-Choose 3-Day or Ground Shipping Method Only-No USPS-U.S.A Only
Saga Musical Instruments has steadily grown to be one of the largest importers and distributors of stringed and fretted musical instruments in the world. For close to 30 years Saga has been a global pioneer in the successful development, marketing and distribution of high-quality student and professional level musical instruments.
You might also like...


text:
...the best surprise of the evening was the Pegu Club...
Pegu Club
2 oz London dry gin
3/4 oz orange curaçao (or Grand Marnier)
3/4 oz lime juice
dash Angostura bitters
dash orange bitters
Shake well with cracked ice. Strain into chilled cocktail glass.
The Pegu has been the drink I've been teaching folks around here lately. It is an excellent drink with wonderful complexities. The recipe above is slightly modified from the traditional/original version. You are using way too much lime juice, I think this would make the drink too tart (although I'll want to try this just to verify). The drink as it is often listed in the older books from the 30's is along the lines of:
2/3 part dry gin
1/3 part Curaçao
1 teaspoon lime juice
1 dash orange bitters
1 dash angostura bitters
(I hate it when recipes mix "parts" with real measurements... but that is the way most of the recipes listed it)


text:
Showing Suppliers 1 - 20 of 52
Perfect Parts Corporation (401388 Products)
Address: 7545 Irvine Center Drive Suite 200, Irvine, CA
Business Type: Distributor
Description: See what Perfect Parts customers are saying about our solutions What Drives Us To Perfection and The Highest Quality Standards in The Industry? Perfect Parts is a distributor of Electronic Components. Whether you are in need of electromechanical devices, passives,... (more)
ROHM Semiconductor USA, LLC (185 Products)
Business Type: Manufacturer
Description: ROHM is the industry leader in system LSI, discrete components and other modules, utilizing the latest in semiconductor technology. ROHM's proprietary production system, which includes some of the most advanced automation technology, is a major factor in keeping it at the forefront of the... (more)
View Catalog
Address: 6020 Cornerstone Court West, Suite 320, San Diego, CA
Address: 2323 Owen Street, Santa Clara, CA
Address: 2323 Owen Street, Santa Clara, CA
4 Star Electronics, Inc.
Address: 930 Calle Negocio, Suite C, San Clemente, CA
Business Type: Distributor
American Accurate Components (AAC)
Address: 188 Technology Center Drive, Suite H, Irvine, CA
Business Type: Manufacturer
Area51-ESG, Inc.
Address: 51 Post St., Irvine, CA
Business Type: Distributor
Aztec Components, Inc.
Address: 1201 Activity Drive, Vista, CA
Business Type: Distributor
BI Technologies Corporation
Address: 4200 Bonita Place, Fullerton, CA
Business Type: Manufacturer
Bourns, Inc.
Address: 1200 Columbia Ave., Riverside, CA
Business Type: Manufacturer, Distributor
C Plus Electronics, Inc.
Address: 17842 Irvine Blvd. #B144, Tustin, CA
Business Type: Distributor
Description: The Leader in Electronic Components Your Global Procurement Specialists, leading the market in innovative approaches to facilitating your supply chain management needs. As a leading independent distributor in the Global Semi-Conductor exchange, C Plus Electronics has forged partnerships with... (more)
Caddock Electronics, Inc.
Address: 1717 Chicago Avenue, Riverside, CA
Business Type: Manufacturer
Central Technologies
Address: One Park Plaza, Suite 600, Irvine, CA
Business Type: Manufacturer
Compostar Inc.
Address: 18019 Sky Park Circle, Suite A, Irvine, CA
Business Type: Manufacturer
Corad Technology, Inc.
Address: 3080 Olcott St., Suite 202B, Santa Clara, CA
Business Type: Distributor
Future Electronics
Business Type: Distributor
Description: More Than a Distribution Company Future Electronics' is a world-class leader and innovator in the distribution and marketing of semiconductors and passive, interconnect and electro-mechanical components. They operate in 169 locations in 41 countries in the Americas, Europe and... (more)
Address: 32 Discovery Suite 170, Irvine, CA
Address: 6256 Greenwich Drive, Suite 250, San Diego, CA
Address: 690 N. McCarthy Blvd., Suite 220, Milpitas, CA
Address: 1640 Lead Hill Road, Suite 200, Roseville, CA
Address: 30851 Agoura Rd., Suite 115, Agoura Hills, CA