ICAT (Inhibitor of β-CAtenin and TCF) is a small acidic protein that negatively regulates β-catenin co-transcriptional activity by competing with TCF/LEF factors in their binding to β-catenin superhelical core. In melanoma cells, ICAT competes with LEF1 to negatively regulate the M-MITF and NEDD9 target genes. The structure of ICAT consists of two domains: the 3-helix bundle N-terminal domain binds to β-catenin Armadillo (Arm) repeats 10–12 and the C-terminal tail binds to Arm repeats 5–9. To elucidate the structural mechanisms governing ICAT/β-catenin interactions in melanoma cells, three ICAT residues Y15, K19 and V22 in the N-terminal domain, contacting hydrophobic β-catenin residue F660, were mutated and interaction was assessed by immunoprecipitation. Despite the moderate hydrophobicity of the contact, its removal completely abolished the interaction. In the ICAT C-terminal tail consensus sequence, neutralization of the electrostatic interactions between residues D66, E75 and β-catenin residues K435, K312, coupled to deletion of the hydrophobic contact between F71 and β-catenin R386, markedly reduced, but failed to abolish the ICAT-mediated negative regulation of M-MITF and NEDD9 promoters. We conclude that in melanoma cells, anchoring of ICAT N-terminal domain to β-catenin through the hook made by residue F660, trapped in the pincers formed by ICAT residues Y15 and V22, is crucial for stabilizing the ICAT/β-catenin complex. This is a prerequisite for binding of the consensus peptide to Arm repeats 5–9 and competition with LEF1. Differences between ICAT and LEF1 in their affinity for β-catenin may rely on the absence in ICAT of hydrophilic residues between D66 and F71.
Copyright: © 2017 Domingues et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by a grant from "La Fondation ARC contre le Cancer", Ligue Nationale contre le Cancer, INCa, Pair Melanoma and Cancéropole IdF, and is under the program «Investissements d'Avenir» launched by the French Government and implemented by ANR Labex CelTisPhyBio (ANR-11-LBX-0038 and ANR-10-IDEX-0001-02 PSL). M.J. Domingues was the recipient of a fellowship from the Ministère de l'Education Nationale de la Recherche et de la Technologie (MENRT) and J. Martinez-Sanz was financed by the French program "Investissements d'Avenir – Institut Carnot" managed by the National Research Agency (ANR-11-CARN-008-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The canonical Wnt/β-catenin signaling pathway is involved in multiple normal and pathological biological processes. This pathway is frequently altered in diverse cancers including carcinoma, pseudopapillary tumors and melanoma [1–4]. In melanoma, two of the main targets of the Wnt/β-catenin pathway are the promoters of M-MITF and NEDD9 genes. The melanocyte specific isoform of the transcriptional factor M-MITF (MIcrophtalmia-associated Transcription Factor) [1, 5] is essential for melanogenesis, and involved in melanoma formation and progression [6, 7]. Variable M-MITF expression has been reported in melanoma cell lines, often correlating with their malignancy [8, 9]. NEDD9 (Neural precursor cell Expressed Developmentally Down-regulated 9) is an adaptor protein transducing signals and playing a key role in cell proliferation and migration. NEDD9 overexpression in human metastatic melanoma is frequent  and the transcriptional activation of the gene was recently found to be β-catenin-dependent [11, 12].
In the presence of a Wnt signal, the multifunctional protein, β-catenin, is translocated to the cell nucleus where it interacts with TCF/LEF transcription factors, displacing the Groucho repressor  and activating multiple target genes [2, 14, 15]. Both positive and negative regulators of the Wnt/β-catenin signaling pathways have been identified including Cadherins, TCFs, Adenomatous Polyposis Coli (APC), Axin and the transcriptional inhibitor ICAT. This highly conserved small protein of 81 amino-acids is encoded by the vertebrate-specific CTNNBIP1 gene . ICAT transcripts are ubiquitously expressed during embryonic development . At the cellular level, ICAT localizes mostly in the cytoplasm but also in the nuclei of normal and cancer cells, sign of a dynamic distribution between the two compartments [12,18]. In the nuclear compartment of colorectal cancer cells, ICAT competes with TCF4 (now referred to as TCF7L2) for binding to β-catenin, thus negatively regulating its co-transcriptional activity . In a panel of human melanoma cell lines we have recently demonstrated variable ICAT expression. High levels of ICAT transcripts were found in metastatic cells with a strong motility, while non-metastatic cells expressed low ICAT mRNA amounts and migrated poorly . Differences in cell migration were ascribed to ICAT-dependent negative regulation of NEDD9 expression. Investigating how ICAT is able to modulate M-MITF and NEDD9 expression in melanoma cells should help clarifying the mechanisms of interaction between ICAT and β-catenin.
β-catenin functions as a scaffold for multiprotein assemblies . It is a 781 amino-acid protein with a central core region composed of 12 Armadillo (Arm) repeats, each one (except repeat 7) consisting of three α helices. Arm repeats 5–9 form a positively charged groove involved in the binding of several ligands . The crystal structure of the full-length zebrafish β-catenin  has allowed the identification of an additional α helix C which docks onto the third helix of Arm repeat 12 through three highly conserved leucine residues (S1 Fig). This helix C has been suggested to mediate the interaction with ICAT , however, its precise role is still unknown since its deletion in cultured cells did not affect β-catenin turnover .
Crystal structures of the ICAT/β-catenin complex at 2.5Å (PDB: 1LUJ, ) and 2.1Å resolution (PDB: 1M1E, ) have shown that the two proteins interact in an anti-parallel orientation (Fig 1A). Two main regions in ICAT have been identified: i) the N-terminal domain consists of a bundle of three α helices, with helix 1 (H1) directly interacting with Arm repeats 10–12 of β-catenin; ii) the C-terminal domain is intrinsically unstructured but adopts a β-strand like structure when bound to β-catenin Arm repeats 5–9. The latter domain contains a β-catenin peptide-binding motif DXθθXΦX2-7E, where θ is an aliphatic hydrophobic amino acid, Φ an aromatic residue (mainly Phenylalanine) and X2-7, two to seven variable residues (Fig 1B). This consensus motif is present in several β-catenin ligands including E-cadherin, APC, Axin and members of the TCF/LEF family, to mediate their interaction with the groove of β-catenin [24–26]. Therefore, the competition between ICAT and these proteins likely occurs in this region. Slight variations in the consensus sequence may account for differences in ligand binding affinities , although the exact contribution of residues D, Φ and E in conferring binding energy to the groove remains debated. Indeed, in TCF7L2, residue F21 was found to be dispensable in one study  or essential in another one  for its binding to β-catenin. Additionally, the disruption of the salt bridge between residue E24 in TCF7L2 and K312 in β-catenin, only mildly affected the affinity . Therefore, the contribution of these residues to the interaction of ICAT with β-catenin needs clarification.
Fig 1. Crystal structure of the β-catenin/ICAT complex.
A Crystal structure of ICAT bound to the core domain of β-catenin (PDB code 1LUJ,). ICAT is shown as yellow ribbons and β-catenin as purple cylinders. The secondary structures were calculated using the program STRIDE . Residues mutated in this study are shown as hard spheres. ICAT residues are colored according to their characteristics: white for hydrophobic, green for polar, red for acidic and blue for basic residues. β-catenin F660 is in pink and the basic residues facing the C-terminal domain of ICAT are in cyan. B. Sequence alignment of the consensus peptide from several β-catenin binding proteins. The conserved acidic residues are in red and the aromatic residue in green. The first X residues, when they are hydrophilic, are boxed. C. β-catenin/ICAT complex showing the interaction between ICAT consensus peptide of the C-terminal domain (ribbon and sticks) and its facing β-catenin residues (surface). All residues are colored according to their characteristics. Figures were drawn using VMD software .
Based on the crystal structure of the β-catenin/ICAT complex [22, 23] several contacts between the ICAT N-terminal domain and β-catenin were assumed to stabilize the two proteins interaction. This included the hydrophobic contacts between ICAT residues Y15, K19 and V22 and β-catenin F660. However, evaluation of their relative contribution is required.
To investigate the importance of the highlighted residues for the ICAT/β-catenin interaction in melanoma cells, point mutations were introduced in ICAT and β-catenin by site-directed mutagenesis. We selected six highly conserved ICAT residues, three being located in the N-terminal domain, and three in the C-terminal domain consensus peptide (Fig 1 and S2 Fig). We also mutated their facing residues in β-catenin. The consequences of these mutations on the ICAT/β-catenin complex and the transcriptional regulation of M-MITF and NEDD9 promoters were studied in two melanoma cell lines expressing opposite amounts of ICAT and β-catenin.
The human melanoma cell lines Lu1205 and Mel501, obtained respectively from Dr M. Herlyn (Wistar Institute, Philadelphia, PA, USA) and C. Goding (Ludwig Institute, Oxford UK) were maintained in RPMI 1640 medium (Gibco) supplemented with 10% foetal bovine serum (Sigma), 5 mM L-glutamine, 100 U/mL penicillin and 100μg/mL streptomycin. All cells were grown at 37°C in a humidified atmosphere containing 5% carbon dioxide.
The human ICAT cDNA (243bp) cloned in the pFLAG-CMV2 expression vector was kindly provided by Dr. C. Gottardi (University of Chicago IL, USA). The expression of ICAT being under the control of the CMV promoter, the vector is referred to as CMV::ICAT WT in the manuscript. A triple mutant form of ICAT in helix 1 (ICAT-DQE mutant) derived from the pFLAG-ICAT-CMV2 construct was created with primers shown in S1 Table. The forward primer LL1806 containing three nucleotide changes (shown in red) when compared to the wild-type sequence was coupled with the reverse primer LL1521. A 186 bp PCR fragment was generated and restriction digested with BspEI and BlpI. This 128 bp sub-fragment was exchanged with the equivalent wild-type fragment isolated from the pFLAG-ICAT-CMV2 vector. Single (D66G, F71A, E75V), double (D66G + E75V = GV; F71A+ E75V = AV and D66G + F71A = GA) and triple (D66G + F71A + E75V = GAV) mutants of ICAT C-terminal domain were created using the Quick-Change Site Directed Mutagenesis kit (Stratagene) and primers listed in S1 Table. The HA-tagged CMV::β-catenin-NLS expression vector was used for overexpression of β-catenin in the nuclei of transfected cells . Single mutants of the HA tagged CMV::β-catenin-NLS vector (K312E, K435E, R386G, F660S, F660A) and the truncated mutant Δ665 (premature stop codon at residue 665) were also created by site directed mutagenesis using primers listed in S1 Table. All constructs were sequenced to validate presence of the different mutations. The M-MITF::Luc promoter (-2293 to + 120) in pGL3 and the NEDD9::Luc promoter (-2011 to + 72) in pGL3 were kindly provided by Dr C. Goding (Ludwig Institute, Oxford UK) and Dr W. Liu (Louisiana State University Health Sciences Center, New Orleans, LA, USA), respectively.
The WT human ICAT cDNA cloned in the bacterial expression vector pGEX-4T3 was provided by C. Gottardi (USA). Mutant cDNAs including ICAT-DQE, ICAT-GV, ICAT-AV, ICAT-GA and ICAT- GAV were subcloned in the pGEX-4T3 vector and used to transform E.coli BL21 competent cells (Stratagene). Positive clones were grown at 37°C for 2–3 hrs to an OD600 of 0.6–0.8 and protein synthesis was induced with 0.5 mM IPTG at 37°C for 3–4 hrs. The bacterial cultures were centrifuged and the cell pellets sonicated. Bacterial lysates were incubated with glutathione Sepharose 4B beads (GE Healthcare) overnight at 4°C. The beads were washed twice with 1X PBS, 0.3M NaCl, Triton 0.1%, protease inhibitors, twice with the same buffer containing 0.15M NaCl, and once with 1X PBS, Triton 0.1%. After re-suspension in one volume of 1X PBS, aliquots were analyzed on polyacrylamide gels and stained with Coomassie blue. A similar procedure was used to produce recombinant human LEF1-GST protein.
ICAT WT and ICAT-DQE recombinant proteins were purified from their GST forms. Cleavage of the GST tag was achieved by the addition of thrombin (1U/μl in PBS) and incubation at room temperature for 16h. Solutions containing the cleaved proteins were loaded on Hi-Trap Benzamidine Sepharose 4 Fast Flow columns (GE-Healthcare) and eluted with phosphate buffer 20mM, 0.15M NaCl, pH 7.3. Collected fractions were spin concentrated on Amicon Ultra 3K centrifugal filter (Millipore) and dialyzed against 10mM Phosphate buffer pH 7.0 containing 100 mM ammonium sulfate. Final concentrations were determined by UV absorption at 280 nm. Aliquots were separated on polyacrylamide gels and stained with Coomassie blue.
Total cell lysates (TCL) were prepared as described earlier  and submitted to WB analysis. Immunoprecipitation (IP) was achieved by incubating TCL with an anti-Flag monoclonal antibody for 2hrs at 4°C. Immune complexes were bound to Protein G agarose beads. After washing with RIPA, beads were boiled for 10 min in loading buffer. For affinity precipitation, TCL in RIPA (500 μg of total proteins) were incubated for 3 hrs at 4°C with 20–30 μL of a 50% suspension of WT or mutant ICAT-GST, or LEF1-GST recombinant proteins coupled to glutathione-Sepharose 4B beads. Affinity precipitated proteins were washed five times and subjected to SDS-PAGE and immuno-blotting analysis. Gel densitometry was performed using ImageJ. Control values were arbitrarily fixed to 1.0.
The previously described rabbit polyclonal antibody raised against a synthetic ICAT peptide was used for WB . Mouse monoclonal antibodies against human ICAT (clone 5C6), anti-Flag M2 and anti-β-actin (clone AC-15) were obtained from Sigma-Aldrich. Mouse anti-HA monoclonal antibody (clone 12CA5) was from Roche Life Science. Rabbit polyclonal antibody raised against β-catenin was from Abcam. The rabbit polyclonal anti-p27kip1 antibody and the rabbit monoclonal antibody (clone D13A1) raised against the non-phospho (active) β-catenin S33/37,T41, were obtained from Cell Signaling. The mouse anti-LEF1 monoclonal antibody (clone 2D12) was from Millipore. The rabbit polyclonal anti-MITF antibody was a gift from Dr S. Saule (Institut Curie, Orsay). Immunofluorescent staining of cultured cells fixed in 4% PFA was achieved as described .
Lipofectamine 2000 (Life Technologies) was used to transiently co-transfect cells with the M-MITF or NEDD9 promoters and expression vectors for LEF1, β-catenin (WT and mutants) and/or ICAT (WT and mutants). The pRL-TK Renilla luciferase reporter construct was used as an internal control for transfection efficiency. Forty-eight hrs post-transfection, cells were lysed with passive lysis buffer and luciferase activities (Firefly and Renilla) were measured using the Dual Luciferase reporter Assay (Promega, Madison WI) in a TriStar luminometer (Berthold, Germany). Firefly luciferase activity was normalized to Renilla luciferase activity.
Prism6 software (GraphPad, La Jolla, CA) was used for statistical analyses. Luciferase activity differences were assessed using two-way analysis of variance (ANOVA) test followed by Tukey's post-test.
So far, β-catenin is the only demonstrated protein known to interact directly with ICAT at the physiological level [18, 12]. However, ICAT may also bind with high or mild affinity to other proteins. To identify interactors in the melanocyte lineage, a yeast two-hybrid screen using human ICAT as bait was performed with a human melanocyte cDNA library as prey. Although various putative partners were detected at low frequencies by sequencing of prey clones, most of the positive clones (80%) corresponded to CTNNB1/β-catenin cDNAs (S2 Table). These results were compared to the interaction data originating from a large-scale mapping in HEK cells, using a mass spectrometry-based approach ; S3 Table). Only two similar proteins, β-catenin and γ-catenin/plakoglobin, were identified in both studies. That ICAT interacts with both Plakoglobin and β-catenin is not unexpected owing to the strong conservation of their Armadillo domains . However β-catenin got the highest confidence score, suggesting a higher affinity for ICAT than plakoglobin. We concluded that β-catenin is the main ICAT-interacting protein in cells of the melanocyte lineage.
Two melanoma cell lines, the non-metastatic Mel501 and the highly metastatic Lu1205, expressing 3 times more ICAT than Mel501  were chosen to assess the effect of ICAT on the co-transcriptional activity of β-catenin. Using WB, we found that in Mel501 cells, total β-catenin was 4 fold more abundant than in Lu1205 cells. Immunofluorescence (IF) staining with an antibody (here referred to as ABC), directed against the active signaling form of β-catenin showed a faint nuclear signal in Lu1205 cells and a strong signal in Mel501 cells. The amount of ABC in Mel501 cells was 7 fold higher than in Lu1205 cells (S3A Fig, S4 Table), indicating constitutive activation of the canonical Wnt/β-catenin pathway in Mel501. Based on these data and in order to minimize the role of the endogenous proteins, Mel501 cells exhibiting a low level of endogenous ICAT were used to assess the consequences of ectopically expressed ICAT mutants. Lu1205 cells exhibiting a low amount of nuclear β-catenin were chosen for the analysis of overexpressed NLS-tagged β-catenin mutants.
M-MITF being a master regulator gene of the melanocyte lineage and melanomagenesis, but also a target of the TCF/LEF/β-catenin complex, we evaluated by WB and IF the expression of MITF in our cell lines. High protein amount was found in the nuclei of Mel501 cells, while in Lu1205 cells, MITF was undetectable (S3B Fig, S4 Table). The M-MITF promoter comprises three TCF/LEF binding sites. To test whether the low level of MITF in Lu1205 cells was related to the inhibitory effect of endogenous ICAT, its level in Mel501 cells was ectopically increased and M-MITF expression was evaluated by four different approaches. 1) Mel501 cells were co-transfected with the M-MITF::luciferase reporter construct and increasing amounts of CMV::ICAT expression vector. The luciferase (luc) activity showed a dose-dependent negative effect of ICAT (Fig 2A). Based on this result, a dose of 250 ng of CMV::ICAT plasmid was used in subsequent experiments. 2) Quantitative RT-PCR in Mel501 cells transiently transfected with ICAT-WT showed a 50% reduction of M-MITF mRNA amount (Fig 2B), revealing the inhibitory transcriptional effect of exogenous ICAT on M-MITF expression. 3) WB analysis showed a marked reduction of MITF protein in the presence of ICAT (Fig 2C). 4) The effect of variable MITF levels on the amount of cyclin-dependent kinase inhibitor, p27Kip1, was used to verify the action of ICAT. Transient ectopic expression of ICAT-WT in Mel501 coordinately reduced MITF and increased p27Kip1 protein levels (Fig 2D). This result is consistent with the previous finding that siMITF treatment increased p27Kip1 expression . Collectively, these data demonstrate a negative regulatory effect of ICAT on M-MITF expression in Mel501 cells. The lack of detectable MITF protein in Lu1205 cells could be ascribed, at least in part, to their high level of endogenous ICAT.
Fig 2. ICAT negatively regulates the M-MITF promoter activity by competing with LEF1.
A. Mel501 cells were transfected with a M-MITF::luciferase vector in the presence of increasing amounts of CMV::ICAT-WT expression vector. Data are presented as means ± SEM of three independent experiments. B. qRT-PCR analysis of M-MITF mRNA levels in Mel501 cells transfected with empty or ICAT-WT expression vectors. C. WB analysis of MITF and ICAT proteins in Mel501 cells transfected with empty or ICAT-WT expression vectors. β-actin = loading control. D. WB analysis of MITF and p27Kip1 protein levels in siRNA and ICAT-transfected Mel501 cells. SiMITF treatment and ICAT overexpression induce respectively a 42% and 35% increase of p27 protein amount; Scrb = control scrambled siRNA. E. Mel501 cells were transfected with M-MITF::luciferase, LEF1 and ICAT-WT expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p< 0.001; ****p<0.0001.
In colon cancer cells, ICAT was found to compete with TCF7L2 for binding to β-catenin . In Mel501 and Lu1205 cells, qRT-PCR evaluation of TCF7L2 transcripts disclosed an almost undetectable mRNA level, whereas LEF1 mRNA was much more abundant especially in Lu1205 where its amount was twice higher than in Mel501 (S3C Fig). This result was confirmed at the protein level (S3D Fig). Since the biological functions of LEF1 and TCF7L2 are not fully redundant , competition between ICAT and LEF1 for regulating transcriptional activity was tested by co-transfecting Mel501 cells with CMV::ICAT, CMV::LEF1 and M-MITF::luc vectors. LEF1 overexpression greatly increased luciferase activity of the M-MITF::luc promoter. This stimulating effect was almost completely abrogated in the presence of exogenous WT ICAT, illustrating the competition between the two β-catenin ligands (Fig 2F).
In an attempt to suppress the binding of ICAT to β-catenin without impairing the intrinsic secondary structure of ICAT, three residues (Y15, K19 and V22) in the helix1 of ICAT N-terminal domain were mutated simultaneously. Aromatic residue Tyrosine 15 was converted into Aspartate (Y15D). The positively charged Lysine 19, which contributes to the hydrophobic packing through the long aliphatic part of its side chain, was mutated into Glutamine, a shorter neutral residue (K19Q). Valine 22 was converted into Glutamate, a negatively charged residue (V22E). The resulting mutant is referred to as ICAT-DQE (Table 1). To determine whether the triple mutation critically affected the secondary structure of the protein, recombinant ICAT-WT and ICAT-DQE were purified from their GST fusion forms (S4A Fig), and their Circular Dichroism (CD) spectra were recorded. The two spectra were similar (S4B Fig) and their deconvolution with the CDSSTR algorithm showed that both proteins were composed of about 40% of α-helices and 17% of β-sheet, in good agreement with the ICAT crystal structure. This indicates that the three mutations did not significantly affect the secondary structure of the isolated protein. Stability of the two proteins was further evaluated by thermal denaturation using CD at 222 nm. Melting temperature (Tm) derived from the curves was 55°C for both the WT and mutant proteins (S4C Fig), showing that these mutations did not alter the thermal stability.
Table 1. List of ICAT and β-catenin mutants created by site directed-mutagenesis.
To test whether residues Y15, K19 and V22, were required for ICAT binding to β-catenin, we transfected Mel501 cells with WT or mutant (DQE) ICAT-Flag vectors and performed IP assays (Fig 3A). As expected, exogenous ICAT-WT protein bound efficiently to endogenous β-catenin present in Mel501 cell lysates, while ICAT-DQE mutant failed to immunoprecipitate β-catenin, revealing a loss of function of this mutant. In a reporter luciferase assay using the M-MITF promoter, ectopic expression of ICAT-DQE had no effect on M-MITF luc activity in Mel501 cells, both in the presence and absence of exogenous LEF1 (Fig 3B and 3C).
Fig 3. Mutations in the ICAT N-terminal domain prevent binding to β-catenin and abolish the negative regulation of the M-MITF and NEDD9 promoter activities.
The requirement of the three ICAT residues mutated above for proper interaction with β-catenin may be ascribed to their contact with the facing residue F660 (S2 Fig), as indicated by the crystal structure of the complex . However, four other β-catenin residues are in close contact with one or the other of the three ICAT residues, and the electrostatic interaction between β-catenin E664 and ICAT K19, may contribute to the affinity. Consequently, β-catenin residue F660 was mutated into serine (F660S) or alanine (F660A) (Table 1) to evaluate its contribution to the interaction with ICAT in Lu1205 cells. The β-catenin F660S mutant failed to interact with ICAT, whereas the interaction with the F660A mutant was impaired but not totally abolished (Fig 4A). By contrast, both β-catenin mutants interacted normally with the recombinant LEF-GST protein (Fig 4B). Since WT β-catenin is a co-transcriptional activator of NEDD9 , we tested using a reporter assay whether the activity of the NEDD9 promoter in Lu1205 cells was affected by the F660S mutation. The activating role of β-catenin F660S in the absence or presence of ectopic LEF1 was similar to WT (Fig 4C). Thus, residue F660 seems dispensable for the interaction with LEF1, although crucial for the interaction with ICAT.
Fig 4. β-catenin residue F660 is critical for ICAT anchoring to Arm repeat 12 but plays no role in the affinity for LEF1.
A. Left: Western blot (WB) analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: pull-down assay of β-catenin WT and mutants F660S and F660A by WT ICAT-GST recombinant protein. B. Left: Input from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: pull-down assay of β-catenin WT and mutants F660S and F660A by LEF1-GST recombinant protein. C. Lu1205 cells were transiently transfected with a NEDD9::luciferase vector. These cells were also transfected with either NLS-β-catenin-WT or NLS-β-catenin-F660S expression vectors in the absence (left) or presence (right) of exogenous LEF1. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.
Implication of the β-catenin helix C, spanning conserved residues 667–683 in the C-terminal domain (S1 Fig), in increasing the interaction with ICAT N-terminal domain has been hypothesized , despite its absence in the crystal structures of the β-catenin/ICAT complex [22, 23]. To test this assertion, we generated a β-catenin truncated protein by introducing a premature stop codon at position 665 (Fig 5A). The Δ665 HA-tagged β-catenin mutant was properly expressed in Lu1205 cells at the same level as the WT. It interacted with the recombinant WT ICAT-GST protein more efficiently (x1.5) than the full-length WT β-catenin (Fig 5B). Contrary to the model prediction , helix C appeared dispensable for an efficient interaction of ICAT with β-catenin Arm repeats 11–12. The observed increased affinity is in keeping with results from Mo et al , suggesting that the absence of the unstructured β-catenin C-terminal domain that normally extends toward the N-terminal Arm repeats, could facilitate the recruitment of various ligands. Moreover, the close contact of helix C with residue F660 (S1 Fig) could decrease the affinity of ICAT helical bundle for Arm repeats 11–12.
Fig 5. The lack of helix C in β-catenin does not prevent interaction with ICAT.
A. Schematic representation of WT and mutant Δ665 HA-tagged β-catenin-NLS proteins. B. Total cell lysates from Lu1205 cells transfected with WT or mutant β-catenin Δ665 were analyzed by WB or affinity immunoprecipitated with WT ICAT-GST recombinant protein and blotted with anti-HA and anti-ICAT antibodies. Numbers represent mean ± SD of normalized densitometry values from three independent experiments, *p<0.05.
Based on sequence alignments, the three conserved residues D66, F71 and E75 in the ICAT consensus peptide (Fig 1B), seemed important for the interaction with β-catenin Arm repeats 5–9 (Fig 1C). According to the crystal structure, residues D66 and E75 formed salt bridges with β-catenin K435 and K312 respectively, while F71 packed against the aliphatic chain of R386. To determine whether these residues were essential for the interaction with β-catenin, we generated various ICAT mutants harboring single, double or triple amino-acid substitutions (Table 1). D66 was changed into Glycine (D66G), F71 into Alanine (F71A) and E75 into valine (E75V). IP of nuclear endogenous β-catenin in Mel501 cells by ectopically expressed ICAT WT or single mutants, showed no differences between WT and mutants (Fig 6A). On the opposite, double and triple mutants had a lower affinity (reduction over 70%) for β-catenin than ICAT WT (Fig 6B). Consistent with these findings, single mutations of conserved ICAT residues did not affect significantly the luciferase activity of the M-MITF promoter in the presence of exogenous LEF1, whereas the double and triple mutations significantly reduced, but did not totally abolish, the negative regulatory role of ICAT (Fig 6C).
Fig 6. Effect of mutations in the C-terminal domain on ICAT binding to β-catenin and regulation of M-MITF promoter activity.
A. Upper: WB analysis of cell lysates (Input) from Mel501 cells transiently transfected with WT ICAT or ICAT single mutants (D66G, E75V and F71A); Lower: Immunoprecipitation (IP) of endogenous β-catenin by WT and single ICAT-Flag mutants. B Upper: WB analysis of cell lysates (Input) from Mel501 cells transiently transfected with WT ICAT or ICAT double (AV, GA) or triple (GAV) mutants. Lower: IP of endogenous β-catenin by WT and double or triple ICAT-Flag mutants. Numbers under each lane represent normalized densitometry values. β-actin = loading control. C. Mel501 cells were transiently co-transfected with M-MITF::luciferase, LEF1, Flag-tagged ICAT WT and ICAT mutants (D66G; E75V; F71A; D66G,E75V; F71A,E75V; D66G,F71A; D66G,F71A,E75V) expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.
In order to validate our findings, the facing β-catenin basic residues (K312, K435 and R386) were converted into Glutamate and Glycine, respectively. The effects of these mutations on the interaction with WT ICAT, was assessed through a GST pull-down assay in Lu1205 cells (Fig 7A). K to E mutations markedly decreased, but did not totally suppress the affinity of β-catenin mutants for WT ICAT-GST protein and R386G conversion had no significant effect on this affinity. Likewise, the interaction between β-catenin mutants (K312E and K435E) and LEF1 was tested using recombinant LEF1-GST. No detectable interaction was observed with these mutants (Fig 7B). Luciferase assays were also performed to monitor the interaction between β-catenin mutants and LEF1. Consistent with pull-down results, β-catenin mutants K312E and K435E had a much lower co-transcriptional activity on the NEDD9 promoter than the WT, both in the absence (Fig 7C) or presence of exogenous LEF1 (Fig 7D), whereas mutant R386G behaved similarly to WT.
Fig 7. Interactions between ICAT and β-catenin mutants, K312E, K435E and R386G in Lu1205 cell extracts: Consequences on NEDD9 promoter activity.
A. Left: WB analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: Pull-down assay of HA-tagged WT and mutant β-catenin (K312E, K435E and R386G) by WT ICAT-GST recombinant protein. B. Left: WB analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: Pull-down assay of HA-tagged WT and mutant β-catenin (K312E and K435E) by LEF1-GST recombinant protein. C. Lu1205 cells were transiently transfected with NEDD9::luciferase and either β-catenin-WT or β-catenin mutants (K312E, K435E and R386G) expression vectors. D. Lu1205 cells were transiently transfected with NEDD9::luciferase vector in the presence of CMV::LEF1. Cells were also transfected with β-catenin-WT or β-catenin mutants (K312E, K435E and R386G) expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.
We conclude that electrostatic interactions between β-catenin K312 or K435 residues, and the ICAT consensus peptide are important but not crucial for regulating β-catenin co-transcriptional function. Converting one of these attractive electrostatic interactions into repulsion was not fully deleterious and removal of arginine side chain in the R386G mutant was ineffective.
Appropriate Wnt/β-catenin signaling in cells and tissues relies on a subtle balance between positive and negative β-catenin regulators, among which ICAT [15, 35, 36]. In this study, ICAT overexpression in melanoma cells was found to negatively regulate M-MITF and NEDD9 promoter activities, both in the presence and absence of ectopic LEF1. This indicates that in Lu1205 and Mel501 cells, ICAT competes with LEF1 for binding to β-catenin and interferes with its co-transcriptional function.
Simultaneous mutation of the three ICAT residues, Y15, K19 and V22, in the N-terminal domain, predicted to form a hydrophobic patch with β-catenin F660, completely disrupted the ICAT/β-catenin interaction, making this mutant unable to compete with LEF1. Other ICAT residues (M29 and E37) were surmised to establish either hydrophobic interactions (M29) or form a salt bridge (E37) between ICAT and β-catenin [22, 27]. However, simultaneous mutation of residues Y15, K19 and V22, proved to be sufficient to completely suppress the high affinity anchor between the two proteins without impairing the ICAT helical secondary structure. This suggests that M29 and E37 as such, are insufficient to stabilize the complex.
Importantly, β-catenin mutant F660S interacted normally with LEF1 and its co-transcriptional activity on the NEDD9 promoter was equivalent to WT even though it did not bind to ICAT (Fig 4). We conclude that residue F660 is essential for ICAT anchoring but plays no role in the β-catenin interaction with LEF1. Support to this assertion arises from the β-catenin/ICAT crystal structure (S5 Fig) indicating that the interaction relies not only on previously mentioned hydrophobic contacts , but also involves a surface complementarity between β-catenin F660 and ICAT Y15, K19 and V22. Residues Y15 and V22 seem to act like pincers around F660, thus rendering this interaction stronger than a mere hydrophobic contact.
Conserved residues (D66, F71, E75) in the ICAT consensus peptide, also present in the catenin-binding domain of TCF7L2 and LEF1 (Fig 1B), have been reported to be crucial for the interaction with β-catenin Arm repeats 5–9. Owing to conflicting reports using mutated TCF7L2 consensus residues  or TCF7L2 synthetic peptides , the precise contribution of residue F21 (F26 in LEF1 or F71 in ICAT) on β-catenin affinity for TCF/LEF factors, was questioned. Our results indicate that the F71A ICAT mutant behaves similarly to the WT protein (Fig 6B). Close examination of β-catenin/ICAT crystal structures (Fig 1C) reveals that the contact between ICAT F71 aromatic ring and the aliphatic portion of the β-catenin R386 side chain is not sufficient to create a stable hydrophobic interaction, owing to partial exposure of F71 to the solvent. These observations bring support to the conclusion that F21 residue from TCF7L2 is not a major player in the binding to β-catenin . However, double or triple ICAT mutants had less affinity for β-catenin than WT, suggesting a cumulative contribution of residues D66, F71 and E75 in stabilizing the protein complex. Although these residues are directly involved in the competition with LEF1, their mutation does not completely restore the co-transcriptional activity of WT β-catenin (Fig 6C). Overall, this indicates that even though residues Y15, K19, V22 in the N-terminal domain do not compete with LEF1 for ICAT binding to β-catenin, they play a more critical role in the anchoring process than residues D66, F71 and E75.
The affinity of β-catenin for ICAT was shown by isothermal calorimetry to be higher than for LEF1 or APC , however when the interaction between the ICAT N-terminal domain and β-catenin is lost, ICAT fails to compete with LEF1 (Fig 3). This suggests that ICAT C-terminal consensus peptide has a weaker affinity for β-catenin than LEF1. Comparison of the consensus peptides from β-catenin partners including TCFs, APC, E-cadherin, LEF1 and ICAT (Fig 1B) reveals the absence in ICAT of hydrophilic residues between D and Φ in the DXθθXΦX2-7E sequence, thus accounting for its lower affinity. When the first X residue is hydrophilic, it forms a hydrogen bond with β-catenin R469 or K508 (Fig 8). The second X is generally hydrophobic, except in APC where H1490 interacts with β-catenin D390. Finally, residues X2-7 do not generally interact with β-catenin, at least in the complexes whose structures are available in PDB (Fig 8). Comparative analysis of the ICAT consensus sequences from several vertebrates reveals that the first X in this sequence, is predominantly a valine (more scarcely a glycine), and the second X is always an alanine. Therefore, unlike TCF/LEF, APC and E-cadherin, the tightening of the interaction between the ICAT C-terminal domain and β-catenin positive groove by additional hydrogen bonds is not possible, resulting in a weaker interaction. This weakness is probably required for efficient competition with other regulators.
Fig 8. The characteristics of the first X residue in the consensus peptide of several β-catenin binding proteins regulate their interactions with β-catenin.
A. Zoom is made on the first conserved Aspartate residue of the consensus peptide and its adjacent non conserved residues (shown as sticks) in ICAT, LEF1, TCF4, APC and E-cadherin (yellow ribbons) and the facing β-catenin Arm repeats 8 and 9 (purple cylinders). The first X residue of the consensus is encircled because residue numbering diverges between various β-catenin regulators, although they are facing the same β-catenin residues forming a basic patch. Hydrogen bonds between basic β-catenin residues and their counterpart in β-catenin regulators are presented as black dotted lines. In ICAT, V67 does not establish any hydrogen bond, whereas in LEF1 and TCF4/TCF7L2, E20 and E17, respectively make an H-bond with the facing β-catenin K508. In APC and E-cadherin, T1487 and S675 respectively form hydrogen bonds with the facing β-catenin R469. The color scheme of stick residues based on their characteristics is the same as in Fig 1. PDB codes: ICAT (1luj), LEF1 (3ouw), TCF3/TCF7L1 (1g3j), TCF4/TCF7L2 (1jdh), APC (1t08) and E-cadherin (1i7w).
Based on these observations, the dynamic process of ICAT binding to β-catenin may involve one or two steps. The one step process is unlikely because the N-terminal domain is intrinsically structured while the C-terminal domain is unstructured. Consequently, the N-terminal domain should bind more rapidly than the C-terminal domain since during this process the loss of entropy of the N-terminal domain alone (TΔS = -1.5 Kcal/mol) was less unfavorable than that of the full length protein (TΔS = -8.0 kcal/mol) . Therefore, binding of ICAT full length to β-catenin would presumably occur in two steps: i) the ICAT helical N-terminal domain first anchors to β-catenin through F660, then, ii) the ICAT unstructured C-terminal domain lies into the groove and become structured resulting in an increased negative entropy change .
The Wnt/β-catenin signaling is regulated, among others, by ICAT. This process implies a competition between ICAT and various β-catenin ligands sharing a consensus peptide that mediates, with variable affinity, the interaction with β-catenin Arm 5–9. Differences in affinity seem to depend not only on conserved residues in ligands but also on non-conserved ones. ICAT being a negative regulator, the affinity of its consensus peptide needs to be lower than that of positive β-catenin interactors to facilitate its removal. However, ICAT, through its N-terminal domain, holds its capacity to bind β-catenin residue F660 located more than 30 Å apart from its consensus peptide. Loss of both hydrophobicity and surface complementarity of this unique residue (F660) completely prevents ICAT from achieving its negative regulation.
S1 Fig. Crystal structure of human β-catenin Arm repeat region and C-terminal domain.
S2 Fig. Model for the competition between ICAT and LEF1 for binding to β-catenin based on structural data.
The central core of β-catenin is made of 12 armadillo (arm) repeats (in blue) and an additional helical domain, helix C (in red) that docks to the 12th arm repeat . The N-terminal and the C-terminal domains (in white) are unstructured. β-catenin residues predicted to form electrostatic (K312 and K435) or hydrophobic interactions (R386) with LEF1 residues D21, E29 and F26 are shown as blue arrowheads. ICAT and LEF1 residues reported to have putatively the most critical contribution to the interaction with β-catenin are shown as arrowheads (blue for β-catenin, purple for ICAT and orange for LEF1). These interactions are either hydrophobic (represented by green dotted lines) or hydrophilic (represented by black dotted lines). The HMG box of LEF1 interacts with the TCF/LEF binding element (TBE) in the promoter of target genes.
S3 Fig. β-catenin, MITF and LEF1 are differently expressed in Mel501 and Lu1205 melanoma cells.
A. WB (upper panel) and IF (lower panel) analyses of Mel501 and Lu1205 cells: The non phospho S33/37/T41 active form of β-catenin (ABC) is much more abundant (7 fold) in Mel501 than in Lu1205 cells and mainly visible in their nuclei. The numbers below each lane represent normalized densitometry values. α-tubulin = loading control; (bars = 20 μm). B. WB (upper panel) and IF (lower panel) analyses of MITF in Mel501 and Lu1205 cells. MITF is present in the nuclei of Mel501 cells. The two bands correspond to at least two different MITF isoforms. Lu1205 cells are devoid of MITF (bars = 10 μm). C. qRT-PCR analysis of LEF1 and TCF7L2 mRNA levels in Lu1205 and Mel501 cells. D. WB analysis of endogenous LEF1 protein levels in Mel501 and Lu1205 cells. Numbers represent normalized densitometry values. β-actin = loading control.
S4 Fig. Circular dichroism (CD) analysis of purified ICAT-WT and DQE recombinant proteins.
A. Gel electrophoresis and Coomassie blue staining of purified proteins. B. Far-UV CD spectra of ICAT WT (in red) and ICAT DQE (in blue) recombinant proteins diluted at 30 μM in 10mM sodium phosphate, 100mM ammonium sulphate buffer pH 7.0. Data were recorded at 20°C. Similar results were obtained with 50 μM protein concentrations. C. Thermal denaturation curves of ICAT WT and ICAT DQE. Tm = melting temperature.
S5 Fig. Embedding of μ-catenin F660 in the ICAT N-terminal domain.
The entire ICAT protein is shown (surface), with its globular N-terminal domain and extended C-terminal domain. The residues are colored according to their characteristics: white for hydrophobic, green for polar, red for acidic and blue for basic residues. β-catenin residue F660, part of Arm repeat 12 helix 3 (purple cylinder) is shown as pink hard spheres. It is embedded in an ICAT niche made of residues Y15, K19 and V22.
S1 Table. Primers used to create the different mutants.
S2 Table. Results of the yeast two-hybrid screening using CTNNBIP1/ICAT as bait and cDNA from human melanocytes as prey library.
* PBS (Prey-Bait-Score) was automatically computed. A and B represent respectively very high and high confidence in the interaction. D represents moderate confidence. N/A = non applicable.
S3 Table. List of CTNNBIP1/ICAT interactors in HEK cells identified by affinity capture coupled to mass spectrometry (MS).
Data were compiled from . Human epithelial kidney (HEK) cells were used for affinity capture experiments. *Interactors identified in both studies (cf S2 Table). **Computed confidence score based on partial least squares model with values between 0 and 1. Values higher than 0.3 are considered as high confidence interactions.
S4 Table. Comparative levels of ICAT, β-catenin, MITF and LEF1 proteins in melanoma cells.
The authors thank Drs. C. Gottardi (University of Chicago, Chicago, IL), Dr C. Goding (Ludwig Institute Oxford UK) and Dr Liu (Louisiana State University Health, New Orleans, Louisiana) for providing biologic materials. They also thank P. Duchambon for technical assistance.
Writing – original draft: MJD JB.
Writing – review & editing: JB LM.


There are a few people who feel that mold remediation and water damage restoration solutions aren't definitely necessary for home and property owners. As opposed to that notion, there are lots of logic behind why it is necessary for people to get mold remediation solutions from professionals, it doesn't matter just how small their own dilemma may be. That's due to the fact these kinds of pros don't simply eliminate the damage that is apparent to the structure, however they may locate the root source of the challenge. This will resolve the issue once and for all.
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Now, after more than four months, I continue neck stretches and have more therapy ahead for my sprained hand. Yet, I'll fully recover. So, how is it I walked out of ER that same day with no concussion and not a single broken bone? For me, the conclusion is inescapable: The unmerited favor of God.
Pete, what a blessing it is to still have you with us, and that you were not more seriously injured. And a tremendous testimony to the glory of the mighty God that we share.


The Federal Reserve Board on Monday announced the approval of fee schedules, effective January 2, 2018, for payment services the Federal Reserve Banks provide to depository institutions (priced services).
The Reserve Banks project that they will recover 100 percent of their priced services costs in 2018. The Reserve Banks expect to fully recover actual and imputed expenses, including profit that would have been earned if a private business firm provided the services. Overall, the Reserve Banks estimate that the price changes will result in a 1.4 percent average price increase. The Reserve Banks estimate that the price changes will result in a 0.4 percent average price increase for check customers. The Reserve Banks estimate that the price changes will result in a 3.6 percent average price increase for FedACH® customers. The FedACH price increase is in response to increasing operating costs associated with an ongoing technology upgrade. The Reserve Banks estimate that the price changes will result in a 1.2 percent average price decrease for Fedwire® Funds customers. The fees will remain unchanged for the Reserve Banks' National Settlement Service and Fedwire Securities Service.
Lastly, the Reserve Banks estimate that the price changes will result in a 4.3 percent average price increase for FedLine® customers. The 2018 fee schedule for each of the priced services is available on the Federal Reserve Banks' financial services website at FRBservices.org.
The Board approved the 2018 private-sector adjustment factor (PSAF) of $18.9 million for Reserve Bank priced services. The PSAF is an allowance for income taxes and other imputed expenses that would have been paid and profit that would have been earned if the Reserve Banks' priced services were provided by a private business. The Monetary Control Act of 1980 requires that the Federal Reserve establish fees to recover the costs of providing priced services, including imputed costs, over the long run, to promote competition between the Reserve Banks and private-sector service providers.


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KTP project takes centre stage at Scotland's recent Can Do Innovation summit
Posted on 18 Dec , 2019
A KTP project between Sanondaf UK and the University of the West of Scotland (UWS) was spotlighted at last month's high profile VentureFest event – the Can Do Innovation Summit at Glasgow's Science Centre – where both Ian Campbell, Interim Executive Chair of Innovate UK and Ivan Mckee, MSP, highlighted the KTP programme.
Sanondaf UK specialises in the disinfection and decontamination markets using revolutionary touch-less disinfection technologies to help eradicate micro-organisms and infections that can cause health issues in everyday life for people and animals. Via this KTP, the company aims to embed engineering and microbiology expertise to facilitate the design, development, testing and launch of the next generation of microbial disinfection and decontamination technology.
Stuart White, Director, commented that "2020 looks set to be a landmark year at Sanondaf. Having now passed the first 6 months of our KTP partnership with the University of West Scotland (UWS) we have already made significant progress in our project to design and build the world's first combination sprayer and fogger for the decontamination market. The technical support and input from UWS has been invaluable in getting Sanondaf to this stage and we have seen massive benefit in being involved with the UWS – KTP family."
All KTPs are a three way partnership between a company, a university and a talented graduate, or post graduate – the KTP Associate – who acts as both project manager and conduit for the knowledge transfer. Here Connor Spiers is the KTP Associate and he echoed the value that most Associates experience as a result of their unique role in a KTP: the opportunity to develop a broad range of skills, and to meet the challenges and responsibilities of working on innovation projects whilst enjoying the support of academic and company teams: " This is a unique opportunity for an early career engineer and has given me exposure to a number of different opportunities that I may not have received elsewhere. Through this I have developed my engineering, project management and business skills while working with a combination of academia and business management."
From UWS, Tony Leslie – the lead Academic – added, "…. Since the start of the project the company has been able to identify a number of additional business opportunities for the technology, which the team was unaware of at the outset. As a supervisory team we look forward to the next period of the project where we anticipate that significant tangible project aims will be realised. This KTP project is an excellent example of the KTP three-way partnership providing solutions to current challenges whilst responding to new opportunities throughout"
UWS' KTP Manager, Dr Stuart McKay, said: ""This is a great example of how a KTP project allows businesses to draw on multi-disciplinary teams from across a University – in this case involving four academics with expertise spanning Mechanical Engineering, Chemical Engineering and Microbiology from our Schools of Computing Engineering and Physical Sciences and from The School of Health and Life Sciences….It was great to see our KTP partner Sanondaf UK front and centre at the recentCan Do Innovation Summit".
UWS' KTP portfolio has grown rapidly in the last 24 months and the University has risen in the UK rankings from outside the top-50 to #7 in the UK, with a current portfolio of 26 KTPs worth in excess of £5.5m.
If you think a KTP might help your organisation innovate for growth, talk to one of our specialist Knowledge Transfer Advisers today.
"Outstanding" KTP delivers safety innovation at Niftylift.
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KTP helps power production of the new Covid-19 vaccine
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redpajama_set_name: RedPajamaCommonCrawl


I've been listening to Tool, a band you will quite often find in any "favourite bands" list that also features Radiohead.
The parallels are fascinating. Both bands emerged – or erupted? – during a moment when rock music was still all about grunge, but just as Nirvana was dissolving. Both Radiohead's first album, Pablo Honey, and Tool's – Undertow – came out in 1993. They were followed by The Bends (1995) and Ænima (1996), respectively. While Ænima was Tool's widely acknowledged "classic" album, Radiohead's classic, OK Computer, was released one year later in 1997.
More recently, Radiohead's fanbase suffered during a hiatus of five years between the release of The King of Limbs (2011) and A Moon Shaped Pool (2016). But spare a thought for Tool's long-suffering fans, who are speculating desperately about the release of the next album by their band, because the last one – 10,000 Days – came out ten years ago, in 2006.
Musical and lyrical parallels between these bands are apparent too. They are more complex, and the differences are just as interesting. But when looking for points of contact between the two bands, I was struck by these two songs – "The Pot", by Tool, and "2 + 2 = 5" by Radiohead – as a case in point. Note the timing again: The Pot is from 2006, 2 + 2 = 5 is from 2003.


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Q: Why are the x-components of the stress tensor ($\sigma_{xx}, \sigma_{yx}, \text{and } \sigma_{zx}$) different? I understand the stress tensor at a specific point $P$ as follows. You draw a cube with its center at $P$ and look at its faces. The face with its normal vector in the positive $x$ direction has a certain force acting on it. $\sigma_{xx}$ is the limit of the $x$-component of that force over the area of the face as the cube shrinks to size $0$. Similarly, $\sigma_{xy}$ is the limit of the $y$-component and so on.
Specifically, the force on a face is essentially the sum of the forces on all the particles on the face. If the force inside the material is roughly continuous (is that true for the insides of materials?) then as you take the limit as the cube shrinks to size $0$, shouldn't the force on all the particles on each face of the cube be essentially the same, thus leading to the forces on each face being the same and thus making, for example, $\sigma_{xx}=\sigma_{yx}=\sigma_{zx}$?
It seems to me somewhat obvious that all stress tensor components whose second element is $x$ should be the same (along with $y$ and $z$) but given how that is not stated in physics books that is definitely not the case.
Where does my logic go wrong? Also, could people give me some examples of where the forces on the different faces of the cube are clearly different even as the cube size gets extremely small? I would like to gain a better understanding of stresses.

A: I think that there is a little confusion here at several places. First, $\sigma_{xx}$ is the normal (not shear) force on the x-face of the tiny cube you mentioned. The corresponding normal force on the y-face of the cube is not $\sigma_{xy}$. $\sigma_{xy}$ is the shearing force on the x-plane in the y-direction (or, equivalently, the shearing force on the y-plane in the x-direction). The corresponding normal force on the y-face is $\sigma_{yy}$ and the corresponding normal force on the z-face is $\sigma_{zz}$. I believe that there are a total of six independent stress components in the stress tensor, 3 normal stress components and 3 shear stress components: $\sigma_{xx}$, $\sigma_{yy}$, $\sigma_{zz}$, $\sigma_{xy}$, $\sigma_{xz}$, and $\sigma_{yz}$.
Now as for your question of why the stress components aren't simply all the same as you shrink the imaginary cube to zero size, it's because these stress components have directionality and solids have strength. Suppose you have a large steel brick and place it into a huge mechanical press that puts an enormous compressive force on the opposite faces of the brick in the x-direction. Now imagine a small, tiny cubical volume element at the center of the steel cube. No matter how small of a tiny cubical volume element you consider, the compressive stress across the x-faces of that tiny imaginary cube is always going to be larger than the compressive stresses on the y- or z-faces of that same cube. (i.e., $\sigma_{xx}$>$\sigma_{yy}$ and $\sigma_{xx}$>$\sigma_{zz}$ no matter how small of a tiny, imaginary cube you are considering for stress analysis). Indeed, even in the limit in which the imaginary cube vanishes to zero size and you are considering the values of $\sigma_{xx}$ and $\sigma_{yy}$ and $\sigma_{zz}$ at the exact same point, $\sigma_{xx}$ will be greater than either $\sigma_{yy}$ or $\sigma_{zz}$. Why? Because they refer to different directions.

A: The stress tensor is a continuum mechanics concept, and you should expect it to break down (or become far more complicated) at the particle scale anyway.
However, a problem with your logic is that stress isn't really concerned with the net force on particles. Assume that your material is stationary: the net force on every single particle is zero, regardless of whether there is stress present or not. (I'm also not sure you're aware that $\sigma_{xy}=\sigma_{yx}$, nor that you appreciate $\vec\sigma[-\hat n]=-\vec\sigma[\hat n]$.)
Instead, stress is all about the relationship between adjacent particles (and how they respond to this deviating from their relaxed or "preferred" configuration). Think of a (stationary) material body that is subjected to large (static) external forces, and imagine surgically removing a cube (or other test volume) from the interior of this body. When you remove this cube, what forces would you have to apply to the cube's sides to keep it a cube (that is, to maintain it in exactly the same configuration as before you removed it from the bulk, and prevent it relaxing or "springing" back to some other shape)? Alternatively, what forces would you have to apply to the internal surfaces of the void (the hole left in the middle of the original body) to prevent it from deforming in response to removal of that piece of material?
A simple example (described in Landau & Lifshitz, and probably any other elasticity textbook) is a block of material in a vice, such that the stress tensor is homogeneous (uniform and constant, and the same for any sized cube) and $\sigma_{xx}$ is its only non-zero component (i.e. $\sigma_{xx}\neq\sigma_{yx}$).

A: I think an analogy is helpful here.
If you had some mass you could discuss the rate at which mass leaves a volume. It could have a mass density $\rho$ and a volume current density $\vec J$ and then the total mass in a region $R$ is $$M=\iiint_R\rho dV.$$
And if the region is fixed you get:
$$\frac{dM}{dt}=\frac{d}{dt}\int_R\rho dV=\int_R\frac{\partial \rho}{\partial t} dV.$$
Alternatively you can use current density $\vec J$
$$\frac{dM}{dt}=-\oint_{\partial R}\vec J\cdot \hat n dS =-\int_R\vec \nabla \cdot \vec J dV$$
And the important thing is that there are three different components to $\vec J$ specifying the direction of the flow of mass as well as the overall rate.
The same happens with charge, you can have a density of charge $\rho$ and a charge current density $\vec J$ and the current has three components specifying the direction of the flow of mass as well as the overall rate.
Now you can think of the stress tensor as three vectors. The first vector tells you the current of x-momentum. The second vector tells you the current of y-momentum. And the third vector tells you the current of z-momentum.
Of you take the divergence of each of those vectors you get a scalar field that tells you the rate at which the momentum density is changing at that point. There are three components of momentum. So three scalar fields telling you how each component changes in time. And each component has a current which has its own three components telling the direction as well as the overall rate of flow.
So basically the $x$ component of momentum $\vec p\cdot \hat x$ can flow in the $\hat x,$ $\hat y,$ or $\hat z$ directions and so we need three components of the stress to tell us about that flow. And we need three more to tell us how $\vec p\cdot \hat y$ can flow in the $\hat x,$ $\hat y,$ or $\hat z$ directions. And three more to tell us how $\vec p\cdot \hat z$ flows in the $\hat x,$ $\hat y,$ or $\hat z$ directions. So nine components are telling us that because we have a 3d flow of three things.
So really it's about how 3d vectors flow in 3d. And sure, momentum changes because of forces, so forces are the reason the momentum is flowing. But stepping back and nothing that a flow of momentum requires three vector fields tells you what is going on.

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Barron couple allegedly caught burglarizing Almena shed
Two people from Barron are facing a burglary charge after they were allegedly burglarizing a shed at a location near Almena on New Year's Day, county Circuit Court records said.
A complaint filed Jan. 4, 2022, identifies the defendants as Amanda M. Thompson, 42, and Joshua W. Thompson, 46, both of 459 W. Division Ave., Barron.
The owner of the shed, identified as T.W.Z., called 911 just after 7 a.m. on New Year's Day to report he'd caught two people, later identified as the defendants, leaving the shed.
Defendant Joshua Thompson allegedly told a Barron County Sheriff's deputy deputy that he was looking for some gas.
In a later interrogation, the defendants said they had been told about the shed from a third party, who wasn't charged in the complaint, and they'd only been there about 10 minutes before the owner showed up.
Defendant Amanda Thompson allegedly said she was told the place was abandoned, so they could "go (in) and look for stuff."
Later, T.W.Z. provided still photos from a trail camera that allegedly shows the defendants going up into the loft and carrying items including an electronic fireplace insert and a chandelier.
T.W.Z. also told officers he got a call from a relative who also had a trail cam, advising T.W.Z about the break-in. T.W.Z. arrived to find the defendants' vehicle, which he blocked from leaving before calling 911.


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Master of Fine Arts Degree Programs
Brandon Woods
Artists who truly want to become experts in their field should consider a Master of Fine Arts degree, which is commonly referred to as an MFA. An MFA degree shows that the artist has gone through a long list of rigorous courses in their particular art form and is adept at their craft.
Master of Fine Arts degrees can be related to any artistic medium. This includes more tech-based careers, such as graphic design or film editing, or more traditional forms of art, such as painting or sculpting. An MFA is a graduate-level program, meaning you must already have a typical liberal arts college degree prior to being admitted.
The length of time it takes to complete an MFA program varies depending on several factors. Some programs can last only a year, but there are others that may take as long as it takes to earn a bachelor's degree. In most circumstances, an MFA program lasts anywhere from two to three year; experts argue that receiving an MFA through accelerated means can sometimes limit its utility.
Best MFA Degree Schools in 2020
Throughout the United States there are incredible MFA programs.
The MFA for studio art at the University of Wisconsin is one of the most competitive and popular fine arts program offerings at the school. There is also an MFA in creative writing, which includes various courses on poetry, fiction, nonfiction and playwriting. Since 1972, Wisconsin's MFA program has become one of the best programs in the nation.
There are a variety of assistance programs for the school as well; the Wisconsin Institute for Creative Writing's post-graduate fellowship gives writers a chance to complete their first book, for example.
» MORE: Best Colleges for a Networking Security Degree
Savannah College of Art and Design, more commonly referred to as SCAD, has several MFA programs. The school has multiple degrees for writing, interior design, painting, photography, film and television, graphic design and more. Many SCAD MFA degrees can even be earned online through their eLearning program.
The School of Visual Arts is located in New York City and offers an MFA degree in Fine Arts. The program has classes taught by professional artists known internationally. The curriculum is also a combination of seminars focused on history, art theory, workshops dedicated to professional development and fieldwork; guest lectures are also offered by various curators and professional artists. Students also have 24-hour access to private artist studios to complete their MFA thesis.
The San Francisco Art Institute is one of the oldest art schools in America west of the Mississippi. The school has participated in several art movements since its inception in 1871. SFAI has attracted several graduate students ranging from Andy Garcia to Paul McCartney and even award-winning cinematographers.
Within its MFA degree program, students create their own curriculum based on the skills they need to succeed in their profession. Students work on their projects independently either in the studio or in the field and receive one-on-one guidance with faculty. SFAI awards more than $8.5 million to students through grants, scholarships, and fellowships each year.
California College of the Arts
The California College of the Arts is among some of the most well-known cultural institutions, companies and underground movements. The CCA offers various MFA degrees in design, fine arts, film, writing and even comics. Each program has a partnership with local organizations and businesses, which include Intel, Google and the Sundance Film Festival.
» MORE: PMI vs PMP: What's The Difference?
CCA has writer forums and mentored study courses, and also provides students with the opportunity to work with local cultural institutions. Students can pursue a large amount of rewarding and valuable internships, such as editing at Eleven Eleven Literary Journal.
MFA Degree Career Prospects
A Master of Fine Arts degree opens up a number of opportunities to you in various creative spaces. Below are some of the career opportunities available to you after earning an MFA degree from a prestigious institution.
MFA in Art Career Prospects
An MFA in art offers several possibilities to you. You can pursue a career in design doing work for various ad agencies, marketing departments or non-profit organizations. You could even start your own design business. Working in local art galleries and teaching are options available to you as well.
MFA in Writing Career Prospects
With an MFA in writing, you can start a career in advertising and marketing. You also have the skills to work for newspapers, magazines or as a freelance writer. Technical writing jobs are also available to you and allow you to be creative while receiving lucrative pay.
You could also work with literary agencies and publishers. It's also possible to work as a teacher and become an educator at any level from elementary to university, depending on the requirements of your state.
When pursuing higher education, many individuals often wonder if they should pursue a master's degree in art or if a bachelor's will suffice for their goals. Choosing to earn an MFA is a significant investment of time and money. It can be rewarding, but it is not ideal for everyone.
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An MFA should not be pursued by someone who is simply seeking to advance their career. A Master of Fine Arts degree is more for people who would like to hone their craft and who are passionate about both the history of their craft and how to improve the overall medium.
So if you want to pursue a career as an artist, earning an MFA may be a worthwhile investment. Not only will new career opportunities be available to you, but you will also expand your knowledge and develop your skillset in a much more significant manner.
While many of these graduate degree programs are in-person, there are several online MFA degree opportunities from prestigious institutions, which means you can earn one remotely.
Earning an MFA could be your first step toward establishing a lifelong career in any number of artistic and creative fields.
As an editor and writer for Career Karma, Brandon covers developments in higher education, job training, and tech careers. After studying media arts at Kennesaw State University, Brandon has drawn on his extensive experience in technology, writing for companies like Udemy, Staples, LG, and a host of small businesses. Since joining the Career Karma team in February 2020, Brandon has used his technological expertise, as well as his exceptional media skills, to allow individuals to connect with schools, careers, and technology.
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text:
58. mistrzostwa świata w łyżwiarstwie szybkim w wieloboju odbyły się w dniach 22–23 lutego 1964 roku w stolicy Finlandii, Helsinkach. Zawodnicy startowali na naturalnym lodowisku na torze na Olympiastadion po raz piąty (wcześniej w 1939, 1948, 1953 i 1959). Łyżwiarze startowali na czterech dystansach: 500 m, 1500 m, 5000 m, 10000 m. Po raz drugi mistrzem został reprezentant Norwegii Knut Johannesen. O tym, które miejsca zajmowali zawodnicy decydowała mniejsza liczba punktów uzyskana z czterech biegów. Do biegu na 10000 m awansowała tylko najlepsza 16 po trzech dystansach.

Uczestnicy
W zawodach wzięło udział 35 łyżwiarzy z 16 krajów. Sklasyfikowanych zostało 34.

Wyniki
 DNS – nie wystartował, NC – nie zakwalifikował się, f – wywrócił się

Linki zewnętrzne
 Wyniki na stronie Schaats Statistieken.nl

Mistrzostwa świata w łyżwiarstwie szybkim w wieloboju
1964 w sportach zimowych
1964 w Finlandii
Łyżwiarstwo szybkie w Finlandii
Sport w Helsinkach

redpajama_set_name: RedPajamaWikipedia


text:
Moldavia: sequestration of personal computers of 12 young people for posting critical comments online
Written bySami Ben Gharbia
Posted 13 June 2008 13:45 GMT
Read this post in 繁體中文, 简体中文, Malagasy
On June 4th, 2008, the Râşcani District Court from the Moldavian capital of Kishinev has ordered the sequestration of personal computers of about 12 young people who expressed critical opinions against the ruling communist party of the Republic of Moldavia on Internet forums and news portals such as forum.md, torrentsmd.com, desteptarea.info and unimedia.md.
According to Curaj.Net. these young people can be charged for making illegal public "calls for the overthrow of the constitutional order" and threatening the " statality and territorial integrity of the Republic of Moldavia."
"We believe that [a Moldavian] IT company gave the IP addresses of its users," the executive Director of New Media Group, Dumitru Ciorici, told us when asked about how did the police find these young people. "Legally, they have no write to do this because it's freedom of expression and these actions are to intimidate the citizens. Now, the young people are very scared and don't want to talk."
Global Voices Advocacy received a copy of a press release titled "The communist governance from Kishinev is censoring the INTERNET" issued on June 11, 2008 by the Moldavian New Media Group and stating that:
on April 15, 2008 New Media Group received a demarche of the General Prosecutor of the Republic of Moldova, in the investigative case nr. 160, started on 15.01.2008, where UNIMEDIA is forced offering IP address of commentators from the news portal forum www.unimedia.md to the Security and Information Service of Moldova.
On April 21 2008, New Media Group responded officially to the Security and Information Service and General Prosecutor with a letter, which states that NMG does not have the IP addresses of commentators on this site www.unimedia.md for the period January 20 – 14 April 2008 because, in accordance with a previous decision of the Governing Board of NMG from January 22, 2008, the IP addresses of commentators are not stored more than 10 days. After this period they are automatically deleted from the database.
In this sense, New Media Group believes that the appreciation of the Court that the young people have place commentaries on UNIMEDIA is erroneous and lacks legal, since there is no evidence, which would submit the statement that from those IP addresses of these young people were published comments on UNIMEDIA. At the same time, we find that the conclusion of the Court affects the company's image and intimidates UNIMEDIA's visitors.
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Correction: they did not express critical opinion. A critical opinion is something else, it's where you express your disagreement with something. What one of them wrote was "kill the communists!", "let's make a revolution!". I read a lot of comments containing messages like those two. Maybe now they don't seem threattening, but in the whole context, where a lot of people write the same things, looks like something that should be examined by the prosecutors. These actions are illegal according to the Criminal Law adopted by the non-communist parliment, that are written to protect the state.
14 June 2008, 10:01 am
Reply to this
as long as it doesn't have any specificity, it doesn't fall under any of the accusation they were brought.
If they'd wrote something like: "let's kill the communists tomorrow in some city region (even more with time indication)" –> that is a threat.
Now, on the other hand, what has been done against national bolsheviks and those who want to beat up/exterminate pro-Romanians? Those who call for unity with mother russia?
IMHO, this is a very well directed action as the authorities fear they'll lose their seats..
14 June 2008, 3:32 pm
those were critical opinions.
in a democratic state the comments to the news on the news portals are very hard even swering politicians, political parties or their attitudes.
that does not mean someone will aply in the reality that swering… it's that simple.
If you put behind bars the youngsters just becouse this then we have a problem… and yes, the communists should go!
14 June 2008, 11:41 pm
Dear Dan, it's not specified anywhere in the Criminal Law, that a threat is considered an expression that contains the time and place. You should know that almost anywhere in the world, if you tell someone you know something like "I will kill you" in a very serious manner, that can be well considered a threat, and will be investigated by the police, and the prosecutors. By these messages these young citizens have violated the rights and freedom of a whole political party and the government itself. Violation must be prosecuted and judged. Now what happenes here: EVERYBODY in the media is saying: The poor youngsters, all they did was comment (not mentioning absolutely anything about the nature of the comments) and they got the prosecutors on their backs.
So big deal! The prosecutors MUST DO THEIR JOB, they DO HAVE EVIDENCE, according to the Criminal Law. The justice mechanism, meaning the Judges, shall decide who is right and who is wrong. What people do now with the protests, with the massmedia, what they do is influence the process, which is again, ILLEGAL.
ugh, it's hard to write long comments on this website :).
Dan, the authority must not fear anything. If they lose their seats before the ellections – it won't be democracy at all! They have been legally ellected by the majority. We, Dan, on the other hand, represent the minority. How can you speak of democracy if you can't accept the majority's choice, and speak of crimes against the state as of pieces of nothings. IMO it's not something that should be punished by death, life in prisson or other punishments. Most likely they'll get out clean after a looong persecution and some trials. That will be done to intimidate them, YES, to intimidate crime, so that people really watch their thumbs when typing "offensless opinions" on forums. Maybe then I'll stop seing in each political topic dozens of messages like "kill the commies", "down with the crow", which did make sense when we were fighting to protect our rights to study the Romanian language, but not years after that, when no major event happened that could determine us to try to overthrow the government (because those are the meanings of those words).
Actually the sad thing about this abuse is that none of the people whose computers were seized dared to participate at a protest in order to challenge the authorities. This means the goal was achieved – people fear to comment and to discuss the incident.
IN STEAD OF GIVING ADVICES OF STEPS THAT CAN BE TAKEN TO HELP THESE YUNG PEOPLE, YOU DEBATE TE CRIMINAL CODE. IS THERE A WAY THAT THIS CAN BE BROUGHT IN FRONT OF CEDO AND IF THERE IS SOMEONE THAT WOULD LIKE TO REPRESENT THESE PEOPLE AT CEDO B/C I CAN TELL YOU THERE ARE A LOT OF US THAT WOULD SUPPORT MORALLY AND NOT ONLY THIS CAUSE.
WHERE AS FOR THE ROMANIAN GOVERNMENT SHOULD GET MORE INVOLVED INTO THIS MATTER, MAKING SURE THE POPULATION OF MOLDOVA (TARA STRAMOSILOR NOSTRI, MICA BUCATICA DIN ROMANIA MARE) IS AWARE OF WHAT IS HAPPENING IN MOLDOVA


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Aspen Institute eyes dining area expansion
The Aspen Institute recently unveiled plans to expand its dining area at the Aspen Meadows Reception Center by 100 seats to meet the demands of its lodge guests.
The think tank filed an application with the city's Community Development Department last month.
It calls for doubling the seating capacity in the lower dining area and adding 1,816 square feet; 1,449 would be for the dining area, the remaining 367 for a glass connector that will separate the expanded dining area from the existing one.
The patio area, which would complement the new dining area, also would be expanded from 2,689 square feet to 3,012 under the proposal.
"The 100-seat expansion does not create any new demand but is proposed to more efficiently handle the lodge guests already staying in the 98 lodge rooms," states the application, which notes the Aspen Meadows lodge, also part of the institute campus, has occupancy rates between 80 and 98 percent during the peak seasons, and "there is the need to provide food service for 120 to 200 people on site."
Currently, the dining area is used primarily for breakfast and buffet-style lunch. It is separate from Plato's Restaurant, a fine-dining establishment also located in the reception center. It opened in 2005.
A spike in traffic or parking is not anticipated with the expansion, the application states, explaining the enlargement is "proposed to provide more efficient food service and a better guest experience for the lodge guests already staying at the Aspen Meadows."
The institute is exempt from the city's growth-management quota system and affordable-housing mitigation because the City Council deemed it an "essential public facility" in 1991.
The reception center building, designed by Aspen visionaries Herbert Bayer and Fritz Benedict, opened in 1958. It is listed on the city's inventory of historic structures.
The city has yet to do a staff review of the application, said Amy Simon of Historic Preservation. It is set to go before the Historic Preservation Commission on March 23.
The Aspen Institute plays host to a number of high-profile events and educational seminars each year, including the Aspen Ideas Festival, Aspen Security Forum, Hurst Lecture Series, McCloskey Speaker Series and Aspen Words. Guests last year included Sen. John McCain, former President Jimmy Carter and U.S. Attorney General Loretta Lynch.


Ayn Rand (1905-1982) was the great white hope of atheists determined to commandeer rationality. Her philosophy, "Objectivism," was unique in its separation from the sensualists and its rejection of relativism. She did, nevertheless, hold man's "own happiness as the moral purpose of his life," and thus hearkens to John Stuart Mill.


text:
Berechinskiy Vyacheslav
Vyacheslav Berechinskiy is an artist, curator. He was born in 1970 in Leningrad. Since the mid-1980s he is engaged in avant-garde experiments in painting. In the 1990s he wrote electronic music. In 2000, the music album "Chekhov" was released in Japan, Europe and the USA. Since 1993 he has been working in the Research Museum of the Russian Academy of Arts. He graduated from St. Petersburg State University of Fine Arts, the faculty of "Museum studies and monuments protection", specialty "Attribution of museum items". In 2011 together with the project "YOU'R ART" made his pictures "Faces" on t-shirts. May 2013 - co-author and curator of the exhibition "Assa: the last generation of the Leningrad avant-garde" (Titian Hall of the Museum of the Academy of Arts). 2015 - co-author and curator of the exhibition of Timur Novikov in the Cast Department of the Scientific Research Museum of the Academy of Arts, 2015-2016 - co-author and curator of the exhibition of Pavel Peppershtein's "Hunters for the marble heads "in the Imagery Department of the Academy's Research Museum Arts. Laureate of the award in the field of contemporary art. S. Kuryokhin 2015 in the nomination "Best curatorial project". Participant in numerous exhibitions, of the last - the exhibition «Faces. 1990-s» in YOU'R ART gallery (2013), "City. Faces "in the gallery Art Muza (2014), exhibition" Meetings" in the museum-apartment of I. I. Brodsky (2015). Works are in private collections in Russia, Europe and the United States. The artist works with a characteristic manner of his work, referring to the traditions of modernism in the first half of the 20th century. Particularly influenced by its formation was the art of post-impressionism, artists of the Paris School and expressionists of the first wave.


They met with the staff at 11 p.m. on Thursday and said that Christine Brennan, executive editor of Voice Media Group, had instructed them to cut five positions from the 20-person staff. Rather than carrying out the cuts, they resigned and left immediately.
Bourne succeeded Tony Ortega as editor of the Voice in late November. We've reached out to him for comment and will update when we hear back.


"What does she want?" Derwen called.
The roommate—Edie still hadn't learned their names, though they had obviously learned hers—turned back to Edie and raised her eyebrows.
"You got that?" the roommate called.
Edie tried to look into the room over Derwen's roommate's shoulder. It seemed very dark in there, especially compared to the corner triple in Sayer, but maybe they just had their blinds all closed. Anyway, with the rain, the triple in Sayer probably wasn't very bright right now either.
"Do you like living in a triple?" she asked.
"Seems like a good idea," Edie said, nodding as Derwen came out of the room.
"Have a good brunch," the roommate said before shutting the door.
"It's raining pretty hard out there." Edie gestured at her rain jacket and boots.
"Raining?" Derwen's eyes opened wide, her whole posture straightening. Without saying anything else to Edie, she ran down the steps. Edie started to go after her, but heard a noise from above, and looked up to see Corrie, Annie, Dawn, and Rico coming down the steps.
"Derwen not coming?" Corrie asked.
She didn't find Derwen until she'd gotten outside, tugging her hood up and almost missing some movement out of the corner of her eye. Derwen was spinning around in the grass, the rain plastering her clothes and hair to her skin. Edie tried not to stare. "Sarah" wasn't her type, but she was certainly not bad-looking with her clothes stuck against her figure.
"What are you doing?" Corrie called to Derwen, raising her voice to be heard over the rain.
"It's rain!" Derwen called back. She spun one more time, her arms outstretched, then skipped back to the path to walk with them.
"What did it look like?" Derwen asked. Edie was curious, too. She remembered that day very well, and she'd never seen Derwen turn into a plant… but she'd also been unable to see any of the things Dawn Saw.
They swiped into the dining hall and followed Corrie to an empty table. Edie hung her jacket on the back of a chair. Derwen seemed to be oblivious to the way she was tracking water and mud all over the floor, or the looks some people were giving her.
She headed toward the food, and Edie followed, deciding what she wanted to eat. It wasn't until she had gotten in line for fresh pancakes that she realized Derwen had never actually answered Dawn's question.
Ha, you've described Derwen well!