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Heinrich Max Imhof (* 14. Mai 1795 oder 1798 in Bürglen, Kanton Uri; † 4. Mai 1869 in Rom; mit vollem Namen Heinrich Maximilian Imhof) war ein Schweizer Bildhauer des Klassizismus.

Leben

Heinrich Max Imhof wurde als Sohn des Kleinbauern Johann Joseph Imhof und der Katharina Barbara Arnold in Bürglen geboren. Seine Kindheit verbrachte er in Bifang bei Bürglen, wo er in einfachen Verhältnissen aufwuchs. Der Lehrer und Vedutenmaler Franz Xaver Triner (1767–1824), der dem jungen Imhof Zeichenunterricht erteilte, erkannte schon früh dessen künstlerische Begabung. Er überzeugte die Eltern davon, den 16-Jährigen 1811 beim Holzbildhauer Franz Abart in Kerns in die Lehre zu schicken. Als sein Lehrmeister die Pfarrkirche in Kerns zwischen 1814 und 1823 mit plastischen Arbeiten ausstattete, fertigte Imhof am Portal seine ersten bekannten professionellen Schnitzarbeiten. 1818 wurde der Geograf Johann Gottfried Ebel auf Imhof aufmerksam und holte ihn nach Zürich, wo er als selbstständiger Bildhauer vor allem Porträtreliefs schuf. Zu den Auftraggebern gehörte unter anderem der preußische Kronprinz Friedrich Wilhelm (IV.), der sich 1819 während seiner Schweizreise von Imhof porträtieren ließ. 1820 protegierte sein Mentor Ebel eine Weiterbildung im Atelier des schwäbischen Bildhauers Johann Heinrich Dannecker in Stuttgart und finanzierte 1824 die erste Studienreise nach Rom, die Imhof zusammen mit seinem befreundeten Bildhauerkollegen Johann Jakob Oechslin antrat. Dort machte er sich in der Werkstatt des dänischen Bildhauers Bertel Thorvaldsen mit dessen klassizistischen Gestaltungsprinzipien vertraut, die die klassische griechische Kunst der Antike zum Vorbild hatte. In der deutschen Künstlerkolonie pflegte er Kontakte zu Oechslin, zu den deutschen Künstlern Ernst von Bandel und Bonaventura Genelli sowie zum Schweizer Bildhauer und Schriftsteller Heinrich Keller.

1827 begann Imhof mit den Modellarbeiten an der Statue des David mit dem Haupt des Goliath, die Kronprinz Friedrich Wilhelm in einer Marmorausführung für die Ausstattung des Potsdamer Sommerschlosses Charlottenhof anfertigen ließ. Neben Statuen erhielt Imhof vermehrt Aufträge für Büsten, unter anderem vom bayerischen König Ludwig I., der 1832 Büsten des bayerischen Kurfürsten Maximilian I. und 1835 des Humanisten Johannes Reuchlin zur Ausstattung der Gedenkstätte Walhalla bestellte. Dessen Sohn Otto I., König von Griechenland, berief Imhof als Professor an die 1836 gegründete Kunstakademie nach Athen, wo er sich zudem mit der Restaurierung ausgegrabener Antiken und der Karyatiden des Erechtheions beschäftigte. Sein schlechter Gesundheitszustand und der Mangel an Aufträgen führten ihn 1838 wieder nach Rom zurück, wo er bis auf einen kurzen Aufenthalt in Zürich und einigen kleineren Reisen bis zu seinem Lebensende am 4. Mai 1869 wohnte und arbeitete. Neben dem deutschen und englischen Adel und Großbürgertum zählten auch der Herzog Maximilian von Leuchtenberg aus dem Geschlecht de Beauharnais und das russische Kaiserhaus zu seinem Kundenkreis. Als Vertreter der Thorvaldsen-Schule hatte Imhof ab 1848 zunehmend Mühe, sich auf dem internationalen Kunstmarkt zu behaupten, wodurch sich auch seine ökonomische Situation verschlechterte. In den letzten Lebensjahren litt er wie viele Bildhauer unter rheumatischen Beschwerden, was ihn bei der Arbeit zunehmend beeinträchtigte. Begraben wurde Imhof auf dem Campo Santo Teutonico.

Der Maler Carl Christian Vogel von Vogelstein porträtierte Imhof in einem Gemälde, das in dessen Sammlung weiterer Künstlerporträts im Kupferstichkabinett Dresden aufbewahrt wird. Imhofs Tochter Mariette heiratete den österreichischen Maler Othmar Brioschi. Zu seinen Schülern gehörten die Freiburgerin Marcello und wohl auch der Basler Ferdinand Schlöth, zu dem das Verhältnis später in eine gehässige Rivalität umschlug.

Familie
Imhof war seit 1849 mit der Protestantin Henriette Ott (1825–1892) aus Zürich verheiratet. Aus dieser Ehe gingen fünf Töchter und zwei Söhne hervor. Die Tochter Franziska Auguste Sophie (* um 1854 in Rom; † 16. März 1944 in Dresden), genannt Fanny, heiratete 1875 den Ingenieur Oskar Ludwig Kummer. Annarella Imhof (1856–1935) heiratete 1881 den Kunsthändler und Galeristen Fritz Gurlitt. Maria Imhof, genannt Mimmy, war seit 1887 mit dem Maler Othmar Brioschi verheiratet. Angela Imhof (1858–1940) heiratete 1878 in Rom den Maler Hermann Knackfuß.

Bedeutung
Die 1828/29 für den späteren preußischen König Friedrich Wilhelm IV. geschaffene Marmorstatue David mit dem Haupte Goliaths ist wahrscheinlich die erste Skulptur, die bewusst den Klassizismus Thorvaldsens mit der Bildsprache der Nazarener zu verbinden suchte. Sie steht am Anfang von Imhofs lebenslanger Beschäftigung mit den Gestalten des Alten Testaments, die er in empfindsamer Schlichtheit darzustellen verstand und denen er einen Teil seines Ruhms verdankte. Eine eigenständige Stellung innerhalb der weitverzweigten Thorvaldsen-Schule erlangte er auch durch die Einbindung von Bewegungsmotiven in die vom dänischen Künstlerunternehmer geprägte, auf Ruhe und verhaltene Gebärden ausgerichtete spätklassizistische Formensprache.

Werke (Auswahl)

 um 1814 Schnitzarbeiten am Portal der Pfarrkirche in Kerns
 1819 Büste oder Relief des preußischen Kronprinzen Friedrich Wilhelm (IV.)
 1820 Profilporträt des Bildhauers Johann Heinrich Dannecker, als Dauerleihgabe der LETTER Stiftung seit 2003 im Württembergischen Landesmuseum, Stuttgart.
 1827 Porträtbüste des Architekten Melchior Berri, Museum an der Augustinergasse, Basel, Treppenhaus
 1828 Marmorstatue David mit dem Haupt des Goliath, für Schloss Charlottenhof, Park Sanssouci, Potsdam
 1832 Büste des bayerischen Kurfürsten Maximilian I., für die Walhalla
 1831/32 Atalante im Wettlauf die goldenen Äpfel des Hippomenes aufhebend, Reste des Originals aus Gips im Kunstmuseum Bern
 1832/33 Büste des Geografen Johann Gottfried Ebel, heute in der Zentralbibliothek Zürich
 1835 Büste des Humanisten Reuchlin, für die Walhalla
 1837 und nach 1850 Madonna mit Jesuskind, Gipsabguss im Historischen Museum Uri, Altdorf
 1838 Büste des griechischen Königs Otto I., heute im Otto-König-von-Griechenland-Museum, Rathaus Ottobrunn
 1838 Büste der Herzogin Amalie von Oldenburg, dann Königin von Griechenland, heute im Stammhaus der Landessparkasse zu Oldenburg
 1841 Rebekka mit dem Armband, Original aus Gips im Kunstmuseum Bern. Marmorausführung von 1867 im Kunstmuseum Basel.
 1842 Hagar mit dem verschmachtenden Ismael, für den Herzog Maximilian von Leuchtenberg, Eremitage, Sankt Petersburg. Eine weitere Gruppe im Royal Museum and Library, Peel Park, Salford
 1845 Die Mutter des Moses setzt ihr Kind auf dem Wasser des Nils aus, für Zar Nikolaus I.
 um 1845 Marmorrelief an der Friedhofsmauer in Altdorf, Kanton Uri
 1845 Miriam, ehemals im Stadtschloss Potsdam, heute im Raffaelsaal des Orangerieschlosses und in der Staatsgalerie Stuttgart
 1846 Büste des Pädagogen Johann Heinrich Pestalozzi, für die Stadtbibliothek, heute Zentralbibliothek in Zürich
 1858 Büste des Zürcher Bürgermeisters Johann Jakob Hess, (ehemals im Haus zum Lindental/Landolthaus Zürich, heute im Kunsthaus Zürich ?)
 1859 Ruth die Ährenleserin, Staatsgalerie Stuttgart
 1865 Eva vor dem Sündenfall, Kunstmuseum Bern
 1866 Jesus vor den Schriftgelehrten, Grabstele von Henriette Imhof-Ott, Protestantischer Friedhof, Rom

Ausstellungen
 1995: Altdorf, Historisches Museum Uri

Literatur
 Nekrologe – Heinrich Max Imhof. In: Kunstchronik. Beiblatt zur Zeitschrift für bildende Kunst.4. Jahrgang, Nr. 20, 6. August 1869, S. 189 (uni-heidelberg.de).
 Edudard Prosch: Heinrich Max Imhof. In: Neujahrsblatt der Künstlergesellschaft in Zürich. 30, 1870.
 Conrad Escher: Heinrich Max Imhof. In: Schweizer Illustrierte. Band 9, 1905, S. 57–62 (e-periodica.ch).

 Carl Franz Müller: Zum 100. Todestag von zwei verdienten Urnern. In: Verein für Geschichte und Altertümer von Uri (Hrsg.): Historisches Neujahrsblatt. Neue Folge, 24–25 (1969/1970), S. 7–13.

 Dieter Ulrich: Die Eva vor dem Sündenfall …. Unveröffentlichte Lizentiatsarbeit, Universität Zürich, 1988.
 Paul-André Jaccard: Skulptur. Disentis 1991 (Ars Helvetica VII)
 Karl Iten: Heinrich Max Imhof, 1795–1869. Ein Urner Bildhauer in Rom. Herausgegeben vom Historischen Verein Uri. Verlag Gisler, Altdorf 1995, ISBN 3-9520570-5-3.

 Helmi Gasser: Der Maler Albert Anker sucht in Altdorf Heinrich Max Imhof auf. In: Historisches Neujahrsblatt / Historischer Verein Uri. Band 105, 2014, S. 121–130 (e-periodica.ch).

Weblinks

Einzelnachweise

Bildhauer (Schweiz)
Bildhauer des Klassizismus
Schweizer
Geboren im 18. Jahrhundert
Gestorben 1869
Mann

redpajama_set_name: RedPajamaWikipedia


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Intelligence, Learning, Neurotech
How 'random noise' stimulation could actually boost intelligence, brain function
by John Anderer
Silence may be golden, but a little bit of background noise may actually unlock hidden learning potential in certain people. Scientists at Edith Cowan University investigated the effects of transcranial random noise stimulation (tRNS), noting the technology may prove useful in a number of different settings.
The name is a bit misleading, as it doesn't actually make use of "noise" in the traditional sense of the word. Instead, the process involves attaching a group of electrodes to the head to facilitate a weak electrical current that passes through specific parts of the brain.
The researchers believe tRNS has serious promise as a potential tool for people with compromised learning capabilities.
"The effect on learning is promising: it can speed up learning and help people with neurological conditions," study leader Dr. Onno van der Groen says in a university release. "So, people with learning difficulties you can use it to enhance learning rate, for example. It's also been trialed on people with visual deficits, such as after stroke and traumatic brain injury. When you add this type of stimulation during learning, you get better performance, faster learning and better attention afterwards as well."
Study authors explain that tRNS helps the brain form new neural connections and pathways, a process formally referred to as neuroplasticity. "If you learn something, there has to be neuroplastic changes in your brain, which allows you to learn this information," Dr. van der Groen explains. "And this is a tool to enhance this neuroplasticity."
Generally speaking, tRNS appears to have two major effects on the mind: an "acute" effect that promotes improved performance while undergoing tRNS, and a "modulating" effect associated with longer lasting results.
"If you do 10 sessions of a visual perception task with the tRNS and then come back and do it again without it, you'll find you perform better than the control group who hasn't used it," Dr. van der Groen adds.
Can tRNS make the average person smarter?
The prospect of expanding one's learning capabilities and limits is certainly attractive. Who doesn't want to become smarter faster? However, study authors explain that technology like tRNS also come with many additional, lingering questions.
While the focus is currently on how this technology can help those with learning difficulties, researchers admit they've wondered what effect tRNS will have on a "neurotypical person." Dr. van der Groen says it's possible that tRNS may be able to take the average individual's intelligence to new heights, but adds it is unlikely the technology will create a "new level of intelligence."
"The question is, if you're neurotypical, are you already performing at your peak," the study author comments. "There's a case study where they tried to enhance the mathematical skills of a super mathematician; with him, it didn't have much of an impact on his performance, presumably because he is already a top performer in that area. But it could be used if you're learning something new."
Is 'random noise' therapy available right now?
Right now, tRNS technology is still very much in its infancy. The only way people can access it is by entering a controlled trial. Still, researchers are optimistic tRNS will one day have a number of different applications in the real world.
"The concept is relatively simple," Dr. van der Groen notes. "It's like a battery: the current runs from plus to minus, but it goes through your head as well. We're working on a study where we send the equipment to people, and they apply everything themselves remotely. So in that regards, it's quite easy to use."
Meanwhile, other scientists all over the world are currently investigating tRNS's effects on perception, working memory, sensory processing, and various other aspects of behavior. The technology has already shown notable promise as a treatment for a wide array of clinical conditions.
"We're still trying to find out how best we can use it," Dr. van der Groen concludes.
The study is published in the journal Neuroscience.
John Anderer
View John's article archive
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These Cornell-built microrobots can walk on their own — using wireless, electronic 'brains'November 9, 2022

redpajama_set_name: RedPajamaCommonCrawl


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Toodles Vox, it was nice working with you. To all my friends on Vox, please don't forget to let me know where you've all moved to.
How long can I hold out for?
've got a couple of bruises to report but other than that, I really had fun. I was still confused and I don't have the rolling quite down yet but managed to learn a couple of moves :) All I'm hoping now is to be able to remember them for next week … so to help me do that, I'm thinking of buying this book.
Anyone else know of any good Aikido books I should be reading?
Winnie enjoyed her last day today 🙂 yes, she leaving for greener pastures. The world is now her oyster! You know, I never quite understood the meaning of that. Anywho, good luck Winnie and I know it's see you soon and not farewell. Later chickie!


Again, a fantastic read by Cassandra Clare. In book #2 there are more uncovered secrets, more adventure and of course more confused feelings. Most surprisingly, Simon steps up and becomes more confident, self assured and well, a bit of a badass considering he won't die!
I loved the scene with the faerie queen, although I sympathise with Simon. He finally thinks there is a chance with Clary considering Jace is her brother, but his hope is short-lived. Although I must say, I am glad Clary gave Simon a chance...We get to see more of Magnus, who is frankly hilarious.
"I didn't know you were bringing the mundane."
His blue eyes flickered uneasily over Simon.
"That's what I like about you people," said Simon. "You always make me feel so welcome."
Jace put his head in his hands. "Tell Isabelle no."
"But she thinks it's a good idea," Alec protested.
"Then tell her no twice."
Something inside Clary cracked and broke, and words came pouring out.
"What do you want me to tell you? The truth? The truth is that I love Simon like I should love you, and I wish he was my brother and you weren't, but I can't do anything about that and neither can you! Or do you have some ideas, since you're so goddamned smart?"
He smiled at her. It was a sad smile with something else underneath. "Ypu know what the worst thing I can imagines is?"
She blinked at him. "No."
"Not trusting someone I love."
She put her hand on his sleeve. He didn't move away, but he didn't respond to her touch either. "Do you mean-"
"Yes," he said., knowing what she was about to ask. "I mean you."
"But you can trust me."
"I used to think I could," he said. "But I get the feeling you would rather pine over someone you can never possibly be with than try being with someone you can."
Infernal Devices book 1 review!


Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1ε). Because CK1ε is a crucial regulator of the Wnt signaling cascades, we determined how these CK1ε mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines.
We performed in silico modeling of various mutations and analyzed the kinase activity of the CK1ε mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1ε affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1ε in these processes.
In silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1ε, is involved in positive regulation of the CK1ε activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1ε failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1ε mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1ε mutants acted as loss-of-function in the Wnt/β-catenin pathway, and that CK1ε mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7.
In summary, these data suggest that the mutations of CK1ε found in breast cancer can suppress Wnt/β-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1ε, which are correlated with decreased phosphorylation activities of mutated forms of CK1ε both in vitro and in vivo, interfere with positive autophosphorylation at Thr 44.
Mammary carcinomas are one of the most common neoplasias in women. Several improvements in understanding the molecular pathology of breast cancer have been achieved in the past decade. In most cases, however, the molecular mechanisms underlying this malignancy are still unknown.
Sequencing of mammary carcinoma samples by Fuja and colleagues revealed that the casein kinase 1 epsilon (CK1ε) gene was mutated in this disease; CK1ε was found to be mutated within its N-terminal region with approximately 15% incidence . CK1ε is a Ser/Thr kinase with many known functions and substrates. CK1ε phosphorylates several regulators of crucial processes, such as cell proliferation, differentiation, migration, and circadian rhythms. The key known targets of CK1ε involve p53, key components of the circadian clock, the Wnt signaling pathway, and cell division machinery (for a review, see ). In the original sequencing study, 19 nonsynonymous mutations were identified in the CK1ε gene in ductal carcinoma samples . The identified mutations were shown to have a significant association with the loss of heterozygosity and decreased staining of CK1ε in the tumor sections. Some of the mutations in CK1ε were found repeatedly in several patients, such as L39Q (detected five times), L49Q (detected three times), and S101R (detected twice) . These observations suggest that these mutations may affect CK1ε function and may be favored during the microevolution of the tumor, and thus may contribute to tumor progression.
Importantly, nothing is known about how these mutations affect the kinase activity and signaling potential of CK1ε and the behavior of mammary cells. In the present study, we characterized three CK1ε mutants that were previously identified in mammary carcinoma. We demonstrated that these CK1ε mutants had limited kinase activity and failed to phosphorylate the physiological targets of CK1ε in vitro and in vivo. The analyzed mutations acted as loss of function in the Wnt/β-catenin pathway and promoted the alternative Wnt/Rac1 pathway, which in turn decreased cell adhesion and promoted cell migration.
ORFs of the wild-type (WT), full-length human CKIε cDNA (residues 1 to 416), two mutants mimicking either nonphosphorylatable Thr 44 (Thr44Ala) or constitutively phosphorylated Thr 44 (Thr44Asp), and three mutated versions (P3, P4, and P6) were cloned into pcDNA3. The truncated versions of CKIεΔC (residues 1 to 315) were cloned into pHAK-B3. Plasmids encoding mDvl2-Myc  and human Dvl3-Flag  have been previously described. Details and bacterial overexpression vectors are presented in Additional file 1.
The three-dimensional model for CK1ε was obtained via template-based homology modeling using the program PHYRE . The mutated sites and kinase-specific functional domains were mapped onto a three-dimensional model of CK1ε using the program CHIMERA . The kinase-specific functional domains in CK1ε were predicted using the NCBI Conserved Domain Database . Predictions of changes in protein stability upon point mutations were conducted using CUPSTAT . (Auto)Phosphorylation sites were predicted using GPS v. 2.1 .
Western blot analysis, immunoprecipitation, and small GTPase activity assays were performed as previously described [10, 11]. The antibodies used for the western blot analysis were as follows: mouse anti-Flag (M2; Sigma, Schnelldorf, Germany), goat anti-CK1e (sc6471; Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-Myc (sc-40) and anti-actin (sc-1615-R) (both Santa Cruz Biotechnology), anti-HA (HA.11; Nordic Biosite, Täby, Sweden), mouse anti-Rac (05-389; Upstate, Waltham, Massachusetts, USA), and mouse anti-Cdc42 (610928, 1:1,000; BD Biosciences, San Jose, California, USA). The antibodies used for immunoprecipitation were as follows: anti-CK1ε (sc-6471; Santa Cruz Biotechnology), anti-MYC (C3965; Sigma), and anti-FLAG (F1804; Sigma).
Transfected cells were grown on glass coverslips, washed with PBS, fixed for 15 minutes in 4% paraformaldehyde, washed with PBS, and blocked in 1.5% BSA, 0.1% Triton X-100 in PBS for 1 hour. After overnight incubation at 4°C with the primary antibody, cells were washed with PBS, 0.1% Triton X-100, incubated for 2 hours at room temperature with secondary antibody, washed with PBS, 0.1% Triton X-100, and counterstained with 4 μg/ml 4'',6 - Diamidino - 2 - phenylindole, dihydrochloride. Phalloidin-Alexa488 (1:66; Molecular Probes, Carlsbad, CA, USA) was added in the last 30 minutes of incubation with the secondary antibody. Samples were analyzed with a FV1000 confocal microscope (Olympus). The following antibodies were used: mouse anti-Myc (1:150) and goat anti-CK1ε (1:100) (both from Santa Cruz Biotechnology), anti-goat-Cy5 (1:500; Molecular Probes), and anti-mouse-Alexa488 (1:1,000; Molecular Probes).
AceaE-plates®96 were used for non-invasive real-time measurements with an xCELLigence RTCA SP system and RTCA software version 1.1 (both Roche Applied Science, Indianapolis, IN, USA). First, a background measurement was performed using 100 μl complete cultivation media with or without CK1 inhibitors incubated for 30 minutes in the incubator (37°C, 5% CO2). MCF7 cells were trypsinized, quantified, and seeded (15,000 cells/well) in an additional 100 μl cultivation media. The impedance was then monitored continually for a period of 20 hours. Data are presented as a cell index. In parallel to xCELLigence measurements, cells were seeded (15,000 cells/well) in 24-well plates, and the effects of IC261 on cell number and cell viability were determined after 20 hours. The cell number was measured using a Coulter Counter (Immunotech a. s., A Beckman Coulter Company, Praque, Czech Republic). Cell viability was determined using eosin staining.
MCF7 cells (4 × 105 cells/ml) with or without CK1ε inhibitors were seeded in 30 μl drops on the inner side of a 10 cm plate lid. The lid of the plate was then turned upside down and placed on top of the plate filled with 10 ml PBS. MCF7 cells under the force of gravity aggregated at the bottom of the hanging drop. After 24 hours, cell clusters were photographed, collected, and resuspended by pipetting up and down seven times, and single cells released from the clusters were counted.
HEK293 and MCF7 cells were transfected in a 24-well plate format with the indicated combinations of plasmids using polyethylene-imine (HEK293) or FuGENE (MCF7). The amounts used per well were as follows: vectors encoding Dvl2, Dvl3, and CK1ε, 300 ng; pRLtkLuc (Renilla Promega, Madison, WI, USA), 50 ng; and firefly luciferase reporters, 200 ng. The following luciferase reporters were used: pSuperTopFlash , p-E-cadherin-Luc , pNFAT-Luc, and pAP1-Luc (Stratagene, La Jolla, CA, USA). The total amount of plasmid DNA per well was kept constant in all conditions. Twenty-four hours post transfection, cells were harvested and processed using the Dual Luciferase kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. To normalize for the efficiency of transfection, firefly luciferase values were normalized to Renilla luciferase in each well.
Each data point was run in duplicate, and at least three independent experiments were performed. Datasets from each experiment were normalized to the control, and all individually normalized experiments were statistically analyzed by analysis of variance and Tukey's multiple comparison post tests (GraphPad Prism software, La Jolla, CA, USA The graphs indicate the mean ± standard deviations from at least three independent replicates.
MCF7 cells (5 × 104 cells) were seeded into the upper chamber of Transwell plates (Transwell 96-well plate, 5.0 μm polycarbonate membrane; Corning (Corning, MY, USA)). The inhibitors (D4476 or IC261; Calbiochem, Darmstadt, Germany) were added into the bottom chamber at the indicated concentrations. Identical amounts of dimethylsulfoxide were used as a control. After 24 hours, cells that migrated through the filter into the bottom chamber were counted with a Coulter Counter. The number of migrating cells was normalized to control conditions and expressed as a migration index.
L39Q is the most frequently occurring mutation found in patients with breast cancer. In these patients, this mutation occurs alone (Patient P6) or in combination with other mutations - L39Q and S101R (Patient P3), or L39Q, L49Q, and N78T (Patient P4) (Figure 1a). Leu 39 and Leu 49, as well as Ser 101 (occupied exclusively by Ser or Thr), are absolutely conserved among CK1 paralogs and CK1ε orthologs (data not shown).
Casein kinase 1 epsilon mutant phosphorylation of Dishevelled. All three mutants of casein kinase 1 epsilon (CK1ε) bind Dvl but are unable to cause the typical phosphorylation-dependent shift of the Dvl protein. (a) Schematic representation of the CK1ε mutants used in this study. (b) HEK293 cells were transfected with a plasmid encoding Dvl2-Myc and the CK1ε variants. CK1ε phosphorylates Dvl proteins, which subsequently exhibit a mobility shift in western blot analysis. The P6 mutant maintains residual activity, while the P3 and P4 mutants are unable to promote a Dvl2-Myc shift. (c) Truncated versions of CK1ε that lack the C-terminal autoinhibitory domain do not differ from full-length proteins in their ability to shift the Dvl2 protein.
One of the best defined functions of CK1ε is the Wnt-induced phosphorylation of Dvl, a crucial component of the Wnt signaling cascades [11, 14–20]. In the first step, we analyzed the capacity of individual CK1ε mutants to phosphorylate Dvl in mammalian HEK293 cells. As shown in Figure 1b, WT CK1ε promoted the formation of phosphorylated and shifted (PS) Dvl2, whereas mutant forms did not. A partial shift was observed with the P6 mutant, but the P3/P4 mutants were indistinguishable when compared with the control-transfected sample. Deletion of the C-terminus of CK1e, which inhibits CK1e kinase activity when phosphorylated , did not affect these results (Figure 1c). Importantly, using immunoprecipitation we were able to detect the presence of overexpressed Dvl2 or Dvl3, and both WT and mutated CK1e kinases in one protein complex (Figure 2a and Additional file 2), suggesting that the CK1e mutants still possess the ability to bind Dvl.
Interaction of casein kinase 1 epsilon mutants and Dishevelled 2. Casein kinase 1 epsilon (CK1ε) mutants associate with Dvl2, co-localize with Dvl2, and promote the punctate cytoplasmic localization of Dvl2. (a) HEK293 cell lysates transfected with either wild-type (WT) CK1ε or the P3, P4, and P6 mutants together with Dvl2-Myc were lysed and immunoprecipitated using an anti-Myc antibody. WT CK1ε and all of the CK1ε mutants efficiently bind the Dvl2 protein. (b) Dvl2-Myc protein localization in transfected COS7 cells was observed by confocal microscopy using an anti-Myc antibody. Dvl2 is either found in cytoplasmic inclusions or evenly dispersed within the cytoplasm. (c) WT CK1ε co-transfected with Dvl2-Myc into COS7 cells dissolves most of the Dvl2 punctae, resulting in the predominance of evenly distributed Dvl2 protein. In contrast, the P3, P4, and P6 mutants enhance the formation of Dvl2 clusters with a punctate appearance in the cytoplasm. A localization pattern of CK1ε was observed with an anti-CK1e antibody. All CK1ε forms show even cytoplasmic distribution when transfected alone. (d), (e) Cells with an even or punctate distribution of Dvl2 and CK1e were counted. For each condition, at least 150 cells were examined.
Phosphorylation of Dvl by CK1ε leads not only to the formation of PS-Dvl but also to changes in the intracellular distribution of Dvl [11, 19]. Dvl is usually present in dynamic multiprotein aggregates [21, 22] called Dvl dots. Based on the cellular context and activity of CK1e, which promotes the dissolution of Dvl aggregates, Dvl is usually present either in dots or in an even distribution (see Figure 2b for COS7 cells; see Additional file 2 for HEK293 cells). WT CK1ε strongly promoted an even localization of Dvl2-Myc (Figure 2c, d and Additional file 2). In contrast, all of the analyzed mutants (P3, P4, and P6) promoted the formation/maintenance of Dvl dots in COS7 cells and co-localized with Dvl in these multiprotein complexes (Figure 2c, d), similar to earlier observations with dominant negative CK1ε or CK1 inhibitors [11, 19]. All CK1ε proteins were evenly distributed in the absence of Dvl (Figure 2c, e). These data together show that despite the fact that CK1ε mutants bind and co-localize with Dvl, they cannot phosphorylate Dvl or efficiently promote its even localization.
To understand how individual mutations are spatially related to functionally conserved regions in CK1ε, we developed three-dimensional models for individual CK1ε mutants. In the single-point mutant (P6), the mutated site (Leu 39) directly adjoins conserved residues that participate in ATP binding (Figure 3a). The CK1ε mutant P3 contains two mutations, L39Q and S101R. These mutated sites are distant from each other not only in sequence but also spatially (Figure 3b). While Leu 39 is located in the β-strand-rich N-terminal lobe, Ser 101 is located at the C-terminal end of helix I in the primarily helical C-terminal subdomain. The S101R mutation is predicted to destabilize and alter local protein structure, thus affecting the structural integrity of the four-helix bundle, which forms the structurally conserved core of the C-terminal subdomain .
Homology model of the catalytic domain of casein kinase 1 epsilon mutants. Three-dimensional representation of a homology model of the catalytic domain of casein kinase 1 epsilon (CK1ε) (residues 1 to 295). Phylogenetically conserved functional sites across the serine/threonine kinase superfamily are indicated: pink, substrate binding pocket; yellow, ATP binding site; red, catalytic loop; blue, activation loop. The individual point mutations that were found in patients (a) P6 (L39Q), (b) P3 (L39Q, S101R), and (c) P4 (L39Q, L49Q, N78T) are indicated in orange. (d) A mutation cluster region in P4. The mutations and ATP binding region are indicated in orange and yellow, respectively. A blue sphere indicates a predicted autophosphorylation site (Thr 44).
The last analyzed mutant, P4, contains three point mutations: L39Q, L49Q, and N78T. Although the individually mutated sites are distant in primary sequence, when mapped onto the three-dimensional structure of CK1ε they cluster into a very small area with a radius <10 Å that is located between strands four and five as well as into α-helix B of the N-terminal lobe (Figure 3c). Interestingly, all of these mutations surround a conserved predicted autophosphorylation site at Thr 44 in the N-terminal catalytic domain. This site is localized within a loop region between the terminus of the fourth β-strand (only five residues upstream from the mutation site L39Q) and α-helix B (five residues downstream from the second mutation site L49Q) (Figure 3d).
To assess kinase activities of the individual CK1ε mutants, we attempted to heterologously express individual mutants of human CK1ε as affinity tagged recombinant proteins lacking the autoinhibitory domain in Escherichia coli. Despite significant efforts, we were unable to obtain soluble overexpression for all of the constructs when we expressed them with the same affinity tag. Soluble expressions were feasible for WT, P3, P4, and P6, however, when the recombinant proteins were tagged to His6, to maltose binding protein, to maltose binding protein, and to Small Ubiquitin-like Modifier (SUMO), respectively. The individual recombinant fusion proteins were assayed for their ability to phosphorylate Dvl in vitro. Although the data from this in vitro phosphorylation assay were in qualitative agreement with our in vivo data (see above), we considered these in vitro data inconclusive since the presence of multiple different tags has made direct interpretation impossible (see Additional files 1 and 3).
To test the role of mutated CK1ε proteins in the canonical Wnt pathway, we induced Wnt/β-catenin signaling via the overexpression of several of the components of this pathway - such as Dvl2, Dvl3, β-catenin, Wnt3a, and the Lrp6 co-receptor - and analyzed TCF/LEF-driven transcription using the TopFlash reporter system . As shown in Figure 4, Dvl2-Myc weakly activated the TopFlash reporter in HEK293 and MCF7 cells, but the signal was boosted when WT CK1e was co-expressed. The CK1e mutants P3 and P4 failed to synergize with Dvl2, and P6 promoted Dvl2-driven TopFlash only moderately (Figure 4a, b). Very similar results have been obtained with Dvl3-Flag (Additional file 4).
Casein kinase 1 epsilon mutants downregulate Wnt/β-catenin signaling at the level of the Dishevelled protein. HEK293 or MCF7 cells were transfected with the indicated plasmids, and after 24 hours TCF/LEF-driven transcription was measured using the SuperTopFlash dual luciferase reporter assay. (a) Dvl2 enhances TCF/LEF-dependent transcription when co-transfected with wild-type (WT) casein kinase 1 epsilon (CK1ε) in HEK293 cells. In contrast, all CK1ε mutants are unable to promote Dvl-mediated TopFlash. (b) Similar results to those obtained for HEK293 cells have been obtained for the breast cancer cell line MCF7. (c) β-Catenin induces the TCF/LEF transcriptional response in the cell by recruiting transcriptional cofactors directly to the TCF/LEF complex. Neither WT CK1ε nor the CK1ε mutants could influence the high TopFlash signal caused by constitutively active β-catenin, confirming that CK1ε and its mutants operate upstream of β-catenin (HEK293 cells). (d) The Wnt signaling pathway was induced by seeding B1A-wnt3a cells (fibroblasts that constitutively produce the Wnt3a protein) over transfected HEK293 cells. Wnt3a induces the Wnt/β-catenin/TCF/LEF pathway in control cells transfected with empty plasmid. The Wnt3-induced signal is enhanced by overexpression of WT CK1ε but is strongly depleted by overexpression of the CK1ε mutants. (e) Constitutively activation of the co-receptor LRP6 in transfected HEK293 cells confirmed the regulatory function of CK1ε downstream of the Wnt-receptor complex. WT CK1ε transduces the signal from the activated receptor towards TCF/LEF-driven transcription, while mutant CK1ε proteins abolish downstream signaling. Neither WT nor mutant CK1ε are able to markedly activate the TCF/LEF signal when Wnt stimulation is blocked by the mutant LRP6. (f) Nonphosphorylatable (T44A) and phospho-mimicking (T44D) mutants of CK1ε were co-expressed with Dvl2-Myc in HEK293 cells. The effects on TCF/LEF-dependent transcription show that T44 D behaves as overactive CK1ε. (a) to (f) Data represent the mean ± standard deviation from normalized values. *P < 0.05, ***P < 0.001; n.s., not significant; one-way analysis of variance, Tukey post-test, n ≥ 3.
Importantly, the effects of CK1e were Dvl dependent because the overexpression of any form of CK1e had only negligible effects (Additional file 4). The inhibitory effects of the CK1e mutants were found at the level of Dvl because the activation of TopFlash by constitutively active (S33A)-β-catenin could not be significantly modulated by the overexpression of any CK1e (Figure 4c). Co-cultivation with fibroblasts producing Wnt3a or overexpression of crucial Wnt co-receptor Lrp6 efficiently induced TCF/LEF-dependent transcription. Co-expression of WT CK1e promoted TopFlash even further, whereas the expression of the P3, P4, and P6 CK1e mutants were able to reduce the Wnt3a/Lrp6-induced signal. This effect was obvious in Wnt-3a-stimulated cells and became statistically significant in cells overexpressing Lrp6 (Figure 4d, e). These analyses demonstrate that the P3, P4, and P6 mutants of CK1e are dysfunctional in the Wnt/β-catenin pathway and act upstream of β-catenin.
Our structural modeling (Figure 3) suggested that CK1e mutations found in breast cancer may affect the predicted autophosphorylation site at Thr 44. To test the function of Thr 44 we generated two mutants mimicking either nonphosphorylatable Thr 44 by replacing Thr 44 for alanine (T44A) or constitutively phosphorylated Thr 44 by replacing Thr 44 for aspartic acid (T44D). As shown in Figure 4f, T44D-CK1e is a more potent activator and T44A a less potent activator of TCF/LEF-driven transcription than WT CK1e when co-expressed with Dvl2-Myc. This finding confirms the positive functional role of Thr 44 phosphorylation in the modulation of CK1e activity, and explains how L39Q mutation contributes to the diminishing of CK1e activity in the Wnt/β-catenin pathway.
Our laboratory and others have previously shown that CK1e can act as a switch that promotes Wnt signaling pathways that are dependent on PS-Dvl (such as the Wnt/β-catenin or PS-Dvl-dependent noncanonical pathways) and blocks the Wnt/Rac1/JNK pathway [10, 19]. We hypothesized that the CK1e mutants may mimic CK1e inhibition. To test this prediction, we tested the effects of WT and P3 CK1e on Rac1 activation. As shown in Figure 5a, WT CK1e slightly downregulated Rac1 activity, whereas expression of the P3 mutant or inhibition of CK1 by D4476 promoted this activity. We were not able to detect any effects of CK1e on the activity of another small GTPase, Cdc42 (Additional file 5), and we also failed to detect active RhoA in HEK293 cells (data not shown).
Casein kinase 1 epsilon mutants act as loss of function in the Wnt/β-catenin pathway. Casein kinase 1 epsilon (CK1ε) mutants activate small GTPase Rac1 and the transcriptional activity of AP1 and NFAT. (a) Lysates from HEK293 cells transfected with Rac1-Myc were subjected to pull-down of the active GTP-Rac1 form with agarose-PAK beads. HEK293 cells were either co-transfected with wild-type (WT) or mutant CK1ε or treated with 100 μM D4476 inhibitor for 4 hours prior to lysis. The P3 mutant promotes Rac1 activation, as a higher amount of GTP-Rac1 was pulled from the lysate (as compared with WT CK1ε). Consistently, D4476 inhibits the function of WT CK1ε and elevates the level of GTP-Rac1 in cells. Rac1 protein was detected with an anti-Myc antibody. Western blots were quantified by densitometry, and the GTP-Rac1/Rac1 ratios are indicated by the numbers below the panel. After probing, membranes were stained with amidoblack to confirm equal protein loading. (b) HEK293 cells or (c) MCF7 cells were transfected with an AP1-luciferase reporter, Dvl2-Myc, and CK1ε as indicated. Cells were lysed, and luciferase activity was analyzed the next day. (d) HEK293 cells were transfected with a NFAT-luciferase reporter and CK1ε as indicated. Cells were lysed, and luciferase activity was analyzed the next day. (b) to (d) Data represent the mean ± standard deviation. *P < 0.05, **P < 0.01; one-way analysis of variance, Tukey post-test, n ≥ 3.
Importantly, the CK1e mutants also promoted Dvl2-Myc-induced AP1-mediated transcription, which is downstream of the Rac1/JNK pathway (Figure 5b, c). Similar results were obtained with Dvl3-Flag (Additional file 4). We also tested the effects of the CK1e mutants on the noncanonical Wnt/Ca2+ pathway. As shown in Figure 5d, WT CK1e did not affect the transcriptional activity of NFAT, a transcription factor that is activated by calcium waves. The activity of the NFAT reporter was promoted by the CK1e mutants, however, suggesting that mutant forms of CK1e can promote the Wnt/Ca2+ pathway.
In summary, our data demonstrate that mutant CK1e, which is present in ductal carcinoma, can act as an inhibitor of the Wnt/β-catenin and an activator of the noncanonical Wnt/Rac1/JNK and Wnt/Ca2+ pathways.
To date, whether mutations in CK1e that are associated with breast cancer have any effect on the behavior of breast adenocarcinoma cells is not clear. Our results show that mutated forms of CK1e behaved identically to CK1 inhibition in all tested assays - compare Figure 2 and , and compare Figure 5 and . Based on these analyses, one could expect that the presence of CK1e mutants is functionally equivalent to the pharmacological inhibition of CK1e.
Taking advantage of this finding, we tested the effects of the CK1 inhibitors D4476 and IC261 on the cell adhesion of MCF7 epithelial breast cancer cells. The MCF7 cell line retains several characteristics of differentiated mammary epithelium, including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes , and is suitable for analyzing cellular changes during tumor progression. D4476 is the most specific inhibitor of CK1 known to date [26, 27], whereas IC261 specifically inhibits the CK1δ and CK1ε isoforms at doses <10 μM [27, 28]. MCF7 cells efficiently aggregated in hanging drops and usually formed one cell cluster per drop (Figure 6a) after overnight aggregation. Inhibition of CK1 interfered with aggregate formation and caused the MCF7 cells to become more loosely attached to each other (Figure 6a, c). To quantify the level of adhesion, we mechanically resuspended the cell clusters and counted the number of single cells, which was increased by CK1 inhibition (Figure 6b, d). Similar results were obtained when we downregulated the levels of CK1δ and CK1ε in MCF7 cells via siRNA-mediated knockdown (Additional file 6). Next, we seeded the MCF7 cells in the presence of 5 μM IC261 and compared the morphology of the adherent cells after 20 hours. Control cells efficiently adhered to the plastic surface, spread out, and formed cytoskeletal actin networks that were visible as stress fibers and cytoplasmic protrusions (Figure 6e, f). In contrast, IC261-treated cells attached only loosely to the surface and exhibited a predominantly rounded morphology with no detectable stress fibers and protrusions (Figure 6e, f).
Casein kinase 1 epsilon inhibition decreases the cell adhesion of MCF7 breast epithelial cells. (a) to (d) MCF7 cells grown in hanging drop culture tend to form compact cell clusters under control conditions (dimethylsulfoxide (DMSO)). With increasing amounts of the casein kinase 1 epsilon (CK1ε) inhibitors - (a), (b) D4476, and (c), (d) IC261 - the large single-cluster formation is disrupted, and cells form smaller and disintegrated aggregates. After resuspending these aggregates by pipetting up and down seven times, a significantly higher number of single cells was observed to be released from clusters in conditions treated with (b) D4476 and (d) IC261, as normalized to control. ***P < 0.001, Student's t test, n = 3. (e), (f) MCF7 cells were seeded in the presence or absence of 5 μM IC261. Cell morphology was analyzed after 20 hours in (e) phase contrast or (f)after staining of actin filaments (phalloidin, green) and cell nuclei (Hoechst, blue). (g), (h) Dynamics of MCF7 cell adhesion was monitored for 20 hours using the xCELLigence System (Roche Applied Science). Cell adhesion is expressed as the cell index for two control wells (DMSO) and two experimental wells (2.5 μM IC261). (h) Treatment with IC261 did not affect the cell number (compared with control) during 20 hours of stimulation.
To quantify the intensity of cell adhesion to the cell surface, we employed direct measurements of impedance caused by cell adhesion using the xCELLigence System (Roche Applied Science). This technology measures changes in the electrical conductivity produced by cells, which attach to golden electrodes that cover the surface of the well. The changes correspond to the area of cell that is in direct contact with the bottom of the well. These parameters allow for quantification of adhesion intensity (termed cell index) given the prerequisite that cell number and cell viability are identical between samples. CK1 inhibition dramatically decreased the cell index (Figure 6g). Because 2.5 μM IC261 did not affect the cell number (Figure 6h) or cell viability (89% vs. 87.6% for dimethylsulfoxide vs. 2.5 μM IC261), we interpreted changes in the cell index as decreased adhesion to the dish surface. In summary, these results suggest inhibition of CK1δ/ε prevents the formation of cell-cell and cell-surface contacts in epithelial MCF7 cells.
The decrease in cell adhesion that was induced by CK1 inhibition and observed in different experimental setups (Figure 6) might be relevant to cell behavior associated with tumor progression, such as epithelial-mesenchymal transition or increased cell migration. This view is supported by our analysis of the E-cadherin promoter (Figure 7a). E-cadherin mediates intracellular contacts and is commonly used as a marker of epithelial fate, whose downregulation contributes to a shift towards mesenchymal fate . Overexpression of WT CK1e in MCF7 cells slightly increased the activity of the E-cadherin promoter. In contrast, mutant forms of CK1e decreased reporter activity, and the effects of the P6 mutant were statistically significant from WT (Figure 7a). Similar results were obtained in HEK293 cells (Additional file 7). These data suggest that mutation of CK1e contributes to decreased adhesion through the regulation of E-cadherin expression.
Blocking casein kinase 1 epsilon function decreases E-cadherin expression and promotes cell migration. (a) Wild-type (WT) and mutant casein kinase 1 epsilon (CK1ε) were expressed in MCF7 cells together with the reporter encoding E-cadherin-promoter coupled to luciferase. Cells were lysed and the activity of luciferase was analyzed next day. (b) MCF7 cells were treated after seeding out by increasing concentrations of IC261 (2.5 μM, 5 μM and 10 μM) for 48 hours and the expression of E-cadherin was analyzed by western blotting. Empty arrowhead, unspecific band, which confirms equal protein loading. (c), (d) MCF7 cells were seeded out in the upper part of the Transwell migration chamber and were stimulated with indicated doses of (c) IC261 and (d) D4476. The number of migrating cells in the bottom chamber was counted next day, and is expressed as the migration index. Data represent mean ± standard deviation. *P < 0.05; one-way analysis of variance; (a), (c)Tukey post test, (d) Student's t test; n = 3. DMSO, dimethylsulfoxide.
Treatment with the CK1e inhibitor IC261 decreased the expression of endogenous E-cadherin in a dose-dependent manner in MCF7 cells (Figure 7b). These changes in cell adhesion translate into changes of the migratory capacity of MCF7 cells in Transwell assays. As shown in Figure 7c, the dose-dependent inhibition of CK1δ/ε by IC261 promoted the migration of MCF7 cells. Similar effects were obtained with 10 μM D4476, which also increased the migration of MCF7 cells (Figure 7d). These results demonstrate that inhibition of CK1e or analogically mutants of CK1e can decrease cell adhesion and promote cell migration via mechanisms involving the activation of Wnt/Rac1 and Wnt/Ca2+ and the repression of E-cadherin expression.
CK1ε is a crucial regulator of both the canonical Wnt/β-catenin pathway [14, 15, 17] and noncanonical Wnt pathways [10, 11, 30–32]. Wnt ligands from both classes - which either activate β-catenin or do not - activate CK1e, which in turn phosphorylates Dvl [11, 20]. Phosphorylated PS-Dvl is thought to mediate downstream signaling, which results in the stabilization and nuclear translocation of β-catenin or in the activation of some noncanonical pathways [11, 32].
Our results demonstrate that the three CK1ε mutants analyzed in this study that were identified in samples of breast cancer efficiently bind, but fail to phosphorylate, Dvl and act as loss of function in the Wnt/β-catenin pathway. We demonstrate that cells expressing these mutants are biased towards Rac1/JNK and NFAT activation at the expense of the Wnt/β-catenin pathway. Importantly, phosphorylation of Dvl by CK1e was shown to be inhibitory for the noncanonical Wnt/Dvl/Rac1/JNK pathway [10, 19]. CK1e can thus act as a switch that directs signaling away from the Wnt/Rac1/JNK branch of noncanonical signaling towards the Wnt/β-catenin and PS-Dvl-dependent noncanonical pathways. Wnt/Ca2+, the other branch of noncanonical Wnt signaling, was shown to be antagonized by CK1 . Our results confirm this observation and show that the CK1e mutants tested here increase the transcriptional activity of NFAT. These effects are not promoted by co-expression of Dvl (data not shown), suggesting that CK1e can repress NFAT directly. Indeed, phosphorylation by CK1e was shown to promote the cytoplasmic localization of NFAT and reduce its transcriptional activity in other experimental systems . Together, our results provide the first evidence that the switch function of CK1ε, which was predicted based on Xenopus and cell culture experiments, is physiologically relevant and may contribute to cancer progression in ductal carcinoma.
The observation that ductal carcinoma-specific mutants of CK1ε promote the Wnt/Rac1/JNK and NFAT pathways and, on the other hand, inhibit the Wnt/β-catenin pathway are in good agreement with some clinical observations. The expression of β-catenin in breast cancer is usually low and was shown by several laboratories to correlate with poor prognosis [35–38]. Moreover, β-catenin is not localized within the cell nucleus in breast tumors [35–38], suggesting that Wnt/β-catenin signaling is in the off state in most breast cancers. Important to note, however, is the fact that there are also reports supporting a positive role for Wnt/β-catenin signaling in breast cancer (for a review, see ).
Wnt itself was first identified as an oncogene that is activated by the insertion of the mouse mammary tumor virus; and the mouse mammary tumor virus-Wnt-1 transgenic mouse is a well-established model for studies of the genetic basis of breast cancer (for a review, see ). Moreover, increased nuclear β-catenin levels, which correlate with cyclin D1 levels and a poor prognosis, were found in a subset of patients . In contrast to other cancers, such as colon or skin cancer, the key components of the Wnt/β-catenin pathway, such as axin, adenomatous polyposis coli, or β-catenin are mutated in only a small portion of cases (for a review, see ). The levels of Wnt pathway components, which are common to both the canonical and noncanonical pathways, are altered more often; Dvl1 is upregulated in 50% of ductal breast cancer cases , and sFRP1 - a soluble Wnt antagonist that can block both Wnt/β-catenin and noncanonical pathways - is repressed in more than 80% of breast carcinomas . These observations suggest that modulators of Wnt signaling, both at the extracellular level (sFRP1) and intracellular level (Dvl and CK1ε), will have a critical role in the biological outcome.
CK1ε has been shown to be highly expressed in non-invading ductal carcinoma in situ , which is also highly positive for β-catenin staining . Based on our findings, we can speculate that the high activity of CK1ε in ductal carcinoma in situ reduces Rac-1/JNK and NFAT activity, keeping the cells tightly attached and blocking tumor invasion. CK1ε function can be compromised by somatic mutations , which dramatically affect CK1ε kinase activity (present study). The functional importance of this event supports the fact that samples with mutations in CK1ε show an increased frequency of loss of heterozygosity at the CSNK1ε locus . Lack of CK1ε activity/expression leads to the activation of the Rac1/JNK/AP1 and NFAT pathways, which mediates the invasion of breast cancer cells and correlates with increased aggressiveness of the breast cancer [33, 44–47]. Based on our data and other published information, we propose that mutation of CK1ε might be important for the transition between ductal carcinoma in situ and invasive carcinoma.
The effect of mutation or lack of CK1ε on Wnt signaling remains to be tested. In this context, it is especially important to determine how the status of CK1ε affects Wnt5a. Although Wnt5a is implicated in breast cancer pathology, its functional mechanism remains unclear. First, Wnt5a expression was shown to positively correlate with disease-free survival , and Wnt5a blocks breast cancer cell invasion [49–51]. On the other hand, Wnt5a is frequently upregulated in breast tumors in comparison with surrounding tissues  and was shown to promote the invasion of MCF7 cells via the JNK pathway . We can speculate in cells with mutated CK1ε that Wnt5a will predominantly signal via the Rac-1/JNK and NFAT pathways, thus promoting breast cancer cell invasion and tumor aggressivity [44, 45]. In contrast, in cells with abundant and intact CK1ε, Wnt5a principally stimulates other noncanonical signaling pathways that involve CK1, such as the Wnt/CK1ε/Rap1  and Wnt/Yes-Cdc42-CK1α  pathways, and promotes cell adhesion .
In summary, our data demonstrate that the CK1ε mutants found in breast cancer act as loss of function, suppress jWnt/β-catenin, and promote Wnt/Rac-1-mediated and NFAT-mediated pathways. Furthermore, we show that inhibition of CK1ε reduces the intracellular adhesion and increases the migration of breast cancer cells. Our findings show that CK1ε has the potential to act as a tumor suppressor in breast cancer via its negative effects on the Wnt/Rac1/JNK and NFAT pathways. These results demonstrate for the first time how mutations in CK1ε affect cell behavior and may provide a general paradigm for consequences of CK1ε alteration in cancer.
In the present study, we functionally analyzed mutations in CK1ε that are frequently found in breast cancer. Our data demonstrate that breast cancer-specific mutants of CK1ε act as loss of function in the Wnt/β-catenin pathway but activate the Wnt/Rac1/JNK and Wnt/Ca2+ pathways. Physiological consequences of these signaling events in MCF7 breast cells include increased migratory capacity and decreased E-cadherin expression and cell adhesion.
Silvie Foldynová-Trantírková, Petra Sekyrová contributed equally to this work.
Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The authors thank Dr Christina Modak for providing cDNA templates for CK1ε and P3, P4 and P6 mutants. They also thank Dr Marek Mlodzik for GST-bPDZ constructs, Dr S Yanagawa (Kyoto University, Japan) for expression vectors encoding Dvl2-Myc, Dr Jan Kitajewski (Columbia University) for HA-Wnt3a-B1A cells, Dr Mikhail Semenov and Dr Xe He (Harvard Medical School) for vectors encoding mLrp6, Dr Saitoh (University of Tokyo, Japan) for E-cadherin reporter, and Dr Randy Moon (University of Washington, Seattle) for SuperTopFlash, ca-β-catenin and hDvl3 constructs. Plasmids for the overexpression of recombinant CK1ε were generated at the Dortmund Protein Facility . The project was supported by the Grant Agency of the Czech Republic (301/07/0814 to LT and TD, 204/09/0498 and 204/09/H058 to VB, 301/09/0587 to PK), the Academy of Sciences of the Czech Republic (KJB501630801, AVOZ50040507, AVOZ50040702), the Ministry of Education, Youth and Sports (MSM0021622430) and by an EMBO Installation Grant to VB.
SF-T, PS, KT, OB, and VB carried out the molecular genetic and biochemical studies. SF-T, EB, WB, and LT participated in cloning, plasmid production and protein purification. PK, AK, and TD designed and coordinated the study. LT and VB designed and coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.


text:
//Write an expression that calculates trapezoid's area by given sides a and b and height h.
//The three values should be read from the console in the order shown below.
//All three value will be floating-point numbers.

using System;

class Trapezoids
{
    static void Main(string[] args)
    {
        double a = double.Parse(Console.ReadLine());
        double b = double.Parse(Console.ReadLine());
        double h = double.Parse(Console.ReadLine());
        double area = ((a + b) / 2) * h;
        Console.WriteLine("{0:F7}",area);
    }
}

redpajama_set_name: RedPajamaGithub


The Journey Is Just Beginning?
We have come through a wonderful journey from Ash Wednesday to Glorious Easter on April 21. My prayer is that the Holy Spirit has brought you closer to Jesus in humility and faithfulness to our Lord. This is but one of many spiritual journeys you will travel. Every time you intentionally draw closer to you Lord and Savior you will be closer to your last breath on this planet.
Faith is to trust God Almighty with every area of our lives and give God the worship and glory God deserves. Works means to serve God and others with all our heart, soul and mind. No one can do this perfectly, but we can make every effort to be the humble, servant minded person God wants us to be.


text:
1MATCH URL: https://assets.rappler.com/165326BC5537484D932E767609095927/img/B0455EE02A874B7E9D0D70DABC17832C/afp-20160420-australia-great-barrier-reef-bleaching-001.jpg
Coral bleaching hits 93% of Great Barrier Reef – scientists
Researchers at James Cook University said only 7% of the huge reef had escaped the whitening triggered by warmer water temperatures
@afp
Published 3:52 PM, April 20, 2016
Updated 6:02 PM, April 21, 2016
BLEACHING. This undated handout photo released on April 20, 2016 by XL Catlin Seaview Survey shows a diver checking the bleached coral at Heron Island on the Great Barrier Reef. XL Catlin Seaview Survey/AFP
SYDNEY, Australia – Australia's Great Barrier Reef is suffering its worst coral bleaching in recorded history with 93% of the World Heritage site affected, scientists said Wednesday, April 20, as they revealed the phenomenon is also hitting the other side of the country.
After extensive aerial and underwater surveys, researchers at James Cook University said only 7% of the huge reef had escaped the whitening triggered by warmer water temperatures.
"We've never seen anything like this scale of bleaching before," said Terry Hughes, convenor of the National Coral Bleaching Taskforce.
The damage ranges from minor in the southern areas – which are expected to recover soon – to very severe in the northern and most pristine reaches of the 2,300 kilometer (1,430 miles) site off the east coast.
Hughes said of the 911 individual reefs surveyed, only 68 (or 7%) had escaped the massive bleaching event which has also spread south to Sydney Harbour for the first time and across to the west.
Researcher Verena Schoepf, from the University of Western Australia, said coral was already dying at a site she had recently visited off the state's far north coast.
"Some of the sites that I work at had really very severe bleaching, up to 80 to 90 percent of the coral bleached," she told Agence France-Presse. "So it's pretty bad out there."
Severe throughout the Pacific
Australia's Environment Minister Greg Hunt said it was "absolutely clear that there is a severe coral bleaching event occurring not just in the Great Barrier Reef but throughout many parts of the Pacific".
Hughes said the bleaching began in Hawaii late last year and had already affected several Pacific islands.
"Right now, New Caledonia, the Coral Sea, the northern half of the Barrier Reef and New South Wales are bleaching severely, and western Australia is quickly catching up," he told Agence France-Presse.
Bleaching occurs when abnormal environmental conditions, such as warmer sea temperatures, cause corals to expel tiny photosynthetic algae, draining them of their color.
Corals can recover if the water temperature drops and the algae are able to recolonize them, but scientists warned last year that the warming effects of a El Nino weather pattern could result in a mass global bleaching event.
Hughes said while bleaching had been linked to El Niños, which generally occur every 4 to 6 years, "it wasn't until 1998 that one finally caused a bleaching event to happen" on the Great Barrier Reef.
"So the issue is global warming," Hughes told Agence France-Presse, saying the link between water temperature and the severity of the bleaching was clear.
Hughes said the impact on the Great Barrier Reef would have been even worse had not a tropical cyclone which smashed into the Pacific island of Fiji in February brought rain and cooler weather to parts of Queensland.
"If you think about it, being rescued by the vagaries of a cyclone is a fairly precarious place to be," he added.
Andrew Baird, from James Cook University's centre for coral reef studies, said he had been surprised by the scale and severity of the event on the major tourist drawcard which is teeming with marine life.
"We've been expecting a really big event for a while I suppose and here it is," he told Agence France-Presse.
Baird said because the bleaching was far less serious in the southern reaches "lots of the reef will still be in good shape".
"But the reef that's been badly affected – which is a third to a half of it – is going to take a while to recover," he told Agence France-Presse.
"And again the big question is how many of these events can it handle? And I think the answer is not many more." – Madeleine Coorey, AFP / Rappler.com
Filed under:El Niño•Great Barrier Reef•climate•climate change•climate science•coral bleaching•coral reefs•environment•Australia


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How Sappi Connects with Its Customers
Sappi understands the importance of creating innovative solutions that meet evolving customer needs. Our future success depends on listening to, communicating with and responding to our customers. From award-winning product development to stunning promotional campaign materials and a groundbreaking retrospective collection, Sappi stands apart.
Customer collaboration is key to future growth
Read more about How Sappi Connects with Its Customers
Tips to Help Your Clothes Last Longer and Reduce Your Footprint
Did you know that the consumer use of products is the largest contributor to the environmental footprint of almost all the products you buy? A recent study by researchers from the Norwegian University of Science and Technology's Industrial Ecology Program1 found that consumer's impact on the environment after the purchase of products such as clothing was responsible for more than 60 percent of that product's greenhouse gas emissions and up to 80 percent of water use.
Read more about Tips to Help Your Clothes Last Longer and Reduce Your Footprint
Little Architects with Big Dreams are Building Sustainable Cities for the Future
Young students all over the world are building sustainable solutions to the world's biggest challenges, and the technology they're using to express their ideas is quite outside the box.
At Escola Ramon Fuster School in Bellaterra, Spain, students created a model for sustainable cities of the future featuring clean transportation, a sustainable architecture, and eco-friendly materials and systems.
Read more about Little Architects with Big Dreams are Building Sustainable Cities for the Future
"Sustainability in Action" Video Series
Whether through environmental, social or economic initiatives, Ingersoll Rand is committed to making the world a better place to live, through sustainability and energy efficiency. A new video series, "Sustainability in Action," shines a light on the many ways in which the company uses creativity, engagement and works with customers to deliver on its global climate commitment.
Read more about "Sustainability in Action" Video Series
More Mining Companies Begin to Share Resources Using Integrated Infrastructure
Traditionally, mining companies have built their own infrastructure at greenfield sites, and due to necessity, constructed their own supporting "pit to port" infrastructure, including power and water supply schemes, roads and railways. However, as a result of growing cost pressures and increasing capital to build new greenfield operations, a trend is developing, whereby mining companies are seeking opportunities to share resources to save on expenses.
Read more about More Mining Companies Begin to Share Resources Using Integrated Infrastructure
Mining's Quiet Sustainability Revolution Offers New Opportunities for Innovative Engineering
A few years ago, when the Rio Tinto decided to expand its iron ore production in Pilbara region of Western Australia, they faced a problem. While the remote region accounts for more than 40 percent of the world's iron ore production, it is also one of the driest parts of the world. Ironically, however, the problem for Rio Tinto's expansion was too much water: the new ore the company wanted to reach lay underneath huge underground aquifers, which meant that the company had to do something with the excess water that came up.
Read more about Mining's Quiet Sustainability Revolution Offers New Opportunities for Innovative Engineering
Siemens Presents New Metro for Riyadh
74 Inspiro type metro vehicles
2 and 4-car trains designed for the region's extreme climate
Certification runs will start in 2016 in Germany
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Sustainable Solutions for the e-Commerce Era
Learn more in this video
Today, 30 percent of global online sales are made through mobile devices and by 2017, 60 percent of U.S. retail sales will involve the Internet. Everything is "on demand" and people are using mobile devices to get what they want, when they want it, where they want it. But there are unintended consequences of everything being on demand.
These residential deliveries typically have more stops, with fewer packages at each stop. More stops, more miles, more fuel, more emissions.
Read more about Sustainable Solutions for the e-Commerce Era
UPS Accelerates Progress Toward Sustainability Goals
Nearly Tripled Number of Miles Driven by Alternative Fuels and Advanced Technology Fleet in 2014
ATLANTA, Ga., July 29, 2015 /3BL Media/ - UPS (NYSE: UPS) today released its 13th annual Sustainability Report, highlighting its growing investment in alternative fuel and advanced technology vehicles and commitment to log 20 million hours of volunteer time by the end of 2020.
Read more about UPS Accelerates Progress Toward Sustainability Goals
Electronics From Milk
By Carol Baroudi
One of my biggest concerns on the road to sustainable IT is that, today, electronics are made almost exclusively from nonrenewable sources. Metals and oil for plastic come from the earth's crust, never to be replenished. Last week, I wrote about new computer chips made from wood. This week, the news is about electronic components made from, yep, milk.
Read more about Electronics From Milk


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Yellowstone landmark's weird movement finally explained
By Laura Geggel published 16 April 20
This landmark has been 'pulsing' up and down for two decades.
Norris Geyser Basin at sunset (Image credit: Shutterstock)
A swath of Yellowstone National Park larger than Chicago has been rising and falling in erratic jitters over the past 20 years, and it's all because of some wayward magma, a new study finds.
This magma has been so active, it's responsible for lifting up the area around Norris Geyser Basin at Yellowstone a total of 5 inches (almost 13 centimeters) since 2000.
The finding sheds light on the enigmatic magmatic system that lies beneath Yellowstone. Researchers first discovered Yellowstone's magma system about 40 years ago in a pivotal study published in 1979 in the journal Science study; in the years since, scientists have learned that Yellowstone's magma system causes the ground to not only rise but also fall.
"That discovery motivated our quest to understand the cause(s) of uplift and subsidence [sinking] near Norris," Daniel Dzurisin, a co-researcher of the new study and a retired research geologist with the U.S. Geological Survey (USGS) Cascades Volcano Observatory in Vancouver, Washington, told Live Science in an email.
Related: Rainbow Basin: Photos of Yellowstone's colorful grand prismatic hot spring
The Norris Geyser Basin isn't one to sit still. From 1996 to 2004, an area extending about 18 miles (29 kilometers) rose 4.7 inches (12 cm). Then, from 2005 to 2013, it sank 2.8 inches (7 cm), National Geographic reported. After that, the land rose again at a rate of 5.9 inches (15 cm) a year between late 2013 and early 2014, the most rapid rate of uplift ever documented at Yellowstone.
A magnitude-4.9 earthquake struck the basin in March 2014, and the land rose and fell like a slow-motion Jack-in-the-box until early 2019. After all of this incredible movement, the basin is now about 5 inches above where it was 20 years ago.
So, what caused all this commotion?
To find out, the researchers used a combination of GPS and satellite data to map and track Earth-changing events at Norris and the Yellowstone caldera (a volcanic crater) over time, Dzurisin said.
"There are about two dozen GPS stations in the park that operate continuously, providing very precise information about ground movements in three dimensions at those locations," Dzurisin said. Meanwhile, the satellite sensed whether the land had moved toward or away from the satellite, which allowed it to produce "a complete map of ground movements over very large areas," Dzurisin said.
Together, the GPS and satellite data showed the culprits for the tumultuous movement at Norris Geyser Basin. First, the data revealed an intrusion of magma about 10 miles (16 km) underground that caused the 1996 to 2001 uplift.
The two recent Earth-moving events in 2013-2014 and again in 2016, can be explained by fluids that bubbled out from the intruded magma. In late 2013, these fluids accumulated into a sealed, underground reservoir that ruptured when the 2014 earthquake hit. Once the reservoir broke, the fluids escaped, causing sinking above the old reservoir as the fluids flowed westward and upward along the region's underground faults and fractures. In 2016, these fluids escaped again and accumulated closer to the Earth's surface.
As these fluids became stuck underground and pressure built up, the ground above them rose, and when the fluids got expelled and moved to a new location, the ground above where they once were sank, National Geographic reported.
The study is the first to identify a complete cycle of magma intrusion, as well as fluids escaping, traveling upward and then accumulating elsewhere, he said.
Furthermore, it's possible that the escape of these fluids to just below the Earth's surface might be related to the increased activity at Steamboat Geyser — located in the Norris basin — starting in early 2018, Dzurisin said.
In 2018, Steamboat erupted 32 times, surpassing its record of 29 eruptions in 1964, the researchers wrote in the study. In 2019, it had erupted 48 times, according to USGS. As of April 10, 2020, the geyser has erupted 11 times.
The study was published online Jan. 28 in the Journal of Geophysical Research: Solid Earth.
Yellowstone National Park: The early years (photos)
Yellowstone's grizzly bear trapping photos
In Photos: Best national parks to visit during winter
OFFER: Save 45% on 'How It Works' 'All About Space' and 'All About History'!
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The rise and fall of the Great Library of Alexandria


In an attempt to be both aware and participate in the progression towards a circular economy, I collect stories from around the world and post them here in a weekly segment. Most have to do with eliminating waste. Some have to do with broader issues. Some with issues that are tied in to how materials are handled. Regardless of these topics, I attempt to tie them all in together and give my readers an accurate snapshot of how resources are used efficiently today.
I went to the Advance Design & Manufacturing Expo in Toronto Canada this week and shared a few things on my Insta, here are a few of those highlights.
Nothing groundbreaking or surprising here. The waste industry continues to promote incineration instead of allegiances with manufacturers to change the way waste is created, disassembled and reused.
An interesting rundown to various ways the culinary team at Asana has committed to "using every component of food, they reduce costs for the company while also doing their part to reduce the amount of waste in the world."
A new dish goes out.
Leftovers are taken back to the kitchen.
Chefs assess the remaining ingredients and use their creative juices to find ways to repurpose them.
Leftovers get used in new dishes and creative recipes.
For example, if asparagus is served at lunch one day, it might get scrambled with eggs the following morning, tossed in a salad at lunch or dinner, or pureed into a soup. It might even go through all these stages!"
This is actually a very effective process in reducing waste and costs. Some creativity is needed by the chefs in creating meals, but a process of planning leftovers into the next meal could yield some very strong savings. Some education and safety is required so people don't think they are getting Bob's leftover rice in their burrito.
I saw this the other day, NYC 'zero waste' progress report gives updates on shift to single-stream, local AD, and was a little surprised. Everyone knows that if you plan on selling your material on the commodities market that seperate clean streams are best as they preserve material quality far better than mixed or single stream. So what is NYC thinking? Well by combining recyclables (plastics, bottles, cans and paper) they save a bin. A bin in a very big city is a very big deal.
"Switching to single-stream will likely increase capture rates, and expanding organics diversion access will help, though a lack of available space makes siting some of the necessary processing infrastructure to handle more material locally a challenge."
"Switching to single-stream recycling by 2020 is seen as one way to reach that goal. The city reports that while progress is slightly behind schedule, a consultant's study is slated for completion this summer..."
... So when I saw that the NRC was hosting a webinar on NYC Zero Waste Program, naturally I signed up. The presentation offered a tad more insight into the grand scale of NYC dilemma but offered really just one additional benefit of the single-stream program - Less trucks on the road. I shouldn't belittle this decision, NYC has a lot of trucks. And getting those off the road is a big deal. I was just hoping they'd share projected gain vs loss revenue by eliminating the paper bin and going with a single stream recycle program. That would've been interesting to see. Here are some screen shots from that webinar.
#zerowaste program in #newyork presentation highlights. Single stream meant to simplify collection and they believe it'll not compromise the marketability of the waste material commodities. #imo It will do just that unfortunately. #organics collection program will cover 18% of city in 2018. Major hurdles ahead for #NYC. #donatenyc meant to reduce how much is disposed in #msw program.
Every Sustainability team of every company, city and country out there right now is trying to align their programs with the UN Sustainable Development Goals.
The best business communication piece I've been able to find belongs to TECK.
The World Bank does a fantastic job at displaying how each country is progressing towards achieving theSDG's.
...and highlights individual aspects of each goal.
"RACING TO ZERO examines our society's garbage practices in terms of consumption, preparation, use and production, and discovers some amazing solutions in San Francisco, which is successfully taking the necessary steps to reach zero waste. Cities all over the United States have instituted zero-waste policies of their own, and it is through these mandates that we are challenged to think differently about not only how we handle our garbage, but what it can become."


This is one I've featured a few times but the host, Alexia Leachman, had taken a bit of a break from podcasting. I was happy to see The Fear Free Childbirth Podcast back in action and thought this episode looked pretty interesting.
The podcast itself is just one of the many fantastic resources on the Fear Free Childbirth website.
Alexia experienced the extreme fear of pregnancy and childbirth that is Tokophobia and has made it her mission to work toward supporting other expecting parents and to take the fear out of birth.


How To | Page 2 of 2 | Dog Training With Canine Perspective, Inc.
This week's WWAPDT vlog: Your Vet Doesn't Know Behavior may ruffle a few feathers, but in my experience, it is completely true. This is something I come across way too often. People who take their dogs to the vet to deal with their aggression, anxiety, fear, nervousness, etcetera.


Panoramic set of fresh bread products.
Burger bread isolated on white background.
Whole grain bread put on kitchen wood plate with a chef holding gold knife for cut.
Fresh rye bread or whole grain bread. Isolated object on white background. Healthy baked bread, whole bread on white background.
The bread and a wheat on the wooden desk.


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Верши́нка — річка у Києві, у місцевості Чоколівка, права притока Либеді.

Опис

Протяжність річки — 2,2 км.

Починається неподалік рогу вулиць Михайла Мішина та Волинської. Перші сотні метрів тече на захід, далі різко змінює напрям на північний. Виходить на денну поверхню поблизу будинку № 26 по вул. Ушинського. Протікає паралельно залізниці, спочатку частково у колекторі (здебільшого під станцією Караваєві Дачі — з початку 2000-х років). Після перетину шляхопроводу над залізничною платформою Караваєві Дачі виходить на денну поверхню, протікає вздовж колій і, повернувши на північ та перетнувши залізничні колії, впадає у Либідь. Впродовж останніх сотень метрів течії приймає 2 ліві притоки.

Остання притока, що впадає у Вершинку за 10 метрів до впадіння у Либідь, бере початок поблизу зупинного пункту Караваєві Дачі, тече весь час паралельно залізниці, у верхній течії може мати воду лише періодично, але останні 300—400 метрів має вигляд струмочка шириною до 1 метра (але найчастіше ширина не перевищує 0,5-0,7 м). Подекуди дещо розширюється, у одному місці над струмком навіть перекинуто кладку. Цікаво, що за 10 метрів до впадіння у Вершинку ширина струмка в одному місці зростає ледь не до 2-2,5 метрів і нагадує крихітне озерце, але перед впадінням у Вершинку ширина струмка знову не перевищує 0,5 м.

Джерела

Річки Києва
Струмки
Солом'янський район


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Holcomb Township est un township du comté de Dunklin dans le Missouri, aux États-Unis.

Le township s'appelait à l'origine Holcomb Island Township. Il est fondé en 1845.

Références

Article connexe
 Missouri

Source de la traduction

Township au Missouri
Comté de Dunklin


The Burwell v. Hobby Lobby decision has now opened the door for business corporations to be identified as "persons." This type of identification allows for the corporations to be recognized in connection with a particular religious faith. This holding has enabled corporate entities to acquire more power than ever before. And giving this authority to corporations has led to opting out of the contraceptive mandate and thus further admonished the health care rights that are given to many female employees employed through Hobby Lobby stores. The power that is now in the hands of business corporations may have drastic impacts on the future of businesses and their employees. The women who work amongst the thousands of male employees of Hobby Lobby stores, Conestoga, and Mardel, will consequently feel the impact of such changes. The importance of the contraceptive mandate was overlooked and the people behind large for-profit corporations fail to see the benefit of promoting public health and gender equality. Furthermore, this also disregards the probability of many negative repercussions this decision may have on women in the work field, the unemployment rates, the power that is overhauling corporations and taking away from the health of their employees, and the impact on a society filled with women in the work force. Thus, making it necessary to analyze this decision, the background of the decision makers, and the possibility of future ramifications. Is this decision going to change women's rights in the land of the free? How much power can the corporate moguls gain while simultaneously diminishing the rights of their employees? This "power," given to for-profit corporations, holds many changes in the future of large corporations and their employees, alongside the impact of the success of their businesses and the repercussions of the employees in the long run.
Does the Mechanical License Provision of the Copyright Act Violate the Copyright Clause?


At EAG Law LLC, we focus on civil litigation – and on providing counseling and advice that may help you avoid litigation.
For plaintiffs involved in a civil litigation matter, the objective is to right a wrong, honor an agreement, or to obtain compensation for an injury. Defendants in a civil litigation matter want to aggressively protect their rights in opposing the plaintiff's claim, or assert claims of their own.


The following bug report applies only to the Hog4PC on a laptop without any programing or playback wings and in keyboard "MAP" mode. I will test tomorrow on a Hog4PC with the wings to confirm if it is isolated only to keyboard shortcuts using keyboard "MAP" mode.
With the scene and list palletes unguarded mouse clicking the selected pallete will go on that scene or list, but I am unable to release the active scenes or lists by clicking and holding on the pallete while pressing the "Z" key for release.
It actually appears that none of the virtual playback functions are working while using the method of "Click and hold selected Scene/List Pallete + Mapped Keyboard Playback Key".
I am seeing the same thing but I am also see that this did not work in Hog 3.
I will log this, thanks for the information!
Thanks Michael, I just left work after running Hog4PC off of a Hog3 programing and playback wing. The virtual go and release functions off the scene and list palletes work normally as they should. So I guess the above mentioned bug is isolated to the keyboard map mode.


I can't say that stunt platformers have necessarily been my thing over the journey, with the likes of Ubisoft's Trials being the closest I've ever come to enjoying them, but rarely have they clicked in a way that might see me commit any more than an hour or so, and certainly not without a friend to make the experience more palatable… and then FutureGrind came into my life. FutureGrind has been one of those "coming soon" projects for several years now, before the development team at Milkbag Games suddenly dropped a Jan 22nd release date upon us; the obvious question now is… have the years in gestation been kind?
Gameplay in titles such as FutureGrind is king, and what a treat it is, to experience all of the subtle (and not so subtle) tweaks that the game throws at you. The difficulty curve is balanced superbly with new platforming factors to consider, gameplay elements and things to consider dropped upon you at a reasonable (but not too slow) pace, but the core premise is simple; get to the end of the level without blowing up. To do so you'll be riding your very uniquely designed bikes, from the Slice to the Left Coast, the Gimbal and a small handful of others across different coloured platforms looking to bounce, hang or grind off of them on your way to the end of level's checkered flag. The idea sounds simpler than it is to execute, of course, each platform is colour coded and if a wheel of your bike (a strangely vertically aligned bike I might add) doesn't match the colour of the platform it's landing on and it's game over for you. The pairing of the old and the new systems with some creative flair works incredibly well to revitalise a formula that was perhaps beginning to stale a bit.
The gameplay loop itself is a fairly simple and repetitive one. Within each "world" that you visit you'll have three different objectives. The first is always the same, safely reach the end of the level, but once that hurdle is overcome, a more devious duo of tasks reveal themselves. Sometimes you're required to hang-grind for a certain distance, others demand that you to avoid platforms of a certain colour, and they've all been developed to fit in with the design of the core level. You'll die often, but it's not a concern with a nearly non-existent death penalty meaning you're back in the fray with a click of a finger, and less than a second of downtime. The fact that death finds you so quickly is the simple product of momentum, and trying to keep up with the increasing speed and demands of the level, but with each death, your familiarity with the track solidifies. Gradually you'll begin to develop proficiency with the particular track and with each attempt you'll slowly inch closer to success. Inevitably, you'll achieve that endorphin rush that only comes with the euphoria of overcoming the puzzle thrown at you. It's an addictive loop and one that I kept craving to return to even when life was pulling me elsewhere. Points are also allocated for tricks and combos that you perform, so chasing down a high score or a new tier of trophy serves as an enticing secondary objective for any player.
If negotiating increasingly difficult and intensely fast platforming puzzles wasn't already enough for you, there's some clever, very cyberpunk inspired storytelling taking place in the background of each challenge. Far from necessary and still fairly basic, the story involves two warring sponsors and corporations, with the tug-of-war pulling you in the middle of it as well. Optional objectives will open up from time to time that dives a bit deeper into this story, fleshing the narrative out whilst simultaneously providing some different objectives to those you typically complete.
Whilst the core play mode provides several hours of fun, and the pursuit of high scores in those levels can add some additional time to the game clock, there is a noticeable lack of content to try in the game. Beyond the main mode, players can have a few practice runs on the variety of different bikes or explore the "Grindpedia" where they can get a quick refresher on all the fundamentals. Competing against friends in a 1v1 points shootout, or even to use their ghost to learn a few things would instantly add hours to the experience but ultimately as fun as the core game is the lack of longevity is a reasonable concern for those looking to immerse themselves further in the brilliance that Milkbag have developed.
For those looking for something a bit different from their stunt platformers, FutureGrind is for you, and if you've grown tired of the same old formula seen from others in the genre, FutureGrind is for you. An intriguing, but not essential plot serves as a solid base for tightly refined, highly polished gameplay systems, making FutureGrind a highly enjoyable experience, just don't go expecting it to consume a whole lot of your time. Milkbag has made me a believer in the genre, and I'm excited to see where the studio can take this concept going forward.


Chuck Westfall is the unassuming and authoritative head media liaison for Canon, USA. Fake Chuck Westfall is an invented anti-hero and the surly voice behind the Fake Chuck Westfall blog, a site that pokes fun at Canon, Nikon and the photo industry in general. After maintaining secrecy for six years the man behind the account has revealed his identity, announcing that he's moving on from the blog.


Rogers & Sons, Inc. uses the latest technique in computer estimating with the Quick Pen Estimating Program, a Wang 2200 system and IBM/Macintosh networking systems. Mr. Rogers developed his "software" for mechanical sheet metal, mechanical piping and fibrous glass duct estimating in the early 70's with progressive current updating. The Quick Pen and Wang systems are used for engineering, design and estimating and the IBM/Macintosh systems serve all functions including accounting.