191 Matching Annotations
  1. Last 7 days
    1. Das globale Durchschnittseinkommen wird bei der jetzt zu erwartenden globalen Erhitzung 2050 fast um ein Fünttel niedriger sein als ohne Erhitzung. Die (nicht mehr zu vermeidenden) Einbußen durch die Erhitzung bis 2050 sind sechsmal so hoch wie die einer Begrenzung des Temperaturanstiegs auf 2°. 2050 ist einer neuen Studie zufolge mit Klimaschäden von etwa 38 Bllionen Dollar zu rechnen. Bis 2100 wird es in einem Business-as-usual-Szenario zu Einkommensverlusten von mehr als 60% kommen. https://www.theguardian.com/environment/2024/apr/17/climate-crisis-average-world-incomes-to-drop-by-nearly-a-fifth-by-2050

  2. May 2024
    1. Der Artikel im Guardian stellt eine neue Studie dar, aus der hervorgeht, wie viel von der bereits existierenden Infrastruktur zur Förderung fossiler Brennstoff stillgelegt werden muss, um das 1,5° Ziel zu erreichen. Dabei geht die Autoren davon aus, dass man CO2 nicht realistisch wieder aus der Atmosphäre entfernen kann, und dass das 1,5° Ziel also nur zu erreichen ist, wenn nicht zu viel emittiert wird. Diese Studie fordert das Gegenteil der Planungen der fossilen Industrien, über der über die der Guardian gerade berichtet hatte. Der Artikel ist auch bemerkenswert, weil er auf eine Reihe weiterer wichtiger Studien zu fossilen Lagerstätten verweist.

  3. Apr 2024
    1. In Norditalien setzt sich die Dürre-Situation des vergangenen Jahres fort, weil - wie in Frankreich - im Winter lange kein Regen gefallen ist. Der Wasserstand des Po ist extrem niedrig. Maßnahmen zur Kontrolle des Wasserverbrauchs sind unter der Rechtsregierung noch schwieriger durchzusetzen als in der Regierungszeit Draghis.

  4. Mar 2024
  5. Feb 2024
    1. Robert Grosseteste, Bishop of Lincoln (face rubbed), in mitre and red cope, with crosier, seated on left speaks to a seated group of five people, mostly women. Tree on right; large bird with long beak at top.

      image of MS 522 f1r Lambeth Palace Library

      Folio 1 of MS 522 of Château d'amour

      Close up of inset image via link close up of image on folio 1r of Château d'amour

      Book and images mentioned in Chapter 2 of @Duncan2022 Index, A History of the

  6. Jan 2024
    1. Der grönländische Eisschild verliert aufgrund der globalen Erhitzung 30 Millionen Tonnen Eis pro Stunde und damit 20% mehr als bisher angenommen. Manche Forschende fürchten, dass damit das Risiko eines Kollaps des Amoc größer ist als bisher angenommen. Der Eisverlust ist außerdem relevant für die Berechnung des Energie-Ungleichgewichts der Erde durch Treibhausgas-Emissionen. https://www.theguardian.com/environment/2024/jan/17/greenland-losing-30m-tonnes-of-ice-an-hour-study-reveals

      Studie: https://www.nature.com/articles/s41586-023-06863-2.epdf?sharing_token=iqz0ns4_X6P1af3896jdntRgN0jAjWel9jnR3ZoTv0Pcew_aMz7qHMDjrF_9OLTexA24mQs8ERV-259eCQry-G1-OcR886jfHOICrWGcm8cGg2VLBlaWiYSzX6VygthHh72iiwkk1tHZcLD1G1oJIqdPha0A1oTMHLlfMAnTQrtd8PDFsj4xKAmTnOSL-6mrcbTbHbswhJaFji9IbAnyGW2pLAYwREeh-QWIL9xUFdsDBojJhNYWYoijtYUQx5YCyfzCJPGOEtlLO_PeIU9Tip8BaF24vqXfHcmad2_vz5eg0jcny8HHzO0uvDtSh_Bhym1eC8D25wZM6uZZ5vH9BA%3D%3D&tracking_referrer=www.theguardian.com

    1. Die Desinformation zur globalen Erhitzung hat sich von der Klimaleugnung hin zum Säen von Zweifeln an möglichen Lösungen verschoben. Einer neuer Studie zufolge sind wichtige Strategien auf Youdas Tube das Herunterspielen der negativen Konsequenzen, Erzeugen von Misstrauen in die Klimaforschung und vor allem die Behauptung, dass vorhandene technische Lösungen nicht praktikabel sind. Außerdem werden Verschwörungstheorien wie die vom Grand Reset bemüht. https://www.repubblica.it/green-and-blue/2024/01/17/news/negazionismo_climatico_youtube-421894897/

      Studie: https://counterhate.com/wp-content/uploads/2024/01/CCDH-The-New-Climate-Denial_FINAL.pdf

    1. Der deutsche Finanzminister Lindner plant Kürzungen bei ursprünglich für die Verkehrswende, vor allem für die Verbesserung der Schieneninfrastruktur vorgesehenen Geldern. NGOs und Verbände protestieren dagegen. Beim Straßenbau sind kaum Kürzungen geplant. https://taz.de/Sparplaene-fuer-den-Bundeshaushalt/!5986400/

    1. Der Ausbau der Windenergie in Deutschland hat sich unter der Ampelkoalition beschleunigt. Es werden aber im Augenblick nur etwa halt so viele Anlagen gebaut, wie es nach den Plänen der Bundesregierung nötig wäre. In den südlichen Bundesländern ist der Ausbau viel zu langsam. Genehmigungsverfahren z.B. für den Transport dauern zu lange. https://taz.de/Ausbau-der-Windenergie/!5983011/

    1. Die europäische Solar-Industrie ist in einer schweren Krise, weil chinesische Unternehmen mit wesentlich billigeren Modulen auf den europäischen Markt drängen. Die Subventionen der USA für die eigene Industrie verstärkt diesen Druck. Wenn die deutsche Regierung ihre Subventionspolitk nicht ändert, wird der Hersteller Meyer Burger sein deutsches waren schließen und nur noch in den USA produzieren. https://www.handelsblatt.com/unternehmen/energie/erneuerbare-energie-solarausruester-meyer-burger-droht-mit-schliessung-der-deutschen-modulproduktion/100007780.html

    1. Eine neue Studie kommt zu dem Ergebnis, dass die Haltung zu fünf großen Krisen das Wahlverhalten der Europäer:innen in diesem Jahr bestimmen wird: der Klimakrise, der Migrationskrise, der Wirtschaftskrise und Inflation, dem Ukraine-Krieg und Covid. Klimakrise und Migration hätten, wie schon bei den Wahlen in der Niederlanden, ide größte Kraft Wähler zu mobilisieren. Die Autor:innen sprechen von einem "Clash zweier 'Extinction rebellions'". Als wichtigste Krisen werden im Durchschnitt der europäischen Länder die Klimakrise und dann Covid bewertet.

      https://www.theguardian.com/world/2024/jan/17/crises-have-split-european-voters-into-five-tribes-survey-suggests

      Report: https://ecfr.eu/publication/a-crisis-of-ones-own-the-politics-of-trauma-in-europes-election-year/

  7. Dec 2023
    1. Le comité départemental de suivi de l'école inclusive encourage le développement des actions de formation croisée en matière d'école inclusive et de coopération. Il en dresse le bilan
  8. Nov 2023
    1. Auf den Öl- und Gasfeldern der Vereinigten Arabischen Emirate, darunter vielen, die der staatlichen Gesellschaft Adnoc gehören, wurde in den vergangenen 20 Jahren in großem Umfang routinemäßig Gas abgefackelt, was zu hohen Methanemissionen führt. Die Emirate hatten sich verpflichtet, das Abfackeln schnell zu reduzieren. Die dieser Selbstverpflichtung krass widersprechende Praxis gilt bei NGO als weiterer Beleg dafür, dass Selbstverpflichtungen der Fossilindustrie nicht getraut werden kann. https://www.theguardian.com/environment/2023/nov/17/cop28-host-uae-breaking-its-own-ban-on-routine-gas-flaring-data-showsactor

    1. key factor behind academic perseverance was students’ academic mindset — the attitudes and self-perceptions

      Academic Mindset = attitudes + self-perceptsion

    2. act gritty

      Interesting distinction - being gritty vs. acting gritty

    3. all students are more likely to demonstrate perseverance if the school or classroom context helps them develop positive mindsets and effective learning strategies.”

      How can we. help students develop positive mindsets and effective learning strategies?

    4. If they hear the message that a failure is a final verdict on their ability, they may well give up and pull back from school. But if instead they get the message that a failure is a temporary stumble, or even a valuable opportunity to learn and improve, then that setback is more likely to propel them to invest more of themselves in their education.

      Are these the only two types of messages a student can receive in response to their failure?

    5. She was particularly interested in what she called the “narrative” that exists within each school with regard to success and failure — the messages, subtle and not so subtle, that students receive when they fai

      What is the "narrative" at your SL school that a student receives when they fail?

    6. 17. Messages

      An examination of the messages teachers send to students - intentionally or not.

    7. institutional structures affect individual behavior and, specifically, how certain educational structures — like school funding mechanisms, teacher contracts, or patterns of segregation — might incline students toward success or failure.

      So Farrington was looking at the issue from a individual level (psych), as well as more societal/institutional level (sociology)

  9. Oct 2023
  10. Sep 2023
    1. Find Alarm now replaces Get All Alarms, retrieving all alarms or only those which match filter criteria

      Can't believe what I long called "by far and away Siri's most important function in my life" must now be intentionally configured and (non-verbally) triggered in the form of a Siri Shortcut.

      Thank you.

    2. Transcribe Audio generates text from an audio file

      This technically works, yes, but - as I noticed on the very first beta build - the actual output has an absurdly low character(?) limit, after which it simply cuts off.

    1. I think it is absolutely absurd that Apple still(?) chooses to publish "complete" ""feature lists"" in Portable Document Format, of all motherfucking things, but - as always - thank fucking God for mr Hypo Thesis over here!

    1. Passengers must have an iPhone with iOS 17 or later, but don’t need to have an Apple Music subscription.

      Well, now I've confirmed the most crucial answer I needed answering about iOS 17...

      ...now just to figure out how I'm going to make this happen.

  11. Jun 2023
    1. gaily

      happily or brightly 歡樂地,喜氣洋洋地;閃亮地,明亮地

    2. mottled

      covered with areas of different colours that do not form a regular pattern 雜色的;斑駁的

    3. elapsed

      If time elapses, it goes past.(時間)流逝,過去

    4. pierced

      to go into or through something, making a hole in it using a sharp point 刺穿,刺透,刺破

    5. slumbe

      sleep

  12. Mar 2023
    1. Great sorrow filled my heart on hearing this,because I knew of people of great worth,who in that Borderland suspended were.

      He still not understand that God is justice itself.

    2. against me this one seemed to be advancingwith head erect and with such raging hunger,that even the air seemed terrified thereby—and of a she-Wolf, which with every lustseemed in her leanness laden, and had caused 50many ere now to lead unhappy lives.

      This imply that lust is the most difficult one for humans to overcome.

    3. Art thou that Virgil, then, that fountain-headwhich poureth forth so broad a stream of speech?”

      Why does Dante choose Virgil?

    4. When half way through the journey of our lifeI found that I was in a gloomy wood,because the path which led aright was lost.

      This is also Dante reminding himself.

    5. I had his hair wrapped round my hand already,and more than one shock had I plucked from him,while he was barking, with his eyes turned down

      Now Dante fully accepted the fact that God is justice. Those people deserve to be punished like this.

  13. Jan 2023
    1. It is thusclear that the biblical author had access to historical information originatingin the 10th and 9th centuries BCE.

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    Annotators

  14. Aug 2022
    1. Benjy Renton. (2021, November 16). New data update: Drawing from 23 states reporting data, 5.3% of kids ages 5-11 in these states have received their first dose. Vermont leads these states so far in vaccination rates for this age group—17%. The CDC will begin to report data for this group late this week. Https://t.co/LMJXl6lo6Z [Tweet]. @bhrenton. https://twitter.com/bhrenton/status/1460638150322180098

  15. May 2022
  16. Nov 2021
  17. Aug 2021
  18. Jun 2021
  19. 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: 1

      AssayResultAssertion: Abnormal

      StandardErrorMean: 0

    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.1653T>A p.(Tyr551Ter)

    1. SUPPLEMENTARY DATA

      AssayResult: 100.7

      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.2896A>G p.(Ile966Val)

    1. Source Data

      AssayResult: 19.46

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardErrorMean: 1.75

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

    2. Source Data

      AssayResult: 7.03

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardDeviation: 2.68

      StandardErrorMean: 1.9

      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.2006delA p.(E669Gfs)

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

      AssayResult: 77.5

      AssayResultAssertion: Normal

      ReplicateCount: 30

      StandardErrorMean: 8.6

      Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (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.2291T>A p.(Met764Lys)

  20. Feb 2021
    1. Supplemental material

      AssayResult: 80, 99

      AssayResultAssertion: Normal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 2.6, 4.8

      AssayResultAssertion: Abnormal

      Comment: See Table S3 for details

    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.393_395del p.(Asn131del)

  21. Jun 2019
    1. The fluctuation maps (Fu) were calculated from the MD trajectories of the 0-chains as described earlier (Hery et al, 1997). The fluctuation value Fy is given by the equation, where dy(t) is the distance between a pair of designated atoms ( ca atoms as used here) at time t and the angle brackets represent time averages. The Fy values are the standard deviation of interatomic distance. The fluctuation maps in Figure 6 has a black dot wherever the Fu value is less than or equal to 0.5A. Thus dark regions of the map indicate those parts of the molecule which undergo strongly coupled movements.
    2. luctuation Maps
  22. May 2019
    1. Gametocyte rich parasite lysate was prepared using lysis buffer containing phosphatase inhibitors (20IlM sodium fluoride, 20llM ~-glycerophosphate, and IOOIlM sodium vanadate). For some experiments, 2mM calcium or 2 mM EGTA was added to the lysis buffer. IOOllg of lysate protein was incubated with PfCDPK4 anti-sera (1:100 ratio) for 12 h at 4°C on an end-to-end shaker. Subsequently, 50 III of protein A+G-Sepharose (Amersham Biosciences) was added to the antibody-protein complex and incubated on an end-to-end shaker for 2 h. The beads were washed with phosphate-buffer saline three times at 4°C and were resuspended in kinase assay buffer that contained phosphatase inhibitors.
    2. mmunoprecipitation of PfCDPK4 from parasite lysates
    1. nzyme-linked immunosorbent assay (ELISA) was performed to detect cytokine secretion from human THP-1 macrophages upon activation with LPS. The ELISAs were performed according to manufacturer's protocol. Briefly, THP-1 macrophages were subjected to various treatments and after appropriate time interval the cell culture supernatants were harvested. The capture antibodies for the individual cytokines were diluted 1:250 in coating buffer (0.1 M Sodium carbonate, pH 9.5) and 100 flL was ali quoted into each well of a 96 well ELISA plate (BD biosciences ). The plates were incubated at 4°C for 16 h following which three washes with 0.05% PBS-Tween-20 were given. Blocking was performed using 200 flL of assay diluent (PBS with 10% FCS) per well for 1 h at room temperature following which 1 00 flL of appropriately diluted standards and samples were added and incubated for 2 h at room temperature. A total of 5 washes with 0.05% PBS-Tween-20 were given and the plates were subsequently incubated with 100 flL of detection antibody and streptavidin-HRP for 1 h at room temperature following which 5 washes were given. 100 flL of tetramethylbenzidine (TMB) substrate was aliquoted into each well and incubated for 15 min at room temperature in dark following which 50 flL of stop solution (2N H2S04) was added to terminate the reaction. The absorbance was read at 450 nm and the cytokine levels in the samples were derived based on the OD45o values obtained with standards of known concentration
    2. Cytokine ELISA
    1. with appropriate antibiotics. Serial dilutions of the toxin, made in 0.2% HSA were added to the cells and incubated for indicated time periods. After incubation, the adherent cells were labeled with 0.25 J!Ci eH] leucine per well and the suspension cells with 0.75 J!Ci eH] leucine per well for 3 hat 37 °C. The cells were harvested on to filtermats using an automatic cell harvester, and the incorporation of the eH] leucine in the newly synthesized proteins was estimated using LKB ~-plate counter. Activity was expressed as percentage of control where no toxin was added to the cells. To check the specificity of chimeric toxins for TFR, 10 J.Lg of anti-TFR antibody (HB21) was added to each well prior to the addition of the fusion protein in the competition experiments.
    2. Cytotoxic activity of restrictocin, its mutants and chimeric toxins was checked on a variety of cell lines. The cellular protein synthesis was assayed in the absence and presence of various concentrations of toxin by measuring eH] leucine incorporation in the newly synthesized proteins. Adherent cell lines namely A431, A549, HeLa, L929 and MCF7 were plated in RPMI 1640 containing 10% FCS at a density of 5x I a:~ cells per well in 96 well culture plates and allowed to adhere for 12 h at 37 °C in the presence of 5% C02• Next day, the medium was replaced with 200 J.LI of leucine free RPMI medium containing 10% serum. The leucine free RPMI containing 1 0% FCS was used for seeding partially adherent cell lines, COL0205 and J774A.1. Cells were allowed to adhere for 12 h and the toxin was added in the same medium. The suspension cell lines, HUT102 and K562 were plated at a density of 5X 103 _cells/well in 80% leucine free RPMI containing 18% complete RPMI and 2% serum immediately before use. The medium used at various stages was supplemented
    3. Assay of Cytotoxicity of Chimeric Toxins
    1. conjugated to fluorescene isothiocyanate ( FITC ) or horse radish peroxidase HRP ) , added. In case of the FITC staining, the cells were finally mounted in a medium containing 0. 1 % para-phenylenediamine PPD and 90 % glycerol in NKH buffer, to retard fading of the fluorescent label. In case of HRP staining, the colour was developed with 0. 05 % DAB ( 3', 3-diaminobenzidine tetrahydrochloride ) in NKH buffer with 0. 002 % hydrogen peroxide. The colour was developed for 15 mins. and the reaction stopped by rinsing in phosphate buffer. The cells were examined under a standard 1 ight microscope for HRP staining or a fluorescent microscope ( for FITC staining ) .
    2. Cells were grown to subconfluence on polylysine coated glass coverslips contained in plastic dishes. After washing with NKH buffer ( 145 mM NaCl, 5 mM KCl, 15 mM Hepes, pH 7. 4 ) , the cells were fixed for 10 mins. in 70 % ethanol at room temperature. The cells were again rinsed extensively in NKH buffer and the appropriate dilution of primary antibody added. Following OIN incubation at 4°c, the cells were washed 3 X with NKH buffer and the appropriate second antibody,
    3. Immunocytochemistry.
    1. phosphoricacidformedisreducedbytheadditionof1-amino2napthol~4-sulphonicacid(ANSA)reagenttoproducethebluecolor.Theactivityofthebluecolorwasreadat680nmagainstreagentblankusingaU.V.Spectrophotometer.Suitablestandardswererunthrougheachbatchofassays.Theenzymeactivitywasexpressedintermsofpgofinorganicphosphorusformedhr'1mg'1protein.
    2. Aftereffluentexposure,thecontrolandeffluentexposedfishtissueswereremovedandplacedinabeakercontainingice-coldSEIbuffer(300mMsucrose,200mMNa2EDTA,50mMimidazole,pH7.23)foranalysisofNa+-K+ATPaseactivity.ThetissueswereimmediatelyfrozeninliquidN2andstoredat-80°Cuntilanalyzed. Thespecificactivitiesofsodium,potassium,magnesiumandcalciumdependentATPaseswereassayedaccordingtothemethodsdescribedbyWatsonandBeamish(1980)and Boeseetal.(1982).AdenosinetriphosphatasecatalysestheconversionofATPandADP.Duringthisconversion,onemolecule ofphosphorusisliberated.ATPaseAdenosinetriphosphate^...^Adenosinediphosphate+PTheinorganicphosphorusliberatedwasassayedaccordingtothemethodofFiskeandSubbarow(1925).Inthismethodtheproteinisprecipitatedwithtrichloroaceticacid.Theproteinfreefiltrateistreatedwithaceticacidmolybdatesolutionandthe
    3. Na+K+ATPase,Mg2+ATPaseandCa2+ATPase
    4. Thereactionproduction,p-nitrophenolinacidphosphatewasmeasuredspectrophotometricallyat415nmagainstreagentblank.Theenzymeactivitywascalculatedfromthestandardcurveandexpressedasmicromolesofp-nitrophenolformedperhourpermilligramprotein.Therateofhydrolysisofp-nitrophenolphophateisproportionaltotheenzymepresentinthetissue.p-nitrophenylphosphate+NaoH—phosphat?-—>p -nitrophenol+phosphateThecolordevelopedinalkalinephosphataseactivitywasreadat410nmagainstreagentblankspectrophotometrically.Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein
    5. ThealkalinephosphataseactivitywasestimatedbythemethodofMorton(1955)usingp-nitrophenylphosphatesocolorlessinsolutionbutuponhydrolysis,thephosphategroupliberatesp-nitrophenylwhichishighlycoloredinalkalinesolution
    6. Alkalinephosphatase(AKP:ortho-phosphoricmonoester-phosphohydrolase;E.C.3.1.3.1)
    7. AcidphosphatasewasassayedfollowingtheprocedureofMorton(1955).Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein.
    8. Acidphosphatase(ACP:acidorthophosphoricphosphohydrolase)(EC3.1.3.2)
    9. LDHisthekeyenzymeinvolvedinglycolysis,andisresponsiblefortheanaerobicconversionofpyruvicacidtolacticacid,theterminalstageintheEmbden-Meyerhofpathway.Theenzymeactivitywasdeterminedinthecontrolandeffluentexposedfishbrain,gill,muscle,liver,heart,kidneyandair-breathingorgansfollowingSrikantanandKrishnamurthi(1955).TheopticaldensitiesweremeasuredinaUVSpectrophotometerusing340 nmfilterandtheresultsare expressedaspmolesofformazanmg'1proteinhr’1
    10. Lactatedehydrogenase(LDH)(L-LactateNADoxidoreductase)(EC1.1.1.27)
    11. Thesupernatant(0.5mlcontaining50mgtissue)wasassayedforSDH.Thereactionwasinitiatedbytheadditionof0.5mlofthesupernatant.Controlsreceived0.5mlsucroseinplaceoftheenzymeextract.Afterincubationfor30minat37°C,thereactionwasstoppedbytheadditionof5mlglacialaceticacidandthederivedformazanwasextractedinto5mloftoluene.Afterkeepingitovernightincold,thecolorwasmeasuredinUV-Spectrophotometerat495mMusingsilicacuvettes.Enzymeactivitieswereexpressedaspmolesofformazanmg'1proteinhr'1
    12. Thisisanimportantenzymeinvolvedinthecitricacidcycle.Thehomogenatesofcontrolandeffluentexposedtissueswerepreparedin0.25MicecoldsucroseusingPotterElvehjemtypeglasshomogenizerandcentrifugedat3000rpmfor15min
    13. Succinicdehydrogenase(SDH)(E.C.1.3.99.1
    14. Theacetylcholineconcentrationinthetissueswasestimatedspectrophotometricallyat540nmbythemethodofHestrin(1949)usingformicacid-acetonemixture(0.15mformicacidacetone,3:17V/V)astheextractionmediumAchconcentrationwascalculatedintermsofnmolAch/mgtissue
    15. Acetylcholine(Ach)
    16. Aftereffluentexposure,thetissuesweredissectedout,weighedandhomogenizedin0.25Msucrose.Thehomogenateswerecentrifugedat10,000rev/minatatemperaturebelow8°C.AcetylcholinesteraseactivityofthesampleswasdeterminedatpH7.0usingafinalhomogenateconcentrationof25mgmf1at10°CwithmMacetylcholineiodideassubstrate and0.001or0.002NsodiumhydroxideastitrantfollowingHestron’smethodasgivenbyMetcalf(1951).ProteindeterminationsforalltheChEanalyseswereconductedonaliquotsofthehomogenatesusingamodificationoftheLowryetal.(1951)method.AchEactivityisexpressedinpmolesofacetylcholinechloridehydrolysedmgtissue'1hr'1
    17. Acetylcholinesterase(AchE
    18. 0.89%salinesolutioninaTejElon-glasshomogenizerat4°C.Thehomogenatewascentrifugedat4000rpm(3500xg)at4°Cfor20minutes.Theclearsupernatant(organextract)wasusedforestimationofenzymes
    19. Aftereffluentexposure,thecontrolandexperimentalfisheswerekilledbyhammeringonheadanddissectedimmediately.Excisedbrain,gill,muscle,liver,heart,kidneyandair-breathingorganswereweighed(about20mg)andhomogenizedin2mlof
    20. Collectionoftissues
    21. Enzymologicalanalyses
    1. The lacZ U118 is an amber nonsense mutation(Am) that confers Lac─phenotype and also polarity of the downstream lacYA genes in the operon due to premature Rho-dependent transcription termination within the untranslated region of lacZ. Melibiose is a sugar which can only be utilized in a lacZ (Am) strain at high temperature (39 ̊C, when the native melibiose permease is inactive) if the downstream gene lacY encoded permease is transcribed and translated. Therefore, in lacZ (Am) strains, growth on minimal melibiose plates (0.2%) at 39°C reflects transcriptional polarity relief at the lac locus, and the same was scored after streaking the relevant strains on such medium
    2. lacZ (Am) assay
    3. dependent transcription termination within the untranslated region of trpE. Anthranilate is a precursor of tryptophan, which is the product of trpE-encoded anthranilate synthase. Therefore, in trpE(fs) strains, growth on minimal glucose plates supplemented with anthranilate (100 μg/ml) reflects transcriptional polarity relief at the trp locus, and the same was scored after streaking the relevant strains on such medium
    4. The trpE9777 is a frameshift (fs) mutationconfers Trp auxotrophy and also polarity on the downstream trpDCBA genes in the operon due to premature Rho-
    5. trpE(fs) assay
    6. The galEp3 (galE490∗)mutation represents a 1.3 kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to rho dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3mutant or its derivatives was monitored by one of two means. In the first, MacConkey galactose indicator plates (with 1% galactose) were used, where Gal+ colonies are red, and Gal− colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression. In the second method, growth of strains on minimal-galactose (0.2%) was used as a test for Gal+ phenotype
    7. galEp3 assay
    8. In vivo transcription termination assays
    1. taining withER Tracker™ Blue White DPX: ER Tracker™ is a dapoxyl dye that specifically stains ER in live cells. Since the dye was not found to retain after fixation, live cell staining was performed when fixation was required before staining with antibodies against the CYP proteins. Once washed, the cells were blocked with 3% NGS prepared in 0.001% Digitonin for 30 min at 4°C. This was followed by washing cells with chilled PBS at centrifugation at 805 x g for 10 min at 4°C. Then the cells were incubated with the appropriate primary antibody prepared in 0.001% digitonin for 1h at 4°C. The unbound primary antibody was washed off with chilled PBS three times by centrifugation at 805 x g for 10 min at 4°C. Cells were then incubated with appropriate secondary antibody again prepared in 0.001 % digitonin for 1h on ice. This was followed by three washes with chilled PBS as before. ER Tracker Blue (1p.M) then added to cells which were incubated on ice for 30mins on ice. The excess dye was washed and the cells viewed under the microscope after mounting on slides with anti-fading mounting media
    2. The protocols for immunostaining cells with different antibodies and dyes were standardized individually for the respective staining procedure: Staining with MitoTracker® Red: MitoTracker® probes diffuse passively across the plasma membrane and accumulate into the active mitochondria. 100]1M stock solutions of MitoTracker Red CMX Ros were prepared in DMSO. Cells were stained at a final concentration of 100nM for 10 min at 37°C, at RT in the dark. The excess unbound dye was washed off and the cells were ready to be viewed under the microscope or processed further for fixation and antibody staining. Staining with antibodies (Table 3.9): Log phase cells were centrifuged at 129 x g for 5 min at RT to remove all dead cells. The live cells were washed 2X with PBS to remove any adherent media and FBS. Fixation was carried out with 2% formaldehyde at RT for 15 min. The fixed cells were washed 2X with PBS by centrifugation at 805 x g at RT for 5 min. They were then blocked using 3% Normal Goat Serum (NGS) prepared in 0.01% Saponin for 30 min at RT. After a wash with PBS, the cells were incubated with appropriate dilution of primary antibody prepared in 0.01% saponin for 1h at RT. The unbound antibody was thoroughly washed off by at least three washes with PBS. Incubation with respective fluorophore conjugated secondary antibody, again diluted in 0.01% saponin, and was carried out for 45 min at RT. The unbound antibody was thoroughly washed off by at least three washes with PBS. The cells were resuspended in a small volume of PBS and mounted on a slide along with anti-fading mounting media (10% glycerol and 0.001% P-phenylenediamine)
    3. Immunocytochemistry
    4. between the 5' and 3' flanking regions using the sites Smai and BamHI to generate pBSK+CYP710C1Hyg
    5. The vector pBlueScript SK+ was used as the backbone to generate the replacement constructs. Standard cloning techniques were used (Sambrook et al., 1989). CYP5122A1 allelic replacement constructs were prepared by inserting ORFs encoding Neomycin or Hygromycin resistance between 0.4 Kb 5' and 0.37 Kb 3' CYP5122A1 flanking regions cloned into the vector pBlueScript SK+, to generate the constructs pBSK+CYP5122A1Neo and pBSK+CYP5122A1Hyg respectively. The following steps were performed. (i) A 416bp 5' flanking sequence of CYP5122A1 was amplified by Hi-fidelity PCR (Table 3.6) and cloned in between unique EcoRV and EcoRI sites of pBSK+ vector. (ii) A 378bp 3' flanking region of CYP5122A1 was amplified using Hi-fidelity PCR and cloned in between BamHI and Xbal sites in pBSK+ with the 5'fragment already cloned in. (iii) ORF for Neomycin resistance was amplified from the vector pXG-GFP+2 and cloned in between the 5' and 3' flanking regions already cloned into pBSK+ vector using the EcoRI and BamHI sites, to generate the construct pBSK+CYP5122A1Neo. (iv) To generate the second replacement construct, hygromycin ORF was amplified from the vector pXG-Hyg (Kindly provided by Dr. Stephen M. Beverley, Washington University) and cloned in between the 5' and 3' flanking regions using the sites Smal and BamHI to generate pBSK+CYP5122A1Hyg. Similarly CYP710C1 allelic replacement constructs were prepared by inserting ORFs encoding Neomycin or Hygromycin resistance between 0.36 Kb 5' and 0.37 Kb 3' CYP710C1 flanking regions cloned into the vector pBlueScript SK+, to generate the constructs pBSK+CYP710C1Neo and pBSK+CYP710C1Hyg respectively. The following steps were performed. (i) A 364 bp 5' flanking sequence of CYP710C1 was amplified by Hi-fidelity PCR (Table 3.7) and cloned in between unique EcoRV and EcoRI sites of pBlueScript SK+ vector. (ii) A 378bp 3 'flanking region of CYP710C1 was amplified using Hi-fidelity PCR and cloned in between BamHI and Xbal sites in pBlueScript SK+ with the 5'fragment already cloned in. (iii) ORF for Neomycin resistance was amplified from the vector pXG-GFP+2 and cloned in between the 5' and 3'flanking region cloned into pBlueScript SK+ vector using the EcoRI and BamHI sites, to generate the construct pBSK+CYP710C1Neo. (iv) To generate the second replacement construct, hygromycin ORF was amplified from the vector pXG-Hyg and cloned in
    6. Generation of allelic replacement constructs for generation of knock outs
    1. A calibration curve of fluorescence intensity values versuspH was prepared for BCECF-AM-loaded wt cells by incubatingcellsin YPD medium containing 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM NaN3, 10 mM 2-deoxyglucoseand5 μM carbonyl cyanide m-chlorophenylhydrazone, titrated to five different pH values in the range of 4.0-8.0. Fluorescence intensity values were measured by excitation at 440and 490 nm with emission at 535 nm and a graph was plotted between the ratio of intensity at 490 to 440 nm versuspH. Similar to pHi calibration curve, a polynomial distribution of fluorescent intensity signal and pH was observedfor BCECF-AMprobe
    2. In vivovacuolar pH calibration curve
    1. CgRTT107(3.3 kb),CgRTT109(1.3 kb),CgVPS15(3.4kb) and CgVPS34(2.4kb) ORFs were PCR amplified from genomic DNA of the wild-type strain using high fidelity Platinum Pfx DNA polymerase with primers carrying restriction sites for SmaI-SalI,BamHI-SalI,XmaI-XhoIandSalI-XmaI,respectively.Amplified fragments werecloneddownstream of the PGK1promoterin the pGRB2.2 plasmid. Clones were verified by bacterial colony PCR, sequencing and complementation analysis
    2. Cloningof C. glabrataORFs
    1. Table 2.4: List of double-stranded oligonucleotides used in the present study
    2. The DNA-protein complexes were then separated from free oligonucleotides on 6.6% native PAGE gel. The samples were loaded into a native PAGE gel, which was pre-run at constant current (40-50 mA) for 15-30 minutes. Electrophoresis was performed at constant current (80-100 mA), till the bromophenol blue dye front reached 1-2 cm from bottom of the gel. The glass plate was carefully removed without disturbing the gel and the Whatmann filter paper no. 3, cut to the size of the gel, and wasplaced over it. The paper was pressed gently and the gel, which was now firmly stuck on the paper, was covered with saran wrap and kept for vacuum drying on the gel-dryer at 80oC for 1 h. After drying, gel was exposed on a Phospho-imager screen for 12-24 h and scanned on Phospho-imager to detect the band of interest.To determine the specificity of the transcription factor binding or sub-unit interacting to the desired oligonucleotide, super-shift assay was performed. For this, 8-10 μg of nuclear extracts were first incubated with desired antibodies (concentration varies for different) or their isotype control for 1h at 25°C, followed by incubation with binding reaction mixture. The various oligonucleotide sequences used in the present study are listed in Table2.4below