Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.
The involvement of SFs in multiple diseases and processes was the driving force behind the idea to develop SF specific inhibitors. SFs bind, in most cases, to a degenerate motif in the pre-mRNA of the target gene and either recruit or repel the spliceosome to/from nearby splice sites1. Many SFs also possess RNA-independent functions, such as protein–protein interactions in cellular complexes, which are essential for proper cellular functions2. Inhibition of SF expression by siRNAs or antisense oligonucleotides could have broad detrimental effects on cell fate3,4,5. Therefore, development of an efficient SF inhibitor should ideally target only the splicing activity of the factor, without interfering with its other activities.
Current oligonucleotides-based technologies include: Antisense GAPmers, which are designed to knockdown gene expression by binding to specific mRNAs and activating their degradation by RNAse H ; Splice Switching Oligos, which hybridize to pre-mRNA molecules, interfere with the binding of splicing factors or spliceosomal components and shift the splicing between splice sites or affect inclusion/skipping of specific exons; and siRNAs, which are designed to knockdown gene expression and are usually double stranded (reviewed in the ref. 6). All of these oligonucleotide technologies are based on binding/hybridization to either pre-mRNA or mRNA. Here we present a technology using sense oligonucleotides that bind to RNA binding proteins rather than RNA. The only known similar approaches of nucleic acids designed to bind proteins are DNA (double stranded) oligonucleotides, that act as transcription factor decoys7,8 and RNA aptamers, which are RNA molecules (sometimes much longer than the RNA oligonucleotides mentioned above) with a specific 3D structure that can bind many types of proteins according to the designed specificity (not only RNA binding proteins)9.
In order to test the feasibility of using decoy RNA oligonucleotides to inhibit splicing factor activity we chose to target three alternative splicing factors; RBFOX1/2, PTBP1, and SRSF1. RBFOX1 and RBFOX2 are members of a splicing factor family known to be involved in multiple diseases. Altered expression of RBFOX2 in ovarian and breast cancer causes altered splicing of specific targets10. RBFOX1, also known as A2BP1, is deleted in 10% of glioblastoma multiforme and can act as a tumor suppressor11. Abnormal expression of RBFOX1/2 may play a role in neuroblastomas and glioblastomas12,13,14,15, epilepsy and mental retardation16. These proteins have also been shown to be involved in heart and muscle development and function in zebrafish muscle development17. Recent studies showed that RBFOX2 is important for myoblast fusion during myogenesis in mice18 and that repression of RBFOX2 is linked to heart failure19. RBFOX1/2 regulate hundreds of splicing events as measured by their direct RNA binding using RNA CLIP and RNA-seq experiments20,21,22. PTBP1 and SRSF1 are two splicing factors known to be involved in cancer. PTBP1 is aberrantly elevated in glioblastomas and serves as a marker for glioblastoma progression23. The oncoprotein splicing factor SRSF1, is amplified in breast cancer and is a potential target for cancer therapy24,25,26. Most splicing factors bind degenerate motifs in the pre-mRNA of their target genes. However, RBFOX1/2 proteins bind a well-defined motif (U)GCAUG12,16, which makes it an ideal target for specific inhibition.
A requirement for any SF inhibitor is that its activity should be limited to targeting only the splicing activity of the factor, without interfering with other activities of the factor. Many splicing factors possess RNA-independent functions. These functions include protein–protein interactions in cellular complexes, which are essential for proper cellular functions. For example, RBFOX proteins are part of a large assembly of splicing regulators (LASR). One of the LASR subunits, hnRNP M, also has a function independent of the complex and RBFOX1/2 can stimulate splicing repression mediated by hnRNP M in an RNA binding-independent manner21. Another example of a splicing factor functioning in protein interactions is the oncoprotein SRSF1. SRSF1 has a role in stabilizing p53 through interaction with RPL5, which induces senescence in cells2. Therefore, simple knock-down or inhibition of the SF would have multiple negative effects on the cell. Ideally, a decoy oligonucleotide should target only the splicing activity of the factor. Moreover, the efficacy of splicing factor knockdown by siRNAs or antisense oligonucleotides is dependent on the half-life of the factor and the integrity of cellular components (e.g., RNAi machinery), while the decoy oligonucleotide can act directly and immediately, independent of any cellular component.
Here we provide a proof-of-principle demonstration that RNA decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1, and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. Decoy oligonucleotides present a methodology to specifically downregulate SF activity and have the potential to treat diseases where SFs are upregulated. Relevant to this, we demonstrate that inhibition of the oncogenic splicing factors SRSF1 and PTBP1 inhibits the oncogenic properties of cancer cells.
We designed RNA decoy oligonucleotides directed against three alternative SFs; RBFOX1/2 (RBFOXi), PTBP1 (PTBP1i) and SRSF1 (SF2i1 and SF2i2). The oligonucleotides were predicted to bind the RRM domains of each SF (Fig. 1a), based on several studies1,12,27,28. Each oligonucleotide contains three/four tandem motif repeats. An oligonucleotide that does not resemble any of the known exonic splicing enhancer motifs29, scrambled (SCRM), was used as a control in all experiments. Several biological experiments were performed with additional control oligonucleotides. The decoy oligonucleotides are single stranded RNA molecules with a 2′-O-methyl modification on the ribose of each nucleotide, which increases the stability of the molecule6. For some in vitro experiments these oligonucleotides were used, with an additional Cy5 or biotin modification on the 5′ end. For in vivo experiments in zebra fish, oligonucleotides were also modified with a phosphorothioate backbone.
Transfection of the Cy5 labeled RBFOXi oligonucleotides into U87MG cells resulted in nuclear localization (Supplementary Fig. 1A). Cy5 RBFOXi oligonucleotides were detected in the nucleus and cytoplasm as early as 1 h post-transfection but were not detected in the endosomes (Supplementary Fig. 2). The amount of oligonucleotides in the cytoplasm and the nucleus increased at 3 h post-transfection (Supplementary Fig. 3A). Even after 11 h post-transfection there was no evidence of co-localization of the oligonucleotides with endosomes (Supplementary Fig. 3B, Supplementary movie 1, 2, 3). Performing quantification on 213 U87MG cells transfected with RBFOXi we estimated that at least 23% ±10 of the oligonucleotides are localized to the nucleus (Supplementary Data 1).
A pull-down assay performed with nuclear extract efficiently pulled-down RBFOX2 protein only with the biotinylated RBFOXi oligonucleotide and not with the SCRM oligonucleotide. In addition, RBFOXi did not interact with other splicing factors that were examined as controls (Fig. 1b). Mass spectrometry-based proteomic analysis of the pulled-down proteins revealed specific binding of only RBFOX1/2 to the RBFOXi oligonucleotide (Fig. 1c and Supplementary Data 2). NMR is a very sensitive approach allowing the detection of weak interactions. We used NMR to compare RBFOX1 RRM (identical to RBFOX2 RRM) interaction with the RBFOXi oligonucleotide compared to a control oligonucleotide (UCAGAGGA), which was shown to be specifically recognized by SRSF125. Only small chemical shifts were observed with the control oligonucleotide compared to the very large perturbations observed with the RBFOXi oligonucleotide (Supplementary Fig. 4). This result further demonstrates the binding specificity of the RBFOX1/2 binding motif and suggests that the SRSF1 motif would not efficiently interact with RBFOX1/2 proteins.
A recent study showed that RBFOX2 is in a complex with hnRNP M, in an RNA independent manner21. Indeed, in the mass-spectrometry analysis of the pull-down assay we identified hnRNP M as a RBFOXi interactor, even though it was not statistically significant (Supplementary Data 2). A co-IP assay showed that the protein–protein interaction between RBFOX2 and hnRNP M remained intact even after transfection of RBFOXi into the cells, re-enforcing the prediction that the decoy oligonucleotide does not interfere with protein–protein interactions (Fig. 1d). In addition, introduction of RBFOXi into cells did not significantly affect the protein levels of RBFOX2 (Supplementary Fig. 1B).
After determining that RBFOXi successfully binds to RBFOX2 (Fig. 1b) we examined if this binding results in changes in splicing of known RBFOX1/2 targets16,22. Splicing of known target genes was affected by introduction of RBFOXi to U87MG cells (Fig. 1e) and MDA-MB-435S cells (Supplementary Fig. 1C) in a dose dependent manner (Supplementary Fig. 1D). Splicing changes of some of the targets were observed as early as 4 h after transfection and persisted up to 96 h, demonstrating the rapid and enduring biological effect of the decoy oligonucleotides (Fig. 2a). These alternative splicing changes are similar to the changes observed in cells upon RBFOX2 knockdown (Supplementary Figure 1E-F) and previous reports16,22. In order to assess the effect of RBFOXi across the entire transcriptome we performed RNA-seq on U87MG cells transfected with either RBFOXi or SCRM. Data analysis with rMATS30 revealed splicing changes (between RBFOXi and SCRM transfected cells) in 73 alternative exons (Table S3); 49 of which were previously reported to have altered splicing in RBFOX1/2 knockdown cells (changes were in the same direction in both studies)21 (Fig. 2b, c and Supplementary Data 3). Moreover, out of the 73 splicing events affected by RBFOXi, 39 of the downstream introns contained the consensus RBFOX1/2 binding motif UGCAUG, while out of the 457 splicing events affected by RBFOX2 knockdown only 133 of the downstream introns contained this motif. These differences are statistically significant (Fig. 2b). The fact that RBFOX1/2 can also affect splicing in an RNA binding-independent manner21 might explain why the decoy oligonucleotide affected the splicing of fewer genes than RBFOX1/2 knockdown.
We used the switchSENSE approach31,32 (Fig. 3a) to investigate whether an oligonucleotide containing multiple tandem motifs would have increased affinity for the RBFOX1/2 RRM. Oligonucleotide sequences containing either, one, two, three or four consecutive binding sites (RBFOXi ×1 to ×4) and random sequences of similar lengths as controls (SCRM×1 to ×4) were first tested for protein binding kinetics. Increased binding caused delay in the apparent fluorescence kinetics (Fig. 3b). We found that the RBFOX1 RRM binds oligonucleotides containing the RBFOX1/2 motif with delayed kinetics, which increased with increasing number of binding sites (Fig. 3b bottom left panel, Supplementary Fig. 5). No binding was detected with the scrambled oligo (Fig. 3b, top left panel, Supplementary Fig. 5). Although the model used to evaluate the data does not allow exact determination of the molecular weight and stoichiometry of protein-RNA complexes, the method does indicate changes in the size of the protein-RNA complexes. Whereas no binding was observed with the scrambled sequences, an increase in size was detected with oligonucleotides with increasing number of binding sites (Fig. 3b, right panel). We then investigated whether an increase in the number of RBFOX1/2 motifs was associated with increased affinity to RBFOX1/2 RRM. We performed switchSENSE titrations of RBFOXi ×1 to ×4 oligonucleotides with increasing concentrations of RBFOX1/2 RRM to determine the Kd values. We observed a positive correlation of affinity of RBFOX1/2 RRMs with increased number of binding sites. While the oligonucleotide with one motif repeat had a Kd of 2.5 nM, the oligonucleotide containing four repeats had a Kd of 0.6 nM (Fig. 3c). Given that we observed an increased number of RBFOX1/2 RRMs bound to the oligonucleotides with multiple motifs (Fig. 3b), we hypothesize that the lower Kd of the oligonucleotide containing four repeats is due to cooperativity in binding of the proteins. In agreement with these results, oligonucleotides containing four or five binding sites had the strongest effect on splicing (Supplementary Fig. 7B).
Gallagher et al.17 have shown that RBFOX1/2 proteins are essential for the development and function of muscle and heart in zebrafish. Likewise, Singh et al. showed that RBFOX1/2 is essential for muscle development in mammals18. We utilized the zebrafish model system to determine if inhibition of RBFOX1/2 proteins with RBFOXi results in a similar phenotype as RBFOX knockdown by morpholino antisense oligonucleotides (which block the expression of RBFOX1/2). Fertilized zebrafish eggs were injected with either RBFOXi or SCRM oligonucleotides (Fig. 4a). We observed changes in alternative splicing of four muscle related RBFOX targets17 in RNA isolated from the larvae derived from the RBFOXi injected fertilized eggs (Fig. 4b). Phalloidin labeling of F-actin filaments of these larvae revealed disorganized and wavy muscle fibers, with a higher dose of RBFOXi (8pg) having a more severe phenotype (Fig. 4c, d). This phenotype is similar to those reported by Gallagher et al. upon RBFOX1/2 knockdown17, demonstrating that inhibition of the RNA binding properties of RBFOX1/2 proteins by the decoy oligonucleotides and the concomitant change in alternative splicing, result in a delayed muscle development phenotype in vivo.
To examine the possible therapeutic potential of decoy oligonucleotides against splicing factors, we designed a decoy oligonucleotide against another splicing factor, PTBP1, which is upregulated in several cancers and promotes pro-oncogenic splicing events23,33,34,35. The decoy oligonucleotide against PTBP1 (PTBP1i) is composed of four repeats of the PTBP1 binding motif, CUCUCU36,37. Pull-down assays using HEK293 nuclear extracts showed specific binding of PTBP1 to biotinylated PTBP1i and not to the SCRM oligonucleotide. Similar to RBFOXi, PTBP1i did not interact with other splicing factors (Fig. 5a). Mass spectrometry-based proteomic analysis of the PTBP1i pulled-down fraction showed strong and significant binding to PTBP1 (Fig. 5b and Supplementary Data 4) with no down regulation of PTBP1 protein levels in cells transfected with PTBP1i (Supplementary Fig. 6A). Furthermore, we observed splicing changes of known PTBP1 targets, PKM233,35, RTN438, SNAP91, and PTBP239, when PTBP1i was introduced into breast cancer cells (Fig. 5c). The known involvement of PTBP1 in cancer motivated us to check if inhibition of its splicing activity could inhibit transformation. Indeed, transfection of breast cancer and glioblastoma cells lines with PTBP1i reduced proliferation (almost two-fold), inhibited the formation of colonies in a clonogenic assay and inhibited colony formation in soft agar (Fig. 5d–f and Supplementary Fig. 6B-C). These results expand the possibility of use of decoy oligonucleotides to inhibit another splicing factor, PTBP1, and demonstrate that inhibition of PTBP1 with a decoy oligonucleotide can inhibit oncogenic properties of breast and glioblastoma cancer cells.
Two decoy oligonucleotides, differing by a single nucleotide in the motif sequence, were designed against SRSF1; SF2i1 (5′ (CACAGGA)n, n = 3 repeats) and SF2i2 (5′ (CGCAGGA)n, n = 3 repeats) (Fig. 1a, Supplementary Table 1). A pull-down assay performed with nuclear extracts showed efficient pull-down of SRSF1 protein with the biotinylated SF2i2 oligonucleotide and not with the SCRM oligonucleotide. In addition, SF2i2 did not interact with other splicing factors that were examined as controls (Fig. 6a). Mass spectrometry-based proteomic analysis of the pulled-down proteins revealed specific binding of SRSF1 to the SF2i2 oligonucleotide. However, many additional proteins were also pulled down in the assay (Supplementary Fig. 7A and Supplementary Data 5). Many of these proteins were previously shown to bind SRSF1 directly in an RNA-independent manner40, thus suggesting that they interact directly with SRSF1 protein and not with the decoy oligonucleotide. In addition to its role in alternative splicing, SRSF1 also functions in an RNA-independent manner, as part of the RPL5-MDM2 complex2. We examined if the interaction between SRSF1 and RPL5 still persists in cells transfected with SF2i2. We found no interference of this interaction in cells transfected with SF2i2 (Fig. 6b). This confirms that the decoy oligonucleotide does not interfere with protein-protein interactions in cellular complexes, which can be essential for cellular functions. Transfection of the two decoy oligonucleotides (SF2i1 and SF2i2) into U87MG and MDA-MB-231 cells resulted in changes in splicing of four known targets of SRSF1, INSR41, U2AF142, MKNK224,43, and USP825 with SF2i2 having a stronger effect than SF2i1 (Fig. 6c and Supplementary Fig. 7C). In an attempt to calibrate the optimal number of motif repeats necessary for the splicing effect, we tested SF2i2 oligonucleotides containing one, two, three, four, or five repeats of the CGCAGGA motif. The effect on splicing of SRSF1 target genes positively correlated with the number of motif repeats (Supplementary Fig. 7B). These results are consistent with the results obtained for RBFOXi binding to RBFOX1 RRM using the switchSENSE approach, where we observed higher affinity for RBFOXi ×4 compared to RBFOXi ×1 (Fig. 3c and Supplementary Fig. 5). Splicing patterns of SRSF1 splicing targets were altered in a dose dependent manner (Supplementary Fig. 7D) and without any effect on SRSF1 protein levels (Supplementary Fig. 7E).
To further evaluate the specificity of all decoy oligonucleotides, two splicing targets of each splicing factor were chosen; FMNL3 and NUMA1 for RBFOX1/2, RTN4, and SNAP91 for PTBP1 and USP8 and U2AF1 for SRSF1. Changes in splicing of each target were evaluated after introduction of each decoy oligonucleotide (Supplementary Fig. 8). Changes in splicing of FMNL3 and NUMA1 were seen only after transfection of RBFOXi but not PTBP1i or SF2i2 (Supplementary Fig. 8A). Changes in splicing of RTN4 and SNAP91 were seen only after transfection with PTBP1i (Supplementary Fig. 8B) and significant changes in USP8 and U2AF1 splicing were observed only after transfection with SF2i2 but not with any of the other decoy oligonucleotides (Supplementary Fig. 8C). In addition, we used three unrelated control oligonucleotides; Cont.1, Cont.2, Cont.3. These control oligonucleotides were designed as antisense oligonucleotides against exon 78 of the dystrophin (DMD) pre-mRNA which is expressed mostly in muscle tissues. Moreover, these antisense oligonucleotides did not affect DMD exon 78 splicing, which has no known effect on tumorigenic properties or splicing. We examined the effect of these control oligonucleotides on splicing of SRSF1 targets; USP8 and U2AF1. There were no detectable splicing changes in these targets after introduction of the control oligonucleotides (Supplementary Fig. 9A). These results further strengthen our conclusion that each decoy oligonucleotide acts specifically on its targeted splicing factor. It is important to note that other splicing events may not behave in a similar manner to these specific splicing targets, since they might contain different cis-elements and be regulated by several different splicing factors simultaneously.
One of the known splicing targets of SRSF1 is the kinase MKNK243. Elevated expression of MNK2a, a result of alternative splicing of MKNK2, is known to activate the p38-MAPK stress pathway43. We examined if inhibition of SRSF1 by the decoy oligonucleotides activates this stress pathway. We observed increased phosphorylation of p38-MAPK and its substrate ATF244 upon transfection with either SF2i1 or SF2i2 (Fig. 6d and Supplementary Fig. 7F) and up-regulation of expression of downstream transcriptional targets of the p38-MAPK pathway; COX2, c-FOS, and IL-643,45 (Fig. 6e and Supplementary Fig. 7G). Activation of the p38-MAPK pathway was specific to SRSF1 decoy oligonucleotides and was not observed after transfection with RBFOXi and PTBP1i or with the additional control oligonucleotides Cont.1-Cont.3 (Supplementary Fig. 9B). The role of SRSF1 in activating the nonsense mediated decay (NMD) pathway is also mediated through its RRM domains46,47. We therefore predicted that inhibition of SRSF1 with decoy oligonucleotides would inhibit NMD. We observed an accumulation of three known endogenous NMD-prone transcripts48,49 after treatment with the SRSF1 decoy oligonucleotides, suggesting that NMD is inhibited (Fig. 6f and Supplementary Fig. 7H). Once again, in agreement with previous results (Fig. 6c–e and Supplementary Fig. 7G), SF2i2 had a stronger effect than SF2i1. These results demonstrate that the SRSF1 decoy oligonucleotides inhibit SRSF1's ability to enhance NMD, to regulate alternative splicing, and lead to activation of the p38-MAPK stress pathway without interfering with non-RNA binding domain mediated properties of the protein.
Given that SRSF1 acts as an oncoprotein and its inhibition by decoy oligonucleotides activated the p38-MAPK stress pathway, we predicted that treatment of cancer cells with SRSF1 decoy oligonucleotides would reverse the transformed phenotype of these cells. Indeed, treatment of U87MG and MDA-MB-231 cells with SF2i1 and SF2i2 inhibited proliferation (3 to 4-fold), colony survival in a clonogenic assay and colony formation in soft agar while RBFOXi had no effect in these assays (Fig. 7a–c and Supplementary Fig. 10A-D). In order to confirm the specificity of this effect we performed the same experiments using three unrelated control oligonucleotides; Cont.1, Cont.2, Cont.3. As expected, the control oligonucleotides had no effect on the transformed phenotype of the cells, while SF2i2 had an obvious effect (Supplementary Fig. 11A-C). To verify that the observed reduced oncogenic activity is due to activation of the p38-MAPK stress pathway, we used the p38-MAPK inhibitor SB203580 (Fig. 7d). Cells transfected with SF2i1 and SF2i2 formed more colonies in a soft agar assay and had increased survival in a clonogenic assay (4-fold) after treatment with SB203580 compared to cells treated with vehicle (Fig. 7e, f and Supplementary Fig. 11D-G). A concurrent reduction in ATF2 phosphorylation and decreased expression of downstream targets COX2, c-FOS, and IL-6 was also observed (Fig. 7g, h and Supplementary Fig. 11H). These results demonstrate that SRSF1 decoy oligonucleotides can inhibit malignant properties of glioblastoma and breast cancer cells through activation of the p38-MAPK stress pathway.
We next wanted to test if SRSF1 decoy oligonucleotides can inhibit glioblastoma growth in vivo. U87MG cells were labeled with mCherry and transfected with SF2i2 decoy oligonucleotides. Twenty-four hours after transfection cells were injected into the murine striatum of both brain hemispheres. Three weeks after the injection the mice were sacrificed and the tumors visualized under a fluorescent stereoscope. The mice injected with cells transfected with the SF2i2 decoy oligonucleotides developed notably smaller tumors than the control group transfected with the SCRM oligonucleotides (Fig. 8a–c). These results suggest that decoy oligonucleotides against SRSF1 can inhibit glioblastoma tumor growth in vivo.
In this report we demonstrate that decoy oligonucleotides designed against specific splicing factors can bind the splicing factor, affect alternative splicing of known targets and have biological effects in line with inhibition of their splicing activity.
There are multiple advantages of the modified RNA decoy oligonucleotides presented here over existing technologies, such as siRNA or antisense oligonucleotides. One of the advantages is that the decoy oligonucleotides target the RNA binding domain of the splicing factor, inhibiting only its RNA binding activities without interfering with its other activities, such as protein–protein interactions. This is in contrast to knockdown technologies, which inhibit the expression of the protein and thereby inhibit all the activities of the splicing factor (some of which can be RNA-independent). Thus, it is expected that decoy oligonucleotides will have fewer side effects than complete knockdown, as suggested by the smaller amount of splicing events affected by the decoy against RBFOX1/2 than RBFOX1/2 knockdown (Fig. 2b). Another advantage is that these oligonucleotides are not dependent on the half-life of the splicing factor (and thus can act immediately as seen in Fig. 2a) or on any cellular mechanism that can be mutated in certain cancer cells (e.g., siRNAs, which depend on the cellular machinery). Moreover, as some splicing factors are highly conserved through evolution, the decoy oligonucleotides might inhibit splicing factors in organisms, other than mammals, where there is no RNAi machinery present (e.g., plasmodium falciparum)50. Finally, the decoy oligonucleotides are single stranded RNA molecules, which have been shown to be successfully delivered into organs of the body51 much more efficiently than double stranded siRNA molecules6.
In our attempt to understand the biochemical basis for decoy oligonucleotide binding to the RRM of a specific splicing factor, we determined that the affinity of a decoy oligonucleotide with four repeats of the binding motif is almost five times greater than an oligonucleotide with a single binding motif. The explanation for this increased affinity probably resides in the fact that the additional motifs within the decoy oligonucleotide increased the number of splicing factor molecules bound to the oligonucleotide, suggesting cooperative binding of these proteins (Fig. 3). It has previously been shown that mutant splicing factors, for which the mutation decreased their affinity to the target RNA motif in vitro by a factor of five, had reduced splicing activity in cells42,52.
In this paper, we demonstrate the successful use of decoy oligonucleotides targeting three different splicing factors; RBFOX1/2, PTBP1, and SRSF1. The in vivo effect of the RBFOX1/2 decoy oligonucleotides on splicing patterns and muscle development in zebrafish were very significant. The changes observed after oligonucleotide introduction into cells did not interfere with protein–protein interactions between RBFOX2 and hnRNP M, emphasizing the preciseness of this method. Similar to the decoy oligonucleotides against RBFOX1/2, we did not observe any interference with SRSF1 protein–protein interactions, such as the SRSF1-RPL5 complex, while other RRM related functions, such as NMD, were inhibited. Taken together, these results show that the mode of action of the decoy oligonucleotides is specific and affects RNA binding-mediated functions of the splicing factor. In contrast to the RBFOX1/2 decoy oligonucleotides, which showed specific binding to RBFOX1/2 proteins in the proteomic analysis, the SRSF1 decoy oligonucleotides bound multiple proteins in addition to SRSF1. This lack of specificity could be attributed to the SR protein family which is known to have redundant activity for some substrates in vitro and in vivo53,54. Another explanation is the limitation of the proteomic assay. This assay cannot distinguish between proteins that bind directly to the decoy oligonucleotides and indirect binders that bind SRSF1 through protein–protein interactions. A large number of proteins identified in the SRSF1 proteomic analysis have been recently demonstrated to bind SRSF1 through protein–protein interactions40. Even though the binding motif of some splicing factors is not well defined, we observed high specificity to specific splicing targets. Several SRSF1 targets were solely affected by the SRSF1 decoy oligonucleotides and not by the PTBP1 and RBFOX1/2 decoy oligonucleotides and vice versa, the SRSF1 decoy oligonucleotides did not affect splicing of some of the PTBP1 and RBFOX1/2 splicing targets tested (Supplementary Figs. 8 and 9). Moreover, only the SRSF1 decoy activated p38-MAPK while all the other decoy and control oligonucleotides did not (Supplementary Fig. 9). The decoy oligonucleotide against PTBP1 was successful in inhibiting the oncogenic properties of the cells, which opens up the possibility of treatment of cancer with these types of decoy oligonucleotides, targeting splicing factors that are upregulated in certain cancer types. We also show that inhibition of SRSF1 with decoy oligonucleotides has an effect on the oncogenic properties of cancer cells and identified that this occurs through activation of the p38-MAPK pathway. Finally, glioblastoma cells transfected with SF2i2 and injected into the mouse striatum developed smaller tumors, or no tumors, compared to SCRM transfected cells. These results demonstrate the clinical potential of the decoy oligonucleotide strategy.
Inhibition of pleotropic splicing factors such as PTBP1, SRSF1, or RBFOX1/2 is expected to affect the splicing of hundreds of genes25. Thus, the question of toxicity of their inhibitors is a major concern for such an approach. However, many oncogenes and oncogenic pathways (e.g., c-Myc, EGFR, Akt, mTORC1, Ras/Raf, and others), which regulate normal growth, are valid targets for cancer therapy. The question of toxicity due to inhibition of each of these splicing factors and others must be evaluated carefully. Another complexity in inhibiting splicing factors is that they function in many steps of RNA processing; splicing, mRNA export, NMD, and translation55. Thus, even a specific inhibition of their RNA binding activity can affect several different functions. Some of these functions may be beneficial for inhibition of their oncogenic activity and others may not. The advantage of competitive decoy oligonucleotides over knockdown methods is that the level of inhibition can be better controlled in a dose-dependent manner. In addition, the decoy oligonucleotides do not interfere with other functions of the splicing factors (not related to RNA processing), a fact that is expected to reduce the toxicity of this approach compared to knockdown of the splicing factor.
In conclusion, the decoy oligonucleotide technology presented here is the first type of intelligence-based design of molecules that can directly and specifically inhibit the activity of RNA binding proteins (RBPs), such as splicing factors; a substantial advantage over traditional knockdown methods.
Fixed cells images were taken using a Zeiss LSM710 confocal microscope and processed using ZEN lite 2011 blue edition (v2.3). Live cells images were taken by Nikon SMZ18 stereomicroscope using NIS–Elements Br software.
U87MG, MDA-MB-435S, MDA-MB-231, and HEK293 cells were grown in DMEM supplemented with 10% (v/v) FCS, penicillin, and streptomycin.
Cells were lysed in Laemmli buffer and analyzed for total protein concentration56. Fifteen microgram of total protein from each cell lysate was separated by SDS-PAGE and transferred onto a PVDF membrane (Invitrogen). The membranes were probed with primary antibodies. Primary antibodies: RBFOX2 (1:4000, Sigma), SRSF1 (1:200, mAb AK96 culture supernatant57), SRSF5 (1:3000, Sigma), SRSF6 (1:500, mAb 8-1-28 culture supernatant), OctA (Flag tag) (1:500, Santa Cruz), hnRNPM (1:500, Novusbio), PTBP1 (1:10,000, Abcam), T7 (1:5000 Novagen), phospho-p38 (Thr180/Tyr182) (1:1000 cell signaling), p38 (1:1000 Santa Cruz), phospho- ATF2 (Thr71) (1:1000 Cell Signaling), ATF2 (1:1000 abcam), β tubulin I + II (1:1000, Sigma), β-actin (1:200, Santa Cruz), GAPDH (1:1000, Santa Cruz), Secondary antibodies: HRP-conjugated goat anti-mouse, goat anti-rabbit, or donkey anti-goat IgG (H + L) (1:10,000, Jackson Laboratories).
HEK293 cells were transfected with pCDNA3-Flag- RBFOX2 (received from the Krainer Lab, Cold Spring Harbor Laboratory), pCDNA3-T7- SRSF158, or pCDNA3(−). Twenty-four hours after plasmid transfection, cells were transfected with oligonucleotides (RBFOXi, SF2i2 or SCRM). Forty-eight hours after oligonucleotide transfection cells were fractionated and lysed with in nuclear extract buffer (described in pull down assay). One microgram of anti-Flag or anti-T7 antibody bound to 50 µl of 50% protein G-sepharose slurry was incubated with 500 mg of nuclear extract overnight. After washing four times with nuclear extract buffer, beads were incubated with 50 µl of 2× Laemmli buffer and separated by SDS-PAGE.
Total RNA was extracted with TRI Reagent (Sigma) and 1 μg of total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was conducted on 1 µl of cDNA by KAPA 2G Fast HS ReadyMix PCR kit (KAPA Biosystems). PCR conditions were as described in manufacturer's protocol of ReadyMix with the addition of 5% (v/v) DMSO for either 30, 35, or 40 cycles. PCR products were separated on 2% agarose gels. Primers listed in Supplementary Table 1.
Total RNA was extracted with TRI Reagent (Sigma) and 1 μg of total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed on the cDNA using SYBR green (Applied Biosystems) and the CFX96 (Bio-Rad) real-time PCR machine. Normalization was performed using either β-actin or RPL14 and SCRM value was arbitrarily set to 1. Samples were compared to a standard curve, which was established by serial dilutions of a known concentration of cDNA. The PCR reaction is composed of the following steps: one cycle for 30 s at 95 °C and 39 cycles of 5 s at 95 °C and 30 s at 59–63 °C according to Tm of primers. Primers are listed in Supplementary Table 1.
Cells were transfected with oligonucleotides using lipofectamine 2000 (Invitrogen) according to manufacturer's protocol.
To generate stable cell lines, U87MG and MDA-MB-231 cells were transduced with MSCV-puro- shRNA retroviral vectors against RBFOX2 (received from the Krainer Lab, Cold Spring Harbor Laboratory). Medium was replaced 24 h after infection, and 24 h later, infected cells were selected with puromycin (2 μg/ml) for 72 h.
Twenty-four hours post-transfection 15,000 cells per well were seeded in duplicates in 6-well plates. Each well was coated with 2 ml of bottom agar mixture (media, 10% FBS, 1% agar). After the bottom layer had solidified, 2 ml of top agar mixture (media, 10% FBS, 0.3% agar) containing the cells was added. After this layer had solidified, 2 ml of media (media, 10% FBS) was added. Plates were incubated at 37 °C with 5% carbon dioxide. Following 10–21 days, colonies from ten different fields were counted and the average number of colonies per well was calculated.
Four thousand cells per well were seeded in 96-well plates 4 h post-transfection in six replicates, fixed and stained with methylene blue. Cell density was determined every 24 h (until 72 h/96 h) by absorbance of methylene blue staining at 655 nm measured on a plate reader (BioRad).
1000, 500, and 250 cells were seeded in 6-well plates. After 10–21 days, cells were fixated with 2.5% glutaraldehyde solution for 10–15 min and stained with 1% methylene blue solution.
In experiments using the MAPK pathway inhibitor, SB203580, the reagent was added 4 h after transfection and remained until the end of the assay. For anchorage independent growth assays and clonogenic assays the concentrations were 0.5 and 1 µM, for Western blot assays and RT-qPCR the concentration was 20 µM.
Nuclear extracts (NE) were lysed in a buffer containing 20 mM Hepes, 1.5 mM McCl2, 420 mM NaCl, 0.2 mM EDTA and 25% (v/v) glycerol. NE was incubated with biotin tagged oligonucleotides. Streptavidin beads (Thermo Scientific), previously blocked with a solution containing heparin, were added after 3 h. The pulled down fraction was lysed in Laemmeli buffer and analyzed by western blot analysis.
Tupfel Long-fin (aka TL) zebrafish breeding and maintenance were performed as previously described59. 5 or 8 pg of oligonucleotides were injected into zebrafish embryos at the 1–2-cell stage. Injected embryos were allowed to develop at 28.5 °C. Forty-eight hours post fertilization embryos were fixed in 4% paraformaldehyde for immunohistochemistry or solubilized in TRI Reagent (Sigma) and analyzed as described for RT-PCR. All experiments complied with ethical regulations for animal testing and research of the Weizmann Institute and IACUC.
U87MG-mCherry cells were injected via stereotactic surgery into the murine striatum in both hemispheres, 2 mm lateral and to a depth of 3 mm to the bregma. Each injection consisted of 2 μl of solution, containing 200,000 cells/μl transfected with decoy oligonucleotides. The mice were sacrificed after three weeks, the brains were extracted and visualized using Nikon SMZ18 stereomicroscope and NIS–Elements Br software.
All animal experiments were performed in accordance with the guidelines of the Hebrew University committee for the use of animals for research. All experiments complied with ethical regulations for animal testing and research, Hebrew University (IACUC) Ethics approval no' MD-15-14634-5. HU is AAALAC approved.
Embryos were fixed overnight in 4% paraformaldehyde and permeabilized with triton. Alexa Fluor 594 conjugated phalloidin (Invitrogen) was used at 1 unit/ml following manufacturer's instructions. Embryos were mounted in 80% glycerol and visualized using fluorescent binocular microscope.
Total of 1 μg RNA was used to prepare libraries using Illumina library kits. 100 × 106 reads of 100 bp from each side (paired-end) were generated for every samples using the Illumina HiSeq machine. STAR aligner (version 2.4.2a) (Dobin et al. 2013) was used to align each read uniquely to the Hg19 human genome (default parameters except alignSJoverhangMin 8, alignSJDBoverhangMin 3, outFilterMismatchNmax 999, alignIntronMin 20, alignIntronMax 1000000, alignMatesGapMax 1000000, outFilterMismatchNoverReadLmax 0.1). To determine differentially spliced exons between the samples we used rMATs (version 3.0.9) using human UCSC known genes annotation. The statistical analysis and comparison with Damianov et al. results were done using R (the R Project for Statistical Computing (http://www.r-project.org/)).
The RNA-seq was performed on two independent biological replicates of cells transfected with SCRM or RBFOXi.
RBFOXi, PTBP1i, and SF2i interacting proteins were subjected to on-bead digestion. The complexes were reduced with 1 mM Dithiothreitol in 2 mM urea for 30 min, followed by alkylation with 5 mM Iodoacetamide in 2 mM urea for 30 min in the dark. Proteins were digested overnight with sequencing-grade trypsin (Promega) and digestion was terminated by adding 0.1% trifluoroacetic acid (TFA). The peptides were desalted and concentrated on C18 stage tips60. Prior to MS analysis, peptides were eluted from the stage tips using 80% acetonitrile, vacuum-concentrated and diluted in loading buffer (2% acetonitrile and 0.1% trifluoroacetic acid) and subjected for MS measurements. LC-MS/MS analysis was performed using nano-ultra high performance liquid chromatography (nano-UPLC; Easy-nLC1000; Thermo Scientific) coupled on-line to a Q-Exactive Plus mass spectrometer (Thermo Scientific). Peptides were eluted using 240-min gradient of water:acetonitrile. Raw files were analyzed with MaxQuant (1.5.3.36) and the Andromeda search engine61,62. MS/MS spectra were searched with reference to human UNIPROT database and a decoy database to determine false discovery rate (FDR). FDR thresholds were set to 0.01 for protein and peptide identification. All the statistical analyses of the MaxQuant output tables were performed with the Perseus software63. For each analysis ProteinGroups file was filtered to include only proteins that were identified in at least two (out of three) replicates of the pull down. We then imputed missing values by replacing them with a constant value that matches the lowest intensity in the dataset (RBFOXi) or by creating a normal distribution with a downshift of 1.8 standard deviations and a width of 0.3 of the original (PTBPi). Different imputation strategies were selected due to the significantly higher purity of the RBFOXi samples. Significant interactors were examined by performing one-sided T-test for RBFOX1/2, PTBP1 against SCRM with permutation-based FDR cut-off of 0.05 and S0 = 0.164 and SRSF1 against SCRM with FDR cut-off of 0.1 and S0 = 0.3.
ORF of RBFOX1 RRM (amino acids 109–208) was cloned in the pET28a expression vector. The protein was overexpressed at 37 °C in E. coli BL21 (DE3) codon plus cells in minimal M9 medium containing 1 g/l 15NH4Cl and 4 g/l glucose, purified by two successive nickel affinity chromatography (QIAGEN) steps using an N-terminal 6× His tag, dialyzed against T40 (10 mM Tris-HCl pH 7.4, 40 mM NaCl, 0.05% Tween20) or T100 (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.05% Tween20) buffer and concentrated to 0.3 mM with a 10-kDa molecular mass cutoff Centricon device (Vivascience).
RNA oligonucleotides were purchased from Dharmacon, deprotected according to the manufacturer's instructions, lyophilized and resuspended in T40 buffer. The NMR titrations were all performed in the NMR buffer at 313°K on a Bruker AVIII-600 MHz spectrometer equipped with a cryoprobe. Data were processed using Topspin 2.1 (Bruker) and analysed with Sparky (http://www.cgl.ucsf.edu/home/sparky/).
fbeq is the fraction bound in equilibrium; fb0 is the fraction bound at lowest concentration; fb1 is the fraction bound at highest concentration; c is the concentration; KD is the equilibrium dissociation constant.
All measurements were performed at 37 °C.
Unprocessed data: Unprocessed images of RT-PCR agarose gels and Western blots appear in Supplementary Fig. 12.
RNA-seq data have been deposited to the GEO repository as GEO submission GSE126503. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner repository with the dataset identifier PXD012564. The authors declare that all the other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. The source data underlying Figs. 1b, 1d, 4b, 5a, 6a, 6b, and 6d, are provided in Supplementary Fig. 12.
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The authors wish to thank Dr. Zahava Kluger for comments on the manuscript and members of the Karni lab for helpful discussions. This work was supported by the Israel Science Foundation (ISF Grants no' 1290/12 to R.K.), Alex U. Soyka Pancreatic Cancer Research Project (to R.K.) and KAMIN (Israel Innovation Authority) grant (to R.K.). We thank the SNF-NCCR RNA and Disease for financial support to F.H.T.A and A.C.
P.D. and R.K. designed the experiments. P.D., M.M., J.B., G.D.B., A.C., and S.K. performed experiments. O.S., E.Y., J.B., G.L., F.H.A., T.W., and T.G. contributed reagents and technical help. M.D.-G. and E.Y.L. analyzed RNAseq data. P.D. and R.K. analyzed the data and wrote the manuscript.


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Sure Dividend
High-Quality Dividend Stocks, Long-Term Plan
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Will Square Ever Pay A Dividend?
Published on December 23rd, 2020 by Aristofanis Papadatos
Square (SQ) has offered life-changing returns since its IPO, in 2015. During the last five years, the stock has rallied nearly 20-fold, from $12.50 to $242.
The surging stock is a result of the exceptional growth of the company and its still exciting growth potential.
Income investors have probably missed the breathtaking rally of Square, as the company does not pay a dividend. The absence of a dividend is fairly common among technology stocks, particularly in the early phases of their growth story.
While Square has yet to pay a dividend, the 30 Dividend Kings have all increased their dividends for 50+ consecutive years. You download the Dividend Kings spreadsheet by clicking on the link below.
Click here to download my Dividend Kings Excel Spreadsheet now.
Income-oriented investors who are attracted by the impressive return potential of Square probably wonder whether the company will pay a dividend anytime soon.
Continue reading this article to learn more about Square and the company's future dividend prospects.
Square is a high-quality financial technology company. It offers payment and point-of-sale solutions in the U.S. and in foreign markets. It also provides hardware products such as the Magstripe reader, which enables transactions of magnetic stripe cards.
Square is an exceptional company. Most companies try to grow by expanding their core business but Square follows a different approach. It continuously tries to add new businesses to its portfolio by expanding the capabilities of its systems in order to grow its total addressable market as much as possible.
Source: Investor Presentation
Square has greatly expanded the capabilities of its two major ecosystems, Seller and Cash App.
Seller was much simpler in the beginning but it now enables merchants to keep track of their payments, invoices, payrolls and inventory. In addition, Square launched a business debit card last year and provides sellers with immediate access to their funds without the need to set up a bank account. The adoption of this debit card has increased every quarter since the launch, with sellers spending more than $250 million on their cards in the latest quarter. This business card has rendered sellers much more dependent on Square.
Moreover, Square is continuously trying to expand the scope of its Cash App ecosystem. About a year ago, the company launched a stock brokerage product, which has enjoyed extremely fast adoption. More than 2.5 million customers have purchased stocks with the use of Cash App in less than a year.
It is also worth noting that Square has proved resilient to the coronavirus crisis, which rendered the online presence of merchants and their immediate access to cash and more important than ever.
In the most recent quarter, Cash App posted impressive gross profit growth of 212% over the prior year's quarter and Square grew its gross profit 59%.
Growth Prospects
Square has an enviable performance record, as it has grown its gross profit at a 40% average annual rate over the last four years.
Square has ample room to continue growing at a double-digit rate for several more years. The company has penetrated less than 3% of its total addressable market of sellers, which is a market worth more than $100 billion.
Square has penetrated less than 2% of the addressable market of individuals, which is worth more than $60 billion. It is evident that the company has room for future growth for many years, particularly if it continues to expand the capabilities of its ecosystems.
The primary growth driver for the company will be Cash App. This ecosystem represented only 25% of the business of Square last year but it is currently representing 50% of the business thanks to its impressive momentum.
Analysts seem to agree virtually unanimously that Square has exciting growth potential. They expect the company to grow its earnings per share by approximately 50% per year over the next three years, from $0.76 this year to $2.83 in 2023.
Square has greatly enhanced the capabilities of its two ecosystems in just a few years. Thanks to its sustained product improvement, it has significantly increased the engagement of its customers and their dependence on its products.
It has also built a strong brand thanks to word-of-mouth. As a result, Square enjoys a material competitive advantage.
On the other hand, investors should never forget that technology companies rarely enjoy a strong competitive advantage, as it is usually inexpensive to imitate their business model. In addition, there is always cut-throat competition in the tech sector and hence many companies run the risk of incurring business deterioration if their competitors achieve major technological breakthroughs. This is certainly the case for Square.
On the bright side, as many customers of Square have engaged in more than one of the applications of its products, they are not likely to migrate to a competitor. Overall, Square enjoys a relatively narrow business moat but its moat is somewhat wider than that of most tech stocks.
In order to pay a dividend, companies need to generate positive free cash flows. In other words, their business should generate cash flows that exceed the capital expenses by a wide margin. Some popular stocks cannot pay dividends to their shareholders due to their negative free cash flows. For instance, Uber (UBER), Lyft (LYFT) and Netflix (NFLX) have not managed to generate positive free cash flows yet.
Square spends a significant portion of its revenues on sales and marketing expenses in order to attract new customers and increase the engagement of existing customers. It also spends appreciable amounts in product development to enhance the value of its products.
Square recently stated that it intends to grow its operating expenses by 40% in 2021 to approximately $850 million. This amount is about one-third of the gross profit of the company in the last 12 months and hence it represents a significant amount. Due to the elevated amounts that Square spends on R&D and on sales and marketing expenses, its free cash flows are negative. As a result, there are no funds available for the initiation of a dividend.
As mentioned above, analysts expect Square to nearly quadruple its earnings per share over the next three years, from $0.76 in 2020 to $2.83 in 2023. However, investors should not expect a dividend from Square as long as the company remains in high growth mode. The company is growing its business at such a high rate that it makes much more economic sense to invest in the business instead of initiating a dividend.
As Square has several years of double-digit earnings growth ahead, it is not likely to initiate a dividend anytime soon. Instead, its management is likely to remain focused on enhancing the scope of its products, improving them and promoting them in order to continue attracting new customers and keep the existing customers as engaged as possible. Therefore, the shareholders of Square should not expect a dividend anytime soon.
High-growth companies do not offer a dividend for another reason as well. Their stocks usually enjoy such a rich valuation that a dividend is meaningless for the shareholders. To provide a perspective, Square is currently trading at 213 times its expected earnings in 2021. Therefore, even if the company distributes 50% of its future earnings in the form of dividends, it will offer just a 0.2% dividend yield. Such a yield would be negligible for shareholders and hence there is no incentive for the company to initiate a dividend.
Square has exhibited an exceptional growth record since its IPO in 2015. Even better, it is still in the early stages of its growth trajectory, with promising growth prospects ahead thanks to the continuous expansion of the applications of its products.
However, the company will continue investing a great portion of its revenues in its business in order to keep growing its customer base and increase the engagement of its existing customers. As a result, Square is not likely to initiate a dividend for the next several years.
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David Feng is a professional magician, with skills in mentalism based out of New York City. Depending on your event needs he can either perform his talents on a stage or in small groups with close-up magic interacting with the crowd. David prides himself on providing professionalism for any event he performs for, always working with each client to make sure their needs are met. David believes anyone can enjoy a magic show, so he is perfect for any event type including birthdays, weddings, corporate events, private parties, and any other type of special occasion. Book him today!
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Electronic Speedometer
Speedometer Parts
Electronic Speedometer Calibration
Digital Speedometer
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Motorcycle Speedometer
Speedometer Calibration
What is an Electronic Speedometer?
Patrick Roland
The speedometer is an essential component of any motor-driven vehicle because it measures its speed. For nearly a century analog equipment was used to gauge how fast a vehicle was moving, until electronic speedometer technology became readily available. These devices provide more accuracy and reliability than older models, but maintain the familiar layout of a traditional speedometer. In addition to the speed, electronic speedometers measure RPMs and even distance driven with digital precision. Today, these devices are found in many wheeled and non-wheeled vehicles.
A speedometer is a measuring device for a vehicle's speed and normally measures that speed by having a needle move along a circular path, like a clock hand, indicating how fast a motor is propelling a vehicle. The first speedometers, used in the early 1900s, had a rotating cable attached to the engine's transmission. The electronic speedometer, first utilized in the 1980s, may look like a traditional speedometer or be an LCD screen of numbers. It also takes readings from the transmission, but in a very different way.
Electronic speedometers also provide odometer reading with great accuracy.
The electronic speedometer depends on an electronic rotation sensor placed near the engine's transmission. The sensor is usually a toothed metal disc set between the coil and magnetic field sensor. As the engine operates, the magnetic field delivers a pulse to the sensor. The pulses given off correspond to the rotational speed of the drive shaft. This data is fed into the speedometer's internal computer and calculated out by converting rotations to miles or kilometers per hour.
The electronic speedometer depends on an electronic rotation sensor placed near the engine's transmission.
Modern speedometers do not depend on analog methods to read the drive shaft's spinning, so these devices provide more accurate readings. Another benefit of the electric sensor, compared to the spinning cable, is longevity because traditional speedometers would wear out much faster. Electronic speedometers also provide tachometer and odometer readings with equal accuracy. For these reasons, many older vehicles exchange analog models for electronic speedometers.
An electronic speedometer may measure air speed in aircraft.
An electronic speedometer is customarily thought of as a part of cars and trucks. This is true, because almost all modern automobiles use electronic speed measuring devices, but is not the only place these tools are utilized. An electronic motorcycle speedometer is another common sight with two-wheeled vehicles. In addition, airplanes accurately measure air speed with these tools, as do motorboats.
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By: Pavel Losevsky


In January 2014, Jason Andrew's work (and my text), on Nigerian soccer players stranded in Istanbul, will be published in LFI Magazine —an imprint run by Leica.
In the summer of 2012, mere months after graduating from Columbia, I was honored to publish my first story —in print— for The New York Times. The piece, Soccer's Lost Boys, which looks at the award-winning photography series by Jason Andrew, ran full page in the Saturday sports section with a longer article published on the New York Times' Lens Blog.
As the plane's landing gear struck the smooth runway of Istanbul's Ataturk airport in August 2010, Akeem looked around the cabin at the 60 other young African players, dressed in matching adidas track suits and toting their cleats. Akeem had paid for his flight, visa, and – more importantly – the opportunity to try out for Turkey's professional clubs. As he glanced out of the plane's small, fogged window onto a city which has served as the heart to four world empires, Akeem wanted to believe that his future started here.
*The January issue is available for download or purchase here.


Every case is unique, and the facts of circumstances of your situation require personal attention. When considering expenses, it is important to distinguish between attorney fees and costs. Attorney's Fees is the fee charged for the attorney to work on your case. Simply put, the attorney's time. Costs represent every other cost associated with your case besides the attorney's fee. The Law Office of W. Seth Mazirow, P.L. bills attorney's fees at a rate of $250.00 per hour. However, not all cases are billed on an hourly basis. The unique facts and circumstances or your legal matter will determine the appropriate, reasonable fee/payment structure.
Costs common to most cases include filing fees (determined by the Clerk of Court), service of process fees, copies, postage, etc. The Law Office of W. Seth Mazirow, P.L. charges a minimum cost upon retention to cover the costs associated with the case. The unique facts and circumstances or your legal matter will determine the appropriate minimum cost.
It is crucial to note that attorney's are prohibited from working on both criminal defense and family law matters on a contingency basis. Thus, no work in those areas may be considered on a contingency basis. However, the Law Office of W. Seth Mazirow, P.L. does offer flat fee retainer agreements for both family law and criminal defense. Additionally, where allowed, the Law Office of W. Seth Mazirow, P.L. will consider contingency agreements on a case by case basis.
Do you offer Flat Fee representation?
Yes. While many attorney's shun the idea of flat fee representation, The Law Office of W. Seth Mazirow, P.L.is proud to offer flat fee agreements on a case by case basis, limitations and restrictions do apply. What is and is not covered in a flat fee agreement will be discussed in detail, prior to retention. Flat fee representation allows our clients to better budget their legal expense, prior to retaining an attorney.
What is No-Fault? And what does a No-fault divorce mean?
Florida is No Fault divorce state, meaning that one side alone can seek a divorce and the Court will not rule that either party is responsible for the divorce. The most common legal basis to obtain a dissolution of marriage from a Florida Court is the testimony of one spouse that the marriage is irretrievably broken and no matter of counseling can save it; and that the party seeking the divorce has been a resident of Florida for the proceeding six months.
What is a "contested" versus an "uncontested" family law matter?
Contested is where the parties do not agree on how to resolve their legal dispute. Unfortunately, family law disputes are often emotionally charged matters where the parties feelings interfere with their decision making. Often times the party who has decided to move on or change the relationship has already overcome the emotional hurdle; whereas the other side is still coming to terms with the situation. However, things often change over time. Thus, just because the parties disagree today, does not mean that a settlement is impossible in the future. In fact, the overwhelming majority of family law cases settle before trial. When is it advisable to settle? When is it advisable to resolve the dispute by trial? The facts and circumstances of your situation are unique; as will be the recommendation(s) of the attorney, only after having discussed and ascertained the facts your situation. The experience, counsel and advise of the attorney is what you are acquiring when your retain an attorney.
Uncontested is when the parties agree on how to resolve their issues, but one side or the other may require legal assistance in court or have questions they want addressed before resolution. A "second opinion" is usually a good idea. Just because an offer seems reasonable, is it? Moreover, the other side does not have to know you've consulted an attorney.
What is time sharing and/or parenting time?
Florida no longer uses the term custody. Instead, the Florida Legislature has created the term "time sharing" to reflect a Parent's time with the minor child(ren). This is also sometimes referred to a parenting time.
The parenting plan is the agreement between the parties as to how/when/where each parent will enjoy time sharing with the minor child(ren). Additionally, the parenting plan should address the responsibilities of each parent. A parenting plan can stem from either the agreement of the parties, or by order of the Court. An agreement of the parties, if acceptable to the Court, will be adopted and ratified by the Court. In effect, the agreement becomes the order of the Court. Hence, failure to abide by a parenting plan ordered by the Court can lead to sanctions, including contempt of court.
Can a parenting plan be changed or modified?
Yes, as long as the party seeking change retains their parental rights. Parenting plans can be changed, regardless of whether there was a trial or agreement. The burden is on the party seeking to modify the parenting plan. First, The party seeking to modify the parenting plan must show that a substantial, material and unforeseen change in circumstances has occurred AND it is in the best interest of the minor child(ren) to modify the parenting plan. Whose life hasn't changed in the past few years? Put differently, a substantial change in circumstances can be economic, social, physical, or emotional. Life is constantly changing, but remember, the Court will seek to do what is in your child(ren)'s best interest when addressing a request to modify a parenting plan.
No, child support is mandatory. An experienced family law attorney can assist their client in understanding how child support is calculated, the basis of the child support guidelines worksheet; and how and when to seek a modification of the child support.
What should you do if you no longer can afford to pay the Child Support you've been ordered to pay?
Seek the advice of an attorney immediately so you may avoid violating the Court's order. If a substantial change in circumstances has occurred, the petitioning the Court to Modify the child support obligation is likely to be recommended.
What can be done if the other parent stops paying child support?
The facts and circumstances of your situation will vary, and so will the proper course. Sometimes a warning letter from an attorney will resolve the problems. Alternatively, seeking relief from the court via a contempt proceeding will oftentimes motivate payment. Discussion of your situation with the attorney is the recommended first step toward addressing the problem.
First Rule: DO NOT MAKE THE SITUATION WORSE. All persons charged or under investigation for the commission of a crime have a right to remain silent - use it!
Second Rule: DO NOT DISCUSS YOUR CASE WITH ANYONE BUT AN ATTORNEY. Discussions with an attorney are protected by attorney-client privilege. Discussions with friends and family are not privileged.
Third Rule: DO NOT DISCUSS YOUR CASE OVER THE JAIL PHONES. The conversations are recorded and can and will be used against a criminal defendant.
Fourth Rule: THE SOONER YOU SPEAK TO AN ATTORNEY, THE BETTER. Call (727) 738-2874 or email the attorney at Seth@MazirowLaw.com today to schedule your free initial consultation.


Science Library DSS catalog › Details for: Health implications of acrylamide in food : report of a joint FAO / WHO Consultation WHO Headquaters, Geneva, Switzerland, 25-27 June 2002.
Details for: Health implications of acrylamide in food : report of a joint FAO / WHO Consultation WHO Headquaters, Geneva, Switzerland, 25-27 June 2002.
By: FAO / WHO Consultation on Health Implications of Acrylamide in Food (2002 : Geneva, Switzerland) .
Contributor(s): World Health Organization. Food Safety Programme Food and Agriculture Organization of the United Nations .


"Know the Value" Glass – Genterie Supply Co.
The "Know the Value" Glass is part of a Cleo Wade Collection(one of our favorite poets). It measures 4.38" tall and 2.25" wide. A Fishs Eddy Exclusive, this juice glass is made of food-safe glass and is restaurant-quality! This juice glass is microwave safe and can hold 6.5 oz. of whatever you want! And the best news yet: This product is made in America!


Dr. Habibpour is a professional psychiatrist with training in Child, Adolescent, and Adult Psychiatry. He began working with the prestigious George Washington Psychiatry Department in 2014 as a Clinical Assistant Professor of Psychiatry and Behavioral Sciences and then started teaching a course in Child Psychiatry.
He has a strong educational background in his field. After attending a top medical school in Iran Dr. Habibpour was able to complete his Adult Psychiatry Residency at the Medical College of Virginia in Richmond, VA. He then continued his training by pursuing a Child and Adolescent Psychiatry Fellowship at the University of Texas Southwestern Medical Center in Dallas, TX. The Fellowship taught Dr. Habibpour about the normal development of children and how childhood experiences can affect us in the future.
While practicing psychotherapy, Dr. Habibpour has mastered treatment with psychiatric medications. Witnessing the many areas of exciting progress in the field of psychiatry over the last decade has given him the chance to familiarize himself with the most up-to-date medications. Dr. Habibpour's approach to helping his patients involves the integration of both medications and psychotherapy for the most efficient and effective results. Often times the emotional and behavioral styles people use to protect themselves during childhood carry over into their adulthood. These behaviors may be helpful during childhood, but they are no longer needed in adulthood. To achieve an effective change, one must first explore those feelings and behavioral styles.
Dr. Habibpour has also developed an interest in helping those with anxiety and mood disorders, and gained experience in treating eating disorders. A major area of interest for him is the role of culture and ethnicity in emotional disturbances. He had a chance to study this overlap and learn about it because of the cultural diversity to which he was exposed over the course of his life. His passion was reflected in the paper he co-wrote: "The Treatment of Depression in Culturally Diverse Children and Adolescents." (Sunita M. Stewart, Alex Simmons, and Ehsan Habibpour. Journal of Child and Adolescent Psychopharmacology. February 2012, 22(1): 72-79. doi:10.1089/cap.2011.0051.). He is pursuing that passion through his involvement with The Global Mental Health Project at George Washington Psychiatry Department.
Over the years of his practice, Dr. Habibpour has experienced and appreciated the importance of therapeutic relationships. Self-determination, resilience and the power of meaningful relationships are the key factors that nourish human growth.
She studied Psychology at Harvard University, earned a Bachelors in Political Science with an emphasis in International Relations from the University of California Berkeley, has a Master's degree in Marriage Family Therapy from the University of San Francisco and earned her Doctorate in Clinical Psychology from the California Institute of Integral Studies in San Francisco, CIIS. At CIIS the curriculum marries eastern philosophy and religion with traditional western empirical psychological training. Her dissertation was a quality of life study on the use of laughter and humor in the treatment of cancer survivors.
Her specialty is working with the psychological ramifications of medical issues, a branch of psychology known as Behavioral Medicine or Health Psychology. She has successfully worked with treatment resistant medically unexplained symptoms as well as chronic migraines, pain, cardiac, auto-immune, eating disorders and cancer. An additional specialty is in the treatment of recent traumatic/adverse events with ongoing traumatic stress. Dr. Roberts also provides clinical consultation to other EMDR clinicians individually and in groups.
For government workers with access to sensitive information or with security clearance there are newer EMDR protocols that do not require disclosure or articulation of material in order for you to receive benefits. This preserves privacy, affords an extra layer of protection and can diminish your fears about receiving psychotherapy.
Our work will focus on building your strengths and resiliency and giving you the tools to make the necessary changes so that you are the author of your own life. Together we will take a problem solving and solution focused approach building upon the resources and strengths you already have. Some of the work will focus on self-regulation strategies and a variety of ways to contain and manage strong emotions. A good amount of time may be spent on psych-education and empowerment. Our goal will be to transform problems into assets and liabilities into strengths. In many instances you already carry the solutions, but merely need guidance to realize this and implement targeted strategies.
The work can be short-term or longer-term according to your needs, motivation and availability.
Dr Roberts is an EMDR International Association and Trauma Recovery Humanitarian Assistance Programs approved consultant, an EMDR trainer in early intervention and former leader of the Western Massachusetts Trauma Recovery Network, a community disaster response program. She has conducted trainings around the US for trauma clinicians and has been instrumental in setting up the Trauma Response Network in Britain, a government sanctioned organization, formed in 2017 in response to terrorism and mass casualty events. She advises them on logistical and clinical issues. Dr. Roberts has an enduring and lifelong passion for global and international humanitarian issues.
She completed three pre-doctoral internships (At Stanford's Drug and Alcohol Clinic, Stanford's Counseling and Psychological Services and San Mateo County Mental Health Services) and two post-doctoral internships.(One in a national employee assistance program and another at the University of Massachusetts Memorial Center Medical school, ranked among the top ten medical schools in the United States) She worked in the Behavioral Medicine Clinic at the University of Massachusetts with rotations throughout cardiac and oncology, pain and bariatric surgery services. She has served on a mobile psychiatric crisis unit, in a full-service hospital emergency room, community mental health clinics and outreach, inpatient, general outpatient and two pain clinics.
She has provided trainings/workshops nationally and internationally on a variety of mental health topics and was on the United Nations working team of EMDR therapists acting to introduce EMDR Therapy as a modality to be used in humanitarian programs around the world. More recently, as an EMDR ambassador, she facilitated, an engagement with the World Bank in the Philippines and has been involved in various capacities with the Global Initiative for Stress and Trauma Treatment, GIST-T, based in Geneva Switzerland.
She is a consummate researcher and launched the world's first EMDR clinical trial looking at the efficacy of the EMDR Group Traumatic Episode Protocol, GTEP with an oncology population. This protocol also has applicability to other populations.
Monique Deely is a licensed clinical social worker and has 18 years of experience in the field of psychiatry and psychology. She has worked with children, adolescents and their families in outpatient behavioral health, residential treatment and hospice care. She has also provided help for adults who need couples therapy and hospice care. Monique is honored to have patients trust her. To be able to help patients in difficult points in their lives is a privilege that has shaped her and her practice.
Monique earned her undergraduate degree in Psychology and graduate degree in Social Work from the University of Georgia. She started her clinical work during the last year of her Master's program in her practicum working in a residential treatment center with children and adolescents. Monique was able to get valuable experience working at this treatment center. It was during this time working with children, adolescents, and families that she honed diagnostic and therapeutic skills.
Working in a hospice and in a hematology/oncology practice were some of the most rewarding years for Monique. She was able to work with many people addressing end of life issues, chronic illness, chronic pain, and grief. This taught her the special value of resilience and how to stay strong when dealing with life challenges. Monique has worked with military families in the Washington, D.C. area for the last 10 years. All of her experience has made her comfortable and highly skilled in working with patients of all ages and backgrounds.
Recently, Monique has been working with children and adolescents dealing with ADHD, anxiety, mood disorders, autism spectrum and trauma through embedded behavioral health services in schools. She has supported children and families by addressing the challenges of military life and service such as deployment, injury, multiple moves, separation, and divorce. Monique also has experience working with adults on their relationships and marriage, life transitions and difficulties with anxiety/depression.
From Monique's vast amount of experience, she strongly believes in the value of a collaborative approach with patients and planning treatment goals. She utilizes multiple evidenced-based treatments, with a strengths-based approach, to include CBT, TF-CBT, DBT, behavior modification, Prolonged Exposure (PE), Cognitive Processing Therapy (CPT), treatment for insomnia (CBT-I), and psychodynamic therapy. Recently, she has begun to integrate Mindfulness and meditation into her work with patients. Monique wants to help patients find an awareness of their inner state of being and how to connect with the stillness.
Monique's goal is to build a meaningful relationship with clients in the therapeutic process and journey. We all have strengths and naturally seek or strive for wellness and Monique is here to walk that path with you.
I am a Licensed Professional Counselor and a Certified Substance Abuse Counselor with several years of clinical experience. I have worked with adolescents and adults in a university mental health facility, outpatient substance abuse clinics, and a residential substance abuse facility. As a Nationally Certified Counselor, Certified Clinical Trauma Professional, and member of the American Counseling Association, I am dedicated to delivering ethical and cutting-edge treatment to my clients.
I believe that everyone is capable of change. I have counseled clients experiencing substance abuse, grief and loss, family and marital discord, stress, anger, depression, mood disorders, trauma, and anxiety. My approach has been to work together with my clients to develop treatment plans that improve healing, self-efficacy, motivation, and success. I truly enjoy empowering others to discover their inner potential and strengths. Throughout my diverse work experiences, I have taken pride in my ability to form connections with others, to anticipate their needs, and to help my clients change in order to attain healthier and more productive lives.
My education includes a Bachelor of Arts in Psychology from the University of Vermont and a Master of Arts in Education and Human Development with a concentration in Clinical Mental Health Counseling from George Washington University. I use a variety of therapeutic treatment approaches, including Cognitive Behavioral Therapy, Dialectical Behavioral Therapy, Motivational Interviewing techniques, and Mindfulness based therapy, to encourage clients to elicit change within themselves. I have focused training and experience with helping clients navigate struggles around grief and loss and major life transitions. Through a variety of settings, I have had the chance to work with treating health care professionals and military personnel. My experience with this particular demographic includes processing their emotional difficulties and burnouts, including PTSD, and how substance abuse has become a self-destructive coping strategy.
I stand with my clients in the midst of their pain as they gain insight and awareness into life experiences past and present. I am dedicated to creating non-judgmental and sacred spaces where true transformations can occur. My personalized, collaborative, and integrative therapeutic approach allows for gentle healing in a safe and supportive environment that focuses on healing the physical, mental, and spiritual well-being.


Ecological restoration projects encourage the reestablishment of natural shoreline, dune, coastal bank, wetland, salt marsh, and buffer zones that have been lost or damaged due to natural forces or human activities. NETCO has completed several projects in sensitive wetland and salt marsh environments where we have applied our expertise in the use of biodegradable materials, plant relocation, bank and dune stabilization, and natural wetland/salt marsh plantings. We work with permitting and other wetland scientists and consultants to provide recommendations for the most economical, effective and "constructable" restoration program.


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Salman Khan's epic reaction on Race 3 trolling is a must watch
Salman Khan was compared with cringe pop singers like Dhinchak Pooja and Taher Shah for Selfish song from Race 3 as it has unorthodox lyrics.
By : Pramod Gaikwad
Salman KhanTwitter
Ever since Salman Khan's Race 3 trailer was released online, many people including the actor's fans, have been trolling the film. The comments are usually over its logic-defying stunts, bizarre dialogues, cringe worthy songs and "bhai ka dance".
Trolls also turned the Race 3 trailer into hilarious jokes and memes on social media. But the Dabangg Khan is unfazed with all those who are making fun of his upcoming film as he thinks people with one or two followers online can't be called trolls.
Salman was compared with cringe pop singers like Dhinchak Pooja and Taher Shah for the Selfish song from Race 3. The song, which has unusual lyrics, has apparently been penned by Salman himself.
When a scribe asked Salman to comment on the negative buzz around Race 3, the actor replied, "I don't know what you're talking about" asking counter question to the journalist to explain what is trolling.
"Are you trolling it? You know who is trolling it?" Salman asked. When the journalist replied to him that a lot of people are trolling it, Salman replied, "Those guys with one follower or two followers? That's trolling? Who's controlling that trolling, you know? No? How sad."
"So, when guys with one, two, three, four followers troll, it's not trolling," Salman concluded.
Watch Salman Khan's reaction when asked about trolling here:
Race 3 trailer may have received mixed reactions from the audience but it negative publicity seems to be actually helping create buzz around the movie.
Directed by Remo D'Souza, the action-adventure film also stars Anil Kapoor, Bobby Deol, Jacqueline Fernandez, Daisy Shah and Saqib Saleem in important roles. The movie will release this Eid on June 15.
Yash-Vijay Devarakonda Drive Fans Crazy at Dear Comrade Musical Event in Bengaluru
Sreekaram opening ceremony: Sukumar launches Sharwanand's film
Vijay Nirmala's death: Pawan Kalyan, Mohan Babu, Charmi and other celebs pay last respects
7 things you didn't know about 2001: A Space Odyssey


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Ash Wednesday is an Australian musician, who played in JAB, Models and Einstürzende Neubauten.

Biography
Wednesday was a founding member of JAB, an Australian punk rock band, in Adelaide in January 1976. The band's original lineup consisted of Bohdan X (Bodhan Kubiakowski) on guitar and vocals, Wednesday playing bass guitar, synthesizer and tapes and Johnny Crash (Janis Friedenfelds) on drums and vocals. The band took its name from the first initials of the founding members. The band relocated to Melbourne in August 1977. JAB signed to Suicide Records in January 1978, and two of their songs, "Blonde and Bombed" and "Let's Go", were included on the Lethal Weapons compilation. The band played their final show in August 1978 at the Crystal Ballroom (then known as the Seaview Ballroom), which was also the first gig ever played at the now iconic venue.

Wednesday, Crash and Pierre Voltaire then joined Sean Kelly (ex-Teenage Radio Stars) to form the first line-up of Models. The initial version of the group did not stay together for long as Voltaire was soon replaced on bass guitar by Mark Ferrie (ex-Myriad), then in August 1979, Wednesday was replaced by Andrew Duffield (ex-Whirlywirld) on keyboards.

In 1980 he released a solo single, "Love By Numbers" and produced four songs with vocalist Karen Marks (the manager of Models and JAB). That year he also joined experimental outfit The Metronomes with rock journalist Al Webb (guitar, synthesiser; ex-Streetlife)and Andrew Picouleau (bass, synthesiser; Secret Police, Sacred Cowboys), having previously been an invited guest with the band.  The Metronomes released two singles, "Saturday Night"/"Sunday Morning" (1979), "A Circuit Like Me"/"Closed Circuit" (1980) and two albums, Multiple Choice (1981) and Regular Guys (1985) on the Cleopatra label. The band was never intended to be a live outfit and only ever came together in the studio. Around 2000 the members began work on a new project and after four years of sporadic effort released a new album, Today.  This was followed by a re-release of the band's first two albums and an EP (comprising the band's two singles), as a single release, The Ballad of the Metronomes, in 2010.

Concurrently Wednesday collaborated with T.E. Power (Nuv Bloc) on a project titled Thealonian Music, using the latest electronic instruments of the time. The concept being to generate music of a spontaneous nature, with the rhythmic foundation being provided by a programmable drum machine, sequence and synthesizer combination.  Thealonian Music only produced one release, the 1982 tape, Project X Chromosome X.  Throughout the early 1980s, he was a member of Modern Jazz, an improvisation group who performed to randomly generated techno beats. Modern Jazz featured a fluid line-up that included Lyn Gordon (synthesiser), Andrew Park (synthesiser) and Ted Thornbury (vocals). The band played 1980s techno music and issued one album, The Nude, on the Cleopatra label in 1986.

In 1988 he and Lyn Gordon formed electronic / industrial band Crashland, composed of Lyn Gordon (vocal, guitar), Wednesday (sampler), Ray Moore (drums) and Phil Sutherland (bass). A year after their formation, Crashland signed a deal with Regular Records which resulted in the release of their first single, "Cherry Bomb"/"Elvis Working" in October 1989. Crashland's self-titled debut album comprised half original material and half covers, was released in April 1990, together with the band's second single, a demented electro remodelling of John Lee Hooker's "Boom Boom"/"On T.V.".

Wednesday moved to Berlin in 1992, where he worked with Nina Hagen, being involved with pre-production and programming for her 1995 album, FreuD euch ("Bee Happy").

In 1997, Wednesday became a touring member of German band Einstürzende Neubauten. He completed several world tours with them and is present on their live albums, such as 09-15-2000, Brussels and numerous recordings from the Perpetuum Mobile tour (2004). Eventually departing in 2013.

In 1999 he returned to Melbourne and formed The Tingler, with Crashland vocalist Lyn Gordon. The Tingler releasing a self-titled album in April 2000. The Tingler also featured on the bill during the Models 2000/2001 reunion shows.

In 2013 he was to perform solo as The Ash Wednesday Effect, a spontaneously performed audio/visual experience, in which improvised soundscapes triggered entrancing visualisation, which were in turn projected onto Wednesday's physical form. The performances at Club Voltaire, North Melbourne were continuous three-hour pieces, throughout which audience members were free to come and go as they chose.

Discography

JAB (1976–1979)

Models (1979–1980)
 Melbourne – Shock (2001)

Solo
 "Love By Numbers"/"Boring Instrumental" – Independent (1980)
 A Place to Live (digital) – Independent (2017)

The Metronomes
 "Saturday Night"/"Sunday Morning" – Cleopatra (CSP 2203) (1979)
 "A Circuit Like Me"/"Closed Circuit" – Cleopatra (CSP 2205) (September 1980)
 Multiple Choice – Cleopatra (CLP 210) (1981)
 Regular Guys – Cleopatra (CLP 212) (1985)
 Today (2004)
 The Ballad of the Metronomes – Mannequin (29 November 2010)
Time Keeping (1979–1985) It records (November 2014)
Going Somewhere – It records (June 2016)

Thealonian Music
 Project X Chromosome X (1982)

Modern Jazz (1983–1987
 The Nude – Cleopatra (CLP 215) (1986)

Crashland (1988–1991)
 "Cherry Bomb"/"Elvis Working" – Regular (K919) (October 1989)
 Crashland – Regular (D30149) (April 1990)
 "Boom Boom'/"On TV" – Regular (K 1096) (April 1990)

Einstürzende Neubauten (1997–2004)
 09-15-2000, Brussels – Caroline (2001)
 Gemini – Caroline (2003)
 Perpetuum Mobile Tour – Caroline (2004)
 04-01-2004, Amsterdam – Caroline (2004)
 10-29-2004, Reggio Emilia – Caroline (2004)

The Tingler (1999–2001)
 The Tingler – Independent (2001)

The Ash Wednesday Effect (2014/15)

References

External links
 Official Ash Wednesday webpage
 Official Einstürzende Neubauten webpage

Einstürzende Neubauten members
Australian rock keyboardists
Place of birth missing (living people)
Year of birth missing (living people)
Models (band) members
Musicians from Melbourne
Living people

redpajama_set_name: RedPajamaWikipedia


*PERISHABLE. Our pizzas are made with imported Neapolitan "00" flour. This white pizza is made with fresh sliced zucchini and fresh onions. All natural, no preservatives. Sold frozen and cooks in about 15 minutes.