text:
- Research article
- Open Access
Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses
© Dibari et al; licensee BioMed Central Ltd. 2012
- Received: 9 February 2012
- Accepted: 6 June 2012
- Published: 6 June 2012
Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY 1-3) that have been widely characterized in rice, maize and sorghum.
In wheat, for which yellow pigment content is extremely important for flour colour, only PSY 1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY 2 has been partially characterized. Here, we report the isolation of bread wheat PSY 3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY 3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY 3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY 3 during abiotic stress.
We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses.
- Carotenoids
- Phytoene synthase
- Wheat
- Intron loss
- Abiotic stress
- Evolution
Carotenoids represent a diverse group of pigments found in bacteria, fungi and plants [1]. In plants, carotenoids have several important functions, essential for plant development as: (i) they serve as accessory pigments to harvest light for photosynthesis and constitute the basic structural units of photosynthetic apparatus and (ii) they also act as photoprotectors for plants to adapt to high light stress [2]. In addition, oxidative cleavage of carotenoids produces apocarotenoids considered as (i) signals in plant development, (ii) antifungal agents, (iii) contributors to flower and fruit flavours and aromas [3]. The phyto-hormone abscisic acid, the most well known apocarotenoid, plays an important role in plant response to stress conditions [3]. In addition, carotenoids have long been considered as essential nutrients as well as important health beneficial compounds. In animals including humans, unable to synthesize carotenoids de novo, dietary carotenoids are essential precursors of vitamin A and retinoid compounds needed in development [4, 5].
PSY genes are of primary importance in wheat due to their impact on flour colour, so that numerous genetics studies have been conducted on yellow pigment (YP) content in wheat grain [13]. A high YP concentration is a desirable trait in durum wheat and is the target of breeding programs worldwide [14]. However, low YP is a breeding target in hexaploid wheat, for which bright white flour is preferred. The inheritance of YP is complex, controlled by additive gene effects and highly heritable [15]. Several QTLs for YP have been identified in both durum and bread wheat on chromosomes 1A [16], 1B [17, 18], 3A [19], 3B [16, 20], 4A and 5A [21], 2A, 4B and 6B [13], 5B [16], and 6A [17]. However, the majority of studies agreed that chromosome group 7 has the greatest impact in controlling YP content in bread wheat [13, 16–19, 22–24]. Elouafi et al.[22] identified two minor QTLs on chromosome 7A and a third major QTL on chromosome 7BL. This last QTL has been corroborated in two other durum wheat populations [13, 24] and is ortholog to the one identified in hexaploid wheat on the same chromosome [25]. A QTL for YP in durum wheat was identified by Patil et al.[16] on chromosome 7A that accounted for up to 55% of the trait variation. Mares and Campbell [20] identified QTLs on chromosome 7A in two hexaploid wheat populations that had a large impact on flour yellowness, and correspond to loci also associated with xanthophyll concentration. Cloning of genes controlling flour pigmentation would be of great help in breeding for genotypes with appropriate levels of yellow pigments in durum and bread wheat. Using a PCR-based approach Cenci et al.[26] identified and mapped clones from a durum wheat BAC library thought to contain genes coding for important enzymes involved in carotenogenesis. In their study, putative phytoene synthase, phytoene desaturase and ζ-carotene desaturase clones were assigned to the chromosome groups 5, 4 and 2, respectively. To date, four PSY genes have been identified in durum wheat, forming two paralogous series on chromosome groups 5 (Psy 2) and 7 (Psy 1) [13]. One of these, Psy 1-B1, co-segregates with a QTL for YP on 7BL [13, 24]. Psy 1-A1 was localized on the distal end of the chromosome 7AL in one hexaploid wheat mapping population, associated with variation for flour colour [18]. Wheat PSY 1 genomic sequences show similar exon-intron characteristic to PSY 1 known in other Grass species, with six exons separated by five introns [18]. A partial sequence of PSY 2 was also obtained and it was mapped on the short arm of the chromosome group 5 [13], with no clear data regarding the impact of PSY 2 on carotenoid accumulation.
In this article, we report evidence that the wheat PSY gene family also consists of three paralogous genes and that PSY 3 is characterized by a novel gene structure, consequence of intron loss events. Since the wheat PSY 3 promoter region is characterized by cis- acting elements involved in plant response to abiotic stress, we studied changes in wheat PSY expression levels in leaves and roots during exogenous treatment with ABA. Finally, after comparative genomic investigation of PSY loci, we reconstructed the paleo-history for this gene family in Grasses identifying evolutionary events that have shaped PSY gene copies in modern species.
PSY3 gene sequencing and mapping in bread wheat
Identification and characterization of PSY3 loci
BAC sequencing and annotation (cf Materials and Methods) made it possible to identify PSY 3 orthologous regions. We selected ungapped contigs of 25 000 bp, 53 411 bp and 46 803 bp harbouring, respectively, PSY 3A, PSY 3B and PSY 3D genes ( Additional file 1: S1S2S3). Figure 2b illustrates BAC annotation results as well as micro-colinearity with rice, Brachypodium, maize and sorghum genome sequences, confirming that PSY 3 and Bradi4g37530.1 genes are at orthologous positions. In the PSY 3 loci we found the insertion of a non-syntenic gene (orthologous to Bradi3g60840). According to paleogenomic data in monocots [29], this gene is located on the syntenic chromosomes 2, 4 and 3 of, rice, sorghum and Brachypodium genomes respectively. In wheat, this gene would be expected to map on the orthologous chromosome group 6 and not group 5, where it was found based on the PSY sequenced region assignation. We can thus suppose that this gene has been specifically transposed in wheat from an ancestral donor region (group 6) to the modern current acceptor region (group 5).
Despite intron/exon shuffling events reported previously, it was possible to predict protein structure. PSY 3A encodes a protein of 413 amino acids, longer than those coded by PSY 3B and PSY 3D homoeologs (401 and 403 amino acids, respectively) ( Additional file 1: S1S2S3). The NCBI Conserved Domain Search software allowed us to identify the trans-Isoprenyl Diphosphate Synthase (Trans-IPPS) domain which includes squalene and phytoene synthases. According to the previous domain characterization, wheat PSY 3 was classed in the IPSS superfamily and Class I terpene cyclise. The conserved trans-IPPS domain was identified at positions: aa123-386, aa112-375 and aa113-376, respectively, for PSY 3A, B and D copies. Protein motifs identified based on ScanProsite are available in Additional file 1: Figure S1. ChloroP1.1 [34] identified transit peptides with 56 and 57 residues for PSY 3B and PSY 3D, but only 6-residues for PSY 3A and a signal peptide of two amino acids for Brachypodium PSY 3. However, according to ProtComp 9.0 [35] prediction, wheat PSY 3 may have a putative chloroplast localization. We also analyzed sequence homology between wheat PSY 3 proteins: PSY 3A and PSY 3B showed 91%, PSY 3A and PSY 3D 90% and PSY 3B and PSY 3D 96% amino acid sequence identity.
Evolutionary history of PSY gene family
PSY genes expression profile and function investigation in bread wheat
Since there is no evidence of PSY 3 expression in the literature, we used the public wheat Affymetrix chip data [38] based on array hybridization with RNA extracted from wheat grains at different development stages (100 Degree Days (DD), 200DD, 250DD, 300DD, 500DD), from cellular division to starch filling, to unravel the expression of PSY genes in wheat. We blast-searched the PSY 3 sequences obtained against Affymetrix chip sequences and found two probes, Ta.20776.1.S1_at and Ta.18880.1.S1_at, homoeologs of PSY 1 and PSY 2, respectively. The expression level of PSY 1 in grains was twice that of PSY 2 during all developmental stages except at 500DD when PSY 1 levels decreased ( Additional file 1: Figure S2). PSY 2 expression was constant during grain development, whereas PSY 1 expression exhibited significant differences between 350DD and 500DD (p-value = 2.22E-10, using the Bonferroni test). Our data showed that PSY 1 increased at 200DD while its level remained constant at 300DD and then decreased at 500DD. No evidence was found for PSY 3 expression in grain at these developmental stages. To gain insight into PSY gene expression during grain development, we used Single-Strand Conformation Polymorphism (SSCP) analysis [27] to screen wheat RNAs (cv Recital) extracted from leaves, roots and grains at different development stages. SSCP offered in this experiment the opportunity to (i) detect minor sequence changes in polymerase chain reaction-amplified samples and (ii) evaluate the expression level of three homoeologous copies of each PSY gene in the same running time [38]. In the same PCR reaction, we amplified specifically one paralogous gene and its homoeologous copies that were successfully separated, by capillary electrophoresis according to their sequence differences and assigned to each genome, using amplification profiling approaches on nullitetrasomic lines as references [38]. PSY 1 expression was evaluated based on the primer pair PSY 1_F1R1 (amplification product of 218 bp) designed on the fourth exon. Its expression in developing grain (100DD, 200DD, 250DD, 300DD and 500DD) was constant and PSY 1-B showed the highest transcript level. PSY 1 expression profile did not change in leaves and roots where PSY 1-B was always the copy most expressed. We also amplified PSY 2 in each tissue using the primer pair PSY 2F1R1, designed on the fourth exon (amplification product of 141 bp). PSY 2 showed a constant expression pattern in developing grains, the B and D copies showing the highest transcript levels. In leaves, all three homoeolog copies presented the same expression profiles; whereas, in roots, the D transcript decreased compared to B and A, whose levels remain unchanged. Using this SSCP approach we were able to detect PSY 3 expression using the primer pair PSY 3F2R1, designed on the third exon (amplification product of 216 bp). During grain development at 100DD the D copy showed the highest transcript level, while, at 200DD, amplification intensity of the B copy increased in contrast with the two other homoeologs, which decreased. For the remaining stages, expression levels of the three homoeologous copies were constant. In leaves, A and D copies had the highest expression levels, while in roots, the D copy showed a greater expression than the A and B copies ( Additional file 1: Figure S3).
To investigate changes in PSY expression during stress, we determined transcript levels in leaf and root samples after stress treatment. PSY responses to the different ABA concentrations tested were constant. PSY 1 mRNA levels, in stressed leaves, underwent to a minimum reduction during the first 2 h to then returned to the initial level at 5 h. PSY 2 transcripts slightly decreased in stressed leaves after 2 h and then remained constant. PSY 3 expression levels increased about 4-fold after 5 h of stress treatment and then returned to initial levels. In stressed roots, differences in expression levels became more obvious: PSY 1 mRNA levels doubled after 5 h of treatment, while PSY 2 transcript levels doubled after only 2 h of stress and then decreased gradually after 5 and 8 h of treatments to initial expression levels (Figure 5c-d-e). PSY 3 expression levels started to increase just 1 h after the ABA application to 18-fold after 2 h and then returned to lower levels (see Additional file 1: Table S2 for ANOVA analysis). Overall, Figure 5f clearly shows the effectiveness of ABA treatment on PSY 3 up-regulation in roots. Normalized expression data of PSY genes obtained in this experiment are available as Additional file 1: Figure S4.
Carotenoids are widely recognized as essential pigments in plants because of their role in development, photosynthesis, photoprotection and in response to stress since they are essential precursors of apocarotenoids, such as the abscisic acid (ABA). The PSY gene family, playing a central role in carotenoid biosynthesis, is characterized by duplicated gene copies. Their characterization in Grasses and the recent identification of a third copy in rice, maize and sorghum raises questions about PSY gene family organization in wheat. The role and function of PSY 1 have been largely investigated because of its association with major QTLs for carotenoid accumulation in wheat grain. Partial sequences of PSY 2 from several wheat varieties are available but little is known about its function and, up to now, there was no evidence of a PSY 3 homolog and associated activity in wheat. Our current analysis has demonstrated that wheat PSY gene family is characterized by the existence of PSY 1 to 3 gene copies. We report here the precise nature of gene family structure, evolution and function.
Because of the polyploid nature of wheat and the lack of a whole genome sequence, we identified orthologous regions in reference genomes such as Brachypodium and developed COS markers to isolate wheat PSY 3 from a BAC library. Wheat PSY 3 is characterized by a novel gene structure due to intron loss events leading to four exons and three introns. Rice and maize PSY 3, whose gene structure is highly conserved, are characterized by a third intron starting with a non-canonical 5′splicing site GC instead of GT. This type of intron is deleted by the same spliceosome mechanism that removes GT-AG introns, mainly involved in alternative splicing rather than in introns loss events [40]. Brachypodium and sorghum show differences in PSY gene structure, characterized by five exons and four introns. The third intron has been deleted but the causal mechanism is not yet clear. The classical model to explain intron loss is based on recombination of a genomic copy of a gene with a reverse-transcribed copy of a spliced mRNA transcript, deleting one or more adjacent introns. A key aspect of this theory is that only multicellular eukaryotes that have genes transcribed in germ cells would be susceptible to mRNA-mediated intron loss [41–45]. We identified inverted and repeated short motifs at deletion breakpoints in the wheat PSY 3 sequence, corresponding to the 5′ and the 3′ splicing sites, which may have driven illegitimate recombination leading to the observed intron loss events. Brachypodium and sorghum PSY 3 sequences are not characterized by such repeated motifs at deletion breakpoint, suggesting that intron loss occurred by simple genomic deletion. Maize PSY3 intron 2 and rice intron 3 and maize intron 3 and rice intron 2 are similar in size, leading to the hypothesis that these introns may have been inverted but this could not be confirmed due to limited sequence similarities between introns. Howitt et al.[46] described the presence within the intron #3 in wheat PSY-D1 of an insertion of 1200 bp of an inverted repeat motif. The characterization of either repeated motifs and/or TEs in this intron in Grasses may have driven intron loss or shuffling events leading to the distinct PSY 3 gene structures observed.
PSY duplicated genes exist throughout monocots and eudicots. Gallagher et al.[5] proposed an evolutionary model for the Grass PSY gene family in which monocots and eudicots share the same ancestral gene and the same duplication event, giving rise to genes PSY 1 and PSY 2. Welsch and collaborators [11] proposed that rice PSY 3 originated from PSY 2 duplication or vice versa, because of their close similarity in the 5′UTR region. We analyzed homology between PSY genes in reference genomes and showed that PSY 3 had greater homology with PSY 1 sequence than PSY 2, suggesting that PSY 3 originated from a duplication of PSY 1. Furthermore, public paleogenomics data in Grasses indicated that PSY genes are not located on known duplicated chromosomes originating from the characterized ancestral WGD event reported by Murat et al.[29]. We suggest, according to Jaillon et al. and Tang et al.[36, 37], that a more ancient WGD event, specific to monocots has shaped the PSY 3 copy as a duplication of PSY 1.
Based on a semi-quantitative approach, such as SSCP strategy, we observed PSY expression in wheat grains, leaves and roots. PSY 1 and PSY 2 homoeologous copies showed stable expression profiles in the different tissues. PSY-B1 was the copy most expressed, while, PSY-B2 and PSY-D2 showed the largest transcript levels in developing grains and, in leaves and roots, PSY-A2 transcripts increased. PSY 3 homoeologous copies showed a variable pattern of expression during the first stage of cellular division during early grain development and we found PSY-A3 and PSY-D3 most expressed in leaves and PSY-D3 in roots. However, with this approach, a statistically basis comparison was not possible between the expression levels of the three paralogous genes because it is semi-quantitative and only the broad transcript levels of homoeologous copies of each gene can be compared when amplified with the same primer pairs and in the same conditions. Access to recently published Affymetrix data [38] provided the opportunity to investigate PSY gene expression pattern and showed that PSY 1 expression level was higher than PSY 2 in developing grains. PSY 1 transcripts increased at 200DD to remain constant until 500DD, when they were reduced. In developing maize endosperm, Li et al.[12] showed that during endosperm carotenogenesis, only PSY 1 transcript increased significantly, starting after 12DAP until 22DAP. Basis on this evidence, we can speculate that wheat PSY 1 in the endosperm follows the same kinetics as the maize PSY 1 ortholog and that apparently there is a correspondence in timing of expression between wheat and maize PSY 1 orthologs. Our results are consistent with the evidence that PSY 1 plays an important role in carotenoid accumulation in the endosperm and with its association with QTLs for YP on chromosome group 7 in wheat [13, 18, 24].
Gene expression is closely associated with cis- regulatory DNA sequences (known as cis-elements) located in its upstream regions [47]. In rice and maize, PSY 3 expression is suggested to be strongly inducible in roots by drought and salt stress and application of ABA, since their promoter regions harbour cis-elements involved in plant response to abiotic stress [11, 39]. We identified several regulatory elements in the wheat PSY 3 promoter region, (i) the ABA-responsive element (ABRE) whose core is represented by the G-box, and (ii) a Coupling Element (CE1 or CE3) that is necessary to obtain ABRE functional [48, 49]. Through a Real-Time PCR approach, we quantified wheat PSY transcript levels in leaves and roots before and after the exogenous application of ABA, observing that PSY 2 represents the most abundant transcript in unstressed leaves, while PSY 1 transcript levels were 3-fold lower than in carotenoid accumulation in grains [13, 18, 24]. Wheat PSY 2 may thus play an important and complementary role in YP accumulation in photosynthetic tissues. PSY 3 expression levels were low in non-stressed leaves compared to PSY 1 and PSY 2 while its levels increased in non-stressed roots. PSY 1 and PSY 2 transcript levels did not show significant changes in leaves after ABA treatment. In stressed roots, PSY 1 and PSY 2 levels doubled after 5 h and 2 h of treatment respectively. PSY 3 showed the most intense response to ABA as in leaves, after 5 h of ABA application, PSY 3 transcript was 4-fold higher, while in stressed roots after 2 h its transcript levels increase about 18-fold. PSY 3 was the only gene that showed significant changes of transcript levels in the studied conditions. Carotenoids provide precursors for ABA which is the main apocarotenoid that plants can accumulate under drought conditions, ABA applications having been shown to affect plant growth and development, mimicking the effect of water stress and improving capacity to escape or tolerate such stress [50]. Since PSY genes are considered as rate-limiting in carotenogenesis, we can explain the up-regulation of PSY 3 by ABA as the necessity of plants to control carotenoid flux to roots during stress conditions to provide ABA precursors and thus activate stress responses. In a previous study, Singh et al.[15], studying the association of PSY 1-A1 allelic variation with YP colour in durum wheat, speculated about the possible existence of a third PSY on the chromosome 7AL to explain the presence of a second QTL near the marker Xwmc116. We identified the chromosomal bin localization for each homoeolog and the expected syntenic relationships were confirmed assigning PSY 3 to chromosome group 5 which has the highest concentration of QTLs and major loci controlling plant adaptation to environment, particularly, those controlling heading date, frost and salt tolerance, whereas a region with a crucial role in drought tolerance is located on chromosome group 7. Multiple-stress QTLs and linked markers have also been detected, suggesting the existence of common mechanisms for different stresses or of gene clusters controlling different stress tolerance processes [51]. For example, in wheat a significant QTL for ABA accumulation in droughted leaves was found on chromosome 5A co-located with the major vernalization responsive gene, Vrn1 [50], and in the same chromosomal bin we mapped PSY-3A. Aprile et al.[52] studied different physiological reactions to water stress and the substantially different molecular responses of bread and durum wheat genotypes demonstrating that there is a difference between durum and bread wheat response to water stress and that deletion of bin 5AL-10 from Chinese Spring determined a lower response to such stresses. They also showed that the different response by durum and bread wheat can be associated with the absence of D genome, where genes or factors that modulate expression of cluster of genes may be localized. PSY 3 localization and function in wheat are consistent with these data, especially the PSY 3D genomic structure, function and expression reported in the current study. Finally, we also showed here that PSY 3s are characterized by intron losses, inverted repeats and up-regulation by abiotic stresses. It is important to note that biotic/abiotic stresses could also activate transposons and retrotransposons [53] whose expression may have played a role in PSY 3 gene structure modification.
PSY gene family plays an important role in plant development (photoprotection and resistance to stress) as well as is a key contributor to flower and fruit flavours and aromas. The current analysis provides relevant structural (sequence, primers), functional (expression patterns) and evolutionary (gene conservation and specificity) informations that can be used as applied tools in wheat breeding programs.
PSY gene orthologs identification
The methodology used to reassess synteny between wheat/rice/Brachypodium/sorghum/maize genomes as well as the identification of intra-chromosomal duplications in wheat was previously described [28, 29, 54, 55]. Briefly, wheat [30, 31], rice (41046 genes), Brachypodium (25504 genes), sorghum (34008 genes) and maize (32540 genes) genomes were aligned with the publicly available PSY gene sequence [56] to identify orthologs. Three parameters were used to increase stringency and significance of BLAST sequence alignment by parsing BLASTN results and rebuilding HSPs (high scoring pairs) or pairwise sequence alignments. The first parameter, AL (aligned length), corresponds to the sum of all HSP lengths. The second, CIP (cumulative identity percentage) corresponds to the cumulative percent of sequence identity obtained for all the HSPs (CIP = ∑ nb ID by HSP/AL) x 100). The third parameter, CALP is the cumulative alignment length percentage. It represents the sum of the HSP lengths (AL) for all the HSPs divided by the length of the query sequence (CALP = AL/Query length). These parameters allow identification of the best alignment, with the highest cumulative percentage of identity in the longest cumulative length, taking into account all HSPs obtained for any pairwise alignment, regarding PSY sequences. These parameters were applied to all the BLAST alignments that were performed in the present study. Based on the genome-wide synteny analysis, gene relationships between species were then referenced as COS (for conserved gene pairs), CNV (for tandem duplicated genes) and PAV (for non –conserved genes).
BAC clone screening, sequencing and annotation
A BAC library from T. aestivum 'Renan' [32] was used in this study, the library has a 6.9x genome coverage. It consists of 812,544 clones organized in 2592 384-well plates pooled in two steps. The first step aims at mixing the 384 clones of each plate; the second step organizes plate pools in two dimensional matrices. For each 384-well plate, 16-line pools and 24-column pools and a total of 40 pools were available. Overall, 274 line and column pools were available to identify BAC clones of interest through PCR screening using PSY primers. Each amplicon (or BAC of interest) in a 384-wells plate was identified by coordinates which identified positive line and column pools in a matrix. By crossing the coordinates of positive pools we determined the plate harbouring the clone of interest. The individual BAC clone of interest was then identified on specific plates by PCR screening. BAC sequencing was performed on the GS Junior (Roche) using the 454 sequencing technology according the procedure described by the manufacturer at the genotyping platform GENTYANE (UMR-1095 GDEC, Clermont-Ferrand).
Genes and repeated elements (TEs) were identified by computing and integrating results based on BLAST algorithms [57], predictor programs and software described below. Gene structure and putative functions were identified by combining results of BLASTN and BLASTX alignments against dbEST [58] and SwissProt databases [59] with the results of predictor program, FgeneSH [60] with default parameters. Known genes were named based on BLASTX results against protein with known functions (SwissProt). Promoter analysis was performed using both TSSP database and Toucan platform [61] to search for cis-elements. PolyA Miner Signal [62] was used in terminator analysis. Characterization of protein functional domain was performed using InterPro [63] and SMART database [64, 65], while protein motifs were analysed with ScanProsite [66]. ChloroP1.1 and ProtComp 9.0 were used, respectively, to identify signal peptide and to predict protein subcellular localization.
Transposable elements (TEs) were detected by comparison with two databases of repetitive elements: TREP [67] and Repbase [68]. TEs boundaries were identified with the REPET package [69]. Insertion profile of TEs is identified using a modified version of svg_ltr.pl script [70].
Stress treatment experimental design and quantitative real-time PCR
Recital seeds were surface sterilized and embryos at 16 days after pollination (DAP) were collected and grown on Murashige and Skoog agar medium with 2% saccharose and indole-3-acetic acid (IAA). In each pot, three embryos were sown in order to obtain three biological replications for each condition. Plants were grown during the first 3 days in the dark at 25 °C and were transferred to continuous light at 22 °C at the two-leaf stage.
To carry out stress experiments, plants were treated with solution of ABA at different concentration: 50 μM, 100 μM and 150 μM. Leaf and root samples were collected after 0, 1, 2, 5 and 8 hours (h) of ABA treatment and were stored at −80 °C. RNA extraction was performed according to TRIzol® Reagent protocol. DNase set (Qiagen) and RNeasy Minelute Cleanup (Qiagen) kits were used for RNA purification. Integrity and quality of mRNA (contamination assessment of genomic DNA) were checked with the A260/A280 ratio with Nanodrop (Thermo Scientific) and on agarose with a classical PCR using a reference gene (see primers below). cDNA synthesis was carried out with a Transcriptor first strand cDNA synthesis kit (Roche) following the procedure described by the manufacturer with 1 μg of RNA (cycling conditions: 10′ at 25 °C/30′ at 55 °C/5′ at 85 °C°). Four primer pairs used for Real-Time PCR analysis ( Additional file 1: Table S1) were designed with primer3 in order to obtain exonic specific amplification of 100pb. Real-time PCR was performed using the LightCycler® 480 (Roche Diagnostics) and all reactions were performed using the LightCycler® 480 DNA SYBR Green I Master (Roche Diagnostics) in 10 μl with 4 ng of cDNA and 55 PCR cycles at 60 °C (hybridation temperature). All samples were prepared in three technical replicates and a negative control using water as a template was included. Quantification cycles (Cq) were analysed using the Light Cycler software version 1.5.0 and normalized with two reference genes (wheat spastin (primers: TGCCACTGCCTGATCCAAAT and AGCAAGCCTCTCCAGATCATG) and RNase L inhibitor-like (primers: TTGAGCAACTCATGGACCAG and GCTTTCCAAGGCACAAACAT) [71]) using 'Advanced Relative Quantification' module to obtain normalized ratio Et-CqT/Er-CqR (with CqT/CqR : Cycle number at target/reference detection threshold (crossing point) and ET/ER : Efficiency of target/reference amplification (10-1/slope)). Specificity of amplification was confirmed via melting curve analysis of final PCR products by increasing the temperature from 65 °C to 95 °C. PCR efficiency was calculated for each gene using a standard curve of serial dilutions and used in relative expression analysis. To calculate transcript abundance under stress conditions, the transcript levels were obtained for the controls, at each considered time point and ABA concentration and tissues. All observations were expressed as mean ± SD; the significance (P ≤ 0.05) of variables studied was assessed by one-way analysis of variance (ANOVA) using R software version 2.13.1.
This work was supported by grants from the Agence Nationale de la Recherche, Program ANRjc-PaleoCereal (ref: ANR-09-JCJC-0058-01) and Program ANR-Blanc-PAGE (ref: ANR-2011-BSV6-00801). The authors would like also to thank Felicity Vear (INRA Clermont-Ferrand, UMR 1095, France) for her fruitful contribution during the manuscript preparation.
Authors' Affiliations
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Epigenetic changes regulate the fate and progression of osteoarthritis
The latest global burden of diseases study reviewed 187 countries and 27 regions. Their report confirms what was already suspected by many clinicians and epidemiologists. Osteoarthritis (OA) is one of the commonest causes of disease burden worldwide, ranking as high as 11th as a contributor to global disability. This burden is increasing exponentially; the years lived with disability (YLD) for hip and knee OA has increased from 10.5 million in 1990 to 17.1 million in 2010. The study also confirms that the disease is ubiquitous and spread all over the surveyed geographical regions and was most commonly seen in females over the age of 50, though no age was exempt (1). It was a pleasure reading an article entitled "Increased DNA Methylation and Reduced Expression of Transcription Factors in Human Osteoarthritis Cartilage", written by Oscar Alvarez-Garcia, which was published in Arthritis & Rheumatology in Aug. 2016 (2). The authors have shown that normal and OA knee articular cartilage can be distinguished by their DNA, methylation profiles. This raises an important issue of regulation of arthritic changes by epigenetic changes. Epigenetics is a complex process of chromatin and gene modulation through DNA methylation, histone deacetylation or microRNA, which might contribute to the inheritability of disease (3). OA is an age old disease of the old age characterized by degradation and calcification of articular cartilage, but the disease also affects the synovial tissue, ligaments as well as the bone; leading to chronic pain, limitation of joint movement and loss of physical function. The patient suffers from stiffness, joint pains, and joint swellings and may be incapacitated and unable to work.
Unfortunately, there is at present no specific molecule which can modify the disease natural history. Many molecules have been proposed and discarded as definitive treatment for OA and treatment is now mostly confined to analgesia, exercise and surgical joint modification including replacement. Much work has been carried out to elucidate the molecular processes which cause the disease in the hope of understanding mechanisms which would lead to the development of better methods of disease control. Studies have elucidated genetic, epigenetic, proteomic and other pathways which increase and modify the disease pathology (4). The pathogenesis of OA is multifactorial, with aging, obesity, and genetic susceptibility being the main risk factors (5). During OA development, articular chondrocytes (the only cell type in articular cartilage that is responsible for maintaining tissue homeostasis) undergo marked transcriptional and phenotypic changes that compromise their function and lead to cartilage degradation. However, genome-wide association studies have shown only a small genetic variance component for OA, and recent evidence has pointed to epigenetic regulation as a key driver of the transcriptional alterations observed in OA chondrocytes (6).
Arthritis & Rheumatology published an editorial on "Is it time for epigenetics in osteoarthritis?" (7), and made a call to the research community to increase efforts concerning the role played by epigenetics in the development of OA. It was put forth that the characterization and analysis of cartilage DNA methylomes not only offers potential therapeutic targets for the treatment of OA, but also enables the design of a map of epigenetic marks that can help develop potential biomarkers for diagnosis, prognosis, drug response, chondrogenesis, or homeostasis from high-resolution screening technologies. In 2 years' time several articles proved association between epigenetics and OA phenotypes. Different stimuli (environmental factors), such as joint injury, oxidative stress, and inflammatory cues, aging, diet, metabolic (Mtb.) disorders, or both nuclear and mitochondrial genetics are prone to alter the DNA methylome in OA, leading to the development of different phenotypes of OA (8).
In recent years the epigenetic mechanisms associated with disease have garnered a lot of attention as evidence has been generated that epigenetic regulation is a key driver of transcriptional alterations observed in chondrocytes of OA patients (9). The term epigenetics was first introduced by Conrad Waddington in the early 1940s. The original definition was broad, but in recent times, epigenetics has been defined more narrowly as the "the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence." (10). To put it very simply, epigenetics allows information in the long DNA sequence to be turned into expressed characteristics. These expressions can alter in each cell or group of cells thus changes can take place in response to environmental influences.
The epigenetic modifications can have many clinical implications. Environmental influences can cause changes which are relevant in disease causation and progression.
The two principal elements which cause these epigenetic modifications are methylation and histone modifications. Basically the process of methylation involves addition of a methyl group to a cytosine base and in mammals this takes place mainly at CpG dinucleotides (4). There is a growing body of evidence which suggests that DNA methylation plays an important part in the pathophysiology of OA (11). It has been suggested that this may occur in two ways: some individuals with poor epigenetic profiles may be susceptible to OA or that age related changes in the epigenome may lead to cartilage damage. There is some evidence for either point of view and it is quite possible that both mechanisms are responsible for the epigenetic changes in OA.
The development of comparatively cheap methylation arrays has now made it possible to develop genome wide DNA methylation profiles of cartilage and to compare diseased with normal cartilage. Studies tend to be focused on cartilage as it is the principal pathological feature of the disease. In addition, it is composed of a single cell type (12). Roach et al. (11) first hypothesized that abnormal gene expressions of clonal OA chondrocytes is associated with heritable epigenetic alterations in the DNA methylation pattern. Their study provided the first direct evidence of an association between the loss of DNA methylation and abnormal expression of MMP 3, MMP 9, MMP 13 and ADAMTS 4 by OA chondrocytes. Several studies subsequently confirmed these findings. However all these studies were unable to demonstrate causality as longitudinal studies of the same cartilage samples were understandably not done.
The advent of high-throughput arrays for the analysis of DNA methylation has significantly boosted the amount of epigenetic data that has emerged in the past few years. OA has taken part in this enhanced activity and is in fact quite well placed to do so, for two principal reasons: the central tissue involved in the disease process, cartilage, is readily accessible via joint replacement surgery; and the cartilage contains only a single cell type, the chondrocyte, thus limiting the scope for confounding heterogeneity that can be encountered when studying the epigenetics of a multicellular tissue in which cell-specific effects are likely.
Five methylation array analyses of cartilage DNA have been published in the recent years, focused on knee and/or hip OA. A number of interesting observations have emerged. For example, knee and hip cartilages are strikingly different, with DNA methylation differences at a number of genes, including homeobox genes, which are key regulators of skeletogenesis. This is reminiscent of the joint-specific genetic effects touched on earlier, and suggests that cartilages from different parts of the skeleton are not only genetically but also epigenetically distinct. The DNA methylation studies have also revealed that genes that have previously been implicated in OA typically harbor CpG sites that are differentially methylated between OA and non-OA cartilage. This implies that the regulation of gene expression via DNA methylation is a major driver of the OA disease process (13).
Genome wide methylome studies have utilized more sophisticated arrays to analyze up to 485,000 sites. The results of these studies have been summarized by Reynard et al. (4).
DNA methylation contributes to the chronicity of OA and could be responsible for the limitation of current therapies. It may predict progression of the disease as well as response. Epigenetic treatments being used in oncology may prove beneficial also in OA (14).
Ayugenomics, a recently evolving novel method combining the Ayurveda doctrine based constitution determination of study subjects with contemporary genome analysis tools, seem to hold promise for complex traits research efforts. A few groups in the country have already provided both preliminary and established evidence to show that personalized, predictive and protective health approach practiced in Ayurveda since 1500 BC can address some of the contemporary limitations and improve our understanding of biology of health and disease in molecular terms (15). Our recent study on DNA methylation changes in asthmatic individuals in response to Ayurvedic intervention using microarrays has shown encouraging results (16,17). Modalities from alternative and complementary medicine like Ayurveda are often pursued by many patients as well (18,19). Aqueous extracts of Withania somnifera root powder and fruit of Phyllanthus emblica have shown chondroprotective activities in an in vitro model of cartilage degradation with explant cultures of articular knee cartilage from OA patients (20,21). The possible mechanism of this unique activity of chondroprotection could be through epigenetic modifications or stem cell differentiation (22,23).
Mesenchymal stem cells (MSCs) have been proposed by many as an optimal regenerative cellular therapeutic for musculoskeletal regeneration, especially in the setting of degenerative pathology like OA (24,25). The translation of MSCs to clinical therapy for OA has been shown; however, signs of progress are evident and ongoing trials may show efficacy to indicate these products can serve as the disease-modifying therapy necessary to stem the tide of OA.
MSCs have been used in several clinical studies, clinical trials and have been shown to have improved symptoms and developed articular cartilage. Two of the most recent studies are summarized here. A single arm open label Spanish study from Barcelona (26) recruited 15 patients with grade II or III OA and injected round 40 million autologous MSCs into the joint. The patients were followed up for 6 months and the efficacy of the procedure was measured by the visual analogue scale for pain, algofunctional Health Assessment Questionnaire, quality of life (QoL) SF-36 questionnaire, Lequesne functional index and WOMAC score. The cartilage integrity was assessed by magnetic resonance imaging (MRI) and quantitative T2-mapping at 0, 6 and 12 months. There were mild side effects, but improvements were noted in all the criteria for efficacy and cartilage growth was also noted by MRI. Another study (27) also from Spain used a multicenter randomized design to compare two doses of cultured autologous MSCs with hyaluronic acid. In this study, similar results were obtained. MRI studies also showed cartilage growth. There are many similar studies on these lines (28). However the clinical improvements are difficult to explain. Earlier hypotheses of cartilage regeneration by the MSCs are not now generally accepted. However it remains to be seen whether autologous/allogeneic MSC therapy works via reversing the epigenetic changes occurring during progression of OA. Since the epigenetic changes are reversible it would be interesting to see whether interventional therapies for degenerative diseases like OA modify the DNA methylation pattern and facilitate cartilage regeneration.
Conflicts of Interest: The authors have no conflicts of interest to declare.
- Cross M, Smith E, Hoy D, et al. The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis 2014;73:1323-30. [Crossref] [PubMed]
- Alvarez-Garcia O, Fisch KM, Wineinger NE, et al. Increased DNA Methylation and Reduced Expression of Transcription Factors in Human Osteoarthritis Cartilage. Arthritis Rheumatol 2016;68:1876-86. [Crossref] [PubMed]
- Gabay O, Clouse KA. Epigenetics of cartilage diseases. Joint Bone Spine 2016;83:491-4. [Crossref] [PubMed]
- Reynard LN. Analysis of genetics and DNA methylation in osteoarthritis: What have we learnt about the disease? Semin Cell Dev Biol 2016. [Epub ahead of print]. [Crossref] [PubMed]
- Lotz M, Loeser RF. Effects of aging on articular cartilage homeostasis. Bone 2012;51:241-8. [Crossref] [PubMed]
- Goldring MB, Marcu KB. Epigenomic and microRNA-mediated regulation in cartilage development, homeostasis, and osteoarthritis. Trends Mol Med 2012;18:109-18. [Crossref] [PubMed]
- Blanco FJ, Rego-Pérez I. Editorial: Is it time for epigenetics in osteoarthritis? Arthritis Rheumatol 2014;66:2324-7. [Crossref] [PubMed]
- Zhang M, Wang J. Epigenetics and Osteoarthritis. Genes Dis 2015;2:69-75. [Crossref] [PubMed]
- Barter MJ, Young DA. Epigenetic mechanisms and non-coding RNAs in osteoarthritis. Curr Rheumatol Rep 2013;15:353. [Crossref] [PubMed]
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- Roach HI, Yamada N, Cheung KS, et al. Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions. Arthritis Rheum 2005;52:3110-24. [Crossref] [PubMed]
- Le LT, Swingler TE, Clark IM. Review: the role of microRNAs in osteoarthritis and chondrogenesis. Arthritis Rheum 2013;65:1963-74. [Crossref] [PubMed]
- Loughlin J. Genetic contribution to osteoarthritis development: current state of evidence. Curr Opin Rheumatol 2015;27:284-8. [Crossref] [PubMed]
- Meda F, Folci M, Baccarelli A, et al. The epigenetics of autoimmunity. Cell Mol Immunol 2011;8:226-36. [Crossref] [PubMed]
- Rotti H, Mallya S, Kabekkodu SP, et al. DNA methylation analysis of phenotype specific stratified Indian population. J Transl Med 2015;13:151. [Crossref] [PubMed]
- Bhat S, Mallya S, Varghese VK, et al. DNA methylation detection at single base resolution using targeted next generation bisulfite sequencing and cross validation using capillary sequencing. Gene 2016;594:259-267. [Crossref] [PubMed]
- Joshi KS, Nesari TM, Dedge AP, et al. Dosha phenotype specific Ayurveda intervention ameliorates asthma symptoms through cytokine modulations: Results of whole system clinical trial. J Ethnopharmacol 2016. [Epub ahead of print]. [Crossref] [PubMed]
- Ernst E. Complementary or alternative therapies for osteoarthritis. Nat Clin Pract Rheumatol 2006;2:74-80. [Crossref] [PubMed]
- Chopra A, Saluja M, Tillu G, et al. Ayurvedic medicine offers a good alternative to glucosamine and celecoxib in the treatment of symptomatic knee osteoarthritis: a randomized, double-blind, controlled equivalence drug trial. Rheumatology (Oxford) 2013;52:1408-17. [Crossref] [PubMed]
- Sumantran VN, Kulkarni A, Chandwaskar R, et al. Chondroprotective Potential of Fruit Extracts of Phyllanthus emblica in Osteoarthritis. Evid Based Complement Alternat Med 2008;5:329-35. [Crossref] [PubMed]
- Sumantran VN, Kulkarni A, Boddul S, et al. Chondroprotective potential of root extracts of Withania somnifera in osteoarthritis. J Biosci 2007;32:299-307. [Crossref] [PubMed]
- Warrier SR, Haridas N, Balasubramanian S, et al. A synthetic formulation, Dhanwantharam kashaya, delays senescence in stem cells. Cell Prolif 2013;46:283-90. [Crossref] [PubMed]
- Joshi KS, Bhonde R. Insights from Ayurveda for translational stem cell research. J Ayurveda Integr Med 2014;5:4-10. [Crossref] [PubMed]
- Barry F, Murphy M. Mesenchymal stem cells in joint disease and repair. Nat Rev Rheumatol 2013;9:584-94. [Crossref] [PubMed]
- Nöth U, Steinert AF, Tuan RS. Technology insight: adult mesenchymal stem cells for osteoarthritis therapy. Nat Clin Pract Rheumatol 2008;4:371-80. [PubMed]
- Soler R, Orozco L, Munar A, et al. Final results of a phase I-II trial using ex vivo expanded autologous Mesenchymal Stromal Cells for the treatment of osteoarthritis of the knee confirming safety and suggesting cartilage regeneration. Knee 2016;23:647-54. [Crossref] [PubMed]
- Lamo-Espinosa JM, Mora G, Blanco JF, et al. Intra-articular injection of two different doses of autologous bone marrow mesenchymal stem cells versus hyaluronic acid in the treatment of knee osteoarthritis: multicenter randomized controlled clinical trial (phase I/II). J Transl Med 2016;14:246. [Crossref] [PubMed]
- Gupta PK, Das AK, Chullikana A, et al. Mesenchymal stem cells for cartilage repair in osteoarthritis. Stem Cell Res Ther 2012;3:25. [Crossref] [PubMed]
Cite this article as: Bhonde R, Joshi K, Das AK. Epigenetic changes regulate the fate and progression of osteoarthritis. Ann Joint 2016;1:28.


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As of version 1.3 it is possible to create a RAC Flex Cluster using ansible-oracle. From a ansible-oracle configuration perspective there is not a huge difference from a normal 'standard' cluster, basically a few new parameters. There are other differences though, specifically in how you have to run the playbook and deal with the inventory configuration.
In a 'standard' cluster, you have your database cluster nodes and that's it (basically). In a Flex Cluster configuration there are 2 types of nodes:
- Hub nodes. These have access to the shared storage and will house your databases
- Leaf nodes. These nodes are connected to the interconnect network, but not to the shared storage. So for instance, you could run your application on these nodes.
So given that, from an ansible-oracle perspective, it presents a few challenges. With a normal cluster, you could just run the playbook against all your cluster nodes as they are all the same.
Now, when building a Flex Cluster, there are a few things that should only be done on the hub nodes (configuring shared storage, install the database server and create the database(s)
And how do we do that? With a little Ansible inventory ninja-ism.


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For decades, researchers have been appraising the use of power-beaming solar-power satellites. But the projected cost, complexity and energy economics of the notion seemingly short-circuited the idea. Now, a unique new approach has entered the scene, dubbed SPS-ALPHA, short for Solar Power Satellite via Arbitrarily Large PHased Array. Leader of the concept is John Mankins of Artemis Innovation Management Solutions of Santa Maria, Calif.
Read the rest here


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In response to the U.S. Bureau of Labor Statistics, an estimated 500,000 registered nurses are expected to retire by 2022. Total, this college affords eight baccalaureate applications and 5 masters applications within the nursing college. The St. David's Faculty of Nursing is a pacesetter in advanced instructional alternatives and comprehensive new program development. Admission Necessities: For the department of nursing, students will need to achieve entrance into the college, complete core curriculum and preserve a excessive GPA, and take science-primarily based conditions earlier than making use of to the nursing program.
Challenging, diverse, and one of many more advanced applications obtainable in the state of Texas, the Baylor College Louise Herrington Faculty of Nursing is a well-respected program for a lot of reasons. A few key things assist the West Coast University stand out as a frontrunner in nursing in Texas. The curriculum focuses on teaching superior nursing purposes, but also teaches management, administration abilities, choice making, vital thinking, and learn how to deal with moral and ethical questions.
About one-third of all students enrolled in the nursing programs will enroll in this particular area. Admission Necessities: College students need to be admitted to the college, full an application, obtain the required 18 credit hours of prerequisite credit, take the Test of Important Educational Abilities and apply directly to the college of nursing. With the power to complete the Bachelor of Science in Nursing program within 39 numbers from start to end, West Coast University is among the best options for students who are searching for a approach to get into the workforce rapidly.
Discovering a nurse practitioner program or RN to BSN program in Texas isn't too difficult because there are quite a few faculties and universities accessible offering an online associates degree in nursing and Bachelor of Science in Nursing; nonetheless, it makes it extra necessary for potential nursing students to carefully consider their choices.
Admission Requirements: College students must first apply to the college and meet those admissions necessities before making use of to the nursing program after maintaining a C" grade on all prerequisite programs. A few of these together with nursing in maternity, pediatrics, psychiatric care, and neighborhood health. This is due partially to the varsity's commitment to offering particular person-centered nursing skills that support in health restoration, health promotion and disease prevention at their coronary heart.


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Yes! I always protect private personal information by not sharing them on social networks.
Jump to navigation
Do the preparation exercise first. Then read the poster and do the other exercises.
Do you know how to stay safe online?
Yes! I always protect private personal information by not sharing them on social networks.
1. Practice Safe Surfing and Shopping
2. Keep up to date
3. Click Smart
4. Be a selective sharer
5. Create complex passwords
6. Protect your mobie life
7. Keep your guard up
That's good idea
I have three tips for this question .first ,Install antivirus software in your PC.second don't use internet explore. third don't access not trusted web site. however Most influential way avoid accident on the internet is don't connect internet .
I've known how to keep private personal information.
Yep! of course Ikr that. I think that everybody gotta knows that!
of course i know how to stay safe online , i usually change my password, but i always forget it!
Yes. I know how to stay safe online but I don't care about it.
Yes , I know. These are the important rules for internet user. U need to know them to use online safely. P
Good topic with a lot of useful information.
This material is good for us. I can be more careful if i use computer online.
Yes,of course~
no comment
Yes, I do.
About Discussion: Do you know how to stay safe online?
Yes, I know.
It's important to know how to stay safe online when you try to follow the 8 tips that you saw.
Yes, of course.
Yes, i dont have to share personal information with people i dont know and i can use the privacy settings to configure it in a way i feel safe
Yes i know. thanks a lot to information about to safe online.
Yes, I know how to stay safe. but sometime I still feel afraid with people in internet.
Hi SarahJ,
Thanks for your comment. You shouldn't have to feel afraid of people so if you have any problems it's always best to tell an adult who you trust about them. Nobody should have to feel upset or afraid by anything they see online. There are lots of ways to get help.
Best wishes, Jo (LearnEnglish Teens Team)
Yes, I know. Don't share personal information is the basic thing that everyone should know and do.
I do not talk with strangers.
Yes! I've just learned from the eight tips! ☆
You know, nowadays have many ways they can cheat to take your information. It'd better to have knowledge about IT.
Yes, I know the internet is not supposed to talk to strangers in chat or video unknown.
keep your passwords safe.
Take care with what your share.
Don't share your passworrds with anyone and always be careful while using the internet, then you are safe in the online world.
Yes, I know.. Don't talk with the people you don't know and don't give them your personal information like: your name,age,address,email etc...
Yes. From now I know how to stay safe online.
© British Council The United Kingdom's international organisation for cultural relations and educational opportunities. A registered charity: 209131 (England and Wales) SC037733 (Scotland).


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Relive your residence experience independently! Alumni House and London Hall are suite-style residences designed for upper-year student living set with a kitchen and living room. Upper-year students can opt to live in any of our other seven residences as well if they choose, or become a student leader.
Just a few minutes away from class
Cook for yourself or enjoy meals in a dining hall
Prompt repairs and & regular housekeeping
Stay connected to the Western community
Great study & learning environment
Leadership opportunities
Independent Living
You know what to expect from university now, but that doesn't mean residence is only for first timers! Living on campus means you're close to your classes, campus services, and extracurricular activities. Alumni House offers easy access to Richmond Row or even Masonville Mall. London Hall is just steps away from the Western Recreation Centre. Even if you try to avoid cooking at all costs or would rather utilize a kitchen, the choice is yours since meal plans are optional!
Building Assignments are First-Come, First-Served
You'll still rank your building choices, but this time, the sooner you apply, the greater chance you'll get your first choice! Applications open in November, so hurry to the myHousing portal before spots fill up.
Quiet Study Spaces
The ease of having your classmates living alongside you is a benefit you've already experienced in first year, but in our Upper-Year Buildings, we have great study spaces and this independent living style boasts quieter floors.
Live With Your Friends, and Make New Ones!
You've met lots of people in first year, so why not continue living alongside them in this apartment style? Whether you have in mind exactly who you want to live with or not, our new online residence booking system has it covered. You can choose your residence building, live with your friends, and reserve your specific suite.
Leadership Opportunities
Become a mentor for incoming first year students while boosting your resume at the same time! We have more than 400 leadership positions in residence that will teach you valuable skills and provide great training opportunities. Don't believe us? See what our previous student leaders have to say for themselves!
Apply for Upper-Year Living!
The university experience is about what you make it, so while you're here make the most of the opportunities we have for you. Apply to live the RezLife at myHousing. Applications available in November.


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Human Rights and Civil Liberties are witnessing a precarious time where the respective Governments of various Nations are least concerned about them. Governments and their Agencies are openly engaging in Illegal and Unconstitutional E-Surveillance and Eavesdropping. Since there is no clear cut "International Obligations" on the part of our respective Governments to the contrary, these Governments are absolutely free to do whatever they want. The only solace can be found in the form of the text approved by the United Nations regarding Right to Privacy in the Digital Age.
This situation is further made worst due to Conflict of Laws in Cyberspace that allows the Nations to adopt and use their own Policies regarding E-Surveillance and Eavesdropping. Take the example of India. There is nothing like Civil Liberties Protection in Cyberspace in India. On the contrary, India is one of the most Endemic E-Surveillance Nation of the world. India has been engaging in numerous Illegal and Unconstitutional E-Surveillance Project that also without any Parliamentary Oversight and Judicial Scrutiny. The Privacy Rights in India in the Information Age have no meaning as we have no dedicated Laws in the fields of Privacy and Data Protection (PDF).
As on date, Mass Surveillance in India is omnipresent. For instance, projects like Aadhaar, Central Monitoring System, Network and Traffic Analysis System (NETRA), National Intelligence Grid (NATGRID), National Cyber Coordination Centre (NCCC), etc are E-Surveillance Projects that are not supported by any Legal Framework and Parliamentary Oversight. In fact, Vodafone has confirmed that India has been using "Secret Wires" in the Telecom Infrastructure to indulge in E-Surveillance. Indian Department of Telecommunications suppressed the whole incidence with a mere assurance of "Investigation" that never took place. As per my personal information, no "Public Report" was made available in this regard by Indian Government so far.
Intelligence Agencies like GCHQ and NSA intercepted and stored webcam images of millions of innocent Internet users. The Command and Control (C&C) Servers of Malware FinFisher were found in 36 Countries, including India. There are also strong grounds to believe that India and United States are collaborating on Illegal and Unconstitutional E-Surveillance and Eavesdropping. There is an urgent need to take up the issues of Civil Liberties Violations at the International Level among various stakeholders.
A good initiative in this regard has been taken by the Hague Institute for Global Justice. The Hague Institute has constituted a High Level Commission known as the Commission on Global Security, Justice and Governance (PDF). The official website of the Commission states that Humanity is facing unique and growing range of challenges like political violence, environmental decay, cyber insecurity and cross-border economic shocks. These issues have global security and justice implications that need urgent attention but far exceed state and global institutional capacities.
To overcome this capacity deficit, The Hague Institute for Global Justice and the Stimson Center have convened a Commission on Global Security, Justice, and Governance. The Commission also brings together a select group of eminent statespersons and public intellectuals to draft and recommend reforms in this regard that would be considered during the 70th Anniversary Summit of the United Nations in September 2015.
This can be a good opportunity to consider and debate about Civil Liberties Protection in Cyberspace by various Nations. There is an urgent need to bring Transparency, Accountability and Parliamentary Oversight of the Illegal and Unconstitutional E-Surveillance activities of various Nations, including India. The United Nations and its Affiliates/Agencies can play a pro active role in this regard.
I hope and wish that the Commission on Global Security, Justice, and Governance would consider all these issues so that Governments can behave in a "Responsible and Accountable Manner".


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Free & unlimited access to our motoring tools
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The Fiat Doblo is no oil painting but the appearance has not stopped the Fiat Doblo becoming a sales success.
Fiat has worked hard on the new version improving comfort levels and making the styling slightly more attractive. Read More


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Having problems with your Sony Cyber-shot DSC-T50 Digital Camera ?
My sony cybershot DSC T50 has a flash problem..its not working. Clicking menu and selecting flash makes no difference. the red eye is working
Answers :
Press RESET button on camera .Its possibly needs repair ..Flash assembly is possibly bad and or flash data is corrupt.Needs service
Repair Help & Product Troubleshooting for Sony Cyber-shot DSC-T50 Digital Camera
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Suggested Questions/Answers :
My sony cybershot DSC T50 has a flash problem..its not working. Clicking menu and selecting flash makes no difference. the red eye is working
Press RESET button on camera .Its possibly needs repair ..Flash assembly is possibly bad and or flash data is corrupt.Needs service ... Sony Cyber-shot DSC-T50 Digital Camera
Displays all white in all modes. Kodak V570. Turn the power on and all I can see is white. I have tried a different battery an no battery, just power cord. I know the camera good enough I can go into the menu options and select some different features without actually seeing the menu. All that seems to work fine and the lens is working ok. I can push the shutter button, the flash goes off. I can't see the photo on the camera, but I can see the photo if I put the SD card into my computer. Please
Displays all white because all the color screen have RGB mode if any of mode's are not work correctlly it's mean's color chip was fail and not working. u have to repair ur camera and change ur display screen. ... Kodak EasyShare V570 Digital Camera
My Canon 40D locks up when I click the shutter and the blue printer light next to the menu button is flashing. All the LCDs go blank and all functions stop working . Originally I was using a vertical grip and it still would not come on when I took the batteries out but after I detached the grip the camera did come back on . Now I'm not useing the grip and I still have take out the batteries and re-insert and turn back on camera to make function work again. Everything looks normal but the same th
... Canon EOS 40D Digital Camera
Flash rebound I am new to digital cameras. So i took a long time searching before deciding on yhe tz3.The camera works great in daylight situations but with night snaps taking shots against glass or neon etc the flash is on the photograph also neon is exagerated spoiling the snaps.I have tried everything on the menu but cant seem to rectify. I am new to this but just back from holiday with mates all with different makes of cameras and not one had same problem. Any ideas. jack.
A camera flash against a glass, or any reflective surface, will bounce the flash light right back at you. Turn off flash when shooting through glass. Note flash is only for shots less than 12 ft distance. It take it you are shooting scenery shots thr ... Panasonic Lumix DMC-TZ3 Digital Camera
I have a Panasonic DMC-TZ1 that I can't get the flash to go off on. The only flash setting that works on my camera is no flash setting and the icon for the no flash symbol appears white. The rest of the settings such as automatic flash with red-eye, and automatic flash, ect... are red. Is this a settings issues or a repair issue? If it is a repair issue could you tell me what on the camera needs to be repaired. Weather it be the LED light is broken or a resistor or transistor on the circuit
... Digital Cameras
Can't get red eye reduction to work. When I hit the red eye reduction button in the menu, it goes to small photographs instead of the one I want to reduce the red eye in.
When the small photos appear, select the one you want to remove the red eye. ... Nikon D40x Digital Camera
My flash quit working on my canon EOS 500D on every mode, when I go to flash control in the menu and click on built-in flash func. settings it says ... this menu canot be displayed. external flash is attached, but there isn't an external flash attached?
This camera is only one model period old, is it still under warranty? If so take it in for service. I'm including a diagram of the manual release button on the side of the camera for the flash. Try pressing this button to see if it pops up. If not th ... Canon EOS 500D / Digital Rebel T1i Digital Camera
The flash has stopped working. It worked fine a couple days ago but now, nothing. I have made sure that the flash is turned on, but its still not working. None of the setting (auto, red-eye reduction etc.)
It seems like a circuit board fault, u will have to give it to the service centre.Thankyou ... Samsung BL103 Digital Camera
Flash is not working, the red eye works but not the flash. Can this be fixed?
... Canon PowerShot SD1100 IS Digital Camera
My CoolPix L10 camera display will not turn on - it sometimes will work as a video recorder, but I cannot get the display to come one. A red light just above the menu button will flash and then go off and the on light flashes. The camera is only about a year old and used to work great. Please help!
Change the battery..........................................sodeep ... Nikon COOLPIX L10 Digital Camera
Green light on camera back to the right of the 'delete' button continually flashes on-off about 1 second intervals as soon as battery is inserted as long as CF card is in place. Switching camera ON produces no visible operational indicators. Depressing "Menu" button flashes: hour glass; menu display; blank screen. Holding button down just cycles as above. Swapped lens, data card, battery, connected EH-5 power source; nothing makes any difference. Checked same lens in another D70 camera; it's OK.
I would start by looking into the memory card slot to see if you have a bent pin. You may need a small light and a magnifying glass to see them. If the pins are okay, then you can try a reset by holding down the BKT and metering buttons toget ... Nikon D70s Digital Camera with 18-200mm lens
I have a sony cybershot DSC-W210 and just recently the flash wont work when its on any mode to take a picture it has the flash symbol on the screen flashing and the code E:91:01 underneath also flashing its takin pictures as normal just without the flash it wont let me change the settings of the flash at all. Apart from this the camera lens shutter seems to be sticking when i turn on the camera it doesnt always open i have tried cleaning the lens but it doesnt seem to make any difference!
I have a sony cybershot DSC-W210 and just recently the flash wont work when its on any mode to take a picture it has the flash symbol on the screen flashing and the code E:91:01 underneath also flashing its takin pictures as normal just without the f ... Sony Cyber-shot DSC-W70 Digital Camera
My flash is not working on my Canon PowerShot SX110 is camera. Help! My boss thinks the flash is turned off, but when I go to flash settings it says that red-eye and sync are both turned on. Basically I tried to use the camera outside the other night and the camera kept telling me to raise the flash even though the flash was up. The pictures were all blurry. I LOVE camera and am hoping for a simple solution.
... Canon PowerShot SX110 IS Digital Camera
Hi, the flash setting on my Vivitar Vivicam 8025 camera won't stay on what I select. It always goes back to "no flash" when I set it to have flash or no red eye flash. Please help! Thanks!
Remove the battery AND the memory card.\015\012Then reinsert both starting with the memory card FIRST.\015\012Doing this, the camera will keep its settings while you take pictures but will revert back to its default settings as soon as th ... Vivitar ViviCam 8025 Digital Camera
My nikon d40 built in flash is not working suddenly. the camera didn't fall. my son took some pictures with it then suddenly the flash stopped firing. everything is ok except the built in flash.the flash lever keeps on clicking everytime i make a shot but the flash is not firing.
... Nikon D40x Digital Camera
My camera was working fine for about a year. Then one day i turned it on and the screen was black but it shows the grid and date and time also it shows the menu and the differnet scene selections. If i take a pic the flash works and the sound works, but no image just black and the time and date. i think its the shutter that is stuck?? How to fix this?? Not under warranty anymore.
Take the camera andturn off the flash. Point it toward you and take a picture. Look in the lens to see if you see the shutter activate. If you dont see this work, then your shutter may very well be stuck. \015\012\015\012There isn't reall ... Olympus FE-310 Digital Camera
Blurred pictures Okay so my pictures just started to get blurry and stuff like that for no reason (i took good care of my camera). and then it got a bit wet i think and now the flash won't work either and when i press the flash button thing it won't let me go to the flash menu. the red hand in the top right hand corner wont go away. What do i do? i just got this camera 2 months ago:(
WATER + CAMERAS = NOT GOOD!!\015\012does anyone besides me see this? ... Nikon COOLPIX L3 Digital Camera
I have got a Panasonic NV EX3EN digital camera.I am experiencing the following problem, can any one solve it out please. menu button not working-wont record or play back....there is a red light flashing o the lCD and a command is comuing up on the LCD screen U 10.when i disconnect the battery and reconnect it , the camera works for few seconds and again stops working.
... Panasonic Digital Cameras
BROKEN FLASH the flash on my samsung camera wont work, it will take a photo when i turn the flasg off but the red eye ( signalling the flas function is on ) just blinks but will not take the picture ?? helpppppppp !! read the manual with camera - not much use !! i can only take pictures during the day whilst the flash is turned off .
The chip set in your camera is damaged.you want to change it. ... Samsung NV10 Digital Camera
Flash does not work. When I select the flash mode "on" and I push the button to take a picture the flash icon turns red and the picture is taken without it.
The flas unit is defective, if the flesh unit is good u will get green led glow,then flash when u click ... Samsung Digimax L80 Digital Camera
Sony DSC-T100 lcd screen is dark and faded My lcd display doesn't seem to be working. It worked fine one day and the next I turned it on and at first glance it looks black. If I move it around I can bareley make out an image or menu options. I charged the battery and it made no difference.
Try to set it on afactory defaults or try to give the lcd a higher contrast or brightness to chekout is the cam still work or its an internal hardware problerm take a shot and see it through th computer. ... Sony Cyber-shot DSC-W55 Digital Camera
White screen? My camera has suddenly stopped working. When I press the red 'shoot' or green 'review' button, all I get on the display is a white screen. Pressing the shutter button does make it shoot, and the zoom and the flash work. I have just taken the card out and examined the jpg files - it is still taking pictures. So it's the display that is busted. I guess my question is: Is it worth the repair bill to get it fixed?
If you cannot find a solution to your problem you may want to email [email protected] He has good advice and good used parts at a fair price. ... Casio EXILIM ZOOM EX-Z850 Digital Camera
My camera worked very recently, as I took pictures on a camping trip. However, a few days later after getting back, I've found that it refuses to turn on. If I turn it to either Video or Photo, the light turns green, but then immediately flashes red several times and shuts off. I've tried different batteries, with and without several SD cards, and nothing works... How do I fix it?
Here are the steps for the no power problem: 1. Make sure you will install a new or fully charged battery/batteries into your camera. Please use lithium batteries Ni-Mh or rechargeable Batteries (2000 M.Amp or higher). 2. Clean the battery contacts. ... Kodak EasyShare CX6200 Digital Camera
When I turn my 550 PowerShot on, I get a blank screen (black for the most part with white camera symbology around the screen, such as the flash symbol, etc). The screen works, as I can click a picture and see the photo momentarily. I can also view my saved pics. When I select the film mode, the screen looks normal.......meaning it displays a color image of what is in the lens. Have I inadvertently selected a mode which is causing this phenomenon?
Think that you might have accidentally pressed the DISP or DISPLAY button on the back of your camera. This manually turns the LCD display on and off to save batteries. You would then use the optical viewfinder in its place. To turn the screen back on ... Canon PowerShot A550 Digital Camera
Intermittent problem The camera works fine for awhile then the 4-way selector suddenly will not change the menu. Or it gets stuck on full flash or no flash. The selections may get mixed up. The review won't activate except for the last shot sometimes. Then if you leave the camera for a couple of days it seems to fix itself. Everything is fine then suddenly all the troubles come back. Although it gets messed up like this you can still take photos with it and you can use the other modes usually to
Sometimes, by changing the swithces of the control panel solves the problem ... Sony Cyber-Shot DSC-S85 Digital Camera


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If you only have a minute...
The best way to describe what Motopi does is through the following Chinese proverb:
"Give a man a fish and you feed him for a day.
Teach a man to fish and you feed him for a lifetime".
At Motopi, we believe that there is a third element to this approach:
"Help a man to secure a market for his fish and you'll feed his family for generations to come..."
Through the BBBEE framework, Motopi assists communities by helping corporates to use:
This is all under-pinned by a strong strategic focus to ensure sustainability and mutual benefit. This approach adheres to the principles of 'self-help community development' and asset based community development, which aim to build autonomy and self-reliance amongst citizens, through community-led development that focuses on what the community has, as opposed to what they do not have or what they need.
This is aligned with the Department of Social Development's Community Capacity Enhancement approach.
Quick facts about
Motopi Consulting:
© Copyright 2012 of Motopi Consulting. All rights reserved.


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Congrats to all the winners. enjoy your prizes !
Thank You Anker! My Wife is going to love it!
Congratulations to the winners!!
This is so awesome. First time winning a contest ever!! Thank you Anker
but as I stated in my original post I was being petty. Disappointed, etc.
This topic is no longer a banner. It will no longer appear at the top of every page.
May b next time
cool! Thanks! I never expected to see my name in lights
@Lisa_Hrabec You never know.
Surprised we didn't see more expensive items in this contest. I would have chosen either the nubuls Mars, or the soundcore liberty. I probably would have gone for the Mars because of the value. 🤷♂ Maybe I'm just greedy....
If I'm gonna be honest, anker gives away so much stuff that everyone will win free stuff. In the last 3 months this is what I've won
That's just three months...
THREE!!!Since they came out, I haven't won any flares.... Keep trying but...
In the last 6 months (and a lot more) I have 2 wins, well one WIN really, as one gift was for being one of the top active members.
I don't do a lot of the power draws, as either for iPhone, USA, or micro-usb and occasionally stuff I already have in one form or another.
Plus, there haven't been the UK/Europe giveaways, like USA.
So consider yourself EXTREMELY lucky.
Sorry, I mistyped that. It was my third prize, I didn't win three. I only won one.
Congrats Winners!
Yea, lol my luck turned around quick! Wait you won that wireless charger with apple watch thing? I didn't even try because I dislike wireless chargers and don't have an apple watch lol
That's still 5 prizes in three months, compared to my 2 prizes in over 6 months.
As I said, you should count yourself extremely lucky.
It's a good job I'm not a really busy regular on here, or I'd be annoyed lol ... Not at you tho.
... and then you go on to list all the prizes you've won in a short space of time.
Congrats on the wins but just be careful some may take this as you rubbing it in their faces.
I mean, when I posted them I didn't realize it was a lot.... I though that was normal.... sorry....
That is great @Anjou1888 ! Get all you can whilst posting reviews for those winnings. I don't see it as rubbing their faces but more of how Anker is just an awesome company that cares about its customers/members.
Thanks! I'll definitely be posting some reviews in the next few weeks!