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The Barley stripe mosaic virus (BSMV)-based vector has been developed and used for gene silencing in barley and wheat seedlings to assess gene functions in pathogen- or insect-resistance, but conditions for gene silencing in spikes and grains have not been evaluated. In this study, we explored the feasibility of using BSMV for gene silencing in wheat spikes or grains.
Apparent photobleaching on the spikes infected with BSMV:PDS at heading stage was observed after13 days post inoculation (dpi), and persisted until 30dpi, while the spikes inoculated with BSMV:00 remained green during the same period. Grains of BSMV:PDS infected spikes also exhibited photobleaching. Molecular analysis indicated that photobleached spikes or grains resulted from the reduction of endogenous PDS transcript abundances, suggesting that BSMV:PDS was able to induce PDS silencing in wheat spikes and grains. Inoculation onto wheat spikes from heading to flowering stage was optimal for efficient silencing of PDS in wheat spikes. Furthermore, we used the BSMV-based system to reduce the transcript level of 1Bx14, a gene encoding for High-molecular-weight glutenin subunit 1Bx14 (HMW-GS 1Bx14), by 97% in the grains of the BSMV:1Bx14 infected spikes at 15dpi, compared with that in BSMV:00 infected spikes, and the reduction persisted until at least 25 dpi. The amount of the HMW-GS 1Bx14 was also detectably decreased. The percentage of glutenin macropolymeric proteins in total proteins was significantly reduced in the grains of 1Bx14-silenced plants as compared with that in the grains of BSMV:00 infected control plants, indicating that HMW-GS 1Bx14 is one of major components participating in the formation of glutenin macropolymers in wheat grains.
This is one of the first reports of successful application of BSMV-based virus-induced-gene-silencing (VIGS) for gene knockdown in wheat spikes and grains and its application in functional analysis of the 1Bx14 gene. The established BSMV-VIGS system will be very useful in future research on functional analysis of genes contributing to grain quality and the metabolic networks in developing seeds of wheat.
Wheat (Triticum aestivum L.) is one of the major staple crops for the human diet. With the increase in the global population, the shortage of foods has become more and more serious. Therefore, wheat yield and quality improvement has always been the most important target in wheat breeding programs. Although conventional breeding approaches via manipulating genetic variation have been very successful in improving the agronomically important traits of cereals, the next wave of crop improvement will require much greater knowledge of gene function [1].
The rapid advance in genomics [2,3] has greatly facilitated gene isolation and manipulation, generating huge quantities of transcript and putative gene sequences (http://www.ncbi.nlm.nih.gov/guide/dna-rna/). In addition, genomes of major crops including rice, maize, and wheat have been or are being sequenced. As a result of rapid advance in structural genomics, the availability of powerful tools for gene function analysis has become a bottleneck, especially for important crops beyond the few model plant species.
In model plants such as Arabidopsis and rice, two methods, i.e. T-DNA knockout libraries [4] and T-DNA activation libraries [5], have significantly accelerated the speed of gene functional identification. Large collections of plants that contain T-DNA insertions have been generated. Once plants have been identified with the desired phenotypes, isolation of relevant genes is readily accomplished by finding the genomic location of the T-DNA associated with the mutant phenotype [1]. However, none of these techniques can be used in wheat due to the large genome size and very low transformation efficiency which pose a technical challenge to produce the number of transformants needed to saturate the wheat genome. An additional complication for determining gene function through the analysis of loss-of-function mutations in wheat is the fact that all cultivated varieties are polyploid; therefore in most cases, expression of homeologous genes could mask loss-of-function phenotypes resulting from the disruption of the other homeologous alleles [1].
In recent years, virus-induced gene silencing (VIGS) has been developed as an effective genetics tool for assessing gene functions in a range of dicot plant species including Nicotiana spp. (tobacco), Pisum sativum (pea), ArabidopsisSolanum lycopersicum (tomato) [6-9], and some monocot species Hordeum vulgare (barley), Triticum aestivum (wheat), and Zea mays (maize) [10-12]. VIGS was a mechanism of defense response naturally present in plants and other organisms through RNA-mediated post-transcriptional gene silencing (PTGS) to fight pathogens [13,14]. In VIGS, viruses trigger defense machinery of the hosts related to post-transcriptional gene silencing, where double stranded RNA is converted into short interfering RNAs (siRNAs). A gene of interest can be introduced into a virus vector and the recombinant virus will trigger the host defense response and both the virus genome and the endogenous mRNAs homologous to the inserted target sequence become the targets for degradation [15,16]. Silencing initiated by VIGS will spread systemically along with the siRNA [17,18], and by this method, it is possible to knockdown almost any gene of interest if a suitable vector is present for the plant species under investigation.
Many viral vectors for VIGS have been developed for various plant species [6,7,10,19-22]. However, so far, most VIGS protocols are established for gene silencing in vegetative tissues, with very limited success in reproductive and other tissues [23-29]. Gene silencing techniques for developing seeds will be very useful for studying gene functions in seed development and for seed quality improvement. For monocotyledons, two vectors, the Barley stripe mosaic virus (BSMV)-based vector for barley and wheat [10,11] and the Brome mosaic virus (BMV)-based vector for barley, rice and maize [12], have been developed and are being widely used for gene silencing in seedlings [30-32]. More recently, Pacak et al. [33] presented the first evidence of BSMV induced gene silencing (BSMV-VIGS) in the roots of a monocotyledonous plant species. To date, however, no reports have been published on BSMV-VIGS in spikes or grains of monocotyledonous species. Genes expressed in spikes or grains are likely involved in important agronomic traits such as yield, quality and disease resistance, and therefore, revealing the functions of these genes will greatly facilitate the improvement of yield, quality and pathogen resistance in cereals [34-37]. Accordingly techniques for analyzing gene functions in spikes or grains are needed for functional analysis.
High-molecular-weight glutenin subunits (HMW-GSs) are a group of endosperm storage proteins in wheat encoded by a multi-gene family. Members in this gene family possess highly homologous sequences, as found in Glu-A1Glu-B1 and Glu-D1 loci on the long arm of chromosome 1A, 1B and 1D, respectively [38]. The genes at the three loci share a high percentage of homologous sequences. At each locus, one x-type and one y-type subunits are encoded. The composition and amount of HMW-GSs have a profound influence on the baking quality of wheat [39,40]. HMW-GS 1Bx14 is encoded by the gene at the Glu-B1 locus. Several studies have shown that the 1Bx14+1By15 subunit pair has a positive influence on the end use quality of wheat varieties [41]. More recently, the 1Bx14 gene has been isolated and sequenced (GenBank accession: AY367771.1). A 1Bx14 knockout mutant derived from an elite Chinese wheat variety Xiaoyan 54 through chemical mutagenesis was generated and characterized, providing an opportunity for identifying the biological function of 1Bx14[42]. We tried to generate transgenic lines with a 1Bx14 over-expressed construct by microprojectile bombardment to assess the gene function, but introduction of this gene led to silence of several other homologous endogenous genes in the common wheat cv. Mianyang 19 [43].
Our long term goal is to elucidate the effect of specific proteins on wheat grain quality and end use. In this study, we first tested the possibility of employing BSMV vector to silence genes in wheat spikes or grains with the marker gene PDS encoding phytoene desaturase, an enzyme required for the biosynthesis of carotenoid pigments that protect chlorophyll from photobleaching. Silencing of PDS can be visualized as white streaks resulting from photobleached chlorophyll [10]. After optimizing the locations and stages for VIGS in wheat spikes and grains, we chose to silence the HMW-GS 1Bx14 gene because the protein products have been shown by our group to participate in forming larger glutenin polymers and greatly contribute to dough strength [44]. We also chose to silence the entire family of HMW-GS genes related to wheat gluten quality for testing the feasibility of BSMV-VIGS for functional analysis in wheat grains (Figure (Figure1).1). The results in this study indicate that BSMV-VIGS system is a useful tool for functional analysis of genes expressed in wheat spikes and grains.
To examine whether BSMV vector can induce gene silencing in wheat spikes and grains, we used the BSMV vectors carrying a 185-bp fragment of barley PDS gene (designated as BSMV:PDS) [11,45] for the initial silencing experiment. Two winter wheat, cvs Xiaoyan 6 and Shaanyou 225, and a spring wheat, cv. Ningchun 16, were used in this experiment. To test the infectivity and the efficacy of BSMV-VIGS in all three wheat cultivars, we conducted a preliminary experiment at the seedling stage to silence the PDS gene. We inoculated ten seedlings of each wheat cultivar with BSMV:PDS and BSMV:00 (for only the BSMV genome without the target gene), respectively, onto the 2nd fully expanded leaves of 10-day-old seedlings. Mosaic and chlorotic stripes were observed on the tip portions of the third leaves of all inoculated plants about 7days post inoculation (dpi). Only the BSMV:PDS inoculated plants (10 out of 10) showed photobleaching starting from the base areas of the third leaves and extending throughout the length of the fourth leaves during 10~30 dpi. Photobleaching was very rarely seen in the fifth leaves of all three cultivars (data not shown). No photobleaching was observed in the plants infected with BSMV:00. These observations indicated that the BSMV:PDS construct could systemically induce gene silencing in all three cultivars and efficiently induce PDS gene silencing in these cultivars.
In order to investigate the effectiveness of BSMV-VIGS in wheat spikes, endogenous PDS was again selected as a target to silence. We inoculated ten plants of each wheat cultivar on their spikes at heading stage by rubbing each spike five times with BSMV:PDS and BSMV:00, respectively. Unlike the progress of BSMV:PDS infection observed on leaves of wheat seedling, no mosaic symptoms and chlorosis were observed on wheat spikes by 13 dpi; only some ring-like or stripe symptoms were visible on the awns of some infected spikes. The symptoms of wheat spikes infected with BSMV:PDS were indistinguishable from those infected with BSMV:00 at 13 dpi or earlier. However, photobleaching on the spikes infected with BSMV:PDS appeared after 13dpi, and persisted until 30dpi, while the spikes inoculated with BSMV:00 remained green during the same period (Figure (Figure2A).2A). Nine of the ten plants (90%) inoculated with BSMV:PDS showed photobleaching throughout the entire wheat spike (Table (Table1),1), and the photobleaching persisted until 30 dpi (Figure (Figure22A).
To confirm that the observed photobleaching was the result of PDS silencing, we conducted quantitative real-time PCR (qRT-PCR) to measure the PDS transcript abundances in the spikes inoculated with BSMV:PDS at different time points. Surprisingly, a 25% reduction in PDS transcript abundance was detected as early as 2 dpi, but a remarkable reduction of PDS was detected between 8–13 dpi. From 4–30 dpi, the PDS mRNA level in spikes treated with BSMV:PDS was only about 60%, or lower than the level in spikes with BSMV:00 treatment (Figure (Figure2B).2B). The most significantly bleached spikes were seen at 22 dpi, much later than the reduction of the PDS transcript level.
The effectiveness of VIGS in wheat grains was then investigated. Grains from BSMV:PDS infected spikes appeared photobleached only when their glumes were taken off and the grains were exposed directly under bright light. The phenotypes of the grains from the heads infected with BSMV:PDS or BSMV:00 at 22 dpi are shown in Figure Figure3A3A with cultivar Xiaoyan 6 as a representative. Chlorosis occurred in the grains of BSMV:PDS infected heads at 22 dpi, while the grains of BSMV:00 inoculated control plants remained green, suggesting photobleaching of these seed coats was due to the silencing of the PDS gene. To confirm this, transcript abundances of the PDS gene in the photobleached and non-photobleached grains were measured by qRT-PCR. The level of PDS transcript abundance in the chlorotic grains was reduced by 92% (P<0.01), compared with the green ones (Figure (Figure33B).
Similar to leaf-rub inoculation applied to wheat seedlings [1,11], spike-rub inoculation on the spikes at heading stage was also effective for gene silencing in spikes and grains. Syringe-injection inoculation onto the spikes at heading stage was also tested, but this method failed to get results as consistent as spike-rub inoculation. Therefore, spike-rub inoculation was used in all silencing assays later. Because photobleached phenotypes of the spikes or grains in BSMV:PDS inoculated plants exhibited a consistent correlation with the reduction of PDS transcript abundances in our experiments, photobleaching was used as an indicator of PDS gene silencing in wheat spikes and grains.
To optimize conditions for efficient and stable silencing of genes in wheat spikes and grains, inoculation location and stage were determined by inoculating BSMV on flag leaves or spikes of ten plants for each wheat cultivar at three different development stages, i.e. booting, heading and flowering, respectively, by rubbing the in vitro synthesized BSMV RNAs three times on a flag leaf or five times on a spike. For simplicity, only the winter wheat cv. Xiaoyan 6 and the spring wheat cv. Ningchun 16 were tested at three different stages, and at each stage, the experiment included three independent biological replicates. As shown in Figure Figure4,4, at 22 dpi, photobleached spikes were observed in BSMV:PDS infected Xiaoyan 6 plants if BSMV:PDS was inoculated onto spikes at booting, heading or flowering stages. But if BSMV:PDS was inoculated onto flag leaves, photobleaching spikes appeared only in wheat plants inoculated at heading stage. These results were reproducible in three biological replicates. Figure Figure44 shows three reproducible phenotypes with different degrees of photobleaching, i.e. green spikes (no photobleaching), partially photobleached spikes (partial photobleaching) and uniform highly photobleached spikes (complete photobleaching). Effectiveness of BSMV-induced PDS silencing in wheat spikes was strongly influenced by the location of the inoculation and the stage of the plant when the inoculation was done. From heading to flowering stage, 90% (9/10)~100% (10/10) of plants inoculated with BSMV:PDS onto spikes exhibited highly uniform photobleaching (Table (Table1).1). Similar results were also observed in wheat cv. Ningchun 16 (data not shown). We also attempted inoculation onto wheat spikes 10days after flowering, but failed to achieve silenced phenotypes.
Taken together, inoculation onto wheat spikes from heading to flowering stage was optimal for efficient silencing of PDS in wheat spikes.
After optimizing the BSMV-based VIGS system in wheat spikes/grains, we tried to explore the usefulness of the system for functional analysis of genes expressed in wheat grains. The 1Bx14 gene (GenBank accession number AY367771.1) was chosen as a gene of interest tested in two wheat cultivars carrying 1Bx14, Xiaoyan 6 and Shaanyou 225. To make gene silencing as specific as possible, a 176-bp fragment of 1Bx14 from the coding region (corresponding to the region of 98 to 273-bp downstream of the start codon) was inserted into the TA cloning site of the BSMV γ vector to generate the construct BSMV:1Bx14 (Figure (Figure1A1A and and1B).1B). The 176-bp insert fragment possesses approximately 60–82% identity to the DNAs of the HMW-GS gene 1Ax1, 1By15, 1Dx2 and 1Dy12 in wheat cvs Xiaoyan 6 and Shaanyou 225 (Additional file 1).
Ten plants of each wheat cultivar were inoculated with BSMV:00 and BSMV:1Bx14 respectively on the spikes at flowering stage. The grains in the middle of the infected spikes were collected at 15, 20, 25 and 30 dpi for RNA isolation. Transcript abundances of 1Bx14 were measured in all the grain RNA samples by qRT-PCR. The data showed that the transcript levels of 1Bx14 decreased by about 97% at 15 dpi and 85% at 20 dpi in the grains of BSMV:1Bx14 infected spikes compared with that of the BSMV:00 infected control. Actin was used as an internal control. The reduction persisted until at least 25 dpi (Figure (Figure5).5). These data were similar to the results of PDS silencing described above. The amount of HMW-GS 1Bx14 of the 1Bx14-silenced grains of Xiaoyan 6 and Shaanyou 225 was detectably decreased on Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure (Figure6A6A and and6B).6B). The scanning profiles of the SDS-PAGE gels shown in Figure 6 A and and6B6B shows the quantitative reduction of protein level; i.e. the peak of HMW-GS 1Bx14 in the 1Bx14-silenced grains was about 64% reduced in cultivar Xiaoyan 6 (Figure (Figure6C)6C) and 67% in Shaanyou 225 (Figure (Figure6D)6D) compared with that in the controls, respectively. The protein levels of 1Bx14 were not reduced as much as the mRNA levels (Figure (Figure6E6E and and66F).
To evaluate the impact of 1Bx14 knockdown in the formation of wheat gluten network, total proteins (TP), total glutenin polymer(TGP) and glutenin macropolymeric protein (GMP) of the grains from BSMV:Bx14 and BSMV:00 infected spikes were extracted and quantified by micro-Kjeldahl method, respectively. The quantity of GMP, TGP or TP in the grains of 1Bx14-silenced plants were significantly decreased, compared with that in BSMV:00 infected controls in both Xiaoyan 6 and Shaanyou 225. The ratio of GMP to TP or the percentage of GMP in TP was significantly reduced (Table (Table2),2), indicating that HMW-GS 1Bx14 is one of the major components participating in the formation of glutenin macropolymers in wheat grains. These results are consistent with our previous conclusion, suggesting the usefulness of BSMV-VIGS system for the functional analysis of genes expressed in wheat grains.
The successful silencing of the 1Bx14 gene by the BSMV-VIGS system encouraged us to silence all the HMW-GS gene family members in wheat grains. A BSMV-derived construct carrying a 184-bp fragment of artificially assembled sequence (asHMW) was developed and named as BSMV:HMW. The 184-bp fragment contained one 85-bp sequence highly homologous to most of the x-type glutenin subunit genes (asX) and one 99-bp sequence identical to most of the y-type glutenin subunit genes (asY) (Figure (Figure1C).1C). Nucleotide sequence alignment of the asX or the asY with the corresponding elements in other x-typeor y-type HMW-GS genes in wheat was shown in Additional file 2.
All three wheat cultivars mentioned above were tested in this experiment. Ten plants of each wheat cultivar were inoculated with BSMV:HMW or BSMV:00 on the spikes at flowering stage. Silencing of HMW-GS genes was monitored by SDS-PAGE on protein levels due to the lack of primers specific to individual target genes for real-time PCR. As shown in Figure Figure7,7, the amount of each x-type or y-type HMW-GS extracted from the seeds of the BSMV:HMW infected spikes of Ningchun 16 showed detectable decrease compared with the BSMV:00 treated spikes, indicating that all the HMW-GS genes 1Ax2*, 1Bx17, 1By18, 1Dx5 and 1Dy10, were down-regulated in the seeds of BSMV:HMW infected spikes of Ningchun 16. However, in the seeds of BSMV:HMW infected spikes of Xiaoyan 6 and Shaanyou 225, no apparent reduction in amount of HMW-GS was observed, suggesting that all the HMW-GS gene family members were not efficiently silenced.
Despite the widespread use of VIGS in seedling plants, its application in reproductive tissues of plants is rather limited. In the review by Scofield & Nelson [46], the authors mentioned that VIGS could be achieved in the flag leaves and floral organs by inoculating upper leaves of older wheat plants with the BSMV RNAs, citing unpublished data without further details or documentation. In wheat, to date, there is no report of the application of VIGS in developing seeds. In this study, we report, for the first time, the successful application of BSMV-based VIGS for gene knockdown in wheat spikes and grains and its application in functional analysis of HMW-GS-encoding gene 1Bx14. Unlike Cucumber mosaic virus (CMV)- or Apple latent spherical virus (ALSV)-vector for VIGS in soybean (Glycine max), with which inoculation onto the first leaf at the seedling stage or two cotyledons at emergence stage of soybean plants could effectively lead to gene silence in seeds [28,29], inoculation with BSMV:PDS recombinant virus onto the first leaf of wheat or barley plants at the seedling stage led to the most efficient PDS silencing in the third leaf, but the silence effect largely disappeared before the onset of flowering in nearly all plants. Occasionally, some degree of photobleaching was observed in the flag leaf and some of the heads [47]. When barley or wheat were infected with the BSMV:PDS construct, the duration of silencing lasted only about 4weeks post-inoculation [11,47].
Considering both the duration of effective BSMV-based VIGS and the temporal and spatial expression of some genes related to wheat quality or yield, the optimal inoculation conditions for VIGS in wheat spikes and grains are needed to be determined for efficient gene silencing in wheat productive organs. In this study, we established a protocol for effective knockdown of genes expressed in wheat spikes and developing seeds using BSMV-based VIGS. We found that efficiency of BSMV-induced gene silencing varied greatly with the locations and the stages of inoculation, and inoculation with BSMV on wheat spikes from heading to flowering stage by spike-rubbing five times showed the most effective silencing of PDS in wheat spikes (Table (Table1).1). Since gene silencing with BSMV-based VIGS vector is generally transient, inoculation on spikes at the appropriate time is crucial for knockdown of genes expressed only in developing seeds.
Compared with BSMV-VIGS experiments with barley or wheat seedlings, the main visual differences distinguishing BSMV-VIGS in wheat spikes are viral symptoms, the phenotypes of plants with gene silencing, and the time period of the effect. Wheat plants inoculated with BSMV:PDS at the seedling stage exhibited patches of lesion in the leaf above the inoculated one[11]. However, for wheat plants infected with BSMV:PDS onto spikes, only ring-like or stripe symptoms appeared on the awns of infected spikes. As for the phenotype of PDS silencing, both leaves and spikes showed apparent photobleaching. However, photobleaching in the leaf often did not cover the entire width of the leaf and was often confined to narrow stripes parallel to the leaf veins [10], while photobleaching in the spike extended to the entire spike (Figure (Figure2A2A and Figure Figure4).4). With regard to the effective time period of BSMV-VIGS, evident photobleaching initially appeared in the third and fourth leaves of the barley seedlings by 7 dpi, and leaves that emerged after 21 dpi were unlikely to have photobleaching when BSMV:PDS inoculation was done on the first leaves [10]. However, our results showed that, when wheat spikes were infected with BSMV:PDS, visual photobleaching appeared on the spikes after 13 dpi, and persisted until 30 dpi. The qRT-PCR data revealed that PDS transcript abundances in BSMV:PDS infected spikes reduced by about 25% at 2 dpi, 95% between 8~13 dpi, and 45% at 22 dpi, compared with those in spikes inoculated with BSMV:00 (Figure (Figure2B).2B). But the most remarkable photobleaching was observed at 22 dpi, much later than the reduction of PDS transcript. The reduction in PDS mRNA levels began earlier and lasted longer than the period of photobleaching (Figure (Figure22). Notably, PDS transcript abundances in BSMV:PDS infected spikes were prone to increase at 22 dpi and 30 dpi. This is due to the instability of recombinant BSMV:PDS leading to deletion of PDS sequences from the virus vector (data not shown). These conditions were also reported previously by Bruun-Rasmussen et al. [47].
After optimization of conditions, we also successfully silenced the gene 1Bx14 that encodes storage protein in wheat endosperm. The correlated reduction in the levels of the mRNA and the protein of HMW-GS 1Bx14 suggested VIGS-treatment can effectively knockdown genes expressed in developing seeds (Figure (Figure6).6). Notably, the reduction in mRNA-level and in protein level of 1Bx14 in Figure Figure66 was not comparable. We speculate that the reason is that the protein abundances shown in Figure Figure6A6A and and6B6B were the consequence of about a 10-day-accumulation because HMW-GS genes initially expressed 9~12days after flowering, while the transcript abundances shown in Figure Figure6E6E and and6F6F were the mRNA level of 1Bx14 in the 1Bx14-silenced grains at 20dpi due to the short lifetime of mRNA. The peak of gene silencing induced by BSMV was at 20dpi according to qRT-PCR results in the BSMV:1Bx14 time course study (Figure (Figure55).
However, when silencing the entire HMW-GS gene family in wheat grains with the BSMV:HMW construct, the results were not the same among the three wheat cultivars. All the HMW-GS genes 1Ax2*1Bx171By181Dx5 and 1Dy10 in spring wheat cv. Ningchun 16 were detectably down-regulated in the seeds of BSMV:HMW infected spikes, but no apparent reduction was detected in the seeds from BSMV:HMW infected spikes of cultivars Xiaoyan 6 and Shaanyou 225. Scofield et al. [11] also reported that only 21% of the wheat plants and 16% of the barley plants displayed any detectable photobleaching when infected with the BSMV:PDSas construct carrying an 80-bp PDS4as derivative fragment, and no photobleaching was observed in any of the plants infected with the 40-bp PDS4as derivative. The sizes of the inserts greatly affect the efficiency of BSMV induced gene silence. Therefore, we think that the inconsistency among the three wheat cultivars for silencing HMW-GS gene combinations may be due to the sizes of inserts--asX (85-bp) plus asY (99-bp) were not long enough for induction of efficient silencing.
It has been postulated based on early genetic studies [34,40] that the composition and the amount of HMW-GS in the grains can greatly affect gluten strength and thereby the end use qualities of both tetraploid and hexaploid wheat. The observation of significant alteration in the structure of gluten complex and the processing properties of wheat grains in a transgenic line that over-expresses a single HMW glutenin subunit reinforces this postulation [48]. However, more systematic analyses of the direct effects of HMW-GS genes on grain properties are difficult due to technical difficulties in development of an ideal transgenic line that has the target HMW-GS gene over-expressed without affecting other endogenous genes' expression in wheat grains. There have been many efforts in developing transgenic wheat lines to confirm the function of HMW-GS genes, but most of them could not reach the final stage due to very low transformation efficiency and un-intended side effects in the transgenic wheat lines generated. For example, introduction of a construct overexpressing the gene 1Ax1 into a commercial spring bread wheat resulted in the inactivation of the 1Ax2* allele. Transformation of constructs overexpressing both 1Ax1 and 1Dx5 resulted in the silencing of all other HMW glutenin subunits [49]. The technical difficulties and phenotypic complication associated with transgenic approaches make the development of VIGS-based techniques to silence genes in grains more useful and urgent. In this study, we successfully silenced the 1Bx14 gene, which is highly expressed in seeds of wheat cvs Xiaoyan 6 and Shaanyou 225 (Figure (Figure6E6E and and6B).6B). Silencing of 1Bx14 led to a significant decrease in the quantity of GMP and the percentage of glutenin macropolymeric protein in total proteins (GMP/TP) of the grains (Table (Table2),2), indicating that HMW-GS 1Bx14 plays very important roles in the formation of glutenin macropolymers (or wheat gluten network). By using purified HMW-GS 1Bx14 as a supplement to base flour to make dough and test its functional properties, our group found that incorporation of the HMW-GS increased the quantity of glutenin macropolymeric protein (GMP) in the dough and enhanced the dough strength [44]. The present results further support this conclusion. Unexpected was that a reduction of 7%~9% in the total protein content was detected in grains of 1Bx14-silenced plants, although an individual glutenin subunit accounts for only about 2% of total grain proteins. The effect of 1Bx14 silencing on total proteins needs to be further investigated. To our knowledge, this is the first report of knockdown genes expressed in wheat developing seeds with recombinant BSMV. The establishment of this effective approach for gene knockdown in developing seeds will allow us to dissect the genetic pathways that control seed development, grain quality and pathogen defense in grain tissue of hexaploid wheat; it will also provide an important reference for silencing genes in spikes or grains of other monocotyledonous species by VIGS.
We first demonstrated the feasibility of using BSMV for gene silencing in wheat spikes or grains with PDS as a marker gene. We determined that inoculation with BSMV onto wheat spikes from heading to flowering stage by spike-rub five times was the optimal condition for silencing PDS in wheat spikes. In addition, we successfully silenced HMW-GS gene 1Bx14 expressing in the developing seeds through the established BSMV-VIGS approach. The BSMV-VIGS system in spikes and grains will be very useful in future research on functional identification of genes contributing to grain quality and controlling the metabolic networks in seed development of wheat.
Three hexaploid wheat (T. aestivum L.) cultivars, two winter wheat cvs Xiaoyan 6 and Shaanyou 225, and one spring wheat cv. Ningchun 16, were used in the experiments. The two winter wheats are elite cultivars with good quality, and both possess identical HMW glutenin subunit genes, i.e. 1Ax1, 1Bx14, 1By15, 1Dx2 and 1Dy12, while Ningchun 16 has HMW glutenin subunit genes 1Ax2*, 1Bx17, 1By18, 1Dx5 and 1Dy10. For VIGS experiments, all three cultivars were grown in a greenhouse with a day/night temperature regime of 20–25°C/15–18°C, a light period of 16h/8h day/night, regulated with supplementary light, and watered as needed. The two winter wheat cultivars were exposed to a temperature of 4°C for 40days to achieve complete vernalization, while the spring wheat cultivar was exposed to 4°C for 14days at the two leaves stage. The temperature of the greenhouse was kept at a constant 22°C after the booting stage of the three cultivars.
The BSMV vectors utilized in these experiments were obtained from Dr. Andrew O. Jackson at UC Berkeley [50]. The γ vector was reconstructed to include PCR-ready cloning sites following a protocol modified from Holzberg et al. [10]. The plasmids utilized in the experiments of silencing PDS in wheat spikes/grains were described in Campbell [45]. In details, the BSMV γ vector was digested with NotI/PacI and inserted the sequence of GGCCCCACTCATGACATGGCGTTAGCCATGGGAAGCTTGGAT, including two XcmI restriction sites. The modified γ vector (named the γ PCR vector) was linearized with restriction enzyme XcmI to produce a TA cloning site for direct cloning of PCR products. Since a BSMV γ RNA construct carrying a 185-bp fragment of the barley (Hordeum vulgare L.), the PDS gene in antisense orientation (BSMV:PDS4as) induced optimal silencing of PDS in wheat leaves as described previously [11], we used the BSMV:PDS4as for initial testing under our experimental conditions. For simplicity, the BSMV:PDS4as construct was named as BSMV:PDS and the BSMV-derived construct with no insert as BSMV:00 in our experiments.
The BSMV construct utilized to silence the 1Bx14 gene, which carrying a 176-bp fragment of 1Bx14 cDNA, was designed as BSMV:1Bx14 and constructed as follows: The 176-bp fragment of 1Bx14 from the coding region (corresponding to the region of 98 to 273-bp downstream of the start point of translation) was generated by PCR amplification from the plasmids containing the cloned 1Bx14 gene with the forward primer P6:5'- GCGAGCTCCGGAAGCGCG-3' and reverse primer P7: 5'- CGAAGGCGTAGTCTCGCTGGGG-3', and inserted into the γ vector (Figure (Figure1A1A and and1B).1B). The analysis of the identity between the 176-bp fragment and the full-length of HMW-GS gene 1Ax1, 1Dx2, 1Bx14, 1By15 and 1Dy12 in wheat cultivars Xiaoyan 6 and Shaanyou 225 was conducted with the DNAMAN software (http://www.lynnon.com).
The BSMV construct carrying a 184-bp fragment of an artificially assembled sequence (asHMW) with one 85-bp sequence identical to most x-type glutenin subunit genes (asX) and one 99-bp sequence identical to y-type glutenin subunit genes (asY), named as BSMV:HMW, was generated (Figure (Figure1C)1C) and used to silence the entire HMW-GS gene family in wheat grains. The BSMV:HMW was constructed as follows: The fragment containing an 85-bp fragment of 1Bx14 from the encoding region (corresponding to the region of 296 to 380-bp downstream of the start codon) and a 99-bp fragment of 1By16 (GenBank accession number EF540765.1) from the coding region (corresponding to the region of 798 to 896-bp downstream of the start codon) were synthesized (by Sangon, Shanghai, China), and inserted into the γ vector. The 85-bp sequence shared a 90.59–100% similarity to the corresponding elements in all x-type HMW-GS genes and the 99-bp sequence shared a 94.95–100% similarity to those in all y-type HMW-GS genes (Additional file 2).
The procedures for in vitro transcription of viral RNAs were the same as described by Scofield et al. [11]. In vitro synthesized BSMV RNAs were rub-inoculated onto flag leaves or spikes at three development stages, i.e. booting, heading and flowering, respectively.
Ten plants/cultivar were infected with each of BSMV+target, and BSMV:00, respectively, with three biological replicates. Inoculation on flag leaves was performed as described by Scofield et al. [11], while inoculation onto spikes was conducted by 5 times sliding with three gently pinched fingers from base to tip of the spikes.
Total RNA was isolated from wheat spikelets or grains by using RNAiso-mate (TaKaRa, Dalian, China) and RNAiso Plus (TaKaRa, Dalian, China) followed by cold phenol/chloroform extraction three times. The quality and concentration of total RNA were determined with a Nanodrop ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). All the RNA samples were treated with RNase-free DNase I (TaKaRa, Dalian, China) prior to synthesizing cDNA as recommended by the manufacturer. For RT-PCR analysis of PDS and 1Bx14 transcript abundances, first-strand cDNA was synthesized using 500ng of total RNA, oligo (dT) primer and MMLV reverse transcriptase (TaKaRa, Dalian, China).
For analysis of BSMV-VIGS in wheat spikes or grains, the spikelets or grains in the middle of BSMV infected spikes were collected at 2, 4, 8, 15, 20, 22 and 30dpi.
Expression of the genes targeted for silencing was quantified by comparative quantitative real-time PCR (qRT-PCR). QRT-PCR was performed in triplicate for each RNA sample/primer combination. The amount of RNA in each reaction was normalized using primers specific for actin. The primer sequences used to detect each gene were as follows: actin forward, 5'-AAATCTGGCATCACACTTTCTAC-3'; actin reverse, 5'-GTCTCAAACATAATCTGGGTCATC-3';PDS forward P3, 5'-TGTCTTTAGCGTGCAAG-3', PDS reverse P4, 5'-GATGATTTCGGTGTCACT-3'; 1Bx14 forward P5, 5'-TAAGCGCCTGGTCCTCTTTGCG-3', 1Bx14 reverse P7, 5'-CGAAGGCGTAGTCTCGCTGGGG-3'. The positions of the primer set used for qRT-PCR are shown in Figure Figure1B.1B. QRT-PCR was operated three times on iCycler iQTM Multi-Color Real Time PCR Detection System (Biorad, Hercules, CA, USA) using SYBR Green Master Mix (Biorad, http://www.bio-rad.com). The cycling conditions were as follows: 2min at 95°C, followed by 45cycles of 30s at 95°C, 30s at 55°C and 30s at 72°C. In all cases, the relative expression of the targeted gene is presented as the expression level of this gene in silenced plants relative to that of the same gene in plants infected with BSMV:00, and the values of gene expression were the averages of three independent biological replicates. For each PCR, the specificity of the amplifications was validated and the threshold cycle above background was calculated using Bio-Rad iCycler software. PCR efficiency was close to 100%. Relative quantification of the gene transcript abundances was carried out applying an improved ΔΔ analysis [51]. Error bars in all figures showing qRT-PCR data indicated the standard deviations calculated from the original CT (cycle threshold) values. The P-values were estimated using hypothesis test (student test).
To determine the efficacy of 1Bx14 silencing at protein level, grains in the middle of wheat spikes from BSMV:1Bx14, BSMV:HMW or BSMV:00 inoculated plants were collected at 20 dpi, respectively, and HMW-GSs were extracted from one grain of each sample with 200ul solution (50%(v/v) 1-propanol, 0.625mol/L Tris–HCl (pH=6.8), 10% SDS, 2% DDT, 20% Glycerin and 0.2% Bromophenyl blue). Equal volumes (20μl) of each protein sample were loaded and separated by SDS-PAGE with 5% stacking and 12% resolving polyacrylamide. Each treatment was performed with three independent biological replicates.
Each grain sample of approximate 0.2g was milled and equipped with a 0.5mm screen to obtain wholemeal flour. Total glutenin polymers free from monomeric proteins and glutenin macropolymeric proteins (50% 1-propanol insoluble fraction) of each wholemeal flour sample were isolated as previously described by Xu et al. [44]. The protein contents of total glutenin polymer, glutenin macropolymer or wholemeal flour were determined by micro-Kjeldahl (Foss Tecator AB, Hoganas, Sweden) analysis. The amount of proteins was estimated as N×5.7. Each treatment was performed with three independent biological replicates.
MM carried out the VIGS, genomic analysis and qRT-PCR, created the figures, and ran the SDS-PAGE. YY supported the VIGS analysis and raised the plant material. LH provided the BSMV vectors, technical advice, and edited the manuscript. MC participated in modification of the manuscript. HZ designed the experiments and coordinated their implementation. MM and HZ participated in drafting the manuscript. All authors read and approved the final manuscript.
MM, YY, and HZ: State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A & F University, Yangling, 712100, Shaanxi, China.
LH: Department of Plant Sciences & Plant Pathology, Montana State University, Bozeman, MT59717, USA.
MC: USDA-ARS and Department of Entomology, Kansas State University, Manhattan, KS66506, USA.
Identity of the 176-bp fragment of1Bx14with the full-length ofHMW-GSgene1Ax1, 1Bx14, 1By15, 1Dx2and1Dy12in wheat cultivars Xiaoyan 6 and Shaanyou 225.
Nucleotide sequence alignment of theasXor theasYwith their corresponding elements in other x-type (upper region) or y-type (lower region)HMW-GSgenes expressed in the wheat variety, respectively;asXandasYrepresent sequences highly homologous to most of all x-type or y-typeHMW-GSgenes, respectively. Nucleotides conserved in all sequences are represented by '*'. The alignment was conducted using the Clustal W program.
This work was financed by Natural Science Foundation of China (30871578). We would like to thank Jackie Campbell, a Ph.D student of Dr. Li Huang in the Department of Plant Sciences and Plant Pathology, Montana State University, for her suggestions and discussions in exploring the technical feasibility of using BSMV for gene silencing in wheat spikes or grains with the PDS marker gene.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Double_deez Model
Ed Vaughn PhotographyPhotographer Male North Las Vegas, Nevada, US
My Website: ED VAUGHN PHOTOGRAPHYMy MM URL: http://www.modelmayhem.com/edvaughn
Mayhem # 1246003
About Me
Professional career started in 1976. Primarily portrait photographer of the usual subjects. Experience working with professional models for several years as part of a team photographing for Hawaiian Tropic, Ujena swimwear, and fitness groups with body shapers or fitness models.
I do not photograph nudes and have guidelines as to the types of images I will not create.
If you want personal attention and a fully focused professional helping you create what YOU want to help possibly get paid to model in some form or fashion, then contact me and I will take as much time with your photography as you need.
Double_deez Model
Kara Camille Delonas Model
Tiffany Nacke Model
SHAYNA ALEXIS Model
Taichi Hatano Retoucher
Steven Fendy Retoucher
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subset: commoncrawl


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Testimonial: The apartment was exactly what I was looking for. It was reasonably spacious, close to all amenities and very quiet!
Testimonial: This apartment was exactly what I was looking for! Practical, centrally located, tastefully decorated. The owner was absolutely charming, as well. I would definitely rent this place again!
Testimonial: Wonderful apartment. Magical location and excellent welcoming. Cannot recommend it highly enough!! Good space for 1 to 2 people.
Testimonial: The flat in Paris was very pleasant and the location was wonderful. A fantastic base to explore Paris. Thanks!!
Testimonial: We had a wonderful stay (my husband and I with our two children), and would definitely consider using the appartment in Paris again.
Testimonial: The one bedroom paris apartment was GREAT!! The building is in a very convenient location (near Le Louvre Museum) to everything and is quite quiet!!
Testimonial: The apartment is well equipped and in a great location, easy walk to Notre Dame, The Cluny, Pantheon, Luxembourg Gardens and the Louvre. Also very close to a metro stop and there are an assortment of eateries steps from the building. The apartment and building are well maintained. Can be noisy outside on weekends because of the many nightspots. Just bring earplugs. Love the convenience of the elevator after a long day of sight seeing. Monique is a wonderful landlord, very responsive if you need anything and brings fresh sheets and towels if you are staying for a long period of time. My security deposit was returned very quickly to me after my stay was over. Thank you for everything Monique. I hope I have the opportunity to return someday.
Testimonial: I truly love the apartment. It was close to a range of fabulous markets and shops. Metro access was very close too which was a great advantage. Valentina the care taker was punctual and welcoming.
Testimonial: An apartment to be in love in...and in love with! Lovely, clean, perfect location. One can lay in bed and watch the clouds and birds through the skylight.
Testimonial: This Paris apartment rental near Le Louvre was more that I ever expected. It is gorgeous and well decorated. The landlord was gracious and extremely helpful. Our flight was delayed and there was still someone to let us in to the flat late at night. The location can not be beat and the owners were amazing. I will be renting this very apartment the next time I visit Paris. I highly recommend staying here!
Testimonial: Very nice clean bright Paris holiday apartment, plenty of room for 2 couples, everything we needed for 5 nights stay. Restaurants and shops a short walk away.


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Honda City helped make these videos
We are still working to obtain content for your car, however the video above is believed to have similar characteristics as your car.
This video shows you how to change the oil and oil filter in your 1999 Chevrolet S10. When you change your own oil, you know that you are putting quality oil in your S10 and that the filter is being changed too. Most importantly, you get a chance to look around under your S10 for potential trouble spots. This video shows you the location of your oil drain plug, oil filter, oil fill cap and dipstick in addition to the steps needed to change the oil and filter in your S10. For most Chevrolets, you can wrap an old belt around the oil filter and unscrew it by hand. If you can't do this, see our parts page to find a Chevrolet S10 oil filter wrench. If your S10 is too low to the ground to access your drain plug and oil filter, be sure to use jack stands and safe jacking procedures before getting under your S10.
Most Chevrolets have the oil type printed on the oil cap - it will likely be 5W-20, 5W-30, 5W-40, 10W-30 or 10W-40. If it isn't printed on the oil cap, check your owners manual for the exact type before adding new oil. We recommend wearing safety glasses whenever you are working under your S10. You never know what could be dripping down from the engine, battery acid, engine coolant, brake fluid, etc. All of these fluids are extremely harmful to your eyes and skin so it is important to protect yourself.
See how to seal up minor power steering fluid leaks1999 Chevrolet Blazer LS 4.3L V6 (4 Door)
If your car is leaking a small amount of power steering fluid - see this video on how to fix the leak1999 Chevrolet Blazer LS 4.3L V6 (4 Door)
Replacing a dirty air filter can improve gas mileage by up to 4%. Isn't efficiency great?1999 Chevrolet Blazer LS 4.3L V6 (4 Door)
Power steering fluid is easy to check and many people (mechanics included) often forget about - learn how here1999 Chevrolet Blazer LS 4.3L V6 (4 Door)
Learn where your air filter is located and how to determine if it needs to be changed.1999 Chevrolet Blazer LS 4.3L V6 (4 Door)
Got a short fuse? Don't blow a fuse, change it!1999 Chevrolet Blazer LS 4.3L V6 (4 Door)


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Northern Kentucky airport terminal shut down temporarily; grenade found
HEBRON, Ky. (AP) — A hand grenade in a passenger's carry-on bag has caused a temporary evacuation at the northern Kentucky airport.
The Delta Air Lines terminal at Cincinnati/Northern Kentucky International was cleared for about 10 minutes Tuesday night after the device was found at about 7:30 during routine baggage screening.
Airport spokeswomen Barb Schempf said Transportation Security Administration officials found no detonator and determined the grenade was no longer an explosive, according to The Kentucky Enquirer (http://bit.ly/wUgPoo).
The passenger who carried it was denied boarding and the grenade was confiscated. No flights were canceled or delayed.
The passenger was released, but is now under investigation by the TSA.


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Raback, I am not sure if you or anyone will see this since the site is currently in disarray but kudos to you my friend. You hit the nail on the head with your statement. I doubted and was wrong. Many +1's to you and I will never doubt you again (until you make another bold ballsy statement but I won't doubt you as much then!)
in town for free camps


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Teletech Park
20 Science Park Road 117674
D05 Clementi, Pasir Panjang, West Coast, Dover, Buona Vista
3-storey building comprising 2 wings and a basement carpark for telecommunication R&D. It is Asia's first R&D facility specially designed to meet the needs of telecommunications R&D companies. Completed in October 1996, it boasts cutting-edge infrastructure and test-bed facilities to make it the R&D venue of choice for companies in the sector.
Property Name:
Property Type:
TOP Year:
Properties For Rent
Posted on 18 May, 2015
by Sales & Marketing Ascendas Pte Ltd
by Sales & Marketing Ascendas Pte Ltd
SGD 4,442 (Negotiable)Price (psf): SGD 4.50
Floor Area: 987 sqft/92 sqm
Lease Term: Flexible


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Handyman and cleaner needed for the next three days!
We need help from a skilled handyman for the next three days to help with some painting and the odd fixing. We also need a general cleaner to help. We pay £10 an hour (£12 an hour for any electrical work). Pls email or text if interested.


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Heres a little more:)
I am a very low maintance high expectation kind of person. I like seeing the beauty that life has to offer and so I try to see as much as possible. I cry during sad movies. I am very spontanious and sometimes end up in places wondering what it was that got me there to begin with. I have won over 3 all you can eat pizza contests. I drank until i blacked out in the middle of brazils carnival. Have smoked pot with law enforcement officals in other countries, had drinks with a king and princesses. I once shot a quarter at 200yrds. I am not a saint. (made my mistakes) Tangoed in the streets of buneo saris, had one night stands and have partyed until the sun came up. I cheated on a test or 2 in high school. I have loved and lost it. Have survived in the deep jungles of peru. I like quiet nights on the sofa. Had sex in the mountains of machu picchu, been scuba diving in honduras and chile. Partyed like a rockstar in santiago, had to spend the night in a jail in fiji. I like to cuddle after sex. I grew up in alaska and think belly chains are sexy has hell! Saw a man get beat so bad he almost died. I love kids. I once wound up in the middle of a gun fight while in bolivia. Got great pictures of the riots in argentina. I think spending the night under the stars is on of the most romantic things. I am a lover not a fighter, Have bungee jumped and really want to skydive. my friends are the most important thing to me. I dont like confertation, but love competion. i love dogs but hate cats. Pray you never have to hear me sing. I can cook but dont like too. I believe that he who dies with the most experience wins. when I am on my death bed, I want to look back and say I did it right!!


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Lady Gaga - ARTPOP (4th studio album)
View Single Post
08-08-2012, 18:08
Kellie B
Inactive Member
Join Date: Jul 2012
Posts: 166
Originally Posted by
I hope this album is good, as I find with Gaga, she has so much hype, but she never really has the material to back it up with. The Fame was a good debut, TFM was great, and then BTW wasn't good at all, asides one or two tracks. Everyone hypes her up so much, but she's a classic case of style over substance.
I do hope she manages to pull it off though, she seems lovely.
Yeah I do agree with this great point. I would even go as far as to say that Katy Perry and Rihanna have had songs that have made a much greater impact on pop culture than Lady Gaga has. So it will be interesting to say what Gaga tries to pull out of the bag this time.
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LAKE FOREST, Ill. (AP) The Chicago Bears could breathe a little easier this week even if their quarterback was having some trouble.
RENTON, Wash. (AP) Leave it to Seattle cornerback Richard Sherman to come up with a new way of talking about an opponent without saying a word.
ALLEN PARK, Mich. (AP) The Detroit Lions are counting on three players to make up for the loss of receiver Nate Burleson.
LAKE FOREST, Ill. (AP) Chicago Bears quarterback Jay Cutler plans to treat Sunday's game against the Carolina Panthers like any other, even with bruised ribs.
MINNEAPOLIS (AP) Minnesota's rebuilding project might eventually prove it needs more work.


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Easy as Tortilla Pie
We've slimmed down this Tex-Mex favorite by using reduced-fat shredded cheese and by packing the filling with low-cal corn and black beans.
Total Time:
Level: Easy
Yield: 4 main-dish servings
Serves: 4
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- 1 c. frozen corn kernels
- 2 green onions
- 1 tsp. ground cumin
- 1.50 c. salsa
- 1 can low-sodium black beans
- 4 burrito-size flour tortillas (96% fat-free)
- 1 package reduced-fat (2%) shredded Mexican cheese blend
- 2 tbsp. chopped fresh cilantro leaves
- Preheat oven to 450 degrees F. Spray large cookie sheet with cooking spray.
- Spray 12-inch nonstick skillet with cooking spray; place over medium heat. Add frozen corn, green onions, and cumin; cook 3 minutes or until corn thaws. Remove skillet from heat; stir in salsa and beans.
- Place 1 tortilla on cookie sheet; top with 1 cup bean mixture and 1/2 cup cheese. Repeat, starting with tortilla, to make 2 more layers. Top with remaining tortilla and cheese.
- Bake pie 10 minutes or until heated through. Carefully transfer pie to cutting board; sprinkle with chopped cilantro. With sharp knife, cut into wedges to serve.
What do you think?
How to Make a Liberty Bell Cake
Let freedom ring with this fun party centerpiece.
How to Make an Uncle Sam Hat Cake
Because America's birthday deserves a little something special.
Wish Bone Recalls Ranch Dressing Due to a Dangerous Mistake
Double-check your dressing, everyone.
More From Food & Recipes


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Outfit your traditional game room with a handsome ceiling fan design.
This game room features a corner bar and bright room accents.
A wine cabinet storage unit is a great idea for creating a conversation area.
* Free shipping and/or free return offers apply only to shipments made to the 48 continental U.S. states. Free returns does not apply to designs shipped by freight, designs with giclee art shades, Color Plus brand and Tiffany Color Plus brand designs, or certain items with designer shades.
Our associates are standing by to assist you.


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Post-Trip Reports
Post trip reports, backyard gear testing fiascos, etc.
Post trip reports, backyard gear testing fiascos, etc.
|Dolly Sods WV
|Lake Basin, Kings Canyon National Park, California
|Porcupine Mountains
|The Obed in East Tennessee - Climbing/Camping trip - Video
|My Daughter's First Overnight Backpack
|Wasatch 11ers, part 1
|Wind River Crest
|St. Vrain's Ridge Walk
|Started a companion trail guide to Sweden's Bohusleden
|Yakutat Bay, August 2012
|Ever get a bruised knee bone from doing a long hike?
|Trip report: Sierra Trip - Shepherd Pass and Piute Pass
|Chilkoot Trail
|Tahoe-Desolation Wilderness Aug 2012
|Crazy Mountains DIAD patrol
|Enchantments 8/15-8/18
|Chelan Summit Trail 8/11-8/14
|Backpacking in the Basin - Andrew Skurka's RMNP Trip
|Rae Lakes loop counterclockwise trip report
|Sanora Pass to Boulder Lake
|Adventure Boy! Summer Adventures 2012.
|Emigrant Wilderness - Kennedy Meadows to Ridge Lake, July 27-29
|Finding Our Way. 8/18 thru 8/19 2012
|Dawson Pass Loop GNP
|South Fork Conejos River Semiloop


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time to buy some more at these prices
nothing but a 3 week wonder down down we go where we stop who knows?
A haircut and shave I did not want over the last few days
orange crush
more volume than TRP and T today good sign + its going up
Holding $12 nice
Big volume today but could not hold $12
the money always runs out before the paper does
1.3 millions shares to eat through at.015c Will any one step up to the plate?
sorry my mistake I know what's its like to get screwed, remember Shear...
Maybe Mr Allan has gone to the UK to try and collect the 1.3 million they are...
broke through $1 and held, nice move up last few weeks good volume things are...
2 cups of coffee from a international chain or 1 Transalta share Hummm...
we did it under $10 now after owning this stock for over 22 years i can...
Maybe we will go under $10 today NICE thanks a lot
an other hair cut and shave today might drop under $50 at this rate
run for the hills the sky is falling
gee thanks did not really want that haircut and shave today
At this rate 2 coffee at Starbucks will cost the same as 1 share of...
a lot of people got a hair cut and shave they never asked for in the last few...


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Ethan Home Dining Room Sets
at Overstock.com
Country visits town in the earthy Vienna Collection. This casual solid wood dining room offering is featured in a warm two-tone bu...ttercream and medium tone burnished oak finish. Set includes: One (1) Table, Six (6) Chairs Materials: Rubberwood Finish: Buttermilk and medium tone burnished oak finish Upholstery materials: Chenille Upholstery color: Walnut Upholstery fill: Polyurethane foam Seat height: 18 inches Butterfly table leaf Table Dimensions: 30 inches high x 54-72 inches wide x 42 inches deep Chair Dimensions: 40 inches high x 19.25 inches wide x 23.75 inches deep read more
at Overstock.com
Bring affordable, luxurious style into your dining room with this faux marble top dining table and faux leather chairs set. This s...et includes one table and four chairs, all of which are constructed out of solid Asianwood with a lovely cherry finish. read more


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* On/off data2sd and hybrid data2sd
* Setup compcache size
* Convert FS tool
g3mod: http://forum.xda-developers.com/showthread.php?t=1252698
GPS Booster
In opposition to 'GPS Opt' it is transparent for user. It works with all GPS launcher (menu Settings, widget...).
Automatically available at phone startup.
It may become obsolete one day if issue is corrected by a manufacturer update.
Copy to Clipboard
For example your location in Maps to insert a message in GTalk.
Program don't HAVE ICON IN APP DRAWER! It's addon to system share menu!
More from developer
Copy to Clipboard
For example your location in Maps to insert a message in GTalk.
Program don't HAVE ICON IN APP DRAWER! It's addon to system share menu!
GPS Locker
Pocket Skip Track
Only guaranteed to work with PowerAmp https://play.google.com/store/apps/details?id=com.maxmpz.audioplayer
For correct operation of the other players the author is not responsible!
Author is also not responsible for the correct operation is not on firmware other than pure Android!
For *ROOT* available switching on long press (only works on rooted devices)!!!
This program is designed to switch tracks in the player is running and off the device screen by double clicking on the volume button.
The program allows you to choose your preferred delay between clicks.
To switch to the next track you need to make two clicks on the "volume up" with a delay of no more than specified in the "Delay". Accordingly, previous track - "volume down".
!!! The program works only when the play player and the screen turns off. Krosfeyd in the player must be disconnected. Probably does not work on runoff firmware HTC (disable a smooth change in the volume, if possible) !!!
Can not work with some devices.
FFmpeg Media Encoder
FFmpeg is already built into the program and does not require download of additional codec.
Main purpose: to convert any video to mp4 or 3gp, understandable to Android devices and resize under the screen resolution of devices. You can also extract the mp3 audio from the video.
Supports: MPEG4, h264, mp3, aac, ogg (vorbis and theora) and many other formats (the list can be found in the program). Optipization for x86 и ARMv7(cortex-a8).
Requirements: Processor ARMv7 and above or x86.
Built-in: FFmpeg v2.3 + libx264 + libmp3lame + libvorbis + libtheora + libfaac + libvpx
More options you will find in FFmpeg's man pages.
Bluetooth PAN for Root Users
Tested on the tablet PiPo U1 (a stock firmware + root) and phones as an access point: Amoi N821 (4.1.1), Samsung Galaxy Ace (CyanogenMod 7.2) and Sony-Ericsson W610i.
PS: If at first you are not connected, try to update (bottom left corner) and to increase the connection timeout in the settings.
Images of the Sun from SOHO
- Available whole archive of images from 1996, ending today.
- Images from the 10 sensors.
- Saving to SD
- Image size 512x512 and 1024x1024
- Animation
Images are available on http://sohowww.nascom.nasa.gov/home.html
Torchwood Triad
* two in one: live wallpaper and stand alone app
* Triad's triangle +5 objects: Pyramid, Droid, Two Triangles, Random Stars, Many Many Lines.
* Time, date and operator name on screen
* Rotation speed may depends on the charge
* 4 colors
* add more objects and features
Twittery - Fortune-telling
In addition you will be able to answer you accidentally fallen tweet :)
Let only the best prediction of the application come true.
* Simple story source code (Lua-based script);
* GUI and text (readline) interfaces;
* GUI interface supports music and pictures;
* Theme support;
* Portability (INSTEAD depends on SDL and Lua).
Hint: for display keyboard in games, you must click anywhere on the screen, hold more than 1 second and release)


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Nolo's Plain-English Law Dictionary
Need Professional Help? Talk to a Lawyer
Enter Your Zip Code to Connect with a Lawyer Serving Your Area
- Griggs V. Duke Power Co. (1971)
The U.S. Supreme Court case in which the Court established that neutral employment practices that have a discriminatory effect can violate Title VII of the Civil Rights Act of 1964, even if the employer did not intend to discriminate.
- Full text: Griggs v. Duke Power Co. (Nolo)


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Policy wonks are debating whether a trillion-dollar platinum coin would be a clever or insane way for President Obama to play hardball with Republicans in the upcoming debt limit battle. Here's what you should know about this crazy-sounding idea:
1. A legal loophole gives the Treasury Secretary apparently unlimited authority to mint platinum coins.
Treasury is forbidden from printing money to cover government deficits. Treasury must issue debt, while the Federal Reserve independently controls our nation's monetary printing press.
That is exactly as it should be. But there is an arcane exception for platinum coins. To serve coin collectors, Treasury can issue platinum coins of any denomination. That creates an intriguing loophole: Treasury could bypass the collector market and mint a trillion-dollar platinum coin. By depositing it at the Federal Reserve, Treasury could keep paying bills after we've fully exhausted our borrowing limit.
[Related -Four Stocks in the Dow Making Fresh 52 Week Lows]
2. Most observers think this is a terrible idea, but the legal arguments against it are weak at best.
A who's who of commentators has already objected to the coin on legal, economic, political, and image grounds (see, for example, John Carney, Matt Cooper, Tyler Cowen, Kevin Drum, Jim Hamilton, Heidi Moore, and Felix Salmon). I'm no lawyer, but the legal arguments seem wholly unconvincing. The language of the statute is clear, and in any case, the executive branch gets away with expansive actions in extreme times. During the financial crisis, for example, Treasury aggressively interpreted its authorities in order to bail out GM and Chrysler and to backstop money market funds. If default became a real possibility, the same expansiveness could easily justify a platinum coin.
[Related -Some Thoughts on Greece's Don't-Call-It-a-Default]
3. The economic arguments against the coin are stronger but manageable.
There's a good reason that Treasury is forbidden from printing money to pay our debts: inflation. Many economies have been ruined when profligate governments turned to printing money. But minting the platinum coin needn't mean monetizing our debt. The Federal Reserve has ample ability to offset any inflationary impact by selling some of the trillions in Treasury securities it already owns. As long as the Fed does its job, inflation would not be a risk.
4. The best arguments against the platinum coin involve image and politics.
Minting a trillion-dollar coin sounds like the plot of a Simpson's episode or an Austin Powers sequel. It lacks dignity. And despite modern cynicism, that means something.
It would also be premature. President Obama and the Republican and Democratic members of Congress have roughly two months to strike a debt limit deal. There is no reason to short-circuit that process, as painful as it may be, with preemptive currency minting as the now-famous #MintTheCoin petition to the White House suggests.
5. Nonetheless the platinum coin strategy might be better than the alternatives if we reach the brink of default.
Analysts have considered a range of other options for avoiding default, including prioritizing payments, asserting the debt limit is unconstitutional, and temporarily selling the gold in Fort Knox. All raise severe practical, legal, and image problems.
In this ugly group, the platinum coin looks relatively shiny. In particular, it would be much less provocative than President Obama asserting the debt limit is unconstitutional. That nuclear option would create a political crisis, while a platinum coin could be a constructive bargaining chip. As Josh Barro notes, President Obama could offer to close the platinum coin loophole as part of a deal to raise or eliminate the debt ceiling.
6. If necessary, Treasury should mint smaller platinum coins, not a trillion-dollar one.
A trillion-dollar coin is eye-catching and ridiculous. That's why it's filled the punditry void left by the fiscal cliff. But a single coin makes no policy sense. No federal transactions occur in trillion-dollar increments.
Among the largest transactions are Treasury bond auctions, which today raise about $25 billion at a time. If necessary, Treasury could issue individual $25 billion coins, each in lieu of a needed bond auction. Still ridiculous, to be sure, but less so as it would calibrate coin issuance to immediate financing needs.
Steve Randy Waldman suggests as even more granular approach: issuing coins denominated in millions not billions. Such "small" denominations would be even less ridiculous and could potentially be used in transactions with private firms, not just Fed deposits.
Of course, the best path would be a bipartisan agreement to increase the debt limit, address spending cuts, and strengthen our fiscal future, all settled before the precipice. If we reach the brink, however, minting million- or billion-dollar platinum coins would be better than default.
"What gets weak tends to get weaker; what gets strong tends to get read on...
Things got even weirder the day read on...
The Greek default is merely the opening act of the worst sovereign-debt crisis in history. By the time it read on...
In the face of historic monetary stimulus from nearly every major central bank in the world over the past read on...


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Top Definition
Mostly an expression of sheer and utter immaturity. A way of showing ones affection in an overly obnoxious manner. A pet name of sorts.
I love you woodletuft!
by Panick from myspace March 03, 2007
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Details Group Tabs
Reviews Sign in to post reviews
Great Initiative
Submitted by Dino Flores on May 19, 2011 - 14:59
This project is a great initiative.
User is presented with a list of Actions, Wizzards, representing Eclipse menu-items, which can be dragged onto a script-file and then executed.
However these is not a (documented) way to create a (new) script file, neither is there a (documented) way to run the file, rendering this initiative useless.


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Blogs Home
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Intellectual Property and the software-defined supply chain
Mark Bell email@example.com Tags: software source copyright printing supply 3d patent open defined chain 8,316 Visits
By Mark Bell IBM Senior IP Strategist - IBM IP Management Solutions Intellectual Property issues are often flagged as an area of concern for the development of the software-defined supply chain, yet the nature of these perceived issues...Modified by Mark Bell firstname.lastname@example.com
3D Printing – Transforming The Supply Chain: Part 2
Leonard Lee email@example.com Tags: digital printing depot chain service supply electronics 3d repair opertions industry 1,188 Visits
What if you could get that critical, custom part the same day that you needed it for an urgent warranty repair for a customer? What if you could avoid the cost of carrying expensive, slow-moving service inventory items and eliminate the lead-time associated...Modified by Leonard Lee email@example.com
Re: 3D Printing – Transforming The Supply Chain: Part 1
Cristene Gonzalez-Wertz firstname.lastname@example.com Tags: manufacturing chain supply collaboration digital printing connected 3d 10 Comments 926 Visits
In response to: 3D Printing – Transforming The Supply Chain: Part 1 Hi Leonard, great post - what's the time line defined in your benefits analysis?
3D Printing – Transforming The Supply Chain: Part 1
Leonard Lee email@example.com Tags: collaboration connected 3d printing digital chain manufacturing supply 7 Comments 3,714 Visits
There is a great deal of buzz around 3D printing (also known as 3D manufacturing or additive manufacturing in its broader terms) in today ' s tech press. 3D manufacturing's increasing prominence is underscored by President Obama ' s inaugural...Modified by Leonard Lee firstname.lastname@example.com


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The Official Welcome Thread
View Single Post
February 22 2009, 01:08 AM
Location: New Zealand
Re: The Official Welcome Thread
Hey All!
Hi! Stating the obvious -I'm new here. I live in New Zealand, and have been a longtime Trek fan. I'm looking forward to the new Trek XI movie.
As much as I love Trek, and all things Trek, my bent is towards Trek starships - from every era and of every design. My all-time favourite design is the Refit Enterprise (TMP). The new Enterprise (as shown in various Trek XI trailers) looks awsome, IMO - I love it's look, most likely because it shares much in common with the refit Enterprise.
Anywho, enough about me! I look forward to participating in this community and engaging in some great discussions!
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Latest Articles
Colchester have signed former Yeovil midfielder Joe Edwards
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Colchester have signed former Yeovil midfielder Joe Edwards, subject to paperwork.
Southampton sign defender Cuco Martina from FC Twente
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Southampton have announced the signing of versatile defender Cuco Martina from FC Twente on a two-year-deal.
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Hugo Lloris' arrival from Lyon means Friedel is no longer Tottenham's first choice, with Andre Villas-Boas preferring the Frenchman for five out of the club's last six games.
Blackburn, where Friedel spent eight years of his career, made an approach for the American two weeks ago, with the npower Championship side thought to be keen to appoint the 41-year-old as a player-coach.
Spurs rejected the bid and manager Villas-Boas insisted Friedel would not leave.
Perhaps to underline how much he wants him to stay, the Portuguese made Friedel captain for the 3-1 win over Panathinaikos in the Europa League.
Although he stopped short of refusing to rule out a move to Ewood Park in January, Friedel claimed following the victory that he is more than content with life at Spurs.
"I cannot answer questions (about a potential January move)," Friedel said.
"It's all hypothetical. Right now I am a Tottenham player and I am very happy here.
"I have no idea (about Blackburn's interest). When I was apparently contemplating doing a medical I was at home having Thanksgiving dinner with my family.
"What happens between club and club a lot of times players don't know. At the end of the day I am contracted to Tottenham and am concentrating on Tottenham."
Friedel insists he has a good relationship with Lloris, whom Spurs signed for £8million on transfer deadline day.
The American, who will be 42 when his contract expires at the end of the season, understands why Tottenham signed Lloris, but has warned the France captain that his performances will only improve now that he has robbed him of his spot in the team.
"There is no rivalry with Hugo, but do I thrive on competition? Absolutely," Friedel said.
"I wouldn't be playing at the age of 41 if I didn't.
"At every club I have been at for the last 21 seasons there has been competition for places.
"When I signed it was a two-year contract. I figured I would play last year. This year was unknown.
"They have been on the look-out for a long-term number one goalkeeper for a couple of years.
"The club is going to be around a lot longer than any of us so they have to get all their ducks in a row.
"This club wants to be in the Champions League. They want to be there every single year and they want to be trying to win trophies all the time so they continuously have to buy good players in every position."
Friedel put in a solid display in last night's victory, which secured Tottenham's passage to the last 32 of the Europa League.
Up the other end of the pitch it was the Jermain Defoe show once again - the England striker taking his season tally to 13 with a well-taken chip.
Alongside Emmanuel Adebayor, who also scored after missing the last three matches through suspension, Defoe looked sharp again, as he has done all season.
Overlooked for the majority of last season, the 30-year-old has blossomed under Villas-Boas this term. His hat-trick against Maribor last month moved him to eighth in Tottenham's all-time top goal scorer list.
Friedel thinks the former West Ham man should now be finally recognised as one of the best strikers in the history of the Barclays Premier League thanks to his exploits this term.
"He reminds me a lot of Robbie Fowler in his finishing because he has a really short back-lift," said Friedel, who played with Liverpool icon Fowler after transferring from Columbus Crew in 1997.
"When you have a really short back-lift, sometimes keepers cannot get their feet set. On top of that, he has got some power behind his shot.
"He is an exceptional finisher. He has got a finish as good as (Alan) Shearer, Fowler... all of them."
Click here for completely free £10 bet with Sky Bet & £5 free every week


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Oceanview Porthole Window (OG): Norwegian Sun Cabin Reviews
Norwegian Sun Cabin Categories
These Norwegian Sun Oceanview Staterooms have room for up to three guests, with two lower beds that convert into a queen-size bed and a convertible sofa to accommodate one more. You'll also have a porthole to enjoy the amazing view.
Oceanview Porthole Window (OG)
Decks: Deck 4
Loved it! sylanVery clean, & loved the towel animals! Our steward taught me how to make a few. We laughed a lot!
Read entire member review >>
Panama Canal Repositioning Cruise Tampa Bay to Vancouver B.C. Crazyforcruising1951Do not book any cabins at the very end of the fourth floor hallway. Noise will deafen you! Above the engine room the minute you put your head on the pillow the viberation and noise is guaranteed to keep you awake!
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Bad food and bad smells wally437We had an ocean view cabin and when we opened the door our first words were 'sure is small'. That evening when the cabin attendant opened the sleeper couch out, it got even smaller. It did have better storage with the baskets in the closet but I would have preferred more space which I suspect was sacrificed to allow more room for all the specialty restaurants.
Read entire member review >>
Excellent Time On Norwegian Sun! penelopemarchClean, roomier than I thought it would be. Plenty of storage and the steward was very attentive.
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SMALL but very accommodating. Cabin stewards were friendly, and our dining room servers were attentive and pleasant.
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Fun in the Sun patmamaJust a long way from the elevator - port view was just a water view as is standard -room size was just fine
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Almost Perfect Cruise lorenzodCabin was quiet and well located.
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Baltic cruise on Norwegian Sun Rascal5107Lots of space for regular outside cabin
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Enjoyed the Sun LosnabThe cabin was well thought out. With perfect cubbies to keep everything organized.
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Perfect Way to Turn 30 HGVGuyWe had an outside porthole. The view was unobstructed. Salt clung to the window, but we could see fine. The room is a bit small, but probably average as cruise lines go. The room was by the elevators and stairs. It was far enough away to not hear the traffic, but close enough for easy access. The 4th floor may be a little low, but it was only a floor or two away from most activities (other than the pool sides stuff). I would choose this room again for it's minimal noise and easy access.
Read entire member review >>
NCL Sun Terrible Cruise myers69Cabin 4211 dirty and odor in hallway
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Thank God for the ports taschrCabin 4011 (Cat OK). Small, no storage or closet space. Inadequate bathroom. Big picture porthole. NIce new carpeting and very comfortable bedding.
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Our Baltic adventure wpj62Why can't there be more electrial outlets? I understand the space limitations.
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Spring Break with friends EMLchascabin # 4011 With the arrangement of the bed, it is too small to have the sofa pulled out for a third person. When the sofa bed is pulled out, it is virtually impossible to get to the bathroom at night (or any other time that is is out) with out either walking on top of the sofa bed or killing yourself trying to get around it. It should not be classed as a three person room. It is dangerous as a 3 person room. Only bigger cabins should have pullout sofa beds and small cabins, if they are to be 3 person cabins, should have a drop-down bed.
Read entire member review >>
Read entire member review >>
Sponsored links
Norwegian Sun DecksNorwegian Sun Deck 4 - Biscayne Deck
Norwegian Sun Deck 5 - Atlantic Deck
Norwegian Sun Deck 6 - Oslo Deck
Norwegian Sun Deck 6 - Promenade Deck
Norwegian Sun Deck 7 - International Deck
Norwegian Sun Deck 8 - Viking Deck
Norwegian Sun Deck 9 - Fjord Deck
Norwegian Sun Deck 10 - Norway Deck
Norwegian Sun Deck 11 - Pool Deck
Norwegian Sun Deck 12 - Sports Deck
Similar Cabin CategoriesFamily Oceanview Picture Window (O1)
Mid-Ship Oceanview Porthole Window (OF)
Oceanview Picture Window (OA)
Oceanview Picture Window (OC)
Oceanview Picture Window (OD)
Oceanview Picture Window (OE)
Partially Obstructed Oceanview Porthole Window (OK)
Partially Obstructed Oceanview Porthole Window (OX)


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G Drive Mini
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autoexec file for apple
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Imac, external Hd boot and Windows image
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Mac's Line-in Built-in Input Sound not working
Keylogger- not hidden
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itunes through studio monitors
You cannot use this version of the application Mail with this version of Mac OS X.
Install fresh OS on a fat32 hdd.. please help
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Mass Email help required
All installs clicks open LogicExpress
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Spotlight showing web history after clearing in Safari
Dock settings to follow users in Windows Server 2003
rip CD to .iso / create .iso image
MS Office windows for mac
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upgrading from tiger 10.4.11 to snow leopard
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Printer Fonts
Writing Southern Kurdish ?
upgrading from 10.4.11 to osx snow leopard
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Snap to Edges
Need Help :(
Restore back to this morning
folder sharing from Win7 without log in
Updating a PowerBook G4
Snow Leopard and microsoft-ds (SMB) traffic
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Family in Sync
Screensaver appears without reason.
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accessing Time Machine backups from a previous computer
Saving to Network Folder, not located in Finder
Macbook pro freezes/locks up when watching streaming video/browsing the web
watching tv in another country


text:
- Shopping Bag ( 0 items )
All (13) from $1.99
Used (13) from $1.99
Posted January 26, 2010
This book's title says it all. It is, indeed, a practical guide. Each chapter contains valuable information to anyone interested in developing a CNL program. After reading it, I not only understand the role much better, I understand the steps required to initiate a program. I especially liked the case studies, and in fact, those convinced me that our students in that new track will need this book.Was this review helpful? Yes NoThank you for your feedback. Report this reviewThank you, this review has been flagged.


text:
Women's & Children's Center Calendar
Women's and Children Center
Childbirth Preparation
- Date(s): Ongoing
- Location: Women's and Children's Services
- Fee: $80 or $100 with Birth Ball
This weeknight series prepares expectant parents for the birth process and parenting skills. Parents will learn; what to expect during childbirth, comfort and breathing techniques, pain management, postpartum issues, parenting.
Cost: $80 per couple or $100 with Birth Ball
Pregnancy & Postpartum Stress Group
- Date(s): Every Tuesday
- Time(s): 12:30 pm
- Location: PVHC at Pomona
- Fee: Free
In this group for Pregnant and New Moms, we will
- Discuss stress, depression, anxiety and difficulty adjusting
- Learn coping skills, relaxation techniques, communications skills
- Support moms to recover from pregnancy and birth
- Help you feel will and be the mom you want to be
Facilitated by Katayune Kaeni, Psy.D.
Lunch and childcare will be provided
Special Instructions
For more information call Anna Pavlov, PhD. at 909.469.9491.
Pomona Valley Hospital Medical Center - 1798 N. Garey Avenue - Pomona, CA 91767 - (909) 865-9500 - Contact Us


text:
English Scones
|8 Ounce
|1⁄2 Teaspoon (Leveled)
|Cream of tartar
|2 Teaspoon (Leveled)
|Soda bicarbonate
|1 Teaspoon (Leveled)
|2 Ounce
|1⁄4 Pint
1 Preheat the oven to 450°F./ gasmark 8
2 Lightly grease a baking tin.
3 In a bowl, measure salt, cream of tartar and bicarbonate of soda and put with flour.
4 Sieve dry ingredients you have mixed together.
5 Add in the fat, cut into small pieces.
6 Rub the fat into the mixture until it resembles fine breadcrumbs.
7 Measure the quarter pint of milk.
8 Create a well in the mixture and gradually add the milk.
9 Mix with a palette knife to form a soft dough.
10 Turn the dough out on to a floured board or clean kitchen table-top.
11 Knead it very lightly.
12 Form the dough into a flat rounds roughly 3/4 to 1 inch in thickness.
13 Cut into rounds with a cutter and put them on to the prepared tin.
14 In a bowl, break an egg and beat it with a fork.
15 Brush the rounds with egg.
16 Bake in a very hot oven for 8-10 minutes.
17 Allow to cool on a rack.
18 Serve as desired.