301 Matching Annotations
  1. Last 7 days
    1. Alguns números da Company Hero

      Se formos usar os Big Numbers na página de cima, talvez podemos eliminar esta seção.

  2. Nov 2022
    1. 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.

  3. Aug 2022
    1. 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

  4. May 2022
    1. 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.

    2. 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?

    1. 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

  5. Mar 2022
  6. Jan 2022
    1. 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)

    1. 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

    1. 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

    1. 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

    1. 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

    1. 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

  7. Nov 2021
    1. 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...

    1. 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

  8. Sep 2021
  9. Aug 2021
    1. 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


      What is this?

  10. Jul 2021
  11. Jun 2021
  12. Mar 2021
    1. 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

    2. 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)

    1. SUPPLEMENTARY DATA

      AssayResult: 94.01

      AssayResultAssertion: Not reported

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    2. 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)

    1. Source Data

      AssayResult: 23.96

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardErrorMean: 7.6

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 7.75

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardDeviation: 2.59

      StandardErrorMean: 1.83

      Comment: Exact values reported in “Source Data” file.

    3. 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)

    1. 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)

    2. 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)

  13. Feb 2021
    1. 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)).

    2. 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)).

    3. 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)

  14. Dec 2020
    1. 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

  15. Oct 2020
    1. 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.

    Tags

    Annotators

  16. Sep 2020
    1. “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.

  17. Jun 2020
    1. 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

  18. Mar 2020
    1. 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

    1. 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

    1. 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

  19. Dec 2019
  20. Nov 2019
    1. 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

    1. 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

    1. 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

    1. 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

  21. Jul 2019
    1. 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.

    2. 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, ...

  22. Jun 2019
    1. 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

    1. 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
    2. Analytical Ultracentrifugation experiments
  23. May 2019
    1. 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)
    2. 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
    3. Preparation of Parasite Cell Lysate
    1. at the acidic and basic endpoints of the titrations. Na+ free and HC03-free buffer were prepared as described by Khaled et al.
    2. 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
    3. Measurement of intracellular pH
    1. 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.
    2. Western Blotting
    1. 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.
    2. 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
    3. Purification of the probe.
    4. 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.
    5. 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
    6. Nick translation.
    7. 32~ -labelling of DNA.
    1. TheO2carryingcapacity(Vol%)ofbloodwascalculatedby multiplyingtheHbcontentwith1.25O2combiningpowerofHb/g(Johansen,1970).
    2. TheO2carryingcapacity
    1. annealing at 25°C for 5 minutes, the reaction was incubated at 42°C for one hour.
    2. 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
    3. Reverse transcri.ptase polymerase chain reaction (RT-PCR)
    1. 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.
    2. 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
    3. Procedure:
    4. 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
    5. Working Solutions:
    6. 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

    7. Stock Solutions:
    8. 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
    9. In situβ-gal staining of Transfected Cells