text:
The Mediator complex transduces regulatory information from enhancers to promoters and performs essential roles in the initiation of transcription in eukaryotes. Human Mediator comprises 26 subunits forming three modules termed Head, Middle and Tail. Here we present the 2.8 Å crystal structure of MED23, the largest subunit from the human Tail module. The structure identifies 25 HEAT repeats-like motifs organized into 5 α-solenoids. MED23 adopts an arch-shaped conformation, with an N-terminal domain (Nter) protruding from a large core region. In the core four solenoids, motifs wrap on themselves, creating triangular-shaped structural motifs on both faces of the arch, with extended grooves propagating through the interfaces between the solenoid motifs. MED23 is known to interact with several specific transcription activators and is involved in splicing, elongation, and post-transcriptional events. The structure rationalizes previous biochemical observations and paves the way for improved understanding of the cross-talk between Mediator and transcriptional activators.
MED23 was originally discovered as a genetic suppressor of a hyperactive ras phenotype in Caenorhabditis elegans1 and identified as the major target of the conserved transactivation region 3 (CR3) of the adenoviral 13SE1A oncoprotein2. MED23 belongs to the Tail module of Mediator, whose main function is to connect Mediator to sequence-specific transcription factors, and is thus of fundamental importance3. In vitro and in vivo studies have demonstrated a specific interaction between MED23 and the ternary complex factor Ets-like protein-1 (ELK1), which is activated by MAPK signaling4. Functional investigation of MED23 and ELK1 in Ras-active lung cancer demonstrated a critical role for MED23 in enabling the Ras-addiction of lung carcinogenesis, providing MED23 as a potential therapeutic target5. ELK1 interacts with MED23 in a complex manner involving many phosphorylation sites6. MED23 also binds to other transcription factors, such as ESX (ESE-1/ELF3/ERT/Jen), RUNX2 or IRF77,8,9. In addition, MED23 plays important roles in postrecruitment steps. It regulates alternative mRNA processing via direct interactions with splicing factors10, promotes Pol II into transcription elongation via a direct interaction with CDK9 in P-TEFb11, or promotes histone post-translational modifications on active genes12. Altogether, these data underlie the important roles of MED23 in the human Mediator complex and its link with several pathologies. Results obtained from structural biology efforts provided important information on the architecture of yeast Mediator and defined the location of core Mediator on Pol II. These efforts culminated in the cryoEM and X-ray crystallographic studies of the Head and Middle modules reported at atomic or near-atomic resolutions13,14,15,16,17,18,19,20,21. However, structural information remains limited to core Mediator and data about the Tail module are needed to expand our understanding of Mediator function. Here we report the crystal structure of MED23, the largest subunit from the human Tail module. MED23 is composed of 25 HEAT repeats-like motifs organized into 5 α-solenoids and forming an arch-shaped conformation, with an N-terminal domain (Nter) protruding from a large core region. MED23 interacts with several specific transcription activators and is involved in splicing, elongation, and post-transcriptional events. The structure thus paves the way for improved understanding of the cross-talk between Mediator and transcriptional activators and represents an important step towards the complete structural characterization of the human Mediator Tail module.
Results and Discussion
Nanobody-assisted crystallization of MED23
Despite many attempts, recombinant human MED23 could not be crystallized alone. We thus generated specific lama nanobodies, one of which, Nb106, assisted in the crystallization process and allowed us to obtain well-diffracting crystals (see Methods and Supplementary Information). The nanobody binds via its complementarity determining region (CDR) 3 to a cavity in MED23 formed at the junction of three structural regions (named hereafter 5-HEAT, 6-HEAT, and C-HEAT). It is also involved in the crystal packing, with its β-sheet stacking against helices H4, H6, and H8 from the N-HEAT solenoid domain of MED23. We solved the structure of the MED23-Nb106 complex de novo by multiple isomorphous replacement. Diffraction data to 2.8 Å resolution resulted in excellent electron density (Supplementary Fig. 1) and a refined structure that has a free R factor (Rfree) of 24.0% (Supplementary Table 1).
Overall MED23 architecture
The MED23 structure shows that the protein is fully folded (with the exception of the last 30 amino acids) and adopts a global form measuring approximately 145 × 55 × 50 Å (Fig. 1a−e). The structure presents an N-terminal domain (N-HEAT: residues 1–220) extending from a core region that encompasses the rest of the protein (Core MED23: residues 221–1334). Core MED23 adopts a compact and flattened triangular form with sides of 80–100 Å in which the protein wraps on itself and positions the C-terminal in close proximity to N-HEAT. Together with N-HEAT, Core MED23 defines an arch of approximately 140 Å (Fig. 1c), with a concave and a convex face (Fig. 1a, d). The structure identifies 71 α-helices encompassing ~65% of the complete sequence (Supplementary Fig. 2). These α-helices engage in 25 heat repeats-like (HR) motifs arranged in 5 right-handed superhelical solenoids. A linker and a bridge domain complete the structure (Fig. 1a−e).
The N-terminal domain comprises five HR-like motifs
N-HEAT folds into a solenoid of five HR motifs, with the last four motifs arranged nearly linearly (Fig. 2a). N-HEAT contacts the Core MED23 via helices H10 and H13 which stack against the C-terminal domain. Solenoids composed of HR motifs are known to play a role in mediating protein−protein interactions and in MED23 the two faces of N-HEAT are properly exposed for such interactions. MED23 binds specifically to the RNA Recognition Motif 2 (RRM2) domain of hnRNP-L, a sequence-specific RNA binding protein which is involved in splicing10. The region of MED23 involved in this interaction maps to a fragment of MED23 covering residues 1–327 10, which corresponds to N-HEAT extended up to the linker and the first HR motif of the next solenoid (3-HEAT, defined below). MED23 also physically associates via this region with the heterodimeric E3-ubiquitin ligase RNF20/40 which functions with the super elongation and polymerase-associated factor complexes to mediate monoubiquitylation of histone H2B on gene bodies of actively transcribed genes12. The 1–327 fragment of MED23 also binds to the RunT domain of the transcription factor RUNX2, and this interaction is involved in the regulation of osteoblast differentiation and bone development9. Our structure thus rationalizes these previous biochemical results and defines the N-terminal domain of MED23 as an interaction site for partner proteins involved in Mediator functions such as recruitment steps in transcription initiation, splicing and histone post-translational modification on active genes.
Core MED23 architecture
Core MED23 contains four different solenoids domains built from HR motifs, that we named 3-HEAT (residues 267–403: 3HR), 5-HEAT (502–751: 5HR), 6-HEAT (752–1034: 6HR) and C-HEAT (1035–1334: 6HR). Their structural organization is illustrated in Fig. 2b−e. Core MED23 also includes a linker region (221–266) which connects 3-HEAT to the N-terminal domain N-HEAT, and a bridge domain which connects 3-HEAT to 5-HEAT (Fig. 1a−d). One face of the 3-HEAT solenoid points towards the center of Core MED23, where it interacts with the bridge domain and 5-HEAT, while the other face of 3-HEAT is solvent exposed (Fig. 2b). 3-HEAT is followed by the bridge domain (residues 404–501), a four helix module (H23-H26) (Fig. 1a) that links 3-HEAT to 5-HEAT and positions these solenoids in a nearly parallel orientation, but with an inverted polarity. The MED23 bridge domain identified here has been shown to be a target for transcriptional regulators. ESX is an Ets transcription factor that is expressed specifically in epithelial cells and that activates the Her2 gene in breast cancers7. MED23 binds to the transactivation domain (TAD) of ESX, via a region covering residues 391−48722, that maps within the bridge domain. Due to its importance in cancerous pathologies, this interaction has been targeted in drug design programs that relied, in absence of structural knowledge about MED23, on the helical conformation adopted by the TAD of ESX upon binding to MED2322,23,24. The high resolution structure of MED23 now provides a way to improve drug design programs aiming to control Her2 expression in cancer cells.
Downstream the bridge domain is 5-HEAT that stacks along 3-HEAT but is tilted by ~90° (Fig. 2c). In this orientation, 5-HEAT contacts 3-HEAT mainly via the tips of helices from HR motifs 1 to 4, as well as the loops connecting them. The 5-HEAT solenoid is interrupted between HR motifs 3 and 4 by helix H34 which protrudes towards the center of Core MED23. This face of the 5-HEAT solenoid is solvent exposed on the inner face of the MED23 arch (Fig. 2c), while the other face is buried and packs against 6-HEAT, the next solenoid. 6-HEAT is connected to 5-HEAT by a short linear linker of seven residues. Motifs HR1−HR3 of 6-HEAT do not participate at any interface within Core MED23 (Figs. 2d, 1a, c, d). They curve to guide the three following HR motifs (HR 4–6) of 6-HEAT in front of the HR motifs 2−5 from 5-HEAT. There is an additional HR motif in 6-HEAT, formed by helices H47−H49 and inserted between HR motifs 4 and 5 (Fig. 2d). This motif extends along the 6-HEAT solenoid and contacts helix H27 as well as HR motif 1 and HR motif 2 of 5-HEAT. H48, a short 310 helix inserted between H47 and H49, is positioned close to the center of Core MED23 (Fig. 2d). Finally, the last solenoid, C-HEAT, completes the roll-up of Core MED23 to contact N-HEAT. C-HEAT connects to and packs against 6-HEAT via its first HR motif and helix 55, contributing to bend MED23 into its arch-shaped conformation (Fig. 2e). This region also contains H57, a small 310 helix that is located at the center of Core MED23 in front of helix H48 from 6-HEAT (Fig. 2e). The HR motifs 2−6 in 6-HEAT form a solenoid that structurally resembles N-HEAT (Supplementary Fig. 3). In MED23 only one face of the C-HEAT solenoid is solvent exposed, while the other face stacks onto the linker and N-HEAT (Fig. 2e).
In metazoans, MED23 belongs to the Mediator Tail module, together with MED15, MED16, MED24, and MED2520. MED16, MED23, and MED24 are hypothesized to form a submodule4 which contacts MED14, a subunit that bridges the three Mediator modules25,26. How MED23 interacts with these subunits is currently unknown. It has been reported that the N-terminal domain of MED23 binds many partner proteins involved in various Mediator functions, such as hnRNP-L, RNF20/40, or RUNX29,10,12, suggesting that N-HEAT is presumably not the primary site of interactions with other subunits of the Mediator complex. We observed that Nb106, the lama nanobody we used to crystallize MED23, can coimmunoprecipitate MED23 incorporated within the Mediator complex (Supplementary Fig. 4a). Nb106 binds via its complementarity determining region (CDR) 3 at an interface within Core MED23, between 5-HEAT, 6-HEAT, and C-HEAT, on one side of the MED23 arch (Supplementary Fig. 4b). In addition, a commercial antibody targeting a region covering amino acids 400 to 450 has been reported to coimmunoprecipate Mediator12 (Supplementary Fig. 4b). It thus appears that the regions or interfaces targeted by these antibodies are not involved in interactions with other Mediator subunits. Collectively, these data suggest that, within Core MED23, the concave (Fig.1a) and convex (Fig. 1d) faces of the MED23 arch are available to interact with other Mediator subunits, in addition to transcriptional regulators.
On the concave face of the MED23 arch, the linker, 3-HEAT, 5-HEAT, and C-HEAT interact and form a complex interface (Fig. 3a). In this interface 5-HEAT is packed along 3-HEAT and the linker, while C-HEAT contributes helices H64, H65, and H67 which interact with the linker and the fourth HR motif of 5-HEAT (Fig. 3a). On the convex face of the MED23 arch, 6-HEAT and C-HEAT interact respectively via their C and N terminal regions (Fig. 3b). These two solenoid domains stack against 5-HEAT and 3-HEAT, creating a funnel-shaped surface bordered by helices from the four domains (Fig. 3b). The bridge domain contributes to both faces of the arch with helices H23, 25, and 26 contacting 3-HEAT and 5-HEAT (Fig. 3a, b). The MED23 arch displays specific structural features. On the concave face, an extended groove runs along the surface between the linker, C-HEAT, 3-HEAT, and 5-HEAT (Fig. 3a). This groove is well exposed, and its accessibility is only slightly limited by the V230-S240 segment of the linker, which is variable among metazoans (Supplementary Fig. 5). The groove defines a positively charged surface propagating from N-HEAT to the center of the concave face. It communicates with the convex face of core MED23 via a buried negatively charged cavity (Supplementary Fig. 6) that joins the funnel-shaped surface, close to helices H48 and H57 (6-HEAT and C-HEAT, respectively). Finally, on the convex face, we observed a partly exposed negatively charged groove near helices H16 and 19 (3-HEAT), H23 (Bridge), and H21 and H27 (5-HEAT) (Fig. 3b and Supplementary Fig. 6). These sequence motifs are highly conserved across metazoans, demonstrating their important structural and/or functional roles (Supplementary Fig. 5). Altogether, they provide very specific interfaces which might be involved in the recruitment of other Mediator subunits and/or the binding of specific transcription factors. They also reveal signaling transmission paths that link all domains within MED23 and connect both faces of the MED23 arch. The presence of buried charges also suggest that MED23 may adopt a dynamical behavior upon effector binding, which could result in important structural changes. Although these molecular properties have still to be deciphered, data agree with the implication of these interfaces in important MED23 functions. It has been shown that the R611Q mutation in MED23 is linked to neurological disorders, highlighting its role in brain development and intellectual diseases27. This mutation specifically impairs the response of JUN and FOS immediate early genes to serum mitogens. In our structure, R611 rims the funnel on the convex face of MED23 and engages in a buried salt bridge with E565 (Fig. 4), an interaction which is conserved in metazoans. R611 and E565 are stacked against an aromatic cluster at the center of Core MED23 involving H613 and 614 (5-HEAT), H911 and W912 (H48, 6-HEAT), F1094 (H57, C-HEAT) and Y278 (H16, 3-HEAT) (Fig. 4). This aromatic cluster surrounds a signature motif composed of residues D1091, W1092, R1093, E1096 (helix H57), and D529 (Fig. 4), that is conserved in metazoans. The aromatic cluster also contacts the concave face of MED23 via H614 and Y278 at the depth of the groove on this face (Fig. 4). The R611 mutation presumably impacts these structural features, thus affecting both interfaces of the MED23 arch. Although these residues are not fully exposed in the structure to engage in direct interactions with partner proteins, they might play a crucial role in contributing to the functionality of the core MED23 interfaces. The MED23 structure thus provides a molecular rationale to further explore the precise downstream factors in the serum response pathway leading to intellectual disability.
The structural characterization of MED23 reported here represents an essential step to understand at the molecular level how this subunit regulates transcription via many factors including ELK1, ESX, RUNX2, IRF7, and how it participates to postrecruitment events in elongation, splicing, and post-translational modification processes on active genes. It also paves the way to the challenging structural characterization of the metazoan Mediator Tail module, which remains mostly unresolved.
Recombinant MED23 protein production
DNA sequence encoding human MED23 isoform 1 (NM_004830.3) was amplified by PCR and cloned into a pFastBac that contains a rhinovirus 3C protease site followed by a 6xHis tag to generate a C-terminal 6xHis-tagged MED23 protein. Recombinant baculovirus were obtained using the Bac-to-Bac expression system (Invitrogen) as specified by the manufacturer. Seventy-two hours after baculovirus infection, the SF21 cells were resuspended in MEDlysis buffer containing 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 1 mM TCEP, 10% (w/v) glycerol, 0.1% Igepal CA-630 and 5 mM Imidazole. After centrifugation, the purification of MED23 was performed on a Talon Resin column (Clontech). The beads were washed with MEDlysis buffer and MED23-6His was eluted using the MEDlysis buffer supplemented with 250 mM Imidazole. The pooled fractions were dialyzed overnight at 4 °C against 20 mM Tris/HCl pH 7.5, 60 mM NaCl, 1 mM TCEP and 10% (w/v) glycerol and further purified by size-exclusion chromatography on a Superdex 200 column (GE Healthcare) pre-equilibrated with 20 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM TCEP and 10% (w/v) glycerol. The MED23-6His protein was then concentrated to 4 mg ml−1 and aliquots were flash-frozen and stored at −80 °C.
Generation, expression and purification of nanobody Nb106
Nanobodies targeting MED23 were generated following established protocols28. The plasmids containing C-terminal His6-tagged Nb were transformed into E. coli strain WK6, grown in TB medium containing 0.1% glucose, 2 mM MgCl2 and 100 mg ml−1 ampicillin at 37 °C until the A600 nm of the sample reached 1.2, and then induced with 1 mM IPTG for 4 h. Cells were collected and the periplasmic fraction was extracted using the modified osmotic shock protocol. The periplasmic extract containing Nb was supplemented with 10 mM imidazole and incubated with Ni-NTA agarose (Qiagen) for 1 h at 4 °C. The beads were washed with 50 column volumes of PBS containing 10 mM imidazole, and Nb were eluted from the resin with a PBS buffer containing 300 mM imidazole. The eluted fractions containing Nb were concentrated and injected into a Superdex 75 (GE Healthcare) size-exclusion chromatography column pre-equilibrated with 20 mM Tris/HCl pH 7, 150 mM NaCl, 1 mM TCEP. Nb were concentrated to 4 mg ml−1, flash-frozen and stored at −80 °C. The nanobody Nb106 successfully crystallized MED23.
Crystallization, data collection, and structure determination
Purified MED23 was mixed with Nb106 in a 1:1.2 ratio in order to obtain final protein concentrations of respectively 4 and 0.4 mg ml−1. The protein mixture was then incubated for 1 h at 4 °C before crystallization trials. Native MED23 crystals were obtained at 20 °C using the hanging drop vapor diffusion method. Crystals were grown in 10% PEG6000 and 100 mM HEPES buffer (pH 7.0). Native crystals were collected and transferred directly into the cryo-solution corresponding to the mother liquor supplemented with 20% glycerol, and subsequently flash-frozen in liquid nitrogen. Orthorhombic crystals were obtained in all crystallization drops, with the exception of one drop where two crystal forms were obtained simultaneously. In addition to the orthorhombic crystals, a few monoclinic crystals appeared in this drop, one of which diffracted to ~2.65 Å resolution (Supplementary Table 1). Both crystal forms contain one complex of MED23 and Nb106 in the asymmetric unit.
For structure determination, native orthorhombic crystals were derivatized. Pt and Au derivatives were obtained by soaking native crystals for 2 min in a solution containing 20% glycerol, 10% PEG6000, 100 mM HEPES buffer (pH 7.0) and 45 mM K2PtCl4 or 45 mM KAu(CN)2, respectively.
Data were collected at 100K at ESRF and SOLEIL synchrotrons in France (Supplementary Table 1). All data were processed with XDS and XSCALE29. Crystals used for phasing and building the initial models were from the orthorhombic forms, which diffracted to resolutions between 3.0 and 3.85 Å. The structure was solved with the SHARP/autoSHARP distribution30 by the multiple isomorphous replacement method with anomalous scattering using the platinum and gold derivatives (Supplementary Table 1). A poly-ala model was manually built with Coot31, corresponding to 80% of the 71 helices, although with errors in polarity. Rounds of refinement at 3.0 Å resolution combining the use of Refmac31 and Buster32 allowed to obtain improved maps, to identify and fit a nanobody (PDB code 5IMK) related to Nb106, which progressively helped to correct and complete the poly-ala MED23 model. We then placed the complex formed by MED23 and Nb106 in the monoclinic crystal form using MOLREP31, which allowed to complete the poly-ala MED23 model and assign the sequence, from residues 1 to 1334. The last steps of refinement were performed at 2.8 Å resolution using PHENIX33, which allowed us to better control the geometry of the model by optimizing the X-ray/stereochemistry weight. The high resolution limit was set up to 2.8 Å resolution to prevent overfitting by using diffraction data with I/σI greater than 2. No water molecule was added to the model, as we did not observe any obvious electron density corresponding to water molecules and their forced addition by PHENIX33 resulted in a worsening Rfree. The final model is complete, with the exception of the last 30 residues, which have no visible electron density. The structure is well resolved, with only a few sequence segments from loops which are poorly ordered (N233-I237; N290-R296; E316-S327; G364-L367; Y403-Y408, D439-Q444; N502-R506; Q940-D942; N1054-E1058; E1182-G1185). These segments were kept in the model to ensure continuity of the chain. The final model has an R and Rfree of 19.9 and 24.0%, respectively. The geometry of the final model agrees well with structures refined to 2.8 Å resolution, with 94.56 and 4.89% of residues respectively in the favored and allowed regions of the Ramachandran plot, and 0.55% of outliers. The cavities in MED23 which are described and illustrated in Fig. 3 have been analyzed using POCASA34, and rendered using PyMol35.
HEK-293 human cells were lysed in a buffer containing 50 mM Tris/HCl pH 8, 250 mM NaCl, 1 mM EDTA, 1 mM TCEP, 10% glycerol, 3 mM MgCl2 and 0.1% Igepal CA-630. After centrifugation, extracted proteins were incubated with 2 µg of Nb106 overnight at 4 °C. The resulting Nb106 immuno-complexes were then fished with 50 µl of CaptureSelect™ C-tag Affinity Matrix (Thermo). After extensive washing, the precipitated proteins were identified by western blot. The antibodies used to detect Mediator subunits were from Santa Cruz (anti-Med7 sc-12457 and anti-Cdk8 sc-1521), BD Pharmigen™ (anti-Med23 550429) or from Bethyl Laboratories (anti-Med1 A300–793A, anti-Med14 A301–044A, anti-Med16 A303–668A, anti-Med18 A300–777A, and anti-Med24 A301–472A). The same blot was successively hybridized with different primary antibodies combinations at 1:1000, Med1-Med23-Med16-Med24, Med14, Cdk8-Med7 and Med18, respectively. Western blot was performed using an ECL kit (Amersham GE Healthcare Life Sciences) based on the manufacturer’s recommendations. HRP-conjugated secondary antibodies were used at 1:10,000 and were purchased from Amersham GE Healthcare Life Sciences. All the membranes were scanned on an ImageQuant LAS 500 (GE Healthcare Life Sciences).
Coordinates and structure factors for MED23 have been deposited in the protein data bank under accession 6H02. Other data are available from the corresponding authors upon request.
Singh, N. & Han, M. sur-2, a novel gene, functions late in the let-60 ras-mediated signaling pathway during Caenorhabditis elegans vulval induction. Genes Dev. 9, 2251–2265 (1995).
Boyer, T. G., Martin, M. E., Lees, E., Ricciardi, R. P. & Berk, A. J. Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein. Nature 399, 276–279 (1999).
Borggrefe, T. & Yue, X. Interactions between subunits of the Mediator complex with gene-specific transcription factors. Semin. Cell Dev. Biol. 22, 759–768 (2011).
Stevens, J. L. et al. Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit. Science 296, 755–758 (2002).
Yang, X. et al. Selective requirement for Mediator MED23 in Ras-active lung cancer. Proc. Natl. Acad. Sci. USA 109, E2813–E2822 (2012).
Mylona, A. et al. Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation. Science 354, 233–237 (2016).
Asada, S. et al. External control of Her2 expression and cancer cell growth by targeting a Ras-linked coactivator. Proc. Natl. Acad. Sci. USA 99, 12747–12752 (2002).
Griffiths, S. J. et al. A systematic analysis of host factors reveals a Med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication. PLoS Pathog. 9, e1003514 (2013).
Liu, Z. et al. Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development. Nat. Commun. 7, 11149 (2016).
Huang, Y. et al. Mediator complex regulates alternative mRNA processing via the MED23 subunit. Mol. Cell 45, 459–469 (2012).
Wang, W. et al. Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb. Transcription 4, 39–51 (2013).
Yao, X. et al. The Mediator subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination. EMBO J. 34, 2885–2902 (2015).
Imasaki, T. et al. Architecture of the Mediator head module. Nature 475, 240–243 (2011).
Larivière, L. et al. Structure of the Mediator head module. Nature 492, 448–451 (2012).
Nozawa, K., Schneider, T. R. & Cramer, P. Core Mediator structure at 3.4 Å extends model of transcription initiation complex. Nature 545, 248–251 (2017).
Plaschka, C., Nozawa, K. & Cramer, P. Mediator architecture and RNA polymerase II interaction. J. Mol. Biol. 428, 2569–2574 (2016).
Robinson, P. J., Bushnell, D. A., Trnka, M. J., Burlingame, A. L. & Kornberg, R. D. Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II. Proc. Natl. Acad. Sci. USA 109, 17931–17935 (2012).
Robinson, P. J. et al. Structure of a complete Mediator-RNA polymerase II pre-initiation complex. Cell 166, 1411–1422 (2016).
Robinson, P. J. et al. Molecular architecture of the yeast Mediator complex. eLife 4, e08719 (2015).
Tsai, K. L. et al. Subunit architecture and functional modular rearrangements of the transcriptional Mediator complex. Cell 157, 1430–1444 (2014).
Tsai, K. L. et al. Mediator structure and rearrangements required for holoenzyme formation. Nature 544, 196–201 (2017).
Jung, D. et al. Wrenchnolol derivative optimized for gene activation in cells. J. Am. Chem. Soc. 131, 4774–4782 (2009).
Asada, S., Choi, Y. & Uesugi, M. A gene-expression inhibitor that targets an alpha-helix-mediated protein interaction. J. Am. Chem. Soc. 125, 4992–4993 (2003).
Shimogawa, H. et al. A wrench-shaped synthetic molecule that modulates a transcription factor-coactivator interaction. J. Am. Chem. Soc. 126, 3461–3471 (2004).
Cevher, M. A. et al. Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit. Nat. Struct. Mol. Biol. 21, 1028–1034 (2014).
Plaschka, C. et al. Architecture of the RNA polymerase II-Mediator core initiation complex. Nature 518, 376–380 (2015).
Hashimoto, S. et al. MED23 mutation links intellectual disability to dysregulation of immediate early gene expression. Science 333, 1161–1163 (2011).
Pardon, E. et al. A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. 9, 674–693 (2014).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D Biol. Crystallogr. 59, 2023–2030 (2003).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
BUSTER v.2.10.2 (Global Phasing Ltd., Cambridge, UK, 2017).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Yu, J., Zhou, Y., Tanaka, I. & Yao, M. Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics 26, 46–52 (2010).
The PyMOL Molecular Graphics System v.2.0.6 (Schrödinger, LLC, New York, NY, 2018).
D.M., B.C., F.D., Z.L., A.V. and V.V. are researchers from the Centre National de la Recherche Scientifique in France (CNRS). This work was supported by the French National Research Agency (Grant ANR-DS0401-2016 MEDNET). We thank INSTRUCT, part of the European Strategy Forum on Research Infrastructures (ESFRI) and the Hercules Foundation Flanders for their support to the Nanobody discovery. We thank Nele Buys for the technical assistance during Nanobody discovery. We thank Ahmed Haouz from the Pasteur Institute in Paris for his help during crystallization trials. Access to synchrotrons has been achieved through the Beam Allocation Group (BAG) LOR. We thank Leonard Chavas, Serena Sirigu, William Shepard, and Martin Savko at beamlines Proxima-1 and Proxima-2 at the synchrotron SOLEIL and Carlo Petosa and Daniele de Sanctis at beamlines ID23 and ID29 at the European Synchrotron Radiation Facility, Grenoble for their assistance during data collection. B.C. and V.V. are indebted to the Research Federation FRABio (Univ. Lille, CNRS, FR 3688, FRABio, Biochimie Structurale et Fonctionnelle des Assemblages Biomoléculaires) for providing the scientific and technical environment conducive to achieving their contribution.
The authors declare no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Monté, D., Clantin, B., Dewitte, F. et al. Crystal structure of human Mediator subunit MED23. Nat Commun 9, 3389 (2018). https://doi.org/10.1038/s41467-018-05967-y
This article is cited by
Structure of mammalian Mediator complex reveals Tail module architecture and interaction with a conserved core
Nature Communications (2021)
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

domain: literature


text:
Maharashtra: 6K Bank Employees Staged Protest
Hyderabad, Feb.23 (Maxim News): Around six thousand employees of the bank of Maharashtra working in around 1900 branches of the country staged powerful demonstrations demanding adequate recruitment in all cadres like Part-time Sub staff, Sub staff and Clerks.
The agitation program was held under the aegis of All India Bank Employees Association (AIBEA). In a statement, the General Secretary of All India Bank of Maharashtra Employees Federation (AIBOMEF) Devidas Tuljapurkar said that the temporary part-time Sub staff were working in more than 1,100 branches and added that more than 600 branches of the bank had temporary Sub staff . He also said that Since last five years the bank had not filled up vacancies which fell vacant due to the death, retirement, resignation or promotion in clerical cadre and the vacancies arising out of branch expansion and increase in business. He said that as per their estimates, more than one thousand clerks were needed to be recruited to man the branches of the bank properly.
He alleged that the bank was resorting to outsourcing of jobs even in core activities while noting that it is inviting fraud risk by doing so in the process. He also alleged that the Bank management was taking all HR and IR decisions unilaterally by setting aside age-old practice of Bi-partisan despite the strong objection by the employees union.
He said that they were holding a Dharna on February 26th infront of 32 zonal offices besides holding another Dharna before Lokmangal Head Office of the bank on March 6th next month. He also said that they would observe a day long nationwide strike on March 12,2021.(Maxim News)

domain: news


This petition was submitted during the 2017–2019 Conservative government
Rejected petition Help to stop the Turkish aggression against Kurdish people in Rojava.
As the UK Government, there should be a public request for Turkish troops to withdraw from Syria. Further, physical support for the Kurdish people should be offered.
This petition was rejected
Why was this petition rejected?
There’s already a petition about this issue. We cannot accept a new petition when we already have one about a very similar issue.
You are more likely to get action on this issue if you sign and share a single petition.
We think this petition is similar:
We only reject petitions that don’t meet the petition standards.


text:
My metasequoia, in its fourth year from seed, lost its leader last fall. It’s trying to make a new one, but appears to have about six to ten contenders.
It frankly has the ugliest shape I have ever seen in a DR. I hope outgrows it.
I’d pick the best and straightest one and prune out the rest.

domain: hobby


text:
Tuesday, 26 February 2013
Pinks and Needles Cute Pins and Cake Toppers
has such cute wee pin toppers including ones for cakes, lovely wee shop!
If you want to see the new added items in the Strawberry Anarchy shop just
Post a Comment
Leave me a comment
Post Comments (Atom)


Meet Herpsilochmus stotzi, one of 15 new bird species recently found in the Amazon. An international team of researchers recently announced this of Amazonian birds previously unknown to science. The formal description of these birds has been printed in a special volume of the Handbook of the Birds of the World series.
EarthSky // Today's Image Release Date: Aug 30, 2013
This bird was unknown to science
Scientists say the discovery of this Herpsilochmus stotzi, and 14 other new Amazonian bird species, highlight how little we really know about species diversity in the Amazon.


This webinar held in September 2022 explored a digital diaries project which was part ofthe COVID-19 Social Science and Public Engagement Action Research (SPEAR) project. It looked at the experiences and impacts of COVID-19 for healthcare workers and vulnerable communities in South and Southeast Asia
In this article, Dr Adebolajo Adeyemo describes his community and public engagement project, supported by the DELTAS Africa CPE Seed Fund, in which he aimed to raise awareness of aminoglycoside-induced hearing loss through the creation of a Nollywood movie, in Nigeria.
This project from Botswana-Harvard AIDS Institute Partnership, supported by the DELTAS Africa CPE Seed Fund, used storytelling and film to explore perceptions and attitudes to HIV/TB in schools in Gaborone, Botswana
This paper describes an International Public Health Film Competition held in 2020 during the COVID-19 pandemic and shares learning from the process for future film festivals
“Right to Food Project” was implemented by the African Population and Health Research Center from 2018 to 2019 in urban Kenya. Different participatory visual methodologies were used to engage with the community members on the right to food. These included graffiti and wall murals, digital stories, photovoice, radio shows, human libraries, participatory educative theaters, open air events and community dialogue.
An international collaborative public engagement partnership using street art to engage local communities in Botswana, India and South Africa.
A film engages Nepalese communities about the health implications of child marriage
Mwangi Chege, participant of the 2017 Wellcome Trust workshop "It’s Complicated: navigating scientific complexity in public and community engagement" reflects on learning from the workshop. In particular, the Kenyan premiere of The Lucky Specials, a feature film designed as both entertainment and engagement with audiences about Multi-Drug Resistant Tuberculosis and the importance of drug adherence.


text:
This website is for sale! is your first and best source for all of the information you’re looking for. From general topics to more of what you would expect to find here, has it all. We hope you find what you are searching for!
Cool looking feather nail art design that is perfect for your summer escapades with friends. Backed with a plain white polish, your nails are then painted with a combination of blue, pink and violet hues. Simple but looks amazing!


text:
BASECAMP – Serial entrepreneur Seth Godin, the scheduled keynote speaker for Denver Startup Week’s kickoff event Monday, has one key piece of advice for those looking to start a business.
“They’re hiding. They’re looking for a reason why this is not the time for them to start something – that Denver’s not in the right frame,” Godin said in an interview Monday. “It’s completely untrue. This is our golden moment.”
The second annual Denver Startup Week will feature more than 100 events over six days, covering a variety of topics geared toward budding entrepreneurs. It is all free.
“This event is pretty rare,” Godin said. “It’s not just rare for me, it’s rare for the world.” Read more…


text:
Loopers stun BUT
Russell Maxwell and Stephen Payne defied the odds, turning into a two-man wrecking crew as Fast Loopers “B” pulled off a 3-1 victory over the Barbados Union of Teachers (BUT) on Tuesday night at the Table Tennis Centre, Nursery Drive, St Michael.
With only two players turning out for the Loopers, BUT would have been optimistic about their chances in the final set of matches in the first round of the Barbados Olympic Association’s Division 2 table tennis club competition.
Theodore Gill benefited from the limited roster with a walkover but none of his BUT teammates could cope with the youngsters on the table.
Maxwell was in particularly dominant mood, taking out Romel Carter 8-11, 11-2, 11-9, 11-8 and Keon Alleyne 11-7, 11-4, 11-7.
Payne gave excellent support, downing Alleyne 13-11, 11-5, 11-3. (PH)


text:
I don't understand the name but the elephant says it all.An upbeat song that I enjoyed The production was very clean which worked well with the instrumentation and song arrangement. I get the vibe that an elephant would enjoy listening to this while chilling out. I would agree with the pop label more than than rock, but maybe that's just me.
WATERFALL DANCEI am loving the eclectic mix of this musical composition. I am not a pro so I can't guess which instruments are played, however, their combination is magical like a Fairie or Nymph's song. I am guessing a Sitar and some unusual percussion instruments. All of Creation echos in these instruments as you portray the heartfelt love-lorn picture of the Song Writer & Singer watching the object of his imagined affection. I would prefer the song to be more celtic instead of electronica. However, altogether, a lovely piece. I found loudness of music made it difficult to hear the words of the 3rd verse where you "wonder if you would ever meet a girl in a waterfall." However, I love this song and would like to hear any future re-mixes.
Waterfall risesInteresting tune with a nice carefree melody and Scottish leanings. It needs some work though the vocals are to muddy and distant from the wrong type of reverb. The tempo is about 5 bpm to slow and needs to be changed out with a real drummer. The electronic drums are dehumanizing a song that has a lot of soul. It sound like the singer is trying to hide and disguise his voice and there is no need to do that. Let the listener hear your true tones
UniqueI really like the song very unique and creative song well composed and well put together keep on making great music hope this been helpful for you have a blessed day and may God bless you in all that you do.
Well-done songThe music is clear and balanced from this songwriter. The lyrics are hidden just enough to provide an feeling. This could be good for a soundtrack in a movie or foreign commercial. The vocals are perfect in the song.
asfut screenOld skool is my first impression. I love this melody and the background give it a holiday feel. I would love to play this live with a deeper bass on the down notes. This a 5 plus for this middle of the road type.
Indus Rush - Waterfall DanceAnother great track from Indus Rush who hail from Scotland. Wonderfully produced with glittering synths, sparkling electric rhythms and an emotive lead vocal. Waterfall Dance has some very interesting synth breaks that counter punch the songs basic melody and the totality is augmented with some great backing vocals. It reminds me of India, Indonesia and Caledonia all at once and neatly packaged into 3 minutes and fifteen seconds. Great song structure, an immediate sound and well worth a listen. Waterfall Dance is a great track.

domain: entertainment


text:
Using the Plant FinderOur Plant Finder is the simplest and fastest way to find your perfect plants. Let it do the work so you don't have to!
You select attributes and it immediately displays the plants that possess all of them. Want to see all the plants that grow in zone 7, bloom red or white, grow on slopes in poor soil and tolerate drought? No problem!
Resist choosing too many attributes (or you will run out of matching plants).
Read an overview of how it works or watch this video tutorial to learn how to use the Plant Finder to quickly locate the perfect plants.

domain: biology


text:
Beautiful sterling silver crystal ring with centre stone setting for ashes/hair
Really lovely sterling silver ring with generous 6mm centre setting for your ashes or hair/fur stone to be created and set into. Sparkling cz crystal shoulders. Up to a size 19mm. All colours available in crushed opals or pearl metallic solid colour, can add sparkles in desired colours also.

domain: artistic


text:
Since I need no reason to make a list, here goes my pledge for summer 2018!
The weather so far this summer has been unexpectedly great in Manchester so touch wood it continues because I love nothing more than a good old picnic in the park.
I’ve already had a few spur of the moment picnics so here’s me pledging to continue making the most of the opportunity to grab a blanket, snacks and entertainment when the sun is out. Maximum relaxation and zero planning? I’m a big fan.
Last summer I did this a few times in a few local bars, but this year I want to find something bigger and more organised; whether that is a drive-in-cinema or a larger park with bean bags and blankets. Or in a jacuzzi anyone?!
I’m annoyed at myself that I’m not doing anywhere enough reading at the moment. It’s not even a time thing; I’m just being lazy. Each summer I love to find an easy to read page turner that’s light and fun and usually set in a destination that I want to travel to. I still remember the summer I discovered the I heart series by Lindsey Kelk. It completely made me yearn to visit New York, and so I did a few years later (read my New York post here).
Staycation/explore the UK
A new one for me I want to actually try to maximise the time I spend in the UK over the next few months. Whilst I’ll definitely travel outside of the UK this summer, and lets face it I’ve already travelled a fair amount this year, I tend to spend the most time in the UK in autumn/winter when its coldest and then grumble at UK weather. This summer the weather has already been sunny & warm so I plan to make the most of my home country whilst it’s at its best. The UK has some amazing places. I want to explore and experience much more here over the summer so I’m thinking new cities, lakes and mini breaks. I welcome any recommendations please 🙂
Dining al fresco
I need no excuses for a BBQ but I want to dine al fresco as much as possible this summer. The weather is completely unpredictable making it hard to plan so in theory this will mean spontaneously buying disposable BBQs whenever possible ❤
One thing I’ve been doing differently this month is avoiding making too many plans. I’ve felt sort of strange but quite freeing when I’ve got to the weekend and have no idea what I’m going to be doing. I know it’s only mid month but I’m already noticing how relaxed I’ve felt. Although admittedly at times I’ve felt a little uncomfortable as it’s very alien to me to have an open diary, but I have to say it’s also been pleasant to wake up and think what do I want to do today? Last weekend I spontaneously went trampolining & had such a laugh & has made me want to have more time to be spontaneous. Sometimes I need to allow myself moments to get bored to become creative.
I love learning and want to try as many classes as possible. I’ve already tried a few new things this year, such as a Thai cooking class this winter, trampolining and bike rides and loved both planning new things as well as the spontaneity. Spontaneous living is becoming a bit of a theme of mine lately! I am now committing to a summer full of new experiences. My first new activity will be aerial yoga; I’ve signed up to a four week class starting at the end of the month. Other things I think I’d like to try: chocolate tasting, clay pigeon shooting, perfume making. Maybe some outdoor sports too. Do you any fun recommendations?!
I will update on how these things go 😎
What are your plans for the summer? Let me know!
Thanks for reading,

domain: geography


text:
Winter is coming
The first trip through Fridaywoods for a while sees major changes. All but the Evergreen Pines are all but leafless. A carpet of leaves lays inches thick. Some of the leaves have begun their decomposition. A squirrel scurries up a bare trunk to escape Stellas attention.
Coppicing the woodland
The summer’s coppicing or whatever it was has certainly opened up the woodland scrub but for why. Cattle, grazing land for the cattle that were escaping tgeur confines back in the summer.
The woods are deathly quiet, the only sound my footfall in the stones as we approach familiar ground, the crater. The hush continues as the only real sound now is some other dogs barking as they too explore the Bare and almost barren woodland.
It’s a real shame to see the woods being stripped out for more capitalism even if It is only for more cows to be able to graze. Is this also the true reason behind the alleged access issues to the woods. I’m not sure and as I’ve said before other equally irresponsible dog owners do us no favours
The thing that I don’t get with this, you’ve done the hard part you’ve picked up your dogs faeces so why not carry to a bin nearby in the local woods parking areas, is it because you dont want to spoil yiur cars seat covers, but then you’ll spoil it for the rest of us who want to walk our dog in Fridaywoods!
The thing with our local woods, especially in the summer, the rich biodiversity of the woods is amazing and according to an Essex wildlife trust employee I was talking to recently we now not only have the presence of Buzzards and Kites but two peregrine falcons, the fastest bird in the sky. Maybe if we do our bit they might breed.


text:
1. Why did the young man have a measuring line? What was the angel told to tell him?
THE prophet Zechariah, an individual used by Jehovah, had a vision of a man with a measuring line. The account, according to the second chapter of Zechariah, tells us that this young man with a measuring line in his hand went out to measure Jerusalem and to see how broad and how long it was. Just as the angel who was talking to the young man was going away, another angel came forth to meet him and said, “‘Run, speak to that young man yonder, saying, “Jerusalem shall be inhabited like unwalled villages, because of the mass of men and beasts within her.” And I will be unto her,’ is the oracle of the LORD, ‘a wall of fire round about her, and I will be the splendor within her.’” (Zech. 2:4, 5, AT) This young man was certainly interested in Jerusalem or he would not have taken time to find out how broad or how long it was.
2. Whom does the young man picture? Why is the city unwalled?
2 Today we find in the earth a group of Christian men and women who, like this young man, are sincerely interested in the Jerusalem which is above and which is the mother of us all. (Gal. 4:26, NW) They are really interested in God’s organization and in its expansion. Walls are not its boundaries. No, it is an expanding city like an unwalled village. It would be hard to take accurate measurements, because of its constant growth due to incoming crowds. The class of faithful Christians prefigured by this young man was quite a small organization during the years from 1914 to 1918, for then there was only a remnant of God’s faithful people who were truly examining the prophecies and trying to ascertain their meaning in order to find out what their work would be in this great organization of Jerusalem. They wanted its measure and they wanted to know more about God’s work.
3. How did the interest of worldly religionists differ from that of the “young man” class?
3 The world had gone to war. Nation had risen against nation and kingdom against kingdom. There was sickness and sorrow and pain, scattered from one end of the earth to the other, as foretold in Matthew, chapter 24. The nations and peoples of the earth were extremely selfish and interested only in getting the things they wanted. They had no time for God nor were they endeavoring to measure up to God’s principles of truth and righteousness. Even the religionists of the great Protestant and Catholic organizations, the Jewish and pagan religious orders, were involved in the affairs of the world, afterward devoting their attention to the League of Nations and the problems of this old, dying system of things. Forgotten was the statement in the Word of God: “Whoever, therefore, wants to be a friend of the world is constituting himself an enemy of God.” (Jas. 4:4, NW) No, the nations of the earth had no time to seek the kingdom of God as the only hope for mankind. This seeking was left to a remnant of God’s people, just a few faithful followers pictured by this young man in the vision of Zechariah. They were willing to use the measuring line in God’s organization.
4. How, following 1918, did expansion begin with the organization as with an unwalled city?
4 This city of Jerusalem was not to be like a walled city, having limited boundaries so no one could get in or out if the gates were closed; but it would be like an unwalled village without limits. It was to be a growing city because of the mass of men and of beasts that would come to inhabit it. It would continue to spread and reach out into new fields. This is just what is now happening to God’s organization. From 1918 onward this little remnant of God’s people persistently continued to proclaim the kingdom of God as at hand. The nations were not interested in that kingdom. They were opposed to it. But, on the other hand, thousands of persons of good will from all these nations hearkened to the message of God’s anointed and they came to God’s organization, Jerusalem, this expanding city, and sought advice and instruction within it. According to the Society’s own reports, back in the year 1918 there were only 3,868 persons who were proclaiming the message of the Kingdom to the peoples of the world, inviting them to seek refuge, comfort and consolation within Jerusalem. The Lord’s spirit was upon this little band of faithful servants as they steadily preached and reached out to the ends of the earth with this good news. It was not long until many people who were listening and studying with Jehovah’s witnesses in order to learn of Jehovah took their stand on his side. The city continued to grow.
5, 6. How did Isaiah 54:2, 3 go on fulfilling despite World War II? How did it become “one flock, one shepherd”?
5 As the nations of the world went into their second world war and brought about greater regimentation and totalitarian rule among the people, still this remnant with the people of good will kept right on preaching the Word. Today we see gathered into this unwalled city even greater numbers of those who Jehovah knows belong to him. Is it not now as it was stated by Isaiah the prophet centuries ago in Isa chapter 54, verses 2, 3, “Enlarge the site of your tent, and stretch without limit the curtains of your home; lengthen your cords, and make fast your pegs! For to right and to left shall you spread abroad, and your descendants shall take over the heritage of the nations, and shall people the desolate cities”? (AT) Jehovah’s witnesses see this expansion work going on. They live in the growing city.
6 With this great “mass of men” there is a multitude of cattle, and this may fitly represent the wealth of God’s organization. Christ Jesus said, ‘Other sheep I have, which are not of this fold; these also I must gather so that there will be one flock, one shepherd.’ Christ Jesus is the Good Shepherd of this great organization which he gathers together in its completion just before the battle of the great day of God Almighty, which is Armageddon.
7. Who will yet be found and ‘marked in the forehead’, and why?
7 Today, in 1951, we find the remnant of God’s people working alongside the great crowd who have been marked in their foreheads by the anointed class. The prophet Ezekiel (9:1-11) gave us a picture of the man with the writer’s inkhorn who went through the city and marked in the forehead those who were sighing and crying because of the abominations that existed in their city or organization. Certainly there are thousands, and it may yet run into the millions, of people who are sighing and crying because of the terrible conditions that exist in Christendom and throughout the whole world and who are seeking consolation, truth and comfort. These will be found; there is no doubt of that, because, as Paul said, “Jehovah knows those who belong to him.”
8. Why do those coming into the organization not fear because it is without material walls?
8 Jehovah God’s Good Shepherd, Christ Jesus, is doing the gathering work today, and he has assigned to his people in the earth the work of preaching the good news of the incoming new world. This good news attracts the meek to God’s Word, and that draws them closer to Jehovah. Having come to this unwalled village, trusting in God and having confidence in his Word, they are not afraid of what might happen to them. They are sure of protection. Was not God able to protect the congregation of Israel from being engulfed in the landslide that covered up the unfaithful opposition? Was not God able to preserve Noah and his family through the flood? So, then, is Jehovah God not able to preserve those who have come into this unwalled village? He certainly is! For according to the vision of Zechariah, “I will be . . . a wall of fire round about her, and I will be the splendor within her.” So, then, those who have come to the Lord’s organization, Jerusalem, recognize Jehovah as their God and Jerusalem as his organization and feel safe because God is their protector. They need not depend on the walls or bulwarks that nations of this world set up to protect themselves, because “Jehovah knows those who belong to him”.
9. Who is the glory in the midst of the organization, and why?
9 The glory of this entire organization, the Jerusalem which is above, his woman, is Jehovah himself, for he said, “I will be the glory in the midst of her.” In these last days of the old world, it is Jehovah God who has built up this organization and brought about Theocracy’s increase. Its growth and splendor are not due to any man or earthly organization. It is he who through his Son is gathering together the “other sheep” by the hundreds of thousands, making a great city. Jehovah gives it his blessing and protection. These people in the great city are not idle, but they continue to spread out and preach the message of God’s kingdom unto the ends of the earth so that all shall know that Jehovah is the Supreme One of the universe and that the vindication of his name and word is near at hand.
FLIGHT FROM GREAT BABYLON
10, 11. What call to his people in Babylon has gone forth? How has this been heeded, and by whom?
10 The call goes forth in the sixth verse of this prophecy of Zechariah, “Ho, ho! flee from the land of the north.” This is an oracle of the Lord or a direct command from him. “For as the four winds of the heavens have I spread you abroad . . . Ho! Zion, deliver thyself,—thou that dwellest with the daughter of Babylon. . . . For his own honour hath he sent me unto the nations that are spoiling you,—Surely he that toucheth you toucheth the pupil of mine eye.”—Zech. 2:6-8, AT, Ro.
11 It is due to God’s mercy and loving-kindness that he has gathered together the remnant from the nation of the north, which is Babylon. He elsewhere stated, ‘Come out of her my people, and be not partakers of her plagues, and do not be contaminated with her sins.’ (Rev. 18:4) He has seen to it that the remnant of his people, Zion, escape from this wicked Devil-ruled world to a place of safety in his city, along with his “other sheep” who are now coming out. The rulers of the world have tried to regiment all peoples of the nations and make them keep in line with their selfish dictates. However, God commands that they shall escape from Babylon and become a free people—if not in body, then in conscience; and they do! They come out from under its oppression and servitude and sinful way of life. No longer are they slaves to that wicked organization. They will render to Caesar the things that are his, but not what belongs to God! Their eyes are opened to a new world and a New World society. “For such freedom Christ set us free. Therefore stand fast, and do not let yourselves be confined again in a yoke of slavery.”—Gal. 5:1, NW.
12. How are they satisfied not with just freedom for themselves?
12 Those who have already been freed from the oppression of Babylon, even though in some cases they are still in jails, work camps or concentration camps, are not satisfied just to hold this freedom to themselves. At every opportunity they will talk to strangers, even though physically still captive in Babylon. They will help strangers to see clearly the way of life by directing them away from this old system and into a new which leads to life eternal. Paul said, at Hebrews 13:1: “Let your brotherly love continue. Do not forget kindness to strangers.”—NW.
13. How does it affect Jehovah for persecutors to touch his people? But why does he permit it?
13 Those who renounce unrighteousness and take their stand wholly on Jehovah’s side, he will look after as his very own because “Jehovah knows those who belong to him”. If anyone touches these that he has chosen and blessed, it is like touching the pupil of his eye. Jehovah’s organization, the free Jerusalem, is our mother and is the closest thing to the Lord. He has created it and made it for his praise. It will be just too bad for the individuals or the nations that fight against God or his witnesses. These belong to him and in due time he will execute judgment against opposers. This does not mean that Jehovah will not allow individuals or part of his organization to suffer hurt. He will; in order that the individuals involved therein may prove their integrity and faithfulness. However, he will take cognizance of the offenders, and at the same time advises us, “If possible, as far as it depends upon you, be peaceable with all men. Do not avenge yourselves, beloved, but yield place to the wrath; for it is written: ‘Vengeance is mine, I will repay, says Jehovah.’”—Rom. 12:18, 19, NW.
14. How did he show foreknowledge of how we would be treated? Despite this what gathering work goes on?
14 Jehovah is watching what is going on throughout his entire organization. He knows how his people will be treated. “Moreover, you will be delivered up even by parents and brothers and relatives and friends, and they will put some of you to death, and you will be objects of hatred by all persons because of my name. And yet not a hair of your heads will by any means perish.” (Luke 21:16-18, NW) Even so, a greater witness than ever in vindication of Jehovah’s name will be given. We are in the last days of this old world and we must expect these things. Now is the time to gather together a great crowd for his name’s sake, and this crowd is being gathered together from the ends of the earth with haste. So a sharp call and a loud one goes out unto the ends of the earth to all the inhabiters of Babylon who are sighing and crying to flee from her and escape from her oppressive acts now and her complete destruction later. If you love the Lord, then seek his protection and come to his city, the unwalled village, for Jehovah is a wall of fire round about it. Those in it he loves, and to touch one of his faithful servants is like touching the pupil of his eye.
15. What will he do shortly to the nations? So what are we commanded to do?
15 The time is not very far distant when Jehovah God will plunder the nations and destroy them. When Jesus was asked what the sign of the end of this world would be, he gave his apostles many things that they could watch for, and these we see in fulfillment today. It will not be a long time, therefore, until Armageddon. Those who have come to know the Lord can do as commanded: “‘Shout with joy, and rejoice, O daughter of Zion! For lo, I am coming to dwell in the midst of you,’ is the oracle of the LORD. Many nations will attach themselves to the LORD on that day; and they will become his people, and he will dwell in the midst of you; and you will know that the LORD of hosts has sent me unto you. And the LORD will take possession of Judah.” (Zech. 2:10-12, AT) Do you believe it? Will you tell others what you know? Will you show patience and endurance while telling them?
16. In what direction will we help individuals to make up their minds? Why, and how?
16 It is a hard thing for an individual to make up his mind as to what he wants to do: stay with this old, dying world with its wealth, pleasures and present popularity, or, as he puts it, “take a chance” on the new world. Will you who know God and who are acquainted with his Word show your faith by good works? When one studies and knows the Word of God and believes that Word, it is not a matter of taking a chance. It is a matter of being sure what the outcome will be. If you are sure, you will try to convince others who are ready to pull away from Babylon. If one flees from the old world and from the nations that are spoiling him, just what will the outcome be? Primarily it means the individual will be in line to receive everlasting life. He will have the opportunity of worshiping the Most High God, his Creator, for eternity. But in order to receive the blessings of this life he must accept the Son Christ Jesus; for the scripture says whosoever believes on Him will be able to get everlasting life. Are you, as a Christian, going to help him learn these things? It will not be easy; it will take patience and time on the part of both.
17, 18. What is the attitude of the world in which we live? Yet for what must we take a stand, and in imitation of whom?
17 All of us must remember we are living in an old world, the world that is opposed to the establishment of God’s kingdom. It has been opposed to the Kingdom for centuries. Even when the King Christ Jesus was upon the earth and did his wonderful preaching, the nations of the world saw to it that he was put to death and destroyed by being staked. He died as a condemned sinner, as a seditionist, a man against the Roman rule of Caesar and a man hated by the very religious Jews who were seeking the Messiah. He was one who went through great persecution. It was not easy for the King Christ Jesus to live in this old world. He knew they were out to kill him, and they finally did, but that did not scare him. He had his work to do and he did it to become the greatest witness of all time in favor of the name and word of Almighty God.
18 The faithful followers of Christ Jesus must take a similar course. There are no two ways about it. They must be for Jehovah’s kingdom. They no longer want to be spoiled by the nations and come under their subjection; so they flee this old world of Babylon with its devilish rule and place themselves under the rulership of the King of heaven, Christ Jesus, who they know now rules in the heavens. He was placed upon his throne by Jehovah God and made ruler in 1914.
A GREAT CROWD ANNOUNCING THE KINGDOM
19. In fulfillment of Revelation 19:6 what is a great crowd now saying, and why?
19 What a marvelous thing has happened! Today we find a great crowd of people who have listened to the message of the Kingdom and who are saying, “Praise Jah, you people, because Jehovah our God, the Almighty, has begun to rule as king.” (Rev. 19:6, NW) Yes, the time has come for the vindication of Jehovah’s name, and those who have fled from the Babylonish organization recognize that Jehovah has become king by placing his Son upon the throne to rule in the midst of his enemies. This is a day in which to rejoice.
20, 21. What was the effect of the miracles done by the apostles and their fellows? How were they even misunderstood and resented?
20 In the early days of the church Jehovah God through his holy spirit or active force gave power to the apostles to perform miracles, and this attracted many people to the message and to what the apostles were saying. However, these special or miraculous gifts came to an end with the death of the apostles, and we do not see them in operation at the present time.
21 You will remember the account in Acts 14 where the apostles Paul and Barnabas were preaching the good news and they had performed a miracle. After seeing the marvels done by these men, the people wanted to make them gods; for they thought that the gods of heaven had come down to the earth and had become men. “However, when the apostles Barnabas and Paul heard of it, they ripped their outer garments and leaped out into the crowd, crying out and saying: ‘Men, why are you doing these things? We also are human creatures having the same infirmities as you do, and are declaring the good news to you, for you to turn from these vain things to the living God, who made the heaven and the earth and the sea and all the things in them.” (Acts 14:14, 15, NW) Paul and Barnabas did not take credit for doing this miracle themselves. They knew it was done by the power of God, so they gave all the honor to him. They told those surrounding them that the one who backed them up in doing this miracle was the Creator of heaven and earth, and as for themselves they were just mere ordinary human creatures. Their whole purpose in preaching and performing the miracle was to turn the people toward the only true God and for nothing else. They wanted to show the people that their heathen gods in the temples where the Gentiles offered sacrifices were not living gods worthy of worship. It was the truth from God’s Word that they were contrasting with false religion. Even though sincere and honest and trying to do the people good, they were greatly persecuted and hounded by them at other places on their travels because of miracles they performed and especially because of the message they preached. Nevertheless, the truth found a sure foothold in those early days.
22. What about such miracles today? What do we undergo from the nations, and yet what are we showing endurance to do?
22 So it is today with the ministers of the gospel. The good news is still being preached, and while they do not have the power to perform miracles, they do have from God the power and the commission to preach this gospel unto the ends of the world. The Lord’s people have been delivered from the Babylonish organization and from the nations that spoiled them. Because they fled from these organizations they are tormented by Babylonians. Oh, yes, they are still in the world, but they are no longer a part of it. They have come to the unwalled city under Jehovah’s protection and they know that “Jehovah knows those who belong to him”. The faithful followers of Christ Jesus, regardless of oppression, will pursue righteousness and godly devotion. They will stand up against all the opposition that is brought to bear against them, because they know that even as Christ and the apostles were persecuted and were permitted to go through many trials and tribulations; so even today they must do likewise, and it is only by endurance that they will be able to gain the victory. It is the aim of Jehovah’s witnesses to help the people; therefore they cannot give up.
23. With what qualities must we endure in preaching work? Why?
23 When Paul wrote to Timothy, he pointed out that we must have faith, love and endurance and must continue in our preaching work with mildness of temper. We are not to get angry and rant and rave as the peoples of the nations of this old world do. We are not to act as their chief representatives do in the conferences of the United Nations. The loud, abusive talk at election time is typical of their lack of love for one another. Christians cannot share in that kind of speech. It is unbecoming. Rather, with a steady, faithful love and devotion they must show their endurance with a mild temper. This kind of disposition they will be able to use when talking to the people in the world, and they will be able to persuade all kinds of people to listen to the message that they have. They may not be proud or haughty, nor do they feel that they are better than anyone else. In order to contend for the victory in this contest of faith, they must preach the Word in a manner pleasing to God and in a manner that will attract the people who are seeking after truth and righteousness. Is it not the desire of God to see all kinds of men saved? and do not Jehovah’s witnesses feel that way about it, too?
24. How are we making a right confession world-wide, and with what results since 1945?
24 Paul said, “Contend for victory in the right contest of the faith, get a firm hold on the everlasting life for which you were called and you declared the right confession publicly before many witnesses.” (1 Tim. 6:12, NW) So then, in order for a Christian to contend for the victory in the right contest of the faith, he must make a public confession and tell all kinds of people what he believes. Jehovah’s witnesses world-wide are doing just that, and the results in the past six years show the great blessing of Jehovah. Below is set out the number of Kingdom publishers or ministers who have been proclaiming the message of the Kingdom every month throughout the years mentioned. The last figure shows the increase in number of publishers over the previous year.
Publishers 1945, 127,478; increase over 1944, 16,973.
Publishers 1946, 158,034; increase over 1945, 30,556.
Publishers 1947, 181,071; increase over 1946, 23,037.
Publishers 1948, 230,532; increase over 1947, 49,461.
Publishers 1949, 279,421; increase over 1948, 48,889.
Publishers 1950, 328,572; increase over 1949, 49,151.
25. So what increase in ministers has there been yearly? How are they contending for victory?
25 It is noted that during the last three years there has been an increase of approximately 49,000 ministers preaching the Word every year. In other words, 49,000 persons have been coming out of the old world each year, taking a definite stand for God’s kingdom and making proclamation of this stand publicly before many witnesses. In this way they are contending for the victory. While they have had many trials and difficulties, “Jehovah knows those who belong to him,” and he who touches one of these witnesses touches the pupil of his eye. Jehovah loves their faithfulness, and he gives the increase! Are you having a share in it?


text:
The Department for Transport revealed that walking trips have decreased 19% between 2005 and 2015. They also disclosed that only two fifths of adults walk for travel at least once a week. Walking is a simple way to become more active and healthier. If the distance between your home and workplace isn’t too far. Then why not walk to work. Here are the benefits of walking to work:
Provides daily exercise
The NHS recommend that adults aged 19-64 should try to do at least 150 minutes of moderate aerobic activity. For example, cycling or brisk walking every week. Walking briskly can help to build stamina, burn excess calories and make your heart healthier. If you aren’t an overly active person walking is a great way to get started.
Improves your wellbeing
A study from the University of East Anglia and Centre for Diet and Activity Research (CEDAR) found that walking or cycling to work is better for people’s mental health than driving. It highlighted that active commuters are better able to concentrate and are under less stress than if they were to have traveled by car.
Opens your eyes
Walking will give you the opportunity to notice your surroundings more and admire the beauty around you. We can get caught up in everyday life and forget to stop to notice the little things, walking to and from work can make you appreciate everything that little bit more.
Decreases risk of chronic conditions
The benefits of walking to work include lowering the risk of diabetes, high blood pressure and heart disease. It can also help with joint pain and The American Center for Disease Control and Prevention recommends walking to improve arthritis pain, fatigue, function and quality of life.
Maintains a healthy weight
Walking can help you to stay in shape as it increases the metabolism by burning extra calories and preventing muscle loss. You may feel worn out the first couple of times you walk to/from work, but that will soon fade and you won’t even notice it.

domain: medical


text:
Benefits of Iberian pig meat Abrasador
Today we'll talk about the benefits you the Scorching Iberian pig, on its nutritional qualities, proven in scientific studies we have done on our own pigs.
Our priority is that what you provide is healthy, without forgetting that the taste is always a priority for you and the Iberian you get it insured.
The Scorching Iberian pig It has a high protein content of high biological value, Around a 23% by weight edible portion, also it contains a good dose of vitamins, highlighting thiamine, vitamin B6 and B12, niacin and riboflavin, all of which are necessary to achieve growth and muscle regeneration. The contribution of minerals such as iron, zinc, match, magnesium and potassium to promote hydration and maintain electrolyte balance.
Its content in polyunsaturated fatty acids and monounsaturated, It helps balance cholesterol levels, improves cardiovascular health and prevents increased blood pressure. These healthy fats also contribute to the own synthesis of lipids as, therefore you do not have to avoid them if you intend to keep your line.
Remember the keys to a healthy life, include daily fruits and vegetables in your diet and accompany of moderate physical activity.
Here are these five tips Iberian pork bait Scorching field

domain: food


text:
The Kapil Sharma Show
starts with a small skit wherein Bhuri (Sumona Chakravarti
) runs outside and tells Chandan that thieves have come to her house. She calls Inspector Shamsher and tells him to help her out. Shamsher tells Chandan that the makers of Super Dancer had called him. He cracks a joke on Shilpa Shetty
and Archana Puran Singh
Bhuri tells Shamsher that the thieves have not stolen anything yet. Shamsher feels proud at him and says that cop arrived before the incident took place. He tells Chandan to call American President, Barack Obama. Chandan tells Shamsher that Obama is not the president of America anymore. Shamsher asks him whether PM Narendra Modi
got elected in America also. The audience thoroughly enjoys the skit. Later Sapna (Krushna Abhishek) joins them and wins hearts with her amazing performance.
Comedian Kapil welcomes popular Bollywood stars Salman Khan
and Katrina Kaif on his show. Kapil asks Katrina if she talks to food. The gorgeous actress confesses she does, specially to the items she cannot eat due to her diet. Archana tells everyone that Katrina had refused to eat on the sets of a movie as she had diet restrictions. Archana added that Katrina refused to drink mango smoothie. Salman tells her Katrina rejected him also.
Kapil asks Salman what kind of dreams he gets while he is sleeping. Salman describes a few. He tells Kapil that he repeatedly gets a particular dream since his childhood wherein he is falling from a mountain or he is running behind someone.
Salman tells Kapil that he used to wear his father Salim Khan
's flip-flops. He said that once he had to step out of the house and he wore the first pair of slippers that he saw. They were his father's. When Salman returned home, he got scolded for the same.
Sapna comes on the stage and starts dancing on Katrina's popular item song ‘Chikni Chameli’. Sapna starts her show ‘Sapna key ajoobey’ and calls a participant on stage. He starts showcasing his talent in calligraphy. Sapna calls another contestant and tells him to enact Salman's dialogue from ‘Dabangg’. Next Sapna calls Lalli (Bharti Singh
) on stage who is dressed as actress Raakhi from a popular movie.
Later, Bharti calls Baccha Yadav (Kiku Sharda
) on stage. Kapil sings songs and both Baccha Yadav and Titli perform on it.


text:
L'ABUS D'ALCOOL EST DANGEREUX POUR LA SANTÉ. À CONSOMMER AVEC MODÉRATION.
LYLEXOX X MAC COSMETICS EVENT
M.A.C invited Lyle Reimer, the multimedia makeup artist, widely adored on Instagram as Lyle XOX for an afternoon of wildly … Read more
12TH EDITION OF THE HEINEKEN – TALENT BIEROLOGY COMPETITION
On the 11 of May, Heineken revealed the winners of the 12th edition of the Heineken – Talent Bierology Competition… Read more
Mizuno 1906 X Shinzo Lab
On Wednesday, February 23rd, Mizuno, the famous japonese brand of sneakers was hosting at Shinzo Lab, the launch of it … Read more


text:
Retail shares ended a two-day winning streak with a 2 percent decline Monday as a group of Citi analysts forecast a tough back-to-school season, although one with some potential upside.
Comparable-store sales at broadline retailers are forecast to fall 3 to 4 percent for August and September combined — a significant drop from the 0.9 percent b-t-s rise last year and the 10-year average increase of 4.3 percent, said Citi analyst Deborah Weinswig on a conference call with fellow Citi analysts Kimberly Greenberger and Kate McShane.
Hermès is launching a Laundromat pop-up shop in NYC - dubbed Hermèsmatic - where customers can bring their old scarves to be dip-dyed by an expert. Get all the details on WWD.com. #wwdnews (📷: @donstahl)


Random thought while reading about THAAD
seojoonho001 last edited by
I have not seen any missiles like SCUD/Lo Dong/ and other short range Ground to ground missiles fired/launched in campaigns.
Is this hard to model in BMS? I was just fantasizing how cool it would be if I saw surface to surface missiles launched or hitting cities/airbases. Even better, if we saw Patriots intercepting those missiles….
Wouldn’t that be cool? Or would it be too much of eyecandies/FPS destroyer? Maybe it’s too hard to code?


text:
Based on real events, this true crime novel tells the story of Mauricia Hernandez, who scandalized Guatemala in 1939 by poisoning her unloving husband. It is both the story of a woman lashing out at an unforgiving macho society and an exploration of life in Guatemala in the 1930s and 1940s, weaving the story of one woman together with the lost stories of thousands of other peasant women who, like her, faced challenges and unhappy realities in their homes, at their churches, and under their governments.
Related collections and offers
|Product dimensions:||5.50(w) x 8.20(h) x 0.80(d)|
About the Author
Oswaldo Salazar teaches philosophy and literature at the Francisco Marroquín University in Guatemala and is the author of several articles on Guatemalan indigenous policy and history.


text:
Profile: Ningbo Sanxing Medical Electric Co Ltd (601567.SS)
601567.SS on Shanghai Stock Exchange
Change (% chg)
Ningbo Sanxing Medical Electric Co.,Ltd., formerly Ningbo Sanxing Electric Co., Ltd., is principally engaged in the research, development, manufacture and distribution of electric power meters and terminals. The Company primarily provides smart power meters, electronic power meters, induction power meters, terminals, transformers and switch cabinets, among others. The Company is also involved in provision of leaseback service. The Company distributes its products within domestic market and to overseas markets.
Ningbo Sanxing Medical Electric Co Ltd
Yinzhou Industrial Park Zone
Jiangshan Town, Yinzhou District
NINGBO ZHJ 315191

domain: economics


text:
Grab your mason jars, mustache comb, and ironic glasses, it’s time to rank the most hipster movies of all time! As any self-respecting hipster knows, liking anything, even a great hipster movie, is inherently uncool. That’s what makes figuring out which of these hipster films is the best especially difficult. Some may be considered among the best movies of all time, but since a real hipster could never like anything someone else does (even another hipster), it’s up to you to decide which one should be at the top.
From Lost in Translation to Garden State to anything made by Wes Anderson (Rushmore, Bottle Rocket, Royal Tenenbaums... the hipster movie list goes on), these great hipster films are fine pieces of movie making that just happen to feature characters who probably wear flannel while listening to Arcade Fire on vinyl. This list of the best hipster movies includes films that are either beloved by hipsters, or feature a hipster as one of the central characters. If your favorite hipster movie isn't on this hipster movies list, make sure to add it to the list yourself.
Despite stereotypes, the hipster subculture is about more than vintage bicycles and ironic T-shirts. They value progressive, independent thinking, and counter-culture tastes. They tend to lean towards androgyny, because classic male/female imagery is considered too mainstream and even offensive. In many ways, they are modern beatniks. If you consider yourself a hipster, then one of these films may well be your favorite movie of all time.If you want more hipster action, check out the lamest celebrity hipster couples, the best jobs for hipsters, essential brands for your hipster lifestyle and the most hipster bands (warning: there are many).
The Big Lebowski is a 1998 stoner crime comedy film written, produced, and directed by Joel and Ethan Coen. It stars Jeff Bridges as Jeffrey "The Dude" Lebowski, an unemployed Los Angeles slacker and avid bowler. After he becomes the victim in a case of mistaken identity, The Dude finds a ...more
Actors: Julianne Moore, Jeff Bridges, Tara Reid, John Goodman, Steve Buscemi, + more
Initial Release: 1998
Directed by: Ethan Coen, Joel Coensee more on The Big Lebowski
Donnie Darko is a 2001 American supernatural drama film written and directed by Richard Kelly and starring Jake Gyllenhaal, Drew Barrymore, Patrick Swayze, Maggie Gyllenhaal, Noah Wyle, Jena Malone, and Mary McDonnell. The film depicts the adventures of the title character as he seeks the meaning ...more
Actors: Drew Barrymore, Ashley Tisdale, Jake Gyllenhaal, Maggie Gyllenhaal, Seth Rogen, + more
Initial Release: 2001
Directed by: Richard Kellysee more on Donnie Darko
Mark Holmes added Fight Club
Fight Club is a 1999 film based on the 1996 novel of the same name by Chuck Palahniuk. The film was directed by David Fincher, and stars Brad Pitt, Edward Norton and Helena Bonham Carter. Norton plays the unnamed protagonist, an "everyman" who is discontented with his white-collar job. He forms a ...more
Actors: Brad Pitt, Jared Leto, Helena Bonham Carter, Edward Norton, Meat Loaf, + more
Initial Release: 1999
Directed by: David Finchersee more on Fight Club
life20191 added Eternal Sunshine of the Spotless Mind
Eternal Sunshine of the Spotless Mind is a 2004 American romantic science-fiction comedy-drama film about an estranged couple who have each other erased from their memories, written by Charlie Kaufman and directed by Michel Gondry. The film uses elements of science fiction, psychological thriller, ...more
Actors: Kate Winslet, Kirsten Dunst, Jim Carrey, Mark Ruffalo, Elijah Wood, + more
Initial Release: 2004
Directed by: Michel Gondrysee more on Eternal Sunshine of the Spotless Mind