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Conceived and designed the experiments: EAM CJR NJK TLK CG. Performed the experiments: EAM TLK. Analyzed the data: EAM CJR NJK TLK CG. Contributed reagents/materials/analysis tools: EAM CJR NJK TLK CG. Wrote the paper: EAM CJR NJK TLK CG.
Eukaryotic gene expression involves tight coordination between transcription and pre–mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA–independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre–mRNA splicing.
Pre-messenger RNA splicing is the process by which an intron is identified and removed from a transcript and the protein-coding exons are ligated together. It is carried out by the spliceosome, a large and dynamic molecular machine that catalyzes the splicing reaction. It is now apparent that most splicing occurs while the transcript is still engaged with RNA polymerase, implying that the biologically relevant splicing substrate is chromatin-associated. Here, we used a genetic approach to understand which factors participate in the coordination of transcription and splicing. Having recently shown that the Npl3 protein is involved in the recruitment of splicing factors to chromatin-associated transcripts, we performed a systematic screen for genetically interacting factors. Interestingly, we identified factors that influence the ubiquitin modification of histone H2B, a mark involved in transcription initiation and elongation. We show that disruption of the H2B ubiquitination/de-ubiquitination cycle results in defects in splicing, particularly in the absence of Npl3. Furthermore, the ubiquitin ligase, Bre1, shows genetic interactions with other, more canonical spliceosomal factors. Taken together with the myriad Npl3 interaction partners we found, our data suggest an extensive cross-talk between the spliceosome and chromatin.
Pre-mRNA splicing is a critical step in gene expression in which non-coding introns are removed from pre-mRNA and protein-coding exons are ligated together. This process is performed by the spliceosome, a dynamic ribonucleoprotein particle that, in yeast, consists of 5 snRNAs and over 80 proteins that cooperate to recognize and splice target mRNAs [1]. Recent evidence reveals that mRNA splicing in vivo is largely co-transcriptional, and occurs while elongating RNA polymerase II (PolII) is still associated with chromatin [2]–[4]. The basic unit of chromatin is 146 base pairs of DNA wound around a histone octamer to form a nucleosome, arrays of which can be further compacted to form higher-order chromatin structure. A plethora of chromatin remodeling and histone modifying machines are now known to be integral parts of the gene expression process [5]. While much has been learned about the molecular mechanisms of pre-mRNA splicing from in vitro systems [6], a full understanding of the regulation of spliceosome assembly and catalysis will require an appreciation of the complex landscape of the chromatinized template, along which splicing occurs.
To approach this question, we built upon our recent observation that the SR-like protein Npl3 promotes efficient splicing of a large subset of genes via co-transcriptional recruitment of U1 and U2 snRNPs [7]. SR and hnRNP proteins in metazoa are best understood for their role in alternative and constitutive splicing, although they have also been implicated in additional steps in gene expression, including mRNA export, translation, and even transcription itself [8]–[10]. Despite the fact that there are few examples of alternative splicing in S. cerevisiae, this yeast contains three genes with a canonical SR protein domain structure: one or more RNA recognition motifs and a domain enriched in arginine-serine dipeptides [9], [11]. We recently demonstrated that deletion of NPL3 specifically, but not the others, impacts splicing; interestingly, the affected genes are almost exclusively those encoding ribosomal proteins, and make up the largest class of intron-containing genes in budding yeast [7]. Npl3 appears to be appropriately poised to coordinate events in gene expression: it is recruited to chromatin early during transcription [12] stimulates transcription elongation [13]–[16] co-purifies with elongating PolII [12], [14] via its interaction with the C-terminal domain [14], and remains associated with mRNA after processing is completed [17], [18].
Here, in order to understand how Npl3 might choreograph gene expression events in S. cerevisiae, we systematically analyzed genetic interactions of a strain lacking Npl3. We uncovered genetic interactions between the npl3Δ allele and genes involved in transcription and chromatin modification, including factors involved in histone H2B ubiquitination: the E3 ubiquitin ligase, Bre1 [19], [20], and corresponding ubiquitin protease, Ubp8 [21]–[23]. In addition, we show that Npl3 physically interacts with Bre1. Splicing-sensitive microarray experiments reveal that disabling the H2B ubiquitination pathway by deletion of BRE1 or UBP8, or point mutation of H2B, exacerbates the known splicing defect of an npl3Δ strain. Furthermore, we observed an Npl3-independent connection between Bre1 and splicing, as deletion of BRE1 impairs the splicing of a subset of pre-mRNAs and, in combination with deletions of individual splicing factors, causes severe synthetic growth defects. Thus, our data functionally link H2B ubiquitination by Bre1 to pre-mRNA splicing and more broadly suggest that the coordination of transcription and splicing may be aided by crosstalk between Npl3 and chromatin metabolism.
The SR-like protein Npl3 has multiple roles in the regulation of gene expression, including in pre-mRNA splicing, 3′ end processing, and mRNA export. To further interrogate this multifunctional factor, we used synthetic genetic array (SGA) technology [24], [25] to screen ~4,800 non-essential yeast genes for those whose deletion conferred synthetic lethality (SL) or very synthetic sick (SS) growth phenotypes in an npl3Δ strain. Since an npl3Δ strain grows more slowly than wild-type at 30°C, and this defect is exacerbated at 37°C (e.g., see Figure 1C, top panels), we performed the screen at both temperatures to maximize coverage. The analysis revealed strong negative interactions between NPL3 and 83 (1.7% of total) and 333 (6.9% of total) genes after growth at 30°C and 37°C, respectively (see Table S1).
To validate a subset of genetic interactions identified by this high-throughput approach, we generated the cognate double mutant strains using tetrad dissection. In order to refine our list of genetically interacting factors, we included additional subunits from complexes represented in the results of the screen. A list of the most stringent synthetic interaction partners (identified in the 30°C SGA and directed genetics) was integrated with those from a previously published quantitative RNA processing Epistatic Mini Array Profile (E-MAP) [26] to generate a more comprehensive set of NPL3 SS/SL genetic interactions (Figure 1A and Materials and Methods). These negative genetic interactions were highly enriched for genes that function in RNA metabolism (Table S2), consistent with what was previously known about Npl3 function in mRNA processing [7], [12], [13], [16]–[18], [27]–[31]. In addition, there was an enrichment of genetic interactions with genes implicated in "chromosome organization" and "transcription," including components of the chromatin remodeling SWR Complex [32]–[34], the transcriptional elongation PAF Complex [35]–[38], and multiple histone modification complexes, including COMPASS [39], SAGA [40], and the SET3 Complex [41] (Figure 1B, 1C and 1D and Table S3). We note that of these, the SWR1 and SAGA Complexes have previously been implicated in pre-mRNA splicing [42], [43], highlighting the ability of the Npl3 screen to identify factors involved in chromatin-splicing crosstalk.
The screens also showed that deletion of either RAD6 (Figure 1E cf. closed triangles) or BRE1 (Figure 1E cf. open triangles) led to synthetic sickness/lethality in an NPL3 deletion strain. These factors catalyze the mono-ubiquitination of lysine 123 on histone H2B; specifically, Bre1 is the E3 ubiquitin ligase and Rad6 is its corresponding E2 ubiquitin-conjugating enzyme [19], [20], [44], [45]. We found that inactivating Bre1 ubiquitin ligase activity via a point mutation in its RING domain (bre1H665A) [19] exacerbated the growth defect of an npl3Δ strain to the same extent as a full deletion of BRE1 (Figure 1E, cf. orange triangles), suggesting that the genetic interaction is connected to the ligase activity of Bre1. Many nuclear enzymes act not only on histones but on other substrates as well, and, in fact, histone H2B is not the only ubiquitination target of Bre1 [46]. To ask whether the Npl3-Bre1 genetic interaction is due to the loss of H2B ubiquitination specifically, we tested whether a mutation of the target residue in H2B would phenocopy a deletion of BRE1. Indeed, the htb1K123R point mutant also profoundly exacerbated the growth defect of npl3Δ (Figure 1E cf. purple triangles). Taken together, these data provide strong evidence that H2B ubiquitination can account for the genetic interaction of the RAD6 Complex with NPL3.
The PAF Complex and COMPASS have previously been shown to function in the same histone modification pathway as the Bre1 [47]–[50]. The PAF Complex is required for H2B ubiquitination [49], [50]; thus, the synthetic lethality we observed between NPL3 and components of the PAF Complex (Figure 1C and 1D) was consistent with the genetic interactions we observed with the Bre1. H2B ubiquitination is, in turn, required for trimethylation of histone H3 lysine 4 (H3K4) by COMPASS [39], [51]–[53] and lysine 79 (H3K79) by Dot1 [54]–[57]. However, we found no genetic interaction between NPL3 and point mutations of H3K4 or H3K79 (data not shown), suggesting that loss of these chromatin marks is unlikely to underlie the synthetic sickness in the npl3Δbre1Δ double mutant.
Given that maintaining H2B ubiquitination is critical in the absence of NPL3, it follows that mutations in genes required for the removal of this chromatin mark might suppress the npl3Δ growth defect. To investigate this in an unbiased fashion, we made use of the fact that NPL3 deletion causes lethality when yeast are grown at 16°C (e.g., see Figure 2B, top panel); this allowed us to screen for mutants that restore growth to an npl3Δ strain at 16°C. This screen identified 105 (2.1% of total) and 699 (14.4% of total) suppressors after 4 and 8 days of growth, respectively (Table S4), and a number of these suppressors have previously been implicated in transcription and chromatin modification (Figure 2A). We then generated a number of the double mutants using tetrad dissection and validated the suppressive genetic interactions using serial dilution (Figure 2B). In agreement with our expectation, the data from this screen showed that deletion of UBP8, which encodes an H2B de-ubiquitinase [21]–[23], restored viability to a strain lacking Npl3 (Figure 2B cf. closed triangles). In further support of these observations, the SGA also identified SGF11 and SGF73 as genes whose deletion suppresses npl3Δ; these factors are part of a module of the SAGA Complex with Upb8, and are also implicated in gene activation by H2B de-ubiquitination [21], [58]–[61]. Taken together, this dataset shows that the npl3Δ strain is particularly sensitive to deletion of genes affecting the H2B ubiquitination pathway (Figure 1 and Figure 2) and opens the possibility that H2B ubiquitination is important for an Npl3-dependent process.
Interestingly, deletions of genes in other modules of SAGA required for either histone acetylation (Ada2 and Gcn5) or for association of the SAGA complex with promoters (i.e., the TBP regulatory module, Spt3 and Spt8; reviewed in [62]) exacerbated, rather than suppressed, the npl3Δ growth defect (Figure 1C and Table S1). The divergent genetic interactions confirm the functionally separable nature of the SAGA sub-modules [58] and highlight that a connection exists between Npl3 and H2B mono-ubiquitination that is functionally distinct from other chromatin marks.
Given the robust genetic interactions we observed between NPL3 and genes involved in H2B ubiquitination, we performed co-immunoprecipitation assays of the corresponding proteins to test if they physically interact. We had previously shown that Npl3 co-immunoprecipitated components of the U1 snRNP [7]. Here, we immunoprecipitated endogenous Npl3 from whole-cell extract using a polyclonal antibody directed against Npl3 [63] and then probed the precipitate for endogenously tagged forms of Bre1, Ubp8, and Sgf11 as well as positive and negative controls (a U1 protein, Luc7, and Nup188, respectively). Although there is precedent for some interaction specificity with the E3 Bre1 over the E2 Rad6 [46], we also tested for an interaction with Rad6. As shown in Figure 3, only Bre1 and Luc7 but not Nup188, Rad6, Ubp8 or Sgf11, co-immunoprecipitated with Npl3.
It is known that Npl3 is an RNA-binding protein, and its interaction with some components of the splicing machinery is RNA-dependent [7]. To test whether the observed interaction with Bre1 is mediated by RNA, we treated the extracts with RNaseA prior to the immunoprecipitation. We consistently found that a population of Bre1 interacts with Npl3 in an RNase-independent manner (Figure 3 cf. lanes 3 and 4, top panel). These data indicate that Npl3 can physically interact with Bre1, consistent with previous data from high-throughput proteomic analyses [64].
The genetic data connecting NPL3 and the H2B ubiquitination machinery lend support for two possible models. One model predicts that Npl3 will affect H2B ubiquitination; we therefore measured the global percentage of ubiquitinated H2B but found the npl3Δ strain indistinguishable from wild-type (Figure S1). An alternative interpretation of the genetic data is that the H2B ubiquitination cycle is important for an Npl3-dependent process. We previously reported [7] that a strain lacking Npl3 accumulates a subset of pre-mRNAs, consisting primarily of the ribosomal protein genes (RPGs), whose splicing efficiency might be expected to affect growth rate. Given that deletion of BRE1 exacerbates the npl3Δ growth defect, we tested whether deleting BRE1 exacerbates the npl3Δ splicing defect.
We used our splicing-sensitive microarray platform [65], which contains oligos that hybridize to the terminal exon, the intron, and the exon-exon junction of each intron-containing gene, in order to detect total mRNA, pre-mRNA, and mature mRNA, respectively (Figure 4A). For each genotype, the heat map (Figure 4B) reports fold changes in signal intensity of these three RNA species for each intron-containing gene as compared to a wild-type strain. As expected, our experiments showed that a strain lacking Npl3 accumulated RPG pre-mRNAs (Figure 4B npl3Δ, see yellow in Intron feature; RPGs highlighted in purple on right). Notably, the pre-mRNA accumulation in the npl3Δ strain was increased at many RPGs when BRE1 was also deleted (Figure 4B cf. npl3Δ and npl3Δbre1Δ, Intron feature), suggesting that Bre1 is important for the splicing of many Npl3-dependent genes. We note that this effect is complex, and is accompanied by changes in total mRNA (Figure 4B cf. npl3Δ and npl3Δbre1Δ, Exon feature). Because both Npl3 and Bre1 have been shown to have effects on transcription itself [13]–[16], [66]–[68], we normalized for changes in exon level by calculating an Intron Accumulation Index [69] (see Materials and Methods) for each intron-containing gene (Figure S2 and Table S5). The histogram of genes with an Intron Accumulation Index of greater than 0.3 (Figure 4C), shows that even when normalized for changes in transcript levels, the total number of genes with a splicing defect, as well as the severity of the defect, is increased in the npl3Δbre1Δ strain as compared to npl3Δ alone.
We also found that in the presence of wild-type Npl3, a strain lacking BRE1 has a mild but reproducible splicing defect (Figure 4B, bre1Δ –shown is an average of 5 biological replicates). While the majority of pre-mRNAs are not affected by the deletion of BRE1, a small subset of pre-mRNAs accumulates in bre1Δ at 37°C (Figure 4B, e.g., DBP2, LSB3, YOP1). This suggests that Bre1 has a role in pre-mRNA splicing, independent of the sensitivity caused when NPL3 is deleted. This finding was confirmed when we calculated Intron Accumulation Indices for a strain lacking BRE1: a small number of genes exhibit defective splicing in the bre1Δ strain (Figure 4C and Figure S2). We validated these splicing defects for several genes using a qPCR assay (Figure S3). The lack of a significant growth defect in the bre1Δ strain (Figure 1E) is consistent with the idea that yeast can tolerate a modest splicing defect at a small number of non-RPGs.
If the splicing defect exacerbation we observed with npl3Δbre1Δ was due to loss of H2B ubiquitination, we would then expect this exacerbation to be phenocopied by a strain with the H2B lysine to arginine point mutant used earlier (Figure 1E). We did, in fact, find that the htb1K123R point mutation exacerbated the splicing defect observed in the npl3Δ mutant at many genes (Figure 4B), further implicating the ubiquitination of H2B in splicing. This is also evident when normalizing for the changes in exon levels in the npl3Δhtb1K123R strain (Figure 4C). In plotting the Intron Accumulation Index values of this strain, we find that the subset of affected genes overlaps extensively with the subset of genes affected in the npl3Δbre1Δ double mutant (Figure S2).
We have shown that deletion of UBP8 partially suppresses the npl3Δ growth defect, and this is most pronounced at 16°C (Figure 2B). We therefore tested whether deleting UBP8 would suppress the splicing defect of a strain lacking Npl3, as predicted by the genetic interaction. Surprisingly, deletion of UBP8 instead exacerbated the splicing defect observed in the npl3Δ strain (Figure 4B cf. npl3Δ and npl3Δubp8Δ), implying that the growth suppression is related to some other function of Npl3. Notably, however, these microarray results indicate that in the absence of Npl3, the complete cycle of H2B ubiquitination and de-ubiquitination is required for efficient splicing.
To begin to investigate how Bre1 affects splicing, we used chromatin immunoprecipitation (ChIP) to test the prediction that Bre1 is required for association of the splicing machinery. However, we did not observe a significant Bre1-dependent decrease in U1 (Prp42), Mud2, or U2 (Lea1) association with genes whose splicing was inhibited in bre1Δ or npl3Δbre1Δ strains (data not shown), suggesting an alternative mechanism by which Bre1 modulates splicing (see discussion).
In light of our data showing that a bre1Δ strain exhibited a mild splicing defect, we carried out directed genetic analyses to test for interactions between BRE1 and genes encoding other splicing factors, particularly those that genetically interact with Npl3 [7]. Just like a deletion of NPL3, deleting BRE1 caused synthetic sickness when combined with deletion of NAM8 (U1 snRNP), MUD2, LEA1 (U2), or SNU66 (U5), further connecting Bre1 functionally with splicing (Figure 5A). Interestingly, the growth of the bre1Δ strain was also compromised by deletion of the U2 snRNP component CUS2, which does not genetically interact with npl3Δ [7]. Thus, although we approached these experiments through the lens of Npl3, these genetic observations provide further support that Bre1 has independent interactions with the splicing machinery. Consistent with a lack of splicing defect upon UPB8 deletion, we and others generally did not observe genetic interactions between UBP8 and early or late splicing factors (Figure 5B and [70]). There is one notable exception however; deletion of UBP8 suppressed the snu66Δ cold-sensitive growth defect (Figure 5B). Taken together, these data highlight the fact that the H2B ubiquitination pathway is linked to splicing, even in the presence of wild-type Npl3.
While the textbook view of gene expression presents transcription, pre-mRNA processing, export, and translation as independent events, they appear to be closely coordinated in the cell. Discerning the mechanism of this coordination within the broader program of gene expression presents a daunting experimental challenge. In multicellular eukaryotes, SR and hnRNP proteins are thought to regulate gene expression, in part, by integrating mRNA biogenesis steps [9], [10], [71]–[73]. In budding yeast, Npl3 has numerous roles, but is the only such protein that affects splicing [7]. Our lab and others have shown that Npl3 facilitates the co-transcriptional recruitment of early splicing factors to nascent transcripts [7] and itself associates with elongating polymerase [12], [14]. Therefore, we approached this complex problem by conducting a systematic screen of non-essential genes to define the interacting partners of this potential coupling factor.
We found that Npl3 genetically interacts with a number of genes implicated in chromatin metabolism and transcription. We further characterized one set of interacting factors, namely those involved in histone H2B ubiquitination, in what is, to our knowledge, the first set of genome-wide splicing experiments on histone modifier mutants in S. cerevisiae. Using splicing-sensitive microarrays, we showed that Npl3 links the H2B ubiquitination cycle to the splicing efficiency of many transcripts. The connection between H2B ubiquitination and splicing also exists independently of Npl3, as a strain lacking BRE1 exhibits both a mild splicing defect and genetic interactions with deletions of genes encoding early and late splicing factors. Finally, the full complement of genetic interactions we describe (Figure 6) provides multiple entry points for future investigation into the coupling of chromatin modification and mRNA processing.
Our genetic screens revealed that a number of Npl3 genetic interactions center on the histone H2B ubiquitination cycle. Specifically, mutant strains that lack wild-type levels of ubiquitinated H2B (rad6Δ, bre1Δ, lge1Δ, htb1K123R, paf1Δ, cdc73Δ, and leo1Δ) exacerbate the growth defect of an npl3Δ strain at all temperatures tested (Figure 1). We also observed a physical interaction between Npl3 and Bre1 by co-immunoprecipitation (Figure 3) and showed that the splicing defect caused by deletion of NPL3 is exacerbated by the additional deletion of BRE1 or mutation of H2B (htb1K123R), thus implicating H2B lysine 123 mono-ubiquitination in splicing (Figure 4). We previously demonstrated that Npl3 primarily affects the splicing of RPGs [7]; here, we see that in the sensitized background of a strain in which RPG splicing is made limiting (npl3Δ), the histone H2B ubiquitination cycle is an important contributor to RPG splicing.
Recent studies have shown that deletion of components of the cap-binding complex (CBC) or commitment complex causes defective splicing of the SUS1 pre-mRNA [70], [74]. Sus1 is a recently discovered component of the histone de-ubiquitination module of SAGA [75] and if the SUS1 transcript is not properly spliced, it leads to elevated levels of ubiquitinated H2B. Given the physical [29], [76] and genetic (Table S1 and [76]) connections between Npl3 and the CBC, we sought to determine whether Npl3 also affects SUS1 splicing and, therefore, H2B ubiquitination. However, Hossain, et al. have recently shown that deletion of NPL3 has no effect on SUS1 splicing [74], a result we independently confirmed in our npl3Δ strain (Figure S4). Furthermore, we extended this analysis and determined that, unlike in cbcΔ strains, global levels of ubiquitinated histone H2B are not discernibly altered in the npl3Δ strain (Figure S1). While we cannot rule out a change in the dynamics of the ubiquitination cycle or gene-specific effects, our microarray results support a model in which the full histone ubiquitination cycle promotes RPG splicing, a process that becomes critical in the absence of NPL3. Along these lines, it is noteworthy that data from Schulze et al. and Shieh et al. have revealed that chromatin over these genes is enriched for ubiquitinated H2B [77], [78].
We also identified suppressive genetic interactions between NPL3 and genes responsible for removal of ubiquitin from H2B (ubp8Δ, sgf11Δ, and sgf73Δ), suggesting that H2B de-ubiquitination is also linked to Npl3 function. Surprisingly, however, deletion of UBP8 did not suppress the splicing defect in npl3Δ, but rather exacerbated it (Figure 4). Thus, it seems the positive genetic interaction may be due to Ubp8 involvement in a splicing-independent function of Npl3.
The exacerbation seen in the microarray experiments shows that both halves of the cycle of H2B ubiquitination and de-ubiquitination are required for optimal splicing, as is the case for transcriptional activation [23]. Likewise, both halves of the H3 acetylation and deacetylation cycle, performed by Gcn5 and Hos2/3, respectively, promote spliceosome assembly at the ECM33 gene [43], [79]. Thus, these two examples point to a general function of dynamic histone modification cycles in maintaining fine control over co-transcriptional splicing, and may explain the synthetic lethality we observed between NPL3 and the acetylation module of the SAGA Complex (Figure 1C and Table S1).
We found that even in the presence of wild-type NPL3, Bre1 has genetic connections to the splicing machinery as a whole. Specifically, we found that deletion of BRE1 causes growth defects in early and late splicing factor deletion backgrounds (particularly at extreme temperatures; Figure 5, 16°C and 37°C), which alone show little to no growth defect. These negative genetic interactions can indicate two alternative but not mutually exclusive models for a functional relationship between the H2B ubiquitination and splicing machineries. One model is based on the fact that deletion of specific splicing factors is known to increase the levels of ubiquitinated H2B [74], a phenotype that should be relieved by deletion of BRE1, the sole H2B ubiquitin ligase [19], [20]. Because this model predicts an epistatic or positive genetic interaction between BRE1 and the genes that encode splicing factors, the negative genetic interactions that we actually observe (Figure 5 and [70]) require an alternative model, perhaps one in which the growth defects are due to poorer overall splicing efficiency in these strains. Indeed, deletion of BRE1 alone caused a modest but reproducible splicing defect, seen in the microarray in Figure 4. A large fraction of Bre1-dependent splicing events involve non-RPGs, and thus define a distinct role for Bre1 in splicing, apart from Npl3. Shieh et al. [78] recently found that the pattern of this modified histone at non-RPGs shows a remarkable demarcation of intron/exon structure: low levels in the intron, followed by a marked increase at the intron – exon boundary. While the functional significance of this pattern of H2B ubiquitination is unknown, we propose that it may be relevant for the splicing of non-RPGs, as gauged by the splicing defect in a strain that no longer has this mark.
We note that the single mutant htb1K123R has a milder splicing defect than the bre1Δ strain (Figure 4C and Figure S2), opening the possibility of an additional role of Bre1 in splicing that is independent of H2B ubiquitination. Indeed, Bre1-dependent ubiquitination of Swd2, a protein in both COMPASS and the Cleavage and Polyadenylation Stimulatory Factor complex [46], has been shown to regulate mRNA export from the nucleus [80]. Npl3 has previously been implicated in mRNA export [17], [18], [27], [28] in a strain background where Npl3 is an essential protein. However, our data argue against the possibility that the genetic interactions we observed here are due to an adverse effect on mRNA export. In the present strain background (S288C), in which Npl3 is non-essential, the npl3Δ strain does not exhibit the nuclear localization of bulk poly-adenylated mRNA characteristic of an export defect (Figure S5); nor does further deletion of BRE1 in an npl3Δ strain cause an export defect (Figure S5). Furthermore, we found that the npl3S411A phosphorylation mutation, which blocks 3′ end formation [13], [14] and mRNA export [17], does not cause a block in pre-mRNA splicing (Figure S6). This argues against the reported splicing defects being the indirect result of feedback from these downstream defects in mRNA processing.
We tested the prediction that Bre1, like Npl3, promotes spliceosome recruitment, but found that deletion of BRE1 did not affect the association of U1 (Prp42), Mud2, or U2 (Lea1) with chromatin at genes whose splicing is dependent on Bre1 (data not shown). It may be that H2B ubiquitination is required for the recruitment of a later splicing factor or, as the H2B ubiquitination cycle regulates PolII passage through a gene [66], [81], it is possible that disruption of this cycle causes a subtle alteration of spliceosome dynamics that is not observable by ChIP. Furthermore, we cannot rule out the possibility that Bre1 has a ubiquitination target within the spliceosome or even ubiquitinates Npl3 itself.
Both splicing and mRNA processing are largely co-transcriptional processes in eukaryotes, from yeast [2] to human [82]–[84]. Our survey of NPL3 genetic interactions has revealed a multitude of chromatin-connected factors with potential links to splicing and mRNA processing; overall, these results are thus consistent with an "integrator" role for Npl3 in gene expression (Figure 6). Our data provide a basis for the further study of the coupling of SR/hnRNP-dependent mRNA processing and transcription within a chromatin context, and have led to the discovery of Npl3-dependent and independent roles for Bre1 and histone H2B ubiquitination in splicing.
A list of strains used and further strain construction details are available in Table S6.
Unless otherwise indicated, yeast were grown as described in [85]. The npl3Δ::NatNT2 "magic marker" query strain used in the SGA was YTK232D, and was previously used in [26]. YTK232D was generated using techniques outlined in [86]. Briefly, the NPL3 open reading frame was replaced with NatNT2 via integration of a PCR product generated with primers (5′- TACTTTTGAAGGAATCAAAATTAAGCAATTACGCTAAAACCATAAGGATAACATGGAGGCCCAGAATACCC-3′) and (5′-GTTTTAAAACAATTCATATCTTTTGTTAATTTCTCCTTTTTTTTTCTCAACCAGTATAGCGACCAGCATTC-3′) into the SGA diploid strain [87]. The diploid was sporulated and the MATα npl3Δ::NatNT2 query strain was isolated by tetrad dissection, followed by re-selection of magic markers on SD medium lacking leucine and arginine but containing canavanine, s-AEC, and clonNAT [SD - LEU/ARG+100 µg/mL canavanine+100 µg/mL S-(2-Aminoethyl)-L-cysteine hydrochloride+100 µg/mL clonNAT]. The NPL3 deletion was confirmed by PCR, and by Western blot for the absence of Npl3 using an α-Npl3 antibody [63].
The Synthetic Genetic Array was performed as described in [24] with the following exceptions: Here the npl3Δ query strain (YTK232D) was mated to the MATa KanMX-marked deletion collection (OpenBiosystems: www.openbiosystems.com; formerly Research Genetics, Huntsville, AL). The collection was arrayed in duplicate in 384-well colony format using automated pinning (Colony Arrayer) and grown at 30°C for 2 days. Mating was carried out at 30°C for 2 days. Sporulation was carried out at 30°C for 7 days. MATa double mutants were selected on SD medium lacking histidine and arginine but containing canavanine, S-AEC, G418, and clonNAT [SD - HIS/ARG+100 µg/mL canavanine+100 µg/mL S-(2-Aminoethyl)-L-cysteine hydrochloride+150 µg/mL G418 and 100 µg/mL clonNAT]. Double mutant arrays were re-pinned in replicate and photographed after the following incubations: 30°C for 5 days, 37°C for 5 days, or at 16°C for 4 and again after 8 days. Photographs were visually inspected for growth at 16°C (to identify suppressive interactions) or lack of colony growth at 30°C or 37°C (to identify synthetic lethal interactions).
The npl3Δ::NatNT2 strain used for directed genetics (YTK234D) was previously used in [7]. Unless otherwise indicated, YTK234D was crossed to a series of MATa KanMX4-marked deletion strains; diploids were selected by plating on YPD plates+100 µg/mL clonNAT+150 µg/mL G418. Double mutants were isolated by tetrad dissection or random sporulation, as indicated in Table S6. All single mutants were validated by PCR for the knockout chromosome prior to crossing to YTK234D. The HTB1-WT (WHY334) and htb1-K123R (WHY326) strains contain htb2Δ::HygX4l and the indicated htb1 allele as the sole copy of H2B (gifts from W. Hwang and H. Madhani). They were mated as above, except the diploid strains were selected on YPD+100 µg/mL hygromycin+100 µg/mL clonNAT. Because the htb1 allele is unmarked, the final npl3ΔHTB1 and npl3Δhtb1K123R strains were confirmed by sequencing the HTB1 gene and Western blot for the Npl3 protein. Genetic interactions with the bre1H665A allele were analyzed using a set of plasmids provided by the Shilatifard lab [19], designed to complement a bre1Δ allele. Complementation was achieved by plasmid transformation into YM1740 (bre1Δ) or YTK391B (npl3Δbre1Δ), which were maintained on SD -LEU plates.
The bre1Δ::NatNT2 (EMy32) and ubp8Δ::NatNT2 (EMy442) strains were created by replacement of the endogenous ORF with NatNT2, as described in [86]. These strains were subsequently mated to nam8Δ, mud1Δ, mud2Δ, syf2Δ, and snu66Δ (for bre1Δ), and lea1Δ (for bre1Δ and ubp8Δ) from the deletion collection and double mutants were isolated via tetrad dissection. For the rest of the ubp8Δ genetics, the ubp8Δ::KanMX4 strain from the deletion collection was mated to MATα NAT-marked "magic marked" splicing factor deletion strains. These splicing factor deletion strains were made by replacing the KanMX-marked ORFs with NatNT2, followed by crossing to a "magic marked" wild-type (YTK609) to isolate "magic marked" NAT-marked MATα spores. The ubp8Δ::KanMX4 strain was mated to each NAT-marked splicing factor deletion strain and MATa double mutants were isolated by tetrad dissection followed by selection on SD medium lacking histidine and arginine but containing canavanine, S-AEC, G418, and clonNAT [SD - HIS/ARG+100 µg/mL canavanine+100 µg/mL S-(2-Aminoethyl)-L-cysteine hydrochloride+150 µg/mL G418 and 100 µg/mL clonNAT].
For individual growth assays, log-phase yeast were diluted to OD600=0.1, spotted onto YPD plates (unless specifically mentioned) in a 5-fold dilution series and grown at the indicated temperatures. For each cross, growth of the double mutant was confirmed for ≥2 double mutant isolates, and a representative isolate is shown. The single mutants and wild-type strains shown are either parental strains, or were re-isolated from tetra-type tetrads. The bre1H665A and BRE1 strains were serially diluted onto SD –LEU plates.
We sought to integrate the diverse sources of genetic interaction information available to us in order to create a comprehensive dataset for statistical analyses. Because the stronger synthetic interactions were identified in the 30°C SGA, we began with this list of genes whose deletion caused lethality in combination with npl3Δ (see Table S1 – 30°C) and added genes identified as causing markedly decreased growth, as gauged by serial dilution, or lethality, as gauged by loss of double mutant spore after tetrad dissection (Figure 1C, 1D, 1E and Table S1). We further added to this list genes identified as synthetic sick or lethal in the E-MAP [26] i.e., having a genetic interaction score of ≤−2.5.
Biological process definitions were obtained from the Gene Ontology annotations maintained at SGD [88] on April 15th 2012. Forty-five high-level (GO Slim) terms were used and are included in Table S7. Protein complex definitions were obtained from a manually curated list, CYC2008 [89], and augmented with the RAD6 Complex (RAD6, BRE1, LGE1), which was not annotated when the list was created. A hypergeometric test was used to identify complexes and processes that were significantly enriched with genetic interactions. Complex enrichment p-values were corrected for multiple testing using the empirical re-sampling method of Berriz et al. [90] (as 409 complexes were assessed for enrichment), while process enrichment p-values were corrected for using the simpler Bonferoni correction. The results of the these analyses are included in Table S2 (by process) and Table S3 (by complex). The network diagram in Figure 1B was drawn using Cytoscape [91]. For Figure 1B, complexes were referred to by their more common names. The Figure 2A diagram was created to highlight a subset of suppressive interactions identified in the 16°C SGA and the full list of suppressors is available in Table S4.
Co-immunoprecipitation assays were performed as in [7] with extracts from the indicated GFP-tagged or HA-tagged strains. The Nup188-HA strain contains a plasmid encoding Nup188-3XHA. The other strains were tagged endogenously. Briefly, samples were separated by 10% SDS-PAGE and probed by Western blot with either monoclonal α-GFP (Roche 1814460), α-HA (12CA5; Roche 11583816001), or polyclonal α-Npl3 antibodies [63]. Total samples equivalent to 1/60th of the input were analyzed in parallel.
Cultures were grown according to standard techniques [85] in rich medium supplemented with 2% glucose. Strains were cultured overnight to saturation and diluted to OD600=0.1 in the morning. The strains were allowed to grow at 30°C until reaching mid-log phase (OD600=0.5–0.7), at which point they were collected (for Figure S6), or rapidly shifted to either 37°C for 30 minutes or 16°C for 2.5 hours, as indicated. Cultures were collected by centrifugation and snap frozen in liquid nitrogen. Total cellular RNA was isolated using hot acid phenol followed by isopropanol precipitation, as outlined in [92] but with modifications detailed in [93]. cDNA from each strain was synthesized, and labeled with Cy3 or Cy5 according to the low-throughput sample preparation method described in [65].
The optimized oligos listed in [65] were robotically arrayed onto poly-L-lysine coated glass slides (slides from ThermoScientific C40-5257-M20) and slides were processed using the protocols detailed in [65], [94] Each biological replicate contains 6 technical replicates for each feature as well as dye-flipped replicates. Microarrays were scanned using Axon Instruments GenePix 4000B at 635 nm and 532 nm wavelengths and image analysis was done using Axon Instruments GenePix Pro version 5.1. Spots were manually removed from analysis if they contained obvious defects or uncharacteristically high background; the ratio of the median intensities for 535 nm and 625 nm was calculated for each remaining spot. Technical replicate spots and dye flipped replicates were combined and normalized as in [65]. The resulting log2-transformed values for each feature were averaged over 2–5 biological replicates. Averaged data were subjected to hierarchical clustering using average linkage, and uncentered Pearson correlation as the similarity metric using Cluster 3.0 [95]. Resulting heat maps in Figure 4, Figure S2, and Figure S6 were created using Java Treeview [96]. To normalize for changes in total expression evident in the microarrays, Intron Accumulation Indices (IAI) were calculated for each intron containing gene as in [69]; specifically, we calculated log2(Intronmutant/IntronWT)-log2(Exonmutant/ExonWT) for each gene. The IAI heat map is shown in Figure S2. These values were converted into a histogram for Figure 4 using the following cutoffs: −0.3≥IAI≥0.3.
The dT50 assay was performed based on the protocol outlined in [97] with the following modifications. Specifically, 2 mL cultures were fixed in 5% formaldehyde for 1.5 hours after having reached OD600=0.2–0.3. Cells were washed 4 times in wash buffer (100 mM Potassium Phosphate, 1.2 M Sorbitol) before a 40-minute treatment with 27 µg zymolyase at 37°C. An additional fixation was performed in 8% paraformaldehyde in PBS+10 mM MgCl2 and spheroplasted cells were applied to poly-lysine-treated chamber slides (LabTek 178599). Attached cells were treated with ice-cold methanol (−20°C) and allowed to dry. Hybridization to digoxin-conjugated dT50 oligo in blocking buffer was performed at 37°C overnight. Chambers were washed with 2× (20 minutes), 1× (20 minutes) and 0.5× SSC (10 minutes at 37°C) before a 30-minute incubation with FITC- conjugated anti-Digoxin Fab fragments (Roche 1207741) in blocking buffer (125 dilution, 37°C). Antibody was aspirated and three 5-minute washes of PBS +10 mM MgCl2 were performed. Chambers were treated with 0.5 mg/mL DAPI for 2 minutes and slides were mounted using ProLong Gold Antifade Reagent (Invitrogen P36934) according to manufacturer instructions. Slides were visualized using an Olympus BX60 microscope equipped with FITC HiQ and DAPI HiQ Filters (Chroma Technology Corporation). The assay was performed on two biological replicates and representative images are shown. Specificity of the probe and FITC labeling was determined by incubation with hybridization mix lacking probe (data not shown).
SUS1 splicing efficiency was measured essentially according to the non-radioactive protocol described in [74]. Specifically, 10 µg RNA from cultures grown at 30°C was treated with DNaseI (Promega) and RNA was converted to cDNA using 1 µg SUS1 Reverse primer [70]. cDNAs were diluted 1200 and 10 µL was used in a 25 µL PCR (BioRad iProof) with SUS1-specific primers [70]. 25 cycles of PCR were performed and the resulting products were separated on an 8% polyacrylamide gel. Gels were stained using SybrGold and bands were quantified using an AlphaImager HP camera and software. 2–3 technical replicates of 2 biological samples were performed. Shown are a representative gel and the average and standard deviations of all technical replicates. A no-Reverse Transcriptase control was performed for each sample and none showed amplification (data not shown).
A TCA precipitation was performed on strains grown at 30°C [98] and samples were run on a 15% SDS polyacrylamide gel and transferred to PVDF membrane. Membrane was blocked using Li-Cor blocking buffer, followed by incubation of a 11000 dilution of α-H2B antibody (Active Motif 39237) overnight at 4°C. Visualization of bands was achieved with a secondary antibody conjugated to infrared dye (LI-COR 926-32211). The membrane was scanned using the LI-COR Odyssey scanner and software. Shown is a representative Western blot. The assay was performed with 3 biological replicates and shown are the average and standard deviation of the three replicates.
RNA was extracted as described above from strains grown under the same conditions as for the microarray experiment. Five µg RNA were treated with DNaseI (Promega) before being primed with random 9-mers and reverse transcribed. Samples were diluted as necessary and 10 µL were used in each qPCR. qPCRs were run on a C1000 ThermoCycler (BioRad) with an annealing temperature of 55°C. Each qPCR run was finished with a melt curve to determine the homogeneity of the amplified product. Starting quantity was calculated using a standard curve for each primer set. 2–4 technical replicates were performed for 1–5 biological replicates. Error bars represent standard deviation for biological replicates. For samples with 1 biological replicate, standard deviation of technical replicates is shown with uncapped error bars (Figure S3). A no-Reverse Transcriptase control was also generated for each RNA sample and these samples yielded negligible amplification (data not shown). Primers used in the qPCR are listed in Table S8. Each gene was measured using intron- and exon- specific primer sets. The Intron/Exon ratio for each mutant was normalized to its corresponding wild-type before averaging.
NPL3 does not affect global H2B ubiquitination levels. (A) Western blot analysis of histone H2B ubiquitination levels. Whole cell extracts from the indicated strains were subjected to electrophoresis to separate the ubiquitinated H2B from unmodified H2B, followed by Western blotting using α-H2B antibody. Shown is a representative blot. (B) Quantitation of H2B ubiquitination levels. Shown are the average percentages of ubiquitinated H2B from the indicated strains. Error bars represent standard deviation of three biological replicates.
Heat map representation of the Intron Accumulation Indices used to generate histogram in Figure 4. Shown are log2-based Intron Accumulation Index scores for each intron-containing gene, generated by normalizing fold intron changes to fold exon changes (see Materials and Methods for details). Genotype of each strain measured is listed above the heat maps. Transcripts that encode the ribosomal protein genes (RPGs) are highlighted in purple to the right of the heat maps. Gene order along the y-axis is the same for all genotypes.
qPCR validation of the microarrays. (A) Shown are log2-based Intron Accumulation Index scores for the indicated genes, as reproduced from Figure S2. The genotype of each strain is listed above the heat map. K123R refers to the htb1K123R allele.(B) RT-qPCR measurements of un-spliced mRNAs using single-locus RT-qPCR. Percent un-spliced RNA was calculated for each mutant and is represented as fold change compared to wild-type. Capped error bars represent standard deviation of biological replicates; uncapped error bars represent standard deviation of qPCR replicates.
SUS1 splicing is not sensitive to deletion of NPL3. (A) Analysis of SUS1 splicing using quantitative PCR on cDNA generated from the indicated strains. To the right of the gel, is a schematic of the SUS1 gene and the predicted mobility of its un-spliced, partially spliced, and fully spliced isoforms. Arrows indicate position of primers used in the PCR. Shown is a representative gel. (B) Quantitation of SUS1 PCR. To obtain the data shown in panel B, we took the average of two technical replicates generated from two separate biological samples. Error bars represent standard deviation of all replicates.
NPL3 and BRE1 do not affect bulk mRNA export. In situ hybridization of poly-dT oligo shows whole cell localization of mRNAs in wild-type, npl3Δ, bre1Δ, and npl3Δbre1Δ strains. The positive control thp1Δ strain is included to show nuclear localization coincident with DAPI (nuclear) staining.
Spicing is not sensitive to mutation of the Npl3 phosphorylation site. (A) Splicing profiles of the npl3S411A and npl3Δ strains grown at 30°C. The heat map shows the log2-ratio for each gene feature of the indicated strain compared to wild-type. Gene order along the y-axis is the same for all arrays. Transcripts that encode the ribosomal protein genes (RPGs) are highlighted in purple to the right of the heat maps. (B) Histogram shows the number of genes with a log2-based Intron Accumulation Index score greater than 0.3 for the npl3S411A and npl3Δ strains. Heat map within histogram bars shows distribution of the severity of splicing defect.
Genes whose deletion caused severe synthetic sickness or lethality with npl3Δ. List of genes whose deletion caused severe synthetic sickness or lethality with npl3Δ at 30°C or 37°C. Included are the systematic and common names, the synthetic genetic array in which each strain was identified, and the putative function as annotated by SGD (yeastgenome.org). Phenotype of the genetic interactions validated using directed genetics is listed under "notes". Any strains added using directed genetics are listed at the bottom of the table. Genes are sorted first by screen in which they were identified, and then alphabetically by common name.
GO-term analysis by Biological Process. Results of GO-term analysis by "Biological Process" with the associated p-values (pre- and post-correction). Biological Process definitions are available in Table S7.
Protein Complex enrichment analysis. Results of protein complex enrichment analysis with the associated p-values (pre- and post-correction for multiple testing). Significantly enriched complexes in bold are shown in Figure 1B using their more common names. Protein complex definitions were obtained from [89] with the addition of the RAD6 Complex containing RAD6, BRE1 and LGE1, which was not annotated at the time.
Genes whose deletion allowed growth in an npl3Δ strain. List of genes whose deletion allowed growth in an npl3Δ strain in the 16°C synthetic genetic array. Included are the systematic and common names, as well as the time-point at which growth was visualized, and the putative function of each gene. Any strains validated using directed genetics (by tetrad analyses or random sporulation) are indicated. Genes are sorted first by interval of time necessary to observe growth, and then alphabetically by common name.
Averaged microarray results used to generate heat map in Figure 4. Listed are log2 ratios of the intensities for each gene feature for the indicated strains compared to wild-type. "_2" refers to the second intron. Also included are the Intron Accumulation Index scores for each gene used to generate the heat map in Figure S2.
Strains used in this manuscript, and more detailed methods for their generation.
Biological Process definitions used to categorize npl3Δ genetic interactions.
Primers used in the qPCR assay.
We are indebted to Joseph DeRisi, Gregg Whitworth, Megan Bergkessel, and Alex Plocik for assistance with the microarray platform; Anne de Bruyn Kops and Gwendolyn Wilmes for assistance with the dT50 assay; and David Pincus for assistance with the fluorescent Western blots. The gifts of strains from William Hwang, Hiten Madhani, and Dale Cameron, and of plasmids from Ali Shilatifard are deeply appreciated. We also thank Michael Shales for guidance with figure design and Hannes Braberg (Krogan lab) and the entire Guthrie lab, in particular Anne de Bruyn Kops, Jaclyn Greimann, Michael Marvin, Kelly Nissen, Kristin Patrick, and Argenta Price, for suggestions on the manuscript.
This work was supported by NIH grant NIHGM21119 to CG and NIH grants GM084448, GM084279, and GM081879 to NJK. CG is an ACS Research Professor of Molecular Genetics. NJK is a Keck Young Investigator and Searle Scholar. EAM was supported by a graduate fellowship from NSF. CJR is supported by IRCSET and Science Foundation Ireland (Grant No. 08/SRC/I1407). TLK was supported by a postdoctoral fellowship from the American Cancer Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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The 2017 Sosland Publishing Purchasing Seminar will feature 20 different presentations and speakers of critical importance to purchasing executives, along with new opportunities for engaging with attendees. A new venue will accommodate this major industry event following years of rapid growth. We are confident that the 2017 seminar will be the best ever. The seminar will be held June 4-6, 2017, at the Sheraton hotel at Crown Center in Kansas City. If you have attended the seminar in the past, you will be receiving information and registration information for the 2017 seminar in January.
Sunday, June 4, 2017 - Tuesday, June 6, 2017
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Developed by the R&D team at ELEKS, the innovative concept app for Apple Watch that allows managing the basic functions of the Tesla electric car received global media coverage
February 12, 2015 - Lviv, Ukraine – ELEKS is proud to announce the release of the concept "Apple Watch. Tesla Car. How Far Can We Drive Them?" showcasing the capabilities of Apple Watch via an application which allows controlling a Tesla Model S. The concept has already received sensational coverage from world's top media including Mashable, The Verge, CNet and many others. Markiyan Matsekh, the leader of the project and ELEKS' Product Manager, was invited to speak at the Exploring Apple Watch meetup in New York, NY on February 25th. The company experts will also visit California on February 16-21.
The concept includes an article on the company's R&D blog, ELEKS Labs, a working application, a screencast of the application and the source code featured on the GitHub code repository. The idea behind the concept was to develop an Apple Watch application that would let the user access basic information about Tesla Model S and allow one to quickly manage it. The ELEKS team was determined to implement the available Tesla control functionality and adapt the UX to the context of Apple Watch. As a result, ELEKS developed an application that allows opening and closing the door, switching on the headlights, beeping and showing an overview of the car, its battery life, temperature, mileage and much more right from your wrist.
The Apple Watch-Tesla concept was extremely well received, being covered by major media including Mashable, The Verge, CNet, MacRumors, Business Insider, USA Today, Epoch Times, BGR, Cult of Mac, iPhone Hacks, Popular Science, Financial Review, Softpedia, Transport Evolved, Ubergizmo, CNN Money and 600 more online magazines and blogs. The ELEKS Labs article was read more than 26,000 times in one week, while the screencast video received more than 230,000 views and growing.
"At ELEKS, we enjoy exploring new technologies and with Tesla and Apple Watch being the hottest tech products, we knew we had to make them work together," says Markiyan Matsekh, ELEKS' Mobility & Wearables Product Manager. "Consequently, we were among the first to generate a reasoned public opinion of Apple Watch capability based on profound research, creative vision and the ability to overcome technical limitations."
The concept was released with the joint effort of Oleksandr Malyarenko (co-author, iOS engineer), Oleksandr Bolhovetskyy (iOS engineer), Roman Kabachenko, Viktor Maleichyk (concept co-authors), Vlad Krichenko (designer) and Markiyan Matsekh (project leader).
After the successful R&D of the Apple Watch-Tesla app, ELEKS is already actively exploring and developing a similar application for Android Wear to compare the two major wearables platforms.
If you want to find out more about ELEKS and the Apple Watch-Tesla concept, Markiyan Matsekh will gladly share some insights during his visit to New York as a speaker at the Exploring Apple Watch meetup on February 25th. Feel free to contact Markiyan for business meetings or interviews in NY at or @MarkiyanMatsekh on Twitter.
If you live in California, Victor Haydin, ELEKS' Head of Products and Services, Roman Pavlyuk, Technical Fellow, and Oleh Hasoshyn, Art Director, will be visiting existing customers in California on February 16-21 and may be available for a meeting. Do not hesitate to contact them now on LinkedIn or Twitter @victor_haydin.
About ELEKS
ELEKS is a global organization providing software engineering, technology consulting and quality assurance services. We help industry leaders and technology challengers turn creative ideas into tangible business value by delivering innovative and reliable solutions for our customers' unparalleled business growth to include Data Science, Mobility, Digital Production and Financial solutions. Since 1991, our software solutions have significantly contributed to the success of our customers, including Fortune 500 companies, and are recognized as a valuable part of international best practices.
ELEKS is a strategic partner with the proven ability to address our customers' most pressing needs. Our delivery organization, consisting of approximately 1,000 professionals in Eastern Europe, is strengthened by a local presence in Europe and the UK.
For more information, please visit: https://eleks.com/.


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Okay I don't need a lecture on smoking. I'm 17 so it's legal. But even though I sort of like smoking, I can't ever do it because it works as a laxative for me. Same goes for coffee. Why do they make me wanna shit?
Most Helpful Guy
they are both diuretics.00|00|0
Thanks for giving a proper answer.
- Show AllShow Less
No idea, why is anything a laxative? You'd probably have to be a biologist to understand what effect laxatives have on the body and why particular chemicals cause it.
@Matt2k yeah but if I knew what was in the laxative, I'd be able to avoid things that have that in it. No matter.
Most Helpful Girl
Because cigarettes are shit. And coffee generally makes you pee, not poop.00|00|0
I don't care what they "generally" do, they "generally" make me poop! I want an answer for my question, not being told what coffee generally does and that cigarettes are shit!!!
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But not answers
Facts are answers in their own rights.


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At one time heart disease was a death sentence. By the middle of the 20th century, it was killing millions, and, as with the Black Death centuries before, physicians stood helpless. Visionaries, though, had begun to make strides earlier. On September 7, 1895, Ludwig Rehn successfully sutured the heart of a living man with a knife wound to the chest for the first time. In 1929 Dr. Werner Forssman inserted a cardiac catheter in his own arm and forced the X-ray technician on duty to take a photo as he successfully threaded it down the vein into his own heart...and lived. And on June 6, 1944 - D-Day - another momentous event occurred far from the Normandy beaches: Dr. Dwight Harken sutured the shrapnel-injured heart of a young soldier and saved his life, and thus the term "cardiac surgeon" was born.
In The Heart Healers, James Forrester, MD, tells the story of these rebels and the risks they took with their own lives and the lives of others to heal the most elemental of human organs: the heart. The result is a compelling chronicle of a disease and its cure, a disease that is still with us but one that is slowly being worn away by the "heart healers".
©2015 James Forrester (P)2015 Tantor
"It's a book of marvels." (Publishers Weekly)
"Great review of the landmark achievements in Cardiology."
This was a great book. The author did a great job presenting his life and the modern development of cardiology. As a nurse practitioner I found all this information very interesting and came away with a much better understanding of why we do what we do in modern medicine.
The performance was great and I wish Jonathan Yen would do more recording.
So informative!
Some ah hah moments
I learned so much from this book. The final chapters were packed with useful information. I plan to read it again.
"one of the best in medical hitory"
every moment in the book was awe inspiring
I loved the narration
It made me laugh and cry
"A Surgeon's Illness"
Compared to Emperor of All Maladies, this book was more people-oriented. It is also more surgery-oriented because heart disease tends to be caused by mechanical defects amenable to surgical interventions. Drugs mostly play a supporting role - dissolving clots and reducing cholesterol. Cellular/molecular details of the illness are mostly neglected as they feature less prominently and more recently.
My impression from both books is that surgeons are, as you would expect, the jocks of the medical profession. Some of them are wonderful humanitarians, but many are also or primarily competitive or even grossly selfish. And as is typical of applied medicine, and especially of applied innovations in medicine, they require a strong stomach. No matter how good a doctor for a frequently terminal illness is, some of his patients will die due to his mistakes, and he or she has to live with that while remaining productive.
The surgical innovators of this book have it even worse than the normal practicing surgeon because they are learning on living subjects. Although these subjects were generally terminally ill, the innovative surgeon may be the immediate cause of many deaths before he perfects his procedure. Further, given the risks and initial outcomes involved, prior to success, he or she is likely to be a pariah to his peers.
Heart disease is a big deal - it and cancer each kill about 600,000 Americans a year. Forrester is optimistic that it will be overtaken by cancer as the leading cause of death in the near future. Maybe so, but the longer we live, the harder it is to treat or prevent such illnesses - hence success may have to be defined by modest improvements in longevity and eliminating young deaths. We are not, after all, immortal.
"Good history of cardiology care"
Good history of how heart disease has been researched and managed over the years. The last chapter was an over the top ego recap of the successes of cardiology, but hopefully we can all look back and feel we have made one of the world's most outstanding contributions to human health and welbeing.
"The Narration seems to ruin this book"
After listening to the Emperor of All Maladies and the superb narration of that book, this narration seemed too sharp and almost sarcastic in tone and that ruined it for me.Almost every sentence was phrased and read as if it were a question which was annoying.
If there was a different narrator
The narration seemed too sharp and almost sarcastic in tone and that ruined it for me.Almost every sentence was phrased and read as if it were a question which was annoying.
I wanted to like this book but couldn't get through a few chapters of the horrendous narration
"A masterclass"
The Heart Healers
Dr. Forrester gives us a first-hand account of the evolution of modern cardiology. There could have been no better person to present such a book as his own career paralleled the tremendous advancements cardiology has seen in the past 50 years. It seems like he was in the thick of it all and was able to give us a front-row account of the developments that have gotten us to where we are now. He calls them " mavericks and rebels" for good reason . We are fortunate to have such stalwarts willing to think outside the box and go all out in pursuit of their dreams and in the process help shape modern-day cardiology. Some of their experiments would have been difficult to conduct in modern-day medicine with its tight regulations and litigation. As we approach the present day we see less of such characters. Fortunately all the groundwork has been done and what we're doing now is fine-tuning the work of our forefathers.
Dr. Forrester presents various legends of the days gone by including Walter Lillehei ,Mason Zone , Andreaz Gruenzig to mention a few not to forget the most amazing story involving the young German Werner Forssmann. The unifying factor amongst them was a desire to achieve their dreams and bring their ideas to fruition . We also get an insight into their private lives. It's clear that most of them don't exhibit characters one would want to emulate but there's no denying their legacy and their immense contribution to mankind.
I listened to the audio version of the book. It was very well narrated by Jonathan Yen. The narration flows smooth and at an appropriate pace for this book.
Dr. Forrester shows his humility by proudly recognizing the achievements and good work of his mentees who continue to refine the trade. We are fortunate that as a result of great work done by the " Mavericks and Rebels ' the advancements in cardiology has caused tremendous decline in morbidity and mortality from cardiovascular disease.
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The Mercure Rio de Janeiro Botafogo Hotel is located in Botafogo, under the arms of Christ the Redeemer, one of Rio de Janeiro's picturesque sites. Easy access to the city center and the Metro and close to Rio Sul, Botafogo Praia Shopping and the UnibancoCinema area. The hotel is a few minutes from the Botanical Garden and Lagoon.
Hotel with 44 apartments. The 3 star Mercure Apt Botafogo is located in a business district where many multinational companies are based. It is only a 10 minute drive to the Jardim Botanico and the Rodrigo de Freitas Lake and downtown Rio and the famous beaches of Ipanema and Copacabana are easily accessible.


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Sean O'Grady: With the rescue Ireland's troubles really begin
The trouble with being rescued, as the Irish may discover, is that the people from the European Union or the International Monetary Fund who fly in to run your economy have no more a monopoly on wisdom than the "Useless gobshites" who have recently been relieved of that duty.
(That description, by the way, of the Taoiseach and his colleagues comes from a recent front page of the Irish Daily Star, and has not been bettered).
The IMF in particular has a poor track record in imposing "solutions" that make matters worse, even though harsh medicine is always expected. Though such judgements are difficult to hand down in the field of economics, the IMF's prescriptions after the East Asian crisis in 1997, the last big trauma before our current travails, were especially short on wisdom, and sowed the seeds for the current crisis. Then, as now, some smaller nations became insolvent and had to be bailed out. Then as now the contagion spread. And, in return for a bailout, the IMF squeezed the life out of the entire region, prescribing public spending cuts, interest rate rises and the usual round of privatisations.
Yet all the IMF did in 1997 and 1998 was to induce a second round of trouble. The contagion spread to Russia, and, in turn, to Long-Term Capital Management, a hedge fund, which had to be rescued by a consortium of Wall Street banks backed by the US Fed. The world was lucky that this was the last domino to fall.
Mercifully, the IMF does not seem to want to repeat that error in Europe. The more lasting damage from the IMF's mistakes in Asia was caused by the lesson that China learned – the need to build up huge trade surpluses and colossal reserves so the IMF would never be needed. When these "global imbalances" were lent back to the West they fuelled the Noughties bubble and, thus, framed the credit crunch and recession we have recently crawled out of.
Then again, older readers may recall the IMF's brief period of suzerainty over the British economy after the sterling crisis of 1976. The IMF asked Britain to do what it ought to have done in any case – to live within its means and stop inflating the currency. The episode wasn't that painful. But it did mark a low point in our fortunes and was no end of a lesson – as one must hope it will be for Ireland.
Belfast Telegraph


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Baingan Ka Bharta (bihari Style)
See Discussion:
Ingredients Nutrition
- 1 large eggplant
- 1 medium onion, chopped finely
- 7 cloves dried garlic
- 1⁄2 inch ginger, finely chopped
- 1 bunch dhania patta, finely chopped
- 1 tablespoon oil (mustard oil preferably)
- salt
- 1 teaspoon pickle masala (optional)
Directions See How It's Made
- Wash the baingan, wipe dry it.
- Apply little oil on its surface all around and roast it over the flame, turning around on the fire, till whole of its outer surface turns blackish and flaky.
- Peel off the burnt skin of the roasted baingan.
- Add onion, ginger, garlic, dhania patta, salt, and uncooked mustard oil.
- Mash everything well, leaving no lumps of unmashed baingan.
- Pickle masala esp.
- mango or red chilly pickle masala is also added to this bharta to enhance the taste.
- Serve with daal-rice, khichdi or stuffed parathas.


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Here we have a great variety of recipes for breakfast, from apple raisin bread to gluten-free sugar cookies, from turkey sausage breakfast to cheesecakes and cupcakes.
Here you're gonna find a great variety of breakfast recipes in order to make any kind of breakfast you want to, you can find a recipe so as to make english breakfast such as Turkey sausage breakfast recipe, then you can choose other breakfast recipes useful to prepare continental breakfast like Apple pie, Whole wheat banana waffles, chocolate shortbread tarts with brown sugar whipped cream and chopped hazelnuts recipe and Gluten free sugar cookies.
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Garlicky Gilroy Chicken Wings
READY IN: 1hr 30mins
Recipe by Mooseybear
I made these for dinner tonight, and they were the best wings I have ever had! I used sour cream as a dip for them, and they were a hit. Recipe is from Pilsbury.
Top Review by Chef MB
Can't believe I never rated these. They are delicious, especially if you're a big garlic fan. When i was out of olive oil once, I used an Italian dressing in place of it and the result was different but still delicious. Messy to make, but so is anything with bread crumbs.
See Discussion:1 Review
Ingredients Nutrition
- 2 lbs chicken wings
- 2 heads fresh garlic, seperated into cloves and peeled
- 1 cup olive oil, plus additional for greasing
- 1 teaspoon hot pepper sauce (to taste)
- 1 cup parmesan cheese, grated
- 1 cup Italian style breadcrumbs
- 1 teaspoon black pepper
Directions See How It's Made
- Preheat oven to 375 degrees.
- For each wing, cut through first and second joints, discarding tips.
- Rinse wings and pat dry with paper towels.
- Place garlic, oil, and hot pepper sauce in food processor, cover and process until smooth.
- Pour mixture into a small bowl.
- Combine cheese, bread crumbs, and pepper in a shallow dish.
- Dip wings into garlic mixture, then roll in the crumb mixture, coat evenly. Shake off excess.
- Grease 13x9 inch nonstick baking pan, and arrange wings in a single layer in pan.
- Drizzle remaining garlic mixture over the wings, sprinkle with remaining crumb mixture.
- Bake 45 to 60 minutes or until wings are brown and crisp.
- Garnish with celery sticks, if desired.


text:
Dubai: Indian premier spinner Ravichandran Ashwin has been named as the 12th man in the ICC Test team of the year, while England's Alastair Cook is named captain of the team for the second time in his career.
In addition to Cook, the list includes two other Englishmen in Stuart Broad and Joe Root. This is the fifth time since 2009 that Broad has been selected in the Test team of the year having previously been included in 2009, 2011, 2012 and 2014. For Cook, this is his fourth selection.
Steven Smith is one of three Australian players selected alongside opener David Warner, for the second successive year, and pace bowler Josh Hazlewood. There are also three Pakistan players in the panel; Younis Khan, wicket-keeper Sarfraz Ahmed and leg-spinner Yasir Shah.
New Zealand duo Trent Boult and Kane Williamson make up the starting team.
The ICC on Wednesday announced its Test team of the year, as chosen by a specially appointed selection panel chaired by its Cricket Committee chairman and former India captain Anil Kumble. (ANI)


text:
Can a girl bring up the what are we conversation? How would you feel if she did? Or is it a case of you'll bring it up when you're ready and the girl should just wait?
Most Helpful Guy
I think asking "what are we" is not the best way to go out it, I think being honest about how you feel and telling your partner what you want gives the best result. Asking "what are we" gives full control to your partner, and when you invest your happiness in things that are out of your control, you are subject to a whirlwind of possibilities. Be honest to yourself, and tell your partner what you want, and have them respond to it. That way they can say "yes I would like that too," or "no I am not ready for that." Either way, it will come from a more honest place because you will have been more honest as well.
You also must understand that giving your partner full control may be a responsibility they are not ready for, or it may have been something they have never thought about and all of a sudden you force them to make big decisions very quickly (not smart).
Defining what you want from the relationship and asking if they too would like that is much less overwhelming because in that case they will have something to respond to, rather than being put in a position that in many ways alienates them. The "what are we" talk is always uncomfortable for this reason. It can be worked around! I just explained how.11|00|0
Totally agree with you - "what are we?" is a mutual decision and should be made together through discussion. Saying "I'd like this, would you like that too?" is MUCH better.
I brought the question up with my current boyfriend and that's basically how I went about it. At that point, though, he had already invited me to meet his parents (which I wasn't comfortable with yet), and it felt very much like we were on the same page, so I didn't have any hesitation about asking.. I knew what the answer was already, just wanted to "make it official".
Most Helpful Girl
Tis like the worst conversation to have :(00|00|0


text:
GONZALES — The Louisiana Attorney General's Office found earlier this month that a future water impact fee in Ascension Parish is a fee the Parish Council can adopt, but not a tax that requires voter approval.
This legal opinion would seem to bolster some parish officials' case that such fees are a legal means of raising new revenue that don't need voters' approval at the ballot box.
But Ascension Parish government is not proposing a water impact fee, a variety of parish officials said, and they never sought the attorney general's opinion or were even aware of that opinion had been issued May 15.
Lester Kenyon, parish government spokesman, said Wednesday that he asked Parish President Tommy Martinez, Chief Administrative Officer Ken Dawson, Planning Director Ricky Compton and Parish Attorney O'Neil Parenton Jr. about the opinion on water impact fees.
"They all gave me that look of 'What are you talking about?'" Kenyon said.
Parish officials added that Ascension already collects a 5 percent franchise fee from the parish's largest private water provider. The fee revenue has been used to install fire hydrants throughout Ascension.
It turns out state Rep. Erich Ponti, R-Baton Rouge, asked for the opinion at the request of the Capital Region Builders and Louisiana Home Builders associations.
But Ponti, who is a builder and a member of both groups, said Friday that the opinion primarily arose from concerns about the city of Gonzales' adoption sewer impact fees.
He said the associations — both have traditionally opposed impact fees — wanted to know what steps had to be taken to implement the fees. That the AG's opinion delves into water impact fees, as opposed to other kinds, was apparently a miscommunication, he said.
Ponti, who does not represent a single voter in Ascension Parish, defended the appropriateness of his interest in Ascension's governmental matters. He said what happens in Ascension affects businesses in the Baton Rouge region and that his role as a legislator is not limited to House District 69 in suburban Baton Rouge.
"I also represent the entire state. All of my votes in the Capitol are not specific to my district, and this is no different," Ponti said.
Impact fees are an attempt to have developers and others pay for stresses put on roads, sewers and other infrastructure. With traffic impact fees, for instance, fees on new housing could go toward increasing road capacity.
Lynda Evans, executive vice president of the Capital Region Builders and a member of an Ascension impact fee subcommittee, said the request from her group was about Gonzales' sewer impact fees.
Under AG's Office rules about the issuance of its opinions, elected state officials can request opinions on legal questions related to state law.
The city of Gonzales adopted sewer impact fees in October 2013 with little opposition from the public. The fees took effect in January 2014.
Ponti said he believed he requested the opinion about nine months ago, but his request letter to the Attorney General's Office is dated March 17, 2015, and does not mention Gonzales at all. The letter delves into Ascension's home rule charter and argues new traffic and water impact fees in Ascension would be illegal taxes without a public vote.
"The Ascension Parish Planning Commission has discussed both implementing traffic impact fees, which actually came to a vote and failed to pass, and water impact fees on new residential and commercial developments," Ponti wrote.
As Louisiana's fastest growing parish and a suburban hot zone for Baton Rouge, Ascension has toyed with the idea of impact fees for roads and even new sewer systems for at least a decade.
Amid opposition from homebuilders and others, though, successive Parish Councils have remained divided over the issue and, in 2006, the council failed to amass the necessary two-thirds majority to adopt the fees.
Gasper Chifici, a parish planning commissioner and head of the parish subcommittee looking at traffic impact fees, said the subcommittee has recently agreed to update a 10-year-old traffic impact fee study and will probably bring back a recommendation on impact fees in 2016.
But Chifici said he also was unaware of any talk of a water impact fee by the subcommittee. He noted most of Ascension is served by private water companies and was puzzled how a service-based water fee could even be used.
Notably, the new AG's Office opinion says Ascension faces no requirement to conduct a study to set fees but must determine that the cost of the fees "does not materially exceed" the cost of the service being provided.
Parish President Martinez said he called Ponti to find out what was happening. Martinez said he told Ponti that he should have given parish officials a courtesy call in advance. He said Ponti apologized.
Follow David J. Mitchell on Twitter @NewsieDave.


text:
Historian to discuss 'Spotsylvania's
Bloody Angle' at Tallmadge Civil War
Society meeting
Some of the Civil War's most intense fighting will be the subject of the Tallmadge Civil War Society's October meeting.
Nationally renowned Civil War author Kristopher White will present a lecture, "Hell's Half Acre: Spotsylvania's Bloody Angle," for the group on Oct. 18 at 7:30 p.m. The lecture will be held in the Old Town Hall on Tallmadge Circle, and is free and open to the public.
The assault on the Bloody Angle was the most significant combat at Spotsylvania Court House in Virginia in May 1864 -- one of the grisliest encounters between Robert E. Lee's Army of Northern Virginia and Ulysses S. Grant. For more than 20 hours in a driving rain, the soldiers battled hand-to-hand for a segment of earthworks. It was the longest sustained combat operation of the entire war. The lecture will revisit the reasons for the battle, the innovative tactics used to break the Confederate lines, and rehearse the stories of soldiers trapped in a no-man's land.
White is a historian for the Penn-Trafford Recreation Board and a continuing education instructor for the Community College of Allegheny County near Pittsburgh, Pa. A graduate of Norwich University with a master's degree in military history, for nearly five years he served as a staff military historian at Fredericksburg and Spotsylvania National Military Park. For a short time, he was a member of the Association of Licensed Battlefield Guides at Gettysburg. White has addressed more than 60 Civil War roundtables and is the author of six books, including "A Season of Slaughter: The Battle of Spotsylvania Court House." His many articles have appeared in America's Civil War, Blue & Gray, Hallowed Ground and Civil War Times. Copies of White's books will be available for purchase and signing.
Founded in 1986, the Tallmadge Civil War Society is an organization devoted to the study of the American Civil War. It hosts monthly programs featuring authors and noted experts in the field discussing the war's social, political and military history. Membership is open to anyone with an interest in the conflict. For more information about the Society or to join, contact President Ralph Jordan at 330-630-2474.


text:
Jean-Daniel BLASCO, 60 years old, is an engineer graduated from the National Institute of Polytechnics of Grenoble, and has a master degree of economics from the French Petroleum Institute. He made his entire career in Oil and Gas industry, more precisely in Exploration and Production.
He joined the group ELF AQUITAINE in 1982 as an economist, and held several positions in France and in Africa, in development and planning, gas marketing and business development.
After the merger between TOTAL, FINA and ELF groups, he was appointed Vice-President of the Shtockman project, then Managing Director of the TOTAL's affiliate in Yemen. He then moved back to the South West of France as Vice-President of TOTAL's Technical and Research Center in Pau. In 2008, he was appointed Managing Director of the TOTAL's affiliate in Bolivia. He came back to Paris as Vice-President E&P North Africa.
He has been in his current position, Managing Director of Total E&P Libye, for 18 months.
Jean-Daniel BLASCO is married with 3 children.
Over 30 Years of Diversified Experience in Oil and Gas Fields.
1985; B.Sc. Petroleum Engineering, Tripoli University, Libya
2003; M.Sc. Field and Well Management, Robert Gordon University, Aberdeen, UK
Mr. Luca Madeddu has been Managing Director of Morocco at Sound Oil Plc since June 2015. Mr. Madeddu served as Managing Director of Italy at Sound Oil Plc until June 2015 and served as its Managing Director of Apennine Energy Srl, its subsidiary since joining on January 2, 2012. Mr. Madeddu is a Member of the executive management team of Sound Oil. He has over 24 years of experience in the upstream oil and gas industry with ENI, the major integrated energy company. He is a reservoir geologist by background and has extensive experience in hydrocarbon production, field development, petroleum engineering, supply chain management and reservoir engineering. Mr. Madeddu served as a Director of Sound Oil Plc since September 8, 2014 until September 28, 2015. He has managed large teams in complex operations in sensitive onshore and offshore environments and has worked in Venezuela, Nigeria, Indonesia, UK, Congo and Italy. Mr. Madeddu holds a Degree in Geology from Milan University.
Over 27 years of diversified geological experience in the oil industry in Egypt.
-Extensive experience in Exploration and Development wells.
-Proven track of play generation and prospect evaluation by using geological and geophysical data.
-Competence in designing and conducting Exploration and development projects.
-Excellent background in regional subsurface geological studies and basin analysis.
-Well oriented and very good experience using the most modern applications technology of 3D Seismic , Petrel software and Hydrualic frac.
Mr. Abdalkader joined NOC in 1999 as Reservoir Engineer; graduated from Bright Start University-Libya with a bachelor of Petroleum Engineering in 1995, continued his education at University of Missouri – Rolla, USA to complete his Masters of Science degree in Petroleum Engineering in 2007.
Has an experience of 15 years in oil & gas sector as a reservoir engineer, divided between NOC and its oil operation companies.
Mr. Abdalkadeer had also published a technical paper titled "Changes in Near Wellbore Stress and Fracture Gradient Due to Cold Water Injection in a Sirte Basin Field, Libya" , Abu Dhabi, UAE 2009.
Ms. Djeraba is a senior associate in King & Spalding's Global Energy Practice Group, where she specializes in oil & gas. Her activities range from acquisitions (SPAs, farm-in, farm-out agreements, etc.) to project development (negotiation and re-negotiation of concession agreements and PSCs, gas sales agreements, joint operating agreements, etc.) advising on the upstream, mid-stream and downstream sectors.
Although she mainly works on transactional matters, Ms. Djeraba is also involved in energy-related disputes.
Her work geographically focuses on North Africa, from Mauritania to Egypt, with a particularly strong expertise in Algeria.
Ms. Djeraba has recently advised on a potential country entry in Morocco, an IOC on the consequences and implementation of a change of control in Algeria, Mauritania and Libya, and the renegotiation of a concession in Tunisia. She is also currently working on a potential arbitration claim in Libya concerning the impossibility to carry out upstream operations.
Concerning Algeria, she advised several companies on the last oil & gas bid round in Algeria, the country entry of some of the winning bidders, and an IOC on the negotiation of a PSC extension. Ms. Djeraba also assists several IOCs in Algeria on their day-to-day operations in the country.
Ms. Djeraba is a French qualified lawyer based in the firm's Paris office. She was educated/grew up in Algeria, has French/Algerian citizenship and is fluent in French, English and Arabic.
David recently retired (March 2106) from Royal Dutch Shell where his ultimate role was VP for Upstream Development Growth and JVs as well as being Shell's Group Chief Reservoir engineer, a role he had held for 10 years.
Prior roles within Shell included VP Upstream Development for the EMEA region overseeing development engineering work for projects in Europe, Russia, the Middle East and Africa. He had also served in the early 2000's as Shell's Global staff manager for Petroleum Engineering. His early career with Shell was spent as a reservoir engineer and development team lead in Malaysia, Oman, Denmark, UK and The Netherlands.
David is British. After obtaining his degree in Applied Physics at Strathclyde University he worked for two years as a Schlumberger engineer in South America. He subsequently gained a Masters in Petroleum Engineering from Heriot Watt and joined Shell in 1979. He is a long standing member of the SPE.


text:
Autometer Black Shift Light
Product Details
Grab that gear with authority! Auto Meter's Pro Shift-Lite is the perfect choice for professional racers who desire a larger sized unit for enhanced visibility. A bright, quick response light system used for the perfectly timed shift, making this light an exciting accessory for the new Sport-Comp Playback tachometer. These great looking shift lights are available in either black or silver, to match any Auto Meter tachometer.
This lightweight aluminum mini warning light operates the same as 3240 Pro-Lite by giving you unmistakable warning during the heat of racing action. A bright red incandescent bulb attracts immediate attention (night cover included). 12-Volt power and switch required
Made from high impact ABS plastic with textures to give OEM look.
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Bestselling Author Paula Wiseman Releases Sanction--Book Four in Her Christian Fiction Series
Retired from teaching and with her cancer gone, Bobbi struggles to find purpose beyond caring for her family. When she proposes taking over Brad's mission, it touches off an unforeseen conflict with her husband. Chuck's quest for justice unexpectedly forces him to reexamine his thinking when he realizes that he must must seek and offer forgiveness and grace from the unlikeliest people.
"I'm so honored to publish another one of Paula's amazing Christian novels here at MindStir Media Christian book publishers USA," said J.J. Hebert. "She's attracted a very large following the last few years, and for good reason: Her Christian books are incredibly real, which is rare in Christian fiction."
Mrs. Wiseman's books have earned literary awards and landed on Amazon.com bestseller lists, even achieving the #1 spot on Amazon's Hot New Release list for Christian book publishers USA fiction. She has also appeared on Lifetime TV's "The Balancing Act" to discuss her inspirational books.
About the author:
Paula Wiseman was born during the Summer of Love in Ashland, KY. She's wife and mom of three, and a former chemist. She homeschools her children, is an avid runner and a huge football and basketball fan. She blogs at http://www.paulawiseman.com.
About the publisher:
MindStir Media is a full-service self-publishing company that offers Mind-Stirringly Easy Book Publishing. MindStir's publishing services include mentoring from a bestselling author; custom book design; royalties up to 100%; publishing in paperback, hardcover, and ebook; book distribution and marketing. Visit the publisher's website at http://www.mindstirmedia.com.
J.J. Hebert
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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Hallo Jotform, my contact form https://form.Asked by trueiconic on September 20, 2016 at 01:47 AM
Hallo Jotform, my contact form https://form.jotform.me/61031112234435
is not received email, email@example.com
This is a re-post of a comment on Why I am not Receiving Email Notifications?
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Your account was in the bounce list, we had removed tour account from bounce list. To prevent it happen again please look at the post below.
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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
PaymentAsked by tepeyac on December 18, 2015 at 12:45 PM
Good morning,
I currently made a payment for my subscription for jobform. You dont offer the premium plan anymore?
thank you!
Yes, as of 2015 we updated our plans. Premium, Economic, and Professional are no longer available. But the equivalent new plans are available Bronze, Silver and gold.
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A shocking look inside the complex and ever changing olive oil industry. Author, Tom Mueller has given us one of the most extensive and informative books ever written in regard to "Extra Virgin" olive oil.
All material contained within this website Copyright ©2013-2015 All Of Oils. Web design by PostIdol Media. Copyright ©2013-2015.


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Date wordingAsked by kingfisherweddings on January 06, 2016 at 06:49 AM
Is there a way I can make the date of say 21/01/2016 appear as the 21st of January 2016 in thank you's and autoresponders?
JotForm Support
I am sorry to say that, it is not possible with the ios7 Time widget. However, we can solve it with DateTime Tool.
Here is how to do:
Now, you can copy the name of your field.
Then, paste it to your Thank you Page.
For autoresponder, make the arrangements as you see below:
Please note that, at that situation, it will be shown as Sat Jan 09 2016.
I hope it helps.
If you have any question or issues, please do not hesitate contacting us. We will be glad to assist you.
I haven't tried this yet as I think I got around it a different way but looking at your method, what makes the date show up as Sat Jan 09 2016 rather than 09/01/2016?
06.01.2016, 15:09, "JotForm" :
JotForm Support
The changes to the fields display settings are what makes the change. The example is using a regular date/time field, rather than the time widget.


text: By The Associated Press___ Facebook unveils social search feature MENLO PARK, Calif. (AP) â¿¿ Facebook CEO Mark Zuckerberg unveiled a new search feature on Tuesday in the company's first staged event at its Menlo Park, Calif., headquarters since its May initial public offering. Called "graph search," the new service lets users search their social connections for information about people, interests, photos and places. It'll help users who, for instance, want to scroll through all the photos their friends have taken in Paris or search for the favorite TV shows of all their friends who happen to be doctors. Until now Facebook users were unable to search for friends who live in a certain town or like a particular movie. With the new feature, people can search for friends who, say, live in Boston who also like the film "Zero Dark Thirty". ___ Wal-Mart to hire vets, buy American NEW YORK (AP) â¿¿ Why wait on Washington to fix the economy when there's Wal-Mart? Wal-Mart Stores Inc., the world's largest retailer and the biggest private employer in the U.S. with 1.4 million workers here, said Tuesday that it is rolling out a three-part plan to help jumpstart the sluggish U.S. economy. The plan includes hiring more than 100,000 veterans in the next five years, spending $50 billion to buy more American-made merchandise in the next 10 years and helping its part-time workers move into full-time positions sooner. The move comes as Wal-Mart attempts to bolster its reputation, which has been hit in the past year by an alleged bribery scandal in Mexico and a deadly fire at a Bangladesh factory that supplies clothes to the company. Wal-Mart, which often is criticized for its low-paying jobs and buying habits in the U.S., said it's plan aims to highlight career opportunities in the retail industry, which supports one in four jobs in the country. The company's plan could have an impact on the U.S. economy. With $444 billion in annual revenue, if Wal-Mart were a country, it would rank among the largest economies in the world.