- Apr 2024
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www.theguardian.com www.theguardian.com
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www.theguardian.com www.theguardian.com
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www.theguardian.com www.theguardian.com
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Amy Westervelt und Kyle Pope fassen kurz und treffend die wichtigsten Desinformationstaktiken der Fossilindustrie zusammen: - Fossilindustrie als Garantin der Energiesicherheit - Gegensatz von Wirtschaft und Umwelt - Verbraucher:innen brauchen fossile Energien für ihren Lebensstandard - Fossilindustrien sind Teil der Lösung, nicht des Problems - Fossilindustrien als Wohltäterinnen und Sponsorinnen.
https://www.theguardian.com/us-news/2024/apr/14/climate-disinformation-explainer
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www.derstandard.at www.derstandard.at
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www.nytimes.com www.nytimes.com
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In den USA führen wachsende Datencenter, die u.a. durch die Industriepolitik steigende Produktion in Fabriken und immer mehr Elektrofahrzeuge zu einem steilen Anstieg des Bedarfs nach Elektrizität. Der zusätzliche Verbrauch wird in fünf Jahren etwa dem jetzigen Kaliforniens entsprechen. Die bestehenden Klimaziele werden dadurch gefährdet. Viele neue Gaskraftwerke werden bereits projektiert, unter anderem, weil die Regulierungen fossile Energien begünstigen. https://www.nytimes.com/interactive/2024/03/13/climate/electric-power-climate-change.html
Tags
- institution: Electric Power Research Institute
- institution: Southern Environmental Law Center
- 2024-03-14
- country: USA
- industry: digital
- expert: Daniel Brooks
- variable: electricity demand
- institution: R Street Institute
- expert: Tyler H. Norris
- expert: Greg Buppert
- expert: Devin Hartman
Annotators
URL
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www.theguardian.com www.theguardian.com
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Eine neue Studie beschäftigt sich mit der zunehmenden Frequenz der Aufeinanderfolge extremer Trockenheit und extremer Niederschläge in Pakistan und in vielen afrikanischen Ländern. Ähnliche Phänomene lassen sich in Norditalien feststellen. https://www.theguardian.com/environment/2023/nov/14/extreme-drought-in-northern-italy-mirrors-climate-in-ethiopia
Tags
- country: Ethiopia
- process: increasing risk of droughts
- NGO: WaterAid
- process. increasing risk of floodings
- expert: Katerina Michaelides
- study: WaterAid Africa Drought Analysis
- country: Mozambique
- expert: Tim Wainwright
- country: Pakistan
- country: Ghana
- country: Burkina Faso
- region: Mediterranean
- country: Uganda
- region: Africa
- by: Sandra Laville
- 2023-11-14
- country: Italy
Annotators
URL
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- Mar 2024
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www.derstandard.de www.derstandard.de
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Der Standard fasst mehrere Studien zum zur Verringerung der Schneedecke durch die globale Erhitzung in den Alpen und auch in anderen Gebirgszonen der Welt zusammen.
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www.derstandard.at www.derstandard.at
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Österreich hat 2023 22% weniger Gas verbraucht als 2021. ür die Hälfte dieser Einsparungen sind die hohen Temperaturen verantwortlich. Rechnet man auch den – teilweise wetterbdingten – höheren Anteil der der Erneuerbaren an der Stromproduktion heraus, liegt die Einsparung nur bei 4,5%, in den letzten Monaten noch deutlich darunter. https://www.derstandard.at/story/3000000207212/echte-einsparung-oder-einfach-wetterglueck-warum-oesterreich-weniger-gas-verbraucht
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www.reuters.com www.reuters.com
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Shell hat seine Emissionsziele für 2030 reduziert und die Ziele für 2035 gestrichen. Strategische Ziele unter dem neuen CEO Wael Savan sind Erhöhung der Profitabilität, Erhaltung des Ölgeschäfts auf dem bestehenden Niveau und Ausbau des Gasgeschäfts. Shell ist der größte LNG-Händler der Welt. https://www.reuters.com/sustainability/climate-energy/shell-loosens-2030-carbon-emissions-target-2024-03-14/
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www.theguardian.com www.theguardian.com
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Shell plant nicht mehr, seine Öl-Produktion bis 2030 wenigstens um 1-2% pro Jahr zu reduzieren. Zwischen 2023 und 2035 will die Firma jährlich 40 Milliarden Dollar in die Öl-und Gasproduktion und 10-15 Milliarden in „Low-Carbon“-Produkte investieren. Die Ölproduktion soll bis 2030 stabil bleiben, die Gasproduktion ansteigen. Shell beruft sich auch darauf, dass man 2021 die Anteile an einem Projekt im Permian Basin verkauft und damit die Ölproduktion früher als geplant bereits gedrosselt habe. https://www.theguardian.com/business/2023/jun/14/shell-drops-target-to-cut-oil-production-as-ceo-guns-for-higher-profits
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www.theguardian.com www.theguardian.com
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Shell will die Emissionsintensität seiner Öl-Produkte bis 2030 nur noch um 15-20% statt bisher um 20% reduzieren. Das verfässerte Ziele ist in der jüngsten Version der Energiewende-Strategie des Konzerns enthalten, die einen Teil des Jahresberichts 2023 bildet. Für Gas wurden keine Angaben gemacht. Auch für die absolute Höhe der Emissionen gibt der Fossilkonzern kein Ziel an. Durch neue Projekte wird Shell seine Öl- und Gasproduktion bis 2025 um 500.000 Barrel täglich steigern.
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www.repubblica.it www.repubblica.it
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Der italienische Energiekonzern Eni will bis 2027 27 Mrd. Euro investieren. Priorität habe dabei die Erhöhung der Rendite. In erneuerbare Energien werde investiert, wenn sie profitable Geschäfte versprächen. Die Originalquelle zeigt: Es wird massiv in die Exploration von Öl-und Gasfeldern und eine Ausweitung der fossilen Produktion investiert, die 2030 ihren Höchststend erreichen soll. https://www.repubblica.it/economia/finanza/2024/03/14/news/eni_il_dividendo_sale_a_1_euro_per_azione_nel_piano_al_2027_investimenti_per_27_miliardi-422310817/
Eni-Strategie 2024-2027: https://www.eni.com/it-IT/investitori/strategia/piano-strategico.html
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- Feb 2024
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www.biorxiv.org www.biorxiv.org
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RRID:ZFIN_ZDB-ALT-171010-14
DOI: 10.1101/2024.02.22.581649
Resource: RRID:ZFIN_ZDB-ALT-171010-14
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-171010-14
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www.theguardian.com www.theguardian.com
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Ausführlicher Bericht über die neue Studie zum Zustand des Amazonas-Regenwalds. Bis 2050 drohen 10-47% einen Kipppunkt zu erreichen, jenseits dessen sie ihre jetzigen Funktionen für Kohlenstoff- und Wasser Zyklen verloren. Die Studie beschäftigt sich mit 5 Treibern für Wasser-Stress. Um den Regenwald sicher zu erhalten, ist der Verzicht auf jede weitere Entwaldung und das Einhalten der 1,5°-Grenze nötig. https://www.theguardian.com/environment/2024/feb/14/amazon-rainforest-could-reach-tipping-point-by-2050-scientists-warn
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Eine Studie des Forums Ökologisch-Soziale Marktwirtschaft und des Ökoinstituts fordert, dass sich Deutschland besser auf die kommende Ausweitung des EU-Emissionshandels auf Tanke und Heizen einstellt, den geltenden CO<sub>2</sub>-Preis erhöht und zugleich Maßnahmen zur sozialen Abfederung wie das Klimageld umsetzt. https://taz.de/Europaeischer-Emissionshandel/!5992314/
Studie: https://foes.de/publikationen/2024/2024-02_KAD_ETS2-KSF.pdf
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www.climatechangenews.com www.climatechangenews.com
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Nigeria investiert große Summen in die schon länger geplante Trans-Sahara Pipeline. Ob sie je zum Transport von Gas bis zum Mittelmeer gebraucht werden wird, ist allerdings unklar, da Europa seinen Gasbedarf größtenteils durch LNG deckt und ihn voraussichtlich schnell reduzieren wird.https://www.climatechangenews.com/2024/02/14/problems-mount-for-sahara-gas-pipeline-leaving-nigerian-taxpayers-at-risk/
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subethaedit.net subethaedit.net
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Similar to hackmd and etherpad... perhaps related to them genealogically?
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en.wikipedia.org en.wikipedia.org
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Conflict-free Replicated Data Type (CRDT) https://en.wikipedia.org/wiki/Conflict-free_replicated_data_type
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www.braneframe.com www.braneframe.com
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www.derstandard.de www.derstandard.de
- Jan 2024
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www.theguardian.com www.theguardian.com
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Edelmann PR hat 2022 intensiv mit der Charles Koch Foundation zusammengearbeitet. Edelmann PR, der weltgrößte PR-Konzerne, hatte sich wiederholt verpflichtet, Klimaneugnung nicht zu unterstützen. Die Koch-Brüder, ein wichtiger Teil der amerikanischen Fossilindustrie förderten und fördern seit Jahrzehnten Klimarleugnung. Es gibt Hinweise auf weitere Verbindungen von Edelman zu Koch Industries. https://www.theguardian.com/us-news/2024/jan/14/edelman-charles-koch-foundation-climate
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www.theguardian.com www.theguardian.com
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www.repubblica.it www.repubblica.it
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www.nytimes.com www.nytimes.com
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www.repubblica.it www.repubblica.it
- Dec 2023
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www.theguardian.com www.theguardian.com
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www.desmog.com www.desmog.com
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Zur PR für die Vereinigten Arabischen Emirate während der COP28 trugen Prominente Influencer: innen bei, die fast alle ihre Auftraggeber nicht nannten. Einige von ihnen erklärten später, die Seiten zum Greenwashing der Emirate missbraucht worden. https://www.desmog.com/2023/12/14/instagram-influencers-paid-to-boost-uaes-climate-credential-over-cop28/
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www.derstandard.at www.derstandard.at
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www.derstandard.at www.derstandard.at
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Die OMV und die Adnoc fusionieren ihr petrochemiegeschäft zu dem neuen Konzern Beruf, an dem beide gleiche Anteile halten. Aus dem Artikel geht auch hervor, dass die adnock durch Indizierung ihrer Anteile mit der OMV auch über die österreichische Politik bei der OMV mitbestimmt. https://www.derstandard.at/story/3000000194416/klimaphaenomen-el-nino-soll-bis-april-zu-hoeheren-temperaturen-beitragen
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www.theguardian.com www.theguardian.com
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franklin.library.upenn.edu franklin.library.upenn.edu
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colenda.library.upenn.edu colenda.library.upenn.edu
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eduscol.education.fr eduscol.education.fr
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ticle R. 511-14 du code de l’éducation
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- Nov 2023
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www.nytimes.com www.nytimes.com
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Hitzebedingte Todesfälle bei über 65-Jährigen haben seit den 90ern um 85% zugenommen. Senior:innen sind – wie kleine Kinder – zweimal soviel Hitzewellen-Tagen ausgesetzt wie 1986-2005. Extreme Hitze führte 2022 zu Produktivitätsverlusten von ca. 863 Milliarden USD. Alle Indikatoren für öffentliche Gesundheit haben sich in den letzten 9 Jahren verschlechtert. – Die NYT stellt den 2023 Report des Lancet Countdown ausführlich dar. https://www.nytimes.com/2023/11/14/climate/climate-change-health-effects-lancet.html
Mehr zum Rreport: https://hypothes.is/search?q=tag%3A%222023%20report%20of%20the%20Lancet%20Countdown%20on%20health%20and%20climate%20change%22
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www.nytimes.com www.nytimes.com
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Vor der Apec-Konferenz haben China und die USA "statements of cooperation" veröffentlicht, die als positive Signale für eine Zusammenarbeit beim Klimaschutz gewertet werden, auch wenn China nicht auf Investitionen in Kohlekraftwerke verzichtet. Beide Seiten wollen die Kapazität bei Erneuerbaren bis 2030 global verdreifachen. Erstmals ist China bereits, Reduktionsziele für alle Treibhausgase festzulegen. https://www.nytimes.com/2023/11/14/climate/us-china-climate-agreement.html
Sunnylands Statement on Enhancing Cooperation to Address the Climate Crisis: https://www.state.gov/sunnylands-statement-on-enhancing-cooperation-to-address-the-climate-crisis/
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www.theguardian.com www.theguardian.com
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Der State of Climate Action Report 2023 zeichnet ein vernichtendes Bild von den Bemühungen, die Pariser Klimaziele zu erreichen. Der Kohleausstieg müsste 7 Mal schneller erfolgen, die Entwaldung 4 Mal schneller gestoppt werden. Außer dem Wachstum bei elektrischen Autos gibt es keinen Parameter, der dem Pariser Abkommen ansatzweise entspricht. 2023 erreichten die fossilen Subventionen nach der Invasion der Ukraine ein Zehnjahreshoch. https://www.theguardian.com/environment/2023/nov/14/world-behind-on-almost-every-policy-required-to-cut-carbon-emissions-research-finds
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www.liberation.fr www.liberation.fr
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Der französische Konzern TotalEnergies hat einen Report über die Zukunft des Energiesystems publiziert, der radikale Einschränkungen vor allem bei der Ölproduktion fordert. Der Bericht widerspricht der aktuellen Monzernpolitik die auf Expansione beim Öl ausgerichtet ist. https://www.liberation.fr/economie/transition-energetique-totalenergies-pris-en-flagrant-delit-de-contradiction-20231114_WMIQ7ZKDCFD2RM5HQGS24GYARQ/
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Die Deutsche Umwelthilfe will durch eine Klage erreichen, dass Mercedes spätestens ab 2030 keine Verbrenner mehr verkaufen darf. Gleichzeitig klagt die Organisation auch gegen BMW und den Gaskonzern WinterShall Dea. Https://taz.de/Umwelthilfe-zieht-Mercedes-vor-Gericht/!5969656/
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www.liberation.fr www.liberation.fr
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Die extremen Niederschläge der letzten Wochen im französischen Nordwesten, vor allem im Departement Pas-de-Calais, führen auch zu einer Überfüllung der Grundwasserspeicher. Das abfließende Wasser kann zu die Überschwemmungen verlängern, nachdem die Niederschläge aufgehört haben. Erklärender Artikel in der Libération.. https://www.liberation.fr/environnement/climat/inondations-dans-le-pas-de-calais-les-nappes-phreatiques-goutte-deau-qui-fait-deborder-la-vase-20231114_LPHYWOAFT5BJNMHJUHWLDG3ZOM/
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Die deutsche Bundesregierung unterstützt Siemens Energy mit einer Bürgschaft von 7,5 Milliarden Euro. Siemens Energy leidet unter Milliarden Verlusten des für Windkraft zuständigen Konzernteils Siemens Gamesa. https://taz.de/75-Milliarden-Euro-Buergschaft/!5973113/
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www.theguardian.com www.theguardian.com
Tags
- expert: Fabian Flues
- expert: Philippe Sands
- 2022-11-14
- treaty: energy charter treaty
- NGO: Powershift
Annotators
URL
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paultough.com paultough.com
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behaviorist approach
If your school using PBIS, it is very much based on behaviorism
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14. Incentives
Incentives = rewards?
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www.legifrance.gouv.fr www.legifrance.gouv.fr
- Oct 2023
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www.liberation.fr www.liberation.fr
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Die Stadt Paris hat zum ersten Mal mit Einwohner:innen und Medien eine Hitzewelle mit Temperaturen bis zu 50 Grad simuliert in den nächsten Wochen soll eine neue Version des Pariser Klimaplans publiziert werden. https://www.liberation.fr/environnement/climat/exercice-de-simulation-bienvenue-a-paris-en-2032-sous-50-c-20231014_YZNQTOTVHZFSJEF3NBTLI5ME5A/
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en.wikipedia.org en.wikipedia.org
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https://en.wikipedia.org/wiki/Shmita
During shmita, the land is left to lie fallow and all agricultural activity, including plowing, planting, pruning and harvesting, is forbidden by halakha (Jewish law).
The sabbath year (shmita; Hebrew: שמיטה, literally "release"), also called the sabbatical year or shǝvi'it (שביעית, literally "seventh"), or "Sabbath of The Land", is the seventh year of the seven-year agricultural cycle mandated by the Torah in the Land of Israel and is observed in Judaism.
Tags
- Jeremiah 34:13-14
- Nehemiah 10:31
- Leviticus 25:5
- sabbaticals
- Deuteronomy 15:1-6
- Deuteronomy 31:10-13
- time in relation to work
- remission year
- 2 Chronicles 36:20-21
- agriculture
- Isaiah 37:30
- 2 Kings 19:29
- Friends of the Link 2023-10-18
- debt
- Jewish law
- jubilee
- halakha
- Leviticus 25
- Exodus 23:10-11
- shmita
Annotators
URL
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www.theguardian.com www.theguardian.com
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www.liberation.fr www.liberation.fr
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claudemariottini.com claudemariottini.com
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There are several occasions where the massebah is not associated with pagan worship. When the massebah is associated with the worship of Yahweh, the massebah is accepted as a valid expression of commitment to Yahweh.
Massebah for pagan worship: - Exodus 23:24 (https://hypothes.is/a/r3m5QmyDEe6SC8eLYcJE1Q) - Hosea 10:1 (https://hypothes.is/a/4PK2GGyDEe6wZg_r2YpVCA ) - 2 Kings 18:4 - 2 Kings 23:14
Massebah for worship of Yahweh: - Genesis 28:18 Jacob's pillow (https://hypothes.is/a/NF5p8Gx6Ee65Rg_J4tfaMQ)<br /> - Genesis 31:44-45 Jacob and Laban's covenant - Exodus 24:4 - Joshua 24:25-27
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in violation of the demands of the covenant, the people of Israel erected sacred stones dedicated to other gods (Hosea 10:1). In their religious reforms, both Hezekiah (2 Kings 18:4) and Josiah (2 Kings 23:14) destroyed the sacred pillars which the people of Israel had dedicated to the worship of Baal.
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During the establishment of the covenant between Yahweh and Israel, the people were commanded to destroy the sacred stones of the Canaanites, “You must demolish them and break their sacred stones (masseboth) to pieces” (Exodus 23:24).
In neighboring cultures in which both have oral practices relating to massebah, one is not just destroying "sacred stones" to stamp out their religion, but it's also destroying their culture and cultural memory as well as likely their laws and other valuable memories for the function of their society.
View this in light also of the people of Israel keeping their own sacred stones (Hosea 10:1) as well as the destruction of pillars dedicated to Baal in 2 Kings 18:4 and 2 Kings 23:14.
(Link and) Compare this to the British fencing off the land in Australia and thereby destroying Songlines and access to them and the impact this had on Indigenous Australians.
It's also somewhat similar to the colonialization activity of stamping out of Indigenous Americans and First Nations' language in North America, though the decimation of their language wasn't viewed in as reciprocal way as it might be viewed now. (Did colonizers of the time know about the tremendous damage of language destruction, or was it just a power over function?)
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Saul used a large stone to build an altar to Yahweh (1 Samuel 14:35).
Tags
- biblical stones
- sacred stones
- Josiah
- Genesis 31:44-45
- Genesis 28:18
- what's good for the goose is good for the gander
- Exodus 23:24
- Exodus 24:4
- massebah
- Baal
- Canaanite religion
- colonization
- The Covenant
- 1 Samuel 14
- Hosea 10:1
- standing stones
- Exodus 23
- 2 Kings 23:14
- masseboth
- hypocrisy
- Indigenous languages
- stone altars
- orality and memory
- Hezekiah
- 2 Kings 18:4
- Joshua 24:25-27
- songlines
Annotators
URL
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www.theguardian.com www.theguardian.com
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Ein Panel von Experten spricht sich in einem Bericht nachdrücklich für ein Moratorium für Geo-Engineering-Maßnahmen aus, die das Klima manipulieren sollen, Die vom früheren WTO-Chef Pascal Lamy geleitetete Climate Overshoot Commission stellt fest, dass die Wirkungen und Risiken solcher Maßnahmen bei weitem nicht ausreichend erforscht sind. https://www.theguardian.com/environment/2023/sep/14/experts-call-for-global-moratorium-on-efforts-to-geoengineer-climate
Report der Climate overshoot Commission: https://www.overshootcommission.org/report
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In der taz formuliert Sara Schurma 7 Schritte zu einer adäquaten Klimaberichterstattung:
- Kontext liefern
- Klima immer und überall mitdenken
- Strukturelle Probleme anerkennen
- Redaktionen mehr Fachwissen ermöglichen
- Verzögerungsnarrative einordnen
- „False Baance“ vermeiden
- Lösungen kritisch mitberichten
Ihr Artikel gehört zu einem „Schwerpunkt Klima und Medien“. https://taz.de/7-Schritte-fuer-Redaktionen/!5956911/
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www.repubblica.it www.repubblica.it
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Das europäische Parlament hat neue Grenzwerte für die Emissionen von Flugbenzin beschlossen. Eine Anpassung an die strengeren Werte der Weltgesundheitsorganisation soll auf Druck von Kritiker:innen im Parlament erst 2030-35 erfolgen. 2050 sollen 70% der Flugzeugtreibstoffe in der EU „nachhaltig“ sein. https://www.repubblica.it/green-and-blue/2023/09/14/news/ue_limiti_inquinamento_aria_biocarburanti_aerei-414423165/
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www.repubblica.it www.repubblica.it
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Die Veränderungen des Klimas und der Verlust ihres Lebensraums bedohen die europäischen Hummelarten. Bis zu 75% der Arten sind in pessimistischen Szenarein in den kommende 40-60% Jahren gefährdet. Hummeln spielen eine entscheidende Rolle bei der Bestäubung von 90% der Wildpflanzeen und bei vielen Kulturpflanzen. https://www.repubblica.it/green-and-blue/2023/09/14/news/bombi_europa_insetti_declino_specie-414449398/
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www.repubblica.it www.repubblica.it
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Durch die ausgedehnten Waldbrände haben die brandbedingten Treibhausgasemissionen in Kanada 2023 mit 410 Megatonnen ein Allzeithoch erreicht. Der zweithöchste Wert waren 138 Megatonnen 2014. Besonders hoch waren die Emissionen durch Waldbrände auch in Griechenland, Spanien und Portugal. Die Werte werden vom EU-Programm Copernicus erhoben. https://www.repubblica.it/green-and-blue/2023/09/14/news/incendi_copernicus_emissioni_co2_2023_record_canada-414437862/
Copernicus-Bericht: https://atmosphere.copernicus.eu/copernicus-record-breaking-wildfires-throughout-2023-boreal-wildfire-season
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www.repubblica.it www.repubblica.it
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Korallen und andere Lebewesen im Mittelmeer sind sowohl von der Erhitzung des Wassers wie vom weiter wachsenden Tourismus bedroht. Die Rebubblica berichtet über Bruno Borelli, der sich um den Schutz des Meeres-Reservats bei Portofino kümmert. https://www.repubblica.it/green-and-blue/2023/09/14/news/bruno_borelli_progetto_ecoefishent_recupero_reti_abbandonate-412745102/
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- Sep 2023
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en.wikipedia.org en.wikipedia.org
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Spiral Dynamics (SD) is a model of the evolutionary development of individuals, organizations, and societies. It was initially developed by Don Edward Beck and Christopher Cowan based on the emergent cyclical theory of Clare W. Graves, combined with memetics as proposed by Richard Dawkins and further developed by Mihaly Csikszentmihalyi.
https://en.wikipedia.org/wiki/Spiral_Dynamics
related to ideas I've had with respect to Werner R. Loewenstein?
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www.repubblica.it www.repubblica.it
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Die Hochseefischerei hat 2018-2022 um 8,5% zugenommen. In den Gebieten , die dem neuen Internationalen Meeresschutzabkommen zu Folge unter Schutz gestellt werden sollen, steigerte sie sich sogar um 22%, wie eine neue Studie von Greenpeace darstellt.Greenpeace begründet mit der Studie die Forderung, den Meeresschutzvertrag schnellstmöglich umzusetzen. https://www.repubblica.it/green-and-blue/2023/09/14/news/pesca_overfishing_trattato_oceani_greenpeace-414483446/
Greenpeace-Bericht: https://www.greenpeace.org/static/planet4-italy-stateless/2023/09/bca4703b-30x30-from-got-to-protection-at-sea-full-report.pdf
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lacountylibrary.libnet.info lacountylibrary.libnet.info
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https://lacountylibrary.libnet.info/event/9097350
https://www.youtube.com/watch?v=xMF37TXAV5w
Presenter Lawrence Mak broke down types of notes into the following three categories:<br /> - general notes (projects, ideas, journals, recipes, budgeting, homework, etc.)<br /> - lists (groceries, reading, gifts, to dos, assignments) - reminders (birthdays, bills, maintenance, health)
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www.liberation.fr www.liberation.fr
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www.liberation.fr www.liberation.fr
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TotalEnergies plant, 9 Milliarden Dollar in ein fossiles Entwicklungsprojekt in Surinam zu investieren. Ab 2028 sollen dort täglich 200.000 Barrel (insgesamt 700 Millionen) gefördert werden. TotalEnergies hat auch Verträge über die Ausbeutung weiterer Lagerstätten in der Nähe. https://www.liberation.fr/environnement/climat/total-se-lance-dans-un-projet-petrolier-a-9-milliards-de-dollars-au-suriname-20230914_3ZQXYGLVHRBZPG2ZQNUC2O2WLY/
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www.liberation.fr www.liberation.fr
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Das französische Batterien-Startup Verkor hat 2 Milliarden Euro Finanzierung aufgestelltt – ein Rekord in Frankreich. Der Staat subventioniert dabei massiv, um die Abhängigkeit von China zu verringern. Hauptkunde wird Renault sein, das auch mitfinananziert. https://www.liberation.fr/economie/economie-numerique/a-dunkerque-levee-de-fonds-record-pour-la-gigafactory-de-verkor-20230914_C3TFUGVMFBBFLMQHNRFIFZQ3BI/
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- Jul 2023
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gateway.ipfs.io gateway.ipfs.io
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In 1904, he made his move (Goodell 2012, 23–5).
Ordained when he was ~14.
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- Jan 2023
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- Sep 2022
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www.yutunox.com www.yutunox.com
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Tutubox installation guide for iPhone and iPad Method 1 – install Tutubox directly to the device As the first step, you need to clear the cache and history in your Safari browser by navigating to the Safari settings.Now you need to go to the general settings and make sure that there are no certificates available in the certificate list.Then go to Wi-Fi settings and then you need to click configure the proxy. Under the configure proxy you need to paste this URL http://ffapple.com in the automatic field. Turn off Wi-Fi for about 10 seconds and enable it again and check if your proxy is still enabled, if not just go and enable it.The next step is to get the latest Tutubox version to your iDevice. You can use the official Tutubox website (tutubox.io). Visit this website from your Safari browser and then download Tutubx iOS for your device and install it.When the installation process is done, you need to go to Settings, General, Device manager, and then click the certificate and trust it. If you do not do this step, you will get a notification when you are trying to open Tutubox that says the term Untrusted Enterprise Developer.Now you have successfully installed the Tutubox latest version on your iPhone or iPad. You are good to go with installing apps, games, and other tools with Tutubox.
Get Tutubox iOS 16 for installing the latest tweaks for iPhones and iPad for free
Tags
Annotators
URL
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www.historyofinformation.com www.historyofinformation.com
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Artykuł przedstawia historię idei Memeksu, autorstwa Vannevara Busha. Autor przedstawia także informacje na temat różnych wydań jego tekstu na ten temat.
W tekście znajdują się także informacje na temat maszyny Rapid Selector, autorstwa Ralpha R. Shawa, powstałej na tym, co pisał Emmanuel Goldberg, który z kolei inspirował się pracą Busha.
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- Aug 2022
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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RRID:ZFIN_ZDB-ALT-171010-14
DOI: 10.1242/dev.198408
Resource: RRID:ZFIN_ZDB-ALT-171010-14
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-171010-14
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- Jun 2022
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www.intendance03.fr www.intendance03.frFAQ-GF41
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L'autorité académique peut-elle suspendre l'application d'une délibération du conseil d'administration d'un EPLE ? Non, l'article L.421-14 du code de l'éducation prévoit que l'autorité académique peut prononcer l'annulation des actes des EPLE relatifs au contenu ou à l'organisation de l'action éducatrice. En revanche, dans une décision du 26 octobre 2007, le tribunal administratif de Grenoble rappelle que « le pouvoir de suspension de ces actes n'est pas prévu expressément par les dispositions [...] du code de l'éducation »
Tags
Annotators
URL
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- May 2022
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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DICER1 syndrome encompasses a variety of benign and malignant manifestations including multinodular goitre
Gene: DICER1 PMCID: PMC8451242 PMID: 34552563 Pathogenic Inheritance Pattern: Autosomal Dominant MultipleDiseaseEntities Disease Entity: DICER1 syndrome, multinodular goitre, cystic nephroma, anaplastic renal sarcoma, Wilms tumour, differentiated thyroid carcinoma, gynandroblastoma, ciliary body medulloepithelioma, embryonal rhabdomyosarcoma, pineoblastoma, pituitary blastoma, kidney cyst, pulmonary cyst, Sertoli-Leydig Cell Tumor. Mutation: Germline MultipleGeneVariants Variant & Clinvar IDs: c.3452_3453del (485534), c.316del (no ClinVar ID), c.171_172insAC (no ClinVar ID), c.3434del (no ClinVar ID), c.988C>T (933007), c.5388dup (no ClinVar ID) Zygosity: None provided. Case: At time of operation, the goitre patients living in Denmark were ages 21, 12, 21, 8, 14, and 16. Four underwent total thyroidectomies, and two underwent partial thyroidectomies. The patient originally aged 21 previously had a kidney cyst at age 14 and a pulmonary cyst at an unknown age. The patient aged 14 at time of partial thyroidectomy later manifested a Sertoli-Leydig Cell Tumor at age 15. All six patients were female. CasePresentingHPO: None provided. CasePreviousTesting: thyroidectomy gnomAD: ENSG00000100697.10, https://gnomad.broadinstitute.org/gene/ENSG00000100697 Mutation Type: Frameshift, Nonsense
Tags
- Germline
- Pathogenic
- Age:21
- gnomAD:ENSG00000100697.10
- female
- Gene:DICER1
- AutosomalDominant
- PMID:34552563
- MultipleDiseaseEntities
- ClinvarID:933007
- Mutation:Frameshift
- Mutation:Nonsense
- goiter
- c.171_172insAC
- thyroidectomy
- Frameshift
- PMCID:PMC8451242
- cancer
- c.3452_3453del
- DICER1
- Age:12
- Age:14
- Cohort
- Age:8
- c.3434del
- Nonsense
- ClinvarID:485534
- ClinvarID:NotProvided
- c.988C>T
- c.5388dup
- c.316del
- MultipleGeneVariants
- goitre
Annotators
URL
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- Mar 2022
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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ZDB-ALT-050916-14
DOI: 10.1016/j.isci.2022.104042
Resource: (ZFIN Cat# ZDB-ALT-050916-14,RRID:ZFIN_ZDB-ALT-050916-14)
Curator: @evieth
SciCrunch record: RRID:ZFIN_ZDB-ALT-050916-14
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www.law.cornell.edu www.law.cornell.eduURN:lex1
- Jul 2021
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www.sciencedirect.com www.sciencedirect.com
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Zebrafish: Tg(kdrl:GFP)s843
DOI: 10.1016/j.celrep.2019.05.052
Resource: (ZFIN Cat# ZDB-ALT-050916-14,RRID:ZFIN_ZDB-ALT-050916-14)
Curator: @ethanbadger
SciCrunch record: RRID:ZFIN_ZDB-ALT-050916-14
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www.sciencedirect.com www.sciencedirect.com
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RRID:ZDB-ALT-070427-14
DOI: 10.1016/j.cub.2017.11.018
Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)
Curator: @evieth
SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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ZDB-ALT-070427-14
DOI: 10.1523/ENEURO.0286-16.2017
Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)
Curator: @evieth
SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14
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ZDB-ALT-070427-14
DOI: 10.1523/ENEURO.0286-16.2017
Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)
Curator: @Naa003
SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14
Curator comments: Allele Name: Hu2098 Danio Rerio ZFIN Cat# ZDB-ALT-070427-14
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www.sciencedirect.com www.sciencedirect.com
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ZDB-ALT-070427-14
DOI: 10.1016/j.neuron.2017.06.001
Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)
Curator: @Zeljana_Babic
SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14
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ZDB-ALT-170522-14
DOI: 10.1016/j.neuron.2017.06.001
Resource: (ZFIN Cat# ZDB-ALT-170522-14,RRID:ZFIN_ZDB-ALT-170522-14)
Curator: @Zeljana_Babic
SciCrunch record: RRID:ZFIN_ZDB-ALT-170522-14
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www.sciencedirect.com www.sciencedirect.com
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RRID:ZDB-ALT-131122-14
DOI: 10.1016/j.neuron.2019.05.038
Resource: (ZFIN Cat# ZDB-ALT-131122-14,RRID:ZFIN_ZDB-ALT-131122-14)
Curator: @ethanbadger
SciCrunch record: RRID:ZFIN_ZDB-ALT-131122-14
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- Jun 2021
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elifesciences.org elifesciences.org
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RRID:ZFIN_ZDB-ALT-050916-14
DOI: 10.7554/eLife.62196
Resource: (ZFIN Cat# ZDB-ALT-050916-14,RRID:ZFIN_ZDB-ALT-050916-14)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-050916-14
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- Apr 2021
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eduscol.education.fr eduscol.education.fr
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La composition ainsi que les missions et modalités de pilotage des comités d'éducation à la santé et à la citoyenneté sont détaillées dans la circulaire n°2016-114 du 10 août 2016.
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- Mar 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).
AssayResult: 7.2
AssayResultAssertion: Normal
StandardErrorMean: 0.01
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A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected
HGVS: NM_024675.3:c.1250C>A p.(Ser417Tyr)
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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SUPPLEMENTARY DATA
AssayResult: 41
AssayResultAssertion: Indeterminate
PValue: < 0.0001
Approximation: Exact assay result value not reported; value estimated from Figure 6C.
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SUPPLEMENTARY DATA
AssayResult: -31
AssayResultAssertion: Abnormal
PValue: < 0.0001
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SUPPLEMENTARY DATA
AssayResult: 82.22
AssayResultAssertion: Indeterminate
PValue: 0.004
Comment: Exact values reported in Table S3.
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To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).
HGVS: NM_024675.3:c.2840T>C p.(Leu947Ser)
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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Source Data
AssayResult: 117.58
AssayResultAssertion: Not reported
ReplicateCount: 2
StandardErrorMean: 0.81
Comment: Exact values reported in “Source Data” file.
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Source Data
AssayResult: 95.02
AssayResultAssertion: Not reported
ReplicateCount: 2
StandardDeviation: 0.08
StandardErrorMean: 0.06
Comment: Exact values reported in “Source Data” file.
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We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.
HGVS: NM_024675.3:c.1676A>G p.(Q559R)
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www.cell.com www.cell.com
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Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function
AssayResult: 0.9
AssayResultAssertion: Abnormal
ReplicateCount: 12
StandardErrorMean: 0.6
Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)
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we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants
HGVS: NM_198056.2:c.2204C>A p.(Ala735Glu)
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- Feb 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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RRID:ZDB-ALT-131122-14
DOI: 10.7554/eLife.54491
Resource: (ZFIN Cat# ZDB-ALT-131122-14,RRID:ZFIN_ZDB-ALT-131122-14)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-131122-14
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elifesciences.org elifesciences.org
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RRID:ZFIN_ZDB-ALT-001205-14
DOI: 10.7554/eLife.53403
Resource: (ZFIN Cat# ZDB-ALT-001205-14,RRID:ZFIN_ZDB-ALT-001205-14)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-001205-14
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jmg.bmj.com jmg.bmj.com
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Supplemental material
AssayResult: 91
AssayResultAssertion: Normal
Comment: See Table S2 for details
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Supplemental material
AssayResult: 4.9
AssayResultAssertion: Abnormal
Comment: See Table S2 for details
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We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.
HGVS: NM_000546.5:c.323G>A p.(Gly108Asp)
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10.11.66.200 10.11.66.200
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the caller is a Conduent CH and the Conduent Calls call type should be used for the remainder of the call
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- Oct 2020
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Local file Local file
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The graphs of ground state confinement energy againstsize (radius) for zinc sulfide nanoparticles in Figure 14 showthe dependence of confinement on the size of quantum dots.The result shows that ground state confinement energy is
Las graficas de la energía de confinamiento en su estado fundamental en contra del tamaño (radio) por nanopartículas de sulfato de zinc en la Figura 14 muestran la dependencia de confinamiento en el tamaño de los puntos quánticos. El resultado muestra que el estado fundamental de energía en confinamiento es inversamente proporciona al tamaño (radio). Por lo tanto, cuando uno incrementa su radio (tamaño) la energía de confinamiento decrece pero nunca llega a cero. Eso es, el energía mas baja posible para el punto quántica de muestra no es cero. El confinamiento comienza cuando el radio del punto cuántico de muestra es comparable o del orden del radio exciton de Bohr.
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- Jul 2019
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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MP14:
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Molecular confirmation of Vibrioby 16S rRNA gene s
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- Jun 2019
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Statistical Analysis
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Low-pH antibody elution buffer
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Cytoskeleton extraction buffer (CSE)
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Semi dry transfer buffer
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GST elution buffer
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Ni-NTA elution buffer
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Lysis buffer for Ni-NTA and glutathione purification
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1X TBST
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1X TBS
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1X PBS
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Composition of buffers and solutions
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Injections/or determining MHC-peptide ligand densities
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Injections/or determining MHC-peptide ligand densities
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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SOS PAGE
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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LV concentration by ultracentrifugation
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CaPO4 method
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Reagents
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Western Blotting
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Third Component Rating
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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The time kinetics of deoxyhemoglobin polymerization were studied in 1.8M, 1.5 and 1 M potassium phosphate buffer (pH 7.25) respectively as described by Adachi and Asakura (1979a, b) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. Deoxygenation of the hemoglobin sample was ensured by passing moist gaseous nitrogen over the sample in an airtight cuvette and by addition of sodium dithionite. The polymerization of the resultant deoxyhemoglobin samples was initiated by a temperature jump from 4 to 30 oc within 10 sec and the progress of the reaction was followed by monitoring turbidity changes at 700 nm. The delay time was calculated from the kinetic traces
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Kinetics of polymerization
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- May 2019
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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A synthetic peptide (KMMTSKDNLNIDIPS) based on the PfCDPK4 sequence was custom synthesized (Peptron Inc.) and conjugated to keyhole limpet hemocyanin via an additional N terminus cysteine residue. It was used to raise polyclonal antisera against PfCDPK4 in rabbit. First immunization was performed using 1 00 ~g of peptide diluted in PBS and mixed 1: 1 v/v with Complete Freund's Adjuvant (CF A). Subsequently, three booster doses of 50 ~g each were given on the 14th, 28t\ 42nd day post first immunization. Blood was collected from animais on 7th, 21 S\ 35th, 49th day. Antibody titers were checked by ELISA using recombinant proteins or ovalbumin conjugated peptides as an antigen. In all cases, pre immune sera from the same rabbit were used as control
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Generation of anti-PfCDPK4 antisera
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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were represented as arbitrary fluorescence units and comparisons were made against the untrea!ed control samples. Exogenous addition of hydrogen peroxide to cells was used as a positive control for the assay
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he generation of reactive oxygen species in macrophages was detected by fluorimetry using the fluorescent dye CM-H2DCFDA, which can detect hydrogen peroxide, hydroxyl radical, peroxyl radical, and peroxynitrite anion (5, 6). To perform the assay, THP-1 macrophages were washed and resuspended in serum and phenol-red free RPMI-1640 medium and incubated at room temperature for 30 min in the presence of CM-H2DCFDA at a final concentration of 1 JiM. Subsequently the cells were washed once with fresh media to remove the excess probe and fluorescence measurements were commenced on a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and an emission of 520 nm. Appropriate treatments were initiated and time-kinetic measurements were carried out and the values obtained
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Detection of intracellular reactive oxygen species generation
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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1.2% acetylphenylhydrazine (APH) solution to render it anemic. The APH solution wa5 prepared in sterile water and pH was neutralized to 7.0 with 1 M HEPES buffer (pH 7.5). The rabbit was allowed to recover for 5 days, after which it was bled. The blood was collected in a sterile tube, containing an equal volume of prechilled salt solution and the mixture was filtered through a cheese cJoth. Filtrate was centrifuged at 2000 g for 10 min. at 4 °C. Supernatant was discarded, the pellet was washed twice with salt solution without heparin and finally, resuspended in equal volume of chilled sterile water. It was kept on ice for a minute and centrifuged at 20,000 g for 20 min. at 4 °C. The supernatant, containing the lysate, was immediately stored in liquid Nitrogen in 0.5 ml aliquots.
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The rabbit reticulocyte lysate was prepared as described by Sambrook et al ( 1989). A young male NZW rabbit weighing 2-2.5 Kg was subcutaneously injected for five consecutive days respectively with 2 ml, 1.6 ml, 1.2 ml, 1.6 ml and 2.0 ml of
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Preparation of rabbit reticulocyte lysate
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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with glutamine. Rat-2 and FWIL cells were cultured in IMDM supplemented with glutamine. All media were supplemented with 10 % fetal calf serum~ The cells were maintained in a 5 % co2 atmosphere and were split after 72 hours in culture, at a ratio of 1 : 15 approximately. For splitting the adherent cells, the cells were washed once with HBSS and 0.1 % trypsin in PBS added to the cells. The flask was shaken briefly to ensure a uniform distribution of trypsin over the cells. The cells were incubated in trypsin for 1 - 2 minutes after which 2 ml of FCS was added to the cells to inactivate the trypsin. The trypsin was carefully aspirated from the flask and fresh culture medium was added into the flask. The cells were dislodged from the bottom of the flask by gently tapping the flask against the working bench. Alternately, the cells were resuspended by vigorous pipetting up and down of the medium. The cells were centrifuged at 1500g for 5 minutes at room temperature and the supernate was discarded aseptically. The cells were resuspended in a known volume of fresh culture medium, an aliquot counted on a haemocytometer, and then accordingly seeded at the desired density in a fresh flask. For long term storage, the cells were frozen in a mixture of 95 ~ 0 culture medium and 5 ~ 0 DMSO in liquid nitrogen.
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eHO-Kl cells were cultured in Ham s F-12 med1a. NIH3T3, mouse LMtk-and HeLa cells were cultured in DMEM supplemented
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Growth and maintenance of cell lines.
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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weight) as a general anesthetic and ovaries were snap frozen in liquid nitrogen. Ovarian sections of 5 J.lm thickness were cut in a cryostat at -20°C and fixed in chilled methanol for 15 min at RT. Sections passing through follicles were selected, washed with 50 mM PBS and blocked with 3% normal goat serum (NGS) in PBS (v/v) at RT for 1 h. Sections were then washed two times with PBS and incubated with 1: 1 0 dilution of immune serum samples. Ovarian sections incubated with 1:10 dilution of mouse preimmune or immune sera from mice immunized with VR1020 vector served as negative controls. After incubation, the sections were washed three times with PBS and incubated with 1:800 dilution of goat anti-mouse IgG conjugated to FITC (Sigma) for 1 hat RT. The slides were washed three times with PBS, mounted in glycerol : PBS (9 : 1 ), and examined under fluorescence microscope (Optiphot, Nikon, Japan).
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Ability of mouse polyclonal antibodies, generated subsequent to immunization with VRbmZP1 and VRdZP3 plasmid DNA, to recognize native ZP was evaluated by indirect immunofluorescence assay. A normal cycling female bonnet monkey and a female dog were ovariectomized after administration of ketamine hydrochloride (5 mg/kg body
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REACTIVITY WITH NATIVE ZP OF THE IMMUNE SERUM SAMPLES OBTAINED FROM MICE IMMUNIZED WITH VRbmZPl AND VRdZP3 PLASMID DNA
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Bloodsampleswereobtainedbycuttingthecaudalpeduncleandanalysedforlacticacidusinganenzymatictechnique(SigmaCo,1974)MeasurementsweremadeinQuartzcuvettesat340nmwithaBeckmanAVSpectrophotometer.Standardcurvesweremadeonthedaythebloodlacticaciddeterminationsweremade.
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Bloodlacticacid
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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ubjected to three washes with PBST and two washes with PBS. The blot was developed using the substrate DAB (Sigma, U.S.A.) or with ECL (Amersham Biosciences, U.K.)
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he protein samples were diluted with 4X sample buffer which is essentially SDS-reducing buffer (O.SM Tris-Cl, pH 6.8, Glycerol, 10% (w /v) SDS, 2-J3-mercaptoethanol, 0.05% (w /v) bromophenol blue). The samples were denatured at 1000C for 10 min and the proteins were resolved on 12-15% SDS-polyacrylamide gel at 25-30mA. For detection, the proteins were transferred on to nitrocellulose (NC) membrane (Hybond-C extra, Amersham, U.K.) at 200mA, for either 1 hr or at 12 rnA, 40C for overnight. After the transfer was over, the NC membrane was washed thrice with PBST (1X PBS with 0.1% Tween 20) and blocked with 2% BSA for 2hrs (in PBST) at room temperature. Primary antibody to HBx/Vif/ APOBEC3G-NT raised in rabbit and were diluted to 1:1,000 in PBST. One hour incubation with the primary antibody was followed by three washes with PBST (10 min each) and then 1 hr incubation with 1:1,000 dilution of the secondary antibody (Anti-rabbit IgG (Fe) HRP conjugate) was carried out. The blot was further
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Westem blot analysis
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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The plates were kept in incubator gently and the colony formation was monitored every week. Media (500μl) was added to the plates every 4th-5th day to avoid drying. Colonies formed in soft agar photographed were taken without staining, under a microscope in light field
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Agar solution was prepared in a sterile 50ml Schott Duran Bottle and boiled in microwave until fully dissolved and kept at 55°C to 65°C. Master Mix with the rest of the components of bottom agar was made in a sterile corning 50ml tube prewarmed at 55°C and agar solution was added. The solution was once vortex briefly and then added (2ml) carefully to each well avoiding air bubbles. The plates were left undisturbed in laminar flow hood until the agar set fully. Two days before final assay, the bottom agar plates were kept in tissue culture incubator for equilibration. On the day of assay the following mix was prepared for Top Agar 4 dishes 5 dishes1.media with FBS, L-glutamine and Pen-Strep 4.8 ml 6 ml 2.fetal bovine serum 1.8 ml 2.5 ml 3.sterile water 1.8 ml 2.5 ml 4.agar 1.8% (1.8 g/100mLs) 1.8 ml 2.5 ml 5. cell suspension 1.0 X 105/ dish 100 to 350 μl 100 to 350 μl 6. Total 10.2 ml 13.5 ml Top agar mix without cells was first prepared and kept at 42°C. The cells were then trypsinized and re-suspended after counting in final volume of 100μl to 200 μl. Cells were then mixed with top agar and solution was quickly poured over the bottom agar.
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For soft agar assays 2x104, (A549) or 1x105 cells (E-10) were used in 1.5ml top agar. For preparing bottom agar plates (0.64% final con. of agar), a following mix was prepared for five dishes. 1.2X media with FBS, L-glutamine and Pen-Strep 10 ml 2.fetal bovine serum 5 ml 3.sterile water 1 ml 4.noble agar 1.8% (1.8 g/100mLs) 9 ml 5.Total 25 m
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Soft Agar Assay
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f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine
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a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.
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Activation of CM10 (weak cation exchange ) array
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d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine
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One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once
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Activation of LSAX (strong anion exchange ) array
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Activation of H50 protein chip array
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b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine
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5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once
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5 μl of 0.1% TFA was applied to the spots on the SEND array and removed after 30 sec using Whatman paper (care was taken not to touch the spot surface). b. 5 μl of cell lysate sample was spotted on the SEND array and incubated in a humid chamber for 10 min. Removed after 30 min. c. 5 μl of 0.1% TFA was then added and removed after 30 sec. d. 2 μl of 25% ACN in 0.1% TFA was added to the spots and allowed to dry. e. The chip was then placed in the SELDI machine
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Activation of SEND arrays for peptide analysis
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Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA
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Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised
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Destaining of gel, trypsinization and peptide extraction
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12% resolving gel (for 25 ml)Water = 8.2 ml 30% Acrylamide = 10.0 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.01 ml 5% stacking gel (for 10 ml)Water = 6.8 ml 30% Acrylamide = 1.7 ml 1.5 mM Tris (pH 6.8) = 1.25 ml 10% SDS = 0.1 ml 10% APS = 0.1 ml TEMED = 0.01 ml
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A double cylinder gradient former was used with 12% poly acrylamide gel mix in the inner cylinder and a 3% polyacrylamide gel mix in the outer cylinder that was stirred using a magnetic bead on a magnetic stirrer. A pump was connected to the flow tube and the flow rate adjusted at 5-8 to cast a 12-3% gradient gel. A 5% stacking gel was used. After the protein samples were run on the gradient gel, it was stained in instant blue over night under shaking. 3% resolving gel (for 25 ml)Water = 15.68 ml 30% Acrylamide = 2.5 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.02 ml
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Casting a gradient SDS-polyacrylamide gel
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1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl
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Protein estimation by Bradford method
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microfuge tubes and snap frozen in liquid nitrogen and were stored at ─80 ̊C. Protein estimation was performed simultaneously with one of these aliquots
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The strains were grown to stationary phase in 500 ml LB supplemented with ampicillin (100 μg/ml) overnight. Cells were pelleted at 2100g for 30 min at 4 ̊C and dissolved in 5 ml of 1X PBS with 2X protease inhibitor and 3 mM DTT. Cells were lysed using French Press at 1500 psi for three cycles. The lysed cells were pelleted at 20,000g for 45 min at 4 ̊C. Clear supernatant was collected in sterile 2 ml
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Preparation of cell extracts
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These experiments were undertaken in the laboratories of Dr. Sylvie Rimsky and Dr. Malcolm Buckle at the Ecole Normale Superioure, Cachan, Paris (France)
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Methods for SELDI (Surface Enhanced Laser Desorption/Ionization)
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A single plaque of λ contains approximately 105-106 pfu/ml. The method of propagation of λ from a single plaque was as follows. The contents of a single isolated plaque were drawn into a 1-ml pipette tip and dispensed into 0.2 ml of LB broth. After addition of a drop of chloroform, the contents were vortexed and centrifuged. The clear supernatant was mixed with 50 μl of λ-sensitive cells and incubated for 20 min at room temperature for adsorption. 10 ml of Z-broth supplemented with 5 mM MgSO4 was then added to the infection mixture, and incubated at 37°C with shaking until lysis. The lysate thus obtained usually contained 109pfu/ml
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Preparation of λ lysate by propagation from a single isolated plaque
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The method used was essentially the same as that described for preparation of P1 lysate, except that the λ-sensitive C600 cells used for infection were grown in LB broth containing 0.4% maltose and 10 mM MgSO4. The lysate thus prepared was checked for supE+revertants by plaquing on both supE (C600) and supE+strains (MG1655) using various dilutions of the lysate. To be used for experimental purposes, a phage titre of the order of 1010 to 1011 on the supE strain and around four orders of magnitude lower on the supE+strain, indicating very less frequency of supE+ revertants in the lysate is ideal
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Preparation of lambda (λ) lysates
Tags
- Md-22-Md-14-Md-6
- Md-14-Md-1
- Md-22-Md-14-Md-3-d
- Md-22-Md-14-Md-8-d
- Md-22-Md-14-Md-3
- Md-22-Md-14-Md-5
- Md-22-Md-14-Md-8
- Md-22-Md-14-Md-2-d
- Md-14
- Md-22-Md-14-Md-7-d
- Md-22-Md-14-Md-6-d
- Md-22-Md-14-d
- Md-22-Md-14-Md-1-d
- Md-22-Md-14-Md-2
- Md-14-d
- Md-22-Md-14-Md-4-d
- Md-22-Md-14-Md-1
- Md-14-Md-1-d
- Md-22-Md-14-Md-7
- Md-22-Md-14-Md-5-d
- Md-22-Md-14-Md-4
Annotators
URL
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30nm corresponds only to the intermediate. Since equal number of cells was taken for estimation, the height of the peak was taken as a measure of the ergosterol content
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Ergosterol content was measured as described by Arthington-Skaggs et al. (1999) with slight modifications. Briefly, equal number of cells were harvested and washed with PBS to remove media and FBS. They were resuspended in 3mL of 25% alcoholic KOH (25g KOH, 35mL sterile distilled water brought to lOOmL with ethanol) and vortexed for one minute. The cell suspension was transferred to a glass vial and incubated at 85°C for 1 hr. The vial was cooled to room temperature followed by the addition of 1mL sterile distilled water and 3mL of n-heptane. The vial was vortexed for 3 mins after which the heptane layer was collected. It was diluted 5 times in absolute ethanol and transferred to a cuvette. A spectrum was recorded between 220nm and 300nm. The peak at 281.5nm corresponds to ergosterol along with some intermediates.
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Spectrophotometric estimation of ergosterol
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Plasmid DNA was isolated at small scale using QIAprep Miniprep kit according to manufacturer's protocol. Briefly, 5mL of overnight E. coli culture was pelleted and resuspended in 250J..LL Buffer P1(50mM Tris-Cl, pH8.0; 10mM EDTA and 100p.g/mL RNase A). To this, 250 J..LL of Buffer P2(200mM NaOH and 1 %w /v NaOH) was added and mixed thoroughly by inverting the tube 4-6 times. 350 IlL of Buffer N3 (proprietary) was added and mixed immediately and thoroughly by inverting the tube 4-6 times. This was followed by centrifugation at 13000 x g for 10min at RT. The supernatant was applied to a QIAspin column and centrifuges at 13000 x g for 30-60s. The column was washed with 0.5mL Buffer PB followed by 0.75 mL Buffer PE. Residual wash buffer was removed by centrifugation for an additional 60s. The plasmid DNA bound to the column was eluted using the elution buffer, Buffer EB (10mM Tris-Cl, pH 8.5) provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration= A260 X SOX dilution factor
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Miniprep to isolate plasmids
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cellswere collected and washed with chilled sterile water.1 OD600cells were resuspendedin 20 μl chilled10%TCA solution containing 8 mM EDTA (pH 8.0) and incubated at room temperature for 15-20 min.Followingincubation, cellsuspension was centrifuged at 12,000 rpm for 5 minat 4 ̊Cand supernatant was transferred to a fresh 1.5 ml microcentrifuge tube. 10 μl of this supernatant fraction was diluted 75-foldwith ATPassay mix dilution buffer provided with the kit. 50 μl of diluted suspension was added to anequal volume of ATPassay mix (Sigma # FLAAM) which containedfirefly luciferase and luciferin with MgSO4, EDTA, DTT and BSA inTricine buffer.Luminescence was measured inluminometer (Varioskan flash-3001,Thermo Scientific). Total ATP was quantified usingpurified ATP as the standardand expressed in moles/OD cells
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ATPconcentrationin yeast cells was measuredby luminometricluciferase-luciferinbased assayusingATPbioluminescent kit(Sigma # FLAA).Briefly, log-phase yeast
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Determination of intracellular ATPlevels
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Estimation of total glycogen in cells was performed asdescribed previously (Parrou et al., 1997) with slightmodifications.Briefly, YPD medium-grown C. glabratacells were harvested, washed once with 1 ml ice-cold waterandresuspendedin 250 μl sodium carbonate(0.25 M)solution. After incubation at95 ̊C for 4 hin water bath with occasional stirring, cell suspension was cooled and pH of the suspension was adjusted to 5.2 by adding 150 μl 1 M acetic acid. Tothis suspension,600 μl 0.2M sodium acetatewas added and cell suspension was incubated with 1-2 U/ml of α-amyloglucosidase from A.niger(Sigma #A7420)at 57 ̊C for overnight with constant agitation.Resultant glucose liberated by α-amyloglucosidase digestion was collected in the supernatant fraction and quantifiedby phenol-sulphuric acid methodof carbohydratedetermination.For quantification, commercially available purified glucose was used as a standard and total glycogen incells was expressed as μg/2 x 107cells tonormalizeagainstcell density
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Estimation of glycogenlevels
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Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells
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Estimation of trehalosecontent
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Estimation oftrehalose, glycogen and ATPlevels
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QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA sample was resolved on 1-1.2% agarose gel and gel piece containing desired fragment was cut ona UV-transilluminator. DNA fragment was purified as per the kit manufacturer’s instructions
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Gel extraction of DNA
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To prepareelectrocompetent cells, a single colony of E. coli BW23473 strain from a freshly-streaked LB agar plate was inoculated in 50 ml LB liquid medium. Culture was incubated at 37°C for 14 h with continuous shaking at 200 rpm. 25 mlovernight-grown E. coliBW23473 culture was transferred to 500 ml LB liquid medium and incubated at 37°C till the OD600 reached to 0.4. Post incubation, cultures were transferredto ice and centrifuged at 1,000g for 15 minat 4°C. Cells were washed twice with 500 ml ice-cold sterile water, three times with 250 ml ice-cold 10% glycerol solution and resuspended in 1 ml 10% glycerol solution. After absorbance measurement, cell suspension was normalized to final cell density of 3X1010cells/ml and dispensed in 50 μl aliquots to sterile ice-cold 1.5 ml microcentrifuge tubes. Aliquots were snap frozen in liquid nitrogen and stored at -70ºC
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Preparation of E. coliBW23473electrocompetent cells
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