text:
nature cell biology
A p53–phosphoinositide signalosome regulates nuclear AKT activation
Mo Chen ORCID: orcid.org/0000-0002-3274-58891,
Suyong Choi1,
Tianmu Wen1,
Changliang Chen1,2,
Narendra Thapa1,
Jeong Hyo Lee ORCID: orcid.org/0000-0002-1848-53471,2,
Vincent L. Cryns ORCID: orcid.org/0000-0003-0355-22682 na1 &
Richard A. Anderson ORCID: orcid.org/0000-0001-6265-83591 na1
Nature Cell Biology volume 24, pages 1099–1113 (2022)Cite this article
Cell signalling
Phosphoinositol signalling
Tumour-suppressor proteins
The tumour suppressor p53 and PI3K–AKT pathways have fundamental roles in the regulation of cell growth and apoptosis, and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear AKT activation through a p53-dependent mechanism that is distinct from the canonical membrane-localized PI3K–AKT pathway. Following genotoxic stress, a nuclear PI3K binds p53 in the non-membranous nucleoplasm to generate a complex of p53 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which recruits AKT, PDK1 and mTORC2 to activate AKT and phosphorylate FOXO proteins, thereby inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear AKT in an on/off fashion following stress, whereas mutant p53 dose-dependently stimulates high basal AKT activity. The p53–PtdIns(3,4,5)P3 complex is dephosphorylated to p53–phosphatidylinositol 4,5-bisphosphate by PTEN to inhibit AKT activation. The nuclear p53–phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, which underscores its therapeutic relevance.
Fig. 1: p53 interacts with nuclear AKT and regulates its activation.
Fig. 2: p53 recruits upstream AKT-activating kinases in the nucleus.
Fig. 3: The nuclear p53–AKT complex targets FOXO proteins.
Fig. 4: p53 binds PtdIns(3,4,5)P3, and IPMK and PTEN regulate the interconversion of nuclear p53–PIPn complexes.
Fig. 5: p53–PtdIns(3,4,5)P3 recruits and activates the nuclear AKT pathway.
Fig. 6: The p53–PIPn signalosome links the nuclear PI3K–AKT pathway to DNA repair and cell survival.
Fig. 7: The interaction of p53 with AKT depends on PIPn and is mediated by the C terminus of p53.
Fig. 8: Regulation of the nuclear AKT pathway by p53 depends on the PIPn-binding motif of p53.
All data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
Choi, S. et al. Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases. Nat. Cell Biol. 18, 1324–1335 (2016).
Thapa, N., Horn, H. T. & Anderson, R. A. Phosphoinositide spatially free AKT/PKB activation to all membrane compartments. Adv. Biol. Regul. 72, 1–6 (2019).
Fruman, D. A. et al. The PI3K pathway in human disease. Cell 170, 605–635 (2017).
Hoxhaj, G. & Manning, B. D. The PI3K–AKT network at the interface of oncogenic signalling and cancer metabolism. Nat. Rev. Cancer 20, 74–88 (2020).
Manning, B. D. & Toker, A. AKT/PKB signaling: navigating the network. Cell 169, 381–405 (2017).
Stokoe, D. et al. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277, 567–570 (1997).
Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor–mTOR complex. Science 307, 1098–1101 (2005).
Ebner, M., Lucic, I., Leonard, T. A. & Yudushkin, I. PI(3,4,5)P3 engagement restricts Akt activity to cellular membranes. Mol. Cell 65, 416–431.e6 (2017).
Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα. Curr. Biol. 7, 261–269 (1997).
Martelli, A. M. et al. The emerging multiple roles of nuclear Akt. Biochim. Biophys. Acta 1823, 2168–2178 (2012).
Meier, R., Alessi, D. R., Cron, P., Andjelkovic, M. & Hemmings, B. A. Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bβ. J. Biol. Chem. 272, 30491–30497 (1997).
Andjelkovic, M. et al. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272, 31515–31524 (1997).
Borgatti, P. et al. Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin. J. Cell. Physiol. 196, 79–88 (2003).
Wang, R. & Brattain, M. G. AKT can be activated in the nucleus. Cell Signal. 18, 1722–1731 (2006).
Bozulic, L., Surucu, B., Hynx, D. & Hemmings, B. A. PKBα/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol. Cell 30, 203–213 (2008).
Boehme, K. A., Kulikov, R. & Blattner, C. p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK. Proc. Natl Acad. Sci. USA 105, 7785–7790 (2008).
Muller, P. A. & Vousden, K. H. p53 mutations in cancer. Nat. Cell Biol. 15, 2–8 (2013).
Kastenhuber, E. R. & Lowe, S. W. Putting p53 in context. Cell 170, 1062–1078 (2017).
Choi, S., Chen, M., Cryns, V. L. & Anderson, R. A. A nuclear phosphoinositide kinase complex regulates p53. Nat. Cell Biol. 21, 462–475 (2019).
Xu, R. et al. Inositol polyphosphate multikinase is a coactivator of p53-mediated transcription and cell death. Sci. Signal. 6, ra22 (2013).
Freeman, D. J. et al. PTEN tumor suppressor regulates p53 protein levels and activity through phosphatase-dependent and -independent mechanisms. Cancer Cell 3, 117–130 (2003).
Blind, R. D., Suzawa, M. & Ingraham, H. A. Direct modification and activation of a nuclear receptor-PIP2 complex by the inositol lipid kinase IPMK. Sci. Signal. 5, ra44 (2012).
Blind, R. D. et al. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1. Proc. Natl Acad. Sci. USA 111, 15054–15059 (2014).
Huang, Y. et al. Global tumor protein p53/p63 interactome: making a case for cisplatin chemoresistance. Cell Cycle 11, 2367–2379 (2012).
Liu, Q., Turner, K. M., Alfred Yung, W. K., Chen, K. & Zhang, W. Role of AKT signaling in DNA repair and clinical response to cancer therapy. Neuro Oncol. 16, 1313–1323 (2014).
Mayer, I. A. & Arteaga, C. L. The PI3K/AKT pathway as a target for cancer treatment. Annu. Rev. Med. 67, 11–28 (2016).
Wang, Y. H. et al. DNA damage causes rapid accumulation of phosphoinositides for ATR signaling. Nat. Commun. 8, 2118 (2017).
Casamayor, A., Morrice, N. A. & Alessi, D. R. Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. Biochem. J. 342, 287–292 (1999).
Moon, Z., Wang, Y., Aryan, N., Mousseau, D. D. & Scheid, M. P. Serine 396 of PDK1 is required for maximal PKB activation. Cell Signal. 20, 2038–2049 (2008).
Liu, P. et al. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discov. 5, 1194–1209 (2015).
Webb, A. E. & Brunet, A. FOXO transcription factors: key regulators of cellular quality control. Trends Biochem. Sci. 39, 159–169 (2014).
Chung, Y. M. et al. FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage. Nat. Commun. 3, 1000 (2012).
Wang, F. et al. Biochemical and structural characterization of an intramolecular interaction in FOXO3a and its binding with p53. J. Mol. Biol. 384, 590–603 (2008).
Fu, W. et al. MDM2 acts downstream of p53 as an E3 ligase to promote FOXO ubiquitination and degradation. J. Biol. Chem. 284, 13987–14000 (2009).
Huang, H. & Tindall, D. J. Regulation of FOXO protein stability via ubiquitination and proteasome degradation. Biochim. Biophys. Acta 1813, 1961–1964 (2011).
Zhang, X., Tang, N., Hadden, T. J. & Rishi, A. K. Akt, FoxO and regulation of apoptosis. Biochim. Biophys. Acta 1813, 1978–1986 (2011).
Semenas, J. et al. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer. Proc. Natl Acad. Sci. USA 111, E3689–E3698 (2014).
Currie, R. A. et al. Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1. Biochem. J. 337, 575–583 (1999).
Calleja, V. et al. Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo. PLoS Biol. 5, e95 (2007).
Chen, M. et al. The nuclear phosphoinositide response to stress. Cell Cycle 19, 268–289 (2020).
Barlow, C. A., Laishram, R. S. & Anderson, R. A. Nuclear phosphoinositides: a signaling enigma wrapped in a compartmental conundrum. Trends Cell Biol. 20, 25–35 (2010).
Boronenkov, I. V., Loijens, J. C., Umeda, M. & Anderson, R. A. Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol. Biol. Cell 9, 3547–3560 (1998).
Sharma, A., Singh, K. & Almasan, A. Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol. Biol. 920, 613–626 (2012).
Liu, L. et al. DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2–ECT2 pathway. Sci. Signal. 15, eabh2290 (2022).
Plo, I. et al. AKT1 inhibits homologous recombination by inducing cytoplasmic retention of BRCA1 and RAD51. Cancer Res. 68, 9404–9412 (2008).
Liu, P. et al. Akt-mediated phosphorylation of XLF impairs non-homologous end-joining DNA repair. Mol. Cell 57, 648–661 (2015).
Xu, N., Lao, Y., Zhang, Y. & Gillespie, D. A. Akt: a double-edged sword in cell proliferation and genome stability. J. Oncol. 2012, 951724 (2012).
Toulany, M. et al. Akt promotes post-irradiation survival of human tumor cells through initiation, progression, and termination of DNA-PKcs-dependent DNA double-strand break repair. Mol. Cancer Res. 10, 945–957 (2012).
Huang, W. C. & Chen, C. C. Akt phosphorylation of p300 at Ser-1834 is essential for its histone acetyltransferase and transcriptional activity. Mol. Cell. Biol. 25, 6592–6602 (2005).
Liu, S. L. et al. Quantitative lipid imaging reveals a new signaling function of phosphatidylinositol-3,4-bisphophate: isoform- and site-specific activation of Akt. Mol. Cell 71, 1092–1104.e5 (2018).
Schober, P., Boer, C. & Schwarte, L. A. Correlation coefficients: appropriate use and interpretation. Anesth. Analg. 126, 1763–1768 (2018).
Miyaguchi, Y., Tsuchiya, K. & Sakamoto, K. P53 negatively regulates the transcriptional activity of FOXO3a under oxidative stress. Cell Biol. Int. 33, 853–860 (2009).
Cocco, L. et al. Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem. J. 248, 765–770 (1987).
Kikani, C. K. et al. Proliferative and antiapoptotic signaling stimulated by nuclear-localized PDK1 results in oncogenesis. Sci. Signal. 5, ra80 (2012).
Shiraishi, I. et al. Nuclear targeting of Akt enhances kinase activity and survival of cardiomyocytes. Circ. Res. 94, 884–891 (2004).
Fu, W. & Hall, M. N. Regulation of mTORC2 signaling. Genes (Basel) 11, 1045 (2020).
Thapa, N. et al. Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4. Nat. Cell Biol. 22, 1357–1370 (2020).
Chen, M. et al. The specificity of EGF-stimulated IQGAP1 scaffold towards the PI3K–Akt pathway is defined by the IQ3 motif. Sci. Rep. 9, 9126 (2019).
Tzivion, G., Dobson, M. & Ramakrishnan, G. FoxO transcription factors; regulation by AKT and 14-3-3 proteins. Biochim. Biophys. Acta 1813, 1938–1945 (2011).
Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li–Fraumeni syndrome. Cell 119, 847–860 (2004).
Muller, P. A. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 25, 304–317 (2014).
Schulz-Heddergott, R. et al. Therapeutic ablation of gain-of-function mutant p53 in colorectal cancer inhibits Stat3-mediated tumor growth and invasion. Cancer Cell 34, 298–314.e7 (2018).
Hanel, W. et al. Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis. Cell Death Differ. 20, 898–909 (2013).
Iggo, R., Gatter, K., Bartek, J., Lane, D. & Harris, A. L. Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet 335, 675–679 (1990).
Yemelyanova, A. et al. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinoma: an immunohistochemical and nucleotide sequencing analysis. Mod. Pathol. 24, 1248–1253 (2011).
Muller, P. A. et al. Mutant p53 drives invasion by promoting integrin recycling. Cell 139, 1327–1341 (2009).
Andre, F. et al. Alpelisib for PIK3CA-mutated, hormone receptor-positive advanced breast cancer. N. Engl. J. Med. 380, 1929–1940 (2019).
Mellman, D. L. et al. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs. Nature 451, 1013–1017 (2008).
Malin, D., Chen, F., Schiller, C., Koblinski, J. & Cryns, V. L. Enhanced metastasis suppression by targeting TRAIL receptor 2 in a murine model of triple-negative breast cancer. Clin. Cancer Res. 17, 5005–5015 (2011).
Asmari, M., Ratih, R., Alhazmi, H. A. & El Deeb, S. Thermophoresis for characterizing biomolecular interaction. Methods 146, 107–119 (2018).
Costes, S. V. et al. Automatic and quantitative measurement of protein–protein colocalization in live cells. Biophys. J. 86, 3993–4003 (2004).
Chen, M., Horn, H. T., Wen, T., Cryns, V. L. & Anderson, R. A. Assessing in situ phosphoinositide–protein interactions through fluorescence proximity ligation assay in cultured cells. Methods Mol. Biol. 2251, 133–142 (2021).
We thank A. Audhya, M. Sussman, A. Gasch and W. Peti for discussions and comments, and L. Rodenkirch for technical support. This work was supported in part by a National Institutes of Health grant (R35GM134955 to R.A.A.), Department of Defense Breast Cancer Research Programme grants (W81XWH-17-1-0258 to R.A.A.; W81XWH-17-1-0259 and W81XWH-21-1-0129 to V.L.C.) and a grant from the Breast Cancer Research Foundation (V.L.C.).
These authors contributed equally: Vincent L. Cryns, Richard A. Anderson.
University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA
Mo Chen, Suyong Choi, Tianmu Wen, Changliang Chen, Narendra Thapa, Jeong Hyo Lee & Richard A. Anderson
Department of Medicine, University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA
Changliang Chen, Jeong Hyo Lee & Vincent L. Cryns
Mo Chen
Suyong Choi
Tianmu Wen
Changliang Chen
Narendra Thapa
Jeong Hyo Lee
Vincent L. Cryns
Richard A. Anderson
M.C., S.C., T.W., C.C., N.T., V.L.C. and R.A.A. designed the experiments. M.C., S.C., T.W., C.C., N.T. and J.H.L. performed the experiments. M.C., V.L.C. and R.A.A. wrote the manuscript.
Correspondence to Vincent L. Cryns or Richard A. Anderson.
Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
Extended data
Extended Data Fig. 1 p53 associates with active Akt in the nucleus and their interaction is enhanced by stress.
a,b, IF staining against AKT and p53 in MDA-MB-231 cells treated with 30 µM cisplatin for 24 h. The nuclear levels of Akt (b) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. c, WB analysis of pAktT308/S473 in MDA-MB-231 cells after 24 h starvation and treatment with 10 µM mTOR inhibitor Torin 1/KU-0063794/Palomid 529 or Akt inhibitor Miransertib/MK-2206/Tricirbine/Capivasertib/Ipatasertib, followed by EGF stimulation. Data shown represent 3 independent experiments. d–g, IF staining of pAktT308/S473 in MDA-MB-231 cells treated with 30 µM cisplatin and the mTOR inhibitor Torin 1 (10 µM) or the Akt inhibitor Miransertib (10 µM) for 24 h. The nuclear pAktT308/S473 levels were quantified (e,g). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. h, WB analysis of pAktS473 in MDA-MB-231 cells after 24 h starvation and treatment with 10 µM PI3Kα inhibitor alpelisib, 2 µM pan-PI3K inhibitor buparlisib, or vehicle, followed by EGF stimulation. N = 3 independent experiments. i,j, IF staining of pAktS473 in MDA-MB-231 cells treated with 30 µM cisplatin and the PI3Kα inhibitor alpelisib (10 µM) or the pan-PI3K inhibitor buparlisib (2 µM) for 24 h. Nuclear pAktS473 levels were quantified (j). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. k–n, IF staining of p53 and pAktS473 in MDA-MB-231 cells treated with 100 µM tBHQ, 100 µM hydroxyurea, 100 µM etoposide, 30 µM cisplatin, or vehicle for 24 h. The nuclear levels of pAktS473 (l) and p53 (m) and their colocalization (n) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. o–r, IF staining of p53 and pAktS473 in A549 and MDA-MB-231 cells treated with 30 µM cisplatin for 24 h. The nuclear levels of p53 and pAktS473 (q) and their colocalization (r) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. s–u, Two-panel section (s) of confocal IF staining against p53 and pAktS473 in A549 cells treated with 30 µM cisplatin for 24 h. The nuclear and cytosolic colocalization of p53 and pAktS473 was quantified (u). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 2 p53 binds nuclear Akt and regulates its activation.
a, PLA of p53-Akt/pAktT308/pAktS473 in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in Fig. 1g. b,c PLA of p53-pAktS473 in MDA-MB-231 cells treated with 100 µM tBHQ, 100 µM hydroxyurea, 100 µM etoposide, 30 µM cisplatin, or vehicle for 24 h. The nuclear PLA foci were quantified (c). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. d, 3D sections of PLA between p53 and pAktS473 overlaid with Lamin A/C in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. Each frame of the 3D sections was over a 0.2 μm thickness. Data shown represent 3 independent experiments. e, Co-IP of p53 and Akt pathway components from MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. N = 3 independent experiments. f, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs against p53 for 48 h. Cell lysates were then analyzed by WB. Representative data of three independent experiments are shown. g,h Confocal IF staining of p53 and pAktT308 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53. The nuclear p53 and pAktT308 levels normalized to siCon transfected cells were quantified (h). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 3 p53 associates with PDK1, FOXOs and IPMK.
a, PLA of p53-pPDK1S241/pPDK1S396 in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in Fig. 2e. b–e, Confocal images of IF staining against PDK1/Sin1/FOXO3 and p53 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclear levels of PDK1 (c), Sin1 (d), FOXO3 (e) normalized to vehicle treated cells were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. f, Quantification of nuclear pFOXO3S253 in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h (levels normalized to vehicle treated cells). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. Representative images are shown in Fig. 3e. g, PLA of p53-FOXOs in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in Fig. 3g. h, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs against p53. 48 h later, cell lysates were analyzed by WB. Representative data of three independent experiments are shown. i, Co-IP of IPMK with wild-type p53 and mutant p53 from HCT116 and Cal33 cells, respectively, treated with 30 µM cisplatin or vehicle for 24 h. Representative data of three independent experiments are shown. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 4 p53 associates with PI4,5P2 and PI3,4,5P3 in the nucleus.
a–c, Confocal IF staining for p53 and PI3,4,5P3 in MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear level of PI3,4,5P3 normalized to vehicle treated cells (b) and its colocalization with p53 (c) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. d,e, PLA of p53-PI4,5P2/PI3,4,5P3 in A549, MCF-10A, MDA-MB-231, and SUM1315 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear PLA foci were quantified (e). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. f–h, PLA of p53-PI4,5P2/PI3,4,5P3 in MDA-MB-231 cells treated with 100 µM tBHQ, 100 µM hydroxyurea, 100 µM etoposide, 30 µM cisplatin, or vehicle for 24 h. The nuclear PLA foci were quantified (g,h). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. i,j PLA of p53-PI3,4,5P3 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. The nuclear PLA foci were quantified (j). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. k, 3D sections of PLA between p53 and PI3,4,5P3 overlaid with Lamin A/C in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin treatment for 24 h. Each frame of the 3D sections was over a 0.2 μm thickness. Data shown represent 3 independent experiments. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 5 IPMK and PTEN associate with and regulate p53-PIPn complexes and FOXO phosphorylation.
a–c, Confocal IF staining against p53 and IPMK in MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear level of IPMK normalized to vehicle treated cells (b) and its colocalization with p53 (c) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. d–g, Confocal IF staining against p53 and PTEN in A549 or MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear level of PTEN normalized to vehicle treated cells (f) and its colocalization with p53 (g) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. h–j, PLA of p53-IPMK/PTEN in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified (i,j). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. k-l, PLA of p53-PI4,5P2/PI3,4,5P3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, or PTEN (k). See quantification in Fig. 4h, i. The knockdown efficiency was validated by WB (l). m,n, Confocal IF staining of p53 and pFOXO3S253 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. The nuclear pFOXO3S253 levels normalized to siCon transfected cells were quantified (n). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. o,p, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. 24 h later, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for PLA between p53 and pFOXO3S253. The nuclear PLA foci were quantified (p). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 6 The nuclear p53-PIPn signalosome regulates DNA damage repair and cell survival.
a, Confocal IF staining against PI4,5P2 and pPDK1S396/pFOXO3S253 in MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. See Fig. 6a, b for the nuclear region and quantification of the nuclear colocalization of PI4,5P2 and pPDK1S396/pFOXO3S253. b, Confocal and STED super-resolution images of IF staining against p53, pAktS473, and the DNA DSB marker γ-H2A.X in MDA-MB-231 and A549 cells treated with 30 µM cisplatin or vehicle for 24 h. See quantification in Fig. 6d. c–f, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, or IPMK. 24 h later, cells were treated with 30 µM cisplatin, 10 µM mTOR inhibitor Torin1, 10 µM Akt inhibitor Miransertib or vehicle for 24 h before being processed for IF staining of p53 and pDNA-PKS2056/phosphor-p300S1834. The nuclear levels of p53, pDNA-PKS2056 and phospho-p300S1834 normalized to vehicle treated cells were quantified (d,f). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. g–i, MDA-MB-231, HS578T, and MDA-MB-468 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. 24 h later, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for MTT cell viability assay. Cell viability was normalized to siCon transfected cells. N = 3 independent experiments. j, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. 24 h later, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for Caspase-3 activity assay. Activity was normalized to siCon transfected cells. N = 3 independent experiments. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 7 p53 interaction with Akt requires the p53 C-terminal PIPn-binding motif and correlates with nuclear Akt activation.
a, Schematic representation of p53 domains (top). 1 or 6 basic residues in the CTD were mutated to glutamine (Q) to generate the PIPn-binding defective 6Q or 379Q mutants, respectively. b,c, The interaction of Akt with mutant p53 is regulated by the PIPn-binding motif of p53. HEK293FT cells transfected with HA-tagged Akt1 and FLAG-tagged mutant p53R175H, p53R248Q, or their corresponding 6Q or 379Q mutants were processed for IP with HA antibody, and then analyzed by WB. N = 3 independent experiments. d–g, A549 cells treated with 30 µM cisplatin for 24 h were processed for IF staining of p53 and pAktS473. The nuclear levels of p53 and pAktS473 were quantified. The low (L) and high (H) levels of nuclear p53 were determined based on the average p53 levels in the corresponding groups (e). The correlation between nuclear p53 and pAktS473 was determined by Pearson's r (f,g). N = 120 cells pooled from 3 independent experiments; 40 cells per experiment. h–k, MDA-MB-231 cells treated with 30 µM cisplatin for 24 h were processed for IF staining against p53 and pAktS473. The nuclear levels of p53 and pAktS473 were quantified. The low (L) and high (H) levels of nuclear p53 were determined based on the average of the p53 levels in the corresponding groups (i). The correlation between the nuclear p53 and pAktS473 was determined by Pearson's r (j,k). N = 120 cells pooled from 3 independent experiments; 40 cells per experiment. l–o, A549 cells were transfected with Flag-tagged p53R175H and then 24 h later treated with 30 µM cisplatin for 4 h. Cells were then processed for IF staining of Flag-tagged p53R175H and pAktS473. The nuclear pAktS473 levels in Flag-negative and Flag-positive cells were quantified (m). The correlation between the nuclear levels of Flag-tagged p53R175H and pAktS473 was determined by Pearson's r (n,o). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. p, A549 wild-type and p53 KO cells were treated with 30 µM cisplatin for 24 h and analyzed by WB. Data shown represent 3 independent experiments. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.
Extended Data Fig. 8 Stress-induced Akt, DNA-PK, and p300 phosphorylation by p53 is dependent on its PIPn-binding motif.
a–c, A549 p53 KO cells were transiently transfected with Flag-tagged p53R175H or the corresponding 6Q or 379Q mutant. 24 h later, cells were treated with 30 µM cisplatin for an additional 24 h. A549 WT cells were treated with 30 µM cisplatin or vehicle for 24 h. The cells were then fixed and processed for IF staining of p53, pAktT308, pDNA-PKS2056, and phospho-p300S1834. The nuclear pAktT308 (a), pDNA-PKS2056 (b), and phospho-p300S1834 (c) levels in A549 WT cells, p53 KO cells, and Flag-tagged p53R175H-positive cells normalized to A549 WT cells treated with cisplatin were quantified as indicated. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. Scale bar: 5 µm. d, A549 p53 KO cells were transiently transfected with Flag-tagged p53R175H, p53R248Q or their corresponding 6Q or 379Q mutant. 24 h later, cells were treated with 30 µM cisplatin or vehicle for an additional 24 h before being processed for crystal violet (CV) viability assay. Cell viability was normalized to untransfected cells treated with vehicle. N = 3 independent experiments. e,f, A549 p53 KO cells were transiently transfected with Flag-tagged mutant p53R175H, p53R248Q or their corresponding 6Q or 379Q mutant. 24 h later, cells were serum-starved for an additional 24 h and then scored for invasion through Laminin-coated transwell inserts with 8 μm pores using 10 ng/ml EGF as a chemoattractant for 16 h. The invading cells at the insert bottom were stained with crystal violet, imaged, and quantified based on the extracted dye using a plate reader. Invasion was normalized to untransfected cells treated with EGF. N = 3 independent experiments. Scale bar: 100 µm. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test.
Reporting Summary
Supplementary Table 1
The binding affinity of the nuclear p53–PIPn signalosome components with p53. The interaction of recombinant fluorescently labelled p53 and non-labelled signalosome components was quantitated by MST assay. A constant concentration of fluorescently labelled p53 (target, 5 nM) was incubated with increasing concentrations of non-labelled ligands to calculate the binding affinity. The binding affinities determined by MST are shown as indicated Kd values. MST was performed using a Monolith NT.115 pico, and the binding affinity was autogenerated by MO. Control v.1.6 software. Data are presented as the mean ± s.d; n = 3.
Three-dimensional (3D) reconstitution of the p53–pAKTS473 complex in the nucleoplasm of MDA-MB-231 cells under basal conditions. MDA-MB-231 cells were treated with vehicle for 24 h before being processed for PLA between p53 and pAKTS473 (red). Lamin A/C stained the nuclear envelopes (green), and the nuclei were counterstained with DAPI (blue). The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2-μm thickness. The video of the 3D reconstitution of a representative cell from three independent experiments was generated using LASX.
3D reconstitution of the p53–pAKTS473 complex in the nucleoplasm of MDA-MB-231 cells under stress. MDA-MB-231 cells were treated with 30 µM cisplatin for 24 h before being processed for PLA between p53 and pAKTS473 (red). Lamin A/C stained the nuclear envelopes (green), and the nuclei were counterstained with DAPI (blue). The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2-μm thickness. The video of the 3D reconstitution of a representative cell from three independent experiments was generated using LASX.
Source Data Fig. 1
Unprocessed western blots.
Statistical source data.
Source Data Extended Data Fig. 1
Chen, M., Choi, S., Wen, T. et al. A p53–phosphoinositide signalosome regulates nuclear AKT activation. Nat Cell Biol 24, 1099–1113 (2022). https://doi.org/10.1038/s41556-022-00949-1
Issue Date: July 2022
Nature Cell Biology (Nat Cell Biol) ISSN 1476-4679 (online) ISSN 1465-7392 (print)
Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.
Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer


text:
GU-Q Faculty Fellowship 2015-2016 CIRS GU-Q Faculty Fellow
Firat Oruc is Assistant Professor of English and Humanities at GU-Q. He received his Ph.D. in Literature from Duke University in 2010. His teaching specialties include contemporary global literature, 20th century Anglophone writing, literatures of the Middle East, and world cinema. Before joining SFS-Q, he taught in the Comparative Literary Studies program at Northwestern University (2011-2013) and the departments of English and Comparative Literature at the University of California, Los Angeles (2010-2011). His scholarly interests center on the intersections of cultural globalization and transnationalism, postcolonial studies, world literature theory, and translation studies. His recent work has appeared in literary criticism journals such as English Language Notes, Criticism and Postcolonial Text. His current book project is a comparative study of world literature and institutions of translation in Turkey, Egypt, and Iran.
My project examines the connections between national identity and translation by investigating the construction of a state-sponsored world literature canon as a national "culture planning" project in Turkey. With the foundation of the Turkish Republic in 1923, President Mustafa Kemal Atatürk introduced a series of radical cultural reforms aimed to leave the Ottoman/Islamic past irrevocably behind. These measures promoted identification with Europe as "the universal civilization" while simultaneously emphasizing Turkish identity as the foundation for the new nation. One of the most significant Kemalist revolutions was dil devrimi, or "the language revolution," which mandated that all "old, fossilized" Arabic and Persian words be abandoned completely and replaced by un-Turkish equivalents. In this project, I focus on the ways in which this architectonic language revolution shaped the translation of "World Classics" into modern Turkish in line with a strong commitment to the ideals of humanism and secularism.

redpajama_set_name: RedPajamaCommonCrawl


As I've said, have never met a FB page or a blog I didn't like. The Inbox is overflowing every morning, but, that is how I like it!
I'm retired now but even when working, the first thing I do in the morning is check email–don't we all.
This is a quick skim; most can be deleted or starred for later.
FYI: Most of my Internet reading is done on the iPad in the Safari browser; I don't care for the Facebook app as I can't do what is shown below.
But how do I keep up with it all ????? "They Say" one shouldn't spend more than 15 minutes a day on Social Media. Yeah, well, that isn't going to happen! So, I try to be smart about how I go about it and weed out the garbage while I'm at it.
Also, I find myself reading fewer and fewer books as my reading is many shorter articles/pdfs from The Web.
2. The post I want to read is now open in a new tab; I'll go back and read later. Meanwhile, I can go back to my Page list (neucarol) and continue skimming.
The examples below are from my email but works same if a web page.
Note: I have two groups that Gmail automatically assigns–Primary & Social. Gmail figures out which is which based on whether it is from FB, or another social media site–probably–and I haven't had an issue. Can also set up more if I want. It's a good way to start the sort/save process.and click on symbol.
Click on the symbol in the lower right corner to save to Safari. Note there are also multiple options to Follow the blog; always a good thing!
Next, in the upper right corner, open the symbol which now gives lots of save options. My fave is the Save PDF to iBooks; it's always there in iCloud; can always delete. Also like Bookmark and Add to Reading List.
Hope this helps you save and bookmark on the web. There's nothing more frustrating than knowing you read it–somewhere–and saved it–somewhere–but Where!
What's your favorite way to save and organize web stuff?
Thanx to Cloning for Couture for serving as an example–Beach Chic Wedding–a good read, fun fabric, sewing techniques, draping–something for everyone!
This entry was posted in GREAT IDEA and tagged add to reading list, bookmark, facebook, iPad, Open page in Safari, Save PDF to iBooks. Bookmark the permalink.


Hello crocheters, today I share this beautiful pattern crochet potholder.
A crochet piece very beautiful and friendly that will leave your table even more delicate and beautiful.
Below you will find two videos with the standard of this piece of crochet, an easy pattern that any beginner will be able to do. Good job!


text:
All Categories Landscapes Wildlife Marine & Coastal Still Life Country Traditions Flowers & Flora British Seasons
Malcolm Coward
Malcolm is internationally recognised as one of England's leading equestrian artists. Winner of numerous awards at the Society of Equestrian Artists in London, he is the only artist to win the coveted President's medal three times. Artists are only allowed to win this award once every three years. He has also had his work accepted at the Royal Society of Marine Artists, the Royal Institute of Oil Painters and the Royal Birmingham Society.
On the continent his work has been selected regularly for the French equestrian exhibition which starts at the Cadre Noir.
In America he has been invited to exhibit at the American Society of Equestrian Artists, at the Centenary Hunting Exhibition and a three page article about him was published in Equestrian Images. He has exhibited in many other countries over the years, including Sweden at the World Equestrian Games in Stockholm, at Lifestyle Europe in Tokyo, at the Salon du Cheval in Paris, at the Feria in Jerez, Spain and at the Fiera di Verona in Italy.
Malcolm has had several very successful one man exhibitions in England and was featured in a TV programme on sporting paintings. He has also had a number of very successful limited edition prints published after his work, many of which are now collectors' items. In 1996 he was chosen as the artist to illustrate the Injured Jockeys' Christmas card. This subsequently appeared in the Sunday Times Style supplement. He also had two paintings selected to appear in the Injured Jockeys' calendar. Malcolm, who was born in Malton, North Yorkshire, lives for painting and his animals, which are constantly observed and contribute to his eye for anatomy and movement. His work is also noted for its tremendous use of colour and light and his powerful, yet sensitive brush strokes. He is able to paint horses in movement equally as well as portraiture. He spends much of his life travelling the world, watching horses of various types.
A book about him and his work titled 'Malcolm Coward, Horse Painter' was published in 2007, illustrating over 100 of his paintings in colour.
Country Traditions
British Seasons
Marine & Coastal
British Seasons 18
Country Traditions 24
Flowers & Flora 1
Marine & Coastal 11
Still Life 9
Most payment methods accepted


text:
History of Mining
Production, Reserves
County Data
Next Page--Table of Contents
Coal Resources of the Wabaunsee Group in Eastern Kansas
By Walter H. Schoewe
Originally published in 1946 as Kansas Geological Survey Bulletin 63. This is, in general, the original text as published. The information has not been updated.
The Wabaunsee coal-bearing rocks constitute the youngest or uppermost group of Pennsylvanian strata in Kansas. The rocks crop out in a northeast-southwest belt, approximately 3,100 square miles in area, in the eastern part of the state, extending from Doniphan, Brown, and Nemaha counties on the north to the Kansas-Oklahoma state line in Chautauqua and Cowley counties on the south. At least 6 of the 12 coal beds in the Wabaunsee group have been mined at one time or another within the last 75 years. Nodaway, the lowermost coal, is by far the most important of the coals mined, and it is the only one of commercial importance at the present time. Including the 1944-45 coal-mining season, a minimum cumulative total of 11,783,464 tons of Nodaway coal had been mined, of which 97.2 percent came from Osage County. The Elmo coal, of which more than 200,000 tons has been produced, is the second most important of the Wabaunsee coals. The cumulative tonnage of all coals of the Wabaunsee group mined is 12,017,737 tons, which is 4.8 percent of the total coal production in Kansas. There are 186,680,000 tons of proved Wabaunsee reserve coal, of which 128,050,000 tons is Nodaway, 47,560,000 tons Elmo, and 11,070,000 tons other miscellaneous coals. The greatest Nodaway proved coal reserve, 77,670,000 tons, is found in Osage County; the greatest Elmo proved coal reserve, in Brown County, amounts to 23,160,000 tons. The proved Wabaunsee coal reserves are sufficient to last about 50 years if mined at the average annual rate of production for the 5-year period 1940-44. The potential Wabaunsee coal reserves are calculated at 3,685,780,000 tons, an amount sufficient to last approximately 250 years at the average rate of production for the last 5 years. Of the potential coal reserve, 1,285,960,000 tons or 34.8 percent is Nodaway coal, 1,515,850,000 tons or 41.1 percent is Elmo coal, and 883,970,000 tons or 24.1 percent is miscellaneous coals.
This report includes a description of the geographic distribution, stratigraphic position, thicknesses, contact rocks, and physical and chemical characters of the Wabaunsee group coals. It also contains information on the history of Wabaunsee coal mining, mining methods, production and coal reserve data, and a short account of the origin of the coal. Each of the mined Wabaunsee group coals is treated separately under each of the 16 counties in which the coal occurs.
Kansas Geological Survey, Geology
Placed on web Jan. 27, 2013; originally published December 1946.
The URL for this page is http://www.kgs.ku.edu/Publications/Bulletins/63/index.html


text:
Razer unveils futuristic goggle with fan, lights and voice amplifier
At the start of the pandemic, when there was a global shortage of masks, many digital players decided to produce protective equipment against the coronavirus. For example, Apple has embarked on the production of visors protection for nursing staff. On the internet, we could also find tutorials for making reusable masks to be printed in 3D.
And for its part, Razer, specialist in gaming devices, has embarked on the production of masks. "This emergency conversion of some of our production lines, and the donation of masks is the first step in many of the actions Razer will take. We are committed to devoting our time, resources, efforts and talents to the fight against the COVID-19 virus ", explained the company in March.
Razer presents its mask of the future
And in early 2021, during CES 2021 (virtual), Razer unveiled a futuristic mask concept called Project Hazel. "Since the onset of the pandemic, Razer has been proactive in the fight against COVID-19, including converting its manufacturing facilities to produce certified medical masks and donating one million surgical masks to the line of healthcare front as part of its Razer Health initiative ", recalls Razer today.
"A natural progression and evolution of this initiative, Razer's Project Hazel Multi-Purpose Smart Mask addresses the increased focus on hygiene and preparedness for unanticipated health risks as well as the environmental issues of disposable masks, while improving characteristics of a reusable personal protective mask ', indicates the company.
An N95 mask with a fan and a microphone
First of all, Razer says the Hazel mask provides N95 level respiratory protection. The company states that its high efficiency bacterial filtration "Smart Pods" filter at least 95% of airborne particles. And to optimize the breathability of this mask, Razer has equipped it with a rechargeable fan.
If you love the cyberpunk aesthetic, the Razer Hazel Protective Mask is for you https://t.co/kJ3kZGmY2b pic.twitter.com/Vsjy6UTq1X
— Chloé Woitier ☕ (@W_Chloe) January 13, 2021
In addition, to facilitate interactions when the user will wear this mask, it has a transparent design, allowing facial expressions to be revealed. The mask also has interior lights that will allow the wearer to show off their facial expressions, even in the dark.
I'll take that Hazel Mask Razer 👀🔥 pic.twitter.com/O63zNQJlOD
— Ian's Tech 📱 (@IansTechYT) January 12, 2021
And since wearing the mask can muffle the user's voice, the Hazel mask is also equipped with a microphone as well as a voice amplifier. In essence, compared to traditional masks, Project Hazel offers something that seems to have come straight from the future.
At the moment, this is only a concept
But unfortunately this is only a concept at the moment. According to Razer, the Hazel goggle will continue to be optimized through rigorous testing and user feedback to ensure standards are met and to improve comfort. Design changes are also planned by the company.
Also, at the moment, it is not known when this mask will be available on the market. "Razer recognizes the uncertainty on the road ahead, and so it was our duty to help protect members of our community and prepare them against unseen threats.", said Min-Liang Tan, co-founder and CEO of Razer. "The Project Hazel smart mask concept is functional, yet comfortable and useful for interacting with the world, while maintaining a sociable aesthetic. "
Categories Technology
Whatsapp-Alternative Signal – How Elon Musk gave Signal stock superpower – News
DFB Cup: FC Bayern Munich sensationally loses at Holstein Kiel
Eminem at the top of a ranking of the 21st – La Nouvelle Tribune
Donald Trump will end his term with several defeats and two impeachments


Aurus is a charming and small, family-run 4-star hotel, occupying a building from the 16th century, a 5-minute walk from Old Town Square and Charles Bridge. The hotel features 8 rooms, breakfast is served daily here and the dedicated staff is really giving their best.
The house on the corner of Seminářská Street and Karlova Street is situated in place of a former Romanic estate, it is Renaissance in core, later modified in Baroque style. The richly figuratively decorated narrow front has two windows on each of the three floors. In the past, the house was also called At the Red Chair (U Červené sesle), probably as a reminder of the fact that the apostolic nuncio Spinelli lived here for some time, who used to sit in a red armchair decorated with gold thanks to the significance of his function. From the beginning of the 15th century, the house was also called At the Half-gold wheel (U Půlzlatého kola), and later on, it was called At the Golden Sun (U Zlatého slunce) for quite a long time, as one such Sun has been installed on the facade between the 1st and the 2nd floors. The name At the Golden Well (U Zlaté studny) has its origin in a legend connected with the house: the home well always had such good water, and there was a belief a golden treasure was hidden on the bottom. A maid once thought that she saw a glitter while fetching water, so she bent forward a bit more, fell in and drowned. Some sources state that a golden treasure really was found once while the well was being cleaned. In the beginning of the 18th century, the house was owned by Jan Wersser and his wife, and they ordered beautiful stucco figural decorations on the facade from a sculptor Jan Oldřich Mayer. It was probably their way of saying thank you for surviving the plague epidemics in 1714. There are seven figures altogether on the front. Alongside the first floor windows on the left, there is a figure of St. Sebastian, who was tortured to death by Emperor Diocletian for being a Christian and helping the persecuted Christians in Rome. When they built him an altar in Rome during the great plague in 680, the plague immediately ceased. He is therefore worshipped as the protector against plague, just like St. Roch, whose figure is to the right from the window. He devotedly helped the people infested by plague until he got the disease himself. He then went into the forests' solitude to die, but a dog found him and brought him bread and water from the stream, so he cured miraculously. On the second floor on the left, there is a statue of St Václav, murdered by his brother Boleslav, and on the right, there is St. John of Nepomuk, tortured to death by Václav IV and then thrown to the Vltava River. In the centre of the front, there is a bust relief of Virgin Mary of Boleslav dated 1701. It is depicted in accordance with the Stará Boleslav's painting named Palladium - the protection of the Bohemian lands. Legend says that St. Ludmila got this picture from St. Methodius upon baptism. Plague processions were held to the picture in Stará Boleslav. On the third floor, to the left from the window, there is the founder of the Jesuit Order and a missionary Ignatius of Loyola, and to the right there is Francis Xavier, perhaps the keenest missionary ever, who worked in India, where they called him the "Apostle of India", and also in Japan. Above the third floor window, there is a lying figure of St. Rosalia, surrounded by roses, famous Italian hermit who lived in a dark mountain cave out of love for Jesus Christ.
The object was restored in the 1950s under the administration of architect Karel Splavec, and a general reconstruction took place in 1983 - 1987, lead by ing. arch. Jan Dvořák. During this reconstruction, the truss was demolished, as well as the top floor, and everything was built anew; preserved restored Renaissance painted beam ceilings were suspended in the interior, contemporary stylish parquet floors and marble tiles were laid down, the stucco reliefs were renewed, everything after the original designs. The house was interconnected with the neighbouring object in Seminářská Street, which was also in a very bad state and was almost entirely rebuilt.


Marc Chagall: "Great art picks up where nature ends".
Born Moishe Shagal on 6th July 1887 in Lioza near Vitebsk Russian Empire. He died on 28th March 1985 in Saint-Paul-de-Vence, France. Chagall was a Russian-French artist of Belarusian Jewish origin. He worked in virtually every artistic format including painting, book illustrations, stained glass, stage sets, ceramic, tapestries and fine art prints. Using the medium of stained glass he produced windows for the cathedrals of Reims and Metz, windows for the UN, and the Jerusalem Windows in Israel. He also did large scale paintings, including part of the ceiling of the Paris Opera.
Before WW1 Chagall travelled between Saint Petersburg, Paris and Berlin. During this time he created his own mixture and style of modern art based on his idea of Eastern European Jewish folk culture. He spent the war years in Soviet Belarus becoming one of the country's most distinguished artists and a member of the modernist avant-garde before leaving for Paris in 1922. He experienced modernism's "golden age" in Paris where he synthesized the art forms of Cubism, Symbolism and Fauvism, and the influence of Fauvism gave rise to Surrealism. Yet throughout these phases he remained emphatically a Jewish artist.
Swept up in the horrors of European history between 1914 and 1945, world wars, revolution, ethnic persecution, the murder and exile of millions, Chagall turned his experiences of suffering and tragedy into images that were simple and symbolic to which everyone could respond. This at a time when other major artists fled reality for abstration.
"When Matisse dies", Pablo Picasso remarked in the 1950s, " Chagall will be the only painter left who understands what colour really is".
On the north side of Chichester Cathedral, West Sussex, there is a stained glass window designed and created by Chagall at the age of 90. The window, his last commissioned work, was inspired by Psalm 150. 'Let everything that hath breath praise the Lord'.


text:
Developer's App Helps Visually Impaired Navigate
The iBeacon hardware uses the Bluetooth Low Power protocol technology to emit radio signals to nearby smartphones and is used by the audible wayfinding app, AWARE, for the visually impaired.
The AWARE wayfinding app for the visually impaired grew out of necessity for Rasha Said, founder of Sensible Innovations in Springfield. Said, who has a background in computer science, finance and business administration, devised a pilot program for her son through a partnership with Glenwood High School in Chatham. Beacons — so called "Smart Landmarks" — installed throughout the school communicate location to the smartphone user through audio cues.
Two years after starting the Glenwood pilot project, Sensible Innovation systems have been installed in a New York transit system, a social service agency in Chicago and a Springfield school for the visually impaired. The company's AWARE app was recognized last week at the Consumer Electronics Show in New York. The system will be showcased at the annual Consumer Electronic Show in Las Vegas.
Said's inspiration began with a trip to Disney World with her son, whose condition has resulted in a gradual loss of vision. The need to explain scenery and sounds the rest of the family experienced led to experiments with audio, wayfinding apps and beacons.
The AWARE app can be downloaded from the App Store for iOS and Play Store for Android for free.
Introducing a mobile app to help visually impaired explore their surroundings independently!
The AWARE app works with Sensible Innovations Electronic Stickers, to provide indoor directions and descriptions crafted with a visually impaired focus. The AWARE app is built with large fonts and high contrast or a Braille display for those who prefer to read, and is completely audible for non-visual users. The audible function is also handy for anyone who wants to receive locations specific information without having to look or swipe a smart phone screen.
YOU MIGHT ALSO LIKE Microsoft Develops Device That Helps Blind and Visually Impaired Navigate Virtual Spaces
Explore new places at your own time and pace!
Embrace the ability to explore your surroundings independently when Aware recites landmarks as you pass them by and never get lost again!
Location awareness crafted with visually impaired focus!
Get more information and detailed description about your location and get real-time promotions and special offers that are announced when and where it's relevant .
Never get lost again!
Navigate public buildings with confidence. All you need is to speak to Aware. Aware has a speech recognition function and will guide you to your destination by giving you an audio turn-by-turn directions. The Aware system can detect when you are going the wrong route and will adjust your route accordingly. It has a built in navigation algorithm that works like GPS system, only beyond!
Please Note: Continued use of GPS running in the background can dramatically decrease battery life
About Nelson Régo
Nelson Régo is the owner and founder of the Cool Blind Tech website since November of 2012. Nelson launched the first show on March 14, 2013. He also directs the website as a whole, hiring staff, originating new shows, and approving all content.
iOS 9 Accessibility
https://dts.podtrac.com/redirect.mp3/media.blubrry.com/cool_blind_tech/p/content.blubrry.com/cool_blind_tech/iOS_9_Accessability.mp3
iOS has transformed the lives of many users with a range of physical and learning disabilities. Learn how to create apps that leverage the power and simplicity of Apple's assistive technologies to make them accessible to the broadest audience possible. Hear about the latest advancements in iOS accessibility and how you can take advantage of them today.
You can support the Cool Blind Tech Community By giving us a 5 star rating in iTunes.
CBT would like to thank our loyal listeners for listening to this podcast by inviting you to join our Community and possibly join the CBT Team, where you can submit your own podcasts or articles.
YOU MIGHT ALSO LIKE Artificial Intelligence and Text Recognition unite in Microsoft's Seeing Ai iPhone App


Continue reading "Things every small business owner should know"
Continue reading "Small business mistakes you need to avoid"
Continue reading "How to avoid small business mistakes"
The great thing about small business ownership is that anyone who wants to can have the opportunity to be their own boss. Unfortunately, the catch is directly related to the duration that these businesses last. Only those who have certain skills and traits can take their business ideas and turn them into a long-standing success. Researchers have looked into what makes a good business person and have come up with a list of traits that most successful business owners have in common.
Continue reading "Traits you need to be a small business success"
You can't please everyone. Criticism is a natural consequence of being a business owner. Whether your critic is an unsatisfied customer or a competitor looking to discredit you, taking harsh words in stride is definitely a challenge. The unfortunate truth is that the bigger your business is and the more successful you become, the more people will try to bring you down. In many respects you put yourself out there to be criticized. Receiving that first letter of dissatisfaction or nasty phone call can upset even the most professional of small business owners. Although the temptation to take criticism personally is definitely an easy thing to do (after all you have poured a lot of yourself into your business) seasoned business owners know that it is better to put personal feelings aside and try your best to take criticism constructively.
Continue reading "Taking small business criticism constructively"
Starting your own small business is no easy task. Keeping it going is even harder. If you are worried about the future of your business, the best place to start is at the beginning. From the point that you start thinking about starting your own businesses and throughout the life of the business there are certain strategies that you can use to ensure that your business can withstand the test of time.
Continue reading "Secrets to keeping your small business going"
While a lot of small businesses believe you should only look to the future, you can actually look to your past for some great tips on how you can help the future. As you create marketing strategies and other business plans for the future, take a look back upon the things that have worked for you in the past and avoid falling into the mistakes that haven't worked in the past.
Continue reading "Using the past to help the future"
Being your own boss may look attractive in theory, but you will quickly find it's not all fun and games all the time. It will require a lot of time and energy and you won't make millions of dollars overnight, in fact you may never get to that point. Hard work will pay off in the long run and you will be able to create a long-lasting company that you can pass on to future generations. To help you with you new small business, we have put together some basic guidelines for you.
Continue reading "The basic requirements of running your own business"
The economy has taken its toll on small business owners and if you'd made it through so far, you aren't doing too bad. Unfortunately small businesses often have everything going against them and only about 2/3rds will survive the first 2 years and after that only half of those businesses will survive 2 more years. So with all of the odds going against you, plus the hardships in the economy, how can you find a way to keep your small business going strong? This article will explore a few things you can do to keep yourself in business.
Continue reading "Surviving in difficult times"
Since small businesses have all the odds going against them within the first 5 years of business, you may be looking for different ways that will help you stay in business for another year. How can you stay in business? It starts with 2 basic components, a good business plan and determination.
Continue reading "Staying in business for another year"
The widespread use of the internet over the last 15 years has made having an online presence almost essential for any small business owner. Since starting a website is relatively simple and cost effective, there are mostly just advantages to getting it done. Below you will find a list of a number of different advantages of having a website for your small business. In reading these advantages hopefully you will join the millions of other small business owners who are reaping the benefits of having an online presence.
Continue reading "The advantages of having a website for your small business"
It is just about impossible for a small business not to have a website these days. Most customers have now come to expect that all of the businesses they use will have a website no matter how big or how small the company is. But even though having a website is just about essential, many business owners may not be sure just how to go about setting up a website for their small business. This article discusses information that can help a small business owner set up a website for their business.
Continue reading "Setting up a website for your small business"
Before you can start your small business, it is important to make sure you have all the permits and your business license. Check with your state and local governments to make sure you have all the right forms completed and you have all the permits you need before you open your doors. Some states require businesses to post certain signs that talk about the different regulations like workers compensation insurance, disability, discrimination, etc. Usually the business license must be displayed in an area that is visible for customers and inspectors to find.
Besides complying with local and state laws, there are also federal regulations your small business may need to comply with. Employment of minors has a lot of stipulations for businesses so it is important to read all the rules before you hire them. Every business type has different regulations it must comply with, which is why it is so important to check them before you open your doors. Some states have newsletters and alerts they will send out whenever there is a change in some of the regulations that impact your business.
Continue reading "How to get your business license and permits"
Most people are feeling the pinch of a bad economy, and small businesses are no exception. With both consumers and businesses looking to save money, it can be difficult for a small business to stay afloat during difficult economic times.
Continue reading "What you can do to help your small business survive economic recession"
In these turbulent economic times, many small businesses are worried about staying in business when many people and businesses are cutting costs, which is turn slows down business.
Continue reading "How to fortify your small business from depression"
Continue reading "Growth tactics for small business"
As a small business owner, particularly a small business owner that's just starting out, you might feel like you're struggling to keep your business alive. No one ever said that starting up a small business was an easy endeavor. Sometimes it can be really difficult, and it's possible that you feel like you're "running out of steam." However, you don't need to sacrifice your business and your dream because you're not sure how to keep your small business afloat! Here are some things that you need to do to Stay in Business with a small business.
	Make goals: If you own a small business, particularly a small business that is based in your home, you can often get distracted from your work responsibilities. Sometimes there are other things that seem easier and more "fun" to do than working. If you are having a hard time staying motivated or keeping on track, then you should make some goals. You should not only make short term goals, but you should make long term goals as well. If you haven't incorporated these goals into a formal business plan, this is another good idea. When you have the goals and vision of your small business in mind, then you will be better motivated to work and Stay in Business! Remember, no business was able to stay alive and keep in business without some consistent, hard work.
Continue reading "What you need to do to Stay in Business with a small business"


Barack Obama's late arriving compassion for Syrian refugees is puzzling. For years, he apparently felt no compassion for the hundreds of thousands of Syrians who were killed or maimed in the bloodiest civil war in memory. The casualties and suffering in Syria are beginning to approach the horrific and storied Spanish Civil war in which an estimated half million military and civilians were killed in combat or in atrocities.
But now that thousands of refugees of the Syrian civil war are arriving on our doorstep, Barack Obama is at last feeling some compassion.
Where the hell was that compassion during the five years that he frittered away while the Syrians were being gassed by the bloodthirsty Syrian dictator Bashar al-Assad and the population was being slaughtered by ISIS fanatics?
Now he's playing the compassion card to ridicule Americans who have real and genuine fears of Islamic terrorists making their way into America with the Syrian refugees.
"Apparently they're scared of widows and orphans coming into the United States of America," Obama said. Mocking GOP leaders for thinking they're tough, Obama said overblown rhetoric from Republicans could be a potent recruitment tool for the Islamic State group.
Americans want to be compassionate, even though the record of allowing Jewish refugees seeking shelter from Hitler before World War II isn't admirable. But the Paris Islamic murderers included at least one who arrived hiding among the Syrian refugees. Americans of either party or political persuasion are justified in worrying about how the the country can weed out the terrorists who arrive at our shores with the Syrian refugees.
I'm not saying we shouldn't accept the refugees, but I'm looking for acknowledgment from a head-in-the-sand White House that this poses a big, big problem. Saying that officials will be aggressive (and successful) in identifying terrorists among the Syrian refugees without the evidence just doesn't cut it, Mr. President.
Read why Americans need to learn about the nation's most ignored war .
This post is useful to me. Your ideas and tips are great to me. The scholarship essay writing service is sponsored the wonderful writing papers to the students.
So you would be ok accepting Syrian refugees if Obama did more than screen them with a fine-toothed comb? What is the"big, big problem" you refer to? The refugee crisis? The screening itself?
By the way, does the Pope "play the compassion card"?


text:
New Malden, Surrey
Cambelts or Timing Belts
Just the Ticket team
MOT Testing: When did your vehicle last have its MOT?
A backlog of vehicles who benefitted from the government's Covid MOT extension, are now due for their MOT test.
According to the latest figures from the Driver and Vehicle Standards Agency (DVSA), only 2.1 million MOTs were carried out in April and May 2020 – compared to the 7.1 million MOTs which were conducted over the same period in 2019.
"Which means around five million vehicles didn't book in for a test," Dave Ellis, manager at Just the Ticket says. "So to avoid disappointment, we're encouraging vehicle owners who are due a MOT, to book their car in sooner rather than later."
'Backlog in vehicles waiting for MOT'
This backlog comes after we saw the government introduce a Covid-19 MOT extension at the beginning of 2020, in a bid to better protect drivers from exposure to the virus.
This granted a six-month extension to all vehicles with MOT certificates expiring between 30 March to 31 July 2020.
While those whose MOTs ran out on or after 1 August 2020, had to book a retest as usual.
"This was something that's never been done before," Dave at Just the Ticket says. "But it now means there's a backlog of vehicles all waiting to have an MOT test, as many of the six-month extensions will come to an end in September."
What is an MOT?
A MOT test checks that your vehicle – whether it's a car, motorcycle or bus – meets all the road safety and environmental standards.
An MOT isn't optional. It's a legal requirement for you to drive on public roads. Without a valid MOT Certificate, your vehicle insurance is invalid.
When should you get an MOT?
If you're unsure about when was the last time your car had its MOT, and when its certificate runs out, you can use this online service. You just need to insert the vehicle's registration number, so make sure you have that at hand.
But generally speaking, any vehicle that is three years old or over, must get an MOT every year.
What happens at an MOT Test?
During an MOT Test, DVSA approved testers will look at all the important functions of your car to ensure that they meet the legal requirements.
Body or vehicle structure and general items
Load security
Registration plates
Wipers and washers
If everything's in safe and working order, our mechanics will provide you with a MOT Certificate, which proves your vehicle is safe to drive.
But if there are any mechanical issues that would prevent a pass, then we will provide you with an itemised account of any work required to bring the vehicle up to standard.
Here at Just the Ticket, we have DVSA approved testers. This means we are accredited as an MOT testing centre and our dedicated team are here to ensure your car is back on the road safety – and quickly.
Along with petrol and diesel cars, we are also able to service or MOT electric and hybrid cars. And we are the first independent car garage in New Malden to be qualified to do so.
So whatever vehicle you have – do charge this way!
Book in for an MOT today – we'd be more than happy to look after your vehicle. You can book online or give us a call on 0208 336 1111
Six quick home checks to avoid MOT failure
Do I really need to service my car?
Do you know the meaning of these warning lights on your car?
© 2021 by Just the Ticket Garage Services Ltd. Unit D, Wellington Crescent, New Malden, KT3 3NE T:0208 336 1111 Email: enquiries@jttmot.co.uk
Just the Ticket is part of JTT Garage Services Limited Registered in England and Wales Reg No 8204765


text:
Man United Announce Ander Herrera's Exit To PSG
Manchester United have verified that Ander Herrera, will be leaving the club this summer. following five years with the Old Trafford side.
The Red Devils announced his departure with a short clip posted on Twitter and captioned: "A goodbye message from @AnderHerrera. #MUFC" Herrera is widely expected to join Paris Saint-Germain on a free transfer this summer.
United made a final offer of over £200,000-a-week to keep him, but the Spaniard wanted more money. The club opened contract negotiations with Herrera's representatives over 18 months ago, but little progress was made and the player's camp felt the club prioritised other players' deals during that period.
Many felt the club simply were not proactive enough in trying to keep Herrera, but it is believed the club made a final offer worth more than £200,000-a-week in a bid to keep the midfielder.
Related Topics:Ander Herrera
Barcelona Coach Valverde Refuses To Resign After Champions League Embarrassment
Chelsea Defender David Luiz Extend His Stay In Chelsea


In "Hard News: Newspapers are dying. At the Washington Post Co., CEO Donald Graham is banking on the Internet to save serious journalism," Fortune's Marc Gunther takes a very interesting look at the need for old media to adapt.
Citing the Post as his "'$0.35 edition' in his blog," a Post reporter is lauded for being "platform-agnostic, which is a nice way of saying that his bosses are no longer big believers in print. Today a small army of bloggers, podcasters, chatroom hosts, radio voices, and TV talking heads, as well as a few old-fashioned ink-stained wretches, populates the newsroom at the 131-year-old Post. "If circulation is dropping," the Post reporter said, "and we're trying to figure out how people are going to get their news, who am I to say no to trying out new avenues?"
One of the secrets of the Post, according to an editor there, "Investigative reporting is our brand."
According to Fortune, "The Post has two goals online. It wants to become a must-buy in Washington while increasing its share of national ad dollars. Right now ads aimed at local readers account for about 60 percent of revenues. The site offers a vast trove of local content - going-out guides, traffic, weather, sports. Says Graham: 'We have a map of D.C. with every school listed, with test scores and other things parents would want to know. It's not all-inclusive, but it's a lot of information, and you couldn't do that in the paper.'"
The implications for those of us in PR is that we need to make sure our information is platform-agnostic, and look to use the Internet in ways that go well beyond the paper-based press release. It is likely to cost more to produce, certainly at first, but be much for effective.


text:
Delonix elata – gatunek z rodziny bobowatych (Fabaceae) z podrodziny brezylkowych (Caesalpinioideae). Występuje w północno-wschodniej i wschodniej Afryce (od Egiptu po Tanzanię i Zambię) oraz w południowo-zachodniej Azji (Półwysep Arabski). Poza tym jest uprawiany, także poza swoim zasięgiem, np. popularnie w Pakistanie i Indiach.

W rodzimym zasięgu gatunek ten występuje głównie na wysokości do 1400 m n.p.m., ale zauważony był od poziomu morza do 2000 m, a sporadycznie nawet do 4000 m n.p.m.

Morfologia
 Pokrój Drzewo o wysokości 2,5–15 m. Korę ma gładką, płowożółtą lub szarą, łuszczącą się.
 Liście Osiągają do 20 cm długości, są dwukrotnie pierzasto złożone, przy czym odcinków II rzędu, osiągających do ok. 6 cm długości, jest 4–8 par. Na odcinkach tych znajduje się od 10 do 20 listków. Ich blaszka jest równowąskopodługowata, u nasady zbiegająca, na wierzchołku stępiona lub krótko, ostro zakończona, osiąga do 15 mm długości i 3 mm szerokości.
 Kwiaty Zebrane w szczytowe grona, przy czym najniższy kwiat ma wyraźnie najdłuższą szypułkę (do 2,5 cm). Kielich jest wąski, skórzasty, jedwabiście owłosiony i osiąga do 3 cm długości. Korona jest początkowo biała, później żółta. Górny płatek jest mniejszy i ciemniejszy od pozostałych płatków (osiąga do 2,5 cm długości). Pręciki osiągają do 10 cm długości, są ciemno zabarwione i owłosione.
 Owoce Długie i smukłe strąki, o wymiarach 13–25 × 2–3,7 cm. Zawierają od 4 do 8 nasion.

Zastosowanie
Drzewa tego gatunku są często uprawiane jako ozdobne. W Indiach gałęzie i liście używane są jako pasza. Drewno wykorzystywane jest do wyrobu mebli. W Pakistanie wierzy się, że dotyk korzenia usuwa ból po ukąszeniu skorpiona.

Przypisy

Bobowate

redpajama_set_name: RedPajamaWikipedia


Canadians will be able to buy and consume cannabis legally as early as this September.
The country is the second worldwide to legalise the drug's recreational use.
Uruguay became the first country to legalise the sale of cannabis for recreational use in December 2013, while a number of US states have also voted to permit it.
The bill will likely receive Royal Assent this week, and the government will then choose an official date when the law will come into force.
Prime Minister Justin Trudeau tweeted that until now, "it's been too easy for our kids to get marijuana - and for criminals to reap the profits".
But some groups objected to the new law, with opposition Conservative politicians and indigenous groups among those voicing concerns.
The government is expected to give the provinces and territories, as well as municipalities, eight to 12 weeks to set up the new marijuana marketplace.
This timeframe will also allow industry and police forces to prepare for the new legal framework.
In 2015, Canadians were estimated to have spent about C$6bn ($4.5bn, £3.4bn) on cannabis - almost as much as they did on wine.
How will legal marijuana in Canada work?
It is likely that by mid-September, Canadians will be able to buy cannabis and cannabis oil grown by licensed producers at various retail locations.
Canadians across the country will also be able to order the drug online from federally licensed producers, and grow up to four plants at home.
Adults will be able to possess up to 30 grams (one ounce) of dried cannabis in public.
The minimum legal age to buy and consume marijuana has been set federally at 18 but some provinces have chosen to set it at 19.
Cannabis and Canada: Why has drug policy changed?
Provinces are in charge of how it is sold and have the power to set various other limits on its use within their jurisdiction - like where it can be smoked.
But the federal government has set guidelines for plain packaging with little branding and strict health warnings. It will also impose restrictions on promotions targeting young people, promotion through sponsorships, or depictions of celebrities, characters or animals in advertisements.
It will be illegal to possess more than 30 grams of cannabis in public, grow more than four plants per household and to buy from an unlicensed dealer.
Penalties will be severe. Someone caught selling the drug to a minor could be jailed for up to 14 years.
What are the rules around the world?
Cannabis is banned in most countries but a number of places have decriminalised its use in recent years. The UK government said recently it would review the use of medicinal cannabis.
It is legal for medicinal purposes in 14 European countries, Israel, Argentina, Puerto Rico, Panama, Mexico, Turkey, Zambia and Zimbabwe.
In the US, medicinal use is allowed in 29 states and the District of Columbia, Nine of the states have legalised both medical and personal cannabis use.
Spain, the Netherlands, Portugal, Slovenia, Jamaica, Chile, Peru, Colombia, Ecuador and Luxembourg are among countries that have relaxed legislation regarding personal use.
The new law has not been met with universal praise.
Conservative Party leader Andrew Scheer has previously said he was apprehensive it would normalise cannabis use and make it more accessible.
Leo Housakos, a Conservative senator from Quebec, tweeted to say that he thought the law would be "catastrophic for Canadian generations to come".
One specific concern being raised is the legal minimum age being set at 18 instead of 25, as was recommended by multiple experts. However, the government's own task force recommended 18, warning that setting the age limit too high could continue to encourage youth to buy marijuana on the illegal market.
Opponents also question the potential public health impact of legalisation.
Meanwhile, indigenous groups and politicians have voiced fears that their communities were not adequately consulted in the run-up to the vote.
Indigenous services minister Jane Philpott and health minister Ginette Petitpas Taylor had to write a letter to the chair and deputy chair of the Senate's Committee on Aboriginal Peoples earlier in June promising a full report to parliament addressing their concerns, narrowly averting a delay to the law's passage.
Why is Canada doing this now?
This legislation fulfils a 2015 campaign promise by Mr Trudeau, the leader of the Liberal Party.
Polls have repeatedly indicated that a solid majority of Canadians are supportive of the move.
The decision to legalise recreational use of marijuana in Canada comes as global trends shift away from criminal prohibition of the widely used drug.
WATCH More and GET Buzzed on WeedTV! Sign Up - It's Free!


text:
"""
This module provides WSGI application to serve the Home Assistant API.

For more details about this component, please refer to the documentation at
https://home-assistant.io/components/http/
"""
import asyncio
import json
import logging
import ssl
from ipaddress import ip_network
from pathlib import Path

import os
import voluptuous as vol
from aiohttp import web
from aiohttp.web_exceptions import HTTPUnauthorized, HTTPMovedPermanently

import homeassistant.helpers.config_validation as cv
import homeassistant.remote as rem
from homeassistant.util import get_local_ip
from homeassistant.components import persistent_notification
from homeassistant.const import (
    SERVER_PORT, CONTENT_TYPE_JSON, ALLOWED_CORS_HEADERS,
    EVENT_HOMEASSISTANT_STOP, EVENT_HOMEASSISTANT_START)
from homeassistant.core import is_callback
from homeassistant.util.logging import HideSensitiveDataFilter

from .auth import auth_middleware
from .ban import ban_middleware, process_wrong_login
from .const import (
    KEY_USE_X_FORWARDED_FOR, KEY_TRUSTED_NETWORKS,
    KEY_BANS_ENABLED, KEY_LOGIN_THRESHOLD,
    KEY_DEVELOPMENT, KEY_AUTHENTICATED)
from .static import GZIP_FILE_SENDER, staticresource_middleware
from .util import get_real_ip

DOMAIN = 'http'
REQUIREMENTS = ('aiohttp_cors==0.5.0',)

CONF_API_PASSWORD = 'api_password'
CONF_SERVER_HOST = 'server_host'
CONF_SERVER_PORT = 'server_port'
CONF_DEVELOPMENT = 'development'
CONF_SSL_CERTIFICATE = 'ssl_certificate'
CONF_SSL_KEY = 'ssl_key'
CONF_CORS_ORIGINS = 'cors_allowed_origins'
CONF_USE_X_FORWARDED_FOR = 'use_x_forwarded_for'
CONF_TRUSTED_NETWORKS = 'trusted_networks'
CONF_LOGIN_ATTEMPTS_THRESHOLD = 'login_attempts_threshold'
CONF_IP_BAN_ENABLED = 'ip_ban_enabled'

NOTIFICATION_ID_LOGIN = 'http-login'

# TLS configuation follows the best-practice guidelines specified here:
# https://wiki.mozilla.org/Security/Server_Side_TLS
# Intermediate guidelines are followed.
SSL_VERSION = ssl.PROTOCOL_SSLv23
SSL_OPTS = ssl.OP_NO_SSLv2 | ssl.OP_NO_SSLv3
if hasattr(ssl, 'OP_NO_COMPRESSION'):
    SSL_OPTS |= ssl.OP_NO_COMPRESSION
CIPHERS = "ECDHE-ECDSA-CHACHA20-POLY1305:ECDHE-RSA-CHACHA20-POLY1305:" \
          "ECDHE-ECDSA-AES128-GCM-SHA256:ECDHE-RSA-AES128-GCM-SHA256:" \
          "ECDHE-ECDSA-AES256-GCM-SHA384:ECDHE-RSA-AES256-GCM-SHA384:" \
          "DHE-RSA-AES128-GCM-SHA256:DHE-RSA-AES256-GCM-SHA384:" \
          "ECDHE-ECDSA-AES128-SHA256:ECDHE-RSA-AES128-SHA256:" \
          "ECDHE-ECDSA-AES128-SHA:ECDHE-RSA-AES256-SHA384:" \
          "ECDHE-RSA-AES128-SHA:ECDHE-ECDSA-AES256-SHA384:" \
          "ECDHE-ECDSA-AES256-SHA:ECDHE-RSA-AES256-SHA:" \
          "DHE-RSA-AES128-SHA256:DHE-RSA-AES128-SHA:DHE-RSA-AES256-SHA256:" \
          "DHE-RSA-AES256-SHA:ECDHE-ECDSA-DES-CBC3-SHA:" \
          "ECDHE-RSA-DES-CBC3-SHA:EDH-RSA-DES-CBC3-SHA:AES128-GCM-SHA256:" \
          "AES256-GCM-SHA384:AES128-SHA256:AES256-SHA256:AES128-SHA:" \
          "AES256-SHA:DES-CBC3-SHA:!DSS"

_LOGGER = logging.getLogger(__name__)

DEFAULT_SERVER_HOST = '0.0.0.0'
DEFAULT_DEVELOPMENT = '0'
DEFAULT_LOGIN_ATTEMPT_THRESHOLD = -1

HTTP_SCHEMA = vol.Schema({
    vol.Optional(CONF_API_PASSWORD, default=None): cv.string,
    vol.Optional(CONF_SERVER_HOST, default=DEFAULT_SERVER_HOST): cv.string,
    vol.Optional(CONF_SERVER_PORT, default=SERVER_PORT):
        vol.All(vol.Coerce(int), vol.Range(min=1, max=65535)),
    vol.Optional(CONF_DEVELOPMENT, default=DEFAULT_DEVELOPMENT): cv.string,
    vol.Optional(CONF_SSL_CERTIFICATE, default=None): cv.isfile,
    vol.Optional(CONF_SSL_KEY, default=None): cv.isfile,
    vol.Optional(CONF_CORS_ORIGINS, default=[]): vol.All(cv.ensure_list,
                                                         [cv.string]),
    vol.Optional(CONF_USE_X_FORWARDED_FOR, default=False): cv.boolean,
    vol.Optional(CONF_TRUSTED_NETWORKS, default=[]):
        vol.All(cv.ensure_list, [ip_network]),
    vol.Optional(CONF_LOGIN_ATTEMPTS_THRESHOLD,
                 default=DEFAULT_LOGIN_ATTEMPT_THRESHOLD): cv.positive_int,
    vol.Optional(CONF_IP_BAN_ENABLED, default=True): cv.boolean
})

CONFIG_SCHEMA = vol.Schema({
    DOMAIN: HTTP_SCHEMA,
}, extra=vol.ALLOW_EXTRA)

@asyncio.coroutine
def async_setup(hass, config):
    """Set up the HTTP API and debug interface."""
    conf = config.get(DOMAIN)

    if conf is None:
        conf = HTTP_SCHEMA({})

    api_password = conf[CONF_API_PASSWORD]
    server_host = conf[CONF_SERVER_HOST]
    server_port = conf[CONF_SERVER_PORT]
    development = conf[CONF_DEVELOPMENT] == '1'
    ssl_certificate = conf[CONF_SSL_CERTIFICATE]
    ssl_key = conf[CONF_SSL_KEY]
    cors_origins = conf[CONF_CORS_ORIGINS]
    use_x_forwarded_for = conf[CONF_USE_X_FORWARDED_FOR]
    trusted_networks = conf[CONF_TRUSTED_NETWORKS]
    is_ban_enabled = conf[CONF_IP_BAN_ENABLED]
    login_threshold = conf[CONF_LOGIN_ATTEMPTS_THRESHOLD]

    if api_password is not None:
        logging.getLogger('aiohttp.access').addFilter(
            HideSensitiveDataFilter(api_password))

    server = HomeAssistantWSGI(
        hass,
        development=development,
        server_host=server_host,
        server_port=server_port,
        api_password=api_password,
        ssl_certificate=ssl_certificate,
        ssl_key=ssl_key,
        cors_origins=cors_origins,
        use_x_forwarded_for=use_x_forwarded_for,
        trusted_networks=trusted_networks,
        login_threshold=login_threshold,
        is_ban_enabled=is_ban_enabled
    )

    @asyncio.coroutine
    def stop_server(event):
        """Callback to stop the server."""
        yield from server.stop()

    @asyncio.coroutine
    def start_server(event):
        """Callback to start the server."""
        hass.bus.async_listen_once(EVENT_HOMEASSISTANT_STOP, stop_server)
        yield from server.start()

    hass.bus.async_listen_once(EVENT_HOMEASSISTANT_START, start_server)

    hass.http = server
    hass.config.api = rem.API(server_host if server_host != '0.0.0.0'
                              else get_local_ip(),
                              api_password, server_port,
                              ssl_certificate is not None)

    return True

class HomeAssistantWSGI(object):
    """WSGI server for Home Assistant."""

    def __init__(self, hass, development, api_password, ssl_certificate,
                 ssl_key, server_host, server_port, cors_origins,
                 use_x_forwarded_for, trusted_networks,
                 login_threshold, is_ban_enabled):
        """Initialize the WSGI Home Assistant server."""
        import aiohttp_cors

        middlewares = [auth_middleware, staticresource_middleware]

        if is_ban_enabled:
            middlewares.insert(0, ban_middleware)

        self.app = web.Application(middlewares=middlewares, loop=hass.loop)
        self.app['hass'] = hass
        self.app[KEY_USE_X_FORWARDED_FOR] = use_x_forwarded_for
        self.app[KEY_TRUSTED_NETWORKS] = trusted_networks
        self.app[KEY_BANS_ENABLED] = is_ban_enabled
        self.app[KEY_LOGIN_THRESHOLD] = login_threshold
        self.app[KEY_DEVELOPMENT] = development

        self.hass = hass
        self.development = development
        self.api_password = api_password
        self.ssl_certificate = ssl_certificate
        self.ssl_key = ssl_key
        self.server_host = server_host
        self.server_port = server_port
        self._handler = None
        self.server = None

        if cors_origins:
            self.cors = aiohttp_cors.setup(self.app, defaults={
                host: aiohttp_cors.ResourceOptions(
                    allow_headers=ALLOWED_CORS_HEADERS,
                    allow_methods='*',
                ) for host in cors_origins
            })
        else:
            self.cors = None

    def register_view(self, view):
        """Register a view with the WSGI server.

        The view argument must be a class that inherits from HomeAssistantView.
        It is optional to instantiate it before registering; this method will
        handle it either way.
        """
        if isinstance(view, type):
            # Instantiate the view, if needed
            view = view()

        if not hasattr(view, 'url'):
            class_name = view.__class__.__name__
            raise AttributeError(
                '{0} missing required attribute "url"'.format(class_name)
            )

        if not hasattr(view, 'name'):
            class_name = view.__class__.__name__
            raise AttributeError(
                '{0} missing required attribute "name"'.format(class_name)
            )

        view.register(self.app.router)

    def register_redirect(self, url, redirect_to):
        """Register a redirect with the server.

        If given this must be either a string or callable. In case of a
        callable it's called with the url adapter that triggered the match and
        the values of the URL as keyword arguments and has to return the target
        for the redirect, otherwise it has to be a string with placeholders in
        rule syntax.
        """
        def redirect(request):
            """Redirect to location."""
            raise HTTPMovedPermanently(redirect_to)

        self.app.router.add_route('GET', url, redirect)

    def register_static_path(self, url_root, path, cache_length=31):
        """Register a folder to serve as a static path.

        Specify optional cache length of asset in days.
        """
        if os.path.isdir(path):
            self.app.router.add_static(url_root, path)
            return

        filepath = Path(path)

        @asyncio.coroutine
        def serve_file(request):
            """Serve file from disk."""
            res = yield from GZIP_FILE_SENDER.send(request, filepath)
            return res

        # aiohttp supports regex matching for variables. Using that as temp
        # to work around cache busting MD5.
        # Turns something like /static/dev-panel.html into
        # /static/{filename:dev-panel(-[a-z0-9]{32}|)\.html}
        base, ext = url_root.rsplit('.', 1)
        base, file = base.rsplit('/', 1)
        regex = r"{}(-[a-z0-9]{{32}}|)\.{}".format(file, ext)
        url_pattern = "{}/{{filename:{}}}".format(base, regex)

        self.app.router.add_route('GET', url_pattern, serve_file)

    @asyncio.coroutine
    def start(self):
        """Start the wsgi server."""
        if self.cors is not None:
            for route in list(self.app.router.routes()):
                self.cors.add(route)

        if self.ssl_certificate:
            context = ssl.SSLContext(SSL_VERSION)
            context.options |= SSL_OPTS
            context.set_ciphers(CIPHERS)
            context.load_cert_chain(self.ssl_certificate, self.ssl_key)
        else:
            context = None

        self._handler = self.app.make_handler()
        self.server = yield from self.hass.loop.create_server(
            self._handler, self.server_host, self.server_port, ssl=context)

    @asyncio.coroutine
    def stop(self):
        """Stop the wsgi server."""
        self.server.close()
        yield from self.server.wait_closed()
        yield from self.app.shutdown()
        yield from self._handler.finish_connections(60.0)
        yield from self.app.cleanup()

class HomeAssistantView(object):
    """Base view for all views."""

    url = None
    extra_urls = []
    requires_auth = True  # Views inheriting from this class can override this

    # pylint: disable=no-self-use
    def json(self, result, status_code=200):
        """Return a JSON response."""
        msg = json.dumps(
            result, sort_keys=True, cls=rem.JSONEncoder).encode('UTF-8')
        return web.Response(
            body=msg, content_type=CONTENT_TYPE_JSON, status=status_code)

    def json_message(self, error, status_code=200):
        """Return a JSON message response."""
        return self.json({'message': error}, status_code)

    @asyncio.coroutine
    # pylint: disable=no-self-use
    def file(self, request, fil):
        """Return a file."""
        assert isinstance(fil, str), 'only string paths allowed'
        response = yield from GZIP_FILE_SENDER.send(request, Path(fil))
        return response

    def register(self, router):
        """Register the view with a router."""
        assert self.url is not None, 'No url set for view'
        urls = [self.url] + self.extra_urls

        for method in ('get', 'post', 'delete', 'put'):
            handler = getattr(self, method, None)

            if not handler:
                continue

            handler = request_handler_factory(self, handler)

            for url in urls:
                router.add_route(method, url, handler)

        # aiohttp_cors does not work with class based views
        # self.app.router.add_route('*', self.url, self, name=self.name)

        # for url in self.extra_urls:
        #     self.app.router.add_route('*', url, self)

def request_handler_factory(view, handler):
    """Factory to wrap our handler classes."""
    assert asyncio.iscoroutinefunction(handler) or is_callback(handler), \
        "Handler should be a coroutine or a callback."

    @asyncio.coroutine
    def handle(request):
        """Handle incoming request."""
        if not request.app['hass'].is_running:
            return web.Response(status=503)

        remote_addr = get_real_ip(request)
        authenticated = request.get(KEY_AUTHENTICATED, False)

        if view.requires_auth and not authenticated:
            yield from process_wrong_login(request)
            _LOGGER.warning('Login attempt or request with an invalid '
                            'password from %s', remote_addr)
            persistent_notification.async_create(
                request.app['hass'],
                'Invalid password used from {}'.format(remote_addr),
                'Login attempt failed', NOTIFICATION_ID_LOGIN)
            raise HTTPUnauthorized()

        _LOGGER.info('Serving %s to %s (auth: %s)',
                     request.path, remote_addr, authenticated)

        result = handler(request, **request.match_info)

        if asyncio.iscoroutine(result):
            result = yield from result

        if isinstance(result, web.StreamResponse):
            # The method handler returned a ready-made Response, how nice of it
            return result

        status_code = 200

        if isinstance(result, tuple):
            result, status_code = result

        if isinstance(result, str):
            result = result.encode('utf-8')
        elif result is None:
            result = b''
        elif not isinstance(result, bytes):
            assert False, ('Result should be None, string, bytes or Response. '
                           'Got: {}').format(result)

        return web.Response(body=result, status=status_code)

    return handle

redpajama_set_name: RedPajamaGithub


Lawrence Frank Lee, age 79, of Des Moines (formerly of Norwalk) died Thursday evening, April 4, 2019 at Mercy Medical Center in Des Moines.
Visitation with the family will be 2 to 3 p.m. Sunday, April 28th at O'Leary Funeral & Cremation Services. A Celebration of Life Service will be at 3 p.m. following visitation. To sign his on-line guestbook and to send flowers (using O'Leary's Flowers in Norwalk - no delivery fees) select the send tributes/flowers tab on this screen or call direct (515) 981-0700.
Lawrence "Larry" was born on August 23, 1939, in the family's home, south of Norwalk. Growing up on his parent's pumpkin farm, he attended Norwalk schools and graduated with the Class of 1957. Following high school, he attended College in Ottawa, Kansas, and then enlisted into the United States Army where he served his country honorably. After serving, he worked at Ford Corporation, Colonial Bakery and other odd jobs as, well as many hours helping on his parents pumpkin farm.
Larry loved to play cribbage, and a variety of card games. He enjoyed being outdoors hunting, and had a love for animals, especially his beloved dog, Lily.
Larry will be remembered for his selfless being, always putting his family and friend's needs before himself. He was always looking to share a meaningful story about the greatness of his father. Although he did not have children of his own, he took care of his brothers, sisters, nieces and nephews like they were his own. He was a true protector of his family.
Lawrence is survived by his sister, Linda Shearer of Norwalk; two aunts, and a host of nieces, nephews, and cousins. He is preceded in death by his parents, Raymond and Fannie Lee; brothers, Duane, Robert and Paul; and a sister, Sharon.
There's still time to send flowers to the Visitation at the O'Leary Celebration of Life Center ~ Funeral & Cremation Services from 2:00 PM to 3:00 PM on April 28, 2019.


text:
The Ghost Is Dancing was a Canadian indie-pop band from Toronto, Ontario. The band had as many as ten members, many of whom were also in other bands.

History
The Ghost Is Dancing started out playing and improvising at a French Avenue, Toronto, student apartment in 2002. Their first show was performed in August 2004 at the time the members where leaving the apartment. The original lineup consisted of Kevin Corlis on drums, Jim DeLuca and Jamie Matechuk on guitar and vocals, Odie Ouderkirk on keyboard, Eric Krumins on bass, Jasmine Landau on trumpet and violin, and Gabrielle Nadeau on accordion. Since then, The Ghost Is Dancing has gathered considerable speed (and size), playing shows with The Hidden Cameras, Rogue Wave and The Most Serene Republic, and adding multi-instrumentalists Alt Altman, Ben Deschamps and backing vocalist Lesley Davies. Davies, Corlis and Matechuk were all childhood friends from Niagara Falls, Ontario; Ouderkirk and DeLuca also hail from there.

Their self-titled EP was released in January 2006. They released their debut full-length album, The Darkest Spark, produced by Dale Morningstar, in June 2007. This album received generally good reviews, but, with so many members with different influences contributing it was criticized as being a little unfocused.

DeLuca, Nadeau and Deschamps left the band before the recording of the 2009 album, Battles On. for which group worked with producer Chris Stringer.  Reflecting the reduction to six band members, and two more years playing together, the band had developed a tighter, more distinctive style.

Discography

EPs
The City Waltz (EP) (2004)
The Ghost Is Dancing [EP] (2006)

Albums
The Darkest Spark (2007)
Battles On (2009)

See also

Canadian musical groups
Canadian rock
List of Canadian musicians
List of bands from Canada

References

External links
 The Ghost Is Dancing official website
 The Ghost Is Dancing at Sonic Unyon
  SoundCloud

Musical groups established in 2004
Musical groups disestablished in 2010
Canadian indie rock groups
Canadian indie pop groups
Musical groups from Toronto
Sonic Unyon artists
2004 establishments in Ontario
2010 disestablishments in Ontario