text:
Not Thriving: Mister Danger get usage counts
Total posts:
Darkness and Disgrace
With only 33 wicks and 2 inbounds for what seems like a common villain type. Frankly, I blame the terrible, ambiguous name for it. It's named for a character in a book we don't even have a page for, and frankly it sounds more like a trope about either action heroes or highly unlucky individuals.
Save a webcomic, read Commander Kitty!
"You got your head all tangled up, but if I could only
make you care..."
Hugo Chavez seems to think it's a pre-existing term...
anyone have ideas for a better name?
edited 26th May '12 5:20:50 PM by abk0100
The Demon Slayer
Evil Imperialist, maybe?
4 32_Footsteps28th May 2012 08:49:38 AM from Just north of Arkham
, Relationship Status: THIS CONCEPT OF 'WUV' CONFUSES AND INFURIATES US!
Think of the mooks!
I think the Venezuelan version of TV Tropes might be able to use this, if Mister Danger has enough Pop-Cultural Osmosis to be easy shorthand there. That said, this clearly isn't the Venezuelan version of TV Tropes. I agree with the rename.
Evil Imperialist seems like a good fit to me.
Reminder: Offscreen Villainy does not count towards Complete Monster.
6 Spark94th Jul 2012 04:08:49 AM from Castle Wulfenbach
, Relationship Status: Too sexy for my shirt
Gentleman Troper!
Well, it's unanimous. Are we okay with calling this now?
Special trousers. Very heroic.
8 32_Footsteps9th Jul 2012 08:57:12 AM from Just north of Arkham
, Relationship Status: THIS CONCEPT OF 'WUV' CONFUSES AND INFURIATES US!
Think of the mooks!
Are there any other suggestions than Evil Imperialist? I certainly can't think of anything more fitting.
Reminder: Offscreen Villainy does not count towards Complete Monster.
It's named for a character in a book we don't even have a page for..."
Should we create one? :P
Hobby writter.
only because im bored.
Evil Imperialist just sounds like a guy who wants to conquer the world to me. Arrogant Colonial Villain, maybe?
12 Fighteer18th Oct 2012 08:36:31 AM from the Time Vortex
, Relationship Status: Dancing with Captain Jack Harkness
Ugh. Character named tropes, how I hate thee. I see a problem with the examples, too, in that they are largely zero context.
What is this, exactly? Villain whose goal is conquest of an "inferior" race or culture? Evil Imperialist or Evil Colonialist would be suitable titles, I guess.
14 Ironeye26th Jan 2013 03:15:01 AM from SoCal
, Relationship Status: Falling within your bell curve
Bumping for a few more votes. If you're looking to free up some space in the TRS, start fleshing out some of the examples on the page—we should be able to get this out and make space pretty quickly once that is done.
I'm bad, and that's good. I will never be good, and that's not bad. There's no one I'd rather be than me.
A Wizard boy
Moved wicks, page and subpage, and made the notes on Renamed Tropes, the FAQ thread and the discussion page.
17 Willbyr28th Jan 2013 06:22:47 AM from North Little Rock, AR
, Relationship Status: Pining for the fjords
The system doesn't know you right now, so no post button for you.
You need to Get Known to get one of those.
You need to Get Known to get one of those.
Alternative Titles: Mister Danger
30th Nov '12 1:13:25 PM |
At issue:
Total posts: 17


text: Traffic forecast AM: October 26th Posted 8:57 am, October 26, 2012, by Amanda Meadows FacebookTwitterGooglePinterestRedditEmail × Traffic forecast AM: October 26th RICHMOND, Va (WTVR) – Commuter should expect delays on Midlothian Tpke as construction continues between Chippeham and German School. Shifts in the traffic pattern may also impact conditions on Warwick. More info below:

subset: commoncrawl


text:
Paris polyphylla var. yunnanensis is an important medicinal plant. Seed dormancy is one of the main factors restricting artificial cultivation. The molecular mechanisms of seed dormancy remain unclear, and little genomic or transcriptome data are available for this plant.
In this study, massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform was used to generate a substantial sequence dataset for the P. polyphylla embryo. 369,496 high quality reads were obtained, ranging from 50 to 1146 bp, with a mean of 219 bp. These reads were assembled into 47,768 unigenes, which included 16,069 contigs and 31,699 singletons. Using BLASTX searches of public databases, 15,757 (32.3%) unique transcripts were identified. Gene Ontology and Cluster of Orthologous Groups of proteins annotations revealed that these transcripts were broadly representative of the P. polyphylla embryo transcriptome. The Kyoto Encyclopedia of Genes and Genomes assigned 5961 of the unique sequences to specific metabolic pathways. Relative expression levels analysis showed that eleven phytohormone-related genes and five other genes have different expression patterns in the embryo and endosperm in the seed stratification process.
Gene annotation and quantitative RT-PCR expression analysis identified 464 transcripts that may be involved in phytohormone catabolism and biosynthesis, hormone signal, seed dormancy, seed maturation, cell wall growth and circadian rhythms. In particular, the relative expression analysis of sixteen genes (CYP707A, NCED, GA20ox2, GA20ox3, ABI2, PP2C, ARP3, ARP7, IAAH, IAAS, BRRK, DRM, ELF1, ELF2, SFR6, and SUS) in embryo and endosperm and at two temperatures indicated that these related genes may be candidates for clarifying the molecular basis of seed dormancy in P. polyphlla var. yunnanensis.
Embryo; Stratification; Seed dormancy; High-throughput sequencing; Paris polyphyllaBackground
Paris polyphylla var. yunnanensis (named "Chonglou" in Chinese) is one of the most famous medicinal plants in China. It is a perennial herbaceous plant of the Trilliaceae family and is found in damp, shady woodlands, forests, and bamboo forests. The rhizome of this plant has been developed into traditional Chinese medicines such as "Yunnan BaiYao" and "GongXueNing", which are used to treat dispersing blood stasis and hemostasis, to activate blood circulation, to alleviate pain, for detoxification, and to reduce swelling, stop bleeding and reduce inflammation [1-3]. P. polyphylla var. yunnanensis is easily grown in moist, humus-rich soil in woodland conditions, in full or partial shade. However, its cultivation is difficult because of long seeds dormancy and very slow growth from seed. At present, the wild plant is the only source of the rhizome. However, the wild plant has become rare and endangered because of over collection in recent decades. To preserve the natural resources and ensure a stable and renewable source of P. polyphylla var. yunnanensis for medical purposes, successful cultivation of seedlings and planting is imperative.
There are two key factors limiting the extended cultivation of P. polyphylla var. yunnanensis. One is the difficulty of obtaining enough seedlings. Development of the seed embryo stops at the globular stage, about 120 days after fertilization [4]. Thus, seed germination requires a long period of embryo development and release from dormancy. Under natural conditions, the seeds are dormant for 18 months (some are dormant for over 2 years) with about 40% of them germinating [5,6]. Some studies indicated that stratification could speed up the breaking of seed dormancy to about 6 months [7,8]. The other factor is that this plant grows slowly, taking four years from seed to flowering and another three or four years to develop enough for herb harvesting.
Freshly harvested P. polyphylla var. yunnanensis seeds consist of a mesophyll outer layer coat (bright red), an inner hardy coat, a large endosperm and a very small, undeveloped embryo [5]. According to Baskin and Baskin [9,10] and Huang et al. [7], the seeds are of the morphophysiological dormancy (MPD) type and need a long stratification period. To date, research on P. polyphylla has focused on seed dormancy release and changes in seed phytohormone content [7,8], seed stratification [4], cultivation methods [11] and phylogeny and classification [12]. There is little research on molecular mechanisms, especially functional gene studies of seed development and dormancy release. In this work, a high-throughput gene mining method using the 454 Genome Sequencer FLX platform was used for embryo transcriptome sequencing of P. polyphylla var. yunnanensis. Karin et al. [13] suggested that a combination of high-throughput sequencing with more classical methods could greatly advance our knowledge of plant developmental processes. Recently, some studies on plant development using 454 sequencing were successfully conducted [14,15], showing the efficacy of 454 transcriptome sequencing for rapid gene discovery. The aim of this study was to identify transcripts of the embryo that are involved in seed development and dormancy release in stratification process and attempt to explore its molecular mechanism. qRT-PCR analysis was used to compare the expression differences of identified genes between embryo and endosperm to gain more insight into the stratification process. The increased genomic information produced in this study will aid our understanding of the molecular basis of seed development and dormancy release in P. polyphylla var. yunnanensis.
Sequencing and assembly
We obtained 393,805 reads totaling 88,618,152 bases using the 454 GS FLX Titanium platform. After filtering out the adaptors sequences and removing the short sequences of less than 50 bases, 369,496 (93.8%) high-quality (HQ) reads with lengths ranging from 50 bp to 1146 bp were obtained. The average read length was 219 bp. Using paired-end joining and gap-filling, these reads were assembled into 47,768 unigenes, including 16,069 contigs and 31,699 singletons. The size distribution of these reads and contigs are shown in Figure 1. There were 2,451 (15.3%) contigs with lengths longer than 500 bp, which are considered large contigs. All HQ reads were deposited in the National Center for Biotechnology Information (NCBI) and can be accessed in the Sequence Read Archive (SRA) under the accession number SRX155369. An overview of the sequencing and assembly is given in Table 1.
Figure 1. Size distribution of the high quality (HQ) 454 reads and the contigs assembly (A: 454 HQ reads, B: contigs).
Table 1. Summary of sequencing and assembly
Annotation of unigenes
To capture the most informative and complete annotations, all unigenes were first used in BLASTX searches against non-redundant protein database at NCBI. This yielded 15,757 well-identified sequences with E values <10-5, which accounted for 32.9% of the total unique sequences. All unique putative transcripts were then subjected to a BLASTX search against the Arabidopsis database. Approximately 32.8% of the unique putative transcripts (15,673) were annotated. Subjecting these sequences to a BLASTN search against the NCBI non-redundant nucleotide database yielded 12,663 well-identified sequences with E values <10-5, accounting for 26.5% of the total unique sequences. Among the contigs, 30 contained more than 1,000 reads, representing the most abundant transcripts in the 454 cDNA library. The 30 most abundant transcripts included one that encoded early flowering protein. Some transcripts were related with phytochromone and seed ripening, including gibberellic acid stimulated-like (GAST-like), gibberellin oxidase, auxin-repressed protein, ABA 8'-hydroxylase (CYP707A), 9-cis-epoxycarotenoid dioxygenase (NCED), seed maturation protein, late embryogenesis abundant (LEA) protein, xyloglucan endotransglucosylase/hydrolase, dormancy-associated protein (DRM). A number of unique sequences related to seed development and ripening were selected and shown in Table 2. Highly expressed transcripts included cytochrome P450 (60 unigenes), metallothionein-like proteins (105 unigenes), glutathione S-transferase (71 unigenes), and PR10 proteins (22 unigenes).
Table 2. Selected genes related to seed embryo development in stratification seeds
Gene Ontology (GO) and Cluster of Orthologous Groups of Proteins (COG) assignments
The unique putative transcripts were compared with the Universal Protein Resource database (UniPort), formed by uniting the Swiss-Prot, TrEMBL and PIR protein database activities. The BLASTX and the Gene Ontology (GO) assignments of the UniPort proteome produced assignments for 14,601 (30.6%) sequences, matching 9,749 unigenes, and then assigned them to 43 major GO categories for molecular functions, biological processes and cellular components (Additional file 1). The overall distributions suggested that our library sampled widely across sub-categories and provided a good representation of the embryo transcriptome of P. polyphylla.
Additional file 1. Functional annotation of putative unique transcripts from P. polyphylla based on GO categories.
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To further evaluate the completeness of our transcriptome library and the effectiveness of our annotation process, we searched the annotated sequences for genes involved in the Cluster of Orthologous Groups of Proteins (COG) classifications. Out of 30,500 nr hits, 5,339 sequences had a COG classification (Additional file 2). Among the 25 COG categories, the cluster for 'translation, ribosomal structure and biogenesis' represented the largest group (852, 16.0%), followed by 'post-translational modification, protein turnover, and chaperones' (630, 11.8%) and 'transcription', which we focused heavily on (207, 3.9%), which were particularly interested in. The categories of extracellular structures (0, 0%), nuclear structures (3, 0.06%) and cell motility (11, 0.2%), represented the smallest groups.
Additional file 2. COG annotations of putative proteins. All putative proteins were aligned to the COG database and could be classified functionally into at least 25 molecular families.
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Functional classification by KEGG
The 47,768 unigenes were compared with the KEGG database using BLASTX with an E-value cutoff of < 10-5. Of these unigenes, 15,628 (32.7%) had significant matches in the database. Among those, 5,962 unigenes were assigned to metabolic pathways with an enzyme commission (EC) number; however, 9,666 unigenes were not assigned to any pathways. Figure 2 shows the features of the pathway assignment based on KEGG. Most of the assigned unigenes are involved in the primary metabolites, such as carbohydrate metabolism, amino acid metabolism, energy metabolism and cell growth. Few unigenes participated in secondary metabolites. In particular, 127 unigenes related to phytohormone, sucrose biosynthesis and catabolism were assigned in the KEGG pathway. Some metabolite pathways related with sugar and phytohormone or phytohormone precursor metabolism, including starch and sucrose metabolism, steroid, terpenoid backbone, brassinosteroid and carotenoid biosynthesis are shown in Additional file 3.
Figure 2. Pathway assignment based on KEGG (A: metabolism; B: genetic information processing; C: environmental information processing; D: cellular processes).
Additional file 3. KEGG pathway involved in phytohormone synthesis such as steroid, terpenoid backbone, brassinosteroid and carotenoid biosynthesis (numbers in red indicate expressed genes).
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Gene expression analyses of P. polyphylla seeds during stratification using qRT-PCR
Using the method of Livak and Schmittgen [16], the level of GAPDH mRNA expression was selected as the internal control from four candidate genes (data not shown). The relative expression levels were calculated by comparing the CTs (cycle thresholds) of the target genes with that of the housekeeping GAPDH gene, using the 2-ΔΔCt method. Using the Student's T-test, differences in relative transcript expression levels were compared at P < 0.05 level between the embryo and the endosperm at two temperature treatments during seeds stratification. Sixteen primer pairs out of the thirty-six designed primers were successfully amplified a product (the primers shown in Additional file 4).
Additional file 4. Primers used in qRT-PCR analysis.
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The hormones abscisic acid (ABA) and gibberellic (GA) are considered central to dormancy and control of germination completion, while auxins are important for plant development and growth. In this work, six ABA and GA related genes (CYP707A, NCED, GA20ox2, GA20ox3, ABI2, PP2C) and five auxins-related genes (IAAH, IAAS, ARP3, ARP7, BRRK) were studied (Figure 3 and Figure 4). According to the previous studies [17,18], CYP707A, GA20ox2, GA20ox3, IAAH, IAAS and BRRK might be positively related to dormancy release, in contrast to NCED, ABI2, PP2C, ARP3 and ARP7 negatively related with dormancy release.
Figure 3. Relative expression levels analysis of transcripts positively associated with dormancy release (CYP707A,GA20ox2, GA20ox3, IAAH, IAAS, BRRK,) in the Embryo (Em□) and endosperm (En■) under warm (20°C)/cold (4°C) seed stratification processes. RS, radical just sprouted; RG, radical growing up to 1.5 cm. Expression is shown relative to GAPDH as the reference gene. RNA was extracted from three biological samples and qRT-PCR was performed. Data represent means ± SD. * indicate a significant difference between Em and En at P < 0.05.
Figure 4. Relative expression levels analysis of transcripts negatively associated with dormancy release (NCED, ABI2, PP2C, ARP3, ARP7, DRM) levels in the Embryo (Em□) and endosperm (En■) under warm (20°C)/cold (4°C) seed stratification process. RS, radical just sprouted; RG, radical growing up to 2 cm. Expression is shown relative to GAPDH as the reference gene. RNA was extracted from three biological samples and qRT-PCR was performed. Data represent means ± SD. * indicate a significant difference between Em and En at P < 0.05.
Figure 3 shows genes that are positively associated with dormancy release. CYP707A, which participates in ABA catabolism, showed higher expression levels in the embryo at two stages (radical just sprout (RS) and radical growing up to 1.5 cm (RG)) and at two temperature treatments than within the endosperm (P < 0.05). This indicates that ABA catabolism is active at low temperature in both the embryo and endosperm. GA20ox2 and GA20ox3, which are involved in GA biosynthesis, were expressed significantly differently (P < 0.05) between the embryo and the endosperm at the two temperature treatments. The mRNA level of the GA20ox2 in the endosperm was higher than that in the embryo, but the GA20ox3 expression was higher in the embryo than the endosperm. The expression levels of five auxin related transcripts (IAA hydrolase (IAAH), IAA synthetase (IAAS), auxin-repressed protein (ARP), and brassinosteroid insensitive 1-associated receptor kinase (BRRK)) were successfully determined using qRT-PCR. IAA is a major plant growth hormone that is important for numerous processes throughout plant growth and development. Both IAAH and IAAS participate in increasing plant IAA levels. In this work, the mRNA levels of IAAH and IAAS were significantly higher (P < 0.05) in both the embryo and endosperm under warm stratification compared with cold stratification. In addition, the IAAH expression level in endosperms was significantly higher (P < 0.05) than in embryos at the two temperatures. The difference in IAAS expression level was negligible between the two plant tissues. The BRRK gene expression level was positively correlated with temperature, but it expressed higher in endosperms than in embryos (P < 0.05).
Figure 4 shows genes that are negatively associated with dormancy release. NCED, an ABA biosynthesis key gene, was expressed at an increased level in the embryo relative to the endosperm. It showed decreased expression at the RG stage compared with the RS stage at both temperatures (P < 0.05). Among the many protein phosphatease 2C (PP2C) family members in plants, the ABA insensitive 2 (ABI2) belong to the subgroup A and acts as a global negative regulator of ABA signaling. In the presented study, the expression of ABI2 and a PP2C gene were determined by qRT-PCR. The results showed that expression of the two genes had a similar mode, being significantly higher (P < 0.05) in the embryo than in the endosperm. PP2C showed higher expression in the embryo at 4°C. The relative expression levels of ARP3 and ARP7 in the embryo were significantly lower (P < 0.05) than in the endosperm at 20°C, but there was no significant difference under cold treatment. These results indicate that the expressions of ARP3 and ARP7 were inhibited by warm stratification. In this work, three unigenes were annotated to the same dormancy-associated protein (DRM; AAW02792) and the mRNA level of the DRM gene in embryos was significantly inhibited (P < 0.05) compared with in the endosperm, under warm stratification, but not under cold stratification. The warm stratification stage is a morphological dormancy release period when the radical, hypocotyls and cotyledon are formed (Figure 5D). Therefore, this result indicates that DRM activity may be highly correlated with morphological dormancy release in P. polyphylla seeds.
Figure 5. Paris polyphylla var. yunnannsis seed (Em, embryo; En, endosperm; Co, cotyledon; Hy, hypocotyls; Ra, radical). A, the ovule 130d after fertilization; B, Freshly harvested seed not at full development (dyed with tetrazole); C, D, Radicle sprout testa after about 60d stratification.
Figure 6 shows the qRT-PCR analysis of four plant circadian rhythms related genes, ELF1 (Early flowering protein), ELF2, SFR6 (SENSITIVIE TO FREEZING-6) and Sus (Sucrose) [19-21]. The relative expression levels of ELF1 and ELF2 were significantly higher (P < 0.05) under cold stratification than under warm stratification, whether in the embryo and endosperm or at RS and RG stages. There were no differences in the relative expression of ELF1 in the embryo and endosperm across RNA samples; however, the expression of ELF2 showed a particular pattern: it was significantly higher expressed (P < 0.05) in the endosperm at 4°C stratification but was expressed at a low level in the endosperm at 20°C stratification. The expression levels of SFR6 and Sus were positively correlated with temperature treatments and they both showed higher expressions in embryos than in endosperms (P < 0.05). These results indicated that ELF1 and ELF2 might participate in different biological clock regulator processes, while SFR6 and Sucrose might have coincident regulator functions.
Figure 6. Relative expression levels analysis of four circadian rhythms related transcipts (ELF1, ELF2, SFR6, SUS) in the Embryo (Em□) and endosperm (En■) under warm (20°C)/cold (4°C) seed stratification process. RS, radical just sprouted; RG, radical growing up to 2 cm. Expression is shown relative to GAPDH as the reference gene. RNA was extracted from three biological samples and qRT-PCR was performed. Data represent means ± SD. * indicate a significant difference between Em and En at P < 0.05.
High-throughput transcriptome sequencing is an effectively gene mining method
Freshly matured seeds of P. polyphylla var. yunnanensis have embryos that are at an undeveloped globular stage. The embryos are very small relative to size of seed, which has a large endosperm (Figure 5A, B). Baskin and Baskin [9] characterized these plant seeds as the Morphophysiological dormancy (MPD) type with a temperature requirement for breaking dormancy and embryo growth. Although cold and/or warm stratification can break dormancy in seeds of many species [9,10], the molecular mechanism of seed dormancy release remains unclear. Nambara and Nonogaki [22] and Mochida and Shinozaki [23] suggested that new methodologies including analysis of transcriptomes, proteomes and metabolomes, might advance our knowledge and understanding of seeds development. Here, we obtained 47,768 unigenes, including 16,069 contigs and 31,699 singletons from the embryo of Paris polyphylla var. yunnanensis seeds that had undergone a stratification process, using the Roche 454-GS FLX Titanium platform. Using BLASTX searches of public databases, 15,757 unique transcripts were annotated with specific biological functions. Some transcripts were assigned to important plant physiological and biochemical processes, such as phytohormone biosynthesis and catabolism, seed maturation, cell wall growth, and circadian rhythms regulator, many of which might participate in seed development and dormancy release (Table 2 and Additional file 5). These results demonstrated that de novo sequencing and analysis of the P. polyphylla seeds during stratification were effective and informative in reflecting the transcript levels in the seed embryo.
Additional file 5. Possible transcripts involved in seed stratification of P. polyphylla var. yunnanensis.
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However, although we obtained abundant transcripts information, there were two aspects that require further study in future research: (1) Among the annotated 15,757 unigenes, some had functions that were obviously related to seed development and dormancy, others had no specific functions related to any particular biological processes; (2) 32,011 (67%) unigenes were not assigned to any specific functions. To further discover valuable genes in our data base, we consider that combinations of high-throughput sequencing, gene-chip and microarray analysis, and digit gene expression tag profiling with more classical methods (for example gene clone and Southern blotting) could greatly advance our knowledge of plant developmental processes. Recently such combinations were used to study seed-specific transcription factors and gene expression [24,25], and determine the roles of genes in seed dormancy in Arabidopsis[26].
Phytohormone related genes involved in P. polyphylla var. yunnanese seeds stratification
Compared with orthodox seeds, recalcitrant seeds like P. polyphylla undergo little or no maturation dehydration and remain desiccation sensitive during development. However, as in orthodox seeds, Huang et al. [7] and Chen et al. [8] indicated that the ABA levels increased during P. polyphylla seed development. According to previous studies [27-29], seed dormancy and germination are controlled primarily by the balance of ABA and GA. ABA is a negative regulator of seed germination, while GA, BR, cytokinins, and ethylene are positively associated with dormancy release. In this work, there were six types of phytohormone-related genes annotated in the database including GA2ox, GA20ox, GA3ox, CYP707A1, NCED, BR, ACC, ACO, and IAA. GA3ox (GA 3-oxidase) and GA20ox (GA 20-oxidase) participated in GA biosynthesis, while GA2ox (GA 2-oxidase) catalyzes the catabolism of biologically active GA and its precursors [29-32]. ABA 8′-hydroxylation (encoded by the CYP707A gene family) was shown to play the predominant role in ABA catabolism [33], while NCEDs (9-cis-epoxycarotenoid dioxygenase genes) are rate-limiting enzymes in ABA biosynthesis [34,35]. Therefore, GA3ox, GA20ox and CYP707A are positively associated with dormancy release, while GA3ox and NCEDs are negatively related. The qRT-PCR analysis indicated that GA20ox2 and GA20ox3 expression patterns were very dissimilar in embryos and endosperm even at both temperatures, suggesting they might be different kinds of GA20ox genes. These observations are consistent with our annotation results for GA20ox2 and GA20ox3 from Zea mays (GenBank: ACG35782) and Glycine max (GenBank: ACJ76438) (Additional file 5). The GA2ox and GAST-like (gibberellic acid stimulated-like gene) [36] are also important GA regulator genes; however, qRT-PCR analysis using the designed primers was not successful. In our cDNA library, four unigenes (two contigs for CYP707A1 and two singletons for NCED) associated with ABA metabolism were obtained. Relative expression analysis indicated that CYP707A and NCED are both expressed both in embryos and endosperm, and are expressed at higher levels in embryos than in endosperm. These result demonstrated that a shift in ABA levels in embryos might be an important factor during seed stratification. However, we only found four transcripts related to ABA metabolism among 47,768 unigenes, which indicated that ABA metabolism is low in the embryos. Footitt et al. [28] suggested that ABA signaling and sensitivity were more likely regulators of dormancy than the absolute level of ABA. We found 22 transcripts of PP2Cs, which are the ABA signaling molecules [37], in our sequence data set. PP2Cs, including ABA-INSENSITIVE1-2 (ABI1, ABI2), are negative regulators of seed germination [27,37]. The expressions of PP2C and ABI2 genes in this work suggested that ABA signals are more active in embryos than in the endosperm and their expression patterns are consistent with the expressions of CYP707A and NCED.
IAA hydrolase contributes free IAA to the auxin pool during germination in Arabidopsis[38], while IAA synthetase may catalyze the entire pathway of biosynthesis of the major plant growth hormone [39]. The relative genes expression ratios of IAAH (IAA hydrolase gene) and IAAS (IAA synthetase gene) were compared between embryos and endosperm in P. polyphylla at two temperatures. The results indicate that higher IAAH and IAAS relative expression levels were correlated with stratification temperature and also related to seed morphological dormancy release. We also found that the two expressions of two ARP genes were decreased in embryos during warm stratification and were negatively related with IAAH and IAAS expressions. Park and Han [40] demonstrated that cold treatment abolished the auxin-mediated repression of RpARP gene expression and that its expression was negatively associated with hypocotyl elongation. The Arabidopsis thaliana SERKs (Somatic embryogenesis receptor kinases) are essential for the early events of BR signaling pathway [41,42]. In the present study, two transcripts of brassinosteroid insensitive 1-associated receptor kinase gene (BRRK) and six transcripts of SERK gene were found. Relative expression analysis of the BRRK indicated differences in transcription levels between embryos and endosperm at two temperatures.
Other genes associated with P. polyphylla var. yunnanensis seeds stratification
Dyer [43] identified two dormancy-associated mRNAs with over 3-fold higher expressions in dormant compared with non-dormant embryos of Avena fatua. Expression of the dormancy-associated genes ATS2 and ATS4 was high in the dry seed and decreased during germination [44]. Using qRT-PCR method, we analyzed a DRM gene that is very similar to the dormancy-associated protein gene (GenBank: AAW02792). The expression pattern of DRM in this work is similar to that reported by Dyer [43]. Toorop et al. [44] reported high expression in dormant seeds and low expression during germination. However, many similar genes were also found, such as Oryza sativa dormancy-associated protein (GenBank: AF467730) and Zea mays auxin-repressed 12.5 kDa protein (Eu967389), in nucleotide database. These findings indicated that although only one dormancy related gene (three unigens with same accession no. GenBank: AAW02792) was sequenced in this work, its specific physiological function must be further investigated in the future.
Several genes, such as CCA1 (Circadian Clock Associated 1), LHY (Late Elongated Hypocotyl), TOC1 (Timing of CAB1), and GI (GIGANTEA) have been identified as major components of the circadian clock [17,45]. In this work, we obtained two genes, ELF (Early Flowering Protein gene) and SFR6 (Sensitive to Freezing 6 gene) that are related to circadian rhythms. However we did not find CCA1, LHY, TOC1, and GI. ELF3 is a novel protein associated with control of plant morphology, flowering time, and circadian rhythms in Arabidopsis. Some studies showed that elf3 mutations cause a light-dependent circadian dysfunction, elongated hypocotyls, and early flowering [20,46]. In this research, 21 unigenes were annotated to ELF, which were found in monocots Elaeis guineensis (16 unigenes matching GenBank: ACF06553) and Asparagus officinalis (five unigenes matching GenBank: AAB09084). Knight et al. [21] indicated that SFR6 is a component of the photoperiodic regulatory pathway. They also observed that clock gene expression and sucrose might act as a regulator of clock function and interacts with SFR6. In the present study, we obtained 20 SFR6 unigenes, which all annotated to the same Arabidopsis SFR6 (NP-192401.5) gene, and 22 unigenes that matched sucrose synthase genes. A decline curve of sucrose content variation was found in the P. polyphylla seeds stratification process from zero to 100 days and this was accompanied by seed dormancy release (data not shown). In this study, the mRNA expression levels of ELF1, ELF2, SFR6 and SUS varied in the embryo and endosperm and at both temperatures. These results suggest that ELF, SFR6 and sucrose, as clock components, may participate in the germination of P. polyphylla seeds during warm/cold temperature stratification.
LEA proteins might be involved in embryo development or genetic diversity [47,48]. The deduced LEA protein sequences are classified into at least five groups according to their conserved motifs or sequence similarity [49]. Group I to IV LEA proteins are highly hydrophilic, and the remainders are hydrophobic. Dehydrins are group II LEA proteins that contain high glycine levels and are highly hydrophilic [50]. Some dehydrins are constitutively present in vegetative tissues during normal growth [51], but others are induced by tissue water-deficits, such as drought, salinity, low temperature, and seed maturation [52,53]. Recently, in wheat, it was demonstrated that Dehydrin is associated with the maintenance and breaking of seed dormancy, and that ABA affected the expression of Dehydrin gene at the transcript level [54]. Sixteen dehydrins transcripts (Additional file 5), which were annotated to seven species of plant dehydrins, were found in this research in our cDNA library.
Cell wall remodeling enzyme, including endo-β-mannanase, β—1, 3- glucanases, expansins, xyloglucan endotransglycosylase, pectin methylesterase, polygalacturonase and cell wall invertase may participate in endosperm weakening and seed germination [55-62]. In the present study, we found 104 unigenes with high similarity to these proteins (Table 2 and Additional file 5). We also found many genes, such as CHS (chalcone synthase), CHI (chalcone-flavanone isomerase), F3H (flavanone 3-hydroxylase), and DFR (dihydroflavonol reductase), which were expressed abundantly in the embryo of stratified seeds. Recent research [63] showed that these genes are expressed in the embryo and in the endosperm at germination stages of Arabidopsis seeds. However, the physiological role of flavonols in the endosperm is still unclear, though such flavonols might protect the embryo or control seed germination by regulating auxin transport or by acting as scavengers [64].
The relative expression levels of analyzed genes in seed stratification
In this study, the expression levels of mRNA in different stratification stages or different treatments were altered by less than twofold. We considered that there might be two explanations. (1) Although these genes (GA2ox, GA20ox, CYP707A1, and NCED) are key enzymes involved in a metabolic network, we cannot determine the amount of the final product. Some studies [7,8] showed that ABA content decreased while GA content increased, and the GA/ABA increased by over tenfold when seed stratification was completed. (2) The embryo and the endosperm are two tissues, but they exist in one organ, the seed. Comparisons of gene expression between the embryo and endosperm might show only subtle differences under the same temperature treatment.
Despite considerable progress in seed biology, the major factors affecting seed dormancy, seed stratification and drying after-ripening remain unclear. Finkelstein et al. [17] and Kucera et al. [18]) have reviewed the role of plant hormones during seed dormancy release and germination, and the molecular aspects of seed dormancy. However, unlike orthodox seeds, the dormancy release of P. polyphylla seed is different because of its undeveloped embryo, which requires morphological (cotyledon, hypocotyls and radicle) establishment and physiological dormancy release during the stratification process.
Post-genome methodologies, such as analysis of transcriptomes, proteomes, metabolomes and bioinformatics have advanced our understanding of seed germination. The present study further demonstrates that the high-throughput embryos transcriptome sequencing of P. polyphylla seeds is a highly effective method for mining genes that may be involved in seed stratification and dormancy release. A large number of transcriptome data of P. Polyphylla seeds have been recently released in the NCBI SRA database under the series identifier SRX155369, which provides an additional resource for the discovery of genes in P. polyphylla. Bioinformatics and qRT-PCR analysis help us to identify many important genes, such as CYP707A, NCED, GA20ox, PP2C, SFR6, ELF, DRM, which should be studied further to clarify their specific function in seed dormancy release. In addition, there were 67.01% (32,011) unique putative transcripts that were not annotated during BLAST searches against NCBI non-reduntant protein database. This large amount of unidentified sequences should also be studied with a view to annotating new transcripts.
Plant material and RNA preparation
Seeds of P. polyphylla var. yunnanensis were harvested from plants growing in Wuding county, Yunnan Province, China in October 2010. The freshly matured seeds were stored in wet sand in a temperature-controlled incubator at 20°C (warm stratification treatment) for 2 months and then at 4°C (cold stratification treatment) for 2 months. The pulpy outer layer of the seeds coat was removed, and then soaked in water for 24 h prior to the temperature stratification. For the construction of the 454-sequencing cDNA library, three embryo samples at different development stages (radical just sprout testa, radical growing to length 0.5 and 1.5 cm) at two temperature treatments (4 and 20°C) were excised, frozen in liquid nitrogen immediately, and then stored at −80°C until RNA isolation. For qRT-PCR analysis, the embryo and endosperm were sampled at two stages RS (Figure 5C) and RG (Figure 5D), and at the two temperature stratifications.
Total RNA was isolated from 2 mg each of the two temperature treatments using the RNeasy plant kit (BioTeke, Beijing, China). The RNA quality was assessed using a 1% agarose gel and quantified by a GeneQuant100 spectrophotometer (GE Healthcare, Chalfont St Giles, UK) before proceeding. After pollination (13d), the ovules were fixed using FAA (95% alcohol: acetic acid: formalin: water = 10:1:2:7) and made into tablets for microscopic inspection.
cDNA library construction and pyrosequencing
Approximately, 2 ug of poly(A) RNA was isolated from equal mixtures of the three total RNAs using an Oligotex mRNA Midi Kit (Qiagen, Shanghai China). The long poly(A/T) tails in cDNA may lead to low-quality sequencing reads from the GS FLX system. To overcome this limitation, we designed a modified poly (T) primer with a BsgI site between the adaptor and the poly(T) 5′-AAGCAGTGGTATCAACGCAGAGTACT(20)VN-3′) [65]. For cDNA synthesis, this poly(T) primer was used in combination with the Clontech SMART IV primer. The cDNA was then treated overnight with BsgI (NEB, MA, USA) at a concentration of 5 units/μg of cDNA. This restriction enzyme cut within the poly(A) tail, and greatly increased the quantity and quality of the sequencing reads. For the library, digested cDNA was amplified using PCR Advantage II polymerase (Clontech, Dalian, Chian) and the following thermal profiles were used: 1 min at 95°C; followed by 16 cycles of 95°C for 15 s, 65°C for 30s, and 68°C for 6 min. PCR products (5 ul) were determined by electrophoresis. Approximately 3 ug ds cDNA was sent to the Beijing Institute of Genomics of the Chinese Academy of Sciences (Beijing, China) for pyrosequencing using the 454-GS FLX Titanium Kit.
Unigene assembly
Using the GS FLX pyrosequencing software, high-quality sequences (> 99.5% accuracy on single base reads) were selected for further processing and assembly. A subsequent filtering step, which included the masking of SMART PCR primer sequences and the removal of sequences shorter than 50 bp, was performed before assembly. The Newbler software 2.3 (provided with the GS FLX sequencer) was used for sequence assembly using the default parameters.
Sequence annotation
The sequence annotation was based on a set of BLAST searches. The sequences were searched using BLASTX against the NCBI non- redundant protein (nr) database with an E-value cut off of 10-5, then the NCBI non- redundant nucleotide (nt) database was searched. The remaining no hit sequences that putatively encoded proteins were searched against the Arabidopsis protein database in the Arabidopsis Information Resource (TAIR 2.2.8); a typical cutoff value of E < 1.0-5 was used. Pathway assignments were carried out according to KEGG mapping. Enzyme commission (EC) numbers were assigned to unique sequences that had BLASTX scores with cutoff values of E < 1.0e5, as determined upon searching the protein databases. The sequences were mapped to the KEGG biochemical pathways according to the EC distribution in the pathway database. We performed a functional classification of the unigenes following the Gene Ontology (GO) scheme. The sequences were annotated by a BLAST search against a series of protein and nucleotide databases, including the curated protein database of Uniprot/SwissProt. The transcripts were classified into 45 GO categories under the major categories of Cellular Component, Molecular Function and Biological Process. A unigene blast was performed using the uniprot database and the annotated GO function. The unigene (congtig and singletons) blast was performed on reference canonical pathways and annotated KEGG pathway.
Real-time PCR analysis
Total RNA was extracted from embryos and endosperms from warm and cold seed stratification stages using the RNeasy Plant kit (BioTeke, Beijing China). Approximately 1 μg of Dnase I-treated total RNA from each was converted into single-stranded cDNA using a PrimeScript™ RT reagent Kit (Takara, Dalian, China). The cDNA products were then diluted 10-fold with deionized water before use as a template for real-time PCR. Each reaction contained 10 μl 2× SYBR Premix DimerEraser (Takala, Dalian, China), 1 μM each of the forward and reverse primers, and 1 μl of template cDNA. The total reaction volume was 20 μl. PCR amplification was performed under the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, using CFX96TM Real-Time system (Bio-Rad, USA). Thirty-six unigenes from the 454 data were selected to design qRT-PCR primers and ten housekeeping gene transcripts were selected for design internal control primers (Additional file 4). Each embryo sample in 4°C stratification was selected as a calibrator and the data were presented as the fold change in gene expression normalized to an endogenous reference gene and relative to the calibrator [16]. The qRT-PCR analyses were performed three times with independent RNA samples.
ABA: Abscisic acid; BR: Brassinosteroid; COG: Cluster of orthologous groups of proteins; EST: Expression sequence tags; GA: Gibberellins; GO: Gene ontology; IAA: Indo-3 acetic acid; KEGG: Kyoto Encyclopedia of Genes and Genomes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JQ conceived this study, designed and built the cDNA library, participated in data analysis, and drafted the manuscript. NZ participated in seeds stratification, RNA extraction, library construction and data analysis. BZ and SH performed the sequence assembly, alignment and annotations. PS and WX participated in sequence data analysis, the real-time qPCR and the corresponding data analysis. QM and TZ carried out the field management, seed collection and seed stratification. LZ and MQ helped to conceive the study and performed the statistical analysis. XL initiated the project, helped to conceive the study, and participated in the design and coordination. All authors read and approved the final manuscript.
We would like to thank Dr. A. Egrinya Eneji of the University of Calabar, Nigeria for helpful comments on the manuscript. This work was supported by the National Natural Science Foundation of China (Grant number 31171623) and Traditional Chinese Medicine Support project (2008) of Ministry of Industry and Information technology of PR China.
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I bought 3 of these stools for my very light granite counter. I really like that they are low profile and do not hide my expensive counter. They look great with the nailhead detailing and the color is just right. My daughter was selling her home and borrowed them for her open house events. She still has them so I bought her two for her birthday...
This plush counter stool gives your kitchen some quality furnishing. The soft beige cushion provides plenty of comfort, and the chair back provides support. Beautiful nailhead trims around the bottom and maple finish complete the piece.
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This Noho counter stool offers simplicity in design. The seat is made of soft beige linen, subtle bisquit tufting and individual nailhead trims around the bottom and back of the barstool.- Materials: Birch wood, linen fabric
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Great, but…. They came damaged and missing metal decorative rivets….had to repair first and glue on the little pieces that were found laying on the bottom of the box. also, the wood legs were scratched badly. Design is great, just a bit shabby on quality. Bought 3 after seeing them in the model home we eventually bought. perfect counter height, not for the taller breakfast bar counters. These are tiny stools. Check dimensions carefully. love the design.
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Love these stools I bought 3 of these stools for my very light granite counter. I really like that they are low profile and do not hide my expensive counter. They look great with the nailhead detailing and the color is just right. My daughter was selling her home and borrowed them for her open house events. She still has them so I bought her two for her birthday...
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Good Seatback is a little small, but overall nice bar stool
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Miniature in size, color is grey, but well made I can't see many homes where the size of these chairs would coordinate with existing décor. They look very miniature despite fitting to counter height (backs are small, seats are small, just appear odd). Grey in color too which I thought was going to be more neutral than it appeared. However, look to be quality made and studs look nice, wish it was just bigger and more appropriate for my kitchen. Be very careful about measuring and envisioning before ordering, returning is $*** to ship back.
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Perfect for our counter height vanity! I want to thank previous reviewers who made a point of detailing the size,height and color of this chair. It was just what I was looking for, and fits perfectly into our newly remodeled bathroom that has a 36" counter height vanity. I sit at it to put on makeup, and even to blow dry my hair. The chair has plenty of cushion to it and is very sturdy. Shipping was perfect. Thanks, Overstock.com.
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Nice stool, but smaller than it looks. The back of these chairs do NOT show above your counter. In other words, if you are wanting the type of counter stool where the back pops up over the counter so you can see the style, these are too short. As far as the height of the seat, it's perfect and works well at the counter. It's just that the backs are much smaller than they look in the photo. Pay close attention to dimensions when you order these. The color is darker than the photo too. I was hoping that they were the traditional light/ivory color of linen and they are definitely darker than that. They are more taupe/dark beige than ivory. All that said, they are nice. They look nice and the style is very current.
Read Morewebbsquirrel Apr 24, 2013 - Helpful 7
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Great styling We ordered these chairs to accompany our counter-height table and they're absolutely perfect in size and styling. The fabric is really nice, tufting is sleek, and it's exactly as it looks on the site. We did have one loose nailhead, but that's easy enough to glue back on. The chair arrived fully assembled and is quite sturdy (yet light enough to maneuver easily). The color is a beige linen, which we incorporated into our living room that is actually painted gray, and it works well. This is a lovely neutral piece that is also quite stylish and comfy. (Make sure you pay attention to the dimensions. The seat back is fairly low, though not uncomfortably so. But if you're expecting a traditional high back dining chair, this isn't it.)
Read Moremissnattymich Sep 4, 2012 - Helpful 2
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LOVE this stool! I absolutely LOVE this counter stool. It is beautiful and well-made. Unfortunately, it did not work with my kitchen due to size requirements. The sizing on the website is accurate, they just didn't look right scale-wise for me, but there was NOTHING wrong with the chairs. If they were a little wider and the back was a few inches taller I would have kept them and bought more. If you think the measurements will work for you and you like them, then I would get them. They are great quality and very sharp looking. I'm so bummed I had to return them but definitely think they are worth the price!
Read Moreamadonia Aug 21, 2012 - Helpful 2
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Questions & Answers
Seems to match our current furniture and room décor.
What finish is the wood? Is it black?
These chairs are upholstered in a lovely beige linen and stands on solid birch wood legs finished in a rich espresso. Thank you for your question. Please feel free to contact us again with any further questions.
Dark brown
What is the seat height of this stool?
The seat height of this item is 23 inches.
Very small and short for counter
Shopping Guides
Why You Should Have a Bar Stool in Your Bathroom
A list of traditional bathroom furniture usually doesn't include a bar stool. However, this traditional bar furniture is slowly gathering popularity for its versatility. A bar stool is good for more than just sitting at a kitchen counter. You can just pull your bar stool right up to the vanity and enjoy the benefits it has to offer. Keep reading for more reasons why you should have a bar stool in your bathroom.
How to Maintain Bar Stools
No home bar can survive without some comfortable bar stools. Bar stools come in a variety of styles and sizes to match any decor and bar height. You can find anything from swivel bar stools and wooden bar stools to high-gloss metal bar stools in any color imaginable. After you've purchased the perfect bar chairs for your room, you'll want to ensure they last as long as possible. Maintaining your bar stool set depends on the material and style that you have purchased. Each bar stool should be checked every few months for loose bolts and connections. A loose bolt can cause the stool to collapse. Other than that, kitchen bar stools and counter stools should be cared for depending on material. Here are a few tips to make sure your bar stools last as long as possible.
Why You Need Bar Stools
Most people think of bars and pool halls when they hear the word bar stools. However, bar stools are natural additions to any home. Bar stools offer more than just seating; they also offer style and functionality. Not only are they extremely convenient for seating extra guests; they also have many day-to-day uses for your home. Keep reading to discover why you need bar stools in your home.
How to Choose the Right Swivel Bar Stool
When you're in the market for a swivel bar stool, your biggest challenge is likely to be choosing just one from hundreds of choices available. A bar stool will lend personality to your space. Spend a few minutes considering the following factors so you can choose a swivel bar stool that fits your lifestyle perfectly. Below, you'll learn how to choose the right swivel bar stool.
Top 5 Materials for Bar Stools
Whether you need seating at a kitchen counter, breakfast bar or bar table, bar stools solve your seating crisis. Bar stools are available in many styles and come in leather, wood, plastic, metal and other materials. You can even find bar stools upholstered in fabrics of every color, which means that you can match any room in your home. With so many possibilities available, it's good to know what the top five materials for bar stools are in order to make a decision on what will work best in your home.
Tips on Buying Bar Stools
You don't sit down on a bar stool; you sit up on a bar stool. And that seems to make all the difference. People sitting on stools seem to be much more lively and talkative. But stools, whether at a bar in your basement or around the center island in your kitchen, still create a casual, relaxed mood. Adding counter stools or bar stools can add a lot of energy to a home. They are well suited for cocktail parties and holiday gatherings when no one wants to establish themselves in one location too long but might want to get off their feet for just a moment. Fortunately, the many styles and colors of stools let you easily find some that are right for you, regardless of your home's decor.
FAQs about Bar Stools
Introduce a casual atmosphere to your countertop or bar with a set of bar stools. Whether you need additional seating or want to create a fun, new gathering place in your home, bar stools can augment every room in your home. Before you find the perfect barstools for your home, consider some common questions that might help you on your way.
Bar Stool Buying Guide
Whether you have a bar-height table or a kitchen counter you want to get more use out of, you need bar stools. But which ones are right for your home? And how will you know if you're buying the right size? This guide has the information you need to confidently buy the best bar stools for your home. <img src="img/mxc/150424_bar-stool-BG-PIN.jpg" class="hidden"> Shop Bar Stools ▸
Where to Use Bar Stools Besides the Bar
Because of their name, bar stools are usually limited to serving as seating at bars, when in reality they are perfect for many other places around the house. Their height makes them an ideal addition to rooms besides the kitchen. Once you see what else you can do with bar stools, you'll wonder why you went so long before placing them everywhere.
How to Decorate with Bar Stools
From swivel seats to adjustable heights, bar stools have features that make them some of the most convenient furniture for your home. The kitchen has become the place where everyone congregates, so you need to have plenty of seating options. Adding bar stools not only gives you additional seating for your family and guests but can also enhance your home's decor.
Bar Furniture Buying Guide
You may love to invite friends for martinis, or you may be having over the guys for the big game. You might need kitchen stools, so you can use a counter as a breakfast bar. Or you may be adding a dining area to a part of your house you didn't think was going to be usable before. If you love to entertain, you know you need a home bar. How far you take it is up to you. The home bar furniture you choose should reflect your style and show your guests that they are welcome in your home. Here are tips to help you buy the right home bar furniture for you.
Plastic Chairs vs Wood Chairs
Whether you're looking for dining chairs, kids' chairs, bar stools, lawn furniture or office chairs, both plastic and wood designs are available. Are you looking for bright colors, smooth curves or ornate styles? Each material has its benefits, so you can select the chairs that are the perfect furniture for your home.
Best Counter Stool Upholstery Materials
Upholstered counter stools allow homeowners to add warmth to their home decoration through the use of different types of fabrics. Counter stools come in a variety of upholstery materials, allowing you to choose from a wide selection for your home. Before you make a choice, though, consider the type of upholstery fabric you'll use and determine whether it is a good fit for your home. This article will help you learn about the best counter stool upholstery materials.
Best Bar Table for Each Room in Your Home
A chic bar table is a crucial piece of furniture for your bar or lounge. However, a bar table isn't just an important piece of bar furniture; it can migrate to every room in your home to create a chic environment. Next time you shop for bar and pub tables, think about the other rooms in your house.
How to Hang a Towel Bar
Properly installing a towel bar can ensure that these essential bath fixtures doesn't damage your wall. A towel bar may be used to display decorator towels to complete the room's decor or hold wet bath towels after bathing or showering. A towel bar might also be subjected to serving as a hanging spot for clothing. The weight of wet towels combined with the action of pulling towels off the bar puts additional pressure on the towel bar. Improper installation of a towel bar can result in the bar separating from the wall and damaging the wall's surface. Follow these simple steps to be sure your towel bar is properly installed.
How to Maintain Bar Tables
Getting new bar tables can breathe new life into your bar or lounge. Even the living room and family room are perfect places for bar tables. However, you probably won't be replacing your bar furniture every year for that same fresh feeling. Instead of letting your bar or lounge area fade with the years, apply some careful maintenance to make sure the appeal of your pub tables stays in full force throughout the years. Here are some tips that will help you keep bar tables in top condition for years to come.
How to Set Up a Cocktail Bar
A well-stocked cocktail bar can be put together with a few common bar and wine tools and a variety of liquors, mixers and garnishes. This type of bar setup is ideal for any party with adult guests. Your cocktail bar can be self-serve or manned by a designated bartender for the evening. Whether you have a full bar available in your home or you are simply using a regular table set with glasses and barware, you can set up a stunning cocktail bar with ease.
Shopping for Bar Tables Checklist
When you are choosing bar tables for your home, you'll want to make sure you're happy with your decision for a long time. Bar tables are perfect pieces of furniture for several rooms in your home, not just your bar. Because it is so flexible, bar furniture takes some consideration before you pick any old pub table to fit your room. Here are a few things to consider before you go shopping for bar furniture.
How to Set Up a Backyard Bar
Whether you are planning a new an outdoor dining area or just want to add socializing space to your backyard, a backyard bar is always a great addition. A bar adds an extra dimension to your patio, and it's a great place to socialize and relax that's just steps from your door. Here is how to create your perfect backyard bar.
Bar Glasses Checklist
Before you purchase a generic bar glass set, you should know the many different shapes and sizes that bar glasses come in, plus the traditional uses for each kind. This type of knowledge will surely impress your friends and help make your gatherings truly special. Once you have collected the right glasses and barware in your home, you'll entertain your guests in style.
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Usually leaves our warehouse in 1-2 business days. *
Standard Return Policy:
Items must be returned in new or unused condition and contain all original materials included with the shipment. More Details
FINAL SALE EXCLUSION: Items marked as FINAL SALE are not returnable unless the problem you experience is the result of our error.
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Our standard shipping method is ground shipping. We also offer expedited shipping for most items. Your final
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* For your protection, all orders are screened for security purposes. If your order is selected for review, our Loss Prevention Team may contact you by phone or email. There may be a two business day delay to process your order.
** Most Oversize orders are delivered within 1-4 weeks. Some orders may take 6 weeks to be delivered.


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Yo, let me know if there's some fire to check out in the 314, 734 or 248
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S-CHI^TOK@ Reply:
October 7th, 2011 at 2:41 am
oh yeah make your way down to the 708 773 312 area shznitts been wow to say the least kinda new to all a this wanna give tips on good signs or growin email@example.com thnks your quite informative peace
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C Unit Reply:
October 31st, 2011 at 10:53 pm
Thanks! Let me know if there's anything you hear that's good and I'll look for it to review
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perk1perk1 Reply:
July 29th, 2013 at 9:11 pm
I ran across some *fire* from one of the *M.O.M.'s(firstname.lastname@example.com) called O.M.F.G.! and it is bad to the bone. it is one of those hybrid afghani/sativa kind as you can smell the afghani and taste it as well. "By the time you feel it you should put it out as you have had plenty" kinda buds….
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arisbens Reply:
January 27th, 2015 at 7:58 pm
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what is the strain blackwater,picked it up yesterday from dispensary.
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perk1perk1 Reply:
July 29th, 2013 at 9:13 pm
is it the bad to the bone kush? that Blackwater o.g. kush is very good stuff… takes care of what ails ya kinda buds,,, I hope all is well with all of you all as my eyes are fading and it is nap time..
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I'm movin back to Michigan how can I get in touch
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Need-Connect Reply:
September 17th, 2013 at 4:45 am
Yo I need high quality dro for the Midwest and its really dry always..anyway u would consider meeting up and getting some business going? I ain't no bitch ass cop or whatever hit me up bro I'd appreciate it
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Anyone interested in plugging the northern Midwest? Need a legit person with high quality willing to meet up..I have lots of needing patients up here but its always dry. Help? Comment back we'll get in touch I can drive to you.
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Hey I really like your review's Man. By far the most legit reviews I have read. Good info and always Linage included and its always spot on.. Good stuff for real. MMS should just higher you to do reviews for there site if they don't already lol.
Stay up
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Damn yall got it for the low yall need to look out im in durham this shit sucks down here
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Location305 Wilsons Road, WHITTINGTON, GREATER GEELONG CITY File NumberB3978LevelState |
St Albans Homestead at Whittington, a single storey 30-roomed brick mansion, was erected in 1873 for the noted race horse breeder and trainer James Wilson. The Melbourne architect James T Conlan designed the extensive homestead in the fashionable polychromatic mode with an elaborate cast iron verandah by Geelong foundry men Humble & Co.
There were additions to the homestead in 1875 designed by Conlan and in 1878, alterations and additions designed by Conlan and Salway. The homestead was identified as a significant building in the Geelong Regional Commission Study. The integrity of the property is high. St Albans has both historical and architectural significance for the following reasons:
(1) it is a most distinctive work of the architect JT Conlan.
(2) it is of significance as one of the leading horse racing studs in Australia in the late 19th century.
(3) many Melbourne cup runners were trained on the St Albans track. The legendary Phar Lap stayed briefly at St Albans stud in 1930 on the eve of his celebrated Melbourne Cup victory.
Adapted from Heritage Victoria Statement: 21/05/2012
Classified: 26/10/1978
Revised: 03/08/1998
Residential buildings (private)


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Peggy V. Helmerich Library will be closed May 1st & 2nd for repairs.
J. S. (Joshua) Cosden was born in Kent County, Maryland in 1882. He came to Bigheart, Oklahoma in 1908 and later relocated to Tulsa. In 1913, Cosden and Company began operating their West Tulsa refinery, two miles upstream from the Texas Co. (later Texaco) refinery. At that time, the refinery had a total capacity of less than 5,000 barrels of crude a day. Two years later, the refinery added 97 miles of pipeline to connect the plant to the famous Cushing oil field.


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Check out some available loans that are similar to this one!
For the past year, Ahmad has had his own business, operating an electrical goods store. His store is located in a good commercial area and he has many loyal customers, which makes his project a profitable one. It is the only source of income for his family, and Ahmad needs more support to cover some costs.
Ahmad is requesting a $1,000 USD Youth loan from FATEN to cover the cost of purchasing more electrical goods for his store. Taking this loan will increase sales and improve Ahmad's income, so he can provide better living conditions for his family.
Additional Information
More information about this loan
This loan is helping support a borrower who is 35 or under. Because many young people lack business experience or established credit histories, it can be even more difficult for them to access financial services. By funding this youth loan, you are expanding opportunity for a young person with limited resources.
About FATEN
Palestinians face unique challenges due to the economic and political environment in the West Bank and Gaza. Click here to learn more about how Kiva and FATEN are working to help Palestinian borrowers overcome these obstacles and how your support can make a difference.About Palestine
66View loans »
US Dollars
Success!! The loan was 100% repaid
A loan of $1,000 helped Ahmad to purchase more electrical goods to sell.
- Repayment Term
- 21 months (Additional Information)
- Repayment Schedule
- Monthly
- Pre-Disbursed:
- Jan 29, 2013
- Listed
- Feb 27, 2013
- Currency Exchange Loss:
- Ended:
- Oct 17, 2014


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Latest 'the invisible man' News
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'Monsters: Dark Continent' screenwriter Jay Basu has joined Universal Studios' brain trust of monster movie writers.
by H. Shaw-Williams | 5 months ago7 Comments
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We now know which characters are planned for Universal's rebooted monster movie universe, and who's writing the films.
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by Sandy Schaefer | 9 months ago72 Comments
David S. Goyer's 'Invisible Man' Remake Is Still Alive
Universal's 'Invisible Man' remake is still moving forward – and screenwriter David S. Goyer says that he is giving the classic H.G. Wells story the 'Mummy' treatment.
by Sandy Schaefer | 4 years ago5 Comments
No New Batman Script For Awhile; David S. Goyer Is A Busy Man
David Goyer talks about his upcoming projects, including a Magneto origin film & a new Invisible Man movie
by Kofi Outlaw | 7 years ago21 Comments
Weekend Movie News Wrap Up: August 10, 2008
Welcome to another fun filled installment of Screen Rant's Weekend Movie News Wrap Up. This week: We see who is penning Flash Gordon, we learn some…
by Niall Browne | 7 years ago8 Comments


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Looks like they beat me to it lol. Posted before I read the comments. Oh well.
Looks like they beat me to it lol. Posted before I read the comments. Oh well.
The entrance to HWL to Hyperion Theater to TOT are all staying. Muppets, Mike and Sulley to the Rescue, Millionaire, Hollywood & Dine will become Monstropolis.
They could be Pixar/Disney version of those monsters. They for sure can't resemble the Universal monsters and probably can't be named.Those are all owned by Universal, that won't happen
The TRON lightcycle ride could be like this but thru TL and maybe an inside section replacing Innovention.
I hope you are correct!Ohh you wait.....Star Wars and Marvel lands are coming...but in a third theme park...where they'll have a big budget and even more land to really make use of these idea's and characters
I am curious as to what the Tron coaster would mean for the future of the Carousel Theater. Unless that segment of track is bypassed it sure would complicate removing the building for Mr. Stark, much less removing even more where the guideway sits above the Submarine Lagoon. I am also curious as to where such an attraction would load. The Rocket Rods entrance is now occupied and the main Tomorrowland promenade is far too small, even if the Astro Orbitor was moved to its proper place. Could the Tron attraction be based in or at the site of the Carousel Theater? The ground floor could be queue space while the upper level could be a show room.
What if they finially did something with the Starcade?
I'm of the opinion that this is a great idea.
For those seriously concerned about DCA becoming Pixar-land, I'd venture a guess that Bugs Land is not long for this world. It combined with the leftover Timon lot seem to be the last logical place for expansion.
I will be upset if the Carousal Theater was only use for the queue and loading. It should be use for a show room. IMO TRON should be completely indoors and the old PM route should be replace by a similar ride to PM. But if the only choice was between TRON Light Cycles on the PM route or nothing, I would foresure pick the Lightcycles
That said, how do you tie it into Pixar plush sales?
I hope they consider building Monstropolis in the third gate. It would be a better fit with Marvel and Star Wars then it is with DCA. It would also be a good land to feature more kids attractions in a park that will need something for kids. You could do a few things for kids with Star Wars, but I assume most of the Marvel attractions will be more aimed at an older audience.
The Mickey audience is not made up of people; it has no racial, national, political, religious or social differences or affiliations; the Mickey audience is made up of parts of people, of that deathless, precious, ageless, absolutely primitive remnant of something in every world-wracked human being which makes us play with childrens toys and laugh without self-consciousness at silly things, and sing in bathtubs, and dream and believe that our babies are uniquely beautiful. You know the Mickey in us.
-Walt Disney


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Snap Wetherill Park, leader in business solutions, digital & offset printing, graphic design, websites & online marketing, a full range of products to support your marketing campaign.
R.Wagner Prestige Labels Pty Ltd built a reputation in quality embossing labels and seals and In 1989, with new owners, brought the introduction to multicolour labels. ..., Aluminium Tags, Barcodes, Labels Adhesive sale, LABEL, LABELS, Screen printing, Labels Adhesive
... to detail that makes us stand apart from our competitors. At BMS we live and breathe letters, printing and postage and you will see the difference when we connect with your business. We really love...
... Printing Inks, printer cartridges, ink jet cartridges, Cartridge services, Cartridges Ink, toner...
D&D s promise is to deliver personal service, where its clients gain both from its business approach as well as the combined skills of D&D personnel The final result is a totally maintenance and ... services, Wrapping, Magazines, laser printing offset printing, guillotining, Packing and filling...
Since 1985, Sainsbury POS Displays has been providing high quality Point of Sale design and manufacture We are situated at Newton Road, Wetherill Park, Sydney ... sydney, corrugated displays, temporary pos, printing, corrugated, lithographic, cash, display, of...
Select Awards located in Wetherill Park, Sydney Range of acrylic awards, crystal awards, glass awards, engraving, trophies, plaques, signs, digital printing.
..., Dura-Wrap, Dura-Flex, Dura-Flex Pro, Tin Copper Braided Sleeving, Stainless Steel, EMI, RFI, Heat Shrink Tubing, Cable Ties, Heat Shrink Printing, Insultherm, ThermaShield, Nylon Braided Sleeving
Printing Pty Ltd has been established for over 60 years We are manufacturers of cards, envelopes and colour printing We are able to convert most papers into Lick & Stik, Peel & Stik, Self Seal in a...
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Le Monde du Silence (The Silent World) is based on the best-selling book of the same name by famed oceanographer Jacques Cousteau. Set on board--and below--the good ship during an exploratory expedition, this feature-length documentary was codirected by Cousteau and Louis Malle, whose first film this was (Cousteau selected Malle for this assingment immediately upon the latter's graduation from film school). Highlights include a shark attack on the carcass of a whale, and the discovery of a wrecked...more
All The Silent World cast + crew photos


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Arora Designs is pleased to announce our new website content management system. We are extremely excited about this brand new feature. While many companies have some kind of CMS available for their clients, we've waited for over a year to unveil this masterpiece of web technology. It is by far the easiest, most intuitive CMS we've ever worked with, and we know our clients will think so too.
From now on, all our web design clients will have access to our powerful and simple content management system that will allow them to add and edit content on their own websites. While many of our clients have expressed worry that this means they will not have a reason to work with us as often (we love you too!), we are absolutely positive this is the best thing for them. This empowers our clients in a way that was previously not possible without a website editor.
We've updated our website to reflect our new website services and pricing, so don't hesitate to look around. If you have any questions, please don't hesitate to ask us - we're here for you all.
Take a look at the Website CMS features!
Keep in mind that even though Arora Designs is a Dallas Web Design company, we offer our content management and website design services to national and international customers. Just shoot us an email or give us a call and we'll get you started with whatever it is you need!
Mon, May 14, 2012 @ 6:14 PM
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