Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.
Peroxisomes are ubiquitous organelles, which carry out a wide variety of metabolic processes in eukaryotic organisms . Therefore, the dysfunction of these organelles in humans results in severe peroxisomal disorders [2–4]. The functionality of these organelles is governed by dynamically operating import machineries for peroxisomal membrane and matrix proteins [1,5]. Peroxisomal matrix proteins are without exception nuclear encoded, synthesized on free ribosomes and subsequently recognized in the cytosol by specific soluble receptors [6,7]. To this end, cargo proteins are equipped with a targeting sequence, either a C-terminal PTS1 (peroxisomal targeting signal type 1) or an N-terminal PTS2, which are recognized and bound by the import receptor Pex5p or Pex7p respectively . Subsequent to the assembly in the cytosol, the receptor ferries its cargo to the peroxisomal membrane, where it associates with the docking complex and then becomes part of a dynamic translocation pore . The molecular mechanism underlying the translocation and release of the cargo remains elusive. Finally, the cargo-free receptors are exported back to the cytosol in an ATP-dependent manner. This step is catalysed by the AAA (ATPases associated with various cellular activities) peroxins Pex1p and Pex6p, which are supposed to function as mechanoenzymes by pulling the receptor out of the membrane [10,11]. Ubiquitination of the receptor serves as a signal for this process [12–14].
Two kinds of ubiquitination of the Pex5p receptor have been reported, namely mono- and polyubiquitination. Monoubiquitination primes the receptor for its export back to the cytosol to allow a new round of matrix protein import [12,14,15]. Pex5p monoubiquitination occurs at a conserved cysteine residue near the N-terminus of Pex5p (Cys6 in Saccharomyces cerevisiae) and is facilitated by the E2 ubiquitin-conjugation enzyme Pex4p in cooperation with the RING (really interesting new gene)-type E3 ligase Pex12p. Pex12p is part of the peroxisome membrane-associated RING sub-complex of E3 ligases (Pex2p, Pex10p and Pex12p) [12,15–17]. Alternatively, in cells lacking a component of the receptor recycling machinery, the receptor is subjected to polyubiquitination, which occurs on conserved lysines near the N-terminus of Pex5p (Lys18/Lys24 in S. cerevisiae) and leads to receptor degradation [18–21]. In S. cerevisiae, polyubiquitination of Pex5p depends on the cytosolic E2 enzyme Ubc4p (ubiquitin conjugating enzyme) and the E3 ligase Pex10p  or Pex2p with the assistance of Pex10p [17,23].
Usually, Gly76 of the ubiquitin is attached to a ε-NH2 group of a lysine residue of the target protein by formation of an isopeptide bond or in some cases to the α-NH2 group of the extreme N-terminus. As an exception to the rule, also cysteine residues can be targeted by ubiquitination machineries. Apart from the PTS-receptors Pex5p, Pex18p and Pex20p [14,16,21,24,25], only a few cases of ester-based ubiquitination substrates are known, like e.g. tBid (truncated BH3 interacting domain death agonist), MHC-I (major histocompatibility complex I) heavy chain or TCRα (T cell receptor alpha chain) . In these cases, the ubiquitination of substrates via esterification serves to provide more target residues in order to enhance degradation. In contrast, the esterification of the PTS1-receptor seems to have a physiologically relevant mechanistic function, because it is required for the recycling and reuse of Pex5p for further rounds of matrix protein import.
In order to elucidate the physiological relevance of the conserved N-terminal cysteine of PTS1-receptor, Pex5p-variants were generated containing substitution of the corresponding cysteine and other known target amino acids for ubiquitination by lysine, alanine or arginine. Our data show that Pex5pC6K is functional in PTS1 import. However, instead of the typical monoubiquitination, the Pex5pC6K is polyubiquitinated. Moreover, Pex5pC6K displays significantly reduced steady state level when the deubiquitinating enzyme Ubp15p (ubiquitin-specific protease) is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for the general function of Pex5p in peroxisome biogenesis. However, the cysteine is required for efficient recycling of Pex5p and prevents the protein from polyubiquitination, which leads to a rapid degradation of the protein.
Wild-type Saccharomyces cerevisiae strain UTL-7A (MATa, ura3-52, trp1, leu2-3/112)  was the isogenic source for the generation of used pex1Δ , pex4Δ , pex5Δ , pex4Δ pex5Δ , ubp15Δ , as well as ubp15Δpex5Δ strains (present study). Deletion strains were generated by the 'short flanking homology' method as described before . Yeast media have been described previously . For induction of the CUP1 promoter, CuSO4 was added according to . When the proteasome inhibitor MG132 was used, the medium was supplemented with 0.1% (w/v) proline and 0.003% (w/v) SDS to allow the uptake of the inhibitor according to .
SDS/0.1M NaOH and 20 μl of SDS loading buffer [5% (w/v) 2-mercaptoethanol, 15% (v/v) glycerol and 0.01% (w/v) Bromophenol Blue].
Used plasmids and oligonucleotides are listed in Tables 1 and 2 respectively. Pex5p was expressed from a low-copy vector under the control of its own promoter (pHP17-PEX5) . Point mutations in PEX5 were introduced using overlap extension PCR leading to the lysine to arginine, cysteine to lysine as well as cysteine to alanine mutant collection of PEX5.
Fluorescence microscopy of live cells for DsRed (Discosoma sp. RFP) expression and localization was performed with a Zeiss Axioplan microscope and AxioVision 4.1 software (Zeiss). Before inspection, cells were grown for 2 days on solid minimal medium containing oleic acid as a sole carbon source. Both fluorescence and optical photographs were taken by using the connected hardware in combination with the Spot RT software version 3.1 (Diagnostics Instruments). Adjustments of contrast and brightness were carried out with Adobe Photoshop software version 13.0 and characteristic cells were cut-out and copied to Adobe Illustrator version CS6.
Immunoblot analyses were performed according to standard protocols . Immunoblots were incubated with polyclonal rabbit antibodies raised against Pex5p  and mitochondrial porin . Primary antibody was detected with an IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR Bioscience) followed by a detection using the 'Infrarot Imaging System' (LI-COR Bioscience).
On the basis of functional and structural data, the PTS1-receptor Pex5p comprises two separate parts (Figure 1A). Whereas the C-terminal TPR (tetratricopeptide repeat) domain is highly conserved and facilitates binding to PTS1-cargo proteins, the N-terminal domain hosts most of the interaction sites for other peroxins but reveals poor conservation . Interestingly, the extreme N-terminal part contains conserved amino acid residues serving as targets for ubiquitination. The conserved lysines at position 18 and 24 of yeast Pex5p are required for polyubiquitination and subsequent degradation [12,19]. In contrast and as an exception to the rule, a conserved cysteine at position 6 undergoes monoubiquitination , which is required for the Pex1p/Pex6p-dependent release of the receptor from the peroxisomal membrane to the cytosol [16,38]. In order to gain more insight into functional relevance of this atypical linkage of ubiquitin to the substrate protein via a thioester bond to a cysteine, we substituted this conserved amino acid by site directed mutagenesis to alanine or lysine.
Wild-type and pex5Δ mutant strain and transformants were tested for their ability to import the fluorescence marker DsRed fused to type I targeting signal Ser-Lys-Leu (SKL). In wild-type cells, DsRed-SKL appeared in punctate structures indicative for a peroxisomal localization (Figure 1B). In contrast, the synthetic peroxisomal matrix protein mislocalized to the cytosol in pex5Δ cells, which is in line with the specific import defect of this mutant . This mutant phenotype was functionally complemented upon expression of wild-type Pex5p, whereas Pex5pC6A failed to restore the import defect of pex5Δ cells. This finding is in line with previous reports, which demonstrated that the corresponding Pex5p-version of yeast and mammals is not monoubiquitinated anymore and therefore blocked for export from the peroxisomal membrane back to the cytosol [16,40]. In contrast, Pex5pK18R/K24R, which lacks the target residues for polyubiquitination, was still functional (Figure 1B; ). Remarkably, when the cysteine at position 6 was replaced by lysine (Pex5pC6K), peroxisomal import of the PTS1-marker protein DsRed-SKL was not affected (Figure 1B). The PTS1-receptor is not only required for the peroxisomal import of typical PTS1-proteins but also for non-PTS proteins like acyl-CoA oxidase, which bind to the receptor on its N-terminal half [5,41]. We extended our functional assay and monitored the strains for growth on plates containing oleic acid as the sole carbon source, which supports cell growth only if peroxisomal β-oxidation is functional. The pex5Δ-strain was unable to grow on this medium in line with previous findings  and typical for peroxisomal mutant strains of S. cerevisiae . Whereas Pex5pC6A failed to restore the growth defect of pex5Δ, the mutant strains expressing wild-type Pex5p, Pex5pK18R/K24R or Pex5pC6K grew at a rate indistinguishable from the wild-type strain (Figure 1C). Accordingly, we conclude that Pex5pC6K can fully restore the function of native Pex5p in peroxisomal protein import.
Inspired by the finding that a lysine residue on position 6 can functionally replace the conserved cysteine residue of Pex5p, we investigated the effect of shifting the conserved cysteine to position 18, thereby replacing a lysine, which is the usual target for polyubiquitination. We also combined this Pex5p-variant with the C6A substitution. The single substitution as well as combinations displayed similar steady-state levels (Figure 2A), indicative for comparable expression and stability. We monitored the capability of the Pex5p-variants to functionally complement the growth defect of the pex5Δ-strain on oleic acid medium and found that the substitution of Lys18 by cysteine has no significant effect on Pex5p function as long as the cysteine at position 6 is present (Figure 2B). However, the Pex5p-variant Pex5pC6A/K18C could not restore the ability of the pex5Δ-strain to grow on oleic acid medium, indicating that the presence of cysteine at position 18 could not compensate for the loss of the conserved cysteine at position 6, (Figure 2B). We conclude that the position of the target residue for ubiquitination is crucial, regardless of whether a cysteine or lysine residue is present.
Mutant pex5Δ cells were transformed with plasmids expressing Pex5p or indicated variants. (A) Immunoblot analysis of equal amounts of whole-cell trichloroacetic acid lysates of indicated strains with antibodies against Pex5p. Detection of mitochondrial porin served as loading control. (B) Indicated strains were spotted as a series of 10-fold dilutions on a medium with oleic acid as sole carbon source and incubated for 4 days at 30°C. Growth of mutant pex5Δ cells expressing Pex5p or Pex5pK18C was indistinguishable from the wild-type, whereas the mutant pex5Δ cells expressing Pex5pC6A/K18C display no growth on this carbon source.
Next, we analysed the consequence of the functional exchange of the conserved cysteine by lysine to the ubiquitination status of Pex5p. Whole-cell lysates of oleic acid-induced wild-type cells or pex5Δ cells expressing wild-type or mutant variants of Pex5p were prepared and subjected to immunoblot analysis. The steady-state level of plasmid-encoded wild-type and Pex5p-variants was similar (Figure 3A). The plasmid-encoded proteins showed an approximately four-fold higher steady-state level than endogenously expressed Pex5p (Figure 3A). Interestingly, Pex5pC6K displayed high molecular mass species, which were not seen for wild-type Pex5p. We suspected that these Pex5p-species represent ubiquitinated forms of the PTS1-receptor, most probably Pex5p polyubiquitination. As Pex5p polyubiquitination is known to depend on the conserved lysine residues at position 18 and 24 , we additionally substituted these amino acid residues by arginine. The resulting Pex5pK18R/K24R remained unmodified even when combined with C6A substitution (Figure 3A). However, combination with the C6K mutation still resulted in the modification of the receptor as observed for Pex5pC6K. Thus, the Pex5p modification is independent of the Lys18 and Lys24. To analyse whether this modification indeed represents ubiquitination, we analysed pex5Δ Pex5pC6K/K18R/K24R cells additionally expressing either the wild-type Ub (ubiquitin) or the mycUb fusion . The mycUb variant is about 1.5 kDa larger than wild-type Ub and leads to a shift of modified proteins . Whole-cell lysates of the transformants were prepared and equal amounts of protein from each strain were subjected to immunoblot analysis. Samples were probed with anti-Pex5p antibodies and slower migrating Pex5p species appeared (Figure 3B). Comparison of samples from pex5Δ Pex5pC6K/K18R/K24R expressing either Ub or mycUb showed that the putative ubiquitinated species of Pex5p were replaced by slightly larger new bands, clearly demonstrating that Pex5pC6K/K18R/K24R is polyubiquitinated at Cys6 in vivo under wild-type conditions.
Mutant pex5Δ cells were transformed with plasmids expressing Pex5p or indicated variants. (A) Immunoblot analysis of equal amounts of whole-cell trichloroacetic acid lysates of indicated strains with Pex5p-specific antibodies. In contrast with the wild-type, pex5Δ expressing Pex5pC6A shows up to three additional αPex5p reactive bands. The third and especially the second band are more pronounced in case of Pex5pC6K and Pex5pC6K/K18R/K24R. (B) The pex5Δ strain expressing Pex5pC6K/K18R/K24R was additionally transformed with a plasmid either encoding ubiquitin (Ub) or myc-tagged ubiquitin (mycUb). The observed shift to a higher molecular mass upon mycUb expression proved that the additional αPex5p-reactive bands represent ubiquitinated Pex5p.
Polyubiquitination of Pex5p was initially observed in strains affected in components of the peroxisomal AAA-(Pex1p, Pex6p, Pex15p) or the Pex4p–Pex22p-complex [18,19]. Pex5p ubiquitination was also seen in strains with unaffected peroxisomal biogenesis but reduced proteasomal function, indicating that Pex5p-polyubiquitination also occurs under wild-type conditions . Driven by the finding that Pex5pC6K/K18R/K24R is polyubiquitinated, we compared the Ub-pattern with those observed in a strain affected in PEX1 or PEX4. In contrast with pex1Δ strain in which Pex5p displays four Ub-species, Pex5pC6K/K18R/K24R pattern was similar to those present in pex4Δ cells, although with different intensity of the appearing bands (Figure 4A). In pex4Δ the fastest migrating Ub-Pex5p species was most prominent, likely to represent monoubiquitinated Pex5p. In contrast, the second Ub-species was most abundant of Pex5pC6K/K18R/K24R, most probably representing di-ubiquitinated Pex5p, which might indicate a role of Pex4p in this modification. Next we analysed the Ub-status of Pex5p-variants in pex5Δ mutants and in pex5Δpex4Δ double mutants. Pex5pC6A and Pex5pK18R/K24R behaved like wild-type Pex5p and thus displayed no significant polyubiquitination pattern in pex5Δ cells. Pex5pC6A exhibited the typical pex4Δ-pattern when expressed in pex5Δpex4Δ cells (Figure 4B). In contrast, Pex5pC6K as well as Pex5pC6K/K18R/K24R displayed the pattern of three Ub-species from which the intensity of the dominant middle band was shifted to the lowest band and thus lead to a typical pex4Δ pattern. From this, we conclude that Pex4p is involved, either directly or indirectly in the ubiquitination of Pex5pC6K.
Immunoblot analysis of whole-cell trichloroacetic acid lysates of indicated strains with specific Pex5p antibodies. (A) Mutant pex5Δ cells were transformed with plasmids expressing Pex5pC6K/K18R/K24R. The ubiquitination pattern of the Pex5p-variant expressed in mutant pex5Δ cells is different from the Pex5p-modification pattern of pex1Δ cells, but resembles the pattern for Pex5p of a pex4Δ strain, although with shifted intensities. (B) Ubiquitination pattern of indicated Pex5p variants expressed in mutant pex5Δ and in double-mutant pex5Δpex4Δ cells. The pex4Δ-ubiquitination pattern is observed for Pex5p variants when Pex4p is absent, but is more pronounced than in the wild-type. In the Pex5pC6K variants, the second Ub-Pex5p band from below shows a small shift to a lower molecular mass.
Polyubiquitinated proteins are in general designated for proteasomal degradation and usually exhibit a high turnover rate, whereas ubiquitin itself is quite stable [43,44]. This is achieved by specific deubiquitinating enzymes, which remove the ubiquitin moieties prior to the actual degradation process [45–47]. Previously, the deubiquitinating enzyme Ubp15p (or ubiquitin-specific protease 9X in mammals, ) was identified as part of the cytosolic Pex1p–Pex6p complex in S. cerevisiae . Ubp15p associates with the AAA-complex by an interaction with Pex6p and the recombinant protein is capable to deubiquitinate Pex5p at isolated membranes. In order to analyse whether the observed higher ubiquitination rate leads to a fast degradation of Pex5pC6K, we analysed the steady-state level of the PTS1-receptor in wild-type cells and in cells lacking Ubp15p. As judged by immunoblot analysis, the Pex5pC6K level was already slightly reduced in comparison with native Pex5p when expressed in pex5Δ cells (Figure 5). The same was observed for Pex5pC6K/K18R/K24R. However, the effect was more significant in a strain lacking Ubp15p. Here, the amount of Pex5pC6K as well as the Pex5pC6K/K18R/K24R was significantly reduced. Quantification of protein amount by densitometry is shown in Figure 5(B). The data suggest that the reduced level of Pex5pC6K variants is the consequence of ubiquitin-triggered increased degradation of Pex5p by the 26S proteasome.
Mutant pex5Δ cells or ubp15Δpex5Δ cells were transformed with plasmids expressing Pex5p or indicated variants. (A) Whole-cell lysates of oleic acid-induced strains as indicated were subjected to immunoblot analysis with Pex5p-specific antibodies. Mitochondrial Porin served as control for equal loading. (B) Signal intensities of Pex5p of three independent experiments were estimated by densitometry. Obtained signal intensities were normalized to signal from plasmid-encoded wild-type Pex5p expressed in pex5Δ cells. The steady-state concentration of Pex5pC6K and especially Pex5pC6K/K18R/K24R was drastically reduced in the absence of Ubp15p. Error bars=S.E.M.
To test this assumption, we analysed whether Pex5p variants can be stabilized by inhibition of proteasomal degradation. To this end, oleic acid-induced pex5Δ or pex5Δubp15Δ cells expressing wild-type Pex5p, Pex5pC6K or Pex5pC6K/K18R/K24R were shifted to fresh medium with and without the proteasome inhibitor MG132. After additional growth for 4 h to allow uptake and functional development of the inhibitor, whole-cell lysates were prepared and analysed for the presence of Pex5p and its variants. As judged by immunoblot analysis and signal quantification, the level of wild-type Pex5p increased only slightly upon proteasomal inhibition (Figure 6). However, when analysed in the pex5Δubp15Δ strain, the Pex5pC6K variant and especially the Pex5pC6K/K18R/K24R form was decreased in the absence of MG132, but proteasomal inhibition did result in a significant stabilization of the protein. Under these conditions about four-fold higher protein level was detected for Pex5pC6K/K18R/K24R. The data show that substitution of the conserved cysteine residue of the Pex5p N-terminus by lysine leads to artificial polyubiquitination followed by a higher degradation rate of the receptor by the 26S proteasome.
Mutant pex5Δ cells or ubp15Δpex5Δ cells were transformed with plasmids expressing Pex5p or indicated variants. Strains were grown on oleic acid containing medium for 16 h. Subsequently cells where shifted to oleic acid containing medium with or without 40 μm MG132 to inhibit the proteasome and grown for additional 4 h. (A) Whole-cell lysates of the strains were prepared and blotted for the presence of Pex5p and mitochondrial porin which served as loading control. (B) Pex5p signal intensities of blots depicted in (A) obtained from samples without (white boxes) and with (black boxes) MG132 treatment was estimated by densitometry. Intensities were normalized to signal from plasmid-encoded wild-type Pex5p expressed in pex5Δ cells or ubp15Δ pex5Δ cells respectively, without MG132 treatment. In contrast with untreated cells, Pex5pC6K and Pex5pC6K/K18R/K24R remained stable when the proteasome was inhibited, indicating that the observed reduced steady-state concentrations are due to a rapid proteasomal degradation. Error bars=S.E.M. with n = 3.
In the present study, we analysed the function of the conserved monoubiquitination site of the PTS1-import receptor Pex5p in S. cerevisiae. In all known species, this position is occupied by a cysteine . Work in yeasts and mammalian cells demonstrated that this cysteine is indeed modified with an ubiquitin-moiety via an uncommon thioester-bond [16,38,40] and that this modification is essential for the recycling of Pex5p and therefore also for peroxisomal matrix protein import in general [12,14,38].
Ubiquitination of substrates via esterification is a rare type of modification, because in most cases the ubiquitin-moiety is attached to a lysine residue via an isopeptide bond [26,50,51]. Therefore, the functional relevance of the ubiquitin esterification of the PTS1-receptor is a central question for the understanding of peroxisomal matrix protein import. Recently, indications for a redox-based regulation of Pex5p in mammalian as well as Pichia pastoris cells were reported, suggesting that the conserved cysteine of Pex5p might function as redox-sensitive module [52,53]. The P. pastoris study suggests that the cysteine may contribute to formation of disulfide-bonds of homodimeric, cargo-loaded Pex5p molecules, which are supposed to be disassembled in the reducing condition of the peroxisomal matrix . In slight contrast, the mammalian study suggests that the ubiquitination-competence of the cysteine is influenced by the redox-state of the cytosol, because oxidized glutathione interfered with monoubiquitination of human Pex5p . An import-competent Pex5p-version harbouring a lysine instead of a cysteine was used as a control, as it can still be monoubiquitinated during oxidized conditions in an in vitro system . However, the present study focused on redox-dependent effects of the wild-type human Pex5p, whereas the detailed functional consequences of the cysteine-to-lysine-mutant were not further addressed in vivo.
In our study, we analysed the role of the conserved mono-ubiquitination site of S. cerevisiae Pex5p. It has been shown earlier that the exchange of the conserved cysteine against a residue that cannot be ubiquitinated, like arginine , inhibits matrix protein import, blocks monoubiquitination and induces proteasomal degradation of Pex5p via polyubiquitination at the conserved lysine residues 18 and 24 . This is also the case for the Pex5pC6A that we have used here. Beyond this, we have exchanged the cysteine against a residue that can still be ubiquitinated, namely a lysine. Substitution of the conserved cysteine residue of mammalian Pex5p by a lysine did result in a quite functional protein . Also, the yeast Pex5pC6K mutant was still able to complement a pex5Δ strain and to facilitate matrix protein import. This finding demonstrates that esterification of the Cys6 is not essential for the function of Pex5p under standard conditions, as long as this position harbours a lysine as target for ubiquitination.
Interestingly, we found that Pex5pC6K was polyubiquitinated. Until now, polyubiquitination of Pex5p has been regarded as part of a quality-control system that is activated when the receptor cycle is impaired. This occurs when constituents of the AAA-complex or the Pex4p-complex are mutated or when the Cys6 as an acceptor site for monoubiquitination is exchanged against an arginine or alanine [12,16,18,19]. With the discussed concepts in mind that the conserved cysteine might be crucial for a possible redox-regulation of Pex5p and PTS1-protein import [52,53], the polyubiquitination of Pex5pC6K observed by us might simply indicate a compromised functionality of Pex5p without its supposed redox-sensor. However, our data also demonstrate that the detected polyubiquitination of Pex5pC6K differs significantly from the one observed in the regular quality-control pathway, because it occurs independently of the Lys18 and Lys24. In marked contrast, the Lys6 itself is modified, which results in a polyubiquitin chain pattern that is distinct from the one found in a pex4Δ strain. The shift of the Ub-pattern intensity detected for the Pex5p of a pex4Δ strain to the pattern of the Pex5pC6K mutant by one ubiquitin-moiety suggests that Pex4p contributes to formation of the polyubiquitin chain, possibly by attaching the first ubiquitin to Lys6, as it would have done in the presence of Cys6. However, it did not escape our attention that the Pex4p-dependent ubiquitination pattern exhibits a slight shift of the second band to a lower molecular mass (Figure 4). Our data demonstrate that the formation of an isopeptide bond instead of a thioester bond to ubiquitin, does not inhibit the function of the PTS1-receptor, but causes an unusual polyubiquitination that might also involve Pex4p. Currently, we do not know whether the polyubiquitinated Lys6 indeed can substitute for the function of the monoubiquitinated Cys6-form. It could well be that an intermediate monoubiquitinated Lys6 performs a so far unknown function prior to its further ubiquitination. The reason for the presence of a cysteine at this position in all species might thus not be the direction of Pex5p to quality-control and degradation. Another possible explanation considers the slightly different ubiquitination/deubiquitination rates of the thioester-linked compared with the isopeptide-linked ubiquitin. Upon Lys6 polyubiquitination the first ubiquitin might be bound too strong via the isopeptide bond and may stay too long at this position. This may result in further ubiquitination and formation of a polyubiquitin chain at Lys6 followed by receptor degradation. In general, the dynamics underlying the receptor cycle of Pex5p seem to be of importance because the import and export rates of Pex5p have to be balanced . The peroxisomal membrane complexes display a limited capacity to bind PTS1-receptor molecules because a block of the export steps results in an accumulation of Pex5p at the membrane and an inhibition of matrix protein import [18–20]. Furthermore, the expression level and functional activity of docking factors and exportomer proteins have to be adjusted . Moreover, both processes seem to be co-operatively coupled [57,58]. Therefore, it is reasonable that also the ubiquitination-event itself is tightly regulated. In the case of cis-acting factors, recent work has demonstrated that the efficient attachment of the ubiquitin-moiety to the cysteine of Pex5p and therefore functional matrix protein import requires a certain threshold of Pex4p activity, which is dependent on the C-terminal domain of Pex22p as co-activator of Pex4p [59,60]. Therefore, it seems reasonable that the retention time, which the ubiquitin-moiety stays on Pex5p, has to be limited and that the trans-acting factors required for the deubiquitination are tightly regulated as well. First indications that the lability of the thioester-bond might indeed be utilized for rapid deubiquitination of Pex5p were indicated by the finding that the ubiquitin-moiety could be removed non-enzymatically by cytosolic glutathione in a mammalian in vitro system . Our data in S. cerevisiae demonstrate that the stronger isopeptide-bond results in an artificial polyubiquitination at Lys6 and therefore support the notion that deubiquitination of Pex5p has to occur relatively fast.
In line with this assumption, we were able to uncover another layer of regulation. The steady-state level of Pex5pC6K is significantly reduced, when in addition the deubiquitinating enzyme (DUB) Ubp15p is deleted. Ubp15p has been identified earlier as a binding partner of the AAA-ATPase Pex6p and was shown to deubiquitinate mono- as well as polyubiquitinated Pex5p in vitro . A functional counterpart in mammalian cells is USP9X, which also deubiquitinates the monoubiquitinated PTS1-receptor in vitro . However, the relevance was not clear in both cases, because both enzymes seem to be partially replaceable by other ubiquitin hydrolases. In the present study, we clearly show that the combination of the ubp15Δ strain with the thioester-bond-replaced Pex5pC6K results in a significantly reduced level of this version of the PTS1-receptor via proteasomal degradation. Taken together, these data demonstrate that under normal conditions the labile thioester-bond to ubiquitin combined with the presence of deubiquitinating enzymes like Ubp15p prevents the PTS1-receptor from being degraded and thus allows efficient recycling. These two layers of protection are quite unique. Ubp15p and its mammalian counterpart USP9X are the only ubiquitin-hydrolases that have been reported to cleave thioester-bonds of a modified substrate [30,48]. Moreover, the reason, why a cysteine of Pex5p and not a lysine is the physiological important ubiquitination site, seems to be in marked contrast with the few other known examples of non-lysine ubiquitination. This non-canonical ubiquitination of internal residues can involve the formation of oxyester-bonds to serine and threonine in addition to the thioester-bonds to cysteines [26,50,51]. Important cellular examples are the rapid degradation of the lysine-less apoptosis-supporting tBid-N after polyubiquitination on cysteine, serine and threonine residues . Also the transcription factor Neurogenin 2 involved in neuronal differentiation [62,63] as well as non-secreted NS-1 (nonstructural-1 protein) Ig light chain  are polyubiquitinated on lysines, cysteines, serines and threonines, resulting in a rapid degradation by the proteasome. Other examples are linked to viral infections. The HIV protein Vpu (viral protein U) interacts with a cellular SCF-type (Skp1, Cullin, F-Box protein) ligase in order to promote ubiquitnation of BST-2 (bone marrow stromal antigen 2), a regulator of immunity, on cysteines, serines and threonines . Moreover, the cytoplasmic tail of MHC-I in the endoplasmic reticulum (ER) is ubiquitinated on lysine, serine and threonine residues by mK3 (mouse γ-herpes virus ligase), which results in proteasomal degradation via the ER-associated degradation (ERAD) pathway [66,67]. At the cell surface, MHC-I can be ubiquitinated on lysines and cysteines by MIR1 (modulator of immune responses) and MIR2 (Kaposi's sarcoma-associated herpes virus ligases), which results in lysosomal degradation [68,69]. A closer investigation of the specificity of the RING-domain of mK3 suggested that no E2 partner specialized only on thioester-bonds is required . Moreover, not the type of amino acid itself, but the position within the substrate is of importance for the ligases MIR1 and MIR2. Both can ubiquitinate cysteines as well as lysines as long as they are at a specific position . This mechanistic aspect is comparable with the situation of Pex5p and the mutants Pex5pC6A and Pex5pC6A/K18C, where a shift of the cysteine to position 18 cannot compensate the loss of the ubiquitination site at position 6. Only the exchange to a lysine at position 6 can compensate for the loss of the cysteine, indicating that the peroxisomal RING-ligase complex as well as Pex4p are not selective for the type of ubiquitinable amino acid, but for its position.
At the end, the major difference in the physiological outcome of the esterification of Pex5p at Cys6 to all other known examples of non-lysine ubiquitination of internal residues is that it is not intended for the degradation of Pex5p. Our data show that this modification contributes to the stability of Pex5p, whereas all other examples aim to enhance the degradation of the substrates.
Further work will analyse if a similar regulatory context exists for the PTS2-dependent matrix protein import pathway, as the PTS2 co-receptors Pex18p [24,71] and Pex20p [25,72] are also monoubiquitinated on a conserved cysteine. This would further support the novel role of ubiquitin-esterification, namely that the labile thioester-bond together with a DUB function to limit the resident time of ubiquitin on a substrate in order to prevent its degradation.
Benjamin Schwartzkopff designed and performed experiments. Sohel Hasan performed experiments. Harald W. Platta wrote the manuscript. Wolfgang Girzalsky and Ralf Erdmann designed experiments and wrote the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft [grant number FOR1905 (to H.W.P. and R.E.)].
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text:
Q: Cron job scheduled not running automatically in Node.js BOT I am working on a Bot, using BotFramework-node.js hosted on Azure.
I am using four cronjobs in my bot to run different functions at different points of a day. The only issue is that the jobs are not running automatically. They will run normally if i compile/run the code, but wont if i don't compile/run it within 12 hours  i.e, if i run the code manually today morning, the jobs will execute fine for today but tomorrow it won't unless i run it again.
Tried changing the modules. I have tried using node-schedule, node-cron. Now using cron
var Name = new CronJob({
    cronTime:'0 15 11 * * 0-6',
    onTick: function(){
        //function call / another js file call
    }
});
Name.start();

redpajama_set_name: RedPajamaStackExchange


One of the benefits of having lived so close to the edge for so long is that I don't take a damn thing for granted. I know what it's like to be facing losing (and then actually losing) my home. I know what it's like to lose a loved one to addiction, to parent alone, to be afraid, to be facing homelessness, to be dependent on the charity of others. I know the sting and humiliation of throwing my lot at the mercy of an overworked DSHS caseworker in hopes of receiving aid. I know what it feels like to have our names on paper ornaments on the Giving Christmas Tree, where a "Boy, Age 8" would like some Legos and a coat. I know well the spaces inhabiting our periphery, the margins of our lives, where we all hope to never go, and where hope is all you've got if you get there.
So when people talk about the state of the world, of the decay of society- it baffles me. The talking points and even the themes I hear sometimes at church and from the news networks just don't fit with my lived experience. Contrary to the obtuse bloviating of pundits and doomsayers, I don't see the hand-basket to hell overflowing- as a matter of fact, I think it's nearly empty.
Whichever direction you face, you can see people doing good in the world. It would be hard to look past the good being done, it is so pervasive.
In my own life, despite the challenges and sometimes near catastrophic consequences of agency, there were mechanisms and safety nets and hands outstretched waiting to help. I lost my home, but I was able to uncouple myself legally to protect myself and my kids. My children's father was swallowed by addiction, but there were laws, judges and courts to assure my children were protected AND that their father was protected- from himself and from doing further damage. Addiction is a nasty beast slouching around the land, but there are programs and therapies dedicated everywhere to slaying him. Yes, I was suddenly impoverished and without any child support and no hope of receiving any- but there were welfare programs in place for people just like me. Yes, it was hard to navigate some of it, and it's difficult to prove qualification- but there are people who dedicate their lives to protecting the poor and needy, and do so without great financial gain themselves. I couldn't provide gifts for children at the holidays, but how wonderful there are people who care enough to make sure children like mine are not forgotten. I was able to qualify for low-interest student loans and get an education, so I would be able to remove myself from desperation. Yes, work was required of me, but it was work I could not have done had there not been help available. My pride? Obliterated. But also obliterated was any notion or lingering idea that the blessings of any life were somehow owed or earned. "There but for the grace of God go I" is more than an idea.
Pulling back from my personal experience, I see these acts of good expanding, like a beautiful fractal, in the world at large. There are people working all over the world to help and aid the disadvantaged, whether it's engineering systems of waste disposal in Central America, or digging wells in African villages in so families can have potable water. There are programs everywhere, addressing every corner of need. NGO's work to increase access to education for girls in central Asia. There are micro lending programs to help women start small businesses in order to support their families. Homeless shelters and rehabilitation programs can be found in every urban area. Organizations provide malarial nets to curb the devastating effects of mosquito born illnesses. Children with disabilities who were once written off as expendable now receive therapy and IEP's and attend classrooms where their needs are not only met, but their lives are expanded and they contribute to the world. Cargo ships and airlifts of food, medication, and aid workers pour into areas devastated by natural disasters. Doctors, nurses and other healthcare workers dot the globe working to vaccinate and eradicate childhood diseases and mortality.
It doesn't matter if this feels inadequate- to quote Mother Teresa of Calcutta, "What we do is nothing but a drop in the ocean, but if we didn't do it, the ocean would be one drop less than it is." We are imperfect, and there is grace in our imperfection.
People care. People are willing to extend themselves perhaps more so and with better results than at any time in history. It wasn't so long ago that the poor were considered expendable. Children were sent to work in coal mines or worse, women could be legally beaten by their spouses and couldn't vote or get an education, and human beings could be bought and sold as property. These things are, of course, still happening in pockets of the globe- but human consciousness and concern is eroding those spaces, like water over stone. I submit that we are more aware of the plight and pain of our brothers and sisters, and willing to do something about it, than at any time in human history.
There is more to do. Ever so. The work will never be done- but I find awe and beauty in the actual actions of so many of my fellow humans, willing to leave their comfort zones and challenge their assumptions, and to roll up their sleeves and get to the real work of a life worth living.
← Are Not We All Beggars?
There are few words for how much I love this post. Sometimes I am concerned by the "the world is terrible and getting worse" mentality held by so many of my beloved fellow Mormons, because while certainly, there is plenty of evil-doing and selfishness in the world, I worry that this doom and gloom mentality completely ignores how much good is being done in the world. This post makes me want to find and flood facebook with accounts of good being done in the world, if only to show my friends that there is good out there–and we should be part of it!
Tracy, I love this. Thanks, my friend.
Tracy, having seen you through some of these situations I commend you for where you are today, your honest attitude & the love you so freely share. My heart & head thank you.


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Geometrics are bigger than ever these days, so this design is even more popular than we could imagine!
It would make some amazing statement cushions, or even an armchair!


This concept hit me the other day and I didn't write it down, but the following is my attempt to reiterate my thoughts: So I was sitting thinking about my life while listening back to the recording of David Miller's sunday morning talk about "The Box of Our Friends", when I began to ask myself questions. Now, when you ask yourself questions, do you ever really get an unbiased answer? Don't answer that. But here's the real deal, I began to ask myself the question of not what kind of friends I had, but what kind of friend I am to each one of them. I thought about a numerous amount of things that I thought could be true about me, but I realized that I love helping my friends.
Occasionally I won't help just ANYONE, but always my friends. I began to ask myself, "Why do you do this? If you do these things all the time, then they'll start to take it for granted, then you'll just be unappreciated for your 'ministry to your friends'." I couldn't help but think, is that really true?
I wil do things for you as long as they are appreciated genuinely, but as soon as they are 'expected' or 'taken for granted' I will pull the plug."
Is that so wrong of me? I mean there may in fact be times when I don't have time to do something in which case I will likely (but not always) say no to your request. There have been times when people are astonished that I said no, and then become cross with me, because I'm unwilling to help them.
Am I fine with that? Yes. I have said for some time now, that my goal in life is to make myself to be useful, relevant, and ideally indispensable in whatever I am involved in. In other words, I want people to come to me, and have them know that I can do it and I'm more than willing to do so. For sometime now, I have been the person that is always looking for the next way to help out a friend in need. I want to aid and assist in anyway that I can because that is why God has given me the abilities that He has. I always want to make time for people as much as possible, but as we all know there are only so many hours in a day, and days in a week. Right now in my life, I am juggling 40 hours a week of work, 18 credit hours of school, church, a housefull of my closest friends, my amazing girlfriend, my family of course, and my cute little dog Maxine. Where in there am I supposed to have time for God, let alone doing things in obedience to him?
Now I have never been the person to complain about much or try to get attention about things happening in my life, and here's why: If you cared, you'd ask. There are people that know me, and care for me and my heart.So with that said, here's the reason for the title:I came up with the analogy/metaphor mid conversation, and now I'm going to share it with you. Sure it's got some things about it that could be questioned or misinterpreted, but it give me a better mental picture, so I hope it helps you.
"The squeeky wheel gets the grease." We hear that phrase a lot and it's meant to mean that if you make a fuss, your problem will be solved quicker. This may work in the business world, but I feel it's a cop-out. If everyone and their pet monkey knows your life's problems, of course someone along the way is bound to step up and try to help you, either because they are able to or they just want to shut you up. So I sort of do the opposite; I look at myself as "The squeeky board in the floor that still gets walked on." What does that mean? Take this example: When I first moved into my nearly 100 year old house on Rivermont in Lynchburg, Virginia there was a loose board in the floor in my room upstairs. At first, when I didn't know the house I gave this board special treatment, and danced around it as to not worsen the condition hoping that it would last just long enough for it not to affect me while I am living there. Basically while I need it, I want to make sure it has everything it needs to keep functioning as long as I need it. With proper care, that board could last a long time.
When you first move in, you notice all the cracks, creaks, squeeks, clanks, and sounds that an old house makes because your not used to them, but once you've been around it for a while you just get used to them and expect them to be there. This, my friends, is where I feel I come in for some (DEFINITELY NOT ALL) people.
You see, I don't even care where that board is now, I just step wherever my foot lands with the assumption of, "It's held up this long, what difference will a little longer make?" If I was that board, the second that I got overlooked, I would snap and send 'me' straight through the floor and on to the downstairs couch (probably with a broken leg). All this to say, I am like every other person, in that I like to be appreciated and checked up on, but I know that because I am giving and willing, I get taken for granted. Oh well such is life. I am content with the role that God has given me, I just let "myself" get in the way of what God has for me.


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Photo: Reynaldo Riobueno/Shutterstock
The Protests From Around the World That Defined 2017
Considering that 2017 began with the question, "is it okay to punch a Nazi?", it should have been no surprise that last year saw unprecedented social upheaval and protests taking place across the globe. It is equally unsurprising that the tone and content of many of these protests both at home and abroad can trace their roots to the immediate aftermath of the election of President Donald Trump.
While many countries saw protests last year, it is certain that no single figure was as galvanizing for political activism across the globe than the US President, who has already triggered new protests in Pakistan after a series of his Tweets suggested that the United States will be cutting aid to the country. It is mainly for this reason that most of the items on this list take place in the US.
1. Women across the globe kick off a year of protest
A running theme for nearly every 2017 protest in the US, and many across the world, is the Trump presidency. Arguably this began during the presidential campaign in 2016, when protestors showed up at Trump rallies and even tried to prevent him from speaking by blocking roads. But the international Women's March on January 21st made it clear that the demonstrations during the election were just the beginning. Indeed, the biggest protests in the United States were in direct opposition to the policies of the Trump administration. Carrying signs in support of women's rights, the rights of LGBTQ citizens, the rights of people of color, and the rights of immigrants, over than three million marched through the streets of DC, New York, Denver, Portland, and elsewhere around the country and the globe the day after President Trump was inaugurated.
The series of marches were collectively the largest protest in US history and pink "pussy hats" became an international symbol for women's protest against misogyny and sexism. Other marches in the winter and spring echoed the model and tone of the Women's March, including the March for Science and the March for Truth. A sequel to the Women's March is scheduled for January 20th in 2018.
2. The NFL takes a knee to protest police brutality
What began with San Francisco 49ers' quarterback Colin Kaepernick protest for criminal justice reform at NFL games grew into a nationwide debate about free speech, patriotism, and the nature of protest in 2017. The string of well-documented and contentious deaths of African-Americans at the hands of police officers, including that of recent high school graduate Michael Brown in 2014, and the Black Lives Matter movement encouraged Kaepernick to pledge donations to activist organizations. During the 2016 preseason he chose to sit rather than stand while the national anthem played before the start of a game.
After a conversation with former NFL player and Green Beret Nate Boyer, Kaepernick chose to kneel during the anthem to avoid seeming disrespectful to members of the armed services. During a postgame interview in August, Kaepernick said, "I am not going to stand up to show pride in a flag for a country that oppresses black people and people of color. To me, this is bigger than football and it would be selfish on my part to look the other way. There are bodies in the street and people getting paid leave and getting away with murder."
While this protest was somewhat overlooked during the 2016 presidential campaign, "taking a knee" triggered a heated national debate after President Trump tweeted in September that NFL owners should fire NFL players for kneeling during the anthem. As the practice of having NFL players stand for the anthem before each game did not begin until 2009, and is not required of the players according to the rulebook, roundtables on ESPN and other sports channels ended up producing in-depth discussions about a tense political topic, something these channels tended to avoid in the past. Despite this, the path for criminal justice reform is unclear at best under the current administration.
3. Venezuelans protest a looming dictatorship
While Venezuela has seen countless protests for and against the national government since the death of President Hugo Chaves in 2013, 2017's Mother of All Marches stood out for the use of violence against participants. Under Chaves' leadership in the early 2000s, the Bolivarian government built the entire Venezuelan economy around oil exports. When the price of oil dropped, many of the socialist policies instituted by Chaves became unsustainable and the effect on the lives of citizens has since been catastrophic. When confronted with a collapsing economy, a climbing crime rate, and growing levels of poverty Chaves' successor, Nicolas Maduro, veered towards an authoritarian government. This was especially evident on March 29th, when a Maduro-friendly Supreme Court stripped the opposition-led National Assembly of its powers and assumed legislative authority.
On April 19th, hundreds of thousands (possibly millions) of anti-Maduro protestors took to the streets of Caracas for the Mother of All Marches. In a disturbing move to observers, Maduro armed militias with rifles to quell the protest. By the end of the day, more than 500 protestors had been arrested and three killed. In 2018 the situation in Venezuela remains volatile, with shortages of food and medicine defining the lives of many, but Maduro has shown no signs of stepping down from power.
4. Death at the hands of American fascism
One of the starkest contrasts in 2017 was between the rallies of white nationalists and their "antifa" counter-protesters. The Unite the Right rally on August 11th saw several hundred white supremacists, neo-Nazis, and neo-Confederates march through Charlottsville, Virginia, carrying torches to protest the planned removal of a statue of Confederate general Robert E. Lee. The two-day event was met with counter-protestors and caused the governor McAuliffe to declare a state of emergency when it became clear that public safety could not be guaranteed. On the second day, state police authorities forced the dispersal of the increasingly violent rally. Shortly after a man in a Dodge Challenger drove into a crowd of counter-protesters, injuring 19 and killing 32-year-old Heather D. Heyer.
After the arrest and charge of 20-year-old James Alex Fields, National Security Advisor H.R. McMaster and Attorney General Jeff Sessions almost immediately labeled the attack an act of domestic terrorism. President Trump, however, was criticized for inadequately condemning the attack in his statement. In his statement he said, "we condemn in the strongest possible terms this egregious display of hatred, bigotry, and violence on many sides, on many sides", which sounded as if he was equivocating Nazis to the counter-protesters.
5. Juggalos outnumber Trump supporter on the National Mall
On September 26th, around 1,500 fans of rap group Insane Clown Posse (ICP) gathered in D.C. to protest being designated a "gang" by the FBI at the same time as a right-wing rally in support of President Trump. The second event, called the "Mother of All Rallies" (in reference to a bomb dropped on ISIS fighters in the spring), featured right-wing militias like the Oathkeepers and Latinos for Trump founder Marco Gutierrez. Despite MOAR's organizers hope to show mass support for Trump through a neither "a left or right rally," the event was overshadowed by the larger group of colorful ICP fans, also known as "Juggalos", who are famous for wearing clown makeup, drinking Faygo, and wearing shirts that say things like, "sorry, I'm not that great at peopling." Speaking in front of the crowd, several Juggalos told stories of losing their jobs, getting pulled over by the police, and even losing custody of their children for displaying ICP merchandise or attending a concert.
Despite the turnout and widespread media coverage of the Juggalo rally, ICP and the ACLU lost a lawsuit against the Department of Justice in December. If nothing else September 26th illustrated that, while President Trump's large and dedicated base of support has rallied in public spaces, pro-Trump demonstrations have not delivered the same numbers that Trump's opposition has across American cities.
6. Zimbabwe ousts its 40-year dictator
In contrast to Venezuela, Zimbabwe saw the relatively peaceful ouster of its 2nd president, who ran the country for the past thirty-seven years as a dictator. Robert Mugabe was first elected Prime Minister at the dawn of Zimbabwe's independence from white minority control in 1980 and was the world's oldest head of state in 2017. A well-educated African Nationalist, Mugabe began his political career in the 1960s as an activist and guerilla fighter. By the late 70s he was one of the key players in creating the future state of Zimbabwe and often called for extreme violence towards white residents, something he would do repeatedly over his political career in times of economic crisis. Under Mugabe's leadership, Zimbabwe entered an unpopular war in Congo, suffered from food shortages and staggering inflation, saw several constitutional crises, and witnessed routine state-sanctioned violence against white and black citizens alike.
By 2017, it became clear that the 93-year old was considering naming his wife, Grace Mugabe, his successor. Mugabe's political party leadership did not approve and a coup d'état began on November 14th, when military officials seized several government buildings and arrested dozens of officials. While gunfire and artillery were heard in Harare, there were few reported deaths. Over the course of ten days the military blockades were removed, Mugabe's party cut his membership, the government drew up impeachment proceedings, Mugabe resigned, Vice President Emmerson Mnangagwa was sworn in as president, and thousands of peaceful protestors filled the streets in support of the change.
Despite the criticism of unconstitutionality, foreign leaders have characterized the end of Mugabe's rule as peaceful and overdue. Mugabe and his wife are currently living under house arrest, while the future of Zimbabwean democracy and its economy remains unclear.
7. Catalonia Votes For Independence
Over the course of the summer the Parliament of Catalonia, under the leadership of Catalan president Carles Puigdemont, worked on a referendum that would create an independent Catalonian Republic. The autonomous community of Spain, with its capital in Barcelona, then joined other provinces across Europe in the divisive struggle to redraw the map of post-war Europe. Similar to the Scottish independence referendum of 2014, the Catalonian referendum immediately drew ire from the national government, which attempted to tamp down a series of marches and rallies of support across Spain.
Despite the occasionally violent crackdown and the arrest of several Catalonian secessionist leaders, a referendum was held on October 1st. The referendum asked Catalonian voters, "Do you want Catalonia to become an independent state in the form of a republic?" More than two million voters said "yes", securing 92% of the total number of votes cast.
Due to a host of flaws in the vote, including changes to the census and Spanish police shutting down some polling stations, the vote was voided. In December, regional elections saw secessionist parties win a majority of the seats in the Catalonian parliament, but the unionist party remains the largest single party. At the start of 2018, things are looking as they did over the summer, with the separatists preventing the unionists from forming a majority government and the Catalonian relationship to Madrid as fraught as ever.
8. Ultra Nationalist March in Poland
The United States was far from the only nation to see ethno-centric nationalist demonstrations in 2017. On November 11th, Poland's Independence Day, tens of thousands of far-right nationalists marched through Warsaw. Burning red smoke bombs and chanting racist, anti-semitic, and anti-refugee slogans such as "pure Poland, white Poland" and "Death to the enemies of the homeland", the estimated 60,000 marchers overshadowed other Independence Day events and counter protesters, including the event attended by Poland President Duda.
Outside observers have commented that this annual march has been seen as a rallying point to far-right movements across Europe since it began in 2009. Leaders of the increasingly conservative Polish government have also turned away from European leadership in Brussels for a more nationalistic agenda. While President Duda and Deputy Prime Minister Glinski condemned the march, Poland's interior minister Mariusz Błaszczak called the event "a beautiful sight", adding that he was "proud that so many Poles have decided to take part in a celebration connected to the Independence Day holiday."
9. Palestinians and Lebanese protest against US embassy move to Jerusalem
Another divisive announcement from the Oval Office was the decision to move the US embassy in Israel to Jerusalem, which Israel considers to be its national capital. While the option to move the embassy has been on the president's desk since the early 90s, the last three presidents have not signed off on the move for a myriad of reasons, including the safety risk for those who work in the embassy and the desire to appear a quasi-neutral negotiator between the Palestinians and the Israel's. Trump's decision fulfills another campaign promise, although it has further alienated him from the international community, which almost unanimously condemned the move.
The immediate outcome of the announcement was a series of protests throughout the West Bank, Gaza Strip and Lebanon. In Beirut, protestors crowded around the US embassy, throwing rocks and other projectiles at its barricades. Whatever the political gains this president will receive at the Knesset, the protests seem to guarantee that the Palestinians and other Arab leaders will no longer see the United States as a fair negotiator.
10. The tide turns against powerful male sexual assaulters
Perhaps the most well-known of 2017 protests with the broadest cultural impact, the #MeToo movement set off a string a high-profile firings of men who were accused of sexual assault and/or harassment. Many of these men are members of the so-called "Hollywood elite", including comedian Louis C.K., actor Kevin Spacey and producer Harvey Weinstein, which in turn triggered discussions about hypocrisy among members of the political left and misogyny's presence at all levels of society. The resignation of Minnesota Senator Al Franken (D) following a series of accusations and alleged sexual predator Judge Roy Moore's (R) loss in Alabama's special senate election kept #MeToo in the national discourse for the entire year, with the more than a dozen sexual assault allegations against President Trump looming over discussions.
#MeToo shows no signs of stopping in 2018, with Oprah Winfrey, Natalie Portman, Jessica Chastain and others making statements against misogyny and sexual misconduct at the 2018 Golden Globes. Eight actresses brought social activists as their guests and many others wore black attire to show solidarity.
11. Iranians defy their theocratic government
In case you thought 2018 would start with a sense of calm to contrast with 2017, a surge of protests is taking place in Iran. The Iranian government has not seen this much criticism from its own citizens since the dawn of the Arab Spring in 2009, when Iran became a staging ground for political activists learning how to use social media as an organizing tool. Back then, reformists called the election of conservative President Mahmoud Ahmadinejad rigged, leading to protest rallies involving thousands that took place in mainly in Tehran and other large cities.
The current protests, which began at the end of December, are more spread out and are the result of a stagnant economy, rising food prices, government restrictions on social media sites like Instagram. While it is still unclear how widespread these protests are, there appears to be a much stronger sentiment against the regime altogether, with a younger population venting frustrations at an increasingly economically stratified system.
ActivismThe Protests That Changed Us

redpajama_set_name: RedPajamaCommonCrawl


If a new building is to be erected, or an existing library building is to be remodeled, repaired, improved or an addition thereto erected, or necessary equipment is to be furnished, or any or all of these things are to be done, the board of trustees shall cause a plan to be prepared and an estimate to be made of the cost. If a site or a building is to be purchased, the trustees shall cause an estimate to be made of the cost of such site or building. The trustees may then determine the funds that will be available from accumulations, and the amount to be raised from a bond issue, by annual certification, or by a mortgage. The trustees shall further determine the term, not exceeding 20 years, over which they shall spread the collection of the cost of erecting a new building, or remodeling, repairing, improving an existing library building or erecting an addition thereto, or furnishing necessary equipment, or purchasing and improving a site or building, or the acquisition of library materials such as books, periodicals, recordings and electronic data storage and retrieval facilities in connection with either the purchase or construction of a new library building or the expansion of an existing library building, or any or all of these things.
For the purpose of Sections 5-1, 5-2, 5-3, 5-4 and 5-5 of this Act, the acquisition of library material such as books, periodicals, films, recordings and electronic data storage and retrieval facilities is considered to be in connection with the purchase or construction of a new library building or the repair, remodeling or improvement of an existing library building or the expansion of an existing library building if the determination of the Board of Trustees to acquire such library materials is made within 5 years from the date that a new library building is purchased, or construction of a new library building or the repair, remodeling or improvement of an existing library building or the expansion of an existing library building is completed.
The board shall make a record of their proceedings and determinations and transmit a copy thereof to the corporate authorities for their consideration and approval.


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American Horror Story: Roanoke: Evan Peters Is Finally Here and Everything Has Changed
by Lauren Piester | Wed., Oct. 12, 2016 8:09 PM
Sarah Paulson Has Not Watched "The People v. O.J. Simpson"
Finally, the face we've been waiting for!
We're not talking about real life historian Doris Kearns Goodwin, who appeared in tonight's American Horror Story to tell us the story of Edward Mott, the builder of the house on Roanoke Island, but we are talking about the guy playing Edward Mott: Evan Peters.
Peters finally showed his face in tonight's episode as Mott, the house's original owner, who ran off, away from his wife and kids, to be with his servants and his boyfriend.
It was Evan in all his glory, complete with a ridiculous accent, a pretty hot bathtub scene, and multiple amazing temper tantrums over the state of his beloved paintings. He even abandoned his boyfriend over one of his paintings being ruined, which would have been super sad if Mott then weren't immediately burned on a spit over the fire (like a pig??) by the colonists.
American Horror Story Characters Ranked (By Actor) From Worst to Best
According to Goodwin, the house stayed in the family "until the last Mott died in Florida in 1952." AKA, Dandy (Finn Wittrock), from Freak Show. Everything has come full circle!
Mott, who's now definitely dead, later showed up in front of Shelby, Matt, and Flora to help them escape both the angry mob of colonists outside and the dead former residents inside. He said he'd keep them alive, and led them through a creepy crawly tunnel underground before leaving them alone in the woods.
Luckily, the redneck guys from the first episode found them, put bags on their heads, and took them to the house where they first found those feral boys. In that house, they found Elias, who was still alive but in very bad shape.
He was bloody and missing limbs, mumbling, "Mama took my leg" and begging for death.
"Mama" turned out to be Frances Conroy, who was clearly in charge of this small band of men.
American Horror Story: Roanoke: You Get a Human Sacrifice, and You Get a Human Sacrifice!
FX/YouTube
Apparently, the Polks (for that is their name, you see) guarded the island and helped make sure the Butcher and the colonists got their human sacrifice every year. They had intended to buy the house in order to keep it empty, so that all this crap wouldn't be happening.
After Mama instructed her boys to smash Elias' head in to end his suffering, the Polks put Shelby, Matt, and Flora in a truck on the way back to the house, and Shelby and Matt stupidly tried to be heroes and escape. This went badly, and Shelby ended up nearly getting her ankle chopped off.
Matt, Shelby, and Flora were dumped back at the house, right in front of the Butcher, and apparently it was Flora's turn to be a human sacrifice first...until Wes Bentley stepped up.
American Horror Story Renewed for Season 7 on FX
He hit his mom on the head and threw her into the fire, saying he didn't want to spill any more innocent blood, and then Lee finally returned from the police station with a car. Edward got Matt and Shelby untied, Lee hit Flora's attacker, the piggy man, with her car, and then they all got in that car and drove away.
They went to the hospital, found the cheapest motel they could, got some food, took some showers, and then had bad dreams for the rest of their lives, but otherwise lived OK.
So...that's the end of Matt and Shelby's story? It kind of seems that way, and we do know that next week's episode changes everything, but this seems sort of sudden.
What could the rest of the season possibly hold? Sound off in the comments with your theories!
American Horror Story airs Wednesdays at 10 p.m. on FX.
TAGS/ American Horror Story , TV , Top Stories , Evan Peters


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The Mystique of Cuba
My wife and I recently returned from a cruise to Cuba. It was a small ship and we were able to circumnavigate the island, stopping in Havana, Cienfuegos/Trinidad, and Santiago. Since our return I've been struggling with how to write about Cuba without being overly trite or over generalizing. On the superficial (or potentially trite) level, there is, of course, the beautiful architecture, the ubiquitous old American cars (much more common than I had thought), and the music. On the more subjective (and subject to potential generalizations), there is the people and the politics.
Judging from the discussions on our ship, which were led by two Cuba "experts," most people's fascination with Cuba is grounded in the mystique of its geopolitical history, the uncertainty of its future, and the indomitable spirit of many of its people. Of course, had that mystic not been clothed in the delightful gifts to the senses, to Americans I suspect Cuba would be just another country with a troubled past and decades of hardship.
If there's one thing I've learned from traveling the world, and particularly Africa, it's that making learned-sounding pronouncements about the history or politics of a particular place is ill-advised and usually only partially correct. I liked the fact that the speakers on our cruise were perfectly willing to answer a question with "I don't know."
The Cuban guides, on the other hand, were armed with statements and answers with which they had obviously been programmed through their education. None of the guides we came into contact with had been alive at the time of the revolution, yet one guide in particular never uttered the word "revolution" without combining it into the phrase, "the triumph of the revolution." All of the guides had adopted Fidel Castro's term "special period" as a euphemism for the period following 1990 when the Soviet Union collapsed and Cuba lost its 'sugar daddy.' The "special period" was a period of virtual starvation.
While on the cruise one of the speakers gave me a novel by a Cuban writer, Leonardo Padura. It was a mystery set in Havana in the late 1980's, a good story, beautifully written. But what struck me was how much more one can learn about a country's culture from a novel versus from a lecture or essay. The day-to-day lives of people can come alive in the course of a well-written novel. And, indeed, the daily hardships of Cubans, even in the years leading up to the "special period," when not romanticized by propaganda, are part and parcel of Padura's description of life in Havana. [I am now reading another Padura novel which takes place during the "special period" and the portrayal of people's struggle to survive is enlightening and compelling.]
The people of Cuba have gone through some seriously bad shit. Assign whatever cause you want to it: America turning its back on Castro following the revolution, Castro turning to the Soviet Union, Communism, Cuban-Americans' stubborn refusal to ease or lift the embargo, Castro himself, etc. The point is, there is more than enough geopolitical blame to go around and a phrase I think would be more applicable than "the triumph of the revolution" would be "the triumph of spirit."


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Stepan Iwanowycz Tarabałka (; ur. 9 stycznia 1993 w Korolówce w rejonie kołomyjskim, obwód iwanofrankowski, zm. 13 marca 2022 w obwodzie żytomierskim) – ukraiński oficer (major), pilot Ukraińskich Sił Powietrznych. Służył podczas wojny rosyjsko-ukraińskiej, w tym podczas rosyjskiej inwazji na Ukrainę w 2022 roku.

Pośmiertnie uznawany przez niektóre media (według sił zbrojnych Ukrainy niesłusznie) za "Ducha Kijowa" (pseudonim nadany hipotetycznemu asowi myśliwskiemu, któremu przypisuje się zestrzelenie ponad 40 rosyjskich samolotów w 2022 roku). Nie wiadomo, czy taki pilot rzeczywiście istniał, czy też był elementem ukraińskiej propagandy wojennej, stworzonej w celu podniesienia morale. Gdyby Tarabałka rzeczywiście był "Duchem Kijowa", byłby pierwszym zarejestrowanym asem myśliwskim XXI wieku.

Dzieciństwo i młodość
Tarabałka dorastał w ubogiej rodzinie w Korolówce. Jego rodzice pracowali za granicą (w Portugalii), gdzie mogli lepiej zarabiać, ale często wracali na Ukrainę. Mieszkał w pobliżu bazy wojskowej i stale widywał lądujące w pobliżu samoloty wojskowe, co od najmłodszych lat zainspirowało go do zostania pilotem. Tarabałka zgłosił się do szkoły wojskowej, a następnie szkoły lotniczej, studiując na Charkowskim Narodowym Uniwersytecie Sił Powietrznych, kończąc go w 2014 r.

Kariera wojskowa

Konflikt rosyjsko-ukraiński w 2014 roku, obejmujący aneksję Krymu przez Rosję oraz konflikt na wschodzie Ukrainy z dwoma terytoriami secesjonistycznymi (tzw. Ługańską i Doniecką Republiką Ludową) był pierwszym doświadczeniem bojowym Tarabałki. Dzięki temu zdobył doświadczenie w lotach z misjami wsparcia nad Donbasem.

Działalność Tarabałki została dostrzeżona na początku wojny pod anonimowym pseudonimem "Duch Kijowa". W tym czasie spekulowano, czy jest to prawdziwy pilot, czy po prostu mityczny bohater, być może ustanowiony w celu podniesienia morale. "Duchowi Kijowa" (niezależnie od tego, czy był to Tarabalka, czy nie) przypisywano zniszczenie dużej liczby rosyjskich samolotów, a jego rola została upubliczniona w mediach. Po pewnym czasie spekulacje ucichły. Tarabałka kontynuował walkę z rosyjskimi samolotami, latającymi nad Kijowem i wschodnią Ukrainą.

Tarabałka został zestrzelony w marcu. Pilotował samolot MiG-29. Legendy na temat "Ducha Kijowa" przez kilka tygodni nie dementowano; międzynarodowe media spekulowały, czy "Duch" i Tarabałka to w rzeczywistości ta sama osoba, czy też "Duch Kijowa" został po prostu wymyślony, a następnie zastosowany do Tarabałki po jego śmierci, kiedy jego sukces w walce z rosyjskimi siłami powietrznymi był znany. W kwietniu 2022 roku opublikowano wywiad z innym pilotem z jego jednostki, który wskazał szczegóły pasujące do Tarabałki jako "Ducha", w tym m.in. fakt, że latał na MiGu-29 i miał 29 lat.

Do czasu śmierci Tarabałki ukraińskie lotnictwo i lądowe wojska przeciwlotnicze zniszczyły ponad sto rosyjskich samolotów i śmigłowców, z których 40 przypisywano Tarabałce (w tym sześciu w ciągu jednego dnia walki, co czyniłoby go z definicji asem myśliwskim). Służby informacyjne dowództwa ukraińskich sił zbrojnych jednak w specjalnym oświadczeniu wydanym 30 kwietnia zaprzeczyły tej pogłosce, pisząc m.in. Герой України Степан Тарабалка НЕ «Привид Києва» і він НЕ збив 40 літаків (pol. "Bohater Ukrainy Stepan Tarabałka NIE JEST «Duchem Kijowa» i NIE zestrzelił 40 samolotów").

Śmierć
Tarabałka zginął w akcji 13 marca 2022 r. w wieku 29 lat, a plotki budziły wówczas podejrzenia, że to on był tajemniczą postacią "Ducha". Według ukraińskich źródeł zginął podczas misji w walce powietrznej przy przewadze liczebnej przeciwnika.

Tarabałkę pochowano na cmentarzu w Korolówce na Ukrainie, niedaleko Kołomyi. Planowano wystawić na aukcję jego hełm i rękawice, aby zebrać fundusze na trwający konflikt.

Przyznano mu pośmiertnie najwyższe odznaczenie Ukrainy za odwagę: tytuł Bohatera Ukrainy z Orderem Złotej Gwiazdy.

Życie prywatne
Tarabałka był żonaty z Ołeną i w chwili śmierci miał 8-letniego syna Jarosława.

Przypisy

Bohaterowie Ukrainy
Uczestnicy walk w trakcie inwazji Rosji na Ukrainę (strona ukraińska)
Żołnierze wojsk ukraińskich polegli w wojnie rosyjsko-ukraińskiej
Urodzeni w 1993
Zmarli w 2022

redpajama_set_name: RedPajamaWikipedia


text:
Q: How to print a specific line in string Python? So I got a string output out of a css selector while web crawling and the string has 7 lines, 6 of them are useless and I only want the 4th line.
The string is as follows:
کارکرد:
۵۰,۰۰۰
رنگ:
سفید
وضعیت بدنه:
بدون رنگ
قیمت صفر : ۳۱۵,۰۰۰,۰۰۰ تومان

Is there a way to print only the 4th line?
The crawling code:
color = driver.find_elements_by_css_selector("div[class='col']")
for c in color:
    print(c.text)

A: If you want items one to four try this:
for idx in range(4):
    print(color[idx].text)

And if you want only 4th try this: (in python index in list start from zero.)
print(color[3].text)

A: Yes, of course! See python documentation about list items
color = driver.find_elements_by_css_selector("div[class='col']")
print(color[3].text)

List items are indexed, the first item has index [0], the second item has index 1 etc.

A: I'm not sure if i understand the problem correctly, but assuming you have a string with multiple lines, a solution could be:
string = '''this string
exists
on multiple
lines
so lets pick
a line
'''

def select_line(string, line_index):
    return string.splitlines()[line_index]

result = select_line(string,3)

print(result)

This function would select the number line you want (index 0 being the first line)


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Select what languages you want to include to build a custom package.
CSS themes are available on GitHub.
It was designed to be lightweight (2.5kb), easy to use, and extendable.
Please consider donating to help support development.
* This is some sample code to illustrate how things look!
1. First include some markup for code you want to be highlighted.
Rainbow will pick up the language based on the data-language attribute. This example shows you how to display python code.
If you want to highlight HTML you should change data-language="python" to data-language="html".
You can see the list of supported languages here.
3. Finally include rainbow js and whatever language(s) you want before the closing </body> tag.
If you are using node.js you can install rainbow from NPM.
Rainbow on its own is very simple. It goes through blocks of code (or text), processes regular expression (regex) patterns, and wraps matching patterns in <span> tags. All the theming is left up to CSS.
If you would like to contribute or submit a bug check out the project on GitHub.


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text:
<menu xmlns:android="http://schemas.android.com/apk/res/android">
    <item
        android:id="@+id/action_about_this_app"
        android:title="@string/action_about_this_app"
        android:orderInCategory="100"/>
</menu>

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