text:
Noncanonical role of Arabidopsis COP1/SPA complex in repressing BIN2-mediated PIF3 phosphorylation and degradation in darkness
Jun-Jie Ling, Jian Li, Danmeng Zhu, and Xing Wang Deng
PNAS first published March 14, 2017 https://doi.org/10.1073/pnas.1700850114
Jun-Jie Ling
aState Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Advanced Agriculture Sciences and School of Life Sciences, Peking University, Beijing 100871, China
Jian Li
Danmeng Zhu
For correspondence: zhudanmeng@pku.edu.cn deng@pku.edu.cn
Xing Wang Deng
Contributed by Xing Wang Deng, February 20, 2017 (sent for review January 19, 2017; reviewed by Meng Chen and Hsu-Liang Hsieh)
Initially discovered as a central repressor of the seedling photomorphogenesis in plants, and later characterized in animals, the E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) has been shown to have conserved biochemical activities in eukaryotes. In this study, we show that to coordinately promote skotomorphogenesis, the COP1/ SUPPRESSOR of phyA-105 (SPA) complex represses the activity of the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) in a nonproteolytic manner to stabilize the key repressor of photomorphogenesis PHYTOCHROME INTERACTING FACTOR 3 (PIF3). The COP1/SPA-BIN2-PIF3 module represents an additional layer of COP1/SPA regulation in Arabidopsis and is essential for shaping the plants when grown in the dark.
The E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) has been known to mediate key signaling factors for degradation via the ubiquitin/26S proteasome pathway in both plants and animals. Here, we report a noncanonical function of Arabidopsis COP1, the central repressor of photomorphogenesis, in the form of a COP1/ SUPPRESSOR of phyA-105 (SPA) complex. We show that the COP1/SPA complex associates with and stabilizes PHYTOCHROME INTERACTING FACTOR 3 (PIF3) to repress photomorphogenesis in the dark. We identify the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) as a kinase of PIF3, which induces PIF3 degradation via 26S proteasome during skotomorphogenesis. Mutations on two typical BIN2 phosphorylation motifs of PIF3 lead to a strong stabilization of the protein in the dark. We further show that the COP1/SPA complex promotes PIF3 stability by repressing BIN2 activity. Intriguingly, without affecting BIN2 expression, the COP1/SPA complex modulates BIN2 activity through interfering with BIN2–PIF3 interaction, thereby inhibiting BIN2-mediated PIF3 phosphorylation and degradation. Taken together, our results suggest another paradigm for COP1/SPA complex action in the precise control of skotomorphogenesis.
photomorphogenesis
COP1/SPA complex
posttranscriptional regulation
In darkness, Arabidopsis seedlings undergo skotomorphogenesis, which is a developmental strategy that postgerminated seedlings use to accelerate their growth in soil and optimize the plant shape before reaching to the light (1). Any premature opening of the cotyledon, reduced apical hook, and hypocotyl elongation would impede the pace of life or be fatal to terrestrial flowering plants. The precise control of skotomorphogenesis has been under intensive investigation.
To date, it has been shown that plants use three different groups of signaling factors to repress photomorphogenesis in darkness: CONSTITUTIVELY PHOTOMOROPHOTENIC/DEETIOLATED/FUSCA (COP/DET/FUS) group proteins and phytochrome-interacting factors (PIFs), and ethylene-insensitive 3 (EIN3)/EIN3-like 1 (EIL1) proteins (2⇓–4). Genetic analyses have indicated that lesions in each of the three group genes cause a defective skotomorphogenesis with different degrees of cop phenotypes, as characterized by shorter hypocotyls, opened cotyledons, and overaccumulated protochlorophyllide (5⇓–7). The COP/DET/FUS group repressors belong to three biochemical entities: the COP1/SPA (SUPPRESSOR of phyA-105) complex, the CDD (COP10-DET1-DDB1) complex, and the COP9 signalosome, known to mediate the proteolysis of key photomorphogenesis-promoting factors via ubiquitin/26S proteasome pathway (8⇓–10). The PIFs, a small subset of basic helix–loop–helix transcription factors, control the expression of thousands genes to promote skotomorphogenesis, exampled by PIF1, -3, -4, and -5 (11, 12). EIN3/EIL1 have been recently shown to promote etiolation in darkness and phytochrome B can interact with and promote the degradation of EIN3 in red light (13, 14). Strikingly, several molecular connections have suggested that the three group genes synergistically repress photomorphogenesis in the dark (5, 15).
Among COP/DET/FUS factors, the central photomorphogenic repressor COP1 and SPA proteins constitute stable heterogeneous complexes, and function as an E3 ligase to target several key transcription factors for degradation, such as LONG HYPOCOTYL 5 (HY5), HY5 HOMOLOG (HYH), LONG HYPOCOTYL IN FAR-RED 1 (HFR1), and LONG AFTER FAR-RED LIGHT 1 (LAF1) (16⇓⇓–19). COP1 is well conserved in vertebrates and its function as an E3 ligase has been indicated in the lipid metabolism, tumorigenesis, and the stress response (20).
The stability of PIF proteins is tightly controlled by regulated protein degradation (21⇓–23). The key factors that are required for the phosphorylation and ubiquitination of PIFs attract much attention. Importantly, two types of E3 ligases have been shown to mediate the degradation of PIF1 and PIF3, respectively, upon light illumination (24, 25). Moreover, casein kinase II (CKII) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) have been identified as the kinase of PIF1 and PIF4, respectively, which promotes their degradation in response to light (26, 27). However, reports on how the PIFs are regulated in darkness are rather limited, although previous studies have suggested that the accumulation of PIF3, the founding member of PIF, is dependent on COP1 and SPA proteins, as well as DET1 (12, 28, 29). In this study, we identify BIN2 as a kinase of PIF3 that mediates its destabilization, most likely through two BIN2 phosphorylation sites in the dark. Importantly, COP1/SPA blocks the interaction between BIN2 and PIF3, which, in turn, promotes the stability of PIF3. Our results illuminate an additional mechanism for the control of key transcription factor stability by the COP1/SPA complex to achieve the accurate regulation of skotomorphogenesis in the model plant Arabidopsis.
COP1/SPA Complex Stabilizes PIF3 in the Dark.
To study the role of COP1 and SPAs in the regulation of PIF3 stability, we verified PIF3 levels in COP1/SPA complex mutants. In agreement with previous reports, levels of PIF3 were nearly undetectable in the dark-grown cop1–4, as well as in the quadruple spa1 spa2 spa3 spa4 (spaQ) mutant (Fig. S1). To investigate the posttranscriptional regulation of PIF3, a 35S:PIF3-HIS-MYC (PIF3-M)/cop1–4 line was generated and examined. Immunoblot analysis showed that the abundance of PIF3-M in cop1–4 was notably lower than the WT, and was significantly increased when treated with the proteasome inhibitor MG132 (Fig. 1A). This finding indicates a higher than normal degradation of PIF3-M by 26S proteasome in cop1–4. Next, the faster degradation of PIF3 in cop1–4 was recapitulated in a cell-free system. The recombinant MBP (MALTOSE BINDING PROTEIN)-PIF3 was quite stable in the extracts from dark-grown WT seedlings, but was degraded rapidly in the extracts from cop1–4 seedlings (Fig. 1B), supporting a stabilizing role of COP1/SPA complex on PIF3 in the dark.
COP1/SPA complex stabilizes and interacts with PIF3 in the dark. (A) Protein levels of PIF3-M in Col-0 and cop1–4 background with or without MG132 treatment. RPN6 was used as a loading control. D, DMSO. (B) Cell-free degradation of MBP-PIF3 protein in the extracts of Col-0 and cop1–4. RPT5 was used as a loading control. (C) PIF3 interacts with SPA1 in yeast cells. C, coil domain; KL, kinase-like domain; SPA1N (residues 1–545 aa); SPA1C (residues 546–1,029 aa); W, WD40 domain. (D) Mapping of SPA1-interacting regions of PIF3 in yeast cells. Full-length and truncated PIF3 were fused with AD. Full-length SPA1 was fused with BD. PIF3-D1 (1–180 aa), D2 (181–338 aa), D3 (339–524 aa). (E) LCI assays showing the interaction of SPA1 and PIF3 in N. benthamiana leaf cells. cLUC, the vector containing C-terminal fragment of firefly luciferase; nLUC, the vector containing N-terminal fragment of firefly luciferase. Empty vectors were used as negative controls. (F) The Co-IP assays showing COP1/SPA1 complex associates with PIF3.
Fig. S1.
Protein levels of PIF3 in Col-0, cop1–4, RLD, and spaQ. RPT5 was used as a loading control.
COP1/SPA1 Complex Interacts with PIF3 via SPA1.
To examine whether the COP1/SPA complex interacts with PIF3, we performed yeast two-hybrid analysis. As shown in Fig. 1C, the representative SPA protein SPA1, but not COP1, interacted with PIF3 in yeast cells. Moreover, the C terminal of SPA1 spanning the coiled-coil domain and WD40 domain was required for its interaction with PIF3 (Fig. 1C). Meanwhile, our results showed that both N- and C-terminal regions of PIF3 were required for its interaction with SPA1 (Fig. 1D). To verify the interaction between PIF3 and SPA1 in vivo, we first took advantage of the firefly luciferase complementation-imaging (LCI) assay. Indeed, when PIF3-nLUC and cLUC-SPA1 were coexpressed in tobacco leaves transiently, the activity of luciferase was reconstituted, indicating that SPA1 directly interacts with PIF3 (Fig. 1E). We then used a coimmunoprecipitation (Co-IP) assay to test the association of the COP1/SPA1 complex with PIF3. As expected, when PIF3-M was immunoprecipitated with an anti-MYC affinity matrix in the extracts supplemented with the recombinant MBP, MBP-COP1, or MBP-SPA1 proteins, both MBP-COP1 and MBP-SPA1 could be retrieved, respectively, but not MBP (Fig. 1F). Next, we examined the possible genetic interaction of COP1/SPA and PIF3. Compared with the fully opened cotyledons of cop1–4 mutants, overexpression of PIF3-M in cop1–4 exhibited much less expanded cotyledons (Fig. S2). Taken together, our results suggest that the COP1/SPA complex functionally acts together with PIF3 in darkness.
Overexpression of PIF3 in cop1–4 results in reduced photomorphogenic phenotype. Images (Upper) and angles of cotyledon opening (Lower) of dark-grown Col-0, cop1–4, PIF3 OX (PIF3-M)/cop1–4, and PIF3 OX (PIF3-M)/Col-0 seedlings. Mean ± SD, n = 20. (Scale bar, 1 mm.) **P < 0.01 (t test).
The Gain-of-Function bin2-1 Mutation Leads to PIF3 Destabilization in the Dark.
The key repressor of brassinosteroid (BR) signaling BIN2 has been shown to be a kinase that phosphorylates PIF4 and mediates its destabilization via the proteasome pathway (27). This result prompted us to examine whether PIF3 is also a phosphorylation target of BIN2. Interestingly, in our protein blot assay we found that the abundance of PIF3 was dramatically decreased in bin2-1 compared with WT (Fig. 2A). The MG132 treatment recovered the PIF3 level in bin2-1, suggesting that PIF3 was regulated by BIN2-mediated degradation through the 26S proteasome (Fig. 2A). Similarly, in the cell-free degradation system we observed that the recombinant MBP-PIF3 protein in WT extracts displayed a slow rate of degradation, whereas it was rapidly degraded in bin2-1 (Fig. 2B). This finding suggests a requirement of BIN2 activation for PIF3 degradation in the dark.
Constitutive activation of BIN2 mediates the proteasomal degradation of PIF3 in the dark. (A) Levels of PIF3 in Col-0 and bin2-1. RPN6 was used as a loading control. D, DMSO. (B) Cell-free degradation of MBP-PIF3 protein was accelerated in bin2-1 mutant extracts. RPT5 was used as a loading control. (C) BIN2 interacts with PIF3 in yeast cells. Full-length and truncated PIF3 were fused with AD. Full-length BIN2 was fused with BD. (D) In vitro pull-down assays showing the direct interaction of MBP-PIF3 with GST-BIN2. MBP-PIF3 was used as the prey molecules and incubated with GST-BIN2 or GST. (E) LCI assays showing the interaction of BIN2 with PIF3 in N. benthamiana leaf cells.
Mutations on Two BIN2 Recognition Motifs of PIF3 Enhance Its Stability in the Dark.
To investigate whether BIN2 physically interacts with PIF3, we performed a yeast two-hybrid assay. As expected, BIN2 had a positive interaction with PIF3 in yeast cells (Fig. 2C). Interestingly, the interaction regions of PIF3 with BIN2 turned out to be the same domains that mediate PIF3–SPA1 interaction (Fig. 2C). Using in vitro pull-down and firefly LCI assays, we further proved the direct interaction of BIN2 with PIF3 in vitro and in vivo (Fig. 2 D and E). Thus, these data indicate that BIN2 modulates PIF3 degradation through their direct interaction.
Next, we conducted in vitro kinase assays with MBP-PIF3 and GST-BIN2, which clearly suggested a direct phosphorylation of PIF3 by BIN2 (Fig. 3A). We therefore searched for the putative serine (S)/threonine (T) recognition site, the short BIN2 consensus motif (S/T)-X-X-X-(S/T) in PIF3. The motif analysis identified 14 putative BIN2-recongnition motifs in the PIF3 protein sequence (Fig. S3). Among them, only two motifs (T220KEKS224, S283SVGS287) were conserved in both dicot and monocot species (Fig. S3). We therefore introduced alanine mutations to replace S/T residues in the two conserved motifs, generating the PIF3-nA4 mutant protein, which led to its reduced phosphorylation in vitro (Fig. 3B). Importantly, the abundance of PIF3-nA4-Flag in the transgenic seedlings was found to be significantly higher than that of PIF3-WT-Flag, when PIF3-WT-Flag and PIF3-nA4-Flag transcript levels are similar (Fig. 3 C and D). These data indicate that mutation of the two BIN2 phosphorylation sites results in the strong stabilization of PIF3 in the dark. Consistent with this result, when 35S:PIF3-WT-Flag and 35S:PIF3-nA4-Flag lines were each crossed to the bin2-1 mutant, we observed that constitutive activation of BIN2 in these transgenic seedlings caused a strong decrease in PIF3-WT-Flag protein level, but had little effect on PIF3-nA4-Flag protein stability (Fig. 3D). These data again demonstrate that these two conserved BIN2 phosphorylation motifs are essential for PIF3 stability regulation in the dark.
BIN2 phosphorylates and modulates PIF3 stability in the dark. (A) BIN2 phosphorylates PIF3 in vitro. MBP or MBP-PIF3 was incubated with [γ-32P] ATP, without or with GST-BIN2: (Left) the autoradiogram, (Right) Coomassie brilliant blue (CBB) staining. BK is an Arabidopsis GSK3-like kinase inhibitor. (B) Phosphorylation of MBP-PIF3 and MBP-PIF3-nA4 by BIN2. Kinase assays were performed with GST-BIN2 and MBP-PIF3 or MBP-PIF3-nA4: (Left) the autoradiogram, (Right) CBB staining of the gel. (C) mRNA levels of PIF3-WT-Flag and PIF3-nA4-Flag in Col-0 and bin2-1. The relative levels were normalized to PP2A. (D) Protein gel blot of PIF3-WT-Flag and PIF3-nA4-Flag fusion proteins in Col-0 and bin2-1. RPN6 was used as a loading control.
Two putative BIN2 phosphorylation motifs are conserved in the PIF3 orthologs from representative plant species. Multiple alignment of the Arabidopsis PIF3 (AtPIF3) with its closest orthologs identified in Camelina sativa (CsPIF3), Populus trichocarpa (PtPIF3), Zea mays (ZmPIF3), Oryza sativa (OsPIF3). Asterisk: putative BIN2 phosphorylation motifs (S/T-X-X-X-S/T). Red rectangular box: the phosphorylation motif conserved in all these proteins.
COP1/SPA Inhibits BIN2-Mediated PIF3 Destabilization.
To further study the opposite effect of COP1/SPA and BIN2 on the regulation of PIF3 stability, PIF3-M/cop1–4 bin2-3 bil1 bil2 was generated by genetic crossing. When BIN2 and its two closest homologs BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2) were depleted in cop1–4 background, the abundance of PIF3-M was recovered to that in WT. This result suggests that BIN2 and its related genes act downstream of COP1 to destabilize PIF3 (Fig. 4A). Meanwhile, when 35S:PIF3-WT-Flag and 35S:PIF3-nA4-Flag lines were each crossed to the cop1–4 mutant, we observed that PIF3-nA4-Flag was strongly stabilized in cop1–4 (Fig. 4B), supporting that the phosphorylation of PIF3 by BIN2 is downstream of COP1 in the regulation of PIF3 stability. Similarly, overexpression of PIF3-nA4-Flag in cop1–4 displayed much less expanded cotyledons (Fig. S4).
COP1/SPA inhibits BIN2-mediated PIF3 destabilization. (A) Levels of PIF3-M in Col-0, cop1-4, cop1–4 bin2-3 bil1 bil2, and bin2-3 bil1 bil2. Actin was used as a loading control. (B) Protein gel blot of PIF3-WT-Flag and PIF3-nA4-Flag (PIF3-nA4) in Col-0 and cop1–4, respectively. RPN6 was used as a loading control. PIF3-F, PIF3-Flag. (C) mRNA levels of BIN2-Flag (BIN2-F) in Col-0 and cop1–4. Data shown are representative of two independent lines. (D) Protein levels of BIN2-F in Col-0 and cop1–4. RPN6 was used as a loading control.
Overexpression of PIF3-nA4 in cop1–4 caused reduced photomorphogenic phenotype. Images (Upper) and angles of cotyledon opening (Lower) of dark-grown Col-0, PIF3-WT/Col-0, PIF3-nA4/Col-0, cop1–4, PIF3-WT/cop1–4, and PIF3-nA4/cop1–4 seedlings. Mean ± SD, n = 20. (Scale bar, 1 mm.) **P < 0.01 (t test).
Next, to check whether COP1/SPA and BIN2 have direct interaction, we performed yeast two-hybrid and in vitro pull-down assays. We detected that COP1, but not SPA1, physically interacted with BIN2 in vitro (Fig. S5 A and B). Moreover, our Co-IP analysis showed that BIN2–Flag fusion protein expressed from the dark-grown 35S:BIN2-Flag/Col seedlings coimmunoprecipitated COP1, further supporting BIN2–COP1 interaction in vivo (Fig. S5C). These results led us to examine whether COP1 affects the accumulation of BIN2 in the dark. To this end, 35S:BIN2-Flag was introduced into WT background, and each of the resulting two independent lines was crossed into cop1–4 mutant to generate the homozygous plants (Fig. 4C). Interestingly, the abundance of BIN2-Flag from both lines in Columbia-0 (Col-0) was comparable to that in cop1–4, suggesting that the level of BIN2 is not affected by COP1 (Fig. 4D).
COP1 interacts with BIN2 in the dark. (A) BIN2 interacts with N-terminal region of COP1 in yeast cells. COP1 (1–675 aa), COP1N (1–127 aa), COP1C (128–675 aa). (B) In vitro pull-down assays showing the interaction between GST-BIN2 and MBP-COP1. (C) Co-IP experiment showing that BIN2-Flag interacts with COP1 in the dark. The anti-Flag conjugated beads were used for the immunoprecipitation experiments with samples of dark-grown Col-0 and 35S:BIN2-Flag /Col-0. Tubulin was used as a loading control.
COP1/SPA Complex Interferes with BIN2–PIF3 Interaction in the Dark.
Given that SPA1 and BIN2 interacts with the same regions of PIF3, COP1 directly interacts with BIN2, and that the COP1/SPA complex and BIN2 have an antagonistic role in regulating PIF3 stability, we speculated that COP1/SPA may interfere with BIN3–PIF3 interaction. Using a modified yeast three-hybrid system, we found that the interaction between PIF3 and BIN2 indicated by the β-galactosidase activity was greatly reduced when COP1 or SPA1, but not the GFP protein, was coexpressed in yeast cells (Fig. 5A). This result suggests that the presence of COP1 or SPA1 alone may interfere with the BIN2–PIF3 interaction. To further investigate whether the BIN2–PIF3 interaction could be interfered by COP1/SPA complex in Arabidopsis, we performed a Co-IP analysis using extracts from transgenic lines expressing 35S:PIF3-HIS-MYC in WT and cop1–4 mutant, each supplemented with the same amount of recombinant GST-BIN2. As expected, although less than threefold of PIF3-M fusion protein was coimmunoprecipitated using anti-MYC conjugated beads in the total extracts of cop1–4 seedlings than the WT, the yields of GST-BIN2 were similar, suggesting that the interaction of PIF3 with BIN2 is stronger in the absence of COP1/SPA complex (Fig. 5B).
COP1/SPA complex inhibits BIN2-mediated phosphorylation and degradation of PIF3. (A) Yeast three-hybrid assays showing the COP1 and SPA1 inhibition of the BIN2–PIF3 interaction in yeast cells. Data are Mean ± SD, n = 3. **P < 0.01 (t test). (B) The Co-IP assays showing a stronger interaction between GST-BIN2 and PIF3 in the absence of COP1. Actin was used as a loading control. (C) COP1/SPA1 inhibits BIN2-mediated phosphorylation of PIF3 in vitro. The quantity (micrograms) of MBP-COP1 or MBP-SPA1 added in the reaction mix was as indicated. (D) Cell-free kinase assays showing the phosphorylation levels of the MBP-PIF3 in the total extracts of dark-grown Col-0, cop1–4, RLD, and spaQ seedlings. (Top) the autoradiogram of MBP-PIF3; (Middle) CBB staining of the gel; (Bottom) RPN6 was used as a loading control. (E) Protein gel blot of PIF3-M in Col-0 and cop1–4 treated with or without 30 μM BK. Actin was used as a loading control.
COP1/SPA Complex Suppresses BIN2-Mediated PIF3 Phosphorylation and Degradation.
To substantiate whether the interference of BIN2–PIF3 interaction by COP1/SPA1 complex would reduce BIN2 kinase activity toward PIF3, we first carried out in vitro kinase assays. When an increased amount of MBP-COP1 or MBP-SPA1 was added in the kinase reaction mix, phosphorylation levels of PIF3 were gradually decreased (Fig. 5C and Fig. S6), supporting the inhibitory effect of COP1 and SPA1 on PIF3 phosphorylation by BIN2. Interestingly, when MBP-COP1 and MBP-SPA1 were assayed together in the molecular molar ratio 1:1, a decrease of the phosphorylation level of PIF3 was further enhanced, compared with that of PIF3 with the addition of the same amount of COP1 or SPA1 alone. This finding suggested the role of COP1 working in concert with SPA1 in repressing the phosphorylation of PIF3 by BIN2.
MBP does not inhibit BIN2-mediated phosphorylation of PIF3 in vitro. The quantity (micrograms) of MBP added in the reaction mix was as indicated.
To determine this inhibitory effect in plant cells, we designed a cell-free kinase assay by incubation of total extracts derived from the seedlings of Col-0, cop1–4, RLD (Rschew–Starize), and spaQ, each supplemented with equal amount of MBP-PIF3 and GST-BIN2. Phosphorylation levels of PIF3 were obviously higher in cop1–4 and spaQ than WT (Fig. 5D), strongly suggesting that the phosphorylation of PIF3 by BIN2 is inhibited by the COP1/SPA complex in the dark. In agreement with this result, the specific GSK3-like kinase inhibitor bikinin (BK) (30) treatment of the transgenic seedlings overexpressing PIF3-M resulted in significantly higher accumulation of PIF3-M in the cop1–4 mutant, but not in WT (Fig. 5E). Taken together, these data suggest that the instability of PIF3 in the mutants of COP1/SPA complex in darkness appears to have resulted from the increased phosphorylation levels of PIF3 by BIN2.
Recent advances have suggested that the COP/DET/FUS group proteins and PIFs act coordinately to repress photomorphogenesis phenotypically and biochemically (15). However, the relationship between factors in those two groups requires further investigation. In this study, our findings provide an insight on the positive regulation of COP1/SPA complex on PIF3 stability bridged by the GSK3-like protein kinase BIN2, which unveils a noncanonical function of COP1/SPA complex in repressing photomorphogenesis in the model plant Arabidopsis.
To date, several lines of evidence have addressed the functional interaction of COP1 and SPA proteins with PIFs in different schemes of light-mediated plant growth and development. For example, CUL4COP1–SPA E3 ligase mediates the degradation of PIF1 upon light illumination (25). Alternatively, PIFs were reported to function as cofactors of the COP1/SPA1 E3 ligase complex to enhance its activity (31, 32). Moreover, to regulate photomorphogenic development, COP1 promotes PIF3-LIKE1 degradation to relieve its inhibition on transcriptional activity of PIFs (33). In addition, under shade conditions, COP1 enhances the activity of PIF family transcription factors by degrading HFR1 (34). Together, all these data pinpoint the function of COP1/SPA as being an E3 ligase in regulating or regulated by PIFs. Interestingly, in this work we discovered that in darkness COP1/SPA complex promotes the stability of PIF3, most likely through a nonproteolytic regulation on BIN2, the kinase of PIF3. From our data, we assumed that COP1 and the representative SPA protein SPA1 may have different role in repressing the BIN2–PIF3 interaction, in which COP1 functions possibly through sequestration of BIN2, whereas SPA1 works via the competitive interaction of the same regions of PIF3 with BIN2.
It's been reported that light-induced PIF3 phosphorylation is a prerequisite for its ubiquitination and degradation in the light (22). Our data suggest that the phosphorylation of PIF3 mediated by BIN2 also drives its proteasomal degradation in the dark. Moreover, the use of different cassettes of phosphorylation sites and likely distinct kinase systems are evident in the PIF3 degradation regulation in the dark or upon light illumination. Therefore, further characterization and comparison of the different kinase systems for PIF3 in darkness and under light conditions will be of particular relevance.
BIN2, the representative GSK3-like kinase in plants, has been found to be involved in many developmental processes, including skotomorphogenesis (35). A previous study and this work indicated that through the typical phosphorylation motifs on distinct sequences, BIN2 phosphorylates PIF3 and PIF4, respectively (27). Because PIF proteins accumulate in the dark, the regulatory mechanism for controlling BIN2 activity requires better exploration. The dephosphorylation or deacetylation of BIN2 has been shown to inhibit its activity in BR signaling (36, 37). Additionally, the relocalization of BIN2 to the plasma membrane restricts its activity during phloem differentiation (38). In skotomorphogenesis, our evidence pinpoints a key role of COP1/SPA complex in repressing BIN2 kinase activity. In mammalian cells, GSK3β, the orthologous of Arabidopsis BIN2, was reported to cooperate with MmCOP1 to inhibit breast tumor progression and metastasis by promoting the degradation of transcription factor c-Jun (39). Further elucidating of the precise mechanism by which the COP1/SPA complex regulates GSK3-like kinases, as represented by BIN2, will provide insights linking the functional interactions between the two types of key regulators in eukaryotes.
Plant Materials and Growth Conditions.
The ecotypes of all WT Arabidopsis thaliana used in this study were Col-0 and RLD. The cop1–4, spaQ, and pif3-3 mutants were reported previously (10, 40). Seed sterilization, stratification, and standard seedling growth experiments were performed according to the methods previously outlined (10). Four-day-old dark-grown seedlings were used in this study unless otherwise stated. For MG132 treatment, seedlings were grown in the dark for 4 d without or with 100 μM MG132 pretreatment for 6 h before harvesting.
Plasmid Construction and Generation of Transgenic Lines.
To generate activation domain (AD)-PIF3 constructs for yeast two-hybrid assays, fragments containing full-length PIF3 coding sequence (CDS) (1–524 aa), and truncated PIF3 CDS including D1 (1–180 aa), D2 (181–524 aa), D3 (181–338 aa), and D4 (339–524 aa) were amplified and inserted into the EcoRI/XhoI sites of the pB42AD vector (Clontech). For AD-COP1 and AD-SPA1 constructs, fragments encoding full-length SPA1 and COP1, COP1N (1–127 aa) and COP1C (128–675 aa) were each amplified and inserted into the EcoRI/XhoI sites of the pB42AD vector. For DNA-binding domain (BD)-COP1 and BD-BIN2 constructs, DNA fragments encoding full-length COP1 and BIN2 CDS were amplified and inserted into the EcoRI/BamHI sites of the pLexA vector (Clontech). For BD-SPA1 constructs, DNA fragments encoding full-length SPA1 CDS (1–1,029 aa), SPA1N (1–545 aa), and SPA1C (546–1,029 aa) were amplified and inserted into the NcoI/XhoI sites of the pLexA vector. For yeast three-hybrid, DNA fragments encoding SPA1 and GFP were amplified and inserted into KpnI/XhoI sites of pGAD-T7 vector (Clontech).
For purification of MBP-PIF3 and GST-BIN2 recombinant proteins, BamHI /SalI DNA fragments encoding full-length or mutated PIF3 were cloned into the pMAL-c2x vector (New England Biolabs); an EcoRI/SalI DNA fragment encoding full-length BIN2 coding sequence was cloned into pGEX-4T-1 vector (GE Healthcare).
To generate nLUC-PIF3, a KpnI/SalI DNA fragment encoding full-length PIF3 coding sequence was cloned into pCAMBIA-nLuc vector. For cLUC-SPA1 and cLUC-BIN2 constructs, a KpnI/SalI and KpnI/BamHI DNA fragment encoding full-length SPA1 and BIN2 coding sequences, respectively, were each cloned into pCAMBIA-cLuc vector.
To generate Flag-tagged BIN2 and PIF3 transgene, DNA fragments containing full-length BIN2 and PIF3 coding sequences were each amplified and inserted into BamHI/SalI sites of pCAMBIA1307-3xFlag. For stable transformation, Agrobacterium tumefaciens bacteria strain GV3101 carrying those constructs were then transformed into Col-0 using the floral-dip method (41). At least two independent transgenic lines were selected. All of the cloning primers are listed in Table S1.
Table S1.
Primers used in this study
Western Blot and Antibodies.
Protein extraction and immunoblots were performed as previously described (29). Antibodies used in this study were anti-COP1 (10), anti-PIF3 (29), anti-RPN6 (42), anti-RPT5 (42), anti-Actin (Sigma), anti-Flag (Sigma), anti-Tubulin (Sigma), anti-MYC (Sigma), anti-MBP (New England Biolabs), and anti-GST (New England Biolabs).
Cell-Free Degradation Assay.
Cell-free degradation analyses were performed as previously described (43). In brief, 200 ng of MBP-PIF3 protein was incubated with individual extracts in the dark at 22 °C for the indicated time before collection. Anti-MBP and anti-RPT5 were used to analyze the results.
Yeast Two-Hybrid and Three-Hybrid Assays.
Yeast two-hybrid assays were performed according to standard protocols (Clontech). Yeast three-hybrid assays were performed as described previously (44). β-Galactosidase activity was quantified with the yeast β-galactosidase assay kit (Thermo Scientific).
In Vitro Pull-Down and Co-IP.
For in vitro pull-down assay, MBP fusion proteins (2 μg) and GST fusion proteins (1 μg) were incubated with 10 μL prewashed GST beads at 4 °C for 3 h. Eluted proteins were analyzed by immunoblotting using anti-MBP and anti-GST antibodies. For semi-in vivo Co-IP assay, 2 μg of recombinant proteins was mixed with 500 μg of total proteins extracted from the dark-grown seedlings in IP buffer (29), and subsequently incubated with 10 μL prewashed anti-MYC conjugated beads for 4 h at 4 °C. Eluted proteins were analyzed by immunoblotting using anti-MBP, anti-GST, and anti-MYC antibodies. For Co-IP assay, 500 μg of total proteins was incubated with 10 μL prewashed anti-Flag–conjugated beads for 3 h at 4 °C. Eluted proteins were analyzed by immunoblotting using anti-COP1 and anti-Flag antibodies.
LCI Assay.
The LCI assay was performed as previously outlined with some modifications (45). In brief, A. tumefaciens bacteria strain GV3101 containing different constructs were injected in 4-wk-old Nicotiana benthamiana leaves. The plants were subsequently grown in darkness for 2 d before harvesting. The luciferase signals were analyzed with Night SHADE LB 985 (Berthold Technologies).
Quantitative RT-PCR.
Total RNA was extracted from dark-grown seedlings by using the mini Plant RNA extraction Kit (Qiagen). One microgram of total RNA was reverse-transcribed using SuperScript II (Life Technologies) for synthesis of first strand cDNA. Quantitative RT-PCR (RT-qPCR) was performed using a 7500 Fast Real-Time PCR machine (Applied Biosystems). Primer sequences used for RT-qPCR are listed in Table S1. The gene-expression results were normalized by PP2A.
Kinase Assays.
The in vitro and cell-free kinase assays were performed as previously described with some modifications (46). For in vitro kinase assay, MBP-PIF3 (1 μg) and GST-BIN2 (0.5 μg) were mixed into 20 μL of kinase buffer (20 mM Tris⋅HCl [pH 7.5], 100 mM NaCl, 12 mM MgCl2, 0.1 mM ATP, 0.2 μCi [γ-32P] ATP) with or without 20 μM BK. For the cell-free kinase assay, total proteins were extracted from Col-0, cop1–4, RLD, and spaQ, respectively, in the kinase buffer with 50 μM MG132 and 1 mM DTT. Five micrograms of total proteins were incubated with 1 μg MBP-PIF3 and 0.5 μg GST-BIN2. RPN6 was used as loading control. After incubation at 37 °C for 30 min, the samples were added into 1× SDS loading buffer and then boiled at 100 °C for 5 min to stop the reaction. Proteins were loaded and separated in 8% g/mL SDS/PAGE gel. Phosphorylation signals were detected using Typhoon FLA7000 (GE Healthcare).
We thank Dr. Jianming Li for providing the bin2-1 and bin2-3 bil1 bil2 mutant seeds; Dr. Giltsu Choi for providing 35S:PIF3-HIS-MYC line; and Dr. Yuqiu Wang, Dr. Fang Lin, Dr. Renbo Yu, and Kunlun Li for providing critical comments and the technical assistance. This work was supported by the National Natural Science Foundation of China Grants 31330048, 31621001, 91540105; National Basic Research Program (973 Program) Grant 2012CB910900; NIH Grant GM47850; and in part by the State Key Laboratory of Protein and Plant Gene Research at Peking University and the Peking-Tsinghua Center for Life Sciences.
↵1J.-J.L. and J.L. contributed equally to this work.
↵2To whom correspondence may be addressed. Email: zhudanmeng{at}pku.edu.cn or deng{at}pku.edu.cn.
Author contributions: J.-J.L., J.L., D.Z., and X.W.D. designed research; J.-J.L., J.L., and D.Z. performed research; J.-J.L., J.L., D.Z., and X.W.D. analyzed data; and J.-J.L., J.L., D.Z., and X.W.D. wrote the paper.
Reviewers: M.C., University of California, Riverside; and H.-L.H., National Taiwan University.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1700850114/-/DCSupplemental.
Von Arnim A,
Deng XW
(1996) Light control of seedling development. Annu Rev Plant Physiol Plant Mol Biol 47:215–243.
Zhong S, et al.
(2014) Ethylene-orchestrated circuitry coordinates a seedling's response to soil cover and etiolated growth. Proc Natl Acad Sci USA 111(11):3913–3920.
Lau OS,
(2012) The photomorphogenic repressors COP1 and DET1: 20 years later. Trends Plant Sci 17(10):584–593.
Leivar P,
Quail PH
(2011) PIFs: Pivotal components in a cellular signaling hub. Trends Plant Sci 16(1):19–28.
(2009) EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings. Proc Natl Acad Sci USA 106(50):21431–21436.
Shin J, et al.
(2009) Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors. Proc Natl Acad Sci USA 106(18):7660–7665.
Deng XW,
Caspar T,
(1991) cop1: A regulatory locus involved in light-controlled development and gene expression in Arabidopsis. Genes Dev 5(7):1172–1182.
Wei N, et al.
(1998) The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex. Curr Biol 8(16):919–922.
Yanagawa Y, et al.
(2004) Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes. Genes Dev 18(17):2172–2181.
Zhu D, et al.
(2008) Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development. Plant Cell 20(9):2307–2323.
Leivar P, et al.
(2009) Definition of early transcriptional circuitry involved in light-induced reversal of PIF-imposed repression of photomorphogenesis in young Arabidopsis seedlings. Plant Cell 21(11):3535–3553.
(2008) Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr Biol 18(23):1815–1823.
Shi H, et al.
(2016) Seedlings transduce the depth and mechanical pressure of covering soil using COP1 and ethylene to regulate EBF1/EBF2 for soil emergence. Curr Biol 26(2):139–149.
(2016) The red light receptor phytochrome B directly enhances substrate-E3 ligase interactions to attenuate ethylene responses. Dev Cell 39(5):597–610.
Xu X,
Paik I,
Huq E
(2015) Illuminating progress in phytochrome-mediated light signaling pathways. Trends Plant Sci 20(10):641–650.
Yi C,
(2005) COP1—From plant photomorphogenesis to mammalian tumorigenesis. Trends Cell Biol 15(11):618–625.
Holm M,
Ma LG,
Qu LJ,
(2002) Two interacting bZIP proteins are direct targets of COP1-mediated control of light-dependent gene expression in Arabidopsis. Genes Dev 16(10):1247–1259.
Seo HS, et al.
(2003) LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. Nature 423(6943):995–999.
Jang IC,
Yang JY,
Seo HS,
Chua NH
(2005) HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling. Genes Dev 19(5):593–602.
Marine JC
(2012) Spotlight on the role of COP1 in tumorigenesis. Nat Rev Cancer 12(7):455–464.
Nozue K, et al.
(2007) Rhythmic growth explained by coincidence between internal and external cues. Nature 448(7151):358–361.
Al-Sady B,
Ni W,
Kircher S,
Schäfer E,
(2006) Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell 23(3):439–446.
Shen H,
Moon J,
(2005) PIF1 is regulated by light-mediated degradation through the ubiquitin-26S proteasome pathway to optimize photomorphogenesis of seedlings in Arabidopsis. Plant J 44(6):1023–1035.
Ni W, et al.
(2014) A mutually assured destruction mechanism attenuates light signaling in Arabidopsis. Science 344(6188):1160–1164.
Zhu L, et al.
(2015) CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1. Nat Commun 6:7245.
Bu Q, et al.
(2011) Phosphorylation by CK2 enhances the rapid light-induced degradation of phytochrome interacting factor 1 in Arabidopsis. J Biol Chem 286(14):12066–12074.
Bernardo-García S, et al.
(2014) BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth. Genes Dev 28(15):1681–1694.
Bauer D, et al.
(2004) Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis. Plant Cell 16(6):1433–1445.
Dong J, et al.
(2014) Arabidopsis DE-ETIOLATED1 represses photomorphogenesis by positively regulating phytochrome-interacting factors in the dark. Plant Cell 26(9):3630–3645.
De Rybel B, et al.
(2009) Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling. Chem Biol 16(6):594–604.
Henriques R,
Nagatani A,
(2010) Arabidopsis PHYTOCHROME INTERACTING FACTOR proteins promote phytochrome B polyubiquitination by COP1 E3 ligase in the nucleus. Plant Cell 22(7):2370–2383.
Xu X, et al.
(2014) PHYTOCHROME INTERACTING FACTOR1 enhances the E3 ligase activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to synergistically repress photomorphogenesis in Arabidopsis. Plant Cell 26(5):1992–2006.
Luo Q, et al.
(2014) COP1 and phyB physically interact with PIL1 to regulate its stability and photomorphogenic development in Arabidopsis. Plant Cell 26(6):2441–2456.
Pacín M,
Semmoloni M,
Legris M,
Finlayson SA,
Casal JJ
(2016) Convergence of CONSTITUTIVE PHOTOMORPHOGENESIS 1 and PHYTOCHROME INTERACTING FACTOR signalling during shade avoidance. New Phytol 211(3):967–979.
Youn JH,
Kim TW
(2015) Functional insights of plant GSK3-like kinases: Multi-taskers in diverse cellular signal transduction pathways. Mol Plant 8(4):552–565.
Hao Y,
Wang H,
Qiao S,
Leng L,
Wang X
(2016) Histone deacetylase HDA6 enhances brassinosteroid signaling by inhibiting the BIN2 kinase. Proc Natl Acad Sci USA 113(37):10418–10423.
Kim TW, et al.
(2009) Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat Cell Biol 11(10):1254–1260.
Anne P, et al.
(2015) OCTOPUS negatively regulates BIN2 to control phloem differentiation in Arabidopsis thaliana. Curr Biol 25(19):2584–2590.
Shao J, et al.
(2013) COP1 and GSK3β cooperate to promote c-Jun degradation and inhibit breast cancer cell tumorigenesis. Neoplasia 15(9):1075–1085.
Monte E, et al.
(2004) The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development. Proc Natl Acad Sci USA 101(46):16091–16098.
Clough SJ,
Bent AF
(1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743.
Kwok SF,
Staub JM,
(1999) Characterization of two subunits of Arabidopsis 19S proteasome regulatory complex and its possible interaction with the COP9 complex. J Mol Biol 285(1):85–95.
Osterlund MT,
Hardtke CS,
Wei N,
(2000) Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405(6785):462–466.
Huang X, et al.
(2013) Conversion from CUL4-based COP1-SPA E3 apparatus to UVR8-COP1-SPA complexes underlies a distinct biochemical function of COP1 under UV-B. Proc Natl Acad Sci USA 110(41):16669–16674.
Chen H, et al.
(2008) Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol 146(2):368–376.
Cai Z, et al.
(2014) GSK3-like kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis. Proc Natl Acad Sci USA 111(26):9651–9656.
You are going to email the following Noncanonical role of Arabidopsis COP1/SPA complex in repressing BIN2-mediated PIF3 phosphorylation and degradation in darkness
Noncanonical role of COP1/SPA in Arabidopsis
Jun-Jie Ling, Jian Li, Danmeng Zhu, Xing Wang Deng
Proceedings of the National Academy of Sciences Mar 2017, 201700850; DOI: 10.1073/pnas.1700850114


Bleacher Report U.K. has experimented with hundreds of different formats on Snapchat Discover over the last two years. The upshot: Don't get too comfortable.
Bleacher Report is somewhat unique among U.S. Snapchat publishers in that it got its start in the U.K. The London team has four people dedicated to creating two English-language Discover editions a day, one for international audiences and another for domestic. It regularly calls on a pool of independent contractors when extra skills like motion graphics are needed.
Football is the main focus for the publisher's U.K. and international Discovers (the U.S. now has a separate channel), meaning there is an infinite stream of updates either relating to matches or football culture, around which the team can create content. Here's what Bleacher Report has learned.
To keep its editions sharp, the team is strict with switching up formats. As soon as a highly popular format starts feeling easy to re-create, it's dropped. That can be as quick as after 12 editions. The goal: to ensure its editions remain unique to Bleacher Report, while keeping its audience highly engaged.
That's not particularly good news for any publisher that hopes to one day reduce the number of editors required to create great Discover content. But it's not so much a problem for Bleacher Report, which takes a more BuzzFeed-type approach to platforms, rather than using them to bring eyeballs back to its own sites.
In the U.S., Bleacher Report is far newer to Discover. It began publishing there in January, but it has an 11-person team. Naturally, the other main difference is that the U.K. and international editions focus on football, while in the U.S. the emphasis is the NFL and the NBA. That also means there are no typical economies of scale when it comes to sharing content between the international teams because the audiences are totally different, according to Walker. Some snaps will generate global interest, such as a recent one listing Real Madrid star Cristiano Ronaldo's cars. But mainly, the U.S. editions will look markedly different from the international ones.
Another core lesson has been to ensure each snap is created for a singular purpose to avoid wasting resources and to keep users' attention. Bleacher Report has three simple rules for achieving this. A snap must be created to do one, rather than multiple things, to drive high usage. When wanting to drive swipe-ups, the team will give teasers of stories that are impossible to tell in one top snap. For example, the snap "The Cristiano Ronaldo story, told through his goal celebrations" drove high swipe-ups, though Walker wouldn't reveal particulars due to its agreement with Snapchat.
The team uses another format when it decides it wants to drive people to share snaps. Users will be asked to pick or vote on something. Questions like, "Who will win the Champions League? Juventus or Real Madrid?" have been particularly successful at driving high shares. The third format is for video, and naturally the purpose is to drive high views and repeat views of videos.
Finding the right video formats is still a work in progress, and some formats have been quickly abandoned. A talking-heads video was used as a top snap to tease a longer-form video. But the format was dropped when it didn't gain traction. Bleacher Report has also worked on a spinoff Snapchat edition called "Saturdays Are Lit" in partnership with youth-focused football publisher Copa90. The aim of the show is to reinvent the traditional TV-style football shows for the Snapchat generation. The series is still ongoing.


Please fill out this form and submit your request.
can be communicated to related groups charged to give specific information and assistance on the service.
I declare that I have acknowledged that my personnel data will be used in order to receive more specific information on the service.
I recognise that I am given the right of knowing, refusing, modifying, up-dating, integrating, opposing the use of my personal data and of exercising the rights stated by the paragraphs 7,10, 13, law no. 675/96. In order to exercise my rights I can apply to the personnel responsible: "L'Orangerie" - Vico de Naro, 5 - 97015 Modica (RG) - Tel. 0039 0932 754840.


Having a special place to communicate with the private spiritual you is often a necessity in our demanding lives. Most people take very little time to listen to their own thoughts which in the long run can develop a sense of confusion, loss of purpose or direction. Those who have created an area of sacred space inside the home, or garden can gain clarity and recharge their energy when it is needed.
Such a space is used for reflection and meditation, but it must be quiet – so enter your space when you have the house to yourself (the babies are asleep, the children are at school). Find a time that suits your lifestyle (early morning, day or night) and try to do this once or twice a week for 30 minutes. Look for a space in your home that feels right for you; maybe it has filtered light that lets the sun in, or a garden just outside a window that brings in the smell of herbs or flowers. If your sacred space is in a lounge room in a comfortable chair you won't be the only one (a popular choice).
To enhance your sacred space decorate it with things that you feel are special even holy. Find things that have meaning; you might have sacred statues or idols that comfort you. You could choose some soft ambient music, something relaxing (without words) or just listen to the birds. Maybe a candle, incense or fresh flowers would be nice. Then put on your comfortable clothes and sit down with a lovely warm blanket if desired. Sit with your eye's closed and listen to your inner chatter and the profound thoughts that follow, but don't try to force anything, just let it happen. Oh, and have a notebook handy in case you have insights that need recording.
The more you come to this sacred place the more empowered it will become – because you are empowering it every time your ritual begins. Use this peaceful time to create a full understanding of yourself and others and you will be a wise and beautiful being, a gift to all.


text:
Samantha Brennan
Holyoke Softball
2020-2021 8 1 1 1 1 2
Career Totals: 8 1 1 1 1 2
Runs Allowed
2020-2021 14 17 13 6 5 16
2021-2022 30 43 27 14 20 28
Career Totals: 44 60 40 20 25 44
05/11/2021 @ South Hadley L 10-16 3 1 — — — — — — 1 — —
05/24/2021 vs. South Hadley W 11-2 3 — — 1 — — — — 1 — —
06/11/2021 @ South Hadley W 11-8 2 — 1 — 1 — — — — — —
Season Totals: 8 1 1 1 1 — — — 2 — —
05/24/2021 vs. South Hadley W 11-2 7 6 2 2 — 8 —
06/09/2021 @ Belchertown L 12-13 4 6 9 2 5 4 —
06/11/2021 @ South Hadley W 11-8 3 5 2 2 — 4 —
Season Totals: 14 17 13 6 5 16 —
What Happened Wednesday: Samantha Brennan's four-goal game leads Holyoke field hockey to win & more
Meredith Perri | mperri@masslive.com |

redpajama_set_name: RedPajamaCommonCrawl


The SPLASH 2019 Programming Languages Mentoring Workshop encourages graduate students (PhD and MSc) and senior undergraduate students to pursue research in programming languages. This workshop will provide mentoring sessions on how to prepare for and thrive in graduate school and in a research career, focusing both on cutting-edge research topics and practical advice, such as a panel of Ph.D. students discussing topics like "What I wish I had known before attending graduate school." The workshop brings together leading researchers and junior students in an inclusive environment in order to help welcome newcomers to our field of programming languages research. The workshop will show students the many paths that they might take to enter and contribute to our research community.
The workshop program will focus on important skills for beginning researchers and will offer mentoring opportunities with senior researchers attending the conference.
How to network effectively at a conference? How to find your cohort? How to find a mentor and what to look for?
What communication skills are essential for a research career? To give a good presentation? A good poster session talk? To write an outstanding paper?
How do people pick their research area of interest or your advisor/mentor?
What are the new, emerging areas in PL/SE?
What are the differences between a research career in industry and in academia?
What are the important work/life issues researchers deal with?
How did PL/SE researchers successfully navigate their training experiences?
We will provide travel grants that will fully support student attendance to PLMW. Information about applying for these travel grants will become available in July, 2019.


text:
Personal Information and Privacy
The Library subscribes to the Privacy Guidelines for British Columbian Libraries adopted by the Library Services Branch, ministry of Education, in May 2006. Public libraries in British Columbia fall under the definition of "local public bodies" under the Freedom of Information and Protection of Privacy Act (FOIPPA). As such, libraries are responsible for protecting personal information in accordance with the provisions of FOIPPA.
http://www.bced.gov.bc.ca/pls/privacy_guidelines.pdf


text:
A Utopian Dream Stood Still: Ricardo Bofill's Postmodern Parisian Housing Estate of Noisy-le-Grand
04:00 - 1 October, 2015
by James Taylor-Foster
Les Espaces d'Abraxas, Noisy-le-Grand, 2014. Image © Laurent Kronental
East of Paris, in Seine-Saint-Denis, sits a "Babel-like" housing estate. Its otherworldly atmosphere—existing somewhere between a 'new world' utopian dream and a postmodern, neoclassical housing estate—has set the scene for two Hollywood films including Brazil (1984) and, more recently, the upcoming second instalment of The Hunger Games: Mockingjay (2015). Parisian photographer Laurent Kronental's photo series, Souvenir d'un Futur (Memory of a Future), is an homage to the senior citizens of the French capital's Grand Ensemble region — not only in Noisy-le-Grand but across the Parisian banlieue. His photographs capture a number of places and their people which, in spite of their often megalomaniacal architectural settings, have been comparatively overlooked.
See Laurent Kronental's photo series—the result of four years of visits—after the break.
Denise, 81 ans, Cité du Parc et cité Maurice-Thorez, Ivry-sur-Seine, 2015. Image © Laurent Kronental
The Espaces Abraxas and Arènes de Picasso in Noisy-le-Grand were designed by Ricardo Bofill (Theatre, Arc and Palacio) and Manuel Nunez-Yanowsky ('The Pie'); both were co-founders of the Taller de Arquitectura. Vast housing estates of this kind were originally built between the 1950s and '80s to house a migrant population of rural and foreign refugees and are today—as is often the preconception of the banlieue—commonly stigmatised in the media and marginalised by resultant public opinion. Bofill's postmodern edifices, constructed between 1978 and 1983, were designed to represent an alternative approach to the modernist estates. In Paris in particular, they sought to directly oppose Le Corbusier's whitewashed visions of the 1950s, which they deemed to be devoid of 'style'.
In an interview in 2014, conducted by Elvire Camus of Le Monde, Bofill stated that [paraphrased from French] the general idea behind the Theatre, the Arc, and the Palacio was to build three 'rooms' of a theatrical space. In retrospect, he acknowledges that the complex has suffered from a lack of community spirit (a typical criticism in the banlieue), emphasised by the closed nature of the developments.
José, 89 ans, Les Damiers, Courbevoie, 2012. Image © Laurent Kronental
Paulette, 83 ans, Les Damiers, Courbevoie, 2015. Image © Laurent Kronental
Indeed, for Wael Sghaier, the exterior of the neoclassical, postmodern Palacio resembles a fortress, barracks, an imperial city, or a prison. Inside, however, all is quiet: time stops and people are scarce [vu de l'extérieur, Le Palacio d'Abraxas ressemble une forteresse, une caserne, une cité impériale ou une prison].
In 2006, Noisy-le-Grand sought to demolish parts of the development. After being met by widespread resentment from the population of Espaces Abraxas, the decision was made to keep them standing — for now. Kronental's photographs demonstrate very clearly that in spite of any assumed architectural or urban failings, these houses have been appropriated by their residents. In spite of the atmosphere of emptiness that Kronental has consciously captured, houses have become homes.
Les Tours Aillaud, Cité Pablo Picasso, Nanterre, 2014. Image © Laurent Kronental
Josette, 90 ans, Vision 80, Esplanade de La Défense, 2013. Image © Laurent Kronental
Jean, 89 ans, Puteaux-La Défense, 2011. Image © Laurent Kronental
Denise, 81 ans, Cité Spinoza, Ivry-sur-Seine, 2015. Image © Laurent Kronental
Jean-Claude, 82 ans, Les Espaces d'Abraxas, Noisy-le-Grand, 2014. Image © Laurent Kronental
Cité Curial-Cambrai, 19e arrondissement Paris, 2015. Image © Laurent Kronental
José, 89 ans, Puteaux-La Défense, 2012. Image © Laurent Kronental
Joseph, 88 ans, Les Espaces d'Abraxas, Noisy-le-Grand, 2014. Image © Laurent Kronental
Lucien, 84 ans, Les Espaces d'Abraxas, Noisy-le-Grand, 2014. Image © Laurent Kronental
Alain, 80 ans, Les Damiers, Courbevoie, 2013. Image © Laurent Kronental
Les Orgues de Flandre, 19e arrondissement Paris, 2014. Image © Laurent Kronental
Le Pavé Neuf, Noisy-le-Grand, 2015. Image © Laurent Kronental
Jacques, 82 ans, Le Viaduc et les Arcades du Lac, Montigny-le-Bretonneux, 2015. Image © Laurent Kronental
These 'monuments', living memories of their time, personify the fragile strength of a youth having blindly aged. —Laurent Kronental
As part of this photo-series, Laurent Kronental photographed a number of other large estates across the Parisian Suburbs alongside Les Espaces d'Abraxas and Le Pavé Neuf in Noisy-le-Grand, including: Les Tours Aillaud in Nanterre, Les Damiers in Courbevoie, La Cité du Parc and the Cité Maurice-Thorez in Ivry-sur-Seine, Les Arcades du Lac and Le Viaduc in Montigny-le-Bretonneux, Les Orgues de Flandre and La Cité Curial-Cambrai in the 19th Arrondissement of Paris.
References: francealumni.fr, Ricardo Bofill, Tourisme93, Le Monde
Futher reading: Le Monde: Lost Illusions of an Urban Utopia, Le Parisien, Wikipedia
James Taylor-Foster
News Architecture News PostmodernismRicardo BofillRicardo Bofill Taller de ArquitecturaSeine-Saint-DenisPhotographyParisFranceLaurent KronentalManuel Nunez-Yanowsky
Cite: James Taylor-Foster. "A Utopian Dream Stood Still: Ricardo Bofill's Postmodern Parisian Housing Estate of Noisy-le-Grand" 01 Oct 2015. ArchDaily. Accessed . <https://www.archdaily.com/774578/a-utopian-dream-stood-still-ricardo-bofills-postmodern-parisian-housing-estate-of-noisy-le-grand/> ISSN 0719-8884


Hi everyone. Slightly different post today, but very important for those who consider having a blog sale, or simply send beauty items via Royal Mail. I personally didn't have a clue about any UK restrictions for sending beauty items like nail polish or perfume, so I thought I should write about it to warn you and help you prevent losing a lot of money. It's not a nice feeling, especially when it's a big amount.
I recently sent a blog sale parcel with 6 bottles of nail polish and I didn't know that it was against the RM regulations. Royal Mail doesn't allow more than 4 bottles to be sent in the same parcel, so they stopped mine and didn't deliver it. As a nice person, I refunded the money to the buyer. It wasn't a big amount, only £5.00. Including whatever I paid for the parcel as well (just under £3.00), my total loss was £8.00. I am totally fine with that by the way, but that's not the problem.
I also sent another parcel, with many beauty products, including expensive perfume and makeup. Thankfully, the parcel got to the recipient safely, but... I was extremely lucky with this one! Why? If you send perfume or nail polish, it needs a special ID label, otherwise it is against the regulations and might not be delivered! God... if that parcel was stopped, I would have lost A LOT more than £8.00.
-Volume per item must not exceed 30ml. No more than four bottles of nail varnish can be sent in any one package.
-Bottles of nail varnish must be placed in strong outer packaging and be so packed, secured or cushioned in such a way that they cannot break, be punctured or leak their contents into the outer packaging.
-An ID8000 label must be applied. The sender's name and return address must be clearly visible on the outer packaging.
Including eau de parfum, eau de toilette and alcohol-free perfumes, but excluding non-flammable perfumed creams, gels, oils or lotions.
-Volume per item must not exceed 150ml.
-No more than four perfumes or aftershaves can be sent in any one package.
-The perfume or aftershave must be within its original retail packaging and then placed in strong outer packaging.
-The inner packagings must be packed, secured or cushioned to prevent breakage or leakage of their contents into the outer packaging.
WOW! I DIDN'T HAVE A CLUE ABOUT ALL OF THIS! Probably, because it is quite a recent change. Perfume in it's original packaging? Yep! Thankfully, the one I sent was still sealed, but... imagine if it wasn't? I was lucky that it got delivered in the first place. I wasn't asked by the Post Office staff about the content of my parcel so they didn't put the special label on it.
I hope you found this post helpful. You can read more about all the restrictions and regulations (within UK) HERE, but there are even more restrictions in place for shipping beauty products internationally.
Let me know, if you experienced any problems with sending nail polish or perfumes etc?


My view from the Mandarin Oriental in Singapore.
11.14.09 Is Today the Day I Worried About Yesterday?
http://giglioco.com/wp-content/uploads/2017/05/giglogo.png 0 0 Steve Giglio http://giglioco.com/wp-content/uploads/2017/05/giglogo.png Steve Giglio2010-03-28 13:49:472010-03-28 13:49:4711.14.09 Is Today the Day I Worried About Yesterday?
Today I'm taking off for Singapore! I'll be working with American Express executives who meet with banks throughout Asia to offer the Amex Card to their high net-worth customers. Blog to you from the Orient!
http://giglioco.com/wp-content/uploads/2017/05/giglogo.png 0 0 Steve Giglio http://giglioco.com/wp-content/uploads/2017/05/giglogo.png Steve Giglio2010-03-28 13:49:472010-03-28 13:49:4711.13.09 Off to Singapore!
"Working with Steve Giglio is always a great investment. He is a master in a field that few have mastered."
Pascal Guignard, Vice President & GM NBP & Airlines, American Express Services Europe Ltd.
Want Some Tips? Sign up for my newsletter.


corrugated metal garden boxes metal garden boxes corrugated metal raised flower bed garden planter garden boxes recycled plastic raised garden how to build corrugated metal raised garden beds corrugat.
corrugated metal garden boxes metal garden beds corrugated steel raised garden bed corrugated metal garden beds corrugated metal garden beds diy corrugated metal safe for raised garden beds.
corrugated metal garden boxes galvanized steel garden beds galvanized steel garden beds corrugated steel garden beds com metal garden beds is corrugated metal safe for garden beds corrugated metal gar.
corrugated metal garden boxes image result for corrugated metal raised garden beds round corrugated metal garden beds is corrugated metal safe for garden beds.
corrugated metal garden boxes raised garden beds with corrugated metal sides galvanized steel bed our easy wood how to galvanized steel raised bed garden corrugated metal safe for raised garden beds h.
corrugated metal garden boxes metal garden boxes build raised garden boxes corrugated metal garden boxes wood and steel raised garden corrugated metal garden beds how to build corrugated metal raised.
corrugated metal garden boxes raised bed planter corrugated metal garden beds diy corrugated metal garden beds.
corrugated metal garden boxes metal garden boxes incredible metal planter boxes intended for planters extraordinary flower idea metal corners metal garden boxes corrugated metal raised garden beds uk.
corrugated metal garden boxes corrugated metal planter box garden planter boxes appealing garden box ideas digital photograph inspiration corrugated metal garden beds corrugated metal raised garden be.
corrugated metal garden boxes metal garden beds corrugated metal raised beds plans for raised garden beds on legs best images round corrugated metal garden beds how to build corrugated metal raised ga.
corrugated metal garden boxes metal garden beds raised garden beds corrugated steel metal garden beds within the categories of garden metal garden beds corrugated metal raised garden beds uk how to bu.
corrugated metal garden boxes corrugated metal raised garden beds best raised beds colors images on corrugated metal raised garden bed corrugated metal raised garden beds uk is corrugated metal safe f.
corrugated metal garden boxes raised garden beds corrugated metal wood galvanized planter box boxes steel and round corrugated metal garden beds corrugated metal safe for raised garden beds.
corrugated metal garden boxes galvanized metal planter box corrugated metal raised garden beds raised garden bed designs beds corrugated metal how to build corrugated metal raised garden beds corrugat.
corrugated metal garden boxes corrugated metal raised beds metal raised garden bed back to build corrugated metal raised garden beds corrugated metal raised garden beds uk corrugated metal garden beds.
corrugated metal garden boxes corrugated metal raised garden beds corrugated metal raised garden beds perth corrugated metal garden beds diy.
corrugated metal garden boxes metal garden boxes rattan outdoor storage box waterproof outdoor storage outdoor box metal garden storage wooden corrugated metal garden beds diy how to build corrugated.
corrugated metal garden boxes steel raised garden beds corrugated metal garden beds galvanized sheet metal raised garden beds how to steel raised garden beds corrugated metal safe for raised garden be.
corrugated metal garden boxes build your own raised herb garden on round corrugated metal garden beds corrugated metal raised garden beds perth.
corrugated metal garden boxes steel raised garden beds galvanized raised bed galvanized steel and cedar raised garden beds galvanized water trough raised bed raised garden beds round corrugated metal.
corrugated metal garden boxes corrugated garden beds metal raised garden beds corrugated garden bed raised garden beds coral coast corrugated metal wood raised corrugated iron garden corrugated metal.
corrugated metal garden boxes corrugated raised garden bed corrugated steel garden beds corrugated raised garden beds raised garden beds instructions corrugated steel garden beds round corrugated meta.
corrugated metal garden boxes corrugated metal raised garden beds with unique best metal mostly garden beds how to build corrugated metal raised garden beds round corrugated metal garden beds.
corrugated metal garden boxes corruted metal raised rden beds u shaped bed plans corrugated metal safe for raised garden beds corrugated metal raised garden beds perth.
corrugated metal garden boxes garden boxes corrugated metal corrugated metal garden beds corrugated metal safe for raised garden beds.
corrugated metal garden boxes corrugated iron planter box copied from corrugated metal safe for raised garden beds corrugated metal garden beds diy.
corrugated metal garden boxes metal garden box raised cedar garden box with corrugated metal sides galvanized metal garden boxes how to build corrugated metal raised garden beds corrugated metal safe.


DeafTEC™: Technical Education Center for Deaf and Hard-of-Hearing Students is a NSF Advanced Technolgoical Education National Center of Excellence (DUE #1104229 and #151756). DeafTEC's goal is to increase the number of deaf and hard-of-hearing (deaf/hh) individuals in highly skilled technician jobs where they are currently underrepresented and underutilized. DeafTEC serves as a national resource for high schools and community colleges that educate deaf/hh students in STEM-related programs and for employers hiring deaf/hh individuals.
DeafTEC is administered by faculty at the National Technical Institute for the Deaf, one of the nine colleges of the Rochester Institute of Technology, and overseen by a National Visiting Committee made up of professionals in academia and industry.
Emphasizing career education, Rochester Institute of Technology is a privately endowed, coeducational university with one of the most accessible communities available for deaf and hard-of-hearing students. Over 1,300 deaf and hard-of-hearing students attend RIT and study, live, and socialize with more than 16,000 hearing students in what is widely regarded as the largest "mainstreamed" program in the world. Deaf and hard-of-hearing students at RIT can pursue associate degree programs in the college of NTID with courses taught using direct instruction or they can pursue bachelor degree programs in the other eight colleges of RIT using a wide range of educational access and support services.


text:
Two Stories Centenary Edition
July 2017 was the 100th anniversary of the publication of Two Stories by Virginia and Leonard Woolf. It was their first attempt at printing and publishing a book of their own and, as is well known, it was also the start of their Hogarth Press, a successful publishing house which continues in name to this day.
Like the original, the pamphlet-style binding has been sewn by hand. The type has been reset but every effort has been made to replicate the original, including the spelling errors as well as the erratic spacing and punctuation, to recreate the charm of the original that was hand set by the Woolfs, who learned as they went along. The text is illustrated by reproductions of four woodcuts by Dora Carrington. To be as true to the first edition as possible, all the publishing details have been consigned to an accompanying booklet which contains an introduction by Stephen Barkway; appendices detail textual variants between the text of 'The Mark on the Wall' and subsequent editions published in 1919 and 1921 by the Hogarth Press. Both booklets are housed in a folder; the edition is limited to 300 numbered copies.
£18.75 (£21 EU, £22 RoW)
For details of how to order, email: sbarkway@btinternet.com
The Voyage Out: Centenary Perspectives
To celebrate the centenary of the publication of Virginia Woolf's first novel, the Virginia Woolf Society of Great Britain has produced The Voyage Out: Centenary Perspectives. This book features a number of articles written by members about the novel, which was published by Woolf's half-brother Gerald Duckworth's company on 26 March 1915. Contributions include essays and memoirs by academics and common readers, with an introduction by Dame Gillian Beer.
The book, with a limited print run of 200 copies (a collector's item!), is on sale at the price of £22 (around £25 EU and £30 RoW, but this may change depending on postal charges and exchange rates). To order, please email Sarah M. Hall at: smhall123@yahoo.co.uk
Gillian Beer
Part I Context
'What a gift for pen & ink!' A Chronology of the Composition of The Voyage Out ….. Mary Ellen Foley
The Contemporary Critical Reception of The Voyage Out….. Lindsay Martin
Part II Memoirs
The Voyage Out, a Society and a Collection ….. Sheila Wilkinson
On First Encountering The Voyage Out ….. Wendy Nakanishi
On Teaching The Voyage Out ….. Janfarie Skinner
Part III Centenary Perspectives
'Tut, Tut': Censorship, Propriety and Obscenity in The Voyage Out ….. David Bradshaw
Classical Shock in The Voyage Out ….. Jim Stewart
Bayreuth and Berlin: The Voyage Out and the Place of German in Woolf's Writing ….. Michèle Barrett
Voyages In? ….. Matthew Macer-Wright
Meeting the Dalloways ….. Priscilla Martin
Wild Nature, Afternoon Teas and Butterflies: Virginia Woolf, Victoria Ocampo and the South-American Dream ….. Giulia Negrello
Translations from the Russian
By Virginia Woolf and S. S. Koteliansky
Introduction by Laura Marcus, edited by Stuart N. Clarke. Limited edition of 300 copies. This omnibus hardback edition consists of the three books translated by Virginia Woolf and S. S. Koteliansky that have never previously been reprinted in the UK.
Stavrogin's Confession and the Plan of the Life of a Great Sinner (Hogarth Press, October 1922, in an edition of 1,050 copies)
Tolstoi's Love Letters (Hogarth Press, May 1923, in an edition of fewer than 1,000 copies)
Talks with Tolstoi (Hogarth Press, June 1923, in an edition of about 1,000 copies)
£18 (£22 EU, £25 RoW). Cheques should be made payable to Virginia Woolf Society (please mark the back of the cheque 'Translations from the Russian') and sent to: Stuart N. Clarke, Fairhaven, Charnleys Lane, Southport PR9 8HJ, UK.
These are large picture postcards, black on cream with Virginia Woolf's signature in purple, with a photograph of the Virginia Woolf memorial erected in Tavistock Square garden by VWSGB members (see upper wrapper of the Virginia Woolf Bulletin, issue 17). Size = 172mm x 126mm.
A set of five costs £2.50 (£3.50 outside UK). Please email Sarah M. Hall at: smhall123@yahoo.co.uk to order.
This is an A5-sized notebook with 48 pages of plain paper and card covers. The front cover features a glossy black-and-white photograph of the garden of Monk's House.
The cost is £3 (£5 outside UK). Please email Sarah M. Hall at: smhall123@yahoo.co.uk to order.
Birthday Lectures
1. Wave, Atom, Dinosaur: Woolf's Science (2000) by Gillian Beer
2. 'This moment I stand on': Woolf and the Spaces in Time (2001) by Julia Briggs
3. Virginia Woolf, Photography and Modernism (2002) by Maggie Humm LAST FEW COPIES
4. Winking, Buzzing, Carpet-beating: Reading Jacob's Room (2003) by David Bradshaw
5. 'The True Nature of Woman' from Wollstonecraft to Woolf (2004) by Lyndall Gordon
6. 'The Exhibition Is in Ruins': Virginia Woolf and Empire (2005) by Anna Snaith
7. The Child of Two Atheists: Virginia Woolf's Humanism (2006) by Sybil Oldfield
8. Composing Oneself: Virginia Woolf's Diaries and Memories (2007) by Alison Light SOLD OUT
9. Leslie Stephen: A Nineteenth-Century Legacy (2008) by Henrietta Garnett SOLD OUT
10. Virginia Woolf and the Clergy (2009) by Jane de Gay
11. Virginia Woolf and the Art of the Novel (2010; published 2015) by Laura Marcus
12. Virginia Woolf and Friends: The Influence of T. S. Eliot and Katherine Mansfield (2011) by Sue Roe
13. Virginia Woolf, Fame and Gloire (2012) by Michael Whitworth LAST FEW COPIES
14. Burns Night/Woolf Supper: Birthday Thoughts on Virginia Woolf and Scotland (2013) by Jane Goldman SOLD OUT
15. 'To pin down the moment with date and season' (2014) by Hermione Lee
16. Woolf in Winter (2015) by Alexandra Harris SOLD OUT
17. Virginia Woolf and Tristram Shandy (2016) by Frances Spalding
18. Woolf and the Essay (2017) by Susan Sellers
19. 'This sheet is a glass': Virginia Woolf, a Woman of Letters (2018) by Stephen Barkway
20. Becoming a Name: Virginia Woolf's Political Reputation (2019) by Stuart N. Clarke
£4 each (£6 outside UK). Cheques in sterling should be made payable to 'Virginia Woolf Society' and sent to: Stuart N. Clarke, Fairhaven, Charnleys Lane, Banks, Southport PR9 8HJ, UK. If you wish to order three or more Birthday Lectures, please email Stuart N. Clarke at: stuart.n.clarke@btinternet.com as there may be a discount.
Virginia Woolf Bulletin
For individual and back issues, and details of contents, please see Virginia Woolf Bulletin page of the website. NB For payments in US$, please email Stuart N. Clarke at: stuart.n.clarke@btinternet.com
Paying online
You can pay for VWSGB publications online (UK customers, sterling only), once you have emailed us with your order. Please use the following details.
Bank: Santander
Account Name: Virginia Woolf Society GB
Account No.: 40411044
Sort Code: 09 06 66
Reference: first six characters of publication title plus your surname (so that we can match up the payment with the contact details provided). If you are unable to pay in sterling, or you live outside the UK, please contact us to confirm the cost.
* Please note that your bank may charge you an additional fee for processing international payments. *


text:
Release: Disney The Lion King
August 4, 2016 TheGravelPit0
Hakuna matata!
The Lion King, the delightful platformer of yore based on Simba's exciting journey towards his birthright, is available now for Windows, Mac, and Linux, DRM-free on GOG.com with a 10% launch discount. Grab this together with the other two Disney classics for a 33% discount.
Let's just admit it: we've all been Scar-red for life by that big dramatic moment in The Lion King, involving a certain stampede of Wildebeests. We have also danced along Timon and Pumbaa in their exhilarating, cathartic song routine that made "hakuna matata" our ultimate childhood battlecry. And of course, we are all looking forward to re-living these iconic moments as Simba in this beautiful platformer tie-in that brings our beloved story and characters to life. So let's jump on top of some feisty blue bugs, roar at indifferent porcupines, and claim the jungle throne that's rightfully ours. It will take some platforming precision but boy, these sniggering hyenas and their deep-voiced master have no idea what's coming to them!
Revisit the story of Simba as he jumps, pounces, and roars his way into becoming The Lion King, DRM-free on GOG.com.
The 10/33% discounts will last until August 8, 12:59 PM UTC.
Release: Grim Dawn: Crucible DLC
In Development: Kenshi
Weekly Staff Picks: The DRODening
March 9, 2015 TheGravelPit
Divinity: Original Sin 2 updated to Definitive Edition
Coming Soon: Tahira: Echoes of the Astral Empire


Today we offer you to watch a horrible scenes from a Bradford tragedy, which took place at the The Valley Parade stadium, May 11 1985. The cause of death of 56 soccer supporters was a terrible fire. It started while game between Bradford City Football Club and Lincoln City. The match had started in a celebratory atmosphere. At 3.40 pm, a small fire was reported by TV commentator John Helm, but in less than four minutes, in windy conditions, it had engulfed the whole stand, trapping some people in their seats.
The blaze began to spread; the roof and wooden stands were soon on fire. One eyewitness, Geoffrey Mitchell, told the BBC: "It spread like a flash. I've never seen anything like it. The smoke was choking. You could hardly breathe."
There were no extinguishers in the stand's passageway for fear of vandalism, and one spectator ran to the clubhouse to find one, but was overcome by smoke and others trying to escape. Supporters either ran upwards to the back of the stand or downwards to the pitch to escape. Most of the exits at the back were locked or shut, so people were standing in the field, feeling the heat and watching as the fire rages on.


Here you can view the MCDOT road closures and restrictions interactive map for a list of roads that will have restrictions and closures.
Here you can view a list of current projects the county is working on including maps and project details.
View County maintained roads, municipal boundaries, parcel information and more.


Harry Giles shot 5-for-11 from the field for 11 points with 12 boards in the Kings' Summer League win over the Grizzlies on Tuesday.
Giles is looking like a smart pick so far for the Kings at 20th overall last season. It is still early, but the 20-year-old Giles is having a solid Summer League so far, proving he can be a force around the rim and should earn some nice minutes on the final roster.


Dear visitors, considering that so many of you leave comment on this post, I have to warn that information written here are from September 2015. Unfortunately these two sites don't work any more!
The first is new revenue share site-REVADBURST,from known admin and registered legal company!
If you had ever joined some rev-share type site-then you know that the most important thing is TO BE FAST AND IF POSSIBLE FIRST!
The other one-ISOOB is a hybrid site- easy to work, similar system as PAIDVERTS, but with very expensive platform,and all important processors, I tried it with just few dollars- to see how it works-and as a 1st level member I got about $0.12 and 8×25 points, 2nd level about $0.25 and third lvl $0.34 daily-it's really excellent for the beggining!
One dollar invested in your campaign, gives you $1.55 in adds and 3100 points.


text:
Arthur Millar (1649 – 9 October 1727) was an Anglican bishop in the first third of the 18th century.

Millar was born in 1649 and educated at the University of Aberdeen. He was the incumbent at Dumbarton, Musselburgh then Leith. He was consecrated a college bishop on 22 October 1718 and became Bishop of Edinburgh and Primus of the Scottish Episcopal Church on 5 May 1727.

He died on 9 October 1727.

References

1649 births
Alumni of the University of Aberdeen
18th-century Scottish Episcopalian bishops
Bishops of Edinburgh
Primuses of the Scottish Episcopal Church
18th-century Anglican archbishops
1727 deaths
College bishops

redpajama_set_name: RedPajamaWikipedia


To save a missing girl, he'll have to explore his dark past. He may not survive the journey…Adam Rutledge is haunted by his childhood. The drifter never forgot how helpless he felt when his best friend was abducted, and his hometown never forgot his involvement. But when a strange dream sends him back to Cold Springs, he realizes that another child has gone missing and he has no choice but to help.Adam knows that to prevent history from repeating itself, he'll need to dive deep into the darkness of his own upbringing. When his efforts unleash dreams and visions he's spent decades burying, he's unsure if his sanity will survive.To bring the missing girl home alive, Adam must solve the riddle of his tragic past. But between his fractured psyche and the schemes of a madman, he may lose himself in the process…Prodigal is the first book in the Dead Hollow psychological suspense trilogy. If you like crime thrillers, authentic characters, and a dash of paranormal, then you'll love Judy K. Walker's haunting series. Buy Prodigal to unlock the mysteries of Dead Hollow today!


text:
Modern Tokyo Times
About Modern Tokyo Times
Sawako Art
Posted on August 16, 2015 by Lee Jay
Libya is Fueling Wars Based on the Intrigues of NATO and Gulf Powers: UN and War Crimes
Murad Makhmudov and Lee Jay Walker
European Union powers and America are currently lambasting the Russian Federation over actions taken in the Ukraine yet people are dying daily in Afghanistan, Libya, Pakistan, Syria – and other nations – based on the destabilization policies of major Gulf and Western powers (Pakistan destabilized itself). Serbians and Africans of course are sent to criminal courts while the nations that have killed in the millions based on destabilization policies literally get away with murder. Even the United Nations can't cover-up the mess of America, France, Qatar, Saudi Arabia, Turkey and the United Kingdom. Therefore, Libya is free of Colonel Gaddafi but to hell with the collapse of the nation state just like in Afghanistan and Iraq – and now Yemen is facing a Saudi Arabia led coalition of mass destruction and sectarianism.
Reuters reported last year: "U.N. experts say Libya has become a primary source of illicit weapons, including shoulder-fired missiles, which have been trafficked to at least 14 countries and are fueling conflicts on several continents, Rwanda's U.N. envoy said on Monday."
However, does the report go deep enough? After all, it is clear that Gulf and NATO powers have utilized Libya in order to send military arms to various terrorist and sectarian forces against the government of Syria. Indeed, NATO Turkey along the border with Syria is a Takfiri and al-Qaeda paradise because weapons pour in via Libya and other sources. Of course, Gulf and Western covert operatives continue to play their part in helping various terrorist and sectarian forces in Syria. Therefore, while Libya may be at the centerpiece of the UN report it is clear that other sinister forces are also at play.
Eugene Gasana, chairperson of the findings by the United Nations Security Council's Libya sanctions committee, says "The panel noted that the control of non-state armed actors over the majority of stockpiles in Libya as well as ineffective border control systems remained primary obstacles to countering proliferation and that Libya had become a primary source of illicit weapons, including MANPADs."
"The panel furthermore noted that investigations relating to transfers to 14 countries reflected a highly diversified range of trafficking dynamics; and that trafficking from Libya was fueling conflict and insecurity – including terrorism – on several continents."
In other words, powerful Gulf and Western powers have enabled various terrorist and anti-government forces to flourish in at least 14 nations based on their destabilization policies in Libya. Therefore, vast numbers of people have perished because of the policies of America, France, Qatar, Saudi Arabia, Turkey and the United Kingdom. These nations not only helped to destroy Libya, whereby various militias cleansed black Africans and slaughtered individuals deemed to be pro-Gaddafi; but the same nations also used ratlines in Croatia, Kosovo (Albanian led Kosovo) and in Libya (and other nations) in order to destabilize Syria and this in turn led to another crisis in Iraq based on the knock on effect.
If the Russian Federation is serious about standing up to nations that are meddling throughout the world, then why not use several UN findings to charge past and current leaders in major Gulf and NATO nations? Of course, this may sound idealistic but until powerful nations are held accountable for the deaths of untold numbers then nothing will change. In other words, after Syria it will be another nation – and then after the next nation, it will be another nation. Indeed, while outside meddling still funds various terrorist and sectarian forces, it is abundantly clear that the same is happening against Yemen based on the brutal Saudi Arabia led bombing of this nation.
The pattern is now clear and this applies to collective chaos; destabilization; the reduction in the rights of women; religious minorities fleeing; terrorism; ethnic cleansing; and sectarianism when applicable. In modern day Kosovo, Orthodox Christians can't travel freely without armed protection and many areas have been cleansed of Christians. Likewise, in Iraq this nation is a failed state whereby sectarianism and terrorism is an ongoing nightmare and now ISIS (Islamic State – IS) is enslaving Christians, Shabaks and Yazidis – while slaughtering Shia Muslims in vast numbers. Similarly, Afghanistan is in constant turmoil and women have been put into the shadows based on intrigues during the Cold War and now it is the time of Yemen to face sinister forces. This pattern is destroying and creating untold misery therefore nations like Libya and Syria, and other parts of the world mentioned above, suffer daily based on the whims of powerful Gulf and NATO powers (Pakistan involved in Afghanistan and other parts of the world).
All the above means that vast numbers of people have been killed because of Gulf and Western meddling – Turkey and Pakistan are also involved when it comes to Syria and Afghanistan respectively. Indeed, Turkey, Gulf and Western powers have now started the second destabilization of Iraq based on destabilizing Syria. At the same time, Lebanon is now extremely fragile. However, where are the war crimes against the usual players that utilize the mass media, slick private propaganda marketing agencies, so-called humanitarian agencies, manipulating the United Nations – and other sinister means that are available.
Gaddafi was brutally killed and his death sums up the inhumanity of nations that will use any means available to them. It matters not if people agreed with Gaddafi or not. Yet his death said much about what would happen to Libya based on the brutality unleashed against him. In time, the same chaos would ultimately be unleashed throughout Libya whereby law is rendered redundant and now ISIS is beheading Christians for fun. Many years later and not only is Libya a failed state but now according to the UN it is a nation that is being manipulated in order to fund international conflicts. This is a million miles away from the mantra of democracy after the demise of Gaddafi.
Business Insider reports: "The administration has said that the previously hidden CIA operation in Benghazi involved finding, repurchasing and destroying heavy weaponry looted from Libyan government arsenals, but in October we reported evidence indicating that U.S. agents — particularly murdered ambassador Chris Stevens — were at least aware of heavy weapons moving from Libya to jihadist Syrian rebels."
"There have been several possible SA-7 spottings in Syria dating as far back as early summer 2012, and there are indications that at least some of Gaddafi's 20,000 portable heat-seeking missiles were shipped before now."
"On Sept. 6 a Libyan ship carrying 400 tons of weapons for Syrian rebels docked in southern Turkey. The ship's captain was "a Libyan from Benghazi" who worked for the new Libyan government. The man who organized that shipment, Tripoli Military Council head Abdelhakim Belhadj, worked directly with Stevens during the Libyan revolution."
The above applies to 2012 and clearly since this period many deals are being done in the shadows. Of course, the UN is fully aware of this but nothing is being done to hold nations accountable for their destabilization policies. Similarly, international criminal courts are just a witch-hunt against nations outside of the loop and to be held against individuals fighting on the wrong side. Therefore, vast numbers of people are dying every day in countless conflicts because of the collective policies of powerful Gulf and Western powers – alongside the respective deeds of Pakistan and Turkey.
http://www.businessinsider.com/obama-admin-admits-to-covertly-sending-heavy-weapons-to-syrian-rebels-2012-12#ixzz2vg72D8Rl
http://www.reuters.com/article/2014/03/10/us-libya-crisis-un-idUSBREA291OV20140310
Modern Tokyo News is part of the Modern Tokyo Times group
http://moderntokyotimes.com Modern Tokyo Times – International News and Japan News
http://sawandjay.com Modern Tokyo Times – Fashion
https://moderntokyonews.com Modern Tokyo News – Tokyo News and International News
http://global-security-news.com Global Security News – Geopolitics and Terrorism
PLEASE JOIN ON TWITTER
https://twitter.com/MTT_News Modern Tokyo Times
PLEASE JOIN ON FACEBOOK
https://www.facebook.com/moderntokyotimes
Africa, Geopolitics, Middle East, Miitary Conflict, NATO, Religious Persecution, Syria, Terrorism
Tagged IS barbarity, ISIS barbarity, Islamic jihad in Libya, islamic jihad in syria, Japan news and terrorism, NATO barbarity, Takfiri barbarity, Takfiri killers, Tokyo News and Terrorism
At least 23 perish after a brutal attack against a Kyoto Animation (KyoAni) studio in Japan
Buddhism and Japan Art: The influence of the Middle Kingdom
Japan touts national security in a spat with South Korea after high-tech restrictions
A Neighbor's Dilemma: The Implications of the Libyan Crisis on Tunisia
Japanese Zen Buddhist poetry by Sesson Yūbai and the impact of the Middle Kingdom (China)
Heavy rainfall in Japan is lashing down on Kyushu
New mass protests in Sudan is met by violence from the military
US and North Korea relationship is given a positive jolt after Trump meets Kim in North Korea
CIA and DGSE
DGSE
Europe: Visegrad
Info and Technology
International Fashion
ISI and MI6
Japan Art
Japan Fashion
Japan Geopolitics
Japan Tourism
Miitary Conflict
Osaka Fashion
Tokyo Art
Tokyo Culture
Tokyo Fashion
Tokyo Gardens
Tokyo Tourism
© Modern Tokyo Times


text:
Volcanoes National Park Rwanda / Parc National Des Volcans (PNV)
Traveler's guide to Volcanoes national park Rwanda featuring all you need to know about trekking safaris to watch gorillas in Rwanda, how to book gorilla permits, other activities in the park, what to wear for gorilla trekking, gorilla groups in Rwanda, best time to visit Rwanda for gorilla trekking, accommodation in Rwanda and so much more. Volcanoes national park in Rwanda is the most rewarding African Safari destination.
Spanning on a 160sqkm area in the northern part of Rwanda, Volcanoes national park is the oldest national park in Africa. It was initially a small area around Karisimbi, Mikeno and Visoke volcanoes which was gazetted to protect the Mountain gorillas which were facing the threat of extinction as a result of poaching.
In 1929, the park was extended into Rwanda and the then Belgian Congo and was named Albert national park managed and run by the Belgian Colonial Authorities. During early 1960s, the park was divided as Rwanda and Congo gained their independence and by the end of that decade, the park was almost half of its original size.
In 1967, the American zoologist Dian Fossey who had been doing research on mountain Gorillas in the forests of Congo fled from insecurity and established her research base at a place between Visoke and Karisimbi volcanoes that was yet to be known as Karisoke research center. She spearheaded the conservation campaign of the mountain gorillas and mobilized resources to fight against poaching in this area, a fight she put up until her murder in 1985. She was buried at the research center next to the grave of her favorite gorilla called Digit.
The park continued to suffer at the mercies of poachers though conservation efforts were also under way. In early 1990s, the park became a battle field for the Rwanda's civil war which paralyzed tourism activities until 1999. In 2005, in a bid to boost conservation and gorilla tours in Volcanoes national park , Rwanda introduced the annual baby naming ceremony for baby gorillas known as 'Kwita Iziina' which has seen great results in as far as gorilla population in volcanoes is concerned.
Volcanoes national park is home to Mountain Gorilla (Gorilla beringei beringei); golden monkeys (Cercopithecus mitis kandti), Spotted Hyena (Crocuta crocuta), buffaloes (Syncerus caffer), elephants , black-fronted duiker (Cephalophus niger), and bushbuck (Tragelaphus scriptus). The park also harbors 178 bird species including at least 29 endemics to Rwenzori mountains and the Virungas.


Sometimes the two sides of Peter Langley's professional life intersect.
That happened last year when Langley was troubled to realize that the legal consequences of mistreating animals could be more severe than those of mistreating humans.
Langley, an attorney who worked for the Jackson County Prosecutor's office and dealt with domestic violence cases, is now the director of the Senate Majority Policy Office in Lansing. His job is to oversee a staff of 11 who review and analyze the legislative bills and counsel the Republican senators.
"You could get more time for beating your dog than for beating your wife," the 37-year-old Jackson man said.
He was referring to the state statute on animal cruelty that can be up to a four-year felony while a first-time domestic violence offense is a 93-day misdemeanor.
"We worked on a package of bills and we got it passed to enhance the penalties," Langley said. "We changed it so you can get more jail time for habitual domestic violence offenders."
Langley's career is now a blend of his political and legal backgrounds.
"It allows me to combine my legal experience and knowledge along with my passion for politics," said Langley, who served last year as the. president of the Jackson County Bar.
Becoming a lawyer was not Langley's first career choice. It wasn't until he saw how volatile a political job could be that he thought of adding "attorney" to his resume.
The uncertainty of politics became a concern in 2003 when Langley worked for Republican State Representative Jerry Kratz, who died one month into his term. He had just had a child and was uncertain about what would happen to his job.
"I wanted something more long term professionally," he said.
So Langley, who had graduated from Northern Michigan University with a degree in public administration, went to Cooley Law School and graduated in 2007.
He was hired by the Jackson County Prosecutor's Office and worked there for three years as an assistant prosecutor who worked with the domestic violence court in Jackson.
He enjoyed the daily interaction with people that came with the job.
"I loved being on my feet in the court room," Langley said. "I like people. I like getting out."
Langley said he enjoyed talking to the judges as well as the defendants.
"It wasn't glamorous," he said. "It wasn't high profile murder cases. It was everyday people who got hit with a drunk driving, for example. You get a different perspective in life when you get a chance to sit down and talk to these folks. These are normal people who just made mistakes. I just dealt with a lot of good people who slipped and made a mistake."
But in 2011, the "political bug" hit Langley again. And he moved on to work for the Senate Republicans.
Langley, who is divorced and has three children, discovered that his political work can sometimes have the same drama as the courtroom.
In December, he was embroiled in the right-to-work controversy when the state legislature passed the law that gives people the option of joining a union.
When thousands of protesters showed up in Lansing, Langley and his staff had to get past them to get to their office.
"My concern was for my staff," Langley said. "It was a powder keg. That day, there was a lot of emotion. I'd never seen so much police in my life."
However, most days are not filled with such drama.
Most of the work by Langley's office is done in committee meetings. Then there are times during an on-floor debate when Langley and his staff will have to answer questions about amendments to bills and how they impact the law.
Down the road, Langley says he may someday return to the courtroom.
"I wouldn't rule it out. I'd love to do it," he said. "I had such a great time with all the judges I was assigned to. I really enjoy being in the courtroom and I would never rule it out."


Increases the life span of all animals and pets, including low-maintenance pets.
Resets your Romantic Reputation. Good if your Sim was a cheating jerk and needs a reset on life.
Sharply decreases the loss rate of the Hunger meter (or Thirst meter for Vampires).
Significantly decreases the chance for a toddler to reject an action by this Sim. This makes toilet training and general caring much easier.


Beech Sr High School is a public high school of the Sumner County School District located in Hendersonville, TN. It has 1,177 students in grades 9th through 12th. Beech Sr High School is the 94th largest public high school in Tennessee and the 4,906th largest nationally. It has a student teacher ratio of 13.4 to 1.
You can also use the form below to search for alumni info and Beech Sr High School students.
Beech Sr High School is ranked 253rd out of 410 ranked schools in Tennessee, for total students on lunch assistance.
The percentage of Beech Sr High School students on free and reduced lunch assistance (21.4%) is significantly lower than the state average of 52.4%. This may indicate that the area has a lower level of poverty than the state average.


Classes - TRUEBODY : Get Fit. Stay Fit.
We have a full spectrum of classes that deliver quality, variety, and individual progress to keep you engaged and energized. Our schedule offers 100+ classes per week, focusing on an integrated approach to fitness, balancing cardio, strength, yoga, circuit training, healing and stress reduction.
We use our mailing list to keep you and others informed about TRUEBODY.
We do not share or sell your information to any third parties. We may use data from the list in the form of aggregate statistics, which do not identify you, for measuring the use of our website only.
©2018 TRUEBODY. All rights reserved.


Thank you to Woking Scouts!
At the beginning of this term, a group of students from the school enjoyed an activity-packed weekend including climbing, canoeing, archery, outdoor cooking and sleeping under canvas thanks to Woking Scouts and the efforts of a huge number of volunteers including school staff. The students who went would like to say a huge thank you to them, for giving up their time and home comforts to give them such a great and valuable experience, and to the sponsors who financed the event, among them our friends from the Children with Special Needs Foundation.