The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in Arabidopsis thaliana; RPT5a and RPT5b. We showed previously that RPT5a and RPT5b are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), RPT5b does not complement the effect of a strong rpt5a mutation in the male gametophyte, and only partially complements rpt5a mutation in the sporophyte. RPT5b Col-0 and RPT5b Ws-4 differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene.
By exploiting natural variation at RPT5b we determined that the SNP located in RPT5b intron seven, rather than the promoter SNP, is the sole basis of this lack of redundancy. In Ws-4 this SNP is predicted to create a new splicing branchpoint sequence that induces a partial mis-splicing of the pre-mRNA, leading to the introduction of a Premature Termination Codon. We characterized 5 accessions carrying this A-to-T substitution in intron seven and observed a complete correlation between this SNP and both a 10 to 20% level of the RPT5b pre-mRNA mis-splicing and the lack of ability to complement an rpt5a mutant phenotype.
The accession-dependent unequal redundancy between RPT5a and RPT5b genes illustrates an example of evolutionary drifting between duplicated genes through alternative splicing.
Gene duplications contribute to evolution . They arose mostly through polyploidy (i.e. genome duplication) that has been a widespread phenomenon among Angiosperms . Arabidopsis thaliana has undergone several rounds of genome duplication and up to 23% of its proteome is encoded by duplicated genes . The evolutionary outcomes of gene duplication were first theorized by Ohno , who predicted that although duplicated genes are redundant at first, they soon evolve and one of the duplicates either accumulates detrimental mutations and is lost, or gains new function(s). As a consequence, over time the duplicated genes become partially or no longer redundant. In some cases, both genes may have retained unequal redundancy: a mutation in one of the two duplicated genes causes a mutant phenotype while a mutation in the other does not; in the double mutant, this phenotype is strongly enhanced  At its utmost, independent evolution of duplicated genes among close accessions is the basis of incompatibility known as the Dobzhansky-Muller type syndrome [6, 7].
Gene evolution can be assessed by studying natural variation in a single species. A recent survey highlighted that nucleotide sequence polymorphism appears in all gene regions, both coding and non-coding . In particular, alternative splicing, that creates multiple mRNA from the same gene unit, has been described recently as a source of variation among natural accessions. For example, this process alters FLC function in the Arabidopsis accession Bur-0  as well as in some Brassica rapa accessions . Although evolution through alternative splicing may contribute to the creation of new functions through exon sliding , it has mainly been described as inducing Premature Termination Codon (PTC). Transcripts affected by PTC are often targets of Nonsense-Mediated Decay (NMD), leading to their degradation [12, 13]. Interestingly, it has been shown that alternative splicing is often conserved between species as diverse as Rice and Arabidopsis, suggesting that this process has an important role in gene function . However, the extent of variation in alternative splicing among accessions in a single species is not known.
In previous work we showed that two Arabidopsis genes, RPT5a and RPT5b, are necessary for the development of both gametophytes . These genes encode an AAA-ATPase subunit that acts as a component of the 26S proteasome machinery. Loss-of-function for both genes is male and female gametophyte lethal. In the Col-0 accession, both RPT5a and RPT5b genes act in a redundant manner. However, in the Ws-4 accession, RPT5b does not complement for rpt5a mutant phenotype in male gametophyte (and to a lesser extent in the sporophyte). Sequencing the 4800 bp RPT5b locus identified only two SNPs that differed between the RPT5b Col-0 and RPT5b Ws-4 genes, one located in the promoter (named SNP1) and the other at the end of the seventh intron (named SNP2). Those polymorphisms will be referred to as SNP1Ws (or SNP1Col) and SNP2Ws (or SNP2Col). These findings led us to suggest that inactivation of the RPT5b Ws-4 allele could be a result of either diminished RPT5b expression related to SNP1Ws, or else due to a mis-splicing event occurring at intron 7 related to SNP2Ws . Here we address this question by looking among 487 Arabidopsis accessions for RPT5b alleles with polymorphisms similar to those detected in the Ws-4 accession. Redundancy between RPT5a and RPT5b genes was then tested regarding each of these two SNPs. This approach allowed us to demonstrate that SNP2 is responsible for RPT5b inactivation through pre-mRNA mis-splicing, leading to PTC. This A-to-T variation in intron 7, which is shared by 5 unrelated accessions, is predicted to create a new branchpoint sequence upstream from RPT5b intron seven 3' splice site, leading to its mis-splicing.
Our previous work focussed on two accessions for which Arabidopsis insertional mutant collections are available, Col-0 and Ws-4. We presumed that the two SNPs highlighted between the RPT5b Col-0 and RPT5b Ws-4 alleles were responsible for their different abilities to complement the male gametophyte lethal phenotype in a rpt5a background.
We previously showed that a strong rpt5a-1 male gametophyte mutation was complemented by a transgene harbouring a 4 kb RPT5b Col construct . Likewise, 3 constructs were transformed into rpt5a-1/RPT5a plants, each harbouring the same 4 kb of RPT5b with different combinations of Col/Ws SNP1 and SNP2. It was tested whether those constructs would allow the transmission of rpt5a-1 through the male gametophyte (Additional file 1: Figure S1). Surprisingly, all 4 RPT5b constructs (SNP1Col SNP2Col, SNP1Ws SNP2Ws, SNP1Col SNP2Ws and SNP1Ws SNP2Col) did complement the rpt5a-1 mutation. The empty binary vector used as a control did not. It has to be noted that those transformed plants expressed the endogenous RPT5b Ws gene as well as the RPT5b transgene. It is very likely that the additive expression of the endogenous RPT5b together with the transgene is sufficient to complement the rpt5a-1 mutation and is therefore masking the partial loss of function of the RPT5b Ws allele.
As a complementary approach, we looked for additional accessions displaying polymorphism at either the promoter (SNP1) or within intron 7 (SNP2) using previously designed dCAPS markers ( and Fig. 1A and 1B). 487 accessions from the Versailles Resource Centre were screened for both dCAPS (Fig. 1B and not shown). Both Col plants (Col-0 and Col-7) contained in the set of accessions displayed a Col/Col digestion profile (dCAPS SNP1/dCAPS SNP2) as well as the majority (468) of tested accessions. All five Ws accessions (Ws, Ws-1, Ws-2, Ws-3 and Ws-4) as well as 6 new accessions were found to display a Ws/Ws profile and 6 new accessions showed a Ws/Col profile. No accession that would present a (Col dCAPS SNP1/Ws dCAPS SNP2) profile was found. To confirm the first screening, 12 accessions were chosen (Table 1) and were grown for new genomic DNA extraction. Two 500 bp regions, each one encompassing one SNP, were sequenced to confirm the initial dCAPS screening (Fig. 1A). This was of particular importance as the dCAPS could only discriminate between non-Ws and Ws status (SNP1) and non-Col and Col status for SNP2. All sequence data showed that among the accessions chosen there were no variations other than those we previously described between Col-0 and Ws-4 (Fig. 1C). The identification of accessions with different haplotypes gives us the opportunity to test which SNP is responsible for the observed phenotype.
Screening for RPT5b polymorphism among Arabidopsis natural variation. A, Graphic representation of RPT5b. Red boxes represent exons. The start of translation is indicated by a broken arrow. Both promoter and intron seven SNPs (respectively SNP1 and SNP2) are indicated. Double-end arrows located below RPT5b cover the 500 bp fragments that have been re-sequenced. B, Two dCAPS markers were used to discriminate SNP1 and SNP2 between Col and Ws accessions (see Methods). Results are also shown for the 8 selected accessions that show a non-Col signature for SNP1 and/or SNP2. C, Display of sequences around SNP1 (in promoter) and SNP2 (in intron 7) following partial RPT5b re-sequencing. The Col sequence is given as reference. Non-coding sequences are italicised while coding sequence (beginning of exon 8) are not. The first base of exon 8 is indicated by a star.
For practical matter relative to plant crosses, we retained 4 accessions for each haplotype displaying about the same flowering time as Col-0 and Ws-4. Those selected accessions are: Bu-11, Cal-0, Da-0 and Wu-0 for the SNP1Ws/SNP2Ws haplotype, and Akita, Bsch-0, Sn5(1) and Uk-4 for the SNP1Ws/SNP2Col haplotype.
We previously showed that the Col-0 allele of RPT5b (RPT5b Col-0 ) rescued the male gametophyte defect due to rpt5a loss of function whereas RPT5b Ws-4 did not, showing that the RPT5b Ws-4 allele is not functional in the male gametophyte. Male gametophytes harbouring an rpt5a ; RPT5b Ws-4 genotype degenerate as shown by the vital marker Alexander staining . Pollen phenotype is readily checked on the F1 progeny from crosses between the rpt5a-4 mutant (in a Col-0 background) and any other accession: 100% of the pollen should be viable if the rpt5a-4 mutation is complemented -by RPT5b x - but only 75% will be viable if there is no complementation (Fig. 2A and 2B). Each selected accession was crossed to wild-type Col-0 to assess any incompatibility between accessions that is unrelated to RPT5. At the same time, each selected accession was crossed to homozygous rpt5a-4 mutants (rpt5a-4/rpt5a-4; RPT5b Col-0 /RPT5b Col-0 ) to test whether the rpt5a-4 mutation could be rescued by the various RPT5b alleles. Ws-4 and Col-0 plants were used as controls. F1 plants were grown and pollen viability was tested (Fig. 2C and 2D). For 7 out of the 8 tested accessions, no pollen incompatibility was observed: consequently the cross with rpt5a-4 mutant could be used to test the (lack of) complementation of the rpt5a mutation. Only one accession, Da-0 (SNP1Ws/SNP2Ws haplotype), was found to trigger incompatibility when crossed to Col-0. F1 Da-0 × Col-0 showed a 28% pollen lethality that would be consistent with 2 independent genes leading to pollen lethality and could correspond to another example of Dobzhansky-Muller type incompatibility .
A SNP in RPT5b intron 7 correlates with unequal redundancy between RPT5a and RPT5b in pollen development. A, Schematic representation of the F1 crosses used in this study. A and a represent wild-type RPT5 and rpt5a-4 mutated allele, respectively. B C represents the Col allele RPT5b while X stands here for a non-Col accession. B, Gamete segregations for the F1 plants obtained in A. Two hypotheses are taken into consideration: that either the non-Col RPT5b allele complements the rpt5a mutation (B X , from the X accession) or that it does not (B Y from the Y accession). For each genotype, the percentages of expected gametes are indicated. Hence if the hypothesis is correct the percentage of dead pollen is either null or indicated by the grey column. C, Mature pollen stained with Alexander's stain in anther locules from a Aa4 B C B Bu-11 F1 hybrid and parents. Only the F1 plants show some dead pollen (indicated by arrows). D, Percentage of dead pollen determined by Alexander staining and subsequent counting in F1 AA B C B X (black boxes) and Aa4 B C B X (blue boxes where X stands for the accession written below the graph) plants. For each genotype, 300 grains were counted per plant. Values are shown as means on 4 independent plants (±SD).
F1 progenies from crosses of rpt5a-4 mutant with Akita, Bsch-0, Sn5(1) and Uk-4 were found to display almost no dead pollen (Fig. 2D). This means that RPT5b Akita , RPT5b Bsch-0 , RPT5b Sn5(1) , and RPT5b Uk-4 alleles complement the rpt5a-4 mutation, in contrast to RPT5b Ws-4 . As these accessions have a SNP1Ws/SNP2Col haplotype, it can be concluded that the SNP1Ws is not involved in the process that impairs the RPT5b Ws-4 allele from complementing the rpt5a-4 mutation. This is consistent with previous data showing that the promoter SNP1 did not seem to modify the GUS gene expression pattern when a 1200 bp promoter, either from Col-0 or from Ws-4, was fused to the GUS gene reporter .
By contrast, in F1 progeny from crosses of rpt5a-4 mutant with Bu-11, Cal-0 or Wu-0, approximately 25% non-viable pollen was observed, as for rpt5a-4/RPT5a; RPT5b Col-0 /RPT5b Ws-4 plants (Fig. 2C and 2D). This pollen lethality shows that RPT5b Bu-11 , RPT5b Cal-O and RPT5bWu-0alleles do not complement the rpt5a-4 mutation. As these accessions have a SNP1Ws/SNP2Ws haplotype, and because SNP1 was shown not to be involved, we conclude that the SNP2Ws is responsible for the lack of complementation of rpt5a mutation. Likewise, rpt5a-4/RPT5a; RPT5b Col-0 /RPT5b Da-0 plants displayed 42% pollen lethality, while only 28% dead pollen was observed in a wild-type RPT5a background, which is consistent with an additive effect of the Col X Da-0 incompatibility described above and a rpt5a-4; RPT5b Da-0 male gametophyte lethality. Taken together, these data strongly suggest that the SNP2Ws, located in intron 7, is responsible for the lack of complementation of the rpt5a mutation by RPT5b loci.
It had been previously shown that RPT5b is subject to alternative splicing at intron 7 . The alternatively spliced mRNA carries a 10 bp deletion at the beginning of exon 8 and consequently a frameshift resulting in a stop codon at the beginning of exon 8, leading to a much shorter predicted protein (35 kDa instead of 47 kDa) (Fig. 3B). In plants, as in yeast and vertebrates, exons are excised by a two-step reaction (Fig. 3A): firstly the 5' splice site is cleaved and there is formation of a lariat with the branchpoint that is usually located 18 to 40 nts upstream of the 3' splice site. Then, in a second step, the 3' splice site is cleaved and both exons are ligated. The 3' splice site selection results from the spliceosome scanning for the first AG dinucleotide downstream of the branchpoint . By analysing the intron 7 sequence in the Col-0 accession, one branchpoint sequence was found (CUCAU) that partially matches the plant consensus YURAY  with an A nucleotide located 18 bp upstream from the 3' splice site (Fig. 3A). By analysing the same intron sequence in the Ws accession, we found that SNP2Ws generated a new putative branchpoint consensus (CUAAC) that with its upstream sequence perfectly matches the yeast highly conserved sequence (UACUAAC). Therefore, although additional experiments would be required to prove that this sequence is a bona fide branchpoint [17, 18], this sequence is very likely to represent a branchpoint sequence that would compete with the upstream one. It has been shown that, particularly in mammalian genes, the 3' splicing site selection is defined by scanning for the first AG located 3' of the branchpoint , unless this AG is too close to the branchpoint (less than 12 nts), in which case steric effects would hamper the use of this AG in favour of a downstream AG . In RPT5b alleles harbouring a SNP2Ws signature, as the new branchpoint adenosine is located 6 nts from the 3' splice sequence, it is very likely that its use will trigger the spliceosome to scan until the next AG dinucleotide, leading to the mis-spliced RNA (Fig. 3B).
Alternative splicing of RPT5b Ws . A, Hypothesis for the cause of alternative splicing at intron seven of RPT5b. Schematic representation of the Col intron with the different stages of splicing depicted: formation of a lariat between the 5' splice site and the branchpoint (1) and cleavage of the 3' splice site (2) before ligation of the two exon sequences. In the Ws intron, the SNP2 variant (U, in bold character) generates a new branchpoint sequence that activates the selection of a downstream 3' splice site. Exons are represented by boxes. The yellow box corresponds to the beginning of exon 8 that is spliced out in Ws. B, Graphic representation of mis-splicing occurring at RPT5b intron 7. Primers used for the cDNA amplification and for the splicing characterisation are represented (See methods). C, Strategy used to assess the level of mis-splicing at intron 7. Normal spliced forms are amplified with primers N and e10 while the mis-spliced form is detected by amplification with primers M and e10, as exemplified in D. E, Plasmids containing both e5-e10 fragments of the normally spliced and mis-spliced form of RTP5b intron 7 were mixed up to 10 ng, as indicated below the graph. The level of mis-splicing was then assessed as represented in Fig. 3C. At least 100 cloned cDNAs were tested per genotype. Results show that our test gives a good estimate of the mis-spliced form vs total form ratio. Hence, in our conditions, there is no significant bias of PCR amplification and/or cloning of one spliced form over the other.
Previously, a test was developed to quantify the amount of RPT5b intron 7 mis-splicing among mRNA ( and Fig. 3C and 3D). Fragments of RPT5b cDNA surrounding intron 7 were cloned before analysis to make sure that the level of mis-splicing could be accurately assessed (Fig. 3E). However no significant difference could be detected in the level of RPT5b intron 7 mis-splicing between Col-0 and Ws-4 inflorescence . One explanation would be that the mis-splicing occurring only in male gametophyte cells or mother cells would be diluted by splicing occurring in surrounding tissues, making it very difficult to analyse at this stage.
For this reason we looked at young seedlings, as these represent another developmental phase where RPT5a and RPT5b unequal redundancy is detected. In the Ws-4 accession, no homozygous plants are recovered for the strong mutant allele rpt5a-1, because it is fully male gametophytic lethal. However, two weaker alleles, rpt5a-2 and rpt5a-3, were isolated that show some residual transmission through the male gametophyte  and for which some homozygous plants were recovered. Those plants, rpt5a-2/rpt5a-2 ; RPT5b Ws-4 /RPT5b Ws-4 and rpt5a-3/rpt5a-3 ; RPT5b Ws-4 /RPT5b Ws-4 , show reduced development and the seedlings exhibit short roots. This phenotype was reversed to wild-type by introgressing the rpt5a-2 (or rpt5a-3) mutation into a RPT5b Col-0 /RPT5b Col-0 background ( and Fig. 4A), proving that the dwarf phenotype resulted from unequal redundancy between RPT5a and RPT5b Ws-4 . Therefore, RPT5b intron 7 splicing was assessed on six-day old RPT5a/RPT5a, rpt5a-2/rpt5a-2 and rpt5a-3/rpt5a-3 seedlings in either RPT5b Col-0 /RPT5b Col-0 or RPT5b Ws-4 /RPT5b Ws-4 background. A very clear correlation was observed between the presence of the RPT5b Ws-4 allele and a ratio of 20 to 25% of mis-splicing among RPT5b RNA, leading to PTC (Fig. 4B).
RPT5b Ws cDNA is partially mis-spliced, regardless of RPT5a status. A, 6-day old seedlings grown on vertical agar plates from wild type RPT5a plants and rpt5a2/rpt5a2 and rpt5a3/rpt5a3 in either RPT5b Col-0 /RPT5b Col-0 or RPT5b Ws-4 /RPT5b Ws-4 background. B, Percentages of RPT5b intron 7 mis-splicing in the same plantlets as in A. At least 100 cloned cDNAs were tested per genotype.
A similar study was carried out on all 8 accessions selected above as displaying a SNP1Ws/SNP2Ws or SNP1Ws/SNP2Col haplotype (Fig. 5A). A perfect correlation was observed between the presence of SNP2Ws and a significant amount of mis-splicing at intron 7 (12 to 20%). Conversely, all 4 accessions with a SNP1Col/SNP2Col haplotype showed a very limited amount or undetectable mis-splicing (0 to 2.8%) (Fig. 5B).
Partial RPT5b mis-splicing correlates with RPT5 unequal redundancy. A, RT-PCR on mRNA isolated from 6-day old seedlings from different accessions. APT was amplified as a control. B, Percentages of RPT5b intron 7 mis-splicing in the same plantlets as above. At least 100 cloned cDNAs were tested per genotype.
This demonstrates that SNP2Ws, located upstream of the 3'intron site consensus, is very likely responsible for a significant amount of mis-splicing that causes the lack of complementation of rpt5a mutation by RPT5b alleles carrying this SNP2Ws.
In the present study, we confirm that RPT5a and RPT5b display unequal redundancy in some Arabidopsis accessions and we characterized 4 new accessions in which the situation is similar. Moreover, by investigating Arabidopsis natural variation, we show that one SNP, located in RPT5b intron seven, causes this unequal redundancy between RPT5a and RPT5b genes. The A to T substitution in position -8 to the intron 3' splice site correlates with a 25% mis-splicing rate in Ws-4 and in 4 other accessions (Bu-11, Cal-0, Da-0 and Wu-0). Sequence analyses reveal that the SNP2Ws generates a new putative branchpoint sequence that probably competes with the original branchpoint. As a result, another 3' splice site is selected downstream, resulting in a 10 bp deletion in the RPT5b mRNA. However, how this competition occurs between both branchpoint sequences, and consequently both 3' splice sites is unclear. In mammalian genes, despite the steric hindrance caused by the closeness between the branchpoint and the next downstream AG dinucleotide, AG sequences located as close as 4 nt downstream of the branchpoint would still be used at some extent . Data suggest that 3' splicing site scanning occurs in plants as in mammals [17, 21], explaining why even in the presence of SNP2Ws, the first AG remains the main 3' splicing site. In the 5 accessions carrying SNP2Ws, only a fraction of RPT5b pre-mRNA is mis-spliced (between 12 and 25%), though our assay might underestimate this percentage if some of the mis-spliced form is degraded through NMD, as it is frequently the case with mRNA displaying PTC [13, 22]. However, semi-quantitative RT-PCR shows that RPT5b mRNAs accumulate similarly in all accessions we tested (Fig. 5A). The subtle defect in RPT5b pre-mRNA splicing may also explain why transgenes expressing various RPT5b constructs all complement a rpt5a strong male gametophyte defect: the additive expression of both the transgenic and endogenous RPT5b expression is probably sufficient to overcome the lack of RPT5a by providing enough functional RPT5b.
Presumably, this SNP2, through causing a "mild" effect on splicing, is likely to have a stronger effect on RPT5b protein accumulation with deleterious consequences on both male gametophyte and sporophyte development. Indeed, F2 plants that segregate with a rpt5a-4/rpt5a-4; RPT5b Col-0 /RPT5b Bu-11 genotype displayed a short root that is typical of mutants partially affected in the proteasome machinery such as rpt2a, rpn10 and rpn12 as well as weaker alleles of rpt5a [15, 23, 24] (Data not shown). This suggests that the unequal redundancy between RPT5a and the other RPT5b alleles bearing the SNP2Ws extend to sporophyte development.
We previously pointed that while the lack of redundancy between RPT5a and RPT5b Ws-4 has a dramatic effect on male gametophyte development (leading to pollen lethality) it has no or very little effect on the female gametophyte development . Several studies point out that alternative splicing can be regulated by hormones, stresses or developmental cues (i.e. the pattern of alternative splicing can be organ-specific) [25–27]. In this regard, it is interesting to note a recently described set of mutants that impair female gametophyte development: LACHESIS, CHLOTO/GAMETOPHYTIC FACTOR1 and ATROPOS are essential for female gamete cell fate and all three genes encode spliceosomal components [28, 29]. The authors speculate that splicing regulation could be required for maturing specific gametophytic factors mRNA. Likewise, it could be that male gametophytes also have a specific spliceosome machinery that would make them more susceptible to SNP2 Ws related-alternative splicing while the female machinery would not. It will be interesting to see whether mutations affecting male-gametophyte development will be discovered by screening for more mutations of the spliceosome machinery.
The RPT5b alternatively-spliced mRNA described here leads to PTC that is supposed to induce NMD , i.e. degradation of the mRNA to prevent the translation of an incomplete protein. Although alternative splicing has been proposed as a way to generate novel proteins and therefore enhance transcriptomic/proteomic plasticity, there are no indications that this is the case here: the present alternate splicing leads to a RPT5b devoid of the conserved AAA-ATPase domains H and K [30, 31] that is likely to be a non-functional protein as is the C-term deleted RPT2a protein in the hlr-2 mutant . Besides, PTC caused by alternative splicing has been demonstrated to act as a way to regulate gene function by the RUST mechanism (Regulated Unproductive Splicing and Translation) : it triggers the production of mRNAs encoding non functional proteins and those mRNAs are the targets of NMD. However, we found that the level of RPT5b Ws-4 mis-splicing was similar in seedlings in both wild-type RPT5a background and in rpt5a-2 or rpt5a-3 mutant background (Fig. 4B), suggesting that there is no feedback on RPT5b mis-splicing based on RPT5 protein content.
Alternative splicing has been extensively assessed in Arabidopsis  showing that up to 42% of intron-containing genes are alternatively spliced. The majority of those alternate transcripts lead to PTC, suggesting it has an important role for gene expression regulation. New sequencing methods as High Throughput Sequencing [33–35] offer the opportunity to assess how alternative splicing varies between accessions and to see how it can contribute to transcriptomic/proteomic diversification among natural variation.
RPT5a and RPT5b both encode the RPT5 subunit, a component of the Regulatory Particle of the 26S proteasome, the complex that degrades ubiquitin-labelled targets as part of the Ubiquitin proteasome System (UPS) . The 26S/UPS is an essential pathway involved in development and regulatory processes in eukaryotes, including plants [37, 38]. In Arabidopsis, most of the 26S proteasome components are encoded by duplicated genes  and it is not clear whether these duplications are the basis of a mere redundancy or of a proteasome plasticity. In this regard, assessing the redundancy between RPT5a and RPT5b among natural variation in Arabidopsis thaliana brings new insights by showing that these genes offer another example of unequal redundancy  through alternative splicing in some unrelated accessions, without any apparent gain of functions. A recent study carried out in Arabidopsis shows that alternative splicing plays an important role for divergence between duplicated genes . It would be interesting to test whether other proteasome subunits display such incompatibilities and therefore determine whether some of those other duplicated genes are drifting away or not.
Arabidopsis thaliana Columbia (Col-0) and Wassilewskija (Ws-4) accessions were used as reference plants. The rpt5a-4 T-DNA insertion mutant allele has been already described . It was isolated in the SALK collection as line S046321 in Col-0 accession (SIGnAL; http://signal.salk.edu/cgi-bin/tdnaexpress). As previously, the rpt5a-4 mutant allele was genotyped with the S46321-L and LBSalk2 primers while the wild-type allele was scored with S46321-U and S46321-L. All primers are described in Additional file 2: Table S1.
The 487-accessions collection (Additional file 3: Table S2.) was provided by the Resource Centre of INRA Versailles http://dbsgap.versailles.inra.fr/vnat. All Col (Col-0 and Col-7) and Ws accessions (Ws, Ws-1, Ws-2, Ws-3 and Ws-4) were found to present the same RPT5b haplotype; therefore only Col-0 and Ws-4 was used in this study. For growth on either plates or soil, seeds were stratified for 2 days at 4°C and grown at 18 to 20°C, with 16 h light (fluorescent lights: 100 μmol photons m-2 s-1) and 8 h dark cycles. Immature flowers were emasculated and manually cross-pollinated for crosses.
Genomic DNA of the 487 accessions was extracted in 96-well plates from seedlings, using a SDS-based buffer . SNP1 and SNP2 were detected as previously reported using dCAPS markers . For the SNP1 located in the RPT5b promoter, a 266 bp fragment was amplified with primers JLV096 and JLV097 and digested with VspI. Only the fragment originating from the Ws allele was restricted into 249 and 17 bp fragments. For the SNP2, located in the RPT5b intron seven, a 280 bp fragment was amplified with primers RPT5b-3369 and RPT5b-Hind3 and digested with HindIII. Only the fragment originating from the Col allele was restricted into 250 and 30 bp fragments.
Pollen viability was assessed using Alexander staining  and observed with a PL Fluotar X25 dry objective on a Leitz Diaplan microscope.
Total RNA was extracted using TRIZOL-reagent (Invitrogen) from 100 mg of tissue (6 day-old plantlets). Contaminating DNA was removed by DnaseI treatment with RNase Free DNase set (Qiagen) using spin columns of the Rneasy Plant mini kit (Qiagen).
RT-PCR was performed with RevertAid H Minus M-MuLV Reverse Transcriptase (Fermentas) on 500 ng RNA according to the supplier's instructions. RPT5b and APT cDNAs were amplified with specific primers: e5 and e10 for RPT5b, APT5' and APT3' for APT.
RPT5b e5-e10 cDNA fragments were amplified by 25 cycles of PCR with e5 and e10 primers. The PCR products were then cloned into pTOPOII plasmid (Invitrogen) and the resulting constructs transformed into DH10B competent cells. A hundred recombinant clones were analysed by PCR using N and e10 primers for normal cDNA detection and M and e10 primers for mis-spliced cDNA.
We thank Christine Camilleri for her very helpful advice on both the project and the manuscript. We are also very thankful to Raphaël Mercier for his advice that helped to improve the manuscript and to Matthieu Simon for his help with the accessions and the use of MSAT markers. Finally, we thank Denis Headon (Roslin Institute, Edinburgh, UK) for editing the manuscript.
AGD and JLG designed the experiments. AGD, AL and JLG carried out the experiments. AGD and JLG wrote the manuscript. SB and PG participated to the writing. All authors read and approved the final manuscript.


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Sex Trafficking and Social Media
Da: Digital Distraction
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Join Dr. Lisa Day, Kristin Sunanta Walker and John W. Whitehead discussing social media and how it has been used to help make sex trafficking a billion dollar industry.
John Whitehead is an attorney and author who has written, debated and practiced widely in the area of constitutional law, human rights and popular culture.
Widely recognized as one of the nation's most vocal and involved civil liberties attorneys, Whitehead's approach to civil liberties issues has earned him numerous accolades ... Maggiori informazioni and accomplishments, including the Hungarian Medal of Freedom and the Milner S. Ball Lifetime Achievement Award for "[his] decades of difficult and important work, as well as [his] impeccable integrity in defending civil liberties for all." As nationally syndicated columnist Nat Hentoff observed about Whitehead: "John Whitehead is not only one of the nation's most consistent and persistent civil libertarians. He is also a remarkably perceptive illustrator of our popular culture, its insights and dangers. I often believe that John Whitehead is channeling the principles of James Madison, who would be very proud of him."
Whitehead's concern for the persecuted and oppressed led him, in 1982, to establish The Rutherford Institute, a nonprofit civil liberties and human rights organization located in Charlottesville, Virginia. Deeply committed to protecting the constitutional freedoms of every American and the integral human rights of all people, The Rutherford Institute has emerged as a prominent leader in the national dialogue on civil liberties and human rights and a formidable champion of the Constitution. Whitehead serves as the Institute's president and spokesperson.
Whitehead has filed numerous amicus briefs before the U.S. Supreme Court, has been co-counsel in several landmark Supreme Court cases and continues to champion the freedoms enshrined in the Bill of Rights in and out of the courts. His law review articles have been published in Emory Law Journal, Pepperdine Law Review, Harvard Journal on Legislation, Washington and Lee Law Review, Cumberland Law Review, Tulsa Law Journal and the Temple University Civil Rights Law Review.Whitehead is also a member of various groups that seek nonpartisan consensus solutions to difficult legal and constitutional issues through scholarship, activism and public education efforts.
John Whitehead is a frequent commentator on a variety of legal and cultural issues in the national media and writes a weekly opinion column, which is distributed nationwide. He has authored more than 30 books on various legal and social issues. His most recent books include the best-selling Battlefield America: The War on the American People and the award-winning A Government of Wolves: The Emerging American Police State. In addition, he wrote and directed the documentary video series Grasping for the Wind, as well as its companion book, which focus on key cultural events of the 20th Century. The series received two Silver World Medals at the New York Film and Video Festival.
Born in 1946, John W. Whitehead earned a Bachelor of Arts degree from the University of Arkansas in 1969 and a Juris Doctorate degree from the University of Arkansas School of Law in 1974. He served as an officer in the United States Army from 1969 to 1971.
www.rutherford.org

redpajama_set_name: RedPajamaCommonCrawl


text:
Atholus gestroi är en skalbaggsart som först beskrevs av Schmidt 1897.  Atholus gestroi ingår i släktet Atholus och familjen stumpbaggar. Inga underarter finns listade i Catalogue of Life.

Källor

Stumpbaggar
gestroi

redpajama_set_name: RedPajamaWikipedia


I really like Mountain Lion, but the "Check spelling while typing" feature has been tempting me to move back to Linux recently. It's bad enough that there is no way to turn it off globally. But even more annoying is that it seems to turn itself back on after being disabled in an application.
Anyway, I think I finally found a solution. This seems to disable it for good.


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Why Russians Are Going to Prison for Memes (Op-ed)
Eventually people will start asking why they are going to prison for memes while corrupt officials receive suspended sentences.
By	Andrei Pertsev
Andrei Lyubimov / Moskva News Agency
Over the past few weeks, the Russian authorities' campaign against extremism has escalated to a new level. Law enforcement agencies have opened numerous extremism cases against not only opposition activists but also ordinary citizens. The case of eighteen-year-old Anna Pavlikova may have received the most coverage—she was a minor at the time of her arrest, and the Moscow City Court approved her detainment despite her age and health problems. After public protests, Pavlikova and nineteen-year-old Maria Dubovik were placed under house arrest, but they and several other young men, who remain in prison, are still charged with creating an extremist movement called New Greatness.
No one actually denies that, technically, the security services are behind this organization. A Federal Security Service agent joined a Telegram chat room involving some young people and proposed that its members meet offline. He rented space where they could meet and offered to write a charter for their group. Ordinary people who had criticized the government on social media platforms were manipulated into meeting in person and adopting a charter composed by agents provocateurs, and promptly arrested. The system has been set in motion, and it is becoming clear to all Russians that any one of them, or their children, or any person who dares to criticize the government, could end up in the shoes of the New Greatness defendants.
How a Teenage Girl's Chat Group Became an FSB Snare
The New Greatness case is hardly unique. Several criminal cases have been opened in various parts of Russia against people who shared memes on Vkontakte, Russia's largest social media platform. One of them, Maria Motuznaya in Altai Krai, was accused of offending the religious and inciting racial hatred. One offensive image depicted Jesus asking Patriarch Kirill of the Russian Orthodox Church what time it was, a reference to a scandal surrounding a luxury watch Kirill sported in a photo. And in Tuva, Oyumaa Dongak, an activist, was arrested for something even more innocuous: reposting a historical article about Germany that happened to contain a photo with a swastika.
The memes at the center of these extremism cases have been unremarkable, the kind shared by almost anyone who is active on social media platforms. Some of those arrested had never attended rallies and were not particularly involved in, or vocal about, politics. They were simply ordinary citizens of various ages and professions.
The strongest impression from these cases, and a mistaken one, is that the government has deliberately and demonstratively escalated repression to a new level. Previously, opposition activists—people who rallied in the streets and tried to create political parties—were the main enemy. Now, perfectly upstanding citizens appear to be fair game. People are going to prison for jokes, as in the Soviet years. Their jailers are making an example of them, torturing them in detention facilities whatever their age or ailments. Russia's repressive machine is running smoothly, and the regime is showing it off, the argument goes.
This idea presumes that the power vertical is exceedingly monolithic and well thought-out, that the system does not make mistakes, that it operates without any hitches, and that it has a decisionmaking center that devises new rules and enforces them effectively. Many in Russia are mesmerized by the ostensible power, shrewdness, and dark might of the Kremlin. However, the numerous extremism cases opened against those who have liked or shared memes point to the contrary: Russia's system of power is unmanageable and unable to predict the potential consequences of its own decisions.
The cases against Russia's so-called extremists do not represent a new Great Terror. They are a system error and an unforeseen result of poorly thought-out decisions. Laws on extremism—in particular, the law protecting the feelings of the religious, passed in response to Pussy Riot's 2012 protest performance at the Cathedral of Christ the Savior—were intended to be a precision instrument for intimidating specific "malcontents" in Russia. They were meant to be applied infrequently and selectively.
However, Russia's law enforcement system is not well suited to precision instruments. Law enforcement officers are supposed to be "productive" in solving crimes and opening criminal cases. If an article exists in Russia's Criminal Code, then cases must be opened under it. Otherwise, why have it there at all? Finding people to prosecute is not a problem. Furthermore, one need not expend much energy to "uncover" extremism. Browse several pages of someone's social media account and you will find plenty that constitutes extremism in the eyes of the law and under the eminently vague articles of Russia's Criminal Code, which could be applied to almost any satire.
Law enforcement agencies have found the struggle against so-called extremism an excellent way to earn points for having solved "serious" crimes. The cruel treatment of Pavlikova only appears to be a deliberate expression of exceptional sadism, but this is just the way that the siloviki operate; they cannot simply release a person charged with extremist from jail.
A 'Mothers' March' in Moscow for Teenage Girls Charged With Extremism
The Kremlin's initiative failed upon contact with the system it created. Its failure is bearing real-life consequences. The government had not planned to punish ordinary citizens and to keep them in constant fear. On the contrary, President Vladimir Putin's regime has always tried to draw a clear distinction between opposition activists and ordinary citizens. The Bolotnaya Square case demonstrated to ordinary citizens that the regime would shut down any activism and it showed exactly what people were not supposed to do: take to the streets and issue demands.
Relevant legislation was passed: laws protecting the feelings of the religious and introducing stricter punishments for participation in unsanctioned protests. Still, the Kremlin tried to signal to society that if you protest or join an opposition movement, you are an activist; if you sit at home, you are an ordinary citizen, even if you criticize the regime a bit.
Sharing memes did not turn ordinary citizens into opposition activists, and it has yet to do so. However, now it is the siloviki who are turning ordinary citizens into opposition activists. Because of this mistake, ordinary citizens are leaving their comfort zones. They see that they and their children can become victims, that the laws are unfair, and that the law enforcement agencies are using these laws in their own interest. Inevitably, a question arises: why are people going to prison for memes while corrupt officials receive suspended sentences? As more and more people pose it, the Kremlin has only itself to blame.
Andrey Pertsev is a journalist with the Kommersant newspaper and a regular contributor to The Carnegie Center Moscow, where this piece was originally published. The views and opinions expressed in opinion pieces do not necessarily reflect the position of The Moscow Times.
The views expressed in opinion pieces do not necessarily reflect the position of The Moscow Times.
Andrei Pertsev
Andrei Pertsev is a political commentator at Cargenie Moscow.
Read more about: Internet
Will Russia Stop Arresting Its Citizens For Posting Memes?
After Putin proposes softening an extremism law, skeptics say the liberalization is a facade.
New Terrorism Suspect Held After Russian Math Lecturer's Acquittal
Math teacher Dmitry Bogatov was acquitted last week of charges that he called for riots on Red Square.
Russia's Tor Node Administrator Acquitted of Terrorism Charges
Bogatov was charged with inciting terrorism for alleged online calls for riots on Moscow's Red Square.
Russian State Censor Blocks Tools for Bypassing Telegram Ban
The virtual private networks and Internet-address anonymizers weren't specified.


For generations, the Grand Marais Art Colony has nurtured a vibrant artistic community. Whether you have experienced the Art Colony as a student seeking creative guidance, as an artist working in our studios, or as an art aficionado appreciating an exhibit, you know this historic organization is a unique sanctuary that brings art to life.
If you appreciate this community nurtured by the Art Colony, we invite you to support us as a Member. Members are the foundation of the Art Colony that keep the mission thriving while providing you unique benefits for individuals and businesses. Help make the mission come alive!


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our work in Curative therapeutics
The need for systems level intelligence, in combination with artificial intelligence.
A working thesis on how, as an industry, we can design more effective personalised therapies that significantly extend healthy lifespan.
The current rate of therapeutic failure is unsustainable and unnecessary.
The reasons are well understood, from poor models to lack of specificity, but often simply put down to "science is hard".
In reality, the knowledge and toolsets are available, the problem remains due to entrenched biases at every stage, from publishing papers to clinician marketing.
The emergence of personalised and precision therapeutics creates the opportunity to address these entrenched and repeated failures.
This includes leveraging computation in combination with human ingenuity to unpick complex dynamic systems, move the computation into the therapeutic itself, address root causes directly and drive the creation of better models and markers.
Areas for venture creation:
We've launched ConcR and ImmTune, and have partnered with Cancer Research UK to continue building new ventures in this space
Neurodegeneration
We've launched Reflection Therapeutics
Microbiome and AMR
We've launched CC Bio and Ancilia (stealth)
Considering the systems level, from biology to payer
At DSV we aim to get back to first principles and understand the root cause and bottlenecks that have led to past failures. Not just failures in the science but perhaps even more importantly, to discover the gaps and biases in the innovation pipeline. Like many industries the drug development process is optimised for short term wins regardless of long term outcomes, due to fragmentation even within the same organisation. This has led to the current situation, in which for example, only 10% of drugs succeed at phase 3 clinical trials and even after that, only work in less than a third of patients. Everyone knows that current outcomes aren't good enough, and aren't simply a fact of the science being 'hard'. Yet, it is almost impossible to perturb a system with such strong incentives to get the paper out, pass the trial, get the deal done and persuade the doctor.
Targeting uniformity in a complex dynamic system
There are only a small number of diseases in which intervention at a single point, common across all patients, can sufficiently and persistently alter the system towards a healthy state. In these cases medicinal chemistry and antibodies have, in many cases, been extremely effective. However, most diseases consist of a complex set of dynamic failures from genetics and epigenetics to neural activity: these differ across patients, within patients and over time. In these cases, incorrectly perturbing the system can either achieve net zero or throw it into a dangerous runaway state. The complete lack of therapeutic outcomes from the isolated study of genetics is a sobering reminder of this challenge.
Unrepresentative models
Trials often fail because the model that was used to demonstrate effectiveness in preclinical represents the end point, but not the underlying cause. This is becoming all the more evident as medicines move towards modifying complex systems such as the immune system, microbiome and nervous system, all of which share many of the same components as mice, but which behave in a vastly different way at the systems level. This drives more hacks like testing microbiome and immune products in germ free mice, where germs are clearly essential to defining the function of the system. Perhaps the most depressing example of this is the seemingly endless series of failures in Alzheimer's drugs that are derived from models which over-express Amyloid rather than accurately represent the disease.
The lack of appropriate biomarkers
The problem is that we all know that they often have no real relation to the primary endpoints and huge variability across disease and patient. However, there is little incentive to fix this at the local level. Trials evaluating new biomarkers appear more likely to fail, not to mention that stratification makes it far harder to find sufficient numbers of patients. This is very clearly the challenge in fields such as neurodegeneration, psychiatric diseases and longevity, but is also one of the key reasons behind failures in many of the most hyped fields at the moment including immuno-oncology.
Hacking rather than addressing specificity
Targets are rarely unique to the system of interest and rarely respond in a simple on / off fashion, but rather along a distribution. Indeed, the number of potential allosteric molecular interactions are in the hundreds: even for approved drugs. Likewise, antibodies bind all over the place and the current animal production method severely limits the options for avoiding this. In combination, this makes it extremely difficult to find the correct dosing that perturbs the system of interest but not others.
Forge a less linear R&D process identifying analytical and model approaches that fully capture complexity and patient specificity.
Forge a truly multi-stakeholder creation process with academics, venture capital, charities and industry engaged in the design process from day one.
Take a wide, international and interdisciplinary perspective, focused on proving out arrays of potential components to successfully modify a system, rather than aiming to prove the usefulness of a given discovery.
Always aim to get as close to the root cause as current understanding and toolsets allow
Effectively leveraging computational approaches to address complexity
Simply applying deep learning to look for correlations rarely leads to meaningful learning in all but the most simple and fully characterised environments. Not least where such correlations are based on published data with at least 30% being unreproducible. The magic comes in combination, starting with the most constrained and fully measurable systems and using computation to pick apart the highly interdependent and dynamic systems whilst using human ingenuity to direct this in meaningful ways. Leading on our work in tackling heterogeneity is ConcR's work in mapping the evolutionary path of a given patient's tumor resistance. They have already shown over 87% accuracy in predicting the most effective treatment regime, something that has the potential to prevent over a third of deaths from cancer.
Simply applying deep learning to look for correlations rarely leads to meaningful learning in all but the most simple and fully characterised environments.
Therapeutics that can compute invivo
There's something strange about taking a snapshot of a dynamic system, averaging it across a heterogeneous population and using this to design an intervention, when the system itself contains all the necessary analysis and computational ability. Whether its a bacterial quorum deciding when critical mass is achieved, or the immune system mounting an attack, a biological system is using some set of inputs and dynamic models to decide when and how to make a therapeutic intervention. We intend to drive the change towards conducting analysis and differential therapy directly within the patient. Already within the portfolio this includes conditional dampening of inflammation in the brain with Reflection Therapeutics and equipping bacteria with the ability to defend against specific phage in IBD led by Ancilia.
Focus on better models and markers
The technology is becoming available to create much more realistic systems-level in vitro and in silico models. This will emerge from a combination of rapidly advancing analytics (eg. single cell sequencing, proteomics and imaging) combined with stem-cell modification, genetic editing and simulation. The challenge is that there's very little money, yet extreme competition, in commercialising models and testing as a service. It's therefore up to biotech companies to have the discipline to build their own tools, and acquirers then to have the confidence to bet outside of the well-accepted paradigms. Note: markers in early diagnostics is covered in our Healthcare Systems thesis.
Getting back to the root cause
Even if cancer was eradicated, the average lifespan would only be extended by 2 years, as people die of heart disease instead. This makes you wonder; why do we spend so much resource addressing the downstream representation of disease when there are common upstream causes which are universally driven by time dependent damage. As such, across every disease our approach is to look for opportunities to address all of the upstream factors in concort, not just one or two at a time. This includes fixing and buffering molecular level damage, fixing broken or unhelpful messaging and signalling pathways, correcting errors at every level of gene expression and modifying the state of cells to drive regeneration.
Even if cancer was eradicated, the average lifespan would only be extended by 2 years, as people die of heart disease instead.
Bringing it all together
This combination of a more joined up approach across the financial and marketing stack, an intellectually honest approach with respect to the chances of recreating results in patients and a step towards far smarter, more specific therapeutics, will finally put us firmly on the road to curing, not patching up, becoming the norm.
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Portal of the STF
Brasília, July 18th, 2019 - 18:26
Compared Case Law
Federal Supreme Court Activity Report - 2017
Agenda of Events 2015 *
Events 2014 *
Teixeira de Freitas legal and academic exchange program within Mercosur and Associate States
The Teixeira de Freitas Exchange Program encourages cooperation towards the creation of a regional legal and cultural dialogue between the countries of Mercosur and Associates.
Its intention is to build and expand a common understanding to help strengthen the economic integration efforts and to promote training of teaching staff and students, as well as to stimulate the development of research and extension in the legal field within Mercosur and associate States.
Cooperation in the academic level is intended to promote mutual understanding of the legal realities of Mercosur countries and to support the development of legal research and to facilitate the creation of a space for reflection on issues which are central to the strengthening of the regional legal sphere.
To this end, the mobility of undergraduate and postgraduate students, teachers and researchers in the field of law among countries in the region becomes reality through the Teixeira de Freitas Exchange Program, which usually provides eight openings for students from MERCOSUR countries a semester, for the academic period of August 15 until December 2. In addition to attending morning courses in law school at the University of Brasilia - UnB, exchange students follow an internship in the Supreme Court in the afternoon.
Joaquim Nabuco
The Joaquim Nabuco Exchange Program is an incentive for cooperation within MERCOSUR countries towards the creation of a regional dialogue on legal issues among the Member- and Associate-States.
This Exchange Program aims at making the Brazilian judiciary in all its instances known to visiting judges and judicial servants, thus contributing to the expansion of the basis for judicial cooperation, the strengthening of MERCOSUR and the promotion of legal security in the region, as a factor of political stability and economic and social development. Planned activities include assistance to hearings and sessions of trial courts, exposure to the functioning of national justice, monitoring of the routine work of judges of both the first and second instance, legal education in local universities, among others things. In the case of servants, they also get to know the administrative structure of the Federal Supreme Court of Brazil (STF) and they go through a period of internship in one of the Court's Departments.
The Supreme Court (STF) provides twice a year,eight openings for judges and judicial servants of MERCOSUR Member- and Associate-States. In addition, the Supreme Court also sends its servants to know the functioning of the neighboring courts. There, the work schedule is determined according to the particularities of each judiciary system and court in particular. Each semester a selection process is conducted to determine the eight servers that will have the privilege to know on site how works the Judiciary of the countries participating in the program - currently Chile, Paraguay, Uruguay, Bolivia and Ecuador.
Praça dos Três Poderes - Brasília - DF - Brazil - Zip Code 70175-900 Phone: 55.61.3217.3000


When your ship docks in the Cadiz port, head out on a shore excursion to Seville, one of the most remarkable cities in Andalusia (Andalucia). On this small-group tour (limited to 15 passengers) you'll follow your local guide around Seville, a city full of history and beautiful Moorish architecture. Explore famous sites like the Cathedral of Seville, the largest Gothic cathedral in the world and a UNESCO World Heritage Site; Plaza de Toros de la Real Maestranza de Caballería (commonly known as La Maestranza), the oldest bullring in Spain; and the Alcazar, a former Moorish fortress. This is a wonderful day trip for history buffs and architecture-lovers!
While you are in port in Cadiz, discover the capital of Andalucia on a shore excursion to Seville. Meet your local guide in front of the Cadiz port and relax on a comfortable minibus during the 60-minute drive to Seville. In Seville, start your small-group walking tour with a visit to the Macarena Walls, and then head to Plaza de España, a jewel of Seville and one of its prettiest plazas. Next, walk down to Maria Luisa Park and admire the mix of 1920s Art Deco and Moorish designs.Continue to the Baroque palace of San Termo, home to wonderful gardens. Then visit the Cathedral of Seville, the largest Gothic cathedral and third-largest of any church in the world. As you walk around this UNESCO World Heritage Site, visit the tomb of Christopher Columbus, the Capilla Real and the Patio de los Naranjos. Enjoy views of the breathtaking Giralda tower, once the tallest tower in the world and today one of only three remaining Almohad minarets (a mosque spire from the 12th-century Almohad Dynasty). Take a short break for lunch (own expense) in the Jewish quarter and wander around the labyrinth of narrow streets, originally designed to protect Jews from oppression. Continue to the Santa Cruz quarter, one of Seville's best-known neighborhoods with big mansions, family palaces and flower-covered patios. Then visit the Alcazar, a royal palace that was originally a Moorish fort. End your walking tour at the oldest bullring in Spain, La Maestranza. Enjoy free time in this area before you make your way back to the Cadiz port.Worry-free Shore Excursion:We will ensure your timely return to the Cadiz port for this activity. In the rare event your ship has departed, we will arrange for transportation to the next port-of-call. If your ship is delayed and you are unable to attend this activity, your money will be refunded. See our terms and conditions for full details.


"We have found the staff at Fridaysmove to be extremely professional and helpful from the initial telephone call to the final email congratulating us on our move. When we experienced a problem just before exchanging contracts, Andy Boast swiftly intervened which allowed us to complete on time. We cannot thank you enough for all that you did - you certainly went beyond the call of duty."
Thank you for your kind words Mrs Prout. We were delighted to be able to help and wish you the very best in your new home.


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Evidence of expansion of Azzopardi Fisheries' tuna cages
Aerial shots reveal Azzopardi Fisheries have 21 tuna cages at Sikka l-Bajda defying permitted allocation
by Caroline Muscat February 9, 2018, 9:45 am
Some of the cages at St Paul's Bay (full picture below).
Aerial shots submitted to The Shift News confirm that Azzopardi Fisheries has added tuna cages beyond its allocation, despite its application to add "another 12 cages" not yet approved.
AJD Tuna-a company, owned by Azzopardi Fisheries, asked for permission in December to extend the existing tuna penning farm "by placing another 12 tuna cages" off Sikka l-Bajda in St Paul's Bay.
The application was presented before the identification of a permanent site for aquaculture establishments in the north.
Cages in Comino and St Paul's Bay were relocated to the temporary site off Sikka l-Bajda – five km from the coast – last year in the wake of a report linking sea sludge on the coastline to tuna penning operations.
The temporary site for the tuna cages was approved in a marine protection area in the vicinity of protected bird colonies. Birdlife had disputed the legality of the permit issued in the absence of environmental impact assessments.
But the relocation to Sikka l-Bajda, supported by both the government and the opposition's representatives on the Planning Authority's board, ensured the continuation of tuna penning operations after the PA had revoked previous permits in 2016. The legality of the revocation was questioned by the operators.
The latest application for 12 new cages presented by Azzopardi in December is still at a preliminary stage and is still being screened by the PA before being issued for public consultation. Yet, new cages have already been placed.
The company argues that these cages do not contain fish, or the biomass of the fish is retained and spread among the cages. Yet, this is an issue about permits. It is the equivalent of building a room Outside Development Zones and then requesting the Planning Authority to sanction it.
In October, the PA had approved an application by Malta Mariculture Ltd – a subsidiary of Azzopardi Fisheries – to relocate eight of its tuna pens previously operating from St Paul's Bay to the new site offshore from Sikka l-Bajda. On that occasion the number of cages was reduced to six but the same volume of tuna was to be retained.
Previously the PA had approved an application by AJD Tuna to relocate six cages from Comino to the new Sikka l-Bajda site.
If approved the new application will increase the number of cages in Sikka l-Bajda from 12 to 24. There are 21 in place at the moment.
Next week the PA board will be deciding whether to confirm the imposition of a € 30,000 contribution to a fund earmarked for environmental projects in St Paul's Bay, which is being contested by Malta Mariculture Ltd.
Azzopardi Fisheries recently contested the imposition of a €30,000 contribution to a fund earmarked for environmental projects in St Paul's Bay. The fee was imposed as a condition for the temporary relocation of the company's cages in St Paul's Bay to Sikka l-Bajda following the sea sludge controversy.
Aerial shot showing Azzopardi Fisheries' 21 cages.
Previous article PD: 'Create wealth without destroying environment'
Next article BREAKING: Pharmacists on strike
Caught in the act: Yet another cage headed to Azzopardi Fisheries' tuna penning site
by Caroline Muscat February 14, 2018, 9:14 am
An allergy to responsibility
by Ryan Murdock May 21, 2019, 9:12 am
The tuna kingpin: No public accounts for 18 years
by Caroline Muscat October 5, 2018, 12:15 pm
Petition objecting to increase of tuna pens in St.Paul's Bay launched
by Jurgen Balzan March 9, 2018, 2:51 pm
PD: 'Create wealth without destroying environment'
BREAKING: Pharmacists on strike


The hound dog was lonely for three long years.
Henry the hound dog had been lonely for the three long years he's lived at Animal Ark Rescue, a no-kill shelter in Columbus, Georgia. So he did what anyone else looking for love does in the modern age. He set up a Tinder account.
With the help of Animal Ark staff member, Miranda Morrison, Henry listed his occupation as Doggo at the Good Boy Corporation, his education at Obedience School, and his hobbies as car rides, swimming, and snuggling on the couch.
Henry, along with being hilarious, is up on all of the dating lingo.
His one flaw, according to Morrison? That he sometimes barks at small children. And do they deserve it, we wonder?
After a month of searching, we're happy to report – Henry found a dad! His name is Alex, and he heard about Henry's quest for love on Sirius XM radio. (Henry's Tinder profile has made him a national celebrity.) Alex was so moved by the hound dog's story that he drove 11 hours from Virginia to pick him up. It's true — love knows no state lines. We're thrilled for Henry, and his new family.


In exquisite detail, Arvid introduces us to a whole new world of wine. Evoking the ethereal beauty of the Willamette Valley, the contrast of soft blues and golden browns echoes the rolling vistas of Oregon vineyards. With three great bottles to share, reminiscing about a great trip is the best part of the evening . . . "O What a Night" indeed!


Spot Rocker, Gates belt drive, Rohloff Speedhub, BBSHD, 52v 17.5 AH.
I took the bike apart and had the frame reinforced (where it was cracked) the dent taken out, sand blasted and re powder coated. Spot sent me new decals at no charge.
I knew beforehand that I would need to do some modifications because the belt drive requires the belt to be in perfect alignment ( + or - 1 mm if possible).
The rear cog on a Rohloff hub is 54.7 mm from the center-line of the frame.
After inserting the motor into the bottom bracket I found that the gear housing on the BBSHD hit the chain stay so that the motor would not sit all the way into the bottom bracket So I needed to add spacers provided in the Luna kit. Thank you Luna!!
Because of the spacers and the thickness of the gear housing, the front sprocket sat way too far out for a perfect belt alignment with the rear cog. It was 67.53mm, so I needed to come in 12.83mm to achieve my goal of 54.7mm.
But the back of the sprocket was already only 11.11mm away from the chain stay. Remember, I needed 12.83mm, what will I do?
I hated to, but the only thing I could do is add an extra dent into my chain stay. I had a hydraulic crimping tool I purchased from Harbor Freight a few years ago and it worked great to press a extra dent into my chain stay.
I had an adapter machined so that a 55 tooth Gates front sprocket could be attached to it. The machinist did a stellar job.
52v 17.5 ah battery pack installation.
My bottle bracket mount was too low, so I drilled new holes into the battery bracket to line up with the existing mount, & installed riv nuts into my frame further above & below the existing mount.
The battery pack feels very solid on the frame now.
Last edited by Buckwheat; 03-25-2017, 03:36 PM.
I installed a Racktime Add-it rack. It works perfectly with my Racktime panniers. Racktime has what they call a "click-it" system where the panniers just slide onto the rack and click into place. They remove by just lifting up the green button and sliding the panniers back 1/2". They are very well made & convenient to use.
I did not want to always lean my bike against something when I wasn't using it, so I installed a kickstand.
The last thing I would like to do is have the sprocket adapter anodized black.
I also want to program the motor different so that it feels more like riding a bike rather than a moped. It feels like the motor just switches to on (full bore) when I pedal. I would like it to be a more gradual ramp up like a variable speed drill. I do have the programming cable and have played around with the programming a little bit, but I don't know what to do to change to make it behave like I want. Any suggestions?
For programing set start current to 1 and slow start mode to 3 for pedal assit, and start current to1 for throttle handle.
Thanks for the suggestion oledave, I will have to try those settings.
I had start current at 10 and slow start mode at 6 for pedal assist. I had start current at 10 for the throttle.
Very nice! Great motor clearance also. I rode my Rohloff equipped BBSHD Montague folder 13 miles the other day, 18 mph average speed, all pavement, on the MINI battery. My voltage was at 52+ upon reaching my destination! As I can easily carry my other 2 11.5 AH batteries also, (one on the rear rack, one frame hung) my range is ....adequate, to say the least! Plus, thanks to the gear spread, it's also a rock crawler/stump puller. Good work on snapping that EBAY find up.
Yes the gear range on the Rohloff hub is truly amazing, and it is built like a tank.
This last weekend I played around with programming the pedal assist (almost all day Sunday) and I am really happy with how the motor responds now. It almost feels like it has a torque sensor in it which is exactly the way I want the motor to respond when pedaling.
My wife & I have owned "off the shelf" torque sensing, under powered e-bikes for a couple of years, and I have always liked the torque sensing aspect of the bikes.
If anyone would be interested I would be happy to publish my settings.
Great work! I'd be quite interested in having a look at your settings.
After I installed my motor I programmed it according to Karl Gesslein's article "A hacker's guide to programming the BBSO2 & BBSHD". I took my bike out for a ride and I was disappointed because when I began to pedal it was like I flipped on a switch, and the bike would just take off. I knew that the problem was in the programming so I spent a better part of a day changing settings,and by trial & error I got the motor to react the way I want, kind of like it had a torque sensor in it. I will try to explain how these settings worked for me.
As you know, a torque sensor works by being sensitive to the pressure we put on the pedals. The more pressure we put on them, the faster the motor will go up to the point of the current set by the assist level we are in and the speed of the motor set at that particular assist level.
In order to have the same sensation, I needed to adjust the current and speed limit of the 5 assist levels I am using (levels 1,3,5,7,&9) so that I can always feel the pressure I am putting on the pedals (unless going downhill or coasting) for the speed limit I want to go. I have set my display to 5 assist levels, and the maximum speed to 60 Km/h which is about 39 Mph. I did hit 40 Mph by the way, It was a little faster than I want to go. Of course all of these settings work hand in hand with the gear we choose to be in. I have 14 choices with the Rohloff hub.
I am sure these are not optimal settings yet, but they are very close.
start current: I tried different settings here, but I found that this was the best setting to get the bike going initially.
slow start mode: This controls how quickly the start current is reached. When I set this lower, it felt like the bike jumped out too quickly.
Crank trigger pulses: This sets how many pulses from the pedal sensor are needed before the motor starts.A full pedal revolution generates 24 pulses. This is a little bit like the pressure needed on a pedal that has a torque sensor installed, a larger number would represent more pressure required.
Current Decay: This sets how fast the current drops after you reach the maximum speed set by the assist level. A lower value means the current will start to drop at a lower speed.
All the above settings are important to make sure a little pedal pressure is always felt.
I really like using the pedal assist best. I have only used the throttle a couple of times.
If you try this please let me know how you like it and if you would like to make any changes and why.
I know that the gearing would make a big difference on different bikes.
Hello Buckwheat. Tell me please, where can i to get the adapter? I need to make a similar adapter for BBSHD. I have Bullitt LvsH. I want to install Alfine Di2 with a belt. Belt line 40 mm. Front sprocket 55 "
Hello Depp, I had the adapter custom machined for my specific needs. It sounds like you may need a deeper offset depending on how far out from the frame center-line your front sprocket sits after your motor is installed into your bottom bracket. 40mm sounds very close to the frame center to me. My rear cog was 54.7mm from the frame center line.
The machinist I went to is in Colorado. It would be best for you to find a machinist in your area to make one for you. Be sure to make careful measurements and take him a lot of pictures (you can use my pictures) so he will understand exactly what you need. Good luck, I hope this helps.
I can give you the name & address of my machinist if you would want to go out of your area.
It looks like the center of the Gates sprocket was machined out and bolt holes were drilled and tapped. Is that accurate?
Just found your build. Wonderful! Great to see someone squeeze a BBSHD belt drive onto a frame pretty cleanly. Nice to know the Rohloff has such a large 54.7mm chain line too. I didn't know it was that far out there. That's larger than most other IGH chain line numbers. Nice job.
Thanks wiGGles, yes you are right. the 55 tooth gates sprocket was machined out in the center, and holes were drilled and tapped for the bolts.
Thanks for your kind words Rider, You have a very nice build yourself.
Yes, when I started my build I was hoping the belt line would have been closer to my rear cog, but the wide chain stay wouldn't let the motor sit close enough without adding spacers. Other than the added cost of having the adapter made, I am real happy with it. I have since had the adapter anodized black.
New pic! That machined spider is a work of art in my book.
Nice build Buckwheat. I tried this exact build except I was going to go with a NuVinci. Gave up and went cheap figuring I would find the solution some day. Well that was a long time ago. So glad you nailed it. How much did the machining cost? Its beautiful. Maybe post the shop where you had it machined. I might buy one to throw in my bottom drawer until I can get the motivation.


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Das Kompostwerk Lobau in Wien-Donaustadt ist die größte Kompostieranlage für getrennt gesammelte biogene Abfälle in Österreich und – zumindest 1993 – die größte derartige Anlage Europas. Gemeinsam mit dem Rinterzelt (Abfallbehandlungsanlage) und der Deponie Rautenweg ist es eine von drei Abfallbehandlungsanlagen in der Donaustadt.

Rechtliche Grundlage
Am 1. Januar 1995 trat eine Verordnung des Bundesministers für Umwelt, Jugend und Familie in Kraft, der zufolge biogene Stoffe, die nicht vom Verursacher selbst verwertet werden, einer getrennten Sammlung bereitzustellen oder zu einer dafür vorgesehenen Sammelstelle zu bringen sind. (BGBl. Nr. 68/1992 idF BGBl. Nr. 456/1994)

Das Wiener Abfallwirtschaftsgesetz (Wr. AWG) (LGBl. Nr. 13/1994 idgF) sieht die stoffliche Verwertung von nicht vermeidbaren Abfällen vor,
 soweit dies ökologisch vorteilhaft und technisch möglich ist,
 die dabei entstehenden Mehrkosten im Vergleich zu anderen Verfahren der Abfallbehandlung nicht außer jedem Verhältnis stehen und
 ein Markt für die gewonnenen Stoffe vorhanden ist oder geschaffen werden kann.

Biogene Abfälle sind je nach Beschaffenheit stofflich oder energetisch zu verwerten.

Die Bereitstellung und Entleerung von Sammelbehältern für die getrennte Sammlung, die Führung der Abfallbehandlungsanlagen sowie Planung und Errichtung von Anlagen zur Verwertung von wieder verwertbaren Stoffen, einschließlich der Kompostierung sind laut Geschäftseinteilung der Stadt Wien Aufgaben der MA 48 – Abfallwirtschaft, Straßenreinigung und Fuhrpark.

Seit dem 1. September 2001 regelt die Kompostverordnung (BGBl. II Nr. 292/2001) die Qualitätsanforderungen an Komposte aus Abfällen, die Art und die Herkunft der Ausgangsmaterialien, die Kennzeichnung und das In-Verkehr-Bringen sowie das Ende der Abfalleigenschaft von Komposten aus Abfällen. Festgelegt wurden drei Qualitätsklassen, die anhand von Anforderungen an Ausgangsmaterialien und an Schwermetallgehalten eingeteilt wurden.

In regelmäßigen Abständen hat der Komposthersteller eine Qualitätskontrolle durch eine externe und dazu befugte Fachperson oder Fachanstalt durchführen zu lassen. Außerdem besteht die Verpflichtung zur Führung eines lückenlosen Aufzeichnungs- und Dokumentationssystems.

Geschichte
Bereits 1986 begann die Stadt Wien – ausgehend von Kleingartenanlagen und Siedlungsgebieten – durch das Aufstellen sogenannter "Biotonnen" kompostierbare Abfälle getrennt vom Hausmüll zu sammeln und einer gesonderten Verwertung zuzuführen. Diese Sammlung wurde immer weiter über das gesamte Stadtgebiet ausgedehnt. Mit den anfangs geringen Mengen an biogenen Abfällen wurden erste Kompostierversuche im Rinterzelt, bzw. auf der Deponie Rautenweg unternommen.

Steigende Sammelmengen machten die Errichtung einer eigenen Kompostieranlage notwendig. Das dazu notwendige Grundstück mit über 77.000 Quadratmetern wurde von einem privaten Besitzer gemäß dem Beschluss des Wiener Gemeinderates vom 19. Juni 1991 in der Nähe des Öllagers Lobau im 22. Wiener Gemeindebezirk in der Lobgrundstraße gepachtet.

Seit dem 16. Mai 2002 ist das Kompostwerk Lobau zur Führung des Kompostgütesiegels berechtigt. Vergeben wird dieses vom Kompostgüteverband Österreich und wird vom Österreichischen Normungsinstitut anerkannt.

1993 wurde auf einem städtischen Grundstück in der Schafflerhofstraße das Kompostwerk Schafflerhof errichtet. Heute dient das Kompostwerk Schafflerhof zur Nachlagerung von fertigem Kompost und zur Vorbereitung von Ausgangsmaterialien für die Herstellung von Garten- und Blumenerden.

Ausstattung
Das Kompostwerk Lobau verfügt über zwei jeweils etwa 26.000 Quadratmeter große befestigte Mietenflächen, unterirdische Auffangbehälter für Regen- und Sickerwasser (Fassungsvermögen rund 1.300 Kubikmeter) und ein offenes Wasserbecken. Das in diesen Speicherbehältern aufgenommene Wasser wird in die öffentliche Kanalisation abgeleitet. Das für den Betrieb notwendige Wasser wird über einen eigenen Nutzwasserbrunnen gewonnen.

Weiters befinden sich auf dem Areal des Kompostwerks Lobau ein Betriebsgebäude mit Labor, eine Garage mit eigener Werkstätte sowie eine Tankstelle. Zur Kontrolle der angelieferten beziehungsweise abtransportierten Mengen ist eine Wiegevorrichtung vorhanden.

Verwertung
Zur Verwertung kommen biogene Abfälle, die von Privatpersonen auf den Wiener Mistplätzen abgeliefert oder in Biotonnen eingebracht werden, von der MA 42 – Wiener Stadtgärten und der MA 49 – Forstamt und Landwirtschaftsbetrieb der Stadt Wien oder von sonstigen Quellen angeliefert werden. Laub, das im Herbst in großen Mengen eingekehrt wird, wird je nach seiner Herkunft getrennt. Stammt es aus Gärten oder Parkanlagen, so kann es in geringen Mengen kompostiert werden. Das übrige Laub wird energetisch verwertet. Eine andere Art von saisonal anfallendem Biomüll stellen um den Jahreswechsel die ausgedienten Christbäume dar. Für diese Sammlung werden im Stadtgebiet rund 375 Christbaumsammelstellen eingerichtet.

Der gesammelte Biomüll wird im so genannten Rinterzelt (Abfallbehandlungsanlage) in der Bioabfallaufbereitungsanlage von Verunreinigungen gesäubert, durchmischt und mit Wasser versetzt. Von der Abfallbehandlungsanlage in der Percostraße 2 wird dieses Kompostrohmaterial per LKW zum Kompostwerk Lobau zur Kompostierung gebracht.

Als Hauptabnehmer des hier hergestellten Komposts treten hauptsächlich Dienststellen des Magistrats der Stadt Wien in Erscheinung. Und hier ist es vor allem die MA 49, die den Kompost zum großen Teil in ihren Landwirtschaftsbetrieben verwendet. Ein Verwaltungsabkommen zwischen der MA 48 und der MA 49 sichert die Abnahme und Ausbringung von 20.000 Tonnen bis 30.000 Tonnen Kompost auf Flächen, die vom Landwirtschaftsbetrieb der Stadt Wien bewirtschaftet werden.

Gratis abgegeben wird Kompost in "Haushaltsmengen" (maximal 2 Kubikmeter) an den Mistplätzen ("Recyclinghöfen") der Stadt Wien. Außerdem wird Kompost an private Abnehmer verkauft.

Seit 2009 wird der Wiener Kompost zur Herstellung von torffreien Garten- und Blumenerden verwendet und als Produkt unter dem Namen "Guter Grund" verkauft.

Fußnoten

Weblinks

 Kompostwerk Lobau – Beschreibung
 Kontrollamtsbericht (PDF-Datei; 79 kB)
 Wienkompost.at der MA 48

Donaustadt
Abfallaufbereitungsanlage
Abfallentsorgungsanlage in Wien


Eileen O'Boyle Bitetto is a partner and concentrates her practice in the area of real estate tax certiorari law and condemnation litigation. This concentration has, as its focus, issues of real property valuation; particularly the review of assessed valuation–seeking opportunities where taxes may be reduced.
She also represents clients whose property has been acquired, or is about to be acquired, in Eminent Domain proceedings–working to ensure that these owners are adequately compensated for their loss of property.
Ms. Bitetto serves on the Board of Directors of Automobile Club of New York, Inc. which is the regional affiliate of AAA. It is a not-for-profit corporation established 100 years ago to maintain a club for auto users and owners, with the primary purpose of advocating law related to the use, rights, and privileges of such owners/users.
Additionally, she volunteers her time as a mentor with the Lawyers in Kids' Education ("LIKE") program, which was created by the partners at Certilman Balin. As part of this team, Ms. Bitetto uses "vocabulary enrichment games" and offers a listening ear to help "at-risk" elementary school students with literacy and life.
A member of the Nassau County, New York State and Nassau County Women's Bar Associations, she earned her undergraduate degree in American Studies from St. John's University and her Juris Doctorate from St. John's University School of Law.
In 2012, Long Island Business News named Ms. O'Boyle Bitetto to the list of "Who's Who in Women in Professional Services," and she has also achieved the highest professional rating by Martindale Hubbell. She is admitted to the New York State Bar.
O'Boyle Bitetto Elected to Board of Directors, Automobile Club of New York, Inc.


text:
Biografia
Tristano De Bonis (conosciuto anche come Trix) è nato a Fondi e  È il fratello maggiore della cantante Erika.

Nel 2000 Trix ha pubblicato il suo primo singolo "Carillon", canzone scritta da lui 11 anni prima, registrata e pubblicata da Spy/TIME.

Nel 2001, il video musicale per il singolo è stato realizzato da TIME Studio.

Nel 2001, "4 Your Love" è stato il secondo singolo di Magic Box. Una canzone molto apprezzata dai fan.

Nel 2003 ha presentato "If You" che lo ha fatto volare ad esibirsi in Brasile,

Nel 2004 arriva "Sorry Marin", il primo singolo che permette al pubblico di conoscere un altro lato della sua voce, senza falsetto.

Nel 2013, dopo anni di assenza dal palco, Magic Box pubblica "Scream My Name" cantata con la sua vera voce,

Nel 2018, Magic Box pubblica "From Now On".

Nel 2019, Magic Box prende parte all'album Back to the Feat di DJ Jump, interpretando i brani "I Play The Piano" e "Baby Queen", quest'ultimo inoltre vede la collaborazione con DJ Ross.

I suoi brani musicali sono stati inseriti in diverse compilation di musica dance, inoltre hanno raggiunto la "top 20" della hit parade in Italia, Francia, Svizzera e altri paesi.

Discografia

Singolo (come Trix)
2001 Bomb Bomb

Singoli (come Magic Box)

Note

Collegamenti esterni