text:
- Research article
- Open Access
Transpositional reactivation of the Dart transposon family in rice lines derived from introgressive hybridization with Zizania latifolia
© Wang et al; licensee BioMed Central Ltd. 2010
- Received: 18 March 2010
- Accepted: 26 August 2010
- Published: 26 August 2010
It is widely recognized that interspecific hybridization may induce "genome shock", and lead to genetic and epigenetic instabilities in the resultant hybrids and/or backcrossed introgressants. A prominent component involved in the genome shock is reactivation of cryptic transposable elements (TEs) in the hybrid genome, which is often associated with alteration in the elements' epigenetic modifications like cytosine DNA methylation. We have previously reported that introgressants derived from hybridization between Oryza sativa (rice) and Zizania latifolia manifested substantial methylation re-patterning and rampant mobilization of two TEs, a copia retrotransposon Tos17 and a MITE mPing. It was not known however whether other types of TEs had also been transpositionally reactivated in these introgressants, their relevance to alteration in cytosine methylation, and their impact on expression of adjacent cellular genes.
We document in this study that the Dart TE family was transpositionally reactivated followed by stabilization in all three studied introgressants (RZ1, RZ2 and RZ35) derived from introgressive hybridization between rice (cv. Matsumae) and Z. latifolia, while the TEs remained quiescent in the recipient rice genome. Transposon-display (TD) and sequencing verified the element's mobility and mapped the excisions and re-insertions to the rice chromosomes. Methylation-sensitive Southern blotting showed that the Dart TEs were heavily methylated along their entire length, and moderate alteration in cytosine methylation patterns occurred in the introgressants relative to their rice parental line. Real-time qRT-PCR quantification on the relative transcript abundance of six single-copy genes flanking the newly excised or inserted Dart-related TE copies indicated that whereas marked difference in the expression of all four genes in both tissues (leaf and root) were detected between the introgressants and their rice parental line under both normal and various stress conditions, the difference showed little association with the presence or absence of the newly mobilized Dart-related TEs.
Introgressive hybridization has induced transpositional reactivation of the otherwise immobile Dart-related TEs in the parental rice line (cv. Matsumae), which was accompanied with a moderate alteration in the element's cytosine methylation. Significant difference in expression of the Dart-adjacent genes occurred between the introgressants and their rice parental line under both normal and various abiotic stress conditions, but the alteration in gene expression was not coupled with the TEs.
- Cytosine Methylation
- Rice Line
- Abiotic Stress Condition
- Tos17
- Introgressive Hybridization
It is widely recognized that hybridization between genetically differentiated natural plant populations may cause structural genomic changes (e.g., via homoeologous or ectopic recombination) as well as perturbation of epigenetic state of the recipient genome (e.g., DNA methylation), and both may result in heritable phenotypic novelties [1–7]. These findings are consistent with Barbara McClintock's insight of "genome shock", which proposed that crossing of different organismal species may cause restructuring of the resultant hybrid genome, and which may represent a facet of adaptive response by plants under specific circumstances [8]. A major cause underlying the genomic shock symptom is transcriptional and transpositional reactivation of otherwise cryptic transposable elements (TEs) in the hybrid genome. The reactivation of TEs is often coupled with disruption of chromatin-based epigenetic controlling mechanisms in the hybrid genome, like loss or re-patterning of cytosine methylation and compromised targeting by small interference (si) RNAs [9–11]. Indeed, several studies in both animals and plants have provided compelling empirical evidence in support of the "TE-epigenetic" basis of genome shock [12–16].
At least circumstantial evidence has indicated that for the hybridization- associated genomic shock to occur, a symmetric hybrid genome is not a prerequisite; instead, introgression or integration of "foreign" chromatin or DNA segments via introgressive hybridization or other means (e.g., transgenic) might as well produce the "shocking" effects on the recipient genome [11]. For example, it was shown in cultured animal cells that random integration of pieces of foreign DNA can cause the host genome to undergo extensive and genome-wide alterations in cytosine methylation of both cellular genes and TE-related DNA repeats [17, 18]. We have demonstrated that introgression of small amount of chromatin of Zizania latifolia (a distantly related species to Oryza) into rice has caused an array of genetic and epigenetic instabilities in the recipient rice genome [19, 20], and in particular, rampant mobilization of a copia retrotransposon Tos17 and a MITE (mPing) [21]. Given the recent finding that the cellular controlling mechanisms for TE activity are likely individualized [22], it is interesting to explore whether TE reactivation in the rice-Zizania introgressants was confined to these two elements or other TEs also experienced reactivation.
The rice Dart transposon family belongs to the hAT superfamily of class II TEs, and which was first characterized by Fujino and colleagues [23]. Dart was found as transcriptionally active in several rice tissues [24]. Moreover, both Dart and its deletion-derivative called nDart can be transpositionally active in certain rice genotypes that harbour active Dart, even under normal growing conditions [25]. In addition, the element's activity was correlated with its cytosine methylation state, and epigenetically silenced Dart copies can be reactivated by 5-azacytidine treatment [25, 26]. Apparently, except for Tos17 and mPing, the Dart/nDart represents another family of highly active TEs endogenous to the rice genome.
The aim of this study was to investigate (1) whether the Dart TE family was transpositionally reactivated in the same set of rice-Zizania introgressants that showed rampant mobilization of Tos17 and mPing [21]; (2) whether the element's activity was correlated with its cytosine methylation state; and (3) whether excision and reinsertion of the element copies impacted expression of their adjacent genes under normal or various abiotic stress conditions.
The Dart transposon family was transpositionally reactivated in the rice-Zizaniaintrogressants
Excision sites of Dart-related TEs identified by transposons-display (TD) (designated as Dart-TDE) in the rice-Zizania introgressants
Excision sites
Excision position§
Locus-specific primers (5'-3')
Excised from
Excision flanks
For: tgcgcacaatcacctctatg
Rev: cttgtggtgcaaccaaccta
Position: 2633169
For: gtgcgcgtaatgctagaaaa
Rev: cagggagggagggattagag
Position: 20137929
For: cccccatacttaccgcatta
Rev: cctaggtgttctgccgactc
For: tgagtgacgtgaagccaaag
Rev: acagatcacggcagggttac
For: cctctaggcacctccctttt
Rev: caggagcaacaattgcatgt
ctccctttttttaa gaactaatgactttt
For: tggtttggaggtcggttaag
Rev: cacatgtcagccaaaaccac
For: gagaagagcacgggaagttg
Rev: aactggctgttcgctcaagt
Position: 15326441
For: tcgtgttcccaaattcacac
Rev: catatatcccgcagaaaagca
Position: 20176647
For: aacgagagcaagggagatgaa
Rev: ttaagccagggcaagtacacg
Position: 21368076
For: gtgcatggattttgaccttta
Rev: ctgtgctcacttcgctactacta
Position: 7832680
For: ggtgtaacgattgctaaggcg
Rev: agtggggggagagtaagatga
Position: 7910449
Os11g0247800 exon
For: agagttcttgccaaccatgc
Rev: ggaagagggaaaaaccaagc
Position: 16720991
For: cgatcgagaatttccgagac
Rev: tggtctgttcgttgtccaaa
Position: 6273602
For: cttttgggctgtgatggagt
Rev: ttaaggacgatgccaaaacc
Position: 26631010
For: cctcggtttccattagca
Rev: gtacggcctggcaagtga
De novo insertion sites of Dart-related TEs identified by transposons-display (TD) (designated as Dart-TDI) in the rice-Zizania introgressants
Insertion sites
Position of insertion sites
Locus-specific primers (5'-3')
Inserted into
Chr.3; position: 2726981;
For: tcacgcagtagatgccaaag
Rev: gcacgtctccgtagctctct
Chr.10; position:4241777;
OSJNAb0015J03.2 exon
For: gtagagggctcaatcgtgga
Rev: ctaaggtctcgaggcacacc
Chr.5; position: 29238540;
For: gcccgtttggccacctctat
Rev: tgtaaaatgaccagcgacga
Chr.5; position:14917879;
For: tacggttcccattgttttcc
Rev: gggtgtgcacgatgttgtaa
Chr.12; position:12726861;
For: ttgttgttagttttgcgtgtaga
Rev: gaaagcaggttggagaggtta
Chr.3; position:27833204;
Os03g0699200 exon
For: taattaagttggaagtgggaca
Rev: tttctgtaagattacaaccagaggt
Chr.3; position:17730615;
For: ctttcgtaggcgaaaagtgc
Rev: ctgcaaccacctgtctctga
Chr.4; position:638561;
For: catgaattgggtgccatgta
Rev: ccccatagggtaggcaaaat
Chr.5; position:10010289;
For: gcccgtttggccacctctat
Rev: ggtggaggacctgctcaata
Chr.12; position:6156546;
For: tgagcacgcctagctcagta
Rev: atgcacggcaactttctctt
Chr.12; position:6345593;
For: gcccgtttggccacctctac
Rev: acaaatggcctcctgtgttc
Chr.12; position:21951729;
For: tccagccaaaccctgttc
Rev: gctcgccagatgtcaggt
Chr.1; position:19308190;
For: tgctacagtagaagggcgtgta
Rev: atgcacatctggtcttttgatg
Chr.2; position:6211564;
For: ggatccgtttggatcagaga
Rev: tgcagcagctgattcatacc
Chr.5; position:10749306;
OSJNBa0037H03.12 intro
For: gagctgctcctgaaaaccac
Rev: gaattttccttgccgtgtgt
Chr.1; position:25299;
For: gtgccggagaatgatttgat
Rev: atttccctcgatgcactgtc
Chr.2; position:10207338;
Os02g0277600 intro
For: gcccatttggccacctcta
Rev: cgaatgagtgtccttgatcg
Chr.12; position:23594158;
Os12g0572000 3'UTR
For: gcccgtttggccacctctac
Rev: agcaacccacagaacagctt
Chr.8; position:23713525;
For: gcccgtttggccacctctac
Rev: tctgcggttgaaacaatgag
Chr.7; position:20028374;
For: gcccgtttggccacctctat
Rev: aaagtcaatggaaaggggaaa
Chr.4; position:25704503;
Os04g0514800 exon
For: gcccatttggccacctcta
Rev: ggcaatgcggttggtttc
Validation and chromosomal location of the Dart excisions and insertions by locus-specific PCR amplification and sequencing in the rice-Zizaniaintrogressants
For each of the putative excised and inserted loci, we designed locus-specific primer pairs specific to the flanks of the Dart elements based on the whole genome sequence of Nipponbare http://rgp.dna.affrc.go.jp (Tables 1 and 2). Note that the design of primers targeting the flanks did not entail that Nipponbare contains a copy of the Dart-related TE at each of the loci. PCR amplification by these locus-specific primers (e.g., Figure 2c) and sequencing of the amplicons verified that all the 15 excisions and 21 insertions were authentic, because the immediate contiguous flanks were intact for all the excisions and typical TSDs were identified for all the insertions (Tables 1 and 2). This locus-specific PCR amplification and sequencing results thus validated transpositional reactivation of the Dart-related elements in the rice-Zizania introgressants.
Cytosine methylation states of the Dart-related elements in the rice-Zizaniaintrogressants and their rice parental line
We next performed the second round restriction by adding each of the pair of isoschizomers, HpaII and MspI, to the BamHI-digests, and using probes specific to each of the three regions to assess their cytosine methylation state in the introgressants relative to Matsumae. We obtained the following results: (1) there had been no detectable internal truncations between the two BamHI sites, as no clear, smaller-sized bands than the expected 2232 bp were detected in this enzyme digest (Figure 4b, the body-region probe); (2) the body-region of the conserved, Dart-related elements was heavily methylated particularly by mCG in all the rice lines, introgressants and parental, as evidenced by the very similar hybridization patterns between BamHI-digest and BamHI+HapII-digest (Figure 4b, the body-region probe), albeit there were 11 5'-CCGG sites within this region of Dart-related elements, and hence several smaller-sized bands would have been detected if the relevant 5'-CCGG sites were hypomethylated (Figure 4a); (3) the only clear difference in the methylation state of the introgressants relative to Matsumae was that one introgressant (RZ35) showed hypermethylation as evidenced by the disappearance of several smaller-sized bands (Figure 4b, the body-region probe; marked by arrows); (4) the 5' region of the Dart-related elements was also heavily methylated by both mCG and mCNG in the all the lines because multiple bands were detected (Figure 4b, the 5' probe), as otherwise we would have predominantly detected a band <678 bp in length (restricted by BamHI at position 678 nt together with restriction by HpaII/MspI at one or more of the six 5'-CCGG sites at the 5'-terminus) (Figure 4a); (5) compared with Matsumae, CG demethylation in RZ1 and concomitant CG hypermethylation and CNG hypomethylation in RZ35 occurred in the 5'-region of Dart-related elements (Figure 4b, the 5' probe); (6) similar to the situation of 5'-region, the 3'-region of the Dart-related elements was also heavily methylated by both mCG and mCNG in all the lines (Figure 4b, the 3' probe), as otherwise we would have predominantly detected a band <656 bp in length (being restricted at position 2910 nt by BamHI together with one or more of the seven 5'-CCGG sites by HpaII/MspI at the 3'-terminus) (Figure 4a); (7) compared with Matsumae, CG demethylation in RZ2 and CNG demethylation in RZ35 occurred in the 3'-region of the Dart-related elements (Figure 4b, the 3' probe); (8) with regard to the methylation state of the 5' and 3' flanks of the Dart-related elements, it was deducible that they had a substantially lower methylation level relative to the Dart-related elements per se, as evidenced by the multiple small-sized bands detected by the Dart 5'- and 3'-region-specific probes (Figure 4b). Taken together the Southern blotting data of all three region-specific probes, it can be concluded that (1) the Dart-related TEs are heavily methylated throughout the entire length, but with their 5' and 3' flanks being relatively less methylated compared with the internal body-region, in all the rice lines studied; (2) to a moderate extent, methylation alteration including both hypo- and hypermethylation occurred in the introgressants relative to their rice parental line Matsumae.
Limited impact by mobility of the Dart-related TEs on expression of their adjacent genes under normal and abiotic stress conditions
Hybridization is prevalent in plants, which plays important roles in genome evolution, and may lead to speciation at both the homoploid level and followed by genome doubling (allopolyploid) [6, 7, 33–37]. Apart from direct transfer and recombinatory generation of genetic variations by hybridization, de novo genetic and epigenetic instabilities can be induced by the process per se, including transcriptional activation and mobilization of cryptic transposable elements (TEs) [15, 16, 29, 38–42]. Several lines of circumstantial evidence have indicated that introgression of DNA or chromatin fragments from an alien species into a recipient genome may also produce similar effects in causing genetic and epigenetic instabilities and generate novel phenotypes [11, 17]. We have reported previously that introgressive hybridization between rice (Oryza sativa) and Z. latifolia had induced rampant mobilization of two TEs, a copia-like LTR retrotransposon Tos17 and a MITE mPing [21, 43]. In this study, we extended the earlier findings and found that the Dart-related TEs were also transpositionally reactivated in the introgressants, although the elements were totally quiescent in the parental rice cultivar Matsumae. We validated the excisions and insertions by transposon-display (TEs) and sequencing, which ruled out genomic rearrangements as the major cause for the dramatically altered hybridization patterns detected by Southern blotting in the introgressants.
Although numerous studies have established correlative or causal links between TE activation and alteration in the element's cytosine methylation state [26, 27, 30, 44, 45], we found that the Dart-related TEs were similarly hypermethylated along their entire length in the introgressants and their rice parental line Matsumae. Nonetheless, moderate alteration in the methylation patterns was discernible in the introgressants. Furthermore, given that the introgressants were at the 9th-selfed generation and stabilized in both phenotype and DNA fingerprinting patterns [20], we could not rule out the possibility that in earlier generations of the introgressants (no longer available for study), more marked methylation remodelling might have occurred in the Dart-related TEs, which however were either largely reverted to the original pattern and/or those individuals with more drastically altered patterns had been purged out during the sexual reproduction, probably due to reduced fitness. Therefore, it remains a formal possibility that alteration in cytosine methylation had been associated with mobility of the Dart-related TEs in the introgressants, a scenario gaining increased empirical support in a vast range of TEs and organisms [9–11, 26, 28, 30, 46].
An array of studies in both plants and animals has established that activity of TEs, particularly LTR retrotransposons, may significantly impact expression and function of their adjacent genes [38, 41]. Nonetheless, other studies have indicated that the majority of newly transposed TEs particularly those with small-sizes like MITEs tended to insert into functionally neutral genomic regions and impose minor effects on their adjacent genes [31]. We found in this study that the six single-copy protein-coding genes adjacent to the newly excised or inserted Dart-related TEs exhibited significantly altered expression in the introgressants relative to their rice parental line under both normal and several abiotic-stress conditions. However, the altered gene expression in the introgressants was not coupled with the TE excisions or insertions, suggesting that other regulatory mechanisms were responsible for the altered gene expression in the introgressants. Because unbiased amplifications between the introgressants and their rice parental line were observed when their genomic DNA was used as templates, it is likely that epigenetic regulation was involved. The observation that for most of the genes, these rice lines exhibited sharply differential response to 5-AC treatment corroborated this possibility, which also accords with our previous results showing that substantial re-patterning of cytosine methylation occurred in the introgressants for amny genomic loci [47]. Further study is required to elucidate the exact molecular basis underlying the dramatically altered gene expression and their phenotypic consequence in these novel rice lines as a result of introgressive hybridization.
Results of this study have extended our previous findings by documenting that introgressive hybridization between rice and Z. latifolia has induced transpositional reactivation of another distinct family of cryptic TEs in the parental rice genome, namely, the Dart-related TEs, suggesting that introgression of chromatin from a realted alien species might have caused a general breakdown of the host cellular machinery responsible for repressive control of TE activity. Transposition of the Dart-related TEs was accompanied with a moderate alteration in the element's cytosine methylation in the introgressants. In addition, results of this study showed that extensive alteration in expression of a set of Dart-adjacent, protein-coding genes occurred in the introgressants relative to their rice parental line, under both normal and various abiotic stress conditions. Nonetheless, the alteration in gene expression was not coupled with excision or insertion of the Dart-related TEs, implicating other regulatory mechanism(s) was underpinning the changes in gene expression in these novel rice introgressants.
Plant lines
Three introgression lines (RZ1, RZ2 and RZ35) derived from a cross between rice (cv. Matsumae) and Zizania latifolia Griseb, were used in this study [21]. The three stabilized introgressants (at the 9th selfed generation) were homogeneous in phenotype and DNA fingerprinting patterns, and exhibited heritable, novel morphological characteristics in multiple traits compared with their rice parental cultivar Matsumae [20, 21]. The introgressants were maintained along with their rice parental line (cv. Matsumae) by strict selfing in our laboratory.
Abiotic stress and 5-azacytidine treatments
Healthy and uniform seeds of three rice-Zizania introgressants (RZ1, RZ2 and RZ35) and their rice parental line cv. Matsumae were disinfected and thoroughly rinsed, and placed on petri-dishes covered with half-strength Murashige and Skoog (MS) medium in darkness at 25°C. For the three kinds of abiotic stress treatments, seedlings were grown to the 3-leaf-stage, and then aqueous solutions respectively containing 5 mM CuSO4 (heavy metal), 5 mM HgCl2 (heavy metal), 10 mM NaCl (salinity) and 10 mM NaHCO3 (alkaline) containing the half-strength MS medium were added, and grown for one more week. For cold stress, the 3-leaf-stage seedlings were grown in the medium at 12°C for one week. The 5-azacytidine (5-AC) treatment was conducted by treating the germinating seeds in the medium containing 50 mM 5-azacytidine (Sigma) for one week and then thoroughly rinsed with ddH2O and allowed the seedlings to grow in the medium up to the same stage as the other treatments. In all cases mock-control seedlings grown in the half-strength MS medium alone was included.
Southern blot analysis
Genomic DNA was isolated from leaf tissue of young seedlings at the same developmental stage from the various treatments and mock of the three rice-Zizania introgressants (RZ1, RZ2 and RZ35) and their rice parental line Matsumae by a modified CTAB method. To assess possible genetic changes in the patterns of the Dart-related TEs, the genomic DNA (~3 μg, per lane) of each line (the mock-control) was digested by HindIII. To test for possible alteration in cytosine methylation of the Dart-related TEs, the genomic DNA of each line (the mock-control) was first digested with BamHI (to delineate the Dart-related TEs into three regions, 5'-, 3'-, and body-regions; see Results), followed by a second round of digestion with a pair of isoschizomers, HpaII and MspI, that recognize the same sequence 5'-CCGG but with differential sensitivity to methylation of the two cytosine residues. Digested DNA were run on 1% agarose gel and transferred onto Hybond N+ nylon membrane (RPN 303B, Amersham-Pharmacia Biotech, Piscataway, New Jersey) by the alkaline transfer method recommended by the manufacturer.
In total, three pairs of primers specific respectively to the 5'-, 3'- and the body-region of the Dart-related TEs were designed to amplify the fragments to be used as hybridization probes. The primers are: (1) for the 5'-region of Dart-related TEs: Dart5'-forward: 5'- aaatagggcatgaaccccagc, Dart5'-reverse: 5'-ggtcgaaatcacccaaggtg; (2) for the 3'-region of Dart-related TEs: Dart3'-forward: 5'-tccagaccaaccccagtagaa, Dart3'-reverse: 5'-aaaaaaagcaaaggaaatgtataagg; (3) for the body-region of Dart-related TEs: Dart-body-forward: 5'-ctagagaggattatcttagcgtagttgtt, Dart-body-reverse: 5'-cttcttcttacctgtagtggggatag. Authenticity of the amplified fragments was verified by sequencing. The fragments were then agarose gel-purified and labelled with fluorescein-11-dUTP by the Gene Images random prime-labelling module (Amersham-Pharmacia Biotech). Hybridizations were done with the Gene Images CDP-Star detection module (Amersham-Pharmacia Biotech). After washing twice with 0.2×SSC, 0.1% SDS for 50 min. The filters were exposed to X-ray films for chemiluminescence signal detection.
Transposon display (TD), locus-specific PCR amplification and sequencing
The genomic DNA (approximately 300 ng per sample) was cut with MseI and subject to transposons display (TD) following the protocol essentially as reported [48]. Three consecutively nested, sequence-specific primers targeting to the 3' end of Dart-related TEs were designed. Two specific primers (TDPrm1/TDPrm2 and MseI+C/G) were respectively combined with the selective-amplification primers targeting the adapters, while the most external primer (TIR+N/MseI+3) was used for further validation of the isolated TD bands. Detailed information concerning adapters and primers are listed in Additional files 1. The PCR amplification conditions and TD amplification products were resolved by PAGE and visualized by silver-staining [20]. Only clear and reproducible variant bands between two technical replications were considered as putative new insertions by the Dart-related TEs, and recovered for sequencing.
Based on the sequencing results by identifying the expected 3'-terminus of the Dart-related TEs, the contiguous flanking regions were extracted and used to query the Nipponbare genome sequence http://rgp.dna.affrc.go.jp by BlastN, and a set of locus-specific primers each being downstream of the Dart but compatible with the Dart-specific TD primers were designed with Primer 3 http://biocore.unl.edu/cgi-bin/primer3/primer3_www.cgi and those produced the expected results were listed in Tables 1 and 2. Each of these primers were then sequentially combined with the nested Dart-specific TD primers to reproduce the putative excisions and insertions identified by TD in the introgressant(s), and the amplicons were then sequenced for validation.
RNA isolation and quantitative real-time-reverse transcriptase (RT)-PCR analysis
Total RNA was isolated from the same young leaf tissue as used for DNA isolation and also from the root tissue of the same seedlings of the various treated and mock lines, with the Trizol Reagent (Invitrogen) according to the manufacturer's protocol. The RNA was then treated with DNaseI (Invitrogen) to eliminate possible genomic DNA contamination before being reverse transcribed with the SuperScript RNase H- Reverse Transcriptase (Invitrogen).
The expression of genes adjacent to the newly excised or inserted Dart-related TEs in the introgressant(s) was studied by quantitative real-time-RT-PCR using gene-specific primers (see Additional file 2). The q-RT-PCR experiments were performed using a Roche LightCycler480 apparatus (Roche Inc.) according to the manufacturer's instruction and SYBR Premix Ex Taq (TOYOBO) as a DNA-specific fluorescent dye. The primers for the six studied genes were designed by the Primer 5 software (see Additional file 2). Expression of a rice β-actin gene (Genbank accession X79378) was used as internal control with the primer pairs 5'-atgccattctccgtctt and 5'-gctcctgctcgtagtc. The choice of the β-actin gene as the internal control was based on previous investigation showing that expression of this house-keeping gene between the tissues and under the various stress conditions was constant [49]. Conditions of q-RT-PCR were as reported [50].
This study was supported by the State Key Basic Research and Development Plan of China (2005CB120805), and the Programme for Introducing Talents to Universities (B07017). We are grateful to two anonymous referees for constructive comments to improve the manuscript.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Welcome, welcome.
I'm pleased you're here. I suppose this means that you either follow my writing, am curious to learn more about me, or want to see me embarass myself (which I am positively inclined to do in some way, shape, or form). Or maybe it's a combination of the above. That's all fine. I'm just happy that you're taking the time out of your day to visit my blog.
This is a story... about stories.
I'm that weird type of person who believes that everything should be experienced through a narrative. That might be why I found history dull in school, but I love historical fiction. If a character or idea is interesting, then I am interested. You'll find that I take character development very seriously. I refuse to bend my fictional reality unless I feel that I am being true to those I am writing about and for.
Writing has become such an integral part of me that not a day goes by without me thinking about a fictional world in some capacity. It makes me tingle with pride and giggle like a little girl; it relaxes me and brings me a sense of accomplishment that I can't find anywhere else. It allows me to master my thoughts, breaking down each tendril of concepts that I could never hope to do otherwise.
Yet, life can be a bit unaccommodating.
I work full time as a financial advisor — basically, I make money by telling others what to do with their money. I never thought of myself much as a technical person, but my career is working out well so far. My customer satisfaction surveys are high, I get along well with my supervisor, and I operate with the highest of ethical standards. The industry's not ideal for me, but I don't think most folks love their jobs. Really, if it puts food on the table, a roof over my head, and I don't loathe it, I'm satisfied. My work environment is excellent. I am blessed to have a far better employer than that of most Americans.
I am engaged, and our wedding is November of this year, which (as of today's blog post) means that I have about three months until the big day. I've loaded myself with tons of DIY projects, and I'm now beginning to realize the extent of the work I've assigned myself. There are still many vendors to contact, preferences to establish, and people to placate. My dog, the non-human love of my life, is destined to be my ringbearer.
My time is scarce. Finding time to write can be challenging. I am a night owl, but our societal standards seem to deem it fit that I wake up at the crack of dawn each morning. Because of this, I get very limited sleep — I'm really just some form of perpetually exhausted pigeon at this point, which is difficult for me to remedy since there's so much to be done and so little time. You can probably understand why I don't update as often as I do.
I treasure your reviews.
I really do. They can make me smile, laugh, blush, and jump for joy all at once. I would argue that there is nothing better than the feeling of getting an email notification and seeing that there is a thoughtful review left for one of my pieces. I try to respond (even though I'm not so great at it sometimes), but I think the best way to show my appreciate is to just keep writing (writing writing).
Truly and sincerely, on some rough days, it's reviews that keep me going, so thank you.
I'm still here.
I promise. I will finish my fics. I don't know when this is going to happen, but you'll notice that I'm pretty persistent. I finished Jaharaan Love, didn't I? Even though it took six years, I finished it and gave it the ending that it deserved. I have that same dedication to my other fics.
You'll see.

subset: commoncrawl


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Speaker has announced that the Supreme Court holds that two clauses of the Office for Reparations Bill are inconsistent with the Constitution and that the Bill has either to be passed with a two-thirds majority in Parliament and a referendum or be amended as per the SC's recommendations to make it consistent with the Constitution.
Speaker Karu Jayasuriya made the announcement at the commencement of Parliament on Tuesday. He stated that the Supreme Court in its determination, sent to Parliament after concluding the hearing of two petitions challenging the Bill, had stated that Clause 27(a) and 27 (a) iii, were inconsistent with the Constitution.
The Bill provides for the provision of individual and collective reparations for aggrieved persons. The Clause 27 (a) deals with the interpretation of "aggrieved persons". The Clause 27 (a) says "aggrieved persons mean persons who have suffered a violation of human rights or humanitarian law, their relatives and missing persons". The Clause 27 (a) iii interprets that "aggrieved persons" also include the individuals, groups or communities of people of Sri Lanka who had faced systematic gross violations of the rights.
The Bill was presented in Parliament for the first reading on July 17. The scope of legislation applies to the conflict in the North and the East, as well as "any political unrest or civil disturbance, systematic gross violations of the rights of individuals, groups or communities of people of Sri Lanka, and enforced disappearances."


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Rest Makes Less Stress
I decided to stir up a batch of cookies today. I used a recipe from our favorite gluten free cookbook, How Can it Be Gluten Free. I was reading over the instructions because it had been a while since I had made them when I came across this note: RESTING TIME Resting the… Continue reading Rest Makes Less Stress


text: "We believe in the resurrection of the body and the life everlasting, Amen." This is our Creed. In eating and drinking with one another we are affirming that we all believe that same promise. We are affirming that full resurrection is promised. But we also affirming that our weak bodies need food. We are affirming that a body without grace is an incomplete body. Therefore, God brings us to taste of his means of grace, and he does it through the act of eating and drinking: the most common of our daily activities. He does this so we may always be reminded that just as food is a daily necessity, so too is the grace of God a daily necessity. Come, and dine in the heavenly places.


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Here are the latest DNA-related news articles and blog posts for the past week. Stay on top of ALL the important DNA news here at National DNA Day!
- 23andMe and Grünenthal to Study the Genetics of Pain: "Pain is complex and personal. You understand the level of your own pain, but it's harder to understand it in others." Click here to read more via 23andme
- AncestryDNA Reaches 4 Million Customers in DNA Database: "Ancestry has surpassed 4 million customers in our DNA database! We're proud to remain the largest consumer genetic testing company." Click here to read more via Ancestry
- DNA Test for Finding Ancestors Raises Privacy Concerns: "A DNA test that's designed to reveal family history also requires users to give up their rights to their DNA data. One consumer says the contract is too broad for him to continue with the service." Click here to view via NBC Bay Area
- Firestorm brewing as scientists work to create synthetic human DNA: "Ethics are being questioned over a project to make synthetic genetic code at Harvard Medical School." Click here to read more via CNBC
- GEDMatch One-to-One Columns Revealed: "Wonder what all those columns on your GEDMatch One-to-One Page mean? Here is a good explanation." Click here to read more via Family Roots and Branches
- I found a Minuteman, a shoemaker and an indentured servant in my DNA: "Clarence Perry Bristol, the columnist's great-great-grandfather, fought in the Civil War with the Illinois infantry. The 1900 census report posted on ancestry.com lists him as a druggist, spelled with one 'g.' The National Genealogical Society's conference in Raleigh on May 10-13 explores this sort of research." Read more here: Click here to read more via The News & Observer (Raleigh, North Carolina)
- I took the Ancestry.com DNA test and was shocked at what I discovered: "I took the Ancestry DNA test about two months ago, figuring it would be helpful to find out where all my Irish relatives traced back to." Click here to read more via Irish Central
- LivingDNA Product Review: DNA expert Roberta Estes offers her review of Living DNA a relatively new DNA vendor based in the UK. Click here to read more via DNAeXplained – Genetic Genealogy
- The Y-DNA Mutation Rate Project: "I've tested myself out to 67 Y-DNA markers (67-111 are pending!), and I've compared that to two 5th cousins. We have some genetic differences, as expected, and I'm always interested in learning more about my Y-DNA. One thing I'd like to do, for example, is compare my results to closer relatives (father and/or son) to look for mutations in father/son pairs." Click here to read more via The Genetic Genealogist
- Thinking of DNA testing? Which one is the best for you?: "DNA testing kits are soaring in popularity. But does it matter which test you choose? We put three to the test." Click here to read more via WGN News
- What can a DNA kit tell you? A CSUF expert has some answers: "Discovering that your German heritage is actually Scottish and turning in your lederhosen for a kilt – that's the premise of a commercial playing a lot these days. It taps into a common human desire: to find out our true identity." Click here to read more via The Orange County Register
Click here to view past DNA News Updates via National DNA Day.
Disclosure statement: I have material connections with various vendors and organizations. To review the material connections I have in the genealogy industry, please see Disclosure Statement.
©2017, copyright Thomas MacEntee. All rights reserved.


text: It's hard to believe that Stephanie and Thiago's Wedding was our last of 2017 and we just kicked off our 2018 Wedding Season last weekend, so it is due time that we share some photos from their lovely wedding celebration in Massachusetts last fall. Stephanie and Thiago had a glorious outdoor ceremony at the Merrimack Valley Golf Club on the North Shore of Massachusetts. It was the kind of fall day that radiates warmth through the cool autumn chill in the air. These two are well suited and full of joy, so it was no surprise that their family and friends also were a fantastic group of people. By the end of the Wedding Day we felt like guests more than vendors. Thiago's family is from Brazil, so it's always a treat to see not only two families coming together to celebrate but two countries! Leaving Stephanie and Thiago's Wedding gave us that full-of-gratitude....pinch-yourself-how-did-we-get-so-lucky feeling to have the job we do and work with wonderful people like these two. Stephanie and Thiago, we know you guys have so many years of happiness in your future! Congratulations!!!


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(11 reviews)
Take a day trip from Seville to explore the ancient city of Ronda, where you'll enjoy tasting local wines and explore Plaza de Toros, a traditional bullfighting ring. See several important monuments, churches and bridges and admire views of El Tajo River gorge. Upgrade your tour to include a visit to Pueblos Blancos, a picturesque area filled with white-washed houses. Here, visit Sierra de Grazalema, the first natural park in Andalusia.
- Day trip (destination A to B)
- Enjoy a day away from the hustle and bustle of the city
- Free hotel pickup and drop-off included
- Informative, friendly and professional guide
You'll be picked up from central Seville or your Seville hotel in the morning and taken on a comfortable drive to Ronda, one of the oldest towns in Spain. When you arrive, take a stroll through the 19th-century gardens in Alameda del Tajo and soak in the wonderful views.
Visit Plaza de Toros, a traditional Spanish bullring made of stone. Behind the stage, learn about the history of bullfighting from your guide and head to the center of the ring to snap some awesome photos.
Continue to several beautiful bridges including the New Bridge, a symbol of the city that connects Ronda´s old and new districts. Enjoy great views of the vast El Tajo River gorge below. Next, visit one of Ronda's most significant monuments, the beautiful San Juan Bosco House, a palace that fuses modern and colonial styles and boasts amazing views of the valley.
Head to the city's historic center to visit the Church of Santa Mari and witness the mix of Christian, Arab, Gothic and Renaissance architecture. Then, immerse yourself in Spanish wine culture with a tour of the Wine Museum of Ronda. Learn about Ronda's long history of wine making and then head to the Sangre de Ronda winery to taste some great local wines. Afterward, enjoy free time to explore Ronda on your own before returning to Seville.
Upgrade: Pueblos Blancos Tour
After Ronda, head to picturesque Pueblos Blancos, a series of towns and large villages in the northern part of the provinces of Cadiz and Malaga.
Learn from your guide about the origin of this area, whose name, meaning 'white houses,' comes from the custom popular among the local inhabitants of whitewashing the outside of their houses and decorating them with brightly colored potted flowers. Combined with the blue sky and the plethora of mountain peaks and green hilltops, Pueblos Blancos creates an explosion of eye-catching color.
Visit Sierra de Grazalema, the first natural park in Andalusia brimming with diverse flora and fauna. See why the park is famous for its spectacularly rugged limestone landscape of cliffs, gullies, caves and gorges.
- Confirmation will be received at time of booking, unless booked within 1 day of travel. In this case confirmation will be received as soon as possible, subject to availability
- A moderate amount of walking is involved
- Due to uneven surfaces, this tour is not recommended for those with walking disabilities or using a wheelchair
- Not recommended for pregnant women
- Occasionally the tour may be offered in two languages at the same time
You can present either a paper or an electronic voucher for this activity.
- Hotel pickup and drop-off
- Local guide
- Wine museum tour and wine tasting
- Entrance fees and guided tour of Plaza de Toros bullfighting ring and museum
- Food and drinks, unless specified
- Gratuities (optional)
8am to 8:30am, hotel pickup
8am from central Seville
Central Seville and centrally located Seville hotels
11 hours
Returns to original departure point at approximately 5pm or 7pm, depending on the tour option selected
Thursday, Saturday


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How it Works
"How does it work?"
A good question, but one that can mean anything from "Are you nice?" to "How much?" , or just "How do things actually get done?". All good, and important questions as well.
Our preliminary phone conversation will answer many of your questions. When you determine it's a good fit, we schedule a consult to answer the rest, and get started.
Consults untangle and evaluate all the intangibles dueling in your brain:
- Ideas and info, logistics and priorities
- Dollars vs dreams, aesthetics vs. strategy
Geared to providing whatever you need to implement on your own; many clients find the consult is all they need to get it done!
- Fee is based on time spent on-site. Pricing quoted when setting an appointment and confirmed in writing, with payment due at time of service.
- Clients are encouraged to take notes, but detailed written recaps can be provided.
Post-consult, you may find you need help to implement. Here are some of the most-requested specialized services …but be in touch if you don't find what you need, we love to find new ways to say YES!


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"Sarika Sahni" <ssa...@simdesk.com>
|Error starting modern compiler on Linux
|Mon, 20 Sep 2004 21:13:23 GMT
Hi, I am new to Ant and am trying to compile some java sources on Linux. I have the ANT_HOME, JAVA_HOME and ANT_OPTS setup correctly on Linux machine but compiling the Java sources keeps giving me the error "Error starting modern compiler" followed by system out of resources: Java.land.outOfMemoryError. I tried changing the heap allocation size via the ANT_OPTS parameter varying from -Xmx256M to -Xmx600M, but to no avail. I can get my Java sources to compile fine on the Windows machine with heap size -Xmx256M. Also, I can compile my CPP sources fine on Windows and Linux setup using Make. Wonder what is wrong with my system configuration for Java compile on Linux. I appreciate any help on this and sorry to repeat it this has already been posted in past. Thanks, Sarika
|View raw message


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Free Online Fax ServicePosted by on March-24-2009 under Free Software
I recently had a need to send a fax from my home. I tried to search on the internet for an online fax service, and found that there are actually many free online fax service! What better way to save some money in this tight economy, than to send free fax online. (This article is a part of my series of articles on FREE Software. Check out other articles in the series here)
Caution: These free online fax services are for casual users, like me. If you have a regular use of an online fax service, you are better off with a full service provider. My favorite one is RingCentral.com . You can Try RingCentral Online FREE for 30 days
1) Faxzero: This is one of the most reliable free online fax service. FaxZero gives you two free faxes a day. Each free fax can be up to 3 pages. They include an ad on the cover page. You can upload a file from your computer, or can write the text that you want to send. Confirmation of successful fax delivery is sent to you by email.
2) : You can Try eFax For 30 Days Free. It lets you send as well as receive fax online FREE for 30 Days! No fax machine or supplies required. You get your free fax number.
eFax has another service as well that lets you receive faxes for FREE. You can only receive faxes using this online free fax service, but cannot send faxes. You can check out the free service here.
3) FreeFaxButton: This is very similar to Faxzero. It lets you send free fax online to over 40 countries! Some of the countries in the list include USA, Canada, UK, Australia, Germany, Hong Kong, Italy, Japan, and many more. You can upload a file from your computer, or compose the fax message yourself. Note: It asks for a Pin to send Fax. Pin is available at the bottom of their screen itself.
4) RingCentral.com : This is one of the best full fledged online fax service out there. This offers 300 Free fax pages every month included with your subscription. You can choose to get a toll free or local number at same price. You can Try RingCentral Online FREE for 30 days


text:
Striving to Suffer
Has Sept. 11 weakened the cult of victimhood? It seems not.
By all accounts Kenneth Feinberg is a man blessed with large reserves of calm and confidence. Heaven knows the man appointed special master of the September 11 Victim Compensation Fund can use all the inner calm and security at his disposal. Mr. Feinberg, who has to decide the amount of the awards to be paid out to the families and victims had an impossible task to begin with--one made the more difficult by the anger and disappointed expectations of the victim families.
There is anger over the amount (roughly $1.85 million...


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Postgraduate Coursework Programs
General Information about our Postgraduate Programs
Aims and objectives
The main objective of the School's programs is to develop a mutual understanding between engineering and medical professionals to facilitate the application of engineering analysis and concepts in medicine. Emphasis is on the life sciences in general but there is a primary focus on clinical medicine.
On completion of a program it is expected that candidates with a medical background will have an appreciation and working knowledge of engineering problem-solving procedures and of the time, effort and cost involved in reaching these solutions.
Similarly, candidates with an engineering background would be expected to understand the ramifications of applying engineering principles, in an appropriate way, to problem-solving in both clinical medicine and basic biology.
Postgraduate Coursework Programs
As a postgraduate coursework student you will gain insight into the application of engineering principles in medicine and healthcare delivery. During your time with us, you will learn to apply the latest engineering techniques to create diverse and innovative solutions, including:
- the design of medical instrumentation and prostheses
- the development, manufacture and testing of medical products
- the management of technology in the health care system.
The application of engineering principles to problems in medicine and biology represents a significant enhancement to learning outcomes attained from an undergraduate engineering, science or medicine qualification. Once accepted into either our Masters or Graduate Diploma programs you may select courses across the four areas below:
Master of Biomedical Engineering (MBiomedE), Program Code 8660
A broad introduction
The Master of Biomedical Engineering (MBiomedE) program offers a broad introduction to the principles underlying biomedical engineering as well as providing an opportunity for more specialised coursework and the scope for original research into the application of engineering principles and technology to medical problems.
Flexible program
The program will allow you to study physiology and/or anatomy, along with Biomedical Engineering courses. Any course substitutions for your program must be authorised in writing by the GSBmE School Office prior to enrolling in the course. The degree is primarily obtained through course work but includes an optional research project conducted at UNSW. The option to undertake a research project will be subject to school approval dependent on your academic progress throughout the degree.
Units of Credit
To graduate from Program 8660 you must successfully complete of 72 units of credit (UOC), normally over three full-time semesters or six part-time semesters. A minimum of 48 UOC must be selected from biomedical engineering electives beginning with the course code BIOM9xxx. The remaining 24 UOC may be from engineering professional electives, or other relevant courses. For all non-biomedical engineering courses, written approval must be obtained from the GSBmE School Office prior to enrolling. The relevance of these courses to your program of study must be indicated in order to gain approval.
Master of Engineering Science (MEngSc), Program Code 8338
NOTE: You must have a Bachelor of Engineering (4 year degree with honours II/2 to be eligible for this program)
The new Master of Engineering Sicence (MEngSc) program code 8338 is designed especially for graduate engineers seeking to develop or enhance their careers through cross-training, re-training and specialisation. An extensive research component ensures every graduating student is armed with advanced practical and analytical skills.
This is a flexible program and students can choose from a variety of specialisation and elective courses. To graduate from Program 8338 you must successfully complete 96 units of credit over 4 full-time semesters, (2 years). In selecting courses students must complete:
- Up to 24 UOC of Disciplinary Courses to provide necessary background. This may include anatomy and physiology.
- A minimum 30 UOC of Advanced Disciplinary Courses taken from Biomedical Engineering specialisation courses (those courses beginning with BIOM9xxx)
- A minimum 12 UOC of Electives chosen from Engineering & Technical Management Courses (those courses beginning with GSOExxxx)
- A minimum 18 UOC of Research including GSOE9010.
For all non-biomedical engineering courses, written approval must be obtained from the GSBmE School Office prior to enrolling. The relevance of these courses to your program of study must be indicated in order to gain approval.
ADVANCED STANDING: Students may be granted credit for some courses. Those with a four year Bachelor of Engineering (honours) may apply for credit up to 24 UOC for the masters degree. This effectively reduces the degree to one year full time. For more details please go to the online handbook.
Graduate Diploma in Biomedical Engineering (GradDipBiomedE), Program Code 5449
The Graduate Diploma of Biomedical Engineering is obtained solely through coursework. Students who perform well in the Graduate Diploma may be able to articulate to the Master of Biomedical Engineering program 8660 after completing the full Graduate Diploma. This program consists of 48 units of credit (uoc) in total and is completed over 2 full time semesters.
Note that the main difference between the Graduate Diploma program 5449 and the Master of Biomedical Engineering program 8660 lies in the breadth of courses students are required to undertake, i.e. 48 uoc vs 72 uoc.
For further information please refer to the online handbook.


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Micro compounder 15 ml HT: MC 15 HT
Melt torque: 40 Nm over the complete 1 - 500 rpm range
Time to explore the benefits of the Xplore MC 15 HT, even more accurate, reliable, faster and stronger.
The Xplore MC 15 HT is our next generation 15 ml compounder that will further simplify and accelerate your R&D: it is stronger in shear, can handle higher viscosities, gives finer dispersion, better mixing and higher operational stability than ever before.
It is twice as fast (rpm), twice as strong and much more stable (screw torque), with less than half the foot print: therefore it gives you more with less.
Although it seems an evolution in look and working with it, it is a revolution in specs:
● double rpm range (1-500), so finer dispersion, better mixing, easier to scale up
● more than double screw torque (40 Nm) at nearly all rpm, more stable extrusion rate
● control over throughput, improved dimensional stability of spun filaments and casted films
● stronger motor drive, so mixing of higher viscous compounds now possible
● less than half the foot print, lower housing, so easier to move and install in a fume hood
● stiffer housing, so less wear of screws and barrel, longer life time, less maintenance
● by design easier to work with, to install add-ons (film or fiber line) and to maintain
● maximum operating temperature 450 C
Much desired options are Xplore's proprietary rheological software, which enables upscaling to large parallel twin screw extruders (not possible? Fake news from competition!), and its Vari-Batch™ technology to easily rebuild the 15 ml into a 3 or 7 ml barrel size for mixing your most precious compounds.
This next generation MC 15 HT is inspired by Xplore's customer's wishes and our drive to continuous improvement. The Xplore MC 15 HT looks nearly unchanged, yet its specs are so much improved that you cannot anymore work without it.
With 40 Nm of torque processing rubbers and elastomers becomes easy, even with fully intermeshing screws, which have better mixing capabilities vs a tangential mixer, commonly used in rubber mixing. As a result, every cm3 has exactly the same properties, as opposed to a tangential screw design, where generating a consistent sample is still a challenge.
Inspired by the need of our existing customers, our engineers designed the best possible to also provide an alternative for a difficult to clean and labor-intensive laboratory kneader or a time consuming "banburry" type internal mixer and subsequent two roll mill process for rubber mixing.
This is not a want to have, but a must have for every R&D and quality control lab working with plastics, resins, compounds, elastomers, or film, filament and reactive extrusion.


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Expanding upon its JZ and JR series chip trimmer capacitors, the new JZL and JRL series maintain the same ±1% capacitance-drift specification as their counterparts but with a 25% price reduction. The new components are available in capacitance ranges from 10 pF to 20 pF. Any model ordered in quantities of 50,000 pieces or greater can be delivered from stock to four weeks ARO.
Company: VOLTRONICS CORP.
Product URL: Click here for more information
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Monday, September 8, 2008
Papa's Soul Food Menu
This menu is rad! It is far from the mundane food list you find at every other restaurant, and best of all, the design fits the style and concept of the restaurant (I know, it doesn't seem that hard but I am amazed at some of the horrible menu designs around). Even though I usually know what I want I still spend a good fifteen minutes staring at their menu, it's great, I especially like the diagram of the chicken sandwich. Every time I go back to my hometown of Eugene I make it a point to go down to 5th and Blair and get the best pulled pork sandwich in Oregon; or if I am feeling like something spicy, the amazing jerked chicken. The menu was designed by Tony Figoli.
1 comment:
- Anonymous said...
This was super helpful seeing as you must have a Facebook account to access their menu now. Wasn't able to find it until I came across your posting. Much thanks!!
- February 25, 2012 at 1:40 PM
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VanDusen Chevrolet Buick GMC
- Business Slogan: We believe in providing an extraordinary customer experience.
- Street: 425 Bayly Street West
- City: Ajax
- Province: Ontario
- Country: CANADA
- Postal Code: L1S 6M3
- Phone Number: ( 905) 427-2500
- Business Email: firstname.lastname@example.com
- Website: http://www.vandusengm.com
- Facebook Profile Link: https://www.facebook.com/VanDusenChevroletBuickGMC/
- Google + Profile Link: https://plus.google.com/114533826728392916294
- Twitter Profile Link: https://twitter.com/vandusengm
- Listed: June 2, 2017 2:38 pm
- Expires: This ad has expired
Business Hours
Mon-Thu 7:30am - 9pm
Fri 7:30am - 6pm
Sat 8am - 6pm
VanDusen Chevrolet Buick GMC in Ajax, serving all of Durham Region. We are located at 425 Bayly Street West. Just a minute south of the 401 at Westney & Bayly. Our goal is to provide you with an exceptional customer experience. We offer a wide selection of New Chevrolet, Buick & GMC cars, trucks, SUVs & vans. We also offer a tremendous variety of GM Certified Pre-Owned vehicles and all other makes of Used vehicles at all times. Our GM Certified Service is here to fix your vehicle whether you need repairs, an oil change, brake service or new tires. Our friendly certified technicians will keep your vehicle in top running condition. We also have an extensively stocked Parts department & an on-site Body Shop/Collision Centre.
Visit our website to view our latest inventory of cars, trucks & SUVs, or to book a service appointment. Visit us at 425 Bayly Street West, Ajax. SouthEast corner of Bayly and Westney. Near the 401, Pickering, Whitby, and Oshawa. Call us at (905) 427-2500 or (888) 486-2963.
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Oshawa Free Business Directory
Place your free business listing in our Canadian directory. This is a clean business directory to help businesses advertise and promote their products & services. Choose the right location to add your free business listing today!


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Far from democracy in Myanmar
Since the military regime in Myanmar initiated a dramatic and positive change of course, the democratic development has now halted. Read more
Since the military regime in Myanmar initiated a dramatic and positive change of course, the democratic development has now halted. Read more
«It is the mismanagement of peace that has led to new conflict in South Sudan» says former head of UNs mission to South Sudan, Hilde Frafjord Johnson. Read more
There is much attention these days to the brutal and unacceptable misbehavior of the terrorist organization IS in Iraq, especially against the religious Yazid minority. Persecution of religious minorities is today extensively worldwide and is also increasing. Millions are displaced. Read more
The recent reports from Iraq has provided us with the dreadful stories on how the Islamic State (IS) is slaughtering people from the Yazidi and Christian minority communities in Iraq. They must be stopped. Unfortunately I do not see a peaceful solution to the tragedy. Read more
Since the adoption of the International Covenant on Civil and Political Rights (ICCPR), and the International Covenant on Social, Economic and Cultural Rights (ICESCR), there has been a longstanding debate over the relationship between the two sets of rights. Are they indeed indivisible and interrelated, or must one set of rights be accorded priority? Read more
Kyrgyzstan consists of a rich diversity of different religious groups and movements. Even though the country's constitution now secures full freedom of religion or belief, the post-Soviet country struggles to find the right balance between religious freedom and state control. Read more
Should political parties compete or cooperate? The answer is both. Read more
This was cried out by a farmer from Lista at the National Assembly at Eidsvoll in 1814 , according to the diary of Magistrate Thomas Bryn. " - which immediately, as by an electric chock , was followed", he told further. It settled the controversy over the so-called Jews' clause, whereby also Jesuits and monastic orders were denied access to the Kingdom. Read more
Kyrgyzstan is facing challenges when it comes to fundamental human rights like freedom of religion or belief. The Oslo Center and the Norwegian Helsinki Committee wish to raise awareness of the importance of human rights obligations. Read more
20 years have passed since the genocide in Rwanda shock the world and reminded us about the importance of international actors acting on behalf of the voiceless. The stateless Rohingya Muslims in Myanmar suffer in silence under similar conditions. Read more


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New pics and bios on 2019 inductees are now up to date.
Huntsville - Madison County Athletic HOF Member
Richard Smock
Ever heard the story about the time Richard Smock, one of Huntsville's best-ever bowlers, went head-to-head against Earl Anthony, maybe the greatest professional bowler of all time? Remember the late Earl Anthony, the smooth left-handed assassin with the crewcut? Won 43 titles on the regular Professional Bowlers Association tour. Won seven more on the Senior Tour. Won 10 major tournaments – the PBA National Championships six times, the Tournament of Champions twice and the ABC Masters twice. His nicknames included "Square Earl," "Earl the Pearl" and "The Doomsday Stroking Machine." Anthony, Dick Weber and Don Carter were household names a generation ago and dramatically increased bowling's profile and popularity with their frequent TV appearances on ABC'S Wide World of Sports. "I can always say I once beat Earl Anthony in a big tournament," says Richard Smock. "Not everybody can say that." More on the Earl Anthony story shortly. At tonight's 26th induction banquet, Richard Smock becomes the 11th bowler from the Huntsville area to be inducted into the Huntsville-Madison County Athletic Hall of Fame. Many local devotees of the sport believe only the late Jimmy Certain, inducted in the inaugural class in 1989, was better. In fact, Certain and Smock are the only bowlers from Huntsville who ever won a PBA Tour tournament. Smock's win came at a PBA regional in Houman, LA, in the early 1990s. "After I won, they made me an Honorary Cajun," he said. "I was glad to win the money and glad to be an 'honorary' anything. Those Cajuns know how to have a good time." Smock considers the Houma tournament and winning the 2005 Eric Defietas tournament in Atlanta a dozen years ago as his top accomplishments in bowling. Smock, the third of Robert and Elva Smock's four children, was born in 1958 in El Centro, Calif., where the senior Smock was working as a civil servant at the Naval Proving Grounds. The family soon moved 2,000 miles eastward when Richard's father took a job at Wright- Patterson Air Force Base in Dayton, Ohio, and they all came to Huntsville in 1963 when Robert Smock went to work at Redstone Arsenal. Richard was 5 at the time. He was "9 or 10, somewhere around 1968," when he discovered bow ling after spending the night at the home of a childhood friend, David Blevins. "David and George Carlyle were the ones who got me started," Smock recalled. "David and I went to Pin Palace the next day and I was just hanging around, watching him bowl. It looked like a lot of fun. George Carlyle came over and told me, 'Don't just stand there. Go get you some shoes and a ball.' I said, 'I don't have any money.' He said, 'I run this place. You don't need any money. Go get some shoes and a ball.' So I did, and that was where it all started. I immediately joined the junior leagues. I liked it and my parents backed me and supported me. For some reason, I excelled at it more than most of the other kids. Guess I just had a knack for it. Nobody in my family bowled except me. But my sister, Mary, was a fan of mine and she kept pushing me to get better. So really, I blame George Carlyle and my sister for me getting hooked on a lifetime of bowling." Incidentally, George Carlyle wasn't kidding about running the place. A year after Pin Palace opened just off North Parkway in 1963, Carlyle leased the snack bar. The following year, after the facility expanded from 24 to 36 lanes, he became the assistant manager, and eventually the manager. Pin Palace was aptly named. For years, it was probably the premier bowling alley in the state. When he was 14, Smock bowled a 298 game at Pin Palace, leaving a 4-6 split on his last ball. The same year, he joined the men's adult league. "I was beating everybody else in my age group," he explained, "and I felt I wasn't going to get any better until the competition was stronger." By then, Smock had already caught the eye of everybody in the local bowling community, including Jimmy Certain. "Jimmy was a mentor of mine – a hero – because I loved bowling so much and because he took me under his wing. Basically, he just told me to do it my way." Certain had been a bowling prodigy himself in the early 1950s. At 13, he won the Tennesssee men's state tournament, singles and all-events, and went on to win virtually every important amateur event in Alabama over the next several years. He joined the professional tour in 1967 and was runner-up to Mike Durbin for Rookie of the Year. He competed off and on the tour for 22 years, and then won first place and $13,000 in his first event on the senior tour, the '89 Showboat Senior Invitational in Las Vegas. "Around here in those days," said Smock, "every bowler wanted to be Jimmy Certain." By his senior year at Johnson High School, lots of bowlers around town were probably saying the same thing about Richard Smock. He and friendly rival Tony Churchy at Grissom High were two of the hottest young bowlers in the northern tier of Alabama at the time. Smock's bowling accomplishments after nearly a half-century include these: Huntsville's Bowler of the Decade (1980-90); winner of the Huntsville Bowling Association city tournament 15 times; HBA Bowler of the Year three times; HBA Hall of Fame inductee in 1990; winner of 15 titles in the Alabama State Bowling Association; ASBA Hall of Fame inductee in 1999; has bowled a perfect 300 game 65 times, including one in the 1993 ABC Masters; also has several 299 games ("I try not to think of those," said Smock). Now for the Earl Anthony story: In 1980 at the age of 22, Smock and Tony Churchy set out on a 13-stop bowling tour, starting on the west coast and working their way east. When they reached Waukegan, Ill., a northern suburb of Chicago, they called Janis and Jan back in Huntsville and invited them to drive up to Chicago for the Waukegan tournament. Jan was Tony's girlfriend and now his wife. Janice was Janice Spray, Richard's girlfriend then and now. "Janis is the love of my life," Smock said. "We've been together 44 year s. Maybe we'll get married someday. But neither one of us want to rush into anything." So Janis and Jan drove to Waukegan to cheer their guys on. About mid-tournament, Smock found himself matched against America's "Mr. Bowling." At the time, Anthony was just two years away from becoming the first bowler to amass $1 million in career earnings – big money in that era. "I was bowling cross-lanes with Mr. Bowling," said Smock. "We had something like 200 or 300 people watching the two of us, and I feel pretty strongly they weren't there to watch me. Earl marked in the 10th frame, finished with something like a 210, and the crowd was clapping and cheering like it was a football game. I'm up in the 10th frame and I wound up with a 279. I heard a little light clapping from the crowd, and most of it was coming from Janice. Afterward, Earl Anthony came over and shook my hand. Then he put his arm on my shoulder and said, 'Don't worry, son. I got the same treatment back when I was a rookie.' "
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A grove of trees in loving memory of André, who left us far to soon.
Nu ben je toch nog een beetje terug in Schotland, xxx
Donated by Eva van Abeelen
5th June 2017
A memory of trees For all our memories, both joy and sorrow For all the lives you touched, and made so much better For all your wisdom and your jokes For all you have given us and shared For all the days and ways we miss you
Sign up to our mailing list to receive our monthly 'Tree News' e-newsletter and other occasional emails about volunteering, events, appeals and fundraising. It's the perfect way to stay up to date with the latest news about the wild forest and it's wonderful wildlife.
Live locally and want to know more about volunteering with us? Get updates about our Conservation Days straight to your inbox.


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You want to send him / her something really sweet and memorable and yet not too bulky. Something that is small enough to be able to carry around but thoughtful enough to show that you really miss him / her!
What about a Miniature Christmas Bonsai tree?
Don't worry, this is not a real tree (so it is postal custom approve!) it is a miniature tree I put together carefully by gluing green bushes onto plastic tree branches (like the model architects use!). The tree is then glued onto a terracotta base, finished by adding a twine bow on the branch.
This sweet and cute Miniature Christmas Bonsai Tree will come packed in a small gift box, with tissue paper underneath. Once the lid is on, it will be wrapped with a red/white twine. (as seen in 2nd and 5th picture)
To see more details, visit my Etsy Shop
Here are some pictures of my friends receiving my mini Bonsai trees :)


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What are the steps in writing an essay?
Refer back to the Research Skills process to ensure that the information you have gathered meets the definition of your task.
Organise the notes you have taken into
Draft the Introduction
The Body of the essay
The conclusion
Present the information in the format required
Assess your areas of strength and difficulty for future reference.
Congratulate yourself on your achievement.


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Connection or Concern?
History of TLC
Connection or Concern?
History of TLC
Tacoma Lesbian Connection
aka Tacoma Lesbian Concern
Who are we?
TLC has been in existence since the 1970's. We are a group of lesbians, primarily from the south Puget Sound area, organized to provide opportunities for socializing and building a supportive network.
To read a history of TLC,
click here
Who Can Participate?
TLC is open to anyone who identifies as lesbian… we welcome adult individuals who are bi, trans, or questioning as well. No litmus test here! While we don't have an age-range membership is primarily women in their 50s, 60, and older… that said, younger women are welcome to give it a try! You never know… some of those old broads have a lot going on.
Why Join?
Members receive six newsletters a year, each covering events, articles and news for a two-month period. Typically, each issue shares the details of 15 to 20 events of which some TLC members plan on participating and encourage you to as well.
Some are events put on by TLC, such as a monthly cards and games day. The newsletter describes the event, sharing any needed details and contact info.
Others are events some of us are planning to attend and are inviting you to join them -- examples would be going to the Grand Cinema to see a documentary, getting together to walk at the Nisqually Wildlife Preserve, or meeting up for Happy Hour at a local restaurant. Those events are hosted by a TLCer who will provide helpful info about the event, and helps to organize those going.
To join TLC, click here
Want to check TLC out before joining? We'd be glad to send you a sample newsletter… come to a few events and then decide.
Friends, Family and Allies?
It isn't unusual for a TLC member to bring along a friend, family member, or ally to a TLC event. We encourage you to check with the event sponsor before hand, but there is seldom a problem as long as the guest is respectful of who we are. For events that we think are the best fit for guests, look in the newsletter… they'll be marked as "Family Friendly."
To contact TLC or request a sample newsletter, email
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Please join us for the Indiana Winter Gathering at CYO Camp Rancho Framasa. Don't miss this top notch day that will allow you to learn, grow, network and explore topics that are applicable to everyone in the camp community. A variety of educational opportunities including breakout sessions, roundtable discussions, and tours of Rancho Framasa will be offered.
Register Today!
Attendee Information
Educational Endorsement
The Indiana Winter Gathering has been endorsed by the American Camp Association. Educational Endorsement means that the program has been reviewed by a qualified panel to verify that the goals, learner outcomes, presenter qualifications, and evaluation requirements are relevant for this educational program. Participants may earn up to 3.5 continuing education credits (CECs) for this endorsed program.
For questions or additional information, contact Betsy Thamert at firstname.lastname@example.com or 765-342-1349.


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Ephedrine (Intravenous route)
Brand Names:
- Akovaz
- Corphedra
Dosage Forms:
- Solution
Uses of This Medicine:
Ephedrine injection is used to treat hypotension (low blood pressure) caused by anesthesia (numbing medicines).
This medicine is to be given only by or under the direct supervision of your doctor.
Before Using This Medicine:
In deciding to use a medicine, the risks of taking the medicine must be weighed against the good it will do. This is a decision you and your doctor will make. For this medicine, the following should be considered:
Tell your doctor if you have ever had any unusual or allergic reaction to this medicine or any other medicines. Also tell your health care professional if you have any other types of allergies, such as to foods, dyes, preservatives, or animals. For non-prescription products, read the label or package ingredients carefully.
Appropriate studies have not been performed on the relationship of age to the effects of ephedrine injection in the pediatric population. Safety and efficacy have not been established.
Older adults—
Appropriate studies performed to date have not demonstrated geriatric-specific problems that would limit the usefulness of ephedrine injection in the elderly. However, elderly patients are more likely to have kidney, liver, or heart problems, which may require caution and an adjustment in the dose for patients receiving this medicine.
There are no adequate studies in women for determining infant risk when using this medication during breastfeeding. Weigh the potential benefits against the potential risks before taking this medication while breastfeeding.
Other medicines—
Although certain medicines should not be used together at all, in other cases two different medicines may be used together even if an interaction might occur. In these cases, your doctor may want to change the dose, or other precautions may be necessary. When you are receiving this medicine, it is especially important that your healthcare professional know if you are taking any of the medicines listed below. The following interactions have been selected on the basis of their potential significance and are not necessarily all-inclusive.
Using this medicine with any of the following medicines is not recommended. Your doctor may decide not to treat you with this medication or change some of the other medicines you take.
- Cyclopropane
- Dihydroergotamine
- Halothane
- Isocarboxazid
- Rasagiline
- Selegiline
- Tranylcypromine
Using this medicine with any of the following medicines is usually not recommended, but may be required in some cases. If both medicines are prescribed together, your doctor may change the dose or how often you use one or both of the medicines.
- Acetyldigoxin
- Clorgyline
- Deslanoside
- Digitalis
- Digitoxin
- Digoxin
- Ergonovine
- Furazolidone
- Iobenguane I 123
- Iobenguane I 131
- Iproniazid
- Methylergonovine
- Metildigoxin
- Midodrine
- Nialamide
- Oxytocin
- Pargyline
- Phenelzine
- Procarbazine
Other interactions—
Certain medicines should not be used at or around the time of eating food or eating certain types of food since interactions may occur. Using alcohol or tobacco with certain medicines may also cause interactions to occur. The following interactions have been selected on the basis of their potential significance and are not necessarily all-inclusive.
Other medical problems—
The presence of other medical problems may affect the use of this medicine. Make sure you tell your doctor if you have any other medical problems, especially:
- Heart disease or
- Hypertension (high blood pressure) or
- Stroke, or history of—Use with caution. May make these conditions worse.
- Kidney disease—Use with caution. The effects may be increased because of slower removal of the medicine from the body.
Proper Use of This Medicine:
A doctor or other trained health professional will give you this medicine. This medicine is given through a needle placed in your veins.
Precautions While Using This Medicine:
Your doctor will check your progress closely while you are receiving this medicine. This will allow your doctor to see if the medicine is working properly and to check for unwanted effects.
Hypertension (high blood pressure) may occur after receiving this medicine. It is very important that your doctor check your blood pressure after receiving this medicine. If you notice any changes to your normal blood pressure, call your doctor right away.
Using this medicine repeatedly may cause a rapid decrease of response to the drug.
Do not take other medicines unless they have been discussed with your doctor. This includes prescription or nonprescription (over-the-counter [OTC]) medicines and herbal or vitamin supplements.
Side Effects of This Medicine:
- Incidence not known
- Blurred vision
- dizziness
- fast, pounding, or irregular heartbeat or pulse
- headache
- nervousness
- pounding in the ears
- slow or fast heartbeat
- Incidence not known
- Nausea or vomiting
- restlessness
Along with its needed effects, a medicine may cause some unwanted effects. Although not all of these side effects may occur, if they do occur they may need medical attention.
Check with your doctor or nurse immediately if any of the following side effects occur:
Some side effects may occur that usually do not need medical attention. These side effects may go away during treatment as your body adjusts to the medicine. Also, your health care professional may be able to tell you about ways to prevent or reduce some of these side effects. Check with your health care professional if any of the following side effects continue or are bothersome or if you have any questions about them:
Other side effects not listed may also occur in some patients. If you notice any other effects, check with your healthcare professional.
Call your doctor for medical advice about side effects. You may report side effects to the FDA at 1-800-FDA-1088.
Last Updated: 6/18/2019


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Bawang Merah dan Bawang Putih is one of the Education games created by the Educa Studio and released on September 29, 2014. This game tells the story of a girl named Bawang Putih lived with her step mother and old sister named Bawang Merah . At first , they lived happily . But when the father away from home , the nature of the mother and her old sister turned into evil . How the fate of Bawang Putih Next? Was she able to get through the day -to-day ?
Game Size : 28MB
Download : Here


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The Latest Trend In Tropical Plants For Sale
Air crops are epiphytes, that means they will grow without soil and making tillandsia one of the best indoor crops to grow. Even handling home crops, he says, could have an impact, either good or unhealthy. Research have proven that home vegetation enhance focus and productiveness (by as much as 15%), scale back stress levels, and boost your temper. Though crops release oxygen in the course of the day, it's price remembering that, at evening, when photosynthesis stops, most vegetation switch issues up and launch carbon dioxide. This understanding is vital when shifting crops into lower mild (acclimation) or when the light levels drop as a result of seasonal adjustments. In the meantime, greenery nyc , a botanic design firm, has increased its clientele by 6,500 percent because it was based in 2010; builders are finding methods to incorporate gardens as an amenity for residents; and extra individuals — like ms.
Oakes are turning what little spare space they've of their apartments into indoor gardens. Also the proud owner of a brand new indoor bonsai tree needs to be according to the care. Regardless of steady enhancements in the technology that may present contemporary drinking water for about a penny per gallon in air water's greatest items, in accordance with zwebner, that rate is still about 3.5 instances dearer than faucet water production, primarily based on the awwa's 2006 survey of 230 utilities throughout the u.s. Gardening know-how: growing jade Tropical Plants For Sale from cuttings starts with taking the cutting. Totally different soil compositions have different results on nutrient levels and effect crops in numerous ways. An indoor backyard can be your refuge from the surface world, and for many people it is a supply of nice joy. They hawk these products and say that home vegetation need food by means of the 12 months. Crops contain chloroplasts within the leaves which convert the power from daylight, carbon dioxide, and water into sugars, which they use as food.
He added that plants may clean the air. Peace lilies are listed by the aspca as one of many frequent houseplants which are toxic to cats. Permitting the soil to dry out between waterings stresses these crops. Vermiculite can also be environment friendly for making certain a well air circulation within the soil, and can be wonderful for holding up water and nutrients to progressively go into the soil, making it wealthy. Truong says the one inform-story sign he is observed is the rash that people get after they have a tendency the plants. In the u.s. And canada, same-day supply is mostly obtainable monday by means of saturday on orders received before 12:00 pm in the recipient's time zone (excluding holidays and peak floral delivery intervals). In the identical family as philodendrons (the araceae family), caladiums, or elephant ears, are additionally thought of stunning houseplants. And i might by no means thought of keeping a begonia as an indoor houseplant. My vegetation have to be simple going like me. Store our choice of indoor plants in the outdoors department at the residence depot.
Due to this fact, shop for fresh indoor vegetation and get them delivered to your home with our express delivery providers. You can even have fun with the pots, and display your plants in beautiful ceramic and copper containers. Next time you get the hiccups, combine a teaspoon of dill leaf with a cup of boiled water, pressure out the leaves, and drink the liquid slowly. I really like gardening and grow as many things as we will in pots ; as a consequence of only having a small paved patio out the again. Plant presents for men are lengthy-lasting and simple to take Buy Indoor Plants Online care of. Plants boost serotonin levels and dissolve risky airborne chemicals," she stated. Your delivered moms day plant comes with a free supply customized card. Add a little bit of pleasure to the winter season with this easy winter growing kit for the paperwhite flower, a fairly bunch of blossoms with white petals and delicate scent. In contrast to an exterior landscape plant, your potted crops rely on you to provide the right amount of water to the foundation system. These plants again need good lighting and are simple to maintain. Whilst you re-pot them you possibly can divide the vegetation and put them in several pots, they make lovely gifts.


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Mail id - info @digikore.com
Digikore Studios Limited
Digikore Studios Limited, a part of the US$ 50 Million Growel Group, is one of the top upcoming animation studios in India. Staffed with top creative professionals, Digikore produces 2D and 3D animated TV Series and Features. IIn the last eight years we have traveled quite a distance to create a mark in the World Animation Industry. Today, Digikore is a very well known company in the International arena and shares fantastic goodwill among Producers Worldwide.
Digikore Studios Limited
"Growel House"
Mumbai - Pune Road,
Pune - 411012
Tel: +91 20 3021 3000
Fax: +91 20 3021 3030
Mail id - info @digikore.com
Mail id - info @digikore.com
The main objective of this blog is to serve animation lovers with various information at one place. All the informations, images, songs & videos contained in the blog are collected from various other websites.This blog operates as an index and database of animation related content found publicly available on the Internet. I do not take responsibility for the content hosted on such third party websites. I do not claim copyright of any informations, images, songs & videos shown on this blog. If you want any content on this blog to be removed due to copyright violation, please feel free to contact me.


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C3 Church is about connecting people to God. The church is full of people who have discovered an incredible richness of life that comes from having a personal relationship with Jesus Christ.
Contrary to a view held by many people, God is not a dreary, depressed or weak person. Rather he is vibrant, joyful and powerful. His presence should always give rise to a sense of excitement and a fullness of spirit.
This understanding of God is reflected in the enthusiastic and upbeat nature of our church services. Jesus said that he came to earth that we might have a life of abundance (John 10:10). Every time we come together to worship God we are celebrating what Jesus has done for us.
C3 Church Global
C3 Church Sydney is a member of C3i our international movement of C3 Churches. Our churches around the world are bright, enthusiastic and relevant to today's world. Our message is one of faith, hope and love.
We have a united vision to build the church by connecting people to God, connecting people to people and also empowering people for effective, joyful service.
To find the locations of all our churches around the world or learn more about c3i globally, you can visit our website at http://www.c3iglobal.org.