- Oct 2023
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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.derstandard.at www.derstandard.at
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Corporate Sustainability Reporting Directive (CSRD)
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www.liberation.fr www.liberation.fr
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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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www.theguardian.com www.theguardian.com
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www.liberation.fr www.liberation.fr
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www.derstandard.at www.derstandard.at
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www.derstandard.at www.derstandard.at
- Sep 2023
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www.repubblica.it www.repubblica.it
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Der revidierte Plan für Energie und Klima (NECP) , den Italien wie die anderen EU-Staaten der EU-Kommission in diesem Jahr vorlegen musste, reicht bei weitem nicht aus, um die EU-Klimaziele zu erreichen. Chiara die Mambo, die für den italienischen think Tank Ecco arbeitet, kritisiert ihn in der Repubblica.
https://www.repubblica.it/green-and-blue/2023/09/12/news/energia_clima_chiara_di_mambro-413616119/
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www.liberation.fr www.liberation.fr
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www.theguardian.com www.theguardian.com
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Die Intensität des Sturms Daniel, der in Libyen Tausende Opfer forderte, geht mit hoher Wahrschilichkeit auf die hohe Temperatur an der Oberfläche des Mittelmeers zurück. Ähnliche Extremereignisse jenseits des bisher Erwartbaren werden folgen. Für die katastrophale Wirkung sind auch schlechte Infrastruktur, ungenügende Vorhersage-Möglichkeiten, mangelhafte Alarmsysteme und falsche Planung verantwortlich. https://www.theguardian.com/world/2023/sep/12/questions-raised-over-deadly-impact-of-storm-daniel-in-libya
Mehr zum Sturm Daniel: https://hypothes.is/search?q=tag%3A%22event%3A%20storm%20Daniel%22
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www.theguardian.com www.theguardian.com
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Die International Energy Agency prognostiziert zum ersten Mal, dass der Verbrauch fossiler Energien vor 2030 seinen Höhepunkt überschreiten wird. Die voraussichtliche Abnahme sei aber bei weitem nicht ausreichend, um das 1,5°-Ziel zu erreichen. IEA-Chef Fatih Birol kündigte in der Financial Times die Kernaussagen des nächsten World Energy Outlook-Berichts der IEA an. https://www.theguardian.com/environment/2023/sep/12/beginning-of-the-end-of-fossil-fuel-era-approaching-says-iea
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www.liberation.fr www.liberation.fr
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Der Sturm Daniel in Libyen Tausende von Todesopfern gefordert, 27 davor in Europa und der Türkei. Der Attributions-Spezialist Davide Faranda stellt ihn im Interview mit der Libération in den Kontext der um 3-4° erhöhten Meerestemperatur und mediterranen HItzewelle im Juli und später Hitzewellen im August und September. Hitzewellen wie Medikane intensivieren sich. Dringend sei eine europaweite Planung von Anpassungsmaßnahmen. https://www.liberation.fr/environnement/climat/evenements-climatiques-extremes-en-mediterranee-cet-ete-on-a-vecu-dans-le-futur-20230912_O7A53XIZKZDPZC2NH4RD355GNA/
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www.liberation.fr www.liberation.fr
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Die Abgeordneten des europäischen Parlaments haben dafür gestimmt, die EU-Länder zu verpflichen, den Verbrauch erneuerbarer Energie bis 2030 von 22% auf 42% zu steigern. Die Renewable Energy Directive muss noch vom Europäischen Rat angenommen werden. Biomasse gilt weiterhin als erneuerbar. https://www.liberation.fr/environnement/les-eurodeputes-votent-pour-faire-doubler-la-consommation-denergie-renouvelable-dici-a-2030-20230912_WMEQYLIL65G6LC7CVDD6CV5AOQ/
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www.liberation.fr www.liberation.fr
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Der Sturm Daniel hat sich, nachdem er Verwüstungen in Griechenland, Bulgarien und der Türkei angerichtet hat, auf dem Weg über das Mittelmeer nach Libyen in einen Medikan, einen „Mittelmeer-Hurrican“ verwandelt. Dort wurden in Al Baida 414,4 mm Niederschläge an einen Tag gemessen.
Mehr zum Sturm Daniel: https://hypothes.is/search?q=tag%3A%22event%3A%20storm%20Daniel%22
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- Aug 2023
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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ZDB-ALT-050414-12
DOI: 10.1371/journal.pbio.3002223
Resource: RRID:ZFIN_ZDB-ALT-050414-12
Curator: @evieth
SciCrunch record: RRID:ZFIN_ZDB-ALT-050414-12
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- Jun 2023
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zettelkasten.de zettelkasten.de
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When I create a new note, I write and link it as usual. Then I call up a saved search in The Archive via shortcut. I then go through the notes of my favorites and see if the fresh note is usable for one of my favorites. In doing so, I make an effort to find a connection. This effort trains my divergent thinking. Afterward, I try to understand the nature of the connection from the fresh piece of paper. In this way, I train my convergent thinking.
Sascha's process of incoporating the problems into the ZK workflow
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Not all favorites are problems! I don’t phrase everything as a problem. For example, I am writing a collection of short stories set in a prison valley. It is also part of my list of favorites. I think Feynman has 12 favorite problems because as a physicist, you mainly solve problems. But as a writer, you don’t only solve problems, you write texts. There are different types of opportunities, not just problems.
Not everything has to be a problem in the literal sense of the word; it's a tool for generating creative insight by means of prompting and relational thinking.
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This technique is another demonstration of Feynman’s genius. It is simple and efficient: Maintain a collection of 12 favorite problems. Whenever you learn something new, check if it helps you with one of your 12 favorite problems. Richard Feynman was fond of giving the following advice on how to be a genius. You have to keep a dozen of your favorite problems constantly present in your mind, although by and large they will lie in a dormant state. Every time you hear or read a new trick or a new result, test it against each of your twelve problems to see whether it helps. Every once in a while there will be a hit, and people will say, “How did he do it? He must be a genius!”
Effective problem-solving method that can be incorporated with ease in the Zettelkasten technique.
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docdrop.org docdrop.org
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learn-us-east-1-prod-fleet01-xythos.content.blackboardcdn.com learn-us-east-1-prod-fleet01-xythos.content.blackboardcdn.com
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The problem with that presumption is that people are alltoo willing to lower standards in order to make the purported newcomer appear smart. Justas people are willing to bend over backwards and make themselves stupid in order tomake an AI interface appear smart
AI has recently become such a big thing in our lives today. For a while I was seeing chatgpt and snapchat AI all over the media. I feel like people ask these sites stupid questions that they already know the answer too because they don't want to take a few minutes to think about the answer. I found a website stating how many people use AI and not surprisingly, it shows that 27% of Americans say they use it several times a day. I can't imagine how many people use it per year.
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www.churchofjesuschrist.org www.churchofjesuschrist.org
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Jesus Christ met with His disciples on the Mount of Olives during His last week in mortality. At that time He prophesied of the destruction of the temple in Jerusalem, and His disciples asked when that destruction would occur and when He would return to the earth (see Joseph Smith—Matthew 1:2–4). In response the Lord revealed the signs that would occur shortly after His death and those that would precede His Second Coming. He repeated this prophecy to His Saints in the latter days, as recorded in Doctrine and Covenants 45:16–59.
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- Mar 2023
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radiolab.org radiolab.org
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Listened to on 2023-03-11
Much like Richard Feynman kept a list of his 12 favorite problems, Maurice Hilleman kept a running list of diseases for which he was working on developing vaccines to remedy.
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- Feb 2023
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www.youtube.com www.youtube.com
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https://www.youtube.com/watch?v=Q2zY7l2tzoQ
Ugh... another short mediocre introduction. Repeats the origin myth.
Seems to take a very Ahrens' based framing, but screws up a few pieces. More focus on "hub notes" and completely misses the idea of an index somehow?!?
The last section of 2+ minutes really goes off the rails and recommends converting notes from other places and muddles about "Favorite problems" (ostensibly a reference to Feynman's 12 Favorite Problems, but isn't direct about it?).
Also encourages the "Feynman technique"...
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- Jan 2023
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zettelkasten.de zettelkasten.de
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When I create a new note, I write and link it as usual. Then I call up a saved search in The Archive via shortcut. I then go through the notes of my favorites and see if the fresh note is usable for one of my favorites. In doing so, I make an effort to find a connection. This effort trains my divergent thinking.
Sascha Fast juxtaposes his new notes with his own favorite problems to see if they have any connections with respect to improving on or solving them.
This practice is somewhat similar to Marshall Kirkpatrick's conceptualization of triangle thinking, but rather than being randomly generated with respect to each other, the new things are always generated toward important questions he's actively working on or toward.
This helps to increase the changes of forward progress in specific areas rather than undirected random progress.
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Richard Feynman was fond of giving the following advice on how to be a genius. You have to keep a dozen of your favorite problems constantly present in your mind, although by and large they will lie in a dormant state. Every time you hear or read a new trick or a new result, test it against each of your twelve problems to see whether it helps. Every once in a while there will be a hit, and people will say, “How did he do it? He must be a genius!”
Gian-Carlo Rota (1997): Ten Lessons I Wish I Had Been Taught, Notices of the American Mathematical Society 1, 1997, Vol. 44, pp. 22-25.
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events.jhu.edu events.jhu.edu
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www.liberation.fr www.liberation.fr
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Im letzten Monat der Amtszeit von Jair Bolsonaro würde 150% mehr Amazonas-Regenwald zerstört als im Dezember 2021.
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- program: Deter
- region: Brasil
- country: Brasil
- time: 2022-12
- Institution: Inpe
- process: deforestation
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- Nov 2022
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www.companyhero.com www.companyhero.com
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Alguns números da Company Hero
Se formos usar os Big Numbers na página de cima, talvez podemos eliminar esta seção.
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billyoppenheimer.com billyoppenheimer.com
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When I come across interesting information, I highlight then comment a corresponding question:
Every studio has a slate.
What is the source for this?
It's highly related to having a direction in life, or the famous example of Feynman's 12 Favorite Problems that he always kept in mind to slowly be working at.
Part of having a list of purpose dovetails to how one builds their identity too.
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- Aug 2022
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www.theguardian.com www.theguardian.com
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Adams, R., & editor, R. A. E. (2021, September 22). Chris Whitty warns MPs it is ‘inevitable’ unvaccinated children will catch Covid. The Guardian. https://www.theguardian.com/society/2021/sep/22/chris-whitty-warns-mps-it-is-inevitable-unvaccinated-children-will-catch-covid
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www.livemint.com www.livemint.com
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Banerjee, P. (2022, February 13). Biological E seeks EUA for its Corbevax vaccine for 12-18 yrs age group. Mint. https://www.livemint.com/news/india/biological-e-seeks-eua-for-its-corbevax-vaccine-for-12-18-yrs-age-group-11644746753338.html
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www.bmj.com www.bmj.com
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Majeed, A., Hodes, S., & Marks, S. (2021). Consent for covid-19 vaccination in children. BMJ, 374, n2356. https://doi.org/10.1136/bmj.n2356
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www.pbs.org www.pbs.org
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American Academy of Pediatrics urges FDA to approve COVID vaccines for children under 12. (2021, September 13). PBS NewsHour. https://www.pbs.org/newshour/show/american-academy-of-pediatrics-urges-fda-to-approve-covid-vaccines-for-children-under-12
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inews.co.uk inews.co.uk
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Gye, H. (2021, November 29). All adults to be offered booster jabs after 3 months, and over-12s to get second doses. Inews.Co.Uk. https://inews.co.uk/news/politics/covid-booster-jabs-to-be-offered-all-adults-after-3-months-and-over-12s-second-doses-1325151
Tags
- is:news
- Omicron
- rollout
- over 12 year olds
- booster
- vaccine
- COVID-19
- health service
- lang:en
- second dose
- United Kingdom
- demand
- NHS
- danger
- 3 month gap
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www.theguardian.com www.theguardian.com
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Adams, R., & editor, R. A. E. (2021, September 15). Covid teams can vaccinate pupils against parents’ wishes, schools told. The Guardian. https://www.theguardian.com/education/2021/sep/15/covid-teams-can-vaccinate-pupils-against-parents-wishes-schools-told
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www.standard.co.uk www.standard.co.uk
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Kirby, B. J. (2022, February 9). UK is ‘past the point’ where vaccinating young children against Covid would help. https://www.standard.co.uk/news/uk/england-children-bbc-radio-europe-pfizer-b981546.html
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twitter.com twitter.com
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Anthony Costello. (2022, February 24). The risks of cognitive symptoms lasting at least 12 MONTHS were much higher in the infected group. 4.8x higher for fatigue, 3.2x for brain fog, 5.3x for poor memory, and an incredible 51x for altered taste and smell. We need data on children, but it could easily be similar. (17) https://t.co/JC1qYyW2Xc [Tweet]. @globalhlthtwit. https://twitter.com/globalhlthtwit/status/1496957266016313348
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twitter.com twitter.com
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ReconfigBehSci. (2022, January 2). RT @trishgreenhalgh: A few tweets on masks for kids (thanks @dgurdasani1). US schools were 3.5 x more likely to have COVID-19 outbreaks if… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1477614232556515331
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www.latimes.com www.latimes.com
- May 2022
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Local file Local file
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As told in Genius: The Life and Science of Richard Feynman byJames Gleick
Forte cleverly combines a story about Feynman from Genius with a quote about Feynman's 12 favorite problems from a piece by Rota. Did they both appear in Gleick's Genius together and Forte quoted them separately, or did he actively use his commonplace to do the juxtaposition for him and thus create a nice juxtaposition himself or was it Gleick's juxtaposition?
The answer will reveal whether Forte is actively using his system for creative and productive work or if the practice is Gleick's.
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new result, test it against each of your twelve problems to seewhether it helps. Every once in a while there will be a hit, andpeople will say, “How did he do it? He must be a genius!”
You have to keep a dozen of your favorite problems constantly present in your mind, although by and large they will lay in a dormant state. Every time you hear or read a new trick or a
Gian-Carlo Rota, Indiscrete Thoughts (Boston: Birkhäuser Boston, 1997), 202.
Richard Feynman indicated in an interview that he kept a dozen of his favorite problems at the top of his mind. As he encountered new results and tricks, he tried applying them to those problems in hopes of either solving them or in coming up with new ideas. Over time by random but combinatorial chance, solutions or ideas would present themselves as ideas were juxtaposed.
One would suspect that Feynman hadn't actually read Raymond Llull, but this technique sounds very similar to the Llullan combinatorial arts from centuries earlier, albeit in a much more simplified form.
Can we find evidence of Feynman having read or interacted with Llull? Was it independently created or was he influenced?
I had an example of this on 2022-05-28 in Dan Allosso's book club on Equality in the closing minutes where a bit of inspiration hit me to combine the ideas of memes, evolution, and Indigenous knowledge and storytelling to our current political situation. Several of them are problems and ideas I've been working with over years or months, and they came together all at once to present a surprising and useful new combination. #examples
Link this also to the idea of diffuse thinking as a means of solving problems. One can combine the idea of diffuse thinking with combinatorial creativity to super-charge one's problem solving and idea generation capacity this way. What would one call this combination? It definitely needs a name. Llullan combinatorial diffusion, perhaps? To some extent Llull was doing this already as part of his practice, it's just that he didn't know or write explicitly about the diffuse thinking portion (to my knowledge), though this doesn't mean that he wasn't the beneficiary of it in actual practice, particularly when it's known that many of his time practiced lectio divina and meditated on their ideas. Alternately meditating on ideas and then "walking away" from them will by force cause diffuse thinking to be triggered.
Are there people for whom diffuse thinking doesn't work from a physiological perspective? What type of neurodiversity does this cause?
Tags
- Richard Feynman
- cross-disciplinary studies
- Llullan combinatorial diffusion
- diffuse thinking
- Llullan combinatorial arts
- quotes
- neurodiversity
- juxtaposition
- lectio divina
- combinatorial creativity
- meditation
- psychology
- commonplace books
- cognitive neuropsychology
- flâneur
- James Gleick
- Feynman's 12 Favorite Problems
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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
- goitre
- ClinvarID:933007
- MultipleDiseaseEntities
- PMCID:PMC8451242
- Cohort
- ClinvarID:NotProvided
- DICER1
- c.316del
- PMID:34552563
- female
- thyroidectomy
- AutosomalDominant
- MultipleGeneVariants
- Nonsense
- Pathogenic
- Germline
- Age:12
- c.171_172insAC
- goiter
- c.3434del
- cancer
- Mutation:Nonsense
- ClinvarID:485534
- Age:21
- Mutation:Frameshift
- c.3452_3453del
- c.5388dup
- Age:8
- Frameshift
- gnomAD:ENSG00000100697.10
- c.988C>T
- Age:14
- Gene:DICER1
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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RRID:ZFIN_ZDB-GENO-131107-12
DOI: 10.7554/eLife.74821
Resource: (ZFIN Cat# ZDB-GENO-131107-12,RRID:ZFIN_ZDB-GENO-131107-12)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-GENO-131107-12
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- Jan 2022
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www.themoviedb.org www.themoviedb.org
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www.themoviedb.org www.themoviedb.org
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www.themoviedb.org www.themoviedb.org
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moviepilot.de 6,7/10
Helge Schneider bringt es wieder einmal, wie so oft, auf den Punkt: „Die schwierigste Zeit im Leben eines Mannes ist die Pubertät, die zweitschlimmste ist die danach.“ Frauen geht es da bestimmt nicht viel besser... wenn der Körper voller Hormone gepumpt wird, die diesen verändern, das Interesse am anderen oder gleichen Geschlecht zunimmt, kurz: die Zeit, in der sich schlicht alles verändert und eine andere Bedeutung bekommt, ist wohl diejenige, die uns alle am meisten prägt. Nachdem in diesem Jahr Gus van Sant mit „Paranoid Park“ einen männlichen Jugendlichen ins Visier nimmt, kontert „Water Lilies“ von Céline Sciamma quasi von weiblicher Seite. filmstarts.de (3,5/5)
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England, 1865: Catherine (Florence Pugh) lebt gemeinsam mit ihrem Gatten Alexander (Paul Hilton) und seinem Vater Boris (Christopher Fairbank) auf dem Land. Liebe ist in dieser Beziehung nicht im Spiel und obwohl Boris beständig darauf pocht, Catherine solle ihre ehelichen Pflichten erfüllen, hat Alexander keinerlei Interesse am Körper seiner Frau. Als ihr Mann eines Tages verreist, nutzt Catherine die Möglichkeit, dem ihr auferlegten Hausarrest zu entkommen und erkundet die Gegend. So lernt sie einen der Landarbeiter, Sebastian (Cosmo Jarvis), kennen. Nach anfänglicher Unsicherheit und trotz einer priesterlichen Warnung gibt sich Catherine schließlich ihrer Leidenschaft hin und beginnt eine Affäre mit Sebastian. Doch Alexanders Rückkehr gefährdet das neu gefundene Glück und Catherine muss eine Entscheidung filmstarts.de 4,5/5
moviepilot.de 6,5/10
Aus einer eingeschüchterten jungen Frau wird eine kaltblütige Mörderin: In dieser stilsicheren, modernen und überaus spannenden neuen „Lady Macbeth“ schlägt Newcomerin Florence Pugh den Zuschauer von der ersten Szene an in ihren Bann. filmstarts.de 4,5/5
„Lady Macbeth“ nimmt die klassische russische Novelle und macht daraus einen Film, der kälter und böser kaum sein könnte. Vor allem die junge Hauptdarstellerin sorgt dafür, dass der Wandel einer unterdrückten Gattin zu einer skrupellosen Herrscherin absolut sehenswert ist. Gleichzeitig lässt sich das Drama aber auch zu allgemeinen Themen aus, gerade in Bezug auf zwischenmenschliche Machtverhältnisse. Oliver Armknecht 8/10
William Oldroyds im viktorianischen England spielende Verfilmung von Nikolai Leskows »Lady Macbeth aus Mzensk« besticht durch formale Virtuosität. epdFilm 6/10
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www.themoviedb.org www.themoviedb.org
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filmstarts.de 4,5/5 moviepilot.de 7,2/10 + diverse Kritiken im Kommentarbereich
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www.themoviedb.org www.themoviedb.orgBergfest1
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filmstarts.de 3,5/5
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Elton John filmstarts.de 3,5/5
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IMDB 7.0/10 · 110
In der DDR lässt es sich für den bekennenden Kommunisten Franz Walter (Lars Eidinger) gut leben. So kommt es für ihn wie gerufen, als er nach seiner Promotion an der Berliner Humboldt Universität ein Jobangebot beim Auslandsnachrichtendienst des Arbeiter- und Bauern-Staats erhält. Der Job bietet viele Vorzüge, also lässt er sich blenden und wird Teil des Geheimdienstes. Gemeinsam mit seiner Freundin Corina (Luise Heyer) genießt er fortan das angenehme Leben und findet in seinem Vorgesetzten Dirk (Devid Striesow) sogar einen neuen Freund und Mentor. Wo es in seiner Tätigkeit anfangs nur um reine Informationsbeschaffung geht, verlangen die Aufträge nach und nach immer mehr von Franz und er ist gezwungen, zu Mitteln zu greifen, die er nicht mehr mit seinem Gewissen vereinbaren kann. Gerade als er sich auf dem Höhepunkt seiner Karriere befindet, kommen ihm moralische Zweifel an seiner Tätigkeit und er will aussteigen. Doch im Netz aus Unterdrückung, Erpressung und Befragung ist er Opfer und Täter zugleich und es gibt für ihn kein Entkommen... filmstarts.de
Ein dunkles Kapitel deutscher Geschichte, beklemmend und hochspannend inszeniert als Mischung aus Politdrama und Psychothriller – ganz weit weg von jedem Lehrstückkino. Als Verkörperungen des Kampfes Mensch vs. unmenschliches System holen Lars Eidinger und Devid Striesow das abstrakte Grauen des DDR-Überwachungsapparates so nah heran, dass es weh tut. filmstarts.de 4,5/5
Angelehnt an eine wahre Geschichte erzählt „Nahschuss“, wie ein Mann für den DDR-Geheimdienst arbeitet und erst zu spät erkennt, worauf er sich eingelassen hat. Spannend ist dabei, wie die widersprüchliche Figur gleichzeitig Täter und Opfer ist und für seine anfängliche Naivität einen hohen Preis zu bezahlen hat. Oliver Armknecht 7/10
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www.themoviedb.org www.themoviedb.org
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www.themoviedb.org www.themoviedb.org
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TMDB (63%) JustWatch (69%) filmstarts.de (–/5) moviepilot.de (6,2/10) IMDB (6,2/10 · 2,5K · Metascore: 54)
Vor einem halben Jahrhundert war Harris Shaw (Michael Caine) mit seinem Erfolgsroman „Atomic Autumn“ alleine dafür verantwortlich, dass ein Verlag erfolgreich wurde. Doch heute sind sowohl beim Autor als auch beim Verlag der Ruhm verblasst. Shaw tippt zwar immer noch fleißig auf seiner alten Schreibmaschine, doch der Raucher und Säufer will nichts mit der Welt zu tun haben. Die junge Verlagserbin Lucy Stanbridge (Aubrey Plaza) greift derweil bei sinkenden Verkaufszahlen und einem von Influencer bestimmten Werbemarkt zum letzten Strohhalm, um ihr Unternehmen zu retten: Einsiedler Shaw soll noch einmal auf eine Lesetour gehen, um sein neues Buch zu präsentieren. Doch der hat wenig Bock... filmstarts.de
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www.themoviedb.org www.themoviedb.org
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www.themoviedb.org www.themoviedb.org
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Originaltitel: Smagen af sult
TMDB (59%) | JustWatch (71%) | filmstarts.de (–/5) | moviepilot.de (–/10) | IMDB (6,4/10 · 405)
Carsten (Nikolaj Coster-Waldau) und Maggi (Katrine Greis-Rosenthal) sind ein Paar, das alles opfert, um die höchstmögliche Auszeichnung in der kulinarischen Welt zu erreichen: einen Michelin-Stern. Eigentlich haben die beiden alles, was man sich wünschen könnte. Sie führen eine liebevolle Beziehung, sie haben zwei wundervolle Söhne und ihr exklusives Restaurant in Kopenhagen, genannt Malus, läuft hervorragend. Doch eines fehlt vor allem Carsten noch zum Glück: Er möchte die offizielle Auszeichnung, die ein Michelin-Stern dem gemeinsamem Restaurant verleihen würde. Nun steht der Besuch eines Restaurantkritikers kurz bevor und die Situation droht Carsten und Maggi über den Kopf zu wachsen... filmstarts.de
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www.themoviedb.org www.themoviedb.org
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TMDB (74%) JustWatch (81%) filmstarts.de (–/5) moviepilot.de (6,6/10) IMDB (6,8/10 · 5,4K)
In naher Zukunft: Cameron (Mahershala Ali) ist todkrank. Dem fürsorglichen Ehemann und Vater wird von seiner Ärztin (Glenn Close) ein Weg aufgezeigt, wie er seine Familie vor dem bevorstehenden Leid bewahrt: Er soll sich durch einen Klon ersetzen lassen, der wie eine Abbild seiner selbst aussieht. Während Cameron mit der Frage konfrontiert wird, ob er das Schicksal seiner Familie ändern soll oder nicht, lernt er immer mehr Wahrheiten über das Leben, den Verlust und die Liebe, als er sich jemals vorgestellt hätte. Cameron erkennt daraufhin immer mehr, was bedeutet, Opfer zu bringen und wie weit ein Mensch in der Lage ist zu gehen, um seinen Liebsten ein glücklicheres, besseres Leben zu ermöglichen... filmstarts.de
„Schwanengesang“ handelt von Klonen todkranker Menschen, welche den Platz ihrer Originale einnehmen sollen, damit die ahnungslose Familie nicht leiden muss. Das überwiegend ruhig erzählte und schön bebilderte Science-Fiction-Drama stellt existenzielle Fragen zu Identität, aber auch moralische, welchen Preis das Glück haben darf. Das richtet sich vor allem an ein Publikum, das gerne nachdenkt, bietet aber ebenfalls emotionale Momente, die auf das Konto von Mahershala Ali gehen. Oliver Armknecht 7,5/10
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- Nov 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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hdac1hi1618Tg
DOI: 10.17912/micropub.biology.000481
Resource: (ZFIN Cat# ZDB-GENO-020426-12,RRID:ZFIN_ZDB-GENO-020426-12)
Curator: @bandrow
SciCrunch record: RRID:ZFIN_ZDB-GENO-020426-12
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www.newscientist.com www.newscientist.com
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Le Page, M., Wilson, C., Hamzelou, J., Wong, S., Lawton, G., Vaughan, A., Quilty-Harper, C., Murugesu, J. A., & Liverpool, L. (2021, November 22). Covid-19 news: Austria goes back into lockdown. New Scientist. https://institutions.newscientist.com/article/2237475-covid-19-news-austria-goes-back-into-lockdown/
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Covid: US approves Pfizer vaccine for children over five. (2021, November 3). BBC News. https://www.bbc.com/news/world-us-canada-59044853
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www.news.com.au www.news.com.au
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Burke, H. (2021, November 10). Covid jab for children as young as six. News.Com.Au — Australia’s Leading News Site. https://www.news.com.au/world/coronavirus/australia/tga-grants-provisional-determination-for-moderna-jab-for-children-under-12/news-story/a6d44efc230e6b86ceb1ef58a06779e4
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www.theguardian.com www.theguardian.com
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Sample, I. (2021, November 2). JCVI failed to back youth Covid jabs despite favourable modelling. The Guardian. https://www.theguardian.com/politics/2021/nov/02/jcvi-failed-back-youth-covid-jabs-despite-favourable-models
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mmcr.education mmcr.education
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Additionally, the constitutionality of this program turns on whether most schools in the program are religious.
Incorrect. The constitutionality does NOT turn on whether and why in a particular area...
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mmcr.education mmcr.education
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the program is not one of true private choice. It is not neutral i
incorrect- the program IS one of private choice, and it IS neutral in respects toward religion
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- Sep 2021
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www.theguardian.com www.theguardian.com
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Walker, P., Davis, N., & Grover, N. (2021, September 3). UK vaccines watchdog fails to back Covid jabs for children. The Guardian. http://www.theguardian.com/society/2021/sep/03/uk-rules-out-covid-vaccinations-for-children
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- Aug 2021
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www.sciencedirect.com www.sciencedirect.com
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ZDB-ALT-070702-12
DOI: 10.1016/j.devcel.2021.07.021
Resource: (ZFIN Cat# ZDB-ALT-070702-12,RRID:ZFIN_ZDB-ALT-070702-12)
Curator: @Naa003
SciCrunch record: RRID:ZFIN_ZDB-ALT-070702-12
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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RRID:ZFIN_ZDB-GENO-060811-12
DOI: 10.7554/eLife.22199
Resource: (ZFIN Cat# ZDB-GENO-060811-12,RRID:ZFIN_ZDB-GENO-060811-12)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-GENO-060811-12
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- Jul 2021
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www.sciencedirect.com www.sciencedirect.com
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ZDB-ALT-170522-12
DOI: 10.1016/j.neuron.2017.06.001
Resource: (ZFIN Cat# ZDB-ALT-170522-12,RRID:ZFIN_ZDB-ALT-170522-12)
Curator: @Zeljana_Babic
SciCrunch record: RRID:ZFIN_ZDB-ALT-170522-12
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www.theguardian.com www.theguardian.com
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the Guardian. ‘Israel Says 600 Children given Covid Jab Had No Serious Side-Effects’, 10 March 2021. http://www.theguardian.com/society/2021/mar/10/israel-says-600-children-given-covid-jab-had-no-serious-side-effects.
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- Jun 2021
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www.sciencedirect.com www.sciencedirect.com
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ZFIN: ZDB-ALT-170119-12
DOI: 10.1016/j.celrep.2021.109255
Resource: (ZFIN Cat# ZDB-ALT-170119-12,RRID:ZFIN_ZDB-ALT-170119-12)
Curator: @Naa003
SciCrunch record: RRID:ZFIN_ZDB-ALT-170119-12
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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: 4
AssayResultAssertion: Indeterminate
StandardErrorMean: 0.32
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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.1226A>G p.(Tyr409Cys)
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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SUPPLEMENTARY DATA
AssayResult: 94.01
AssayResultAssertion: Not reported
PValue: > 0.9999
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.280G>A p.(Glu94Lys)
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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Source Data
AssayResult: 23.96
AssayResultAssertion: Abnormal
ReplicateCount: 2
StandardErrorMean: 7.6
Comment: Exact values reported in “Source Data” file.
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Source Data
AssayResult: 7.75
AssayResultAssertion: Abnormal
ReplicateCount: 2
StandardDeviation: 2.59
StandardErrorMean: 1.83
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.1592delT p.(L531Cfs)
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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: 68.1
AssayResultAssertion: Indeterminate
ReplicateCount: 18
StandardErrorMean: 8.7
Comment: This variant had mild loss of function (peak current >50% and <75% of wildtype), therefore it was considered inconclusive and neither abnormal nor normal in vitro function. (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.2200A>G p.(Met734Val)
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- Feb 2021
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jmg.bmj.com jmg.bmj.com
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Supplemental material
AssayResult: 94
AssayResultAssertion: Normal
Comment: See Table S3 for details; This variant was reported as c.323_235del but assumed to be c.323_325del, which corresponds to the reported protein change (p.(Gly108_Phe109delinsVal)).
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Supplemental material
AssayResult: 3.8
AssayResultAssertion: Abnormal
Comment: See Table S3 for details; This variant was reported as c.323_235del but assumed to be c.323_325del, which corresponds to the reported protein change (p.(Gly108_Phe109delinsVal)).
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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.323_325del p.(Gly108_Phe109delinsVal)
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10.11.66.200 10.11.66.200
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refer to the New Enrollment Card Not Received call type in the DCFC
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- Dec 2020
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www.defenseurdesdroits.fr www.defenseurdesdroits.fr
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L’adoption de l’article 12 de la CIDE a marqué un tournant décisif dans l’avènement des droits de l’enfant. Il impose aux États une obligation de considérer l’enfant comme un acteur en capacité de contribuer à sa propre protection en participant aux décisions le concernant.
à mettre en relation avec le rapport du DD sur la JADE
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- Oct 2020
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Local file Local file
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For many amorphous and crystalline semiconductors,an exponential dependence of absorption coefficientα<104cm−1may take Urbach’s empirical formula [28]:av=a0exphv/EU,wherea0is a constant andEU(Urbachenergy) is an energy characterizing the degree of disorderintroduced from defects and grain boundaries; also, it is inter-preted as the width of the tail of localized states associated withthe amorphous states in forbidden band. Figure 12 representsthe logarithm of absorption coefficient as function of thephoton energy at different deposition times 20, 30, 40, and50 min. The value ofEUis calculated from the inverse slopeof the linear part of curves and also listed in Table 3
Para muchos semiconductores amorfos y cristalinos, una dependencia exponencial del coeficiente de absorción puede tomar la formula empírica de Urbach [28]:
donde es una constante y (la energía de Urbach) es una energía caracterizada por el grado de desorden introducido de defectos y los limites de grano; también es interpretado como el amplio de la cos estados localizados de la cola asociados con los estados amorfos en la banda prohibida. La figura 12 representa el logaritmo del coeficiente absorción como una función del fotón de energía a diferentes tiempos de deposición (20,30,40 y 50 min). El valor de la energía de Urbach es calculado desde la pendiente inversa de la parte linear de las las curvas y también enlistado en la Tabla 3.
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www.youtube.com www.youtube.com
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à 21.20 les écrans à tous les âges résultats de l'étude en 2027 https://youtu.be/ovbeMGfSO2M?t=1284
Tags
Annotators
URL
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- Sep 2020
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www.biblegateway.com www.biblegateway.com
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“You are free to eat from any tree in the garden;(W) 17 but you must not eat from the tree of the knowledge of good and evil,(X) for when you eat from it you will certainly die.”
God instructed Adam and Eve to eat whatever they desired, though prohibited them to eat from the tree of the knowledge of good and evil. In Robin Wall Kimmerer's "Skywoman Falling", she explained how in Indigenous culture, they follow Original Instructions. These "instructions" are not rules but rather guidelines for each person. Kimmerer explains how during Skywoman's time, the first people's understanding of the Original Instructions were to care for the and have respect for hunted animals, value family, and hold respectful ceremonies for their beliefs.
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news.bloomberglaw.com news.bloomberglaw.com
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Trump Back-to-School Threats Pressure Educators Fearing Covid. (n.d.). Retrieved September 3, 2020, from https://news.bloomberglaw.com/coronavirus/trump-back-to-school-threats-pressure-educators-fearing-covid-1
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www.sciencedirect.com www.sciencedirect.com
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ZFIN: ZDB-ALT-010411-12
DOI: 10.1016/j.cub.2020.04.020
Resource: (ZFIN Cat# ZDB-ALT-010411-12,RRID:ZFIN_ZDB-ALT-010411-12)
Curator: @ethanbadger
SciCrunch record: RRID:ZFIN_ZDB-ALT-010411-12
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- Jun 2020
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louisematic.free.fr louisematic.free.fr
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vous
Pourquoi est ce que pour cette question il a fallu calculer a nouveau le rayon de la terre alors qu'elle était déjà donnée dans un document et que nous l'avons calculer dans la question 13 ? Et que les 3 valeurs ( valeur donnée dans le doc et dans les calculs ) ne sont pas les mêmes
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- Mar 2020
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services.google.com services.google.com
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This source is presented by Google who is well-known by its platform to inform and provide technological advancers for others. This particular report highlights the major trends for K-12 classrooms worldwide. This analysis provides data and interviews gathered from students and teachers about the evolution of K-12 classrooms. Rating: 9/10
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tech.ed.gov tech.ed.gov
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Roles and Practices of Educators in Technology-Supported Learning
This article examines how technology is used in classrooms by educators and students in the every-changing technology realm. I liked how this article addressed the professional development educators need in K-12 classrooms. There are multitude of ways educators can gain additional learning opportunities for themselves or their students via technology. K-12 Educators can share their skills with other educators via online platforms. Rate: 7/10
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Annotators
URL
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education.mit.edu education.mit.edu
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This article explores how technology can be utilized in the K-12 classroom. Educators can consider this a professional development due to the content it covers. Technological language can be decoded and evaluated through instruction. Teacher can use what children, adolescents, and teenagers are interested in technology wise. Incorporating this into the classroom will be engaging for learners. Rate: 7/10
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- Dec 2019
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- Nov 2019
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education.asu.edu education.asu.edu
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Mary Lou Fulton Teachers College
This site is the homepage of the Tech Infusion program at Arizona State University (ASU). Housed within ASU's Mary Lou Fulton Teachers College, Tech Infusion is a technology integration curriculum for Pre-K-12 teacher candidates. Through coursework and hands-on practices, teacher candidates are prepared to use technology fluently and innovatively for teaching and learning. The program integrates research, ISTE Standards, and the TPACK (Technological Pedagogical Content Knowledge) framework around effective technology integration. This website provides technology-infusion resources for course developers, instructors, and current and future educators. Examples include research articles, edtech tool tutorials, lesson plans, and outlines of the curriculum. Rating: 9/10
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Local file Local file
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Coffey argues the effectiveness of online literature discussions in the elementary classroom. Addressing both synchronous and asynchronous environments, she suggests that computer-based discussions can enhance understanding of literature and promote community within the classroom.
10/10
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Annotators
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ts.madison.k12.wi.us ts.madison.k12.wi.us
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This manual, published by Hanover Research, outlines theory and practice of technology integration in the K-12 classroom and provides key strategies for professional development to equip teachers to successfully integrate technology.
10/10
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digitalpromise.org digitalpromise.org
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The authors present the benefits of coaching in professional development for educators in today's technologically advanced classrooms. Of particular interest is the explanation of the different methods of coaching: executive, coactive, cognitive, and instructional. They suggest that coaching provides more successful outcomes than single workshops and stress that finding the correct method for each situation and organization is crucial.
10/10
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- Jul 2019
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www.gatesfoundation.org www.gatesfoundation.org
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regularly use data to continuously improve the supports, instruction, and learning students experience.
Data from annotation informing teaching practices, understanding of learning, success, at admin level.
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real-time assessments for gauging student progress
Real-time grading/assessment for reading = annotation
And note just assessment, but presence: peer learning; teacher-student feedback, ...
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Testing of bioformulations
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Preparation of bioformulations and determination of
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Validation of bioformulations under laboratory conditions (in vitro
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Sequence analysis
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Citrate utilisation tes
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Citrate utilisation tes
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Cis-regulatory element analysis
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Mapping of SbDof genes on sorghum chromosomes and its intron/exon gene structure prediction
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In silico prediction of Dof gene family members in S. bicolor (L
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- Jun 2019
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www.parkerphonics.com www.parkerphonics.com
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A Brief History of Reading Instruction. Includes references to studies that support phonics as the best method for teaching reading and writing. Free textbook for phonics instruction: https://elink.io/p/free-phonics-books-98c2d4e
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krishikosh.egranth.ac.in krishikosh.egranth.ac.in
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Spike length (cm)
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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CD107a Degranulation Assay
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Surface plasmon resonance (SPR) measurements
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Bleeding of mice and Collectiml of Sera
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Ion Exchange Column Chromatography
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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ACK lysis Buffer
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ELISA dilution buffer
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ELISA blocking buffer
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ELISA washing buffer
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ELISA binding Buffer
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FACS buffer
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Youman's modified medium
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Phosphate buffered saline (PBS), 0.15M, pH 7.2
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Reagents Used
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Routine maintenance of cell lines
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Dulbecco’s Modified Eagle Medium
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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MTT Assay
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Mesomorphic ectomorph:
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First Component Rating
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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All experiments were carried out on a Beckman XL-A analytical ultracentrifuge, equipped with absorbance optics, and an An60-Ti rotor, at 20 °C. Sedimentation velocity experiments were performed at 40,000 rpm. Data were collected at 540 nm and at a spacing of 0.005 em with three averages in a continuous scan mode. The protein concentration varied in the range 4-40 IJ.M (heme) in 50 mM phosphate buffer, pH 7.2
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Analytical Ultracentrifugation experiments
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link.springer.com link.springer.com
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Expert Rev Med Devices
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J Plast Reconstr Aesthet Surg
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Dermatol Surg
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Lasers Surg Med
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Plast Reconstr Surg Glob Open
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Rev Soc Bras Cir Plast
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J Cutan Aesthet Surg
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Aesthet Surg J
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Plast Reconstr Surg
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J Cosmet Laser Ther
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Rom J Morphol Embryol
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Aesthet Plast Surg
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Plast Reconstr Surg
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- May 2019
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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suspension through a 26 gauge needle. Lysates were cleared by centrifugation at 14,000 g for 30 min at 4°C and supernatant was used for protein estimation using BCA protein estimation kit (Pierce)
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P. Jalciparum infected erythrocytes were lysed by the addition of 0.05 % (w/v) saponin to release parasites, followed by a 30 minute incubation on ice. To remove debris and lysed RBCs were washed with cold PBS followed by centrifugation at 8000g. The lysis buffer containing 10 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, and Ix complete protease inhibitor cocktail (Roche Applied Science) was added to the parasite pellet and homogenized by passing the
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Preparation of Parasite Cell Lysate
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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at the acidic and basic endpoints of the titrations. Na+ free and HC03-free buffer were prepared as described by Khaled et al.
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Intracellular pH measurement was performed using the long-wavelength fluorescent pH indicator carboxy SNARF-1 AM. THP-1 macrophages were resuspended in serum-free and phenol-red free RPMI-1640 medium (106 cells/mL) and incubated at room temperature for 15 min with SNARF-1 AM at a final concentration of 1 f.!M. The cells were washed once in fresh serum-free media and incubated for 20 min for complete de-esterification of intracellular acetoxymethyl esters. In situ calibration ofSNARF-1 AM was performed to determine the pKa of the dye at 3 7°C by using the ionophore Nigericin (1 0 f.!M), which maintains the intracellular pH the same as that of the controlled extracellular medium in a buffer containing high-K+. Appropriate groups were subjected to different treatments and fluorescence measurements were commenced in a spectrofluorimeter (Perkin Elmer, Waltham, MA, USA) followed by kinetic analysis. The pH was calculated from the fluorescence measurements using the following formula: where pKa of carboxy SNARF-1 AM is 7.5 at 37 °C. R is the ratio of fluorescence intensities (F) measured at two emission wavelengths, 580 nm (AI) and 640 nm (A.2), with fixed excitation at 514 nm. The subscripts A and B represent the limiting values
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Measurement of intracellular pH
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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The proteins were resolved on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane, in the transfer buffer, at a constant current of 300 rnA for 2 h. The membrane was incubated in blocking buffer for 45 min. at room temperature with continous shaking. The membrane was further incubated in anti-restrictocin antibody diluted in PBS, pH 7.4, containing 0.1% Tween 20 (PBST), for 45 min .. The membrane was washed thrice with PBST, followed by incubation in anti-rabbit IgG-HRP conjugate diluted in PBST for 30 min. with shaking. After repeated washings with PBS, colour was developed by incubating the membrane with the chromogenic substrate 0.5 mg/ml of DAB.4HCI (diaminobenzidine tetrahydrochloride dihydrate) and 1 f..ll/ml of H202 in PBS.
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Western Blotting
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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eppendorf tube was put at the bottom of the column to collect the eluate. The column was respun as before and the purified probe collected in the eppendorf tube, the unincorporated nucleotides remaining within the column. One ul aliquot from the purified probe was diluted 100 fold, mixed well and 1 ul aliquots were put in triplicate into 3 ml scintillation fluid containing vials which were counted in a Beckman Liquid Scintillation Counter. The total radioactivity of the probe was calculated by multiplying the mean radioactivity of the three diluted samples with a factor of 104 ( dilution factor, 102, total reaction volume, 102 ). The specific activity of the probes ranged from 1 X 107 to 5 x·1o7 cpm 1 ug DNA. The probe purified by the above method did not require any further purification.
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The nick translated probe was purified by a spun column procedure to remove the unincorporated nucleotides. A sterile 1 ml syringe was plugged at the lower end with siliconised glass wool. The syringe was then filled with Bio-gel P-4 Bio Rad Laboratories, USA ) equilibrated in advance with TE. For doing this, 30 grammes of Bio-gel P-4 was slowly added into 250 ml of TE ensuring a good dispersion of the powder. This was then autoclaved at 15 psi for 20 minutes. After cooling, the supernate was decanted and replaced with an equal volume of sterile TE. The slurry was stored at 4°C. The slurry was poured upto the 1 ml mark in the syringe. The syringe was placed into a centrifuge tube and spun at 2000 · rpm for 3 minutes. The column was packed by repeating this process till the packed column volume reached 1 ml mark. Next, 50 ul of 2 mg 1 ml denatured salmon sperm DNA was loaded as carrier and the column spun as before. 100 ul of TE was then added to the column and it was respun as before. Finally, the nick translation reaction was diluted to 100 ul with TE and loaded on to the column. A sterile 1.5 ml
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Purification of the probe.
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400 ci 1 mmole to 3000 ci 1 mmole. The nick translation reaction was set up as recommended by the manufacturer of the kit, using about 0.5 ug DNA. The reaction was incubated at 12 -14 °C for 90 minutes, except in the case of small fragments ( 500 bp ) when the reaction was incubated for 45 minutes only. The reaction was terminated by the addition of stop buffer supplied with the kit.
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DNA was labelled using the nick translation kits supplied by BRL or NEN, USA, or Amersham, UK. The 32P-deTP was from either NEN or Amersham, UK, at a concentration of 10 mei I ml. The specific activity of the label ranged from
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Nick translation.
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32~ -labelling of DNA.
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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TheO2carryingcapacity(Vol%)ofbloodwascalculatedby multiplyingtheHbcontentwith1.25O2combiningpowerofHb/g(Johansen,1970).
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TheO2carryingcapacity
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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annealing at 25°C for 5 minutes, the reaction was incubated at 42°C for one hour.
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series of primers were designed to detect the levels of intact gene of interest or Rz in the cell lysate. The levels of RNA were quantitated by carrying out reverse transcriptase based-PCR using the Im.Prom-11™ Reverse Transcriptase system (Promega, U.S.A.). 1}lg of template RNA and 1}lM terminal primers were combined in 5pl reaction volume and the primer I template mix was thermally denatured at 70°C for 5 minutes and chilled on ice. A reverse transcription reaction mix of volume 15 pl was assembled on ice to contain nuclease-free water, 1X reaction buffer, 1pl reverse transcriptase, 6 mM magnesium chloride, 0.5 mM dNTPs and 1 U ribonuclease inhibitor RNasin. As a final step, the template-primer combination was added to the reaction mix on ice. Following an initia
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Reverse transcri.ptase polymerase chain reaction (RT-PCR)
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and fixed with 100μl of fixative solution per well, for 10 minutes at room temperature. The cells were then washed twice with PBS and 100μl of staining solution was added to each well. The plate was kept at 37° C, until the color development.
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4x103-5x103 cells were plated in 96 well plate, well. Cells were transfected with reporter plasmid 18 -24 hrs after plating. After 48 hrs, cells were washed once with PBS
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Procedure:
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1X PBS diluted in distilled water 1X fixative solution diluted in distilled water 2.4.12.3 Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF
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Working Solutions:
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20 mg/ml X-gal in dimethylformamide Solution A as 40 mM potassium ferricyanide. Solution B as 40 mM potassium ferrocyanide. Solution C as 200mM magnesium chloride. 10X fixative (20% formaldehyde; 2% glutaraldehyde in 10X PBS) 10X PBS as 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7
.4
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Stock Solutions:
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This protocol is for the detection of β-gal expression in fixed cells. It was performed on 96-well plates for initial screening of tTA transfected clone, and is a modification of Sanes et al., 1986
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In situβ-gal staining of Transfected Cells
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(ii) Stacking gel buffer: 1.0 M Tris-Cl pH 6.8 (iii) Resolving gel buffer: 1.5 M Tris-Cl pH 8.8 (iv) SDS stock: 10% (w/v) solution (v) Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh (vi) Gel running buffer (1X) (vii) Loading dye (6X): (viii) Lysis buffer (RIPA) Gels of 1.5 mm thickness were cast in the Biorad small gel apparatus. Resolving gel of 10% (10 ml) was made by mixing 4.2 ml 10% acrylamide, 3.1 ml water, 2.5 ml of 1.5 M Tris-Cl pH 8.8 and 0.1 ml of 10% SDS. Stacking gel (2 ml) was made by mixing 0.33 ml of 30% acrylamide, 1.4 ml of water, 0.25 ml of 1 M Tris-Cl pH 6.8 and 0.02 ml of 10% SDS. Gels were polymerized by the addition of TEMED (N,N,N′, N′-tetramethyl ethylene diamine) and APS (1/100th volume of gel mix). Sample preparation for gel loading was done as follows. Mid log and late log phase 10 ml cultures were centrifuged at 26000g and the cell pellet was resuspended in 0.5 ml RIPA buffer. Cells were sonicated on ice for 1 min at output power of 5 to get a cleared lysate. The culture lysate was centrifuged at 26000g to recover the clear supernatant. Total cell protein was quantified in the lysates using BCA kit reagents (BioRad) using the manufacturers protocol. Appropriate volume of cell lysate was mixed with the loading dye in a final concentration of 1X and loaded onto the gel. The gel was run at constant voltage of 60 V for stacking and 80 V for resolving gel
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The method followed was as described in Sambrook and Russell (2001). The following solutions were used to cast and run SDS-PAGE gels. (i) Acrylamide stock: 29% (w/v) acrylamide and 1% N,N′-methylene bisacrylamide
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Sodium dodecyl sulphate-polyacrlyamide gel electrophoresis (SDS-PAGE)
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the phage (λ1098 for Tn10dTet transpositions and λNK1324 for Tn10dCm transpositions) at a multiplicity of infection (moi) of 0.05 in the presence of 5 mM MgSO4. This mixture was incubated for 15 min at 37°C to allow for phage adsorption. The unadsorbed phage was then removed by centrifugation and the pellet was resuspended in 10 ml of LB broth containing 5 mM sodium pyrophosphate. It was incubated without shaking at 37°C for 30 min for phenotypic expression. The rest of the mixture was diluted into 100 ml of LB broth with 5 mM sodium pyrophosphate carrying the required antibiotic and amplified overnight by growth at 30°C. This population of cells was used as a source of random transposon insertions. The λ lysates used for the transposition experiments carry amber mutations, and were propagated on a supE strain C600 by the protocol described below in section 2.14
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The method used was essentially the same as that described by Miller (Miller, 1992). The strain to be used for obtaining random Tn10dTet or Tn10dCm insertions was grown overnight in Z-broth containing 0.4% maltose. The culture was then diluted 50-fold in the same medium and grown to an A600 of 0.8. Two ml of the culture was infected with 107 pfu of
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Generation of random Tn10 insertions into the genome of E.coli
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he MTT Lysis buffer (20% SDS , 50% Dimethyl formamide) and the O.D.s7onm was measured. The standard curve was plotted and the equation derived, used to calculate the number of metabolically viable cells in experimental groups. Percentage of viable cells was calculated by comparing the number of viable cells in treated wells with that of untreated wells
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MTT micromethod is a colorimetric assay based upon the conversion of the (yellow) MTT reagent (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), a tetrazole to purple formazan in the mitochondria (succinate dehydrogenase) of the living cell which is quantified by measuring the optical density at 570nm. When the amount of purple formazan produced by untreated control cells is compared to that of treated cells, the effectiveness of the agent can be deduced through the production of a dose-response curve. Parasites in their log phase were harvested and the dead cells removed at 129 x g for 5min at RT. The live cells were resuspended in phenol red-free DMEM containing 10% FBS to a cell count of 2.5 X 107. 1mL of above culture was plated into each well of a 24 well plate and appropriate treatments were given for desired duration of time. Some untreated cells were also kept aside for generating the standard curve. After appropriate treatment, from each well of the 24 well plate 200pL was aliquotted into 4 wells of a 96 well plate. To each well of the 96 well plate, 10pL of MTT solution (5mg/ mL prepared in PBS and filtered with 0.22p filter) was added and the plate incubated at 23°C for 2-3 hours till colour develops in the control cells. A standard curve was also plated by taking different dilutions of untreated cells and processed similarly. Once colour developed, the reaction was stopped by lysing the cells using
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MTT assay to assess viability of Leishmania parasites
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Plasmid/Ligation mix was incubated with ultra-competent cells for 30 min on ice. This was followed by a heat shock at 42°C for exactly 45s after which the cells were chilled on ice for 2 min. lmL of LB (Luria Bertani) medium was added to the cells and incubated at 37°C in a shaker incubator for 45 min. Cells were then plated on LB agar plate containing appropriate antibiotic and/ or X-gal solution and incubated at 37°C overnight
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Transformation of competent cells with DNA
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Cells grown overnight in YNBmedium wereinoculated in fresh YNB mediumand incubated at 30 ̊C with shaking at 200 rpm. Cells were harvested when the cell density reached to an OD600of 0.6-0.8.Cells were consecutively washed with sterile MQ water and YNB without phosphate (YNB-Pi) medium. Washed cells were inoculated either in YNB orYNB-Pimediumto the initial OD600of 0.1. Cells were incubated at 30 ̊C for 3-4 h, harvested and resuspendedin 100 μlYNB-Pimedium. Radioactive P32-labelled o-phosphoric acid(Jonaki# LCP 32)was added to the cell suspension to a final concentration of 1 μCi/mlandcells were incubated for 30 min.For determining phosphate uptake, a10-12 μl cell suspensionaliquot,after every5 min,was removed and kept on ice.To this cell suspension, 500 μl ice-cold YNB-Pimediumwas addedand cells were harvested by centrifugation at 5,000 g for 5 minat 4 ̊C.These cells were washed with ice-cold YNB-Pimedium thrice and resuspendedin 100 μlPBS(1X). 10-20 μl of this cell suspension was added to5 ml scintillation fluid and β-decay counts were measured in ascintillation counter(Tri-Carb 2910 TR Liquid Scintillation Analyzer, PerkinElmer).Scintillation counts were normalized to total cell number and plotted with respect to time. Total phosphateuptake was expressed as P32c.p.m/OD600cellswhere c.p.m refers tocounts per min
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Phosphate uptake assay
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To preclude the possibility of human RNA contamination, cDNA prepared from internalized yeast was examined for the presence of human transcripts encoding Ccl5 and histone H3. However,no amplification forhuman genes was observed, thus, eliminating any possiblecontamination of THP-1 RNA with yeast RNA
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Primersfor real-timePCR analysisweredesigned using Primer3 plus software and are listed in Table 4. To extractRNA from macrophage-ingested C. glabratacells, infected THP-1cells were washed twice with PBS and lysed in 1 ml ice-cold water. Lysate was centrifuged followed by two quick washes with DEPC-treated water andwashed yeast cell pellets were frozen on dry ice.For RNA extraction, yeast cells were disrupted with glass beads in trizol and total RNAwas isolated usingacid phenol extraction method described above.Optimal primer and cDNA concentrations were standardizedand qRT-PCR was performedusing ABI 7500 Fast Real-Time PCR System (Applied Biosystems).In brief, 0.5 μl cDNA,0.1 to 0.2picomoles of gene specific primers and 10 μl 2X MESA GREENqPCR™ Mastermix Plus containing SYBR green dye (Eurogentec)were mixed in thewellsof a 96-well PCRplate (Axygen). Final reaction volume was adjusted to 20 μl with DEPC-treated water. Transcript levels were quantified with an end-point value known as Ct(cyclethreshold). Expression of TDH3, which encodes CgGapdh,was used asaninternal control. The Ct defines the number of PCR cycles required for the fluorescent signalof SYBR green dye to cross beyondthe background level.Fold-change in transcript expression was determined usingfollowing formula.Fold change in expression = 2-ΔΔCtΔΔCt= ΔCttreated -ΔCtuntreatedΔCttreated = Ctvalue forthe gene of interest under treated condition -Ctvalue forthe internal control gene (TDH3) under treated conditionΔCt untreated = Ctvalue forthegene of interest under untreated condition -Ctvalue forthe internal control (TDH3) gene under untreated condition
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Quantitative real-timePCR
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10 min at 4 ̊C and gently resuspended in 20 ml ice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80 ̊C
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A single colony of E. coli DH5α strain was inoculated in 10 ml LB medium and incubated at 37 ̊C for overnight. 4 ml overnight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till the OD600 reached to 0.5. Cultures were centrifugedat 2,500 g for 10 min at 4 ̊C and harvested cells were washed gently with80 ml ice-cold Inoue transformation buffer. Cells were collected by centrifugation at 2,500 g for
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