238 Matching Annotations
  1. Jan 2026
  2. Dec 2025
  3. Aug 2025
  4. Jul 2025
  5. Jun 2025
  6. May 2025
    1. Die Studie des Potsdam-Instituts für Klimafolgenforschung zeigt, dass Bioenergy with Carbon Capture and Storage (BECCS) theoretisch bis 2050 jährlich 7,5 Milliarden Tonnen CO₂ entfernen könnte. Allerdings würde dies die planetaren Belastungsgrenzen stark überschreiten, insbesondere in Bezug auf Stickstoffeintrag, Süßwasserverbrauch, Entwaldung und Biosphärenintegrität. Unter Berücksichtigung dieser Grenzen reduziert sich das Potenzial auf nur 200 Millionen Tonnen CO₂ jährlich. Die Studie betont die Notwendigkeit, neben der CO₂-Bilanz auch andere ökologische Faktoren zu berücksichtigen und schlägt vor, durch weniger Fleischkonsum Flächen für Klimaplantagen freizumachen. [Zusammenfassung generiert mit Mistral]

      https://www.derstandard.at/story/3000000257365/kein-platz-fuer-klimaplantagen

  7. Apr 2025
    1. 2024 verzeichnete die zwölf bis ihr heißesten Monate in Europa. Über 400.000 Menschen waren direkt von den Folgen von Extremwetterereignissen betroffen. Über 30% der Flussgebiete In über 30% der Flussgebiete gab es schwere Überschwemme. Ausmaß und Erhezung, Ausmaß und Folgen der Erhezung in Europa werden systematisch in dem Berichtsteht auf Sie klimat 2024 erfasst, der von Copernicus und der WMU veröffentlicht wurde. https://www.theguardian.com/environment/2025/apr/15/europe-storms-floods-and-wildfires-in-2024-affected-more-than-400000

    1. "Scholar of racial difference in the early modern Atlantic world have detected a decline in the contradictions after about the mid-seventeenth century and a rise of more uniformly negative appraisals of Africa and Africans." p. 15

      What lead to this shift? Did it have some connection to the need of dehumanization to condone the act of slavery in the context of the Atlantic Slave trade?

  8. Mar 2025
    1. Dem amerikanischen Historiker Künstlubodien zufolge ist ein Vorbild für die Trump-Administration weniger der historische Faschismus in Italien und Deutschland als die kapitalistische oligarchien Herrschaft in Saudi Arabien. Tramp Wolle die USA zu einem modernen Petro stattmachen. Er wolle dazu die Weltweite Abhängigkeit von den USA nicht nur beim Öl ausnutzen. Außerdem stütze er sich auf die enorm finanziellen Ressourcen Saudi-Abiens. Der S.A. in den New York Times stützt diese Tese mit einer Vielzahl von Einzelbeobachtungen. https://www.nytimes.com/2025/03/15/opinion/trump-saudi-arabia-america.html

  9. southtexascollege.blackboard.com southtexascollege.blackboard.com
    1. Though parents grudge, and you, w'are met, <br /> And cloistered in these living walls of jet.

      The speaker tells the listener to forget societal norms and barriers, telling them that they have already met in a sacred place, a reference to the temple or flea. The flea creates a close, private area for the two of them to be free from the view of others.

  10. Feb 2025
  11. Jan 2025
    1. Roscoe: A suite of metrics for scoring step-by-step reasoning.

      这篇论文介绍了一个名为ROSCOE的度量标准套件,用于评估逐步骤推理的性能。ROSCOE是一套可解释的、无监督的自动评分系统,旨在改进和扩展之前的文本生成评估指标。该研究通过设计一个推理错误的分类学,并在常用的推理数据集上收集合成和人类评估分数,来评估ROSCOE相对于基线指标的表现

    1. history of labor

      for - paraphrase - history of labor - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2 - to - stats - Gallup Chairman's Blog - world poll 2024 - 15% of employees worldwide are engaged - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

      paraphrase - history of labor - Michel gives a nice succinct summary of the broad strokes of the history of labor over the last few millennia: - Civilizations have begun as slave-based societies first - Then when the Christian revolution occurred after the fall of the Roman Empire, "Ora et Labora (Pray and Work)" was adopted to transform work into a spiritually meaningful endeavor - Then in the 16th century, this philosophy was replaced by turning labor into a commodity, where it has remained ever since, - resulting in a world where 85% of those surveyed say they are not engaged with their job

      to - stats - Gallup Chairman's Blog - world poll 2024 - 15% of employees worldwide are engaged - https://hyp.is/iOlXbNBOEe-t6hdOWtvTYw/news.gallup.com/opinion/chairman/212045/world-broken-workplace.aspx

    1. their kids aren’t interested in the grueling work of farming.

      for - question - what if the children were identified to come back? - source - article - Substack - One of the biggest wealth transfers in U.S. history just commenced. Are you aware of it? - Alexandra Fasulo - 2024, Oct 15

    2. for - article - Substack - One of the biggest wealth transfers in U.S. history just commenced. Are you aware of it? - Alexandra Fasulo - 2024, Oct 15

      • opportunity - regenerative agriculture and rewilding - US farmers retiring in the next 20 years - largest transfer in US history - land trusts ?

      • referred by - Kim Chapple

  12. Dec 2024
  13. Nov 2024
    1. Most people in America today (85–90%) agree on most issues and topics (85–90%). The so-called polarization is the result of a media landscape that amplifies the voices of the 10–15% that keep constantly talking about the 10–15% of topics on which people are not on the same page.

      for - stats - most people in America agree on 85 - 90% of issues - unpack why and how the 10 - 15% is made so divisive

  14. Oct 2024
    1. Finnland hatte sich beim Ziel der CO2-Neutralität 2035 darauf verlassen, dass große Mengen von CO2 von Wäldern, Böden und Feuchtgebieten absorbiert werden. Inzwischen ist das Land dort keine Kohlenstoffsenke mehr. Dazu trägt die globale Erhitzung selbst bei, durch die viele Bäume sterben, aber auch die Abholzung des Waldes. Finnland ist ein Beispiel für die Schwächung der ländlichen Kohlenstoffsenken, von der viele Länder betroffen sind. https://www.theguardian.com/environment/2024/oct/15/finland-emissions-target-forests-peatlands-sinks-absorbing-carbon-aoe

  15. Sep 2024
    1. The amount of intact natural land as of 2018 was around 15% below this ESB, but could be increased through restoring degraded ecosystems or previously converted ecosystems,102,103,106102.Strassburg, BBN ∙ Iribarrem, A ∙ Beyer, HL ∙ et al.Global priority areas for ecosystem restorationNature. 2020; 586:724-729CrossrefScopus (536)PubMedGoogle Scholar103.Jung, M ∙ Arnell, A ∙ de Lamo, X ∙ et al.Areas of global importance for conserving terrestrial biodiversity, carbon and waterNat Ecol Evol. 2021; 5:1499-1509CrossrefScopus (162)PubMedGoogle Scholar106.Wolff, S ∙ Schrammeijer, EA ∙ Schulp, CJE ∙ et al.Meeting global land restoration and protection targets: what would the world look like in 2050?Glob Environ Change. 2018; 52:259-272CrossrefScopus (72)Google Scholar with conservation efforts distributed across all ecoregions.

      for - stats - earth system boundary - biodiversity - intact natural systems - 15% below ESB in 2018

  16. May 2024
    1. WirtschaftsWoche: Hintergrundbericht zur geplanten Fusion der Petrochemie-Sparten von OMV und Adnoc. Die Adnoc will mit #Bourouge einen Petrochemie-Großkonzern aufbauen, der dann statt Treibstoffe scheinbar saubere Produkte wie Kunstdünger und Plastik verkauft und auf Recycling und Kreislaufwirtschaft ausgerichtet ist. Dazu ist man auch an einer Übernahme von Covestro und Wintershall interessiert. Der Konzern soll zur Hälfte der OMV gehören. https://www.wiwo.de/unternehmen/industrie/chemieindustrie-was-hinter-dem-megageschaeft-zwischen-omv-und-adnoc-steckt/29560900.html

    1. Neue Daten der "Global Oil and Gas Exit List" zeigen, dass die Adnoc, die Öl- und Gasgesellschaft der Vereinigten Arabischen Emirate, die weitreichendsten Expansionspläne aller Ölfirmen hat. Die rolle des Adnoc-Chefs Sultan Al Jaber als Präsident der #COP28 sei "lächerlich". Seit 2021 sind weltweit 140 Milliarden Dollar in die Exploration neuer Öl- und Gasfelder geflossen. 96% der 700 im Explorationsgeschäft tätigen Firmen explorieren weiter, 1000 Firmen arbeiten an neuen Pipelines, LNG Terminals und anderer Infrastruktur zur Distribution von Öl und Gas. https://www.theguardian.com/environment/2023/nov/15/cop28-host-uae-oil-plans-data

  17. Apr 2024
    1. Aufgrund der Dürren und anderer Extrem-Ereignisse wird die Trinkwasser-Versorgung in vielen Communities im Einzugsgebiet des Mississippi prekär. Der Süden Louisianas wird von eindringendem Salzwasser bedroht, Dabei sind die Folgen der schweren Hurricanes der vergangenen Jahre noch nicht überwunden. Immer mehr Menschen wollen die Gegend verlassen. https://www.nytimes.com/2023/11/15/us/louisiana-saltwater-climate.html

  18. Mar 2024
    1. As soon as he was born, he cried not as other babes use to do, Miez, miez, miez, miez, but with a high, sturdy, and big voice

      Showing the parallel between him and other babies of his age, he describes that he did not shout "Miez, Miez, Miez, Miez", which translates to "no, no, no, no" in old germanic dialects like the other children. But instead, in a polar opposite manor, yelled "high, sturdy and big voice" shouted drink, drink, drink. Showing from an early age leadership, confidence, and "greatness".

  19. Feb 2024
    1. Interview mit dem Blackrock-Portfoliomanager Evy Hambro zu den Folgen der Energiewende für die Rohstoff-Märkte und einzige ihrer geopolitischen Implikationen. Grundaussagen: Durch erneuerbare Energien wird die Wirtschaft rohstoffintensiver und die Rohstoffpreise steigen. Auch der Bergbau ist bisher CO2-intensiv. Das Festhalten an Wachstum führt zu Investitionen in Kerneenergie und Uran-Bergbau. China beherrscht vor allem die Mitte der Lieferkette für wichtige Rohstoffe. https://www.handelsblatt.com/finanzen/maerkte/devisen-rohstoffe/blackrock-experte-energiewende-koennte-mineralienpreise-in-die-hoehe-treiben/100013162.html

    1. In Barcelona und 200 anderen katalanischen Städten wurde Anfang Februar der Wassernotstand ausgerufen. An der spanischen Mittelmeerküste, in Sizilien und Nordafrika herrscht starke Dürre. Südandalusien leidet set 2016 kontinuierlich unter Trockenheit. Der Kommentar im Guardian beschhäftigt sich vor diesem Hintergund mit dem Widerstand gegen Klima- und Wasserschutmaßnahmen, der populistisch angeheizt wird. Es sei noch nicht ausgemacht, dass auch in der Landwirtschaft das Verständnis für die Gründe der Krise wachse. https://www.theguardian.com/commentisfree/2024/feb/15/spain-water-barcelona-farmers-tourism-catalonia-drought

      Karte des European Drought Observatory: https://edo.jrc.ec.europa.eu/edov2/php/index.php?id=1052

  20. Jan 2024
    1. Die Repubblica interviewt Carlo Buontempo, den Leiter des europäischen Klimaservice Copernicus. 20 23 wurden viele Anomalien beobachtet. Jeder Tag war mindestens ein Grad wärmer als in der Vergleichsperiode, fast die Hälfte der Tage 1,5 und zwei sogar zwei Grad. Der Juli war der heißeste je gemessene Monat. Es sei noch unklar, ob es sich dabei um Ausnahmen handelt oder um den Beginn einer neuen Phase. https://www.repubblica.it/economia/2024/01/15/news/clima_cambiamenti_climatici_caldo_record_carlo_buontempo_copernicus-421876070/

  21. Dec 2023
    1. Die afrikanischen Politiker:innen Nathaniel Mong’are und Abdoulie Ceesay bewerten das COP28-Ergebnis und die Ktivitäten der Emirate vor und während der Konferenz als positiv. Maßgeblich für sie ist die Finanzierung des gerechten Übergang; hier sei viel, wenn auch bei weitem nicht genug, Geld mobilisiert worden.

      https://foreignpolicy.com/2023/12/15/cop28-fossil-fuel-nations-sabotage-climate-deal-africa/?utm_source=Sailthru&utm_medium=email&utm_campaign=Editors%27%20Picks:%2012152023&utm_term=editors_picks

  22. Nov 2023
    1. Das deutsche Bundesverfassungsgericht hat untersagt, dass 60 Milliarden Euro, die ursprünglich für Corona-Hilfen vorgesehen waren aber nicht abgerufen worden, für Klimaschutz- und andere Projekte benutzt werden. Die im deutschen Grundgesetz festgelegte Schuldenbremse dürfe so nicht umgangen werden. Damit ist die deutsche Bundesregierung zu einer Neu-Festlegung ihrer Klimapolitik gezwungen, die zu erheblichen Konflikten zwischen den Ampelparteien führen dürfte.https://www.derstandard.de/story/3000000195316/gericht-stoppt-finanztrick-der-deutschen-ampel

    1. And they feel a sense of relatedness when they perceive that their teachers like and value and respect them.

      relatedness is only about relationship between teachers and students - what about student to student relationships?

    2. Deci and Ryan acknowledge that many of the tasks that teachers ask students to complete each day are not inherently fun or satisfying; it is the rare student who feels a deep sense of intrinsic motivation when memorizing her multiplication tables.

      What difference would it make if the things students were asked to do was inherently fun and satisfying?

  23. Oct 2023
    1. https://en.wikipedia.org/wiki/Shmita

      During shmita, the land is left to lie fallow and all agricultural activity, including plowing, planting, pruning and harvesting, is forbidden by halakha (Jewish law).

      The sabbath year (shmita; Hebrew: שמיטה, literally "release"), also called the sabbatical year or shǝvi'it (שביעית‎, literally "seventh"), or "Sabbath of The Land", is the seventh year of the seven-year agricultural cycle mandated by the Torah in the Land of Israel and is observed in Judaism.

  24. Sep 2023
  25. Jun 2023
  26. learn-us-east-1-prod-fleet01-xythos.content.blackboardcdn.com learn-us-east-1-prod-fleet01-xythos.content.blackboardcdn.com
    1. 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.

  27. May 2023
    1. Gold give us, God forgive us,And from all woes relieve us;That we the treasureMay reap of pleasure,And shun whate’er is grievous,Gold give us, God forgive us.

      Why did he asked for god's forgiveness? Does that mean that he admits his request are greedy and not inclusive?

  28. Apr 2023
    1. Given the broad aim of the right of access, the aim of the right of access is notsuitable to be analysed as a precondition for the exercise of the right of accessby the controller as part of its assessment of access requests. Thus, controllersshould not assess “why” the data subject is requesting access, but only “what”the data subject is requesting (see section 3 on the analysis of the request) andwhether they hold personal data relating to that individual (see section 4).Therefore, for example, the controller should not deny access on the groundsor the suspicion that the requested data could be used by the data subject todefend themselves in court in the event of a dismissal or a commercial disputewith the controller9.

      This is very interesting, and the FT (Copies du dossier médical) case will be one to watch.

      Details: https://www.dpcuria.eu/details?reference=C-307/22

  29. Mar 2023
  30. Jan 2023

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  31. Dec 2022
  32. Aug 2022
  33. Mar 2022
  34. Nov 2021
  35. Sep 2021
  36. Aug 2021
  37. Jun 2021
  38. May 2021
  39. Apr 2021
  40. 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: 5.1

      AssayResultAssertion: Normal

      StandardErrorMean: 0.7

    1. Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 41.7

      AssayResultAssertion: Abnormal

      ReplicateCount: 15

      StandardErrorMean: 10.8

      Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

  41. Feb 2021
  42. Oct 2020
    1. he extinction coefficient could be calculated using therelation:k=αλ/4π. Figure 15 shows the variation of extinc-tion coefficient as a function of wavelength; it shows a

      El coeficiente de extinción podría ser calculado usando la relación: (ecuación). La figura 15 muestra la variación del coeficiente extinción como una función de la longitud de onda; esto muestra un agudo/fuerte incremento en la región ultravioleta debido a la alta absorbancia fotones incidentes cerca del band gap.

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    1. Une authentique dérogation est à noter: la loi du 15 mars 2004 a introduit l’interdiction du « port de signes ou de tenues » manifestant « ostensiblement une appartenance religieuse » pour les élèves des écoles, collèges et lycées publics, qui sont des usagers du service public de l’éducation. Ainsi, les signes et tenues interdits sont ceux dont le port conduit à se faire immédiatement reconnaître par son appartenance religieuse, tels que le foulard, la kippa ou une croix de dimension manifestement excessive. La loi ne remet pas en cause le droit des élèves de porter des signes religieux discrets.Il est à noter que cette interdiction, strictement limitée aux écoles collèges et lycées publics, ne s’applique pas dans l’enseignement supérieur,et notamment au sein de l’université, que les élèves, usagers, sont libres de fréquenter en revendiquant leur religion, sous réserve, classiquement, de ne pas troubler l’ordre public.

      vidéo

  43. Aug 2020
  44. Jun 2019
    1. Electrostatic potentials were calculated by the Finite Difference Poisson-Boltzmann (FDPB) method using the program MEAD running within the PCE web server (http://bioserv.rpbs.jussieu.fr/PCE) (Miteva et al, 2005; Bashford et al, 1992). Additions of hydrogen atoms as well as assigning of atomic radii and charges were performed automatically within the server. MEAD numerically solves the Poisson-Boltzmann equation to yield the distribution of electrostatic potential on the protein surface. Calculations were performed on one of the native a-chains of the 2HbS crystal structure (Harrington et al, 1997) as well as its SCWRL (Dunbrack et al, 1993) generated mutants. All calculations were performed by setting the internal protein dielectric constant to 4 and the external solvent dielectric constant to 80. The ionic strength parameter was held at 0.1
  45. May 2019
    1. A 96-well microplate was coated overnight at 4°C with ovalbumin conjugated peptide in 100 mM carbonate buffer, pH 9.5 (2 /J-g/well). The plate was washed 3 times with PBST and blocked with PBS containing 2% BSA (200/J-l/well) at 37°C for 1 h. Serum samples (diluted in PBS) were added in duplicates (50 (/J-lIwell) at different dilutions (1: 1 00, 1: 1000, 1: 10,000) and the plate was incubated at 37°C for 1 h. The plate was washed and incubated with HRP-conjugated appropriate antibody (1: 1 0,000 dilution in PBS containing 2% BSA) at 37°C for 1 h. The plate was washed thoroughly with PBST and freshly prepared TMB substrate (100/J-lIwell) was added and the reaction was stopped with 2 N H2S04 (50 (/J-l/well) and the absorbance at 450 nm was recorded in an ELISA reader
    1. Nitric oxide (NO) generation within the macrophage was detected using the fluorescent NO-sensitive probe DAF-FM diacetate (7). THP-1 macrophages were harvested and resuspended in serum and phenol-red free RPMI-1640 medium and incubated at room temperature for 30 min in the presence of 1 llM DAF-FM diacetate dye. The cells were washed once with fresh medium to remove the excess probe and kinetic fluorescent measurements were performed on a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and emission of 520 nm. Time kinetic measurements were performed after appropriate treatment and the values were represented as arbitrary fluorescence units with the comparisons being made against the fluorescence of the control cells. SNAP (S-nitroso-N-acetylpenicillamine), a photoactivatable nitric oxide donor (8) was used as positive control in the assay.
    1. The specific ribonucleolytic activity of restrictocin and its mutants was followed by detecting the release of the characteristic 400 nucleotide long a-fragment from 28S rRNA of eukaryotic ribosomes. All the reagents, water and glassware used during the experiment were treated with O.I% DEPC to get rid of contaminating ribonucJeases. Rabbit reticulocyte lysate (30 J.Ll) was incubated with different concentrations of the toxin in 40 mM Tris-HCl (pH 7.5) containing 10 mM EDTA at 37 °C for 30 min. in a 50 J.Ll reaction volume. The control reaction did not contain an)' toxin. The reaction was stopped by adding 2 J.Ll of I 0% SDS and incubated at ambient temperature for 5 min. Total RNA was extracted using Trizol reagent. 200 J.LI of the reagent was added to the reaction mixture, mixed well and incubated at room temperature for 5 min. Subsequently, 50 J.LI chloroform was added to each tube, mixed thoroughly and incubated at ambient temperature for 2 min. followed by centrifugation at I2,000 rpm, at 4 °C, for I5 min. in a microfuge (Plastocraft). The aqueous phase was mixed with 125 J.Ll isopropanol to precipitate the RNA, allowed to stand at ambient temperature for I 0 min. and centrifuged at 12,000 rpm at 4 °C for I5 min. The RNA pellet was washed with 75% ethanol, dried in air, dissolved in 10 J.Ll of 0.5% SDS solution and electrophoresed on a 2% agarose gel after heating at 65°C for 2 min. The RNA was visualized by ethidium bromide staining and photographed using Polaroid camera. The photographs were scanned, printed using a laser printer to present as figures in this thesis.
    1. Lipofectin was kindly provided by Syntex, Inc., USA as an aqueous solution containing 1 mg I ml of 1 ipid ( DOTMA DOPE; 50 50 ). The procedure used was as described by Feigner et al., 1987 with appropriate I modifications as suggested in the user s notes. Lipofection was done with 0.5 x 106 cells seeded on a 60 mm plate. For each plasmid, the lipofection was performed in duplicate. The amount and quality of the plasmid DNA used ranged from 400 ng of crude DNA prepared by the mini prep method, to 5 ug of highly purified, cesium banded DNA. The appropriate amount of DNA was suspended in 1.5 ml of serum free DMEM. In another tube, 30 ug of lipofectin was suspended in 1.5 ml of serum free DMEM. The two solutions were mixed. The cells were washed twice with HBSS to totally wash off all traces of serum. The DNA 1 lipofectin mix was then applied to the cells and the cells incubated for 4 hours at 37°C. Next, 3 ml of media containing 10 % FCS was added and the incubation continued at 3 7°C for 16 hours. The culture supernate was then aspirated off and fresh medium added to the cells. The selection for stable clones was started after 48 hours by the procedure described below.
    2. Calcium phosphate mediated stable transfections were performed by the method of Graham and Van der Eb ( 1973 with modifications as described by Gorman ( 1986 ). For each plasmid, two petri dishes each containing 0. 5 x 106 CHO-K1 cells were used, with 10 ug of cesium purified DNA for each transfection. A mock transfection which did not contain any DNA, was performed simultaneously as negative control. Precipitation of the DNA was done with great care to ensure the obtention of a fine, translucent precipitate rather than a dense and opaque precipitate. The calcium phosphate I DNA precipitate was added in 4 ml medium to the cells and the cells incubated for 3 hours at 37°C. At this stage, the cells were examined under the microscope and a fine precipitate appeared as small grains all over the cells. The cells were washed once with serum free medium and a glycerol shock given for 3 minutes at 37°C. The cells were washed twice again with
    3. rinsed twice with serum free medium and replenished with 4 ml of DMEM containing 10 % FCS and 100 uM chloroquine. The incubation was continued for another 3 hours at the cells were washed and fed with the normal growth medium containing 10 % FCS. As in the case of FWIL cells, the supernate was collected after 72 hours of transfection and assayed for BhCG activity by RIA.
    4. ayed for BhCG activity by RIA. In case of the other five monolayer forming cell lines, a slightly different protocol was used. Only 1.8 ug plasmid DNA was used for each transfection using 0.5 x 106 cells, and 70 uM chloroquine was included in the DNA 1 DEAE-dextran mixture. Cells were fed 3 hours prior to transfection and washed twice with serum free medium just before exposure to DNA. Cells were exposed to DNA 1 DEAE-dextran mix for approximately 3 hours at 37°C. Following this, the cells were
    5. 1 - 5 ug of plasmid DNA using the DEAE -dextran procedure. DEAE dextran M.Wt. 500,000 was used to perform transient transfection by the method of Luthman and Magnusson 1983 ) , with modifications as described by Gorman ( 1986 ) . Six cell lines ( described above ) with two petri dishes ( 60 mm ) for each cell line were used. In case of FWIL, 5. 4 ug plasmid DNA was used to transfect approximately 5 x 106 cells. No exposure to chloroquine was given. The cells were treated with the DNA 1 DEAE -dextran mixture for 20 minutes at 37°C in a tightly capped tube, mixed gently and reincubated at 37°C for 10 minutes. The sample was then diluted with 3 ml of IMDM supplemented with 10 % FCS, centrifuged and the pellet washed once with normal growth medium. Finally, the pellet was resuspended in 4 ml of growth medium and transferred to a T-25 flask. After incubating for 24 hours at 37°C, 3 ml of fresh medium was added to the cells. The cells were harvested after 72 hours post transfection and the culture supernate was ass
    1. Single cell suspensions of splenocytes in RPMI-1640 medium were prepared from plasmid DNA immunized mice, on day 45, by mechanical disruption of the spleen. Red blood cells were lysed by exposing the cell pellet to 1 OX concentration of 50 mM PBS and immediately bringing the concentration to IX PBS by addition of water. Cells were diluted to a final concentration of 3 x 106 cells/ml in RPMI-1640 medium supplemented with l 0% FCS. A 100 J.ll aliquot of splenocytes was added to each well of a 96-well microtitration plate containing serial dilutions of refolded recombinant proteins (r-bmZP1, r-dZP3 orr-rG), diluted in the same medium, as a source of antigen. All assays were carried out in . triplicates. Three days after the addition of the cells, culture were pulsed with 1 J.lCi/well of eH] thymidine (NEN, Life Science Products, Boston, MA) for 16 h. Cells were lysed and harvested onto glass fibre filaments for liquid scintillation counting (Betaplate; Wallac,
    1. Aftertheperiodofexposure,thecaudalfinwasseveredtogetthebloodforsmearing.BufferedLeishman'sstainofpH6.8gaveexcellentresults.Theworkreportedhereisbasedontheanalysisofslidesoffishestreatedwith7%effluentconcentrationsastheobservedchangesaremaximuminthesefishes.
    1. otal RNA was isolated from cell lines after 48 hrs of transfection using trizol (Invitrogen, U.S.A.). 32p labeled antisense HBx mRNA was in vitro transcribed using T7 RNA Polymerase and Riboprobe kit (Promega, U.S.A.), as described earlier. For generating antisense HBx probe, plasmid DNA was linearized with Bam HI and subjected to transcription. Total RNA was quantitated and equal concentration (15-20 pg) was loaded after adding loading dye (50%glycerol, 1 mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol FF) on 1% formaldehyde-agarose gel and 1X MOPS was used as the running buffer. The gel was then run at 5 V /em length of the gel. The gel was then treated with 2.5% HCl for 15 min for depurination, 0.4N NaOH for another 15 min and then in 3 M sodium acetate for 15 min, before the transfer was set. Also the nylon membrane prior to transfer was first treated with distilled water for 5 min and then in 0.4 N NaOH for 20 min. The overnight transfer was set up using 20X sse buffer as the transfer buffer at room temperature. Thereafter, the membrane was cross-linked by uv and then dipped in 2X sse for 20 min. For pre-hybridization the membrane was soaked in Rapid hybridization buffer (Amersham Biosciences, U.K.) for 2 hr at 65oC in the hybridizing oven. The probe was then added and further incubation for 4 hrs was carried out. Post hybridization the membrane was washed thrice with 6X sse at 37oC on a shaker. The membrane was then dried on a filter paper and wrapped in a saran wrap. The membrane was then analyzed by autoradiography. For ensuring the equal loading, the formaldehyde-agarose gel was also stained with EtBr for 23s and 18s rRNA
    1. Cells growing in culture medium were harvested by trypsinization and washed twice with ice cold PBS. Cells were fixed by adding ice cold 70% ethanol and stored at 4°C. Before harvesting cells were washed twice with PBS and re-suspended in adequate amount of PBS containing Propidium Iodide (PI) to a final concentration of 50μg/ml and RNase to a final concentration of 10μg/ml. Thereby the cell suspension was incubated at 37°C for 30 minutes in dark. Analysis was done by running the samples in BD FACS Vantage System according to the standard procedures after calibration of instrument with Calibrite beads
    1. Chromatography is the technique of separation of compounds on the basis of their distribution/ partition between two phases. Thin Layer Chromatography (TLC) is a solid-liquid form of chromatography where the stationary phase is normally polar absorbent and the liquid phase is the mobile phase made up of a single or combination of solvents depending on the solutes to be separated. The sterol isolated by method described in 3.2.C.14, were also run on a Thin Layer Chromatogram. Standard ergosterol dilutions and samples from wild-type and half knock out parasites were spotted on a Silica Gel G plate. The sterols were resolved using a binary solvent [hexane/ ethyl acetate (75/25)]. The sterols were visualized using Mo' s stain (12.5g Ammonium molybdate (VI) tetrahydrate, 5.0g Ammonium cerium (IV) sulphate, 50mL concentrated sulphuric acid, water upto 500mL
    2. thoroughly by inverting the tube 4-6 times before keeping at RT for 5 min. 4mL of chilled Buffer P3 was added and mixed immediately and thoroughly by inverting the tube 4-6 times. A cartridge was capped and the entire contents were poured into it and allowed to settle for 10 min at RT. In the meantime, a Qiagen tip was equilibrated with 20mL of buffer QBT (750mM NaCl; SOmM MOPS, pH 7.0; 15%v /v isopropanol and 0.15% triton X-100). After the 10 min incubation, using a plunger, the contents of the cartridge were transferred into the equilibrated tip and allowed to drain by gravity. The tip was then washed with lOmL of Buffer QC (1M NaCl; SOmM MOPS, pH 7.0 and 15%v /v Isopropanol). The DNA was then eluted using SmL Buffer QF (125mM NaCl; SOmM Tris-Cl, pH 8.5 and 15%v /v Isopropanol) into a corex (glass) tube by gravity flow. 3.5mL of isopropanol was added to the eluted DNA and incubated at RT for 30 min. The DNA was then precipitated at 16000 x g at 4°C for 30 min. The supernatant was discarded and the pellet was washed in 2mL 70% ethanol at 16000 x g at 4°C for 10 min. The supernatant was gently decanted; the pellet was dried to remove any traces of alcohol. Then the DNA was resuspended in ~200]lL of Buffer EB (10mM Tris-Cl, pH 8.5) provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration = A260 X SOX dilution factor. Purity of DNA was monitored by looking at the A26o/ A2so ratio (should be above 1.6)
    3. Plasmid DNA was isolated in large scale using QIAprep Midiprep kit according to manufacturer's protocol. Briefly, 100mL for a high copy number plasmid and 200mL for a low copy number plasmid was cultured overnight followed by centrifugation at 4629 x g for 15 min at 4°C. The pellet was washed once with PBS and then resuspended well in 4mL Buffer P1 by vortexing. To this, 4mL of Buffer P2 was added an
    1. colonies come up after 48 hr. A freshly grown overnight culture of the TetR strain was washed once with an equal volume of citrate buffer and resuspended at 10- or 100-fold dilution in the same buffer. 0.1 ml aliquots were then spread on Maloy plates. Colonies were obtained at a frequency of ~ 4 x 10−5/plated cell. The colonies from the selection plate were purified on medium of the same composition and then scored for the Tets phenotype
    2. The method described by Maloy and Nunn (Maloy and Nunn, 1981) was followed for obtaining spontaneous TetS mutants of a TetR strain. Freshly grown cells of the TetR strain of O.D 0.7-0.8 was washed once with an equal volume of citrate buffer and resuspended at 10- or 100-fold dilution in the same buffer. 0.1 ml aliquots were then spread on Maloy agar plates and TetS colonies, which came up after 48 hr of incubation at 37 ̊C with a frequency of 5-8 big colonies/106-107 cells plated were purified on the same medium. This is not a clean selection since in a background lawn of slow growing TetR colonies, few faster growing TetS
    1. Vacuolar morphology of C. glabratacells was examinedby staining vacuoleswith FM4-64 (Molecular Probes, Invitrogen). FM4-64 is a lipophilic dye that exhibits long wavelength red fluorescence when boundto lipids. FM4-64 binds to the plasma membrane and follows the endocytic pathway to reach the vacuole(Vida and Emr, 1995).Log-phase,YPDmedium-grown cells were harvested and washed with 1X PBS. 1 ODcells were resuspendedin 50 μl YPDmedium containing 30 μM FM4-64 andincubated at 30 ̊C for 30-45 min. After incubation, cells were washed thricewith YPD mediumand resuspendedin 100 μl of the samemedium. Cells were observed under confocal laser scanning microscope(Zeiss LSM 510 Meta)with 63X objective lens,2.5X final zoom, pinhole set at 108 μm and emission filterset to LP 565nmto capture fluorescence image.Along with the fluorescenceimage, aphase contrastimage was alsocaptured for each sample
    1. E. coli BW23473 electro-competent cell aliquots were taken out from -70ºC freezer, thawed on ice and were mixed with 1-2 lplasmid DNA. Mixture was pulsed with the Gene Pulser® electroporation apparatus (Bio-Rad),set at 1800 Volts, 25 μF and 200 Ω,in a chilled 0.1 cm electroporation cuvette. After electric pulse, 1 ml LB medium was immediately added to the cuvette and suspension was transferred to a 1.5 ml sterile microcentrifuge tube. Cells were incubatedat 37°C and 200 rpm for 1 h, centrifuged and were plated on LB-agar plates containing kanamycin (30 μg/ml). Transformants were colony purifiedon LB-kanamycin plates. Positive clones were verified by colony PCR and inoculated in LB-liquid medium containing kanamycin (30 μg/ml) for plasmid isolation