266 Matching Annotations
  1. Mar 2022
    1. Ce programme nommé « I can word it too », disponible en hébreu et arabe, a été spécialement créé pour cette étude. Il reproduit les activités quotidiennes (jouer à des jeux, prendre les repas, faire sa toilette…) et demande à l’enfant ce à quoi il veut jouer, en lui présentant un choix de jeux sur l’écran

      ==>il s’agirait d’une déclinaison sur écran des outils et méthodes de communication améliorée et alternative (CAA), comme le PECS ou le Makaton. déjà existants, IDEOPICTO ou le langage conceptuel SACCADE

    2. L’imitation et l’influence du jeu interactif sont bien mises en évidence dans une étude de Orit Hetzroni et Juman Tannous, de la Faculté des Sciences de l’éducation de l’Université de Haifa (Israël)

      ==>l’échantillon de l’étude est est extrêmement limité, l’étude n’est pas répliqué et elle ne permet pas de retirer de résultats concluants

    1. in which we retaliated for an attack on our soil

      Retaliation should have been directed against wahabis in S>Arabia - occupying a whole nation to hunt down a person is a sentimental but flimsy pretext.

  2. Feb 2022
  3. Jan 2022
    1. הנאורות הוגדרה בדרכים רבות ושונות

      הגדרות שונות של..... לממ"ן 11

  4. Dec 2021
  5. Nov 2021
    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

    1. RRID:ZFIN_ZDB-GENO-080326-11

      DOI: 10.1016/j.xpro.2021.100947

      Resource: (ZFIN Cat# ZDB-GENO-080326-11,RRID:ZFIN_ZDB-GENO-080326-11)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-080326-11


      What is this?

    1. the program most likely violates the establishment clause.

      The program most likely DOES NOT violate the establishment clause.

    1. the program most likely violates the establishment clause.

      Incorrect - the textbooks states reasoning as "the program is not readily subject to challenge under the establishment clause"

  6. Sep 2021
    1. Happiness

      Rejoice, O young man, in your youth, and let your heart cheer you in the days of your youth. Walk in the ways of your heart and the sight of your eyes. But know that for all these things God will bring you into judgment.

    1. If you are running Windows 11 and want to enter Safe Mode to troubleshoot some issues with Windows 11. Then here is how to boot Windows 11 in Safe Mode. First, go to the 'System interface,' click 'Recovery.' Now, some recovery options will be seen. Click the ‘Restart Now' button. Then, click Restart now again, and Windows will enter the recovery environment. Now in Restart, press F4, F5, or F6 based on your need to enter Safe Mode.

    2. If you want to make Windows 11 bootable USB drive first, download Rufus and open it up. Now, select the Windows 11 ISO file. If everything is perfect, you will see the status as ready. Now, click 'Start' to begin the formatting and the writing of the Windows 11 ISO file onto your USB drive. This should take a few minutes, depending on your USB drive and the port to which it is connected. And that's it.

  7. Jul 2021
    1. ZDB-ALT-170522-11

      DOI: 10.1016/j.neuron.2017.06.001

      Resource: (ZFIN Cat# ZDB-ALT-170522-11,RRID:ZFIN_ZDB-ALT-170522-11)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-170522-11

      Curator comments: Ct842 Danio Rerio ZFIN Cat# ZDB-ALT-170522-11


      What is this?

    2. ZDB-ALT-170522-11

      DOI: 10.1016/j.neuron.2017.06.001

      Resource: (ZFIN Cat# ZDB-ALT-170522-11,RRID:ZFIN_ZDB-ALT-170522-11)

      Curator: @Zeljana_Babic

      SciCrunch record: RRID:ZFIN_ZDB-ALT-170522-11


      What is this?

    1. RRID:ZFIN_ZBD-GENO-100809-11

      DOI: 10.7554/eLife.16415

      Resource: (ZFIN Cat# ZDB-GENO-100809-11,RRID:ZFIN_ZDB-GENO-100809-11)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-GENO-100809-11

      Curator comments: Trps1j1271aGt Danio Rerio ZFIN Cat# ZDB-GENO-100809-11


      What is this?

  8. Jun 2021
    1. The European Union has strong programs to invest in scientific research. Researchers Abraham García and Pierre Mohnen demonstrate that firms who received support from the Austrian government actually increased their research intensity and had more sales. Governments can support scientific research and technical training that helps to create and spread new technologies. Governments can also provide a legal environment that protects the ability of inventors to profit from their inventions.

      From OS

    2. Scientific Research

      This would be an excellent place to mention the US funding of COVID vaccine research--maybe a link to learning after this card?

  9. May 2021
  10. Apr 2021
  11. Mar 2021
    1. Muse

      Reminded of Chapter 11 in The Odyssey:

      I am likely going to retire this year and I find resonance in this as it appears that I will be accepting a "voluntary" buyout at the end of this fiscal year. My long sea journey, 25 years worth in teaching, will be officially over. Hence...the appeal to propitiate the gods, to let all the pain go, to ask forgiveness of the implacable Poseidon.

    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: 4.9

      AssayResultAssertion: Normal

      StandardErrorMean: 0.27

    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.1222T>C p.(Tyr408His)

    1. SUPPLEMENTARY DATA

      AssayResult: 95

      AssayResultAssertion: Normal

      PValue: Not reported

      Approximation: Exact assay result value not reported; value estimated from Figure 6C.

    2. SUPPLEMENTARY DATA

      AssayResult: -16

      AssayResultAssertion: Normal

      PValue: Not reported

    3. SUPPLEMENTARY DATA

      AssayResult: 102.1

      AssayResultAssertion: Normal

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    4. 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.2794G>A p.(Val932Met)

    1. Source Data

      AssayResult: 120.54

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 11.09

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

    2. Source Data

      AssayResult: 75.45

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 4.03

      StandardErrorMean: 2.85

      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.1544A>G p.(K515R)

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

      AssayResult: 101

      AssayResultAssertion: Normal

      ReplicateCount: 41

      StandardErrorMean: 8.9

      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.2141T>C p.(Val714Ala)

  12. Feb 2021
    1. He was so deeply offended and concerned by the notion that somehow it was America’s fault that a group of radical, violent Islamist terrorists killed nearly 3,000 Americans that it opened his eyes to the indoctrination that was happening in his classes.

      This seems to be true revisionist history. No one I've come across took a "blame America first" approach. Gingrich and Miller would be incredibly hard pressed to come up with contemporaneous statements that back up this proposition.

    1. Supplemental material

      AssayResult: 86

      AssayResultAssertion: Normal

      Comment: See Table S2 for details

    2. Supplemental material

      AssayResult: 5.4

      AssayResultAssertion: Abnormal

      Comment: See Table S2 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.31G>C p.(Glu11Gln)

    1. RRID:ZFIN_ZDB-ALT-190814-11

      DOI: 10.7554/eLife.53403

      Resource: (ZFIN Cat# ZDB-ALT-190814-11,RRID:ZFIN_ZDB-ALT-190814-11)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-190814-11

      Curator comments: allele name: re03 Danio rerio ZFIN Cat# ZDB-ALT-190814-11


      What is this?

  13. Jan 2021
    1. RRID:ZFIN_ZBD-GENO-100809-11

      DOI: 10.1242/dev.193409

      Resource: (ZFIN Cat# ZDB-GENO-100809-11,RRID:ZFIN_ZDB-GENO-100809-11)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-GENO-100809-11

      Curator comments: trps1j1271aGt Danio rerio ZFIN Cat# ZDB-GENO-100809-11


      What is this?

  14. Nov 2020
    1. Tg(actb2:loxP-DsRed-loxP-dnstat3 EGFP)s928

      DOI: 10.1016/j.celrep.2020.108404

      Resource: (ZFIN Cat# ZDB-ALT-110210-11,RRID:ZFIN_ZDB-ALT-110210-11)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110210-11

      Curator comments: allele name: s928Tg Danio rerio ZFIN Cat# ZDB-ALT-110210-11


      What is this?

    1. The truce is over. The failure of the party to operate an online strategy “in a real way that exhibits competence”, Ocasio-Cortez told the Times, made it hypocritical for the party to advance criticism of progressive messaging.

      Sie sagt auch, dass Obama seinen ganzen Kampagnen-Apparat neben der demokratischen Partei aufgebaut hat. Die Argumentation ist: Biden hat vor allem durch Mobilisierung unten, besonders bei schwarzen Wählern gewonnen. Die Basis muss aber spüren, dass sich für sie etwas verändert, damit die Mobilisierung gelingt.

  15. Oct 2020
    1. In Figure11, we have reported the variation of bandgapenergy as a function of diameters of deposited ZnS-NPs. Itis noticed the increment of the energy gap with decreasinggrain size; this is due to the quantum size effect whereas theenergy levels are confined to potential wells of small dimen-sion. The distance between energy levels increases as thecrystal size becomes smaller [27].

      En la figura 11, hemos reportado la variación de la energía del band gap como una función de los diámetros de deposición del ZnS-NPS. Se puede notar el incremento de energía band gap con la decreció del tamaño de grano; esto es debido al efecto de tamaño cuántico mientras que los niveles de energía son confinados a pozos potenciales de pequeña dimensión. La distancia entre los niveles de energía incrementan mientras el tamaño del cristal se vuelve más pequeño [22].

    Tags

    Annotators

    1. See the sun gods, gods of light,howling storm gods, twin gods of dawn,and gods of wind, Arjuna,wondrous forms not seen before

      In this part of statement, it shows that Indian people believe in different gods.

    2. your form is no different, you are just like me,

      Gilgamesh did not mean that He looks as Uta-napishti as appearance, he meant that you are same as me you have the same power as me. it means that i can be immortal as well.

    3. His hopes are destroyed

      Gilgamesh always have the dream to be powerful than every one in the Uruk city. and it leads him to challenge his soul to become immortal and live forever but he could not and finally he fails.

  16. Sep 2020
    1. Let me disclose, 0 Gilgamesh, a matter most secret, to you 1 will tell a mystery of gods.

      Why Ut - napishti wants to give him the secret?

    2. Gilgamesh go without sleep for a week.

      Why Uta - napishti suggest him to sleep for a week?

  17. Aug 2020
  18. Jul 2020
  19. Nov 2019
    1. Überwachung

      Abseits vom Kapital, welches aus solcher Überwachung geschlagen wird, erscheint mir die Überwachung an sich als ebenso interessant. Beispielsweise die staatliche Überwachung Aller aus Gründen der "inneren Sicherheit". Diese wir am Beispiel der Folgen von 9/11 und der Snowden-Affäre von eben diesem in der 1368. Folge des Podcasts "The Joe Rogan Experience" im Detail aufgeschlüsselt.

    1. Überwachungs

      Abseits vom Kapital, was aus solcher Überwachung geschlagen wird, erscheint mir die Überwachung an sich als ebenso interessant. Beispielsweise die staatliche Überwachung Aller aus Gründen der "inneren Sicherheit". Diese wird am Beispiel der Folgen von 9/11 und der Snowden-Affäre von eben diesem in der 1368. Folge des Podcasts The Joe Rogan Experience im Detail aufgeschlüsselt.

  20. Jul 2019
    1. Sequence analysis
    2. DNA sequencing of the 18S rDNA fragment
    3. Purification of PCR product
    4. Analysis of internal transcribed spacer region
    5. RAPDand SSRscoring and data analysis
    6. PCR amplification
    7. Running of gel and visualization of DNA
    8. Determination of the yield
    9. Agarose gel electrophoresis
    10. Qualitative and quantitative estimation of DNA
    11. Determination of the yield
    12. Procedure for DNA isolation
    13. Reagents required for fungal DNA isolationand p
    14. Effect of temperature on xylanase activity
  21. Jun 2019
    1. Flow cytometric analysis of cytokine production (IFN-γ)by intracellular staining
    1. acid. The respective buffer baselines were subtracted from the sample CD data. The ellipticity of the protein samples is reported as mean residue ellipticity (MRE) in deg/cm2/dmol units. The first derivative UV spectra of the oxy and deoxy-HbS were recorded on a Lambda Bio20 spectrophotometer (Perkin Elmer Life ScieAces). The hemoglobin concentration used for the spectral measurements was approximately 50 )!M on heme basis.The spectra of unliganded proteins was recorded subsequent to deoxygenating the hemoglobin samples by passing moist gaseous nitrogen extensively over the sample in an airtight cuvette. Completion of deoxygenation was ascertained by recording the visible spectrum of the deoxygenated Hb sample.
    2. Circular dichroism (CD) spectra were recorded on a J71 0 Spectropolarimeter (Jasco, Japan) fitted with a Peltier type constant temperature cell holder (PTC-348W). The calibration of the equipment was done with (+)-! 0-camphorsulfonic
    3. Spectroscopic studies
  22. May 2019
    1. containing 50 mM Tris, pH 7.5, 10 mM magnesium chloride, 1 mM dithiothreitol, and 100 11M p_32p] ATP (6000 Cilmmol) using 6 Ilg of Myelin Basic Protein. Kinase assays were also performed using "syntide-2" a small peptide substrate (PLARTLSV AGLPGKK) custom synthesized by Peptron, South Korea, and has been used as a substrate for plant CDPKs and CaMKs (Harmon et al., 1994; Hashimoto and Soderling, 1987; Yoo and Harmon, 1996). Reactions were performed in the presence of 2 mM calcium chloride or 2 mM EGTA (0 mM Ca2+) for 40 min at 30°C. When MBP was used as the substrate, reactions were stopped by boiling the assay mix for 5 min in Lammeli's buffer followed by SDS-PAGE. Phosphate incorporation was adjudged by autoradiography of SDS-PAGE gels. When Syntide-2 was used as substrate, reactions were stopped by spotting the reaction mix on P81 phosphocellulose paper (Millipore). The paper strips were air dried followed by washing with 75 mM ortho-phosphoric acid. Phosphate incorporation was assessed by scintillation counting of the P81 paper. In PfCDPK4 inhibition assays, peptide inhibitors were preincubated with proteins in a kinase assay buffer at 25°C for 30-60 min prior to the addition of substrate and ATP
    2. The catalytic activity of recombinant PfCDPK4 (and its mutants), as well as irnrnunoprecipitated PfCDPK4 from parasite lysate, was assayed in a buffer
    3. ssay of Protein Kinase Activity
    1. esterification of the dye. Basal fluorescence was measured in a fluorimeter (BMG Fluostar Optima spectrofluorimeter) at an excitation of 480 nm and an emission of 520 nm. Appropriate treatments were initiated and kinetic fluorescence measurements were performed with the temperature being maintained at 3 7°C. At the end of each experiment, a calibration was performed to convert the fluorescence values into absolute calcium concentration using the following formula: where, Kt is the dissociation constant of Ca2+ -Fluo3-AM complex (325 nM), and F represents the fluorescence intensity of cells, Fmax represents the maximum fluorescence (obtained by treating cells with 1 f.!M Ca2+ ionophore A234187 in the presence of 4 mM CaCh), and Fmin corresponds to the minimum fluorescence (obtained by treating cells with 4 mM EGTA)
    2. Cytosolic free Ca2+ was measured using the fluorescent Ca2+ indicator Fluo3-AM. THP-1 macrophages were harvested and resuspended in Kreb's buffer (118 mM NaCl, 25 mM NaHC03, 4.8 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 11 mM glucose, 1.5 mM CaCh.2H20). Fluo3-AM was added at a final concentration of 0.5 J.LM alongwith 1 J.LM Pluronic acid F-127 to aid in dispersal of the dye. The cells were subjected to constant mixing by end-to-end rotation and incubated with the dye for 20 min at room temperature following which the cells were pelleted and resuspended in fresh Kreb' s buffer and incubated for further 15 min to allow complete de-
    3. Intracellular free Ca2+ assay
    1. M Tris-HCI pH 8.8, containing 4% stacking gels in O.I25 M Tris-HCI pH 6.8. The gels were run in SDS-PAGE running buffer at a constant current of 40 rnA Proteins were visualized by staining the gels with Coomassie brilliant blue.
    2. The purified proteins were analysed by SDS-PAGE as described by Laemmli ( 1970). Restrictocin and its mutants were analysed on 12.5% resolving gels in 0.375
    3. SDS-PAGE
    1. Following electrophoretic resolution of total RNA, the gels were blotted on to GeneScreen membrane as described by Maniatis et al., 1982 ) .. The RNA gel to be used for blotting was not stained with ethidium bromide. The blotting was performed in 20 X sse or 20 X SSPE, OIN.
    2. Northern blot.
    3. bands seen in the DNA size marker, were marked with a ball -point pen at the places where small holes had been pierced in the gel earlier ( see above ). Thus it was easy to monitor the size of the fragments showing hybridisation to the probe. The gel was then peeled off and the membrane w~shed in 6 X sse with gentle rocking for 10 minutes to wash away any residual agarose sticking to the membrane. After air drying at room temperature, the membrane was baked at so0e for two hours. The baked filter was stored at room temperature in a dessicator, if not used immediately. The dehydrated gel was restained in water containing 0.5 ug I ml ethidium bromide for 30 minutes and examined on a short wave UV transilluminator to check for the presence of any DNA fragments that escaped blotting. The absence of any residual bands indicated that the transfer was complete.
    4. Restriction fragments of DNA resolved on agarose gel were transferred to nylon membrane ( GeneScreen or GeneScreen Plus by the capillary blotting procedure of Southern ( 1975 ) as described by Maniatis et al., ( 1982 ) . After the completion of electrophoresis, the gel was stained and photographed as described earlier. Position of the various bands obtained in the DNA size marker lane were marked by piercing small holes at the two ends of each band in the gel with a yellow tip. The gel was then denatured, neutralised and blotted essentially as described by Maniatis et al., ( 1982 ) . Locally available coarse absorbent paper was used to make the paper towels of the appropriate size. In case of genomic DNA from mammalian cells, the agarose gel was first treated with 0.25 M HCl for 10 minutes, followed by the rest of the procedure as mentioned above. The transfer buffer was 20 X SSPE in all cases. To prevent the absorption of fluid from the 3 MM paper under the gel directly to the blotting paper atop the nylon membrane, the gel was surrounded with polythene sheets to minimise the direct contact between the blotting paper and the 3 MM paper placed under the gel. The blotting was performed for 18 -24 hours. After the transfer was over, the paper towels and the 3 MM papers on top of the nylon filter were peeled off. The gel along with the attached membrane, was turned over and kept on a clean sheet of 3 MM paper with the gel side up. The position of the gel slots was marked with a ball -point pen. Also, the positions of the
    5. southern blot.
    6. Colony lifts were performed essentially as described by Maniatis et al. , 1982 ) . Recombinant colonies were grown 0/N at 37°C to have well separated colonies. The colonies were overlaid with 80 mm diameter nitrocellulose filter circles BA 85, S & S and after the filter became wet throughout, it was peeled off in a single, smooth motion, avoiding the smearing of the bacterial colonies. The plate was reincubated at 37°C for a few hours to regenerate the colonies. The colonies transferred to the filter were lysed to bind the liberated DNA to the nitrocellulose.
    7. Colony lifts.
    8. Transfer of DNA.
    9. Imrnobilisation of DNA L RNA on~ solid support.
    1. incubations were carried out for I h at RT and each incubation was followed by three washings with PBS containing 0.1% Tween-20 (PBST). Post-blocking, the membranes were incubated with 1:1000 dilution ofMA-813 ascites (for detection ofr-bmZP1), MA-451 ascites (for detection of r-dZP3) or rabbit polyclonal anti-r-rG antibodies (for detection of r-rG), followed by an incubation with 1:5000 dilution of goat anti-mouse or goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase (HRPO) (Pierce) respectively. The blots were developed with 0.6% (w/v) 4-chloro-1-naphthol in 50 mM PBS containing 25% methanol and 0.06% H202• The reaction was stopped by extensive washing with double distilled water
    2. The cells (2 - 4 x 1 06) transfected with plasmid DNA were resuspended in minimum volume of 2X sample buffer (0.0625 M Tris, pH 6.8, 2% SDS, 10% glycerol, 5% P-mercaptoethanol, and 0.001% bromophenol blue). The samples were boiled for 10 min and resolved on a 0.1% SDS-1 0% PAGE (Laemmli, 1970). The expression of recombinant proteins was analyzed by Western Blot. The proteins were electrophoretically transferred to 0.45 J.lm nitrocellulose membrane 0/N at a constant current of 30 rnA (milliampere) in Tris-Giycine buffer (25 mM of Tris-HCl and 200 mM glycine) containing 20% methanol (Towbin et al., 1979). Post-transfer, the membranes were washed once with PBS and non-specific sites were blocked with 3% BSA in PBS for 90 min at RT. All the subsequent
    3. Analysis of expressed recombinant protein by immunoblot
    4. COS-I cells were seeded at a density of 2.5 x 105 cells per well in a 6-well tissue culture plate and transfected with plasmid DNA essentially as described above. After 48 h incubation, cells Were trypsinized and counted in a hemocytometer. Cells ( ~ 1 06) were washed twice with PBS and fixed with 0.4% paraformaldehyde in PBS followed by all washings and incubations with respective primary and secondary antibodies in presence of 0.1% Saponin. Antibody concentrations used were same as in indirect immunofluorescence assay. After the final wash, cells were resuspended in PBS and samples were run on an Elite ESP flow cytometer (Coulter Electronics, Hialeh, FL, USA) and data analyzed using WinMDI (version 2.8) software. Cells stained with just secondary antibody were used to account for the background fluorescence. Cells tranfected with VR 1020 vector and probed with primary antibody were used as negative control.
    5. Analysis of mammalian cells, transfected in vitro with the plasmid DNA, by flow cytometry
    6. To investigate if the expressed protein was membrane bound or cytosolic, cells were fixed in 3. 7% paraformaldehyde followed by all washings and incubations with primary and secondary antibodies either in presence or absence of 0.1% Saponin and processed for indirect immunofluorescence as described above.
    7. Localization of the expressed recombinant protein in COS-1 cells
    8. albumin (BSA) in PBS for 2 hat 4°C. For detection of r-bmZPI, a murine monoclonal antibody (MAb), MA-813, generated against E. coli expressed r-bmZP1 (Govind et al., 2000), was used as the primary antibody. The cells were incubated with 1 :500 dilution of MA-813 ascites fluid for 2 hat 4°C. Cells were washed 5 times with PBS and incubated for 1 h with a 1:800 dilution of goat anti-mouse Ig-fluorescein isothiocyanate (FITC) conjugate (Sigma) at 4°C. After washing with PBS, coverslips with the cells were mounted in glycerol : PBS (9 : 1 ), and examined under an Optiphot fluorescent microscope (Nikon, Chiyoda-Ku, Tokyo, Japan). For detecting r-dZP3, MAb, MA-451 (1 :500 dilution of ascites fluid), generated against porcine ZP3f3 (a homologue of dZP3) and immunlogically cross-reactive with dZP3 (Santhanam et al., 1998) was used. For detecting r-rG, rabbit polyclonal antibodies (1:1000 dilution) against E. coli expressed r-rG, was used as primary antibody. The polyclonal antibody was provided by Dr. Sangeeta Choudhury, Project Associate, Gamete Antigen Laboratory, National Institute of Immunology, New Delhi. Goat anti-mouse immunoglobulins-FITC conjugate (1 :800) and goat anti-rabbit immunoglobulins-FITC conjugate (1 :2000; Pierce) were used for detecting anti-dZP3 and anti-rG antibodies respectively
    9. Initial standardization of transfection conditions was done using VRbmZPl plasmid DNA and COS-I mammalian cell line. In brief, cells were cultured in T-25 tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C with 5% C02. For subculturing, cells were trypsinized (0.5% trypsin + 0.2% EDTA in DMEM without FCS), centrifuged at 250 X g for 10 min, resuspended in DMEM supplemented with 10% FCS and aliquoted into T-25 flasks. For transfection, cells were seeded on coverslips in a 24-well tissue culture plate at a density of 5x 104 cells/well, a day prior to transfection. To standardize in vitro transfection conditions for optimum expression of bmZP1, varying amount of plasmid DNA was mixed with lipofectamine in DMEM devoid ofFCS (final reaction volume 200 f.!l) and incubated at RT for 45 min. The cells on the coverslips were washed twice with plain DMEM devoid of FCS. DNA-Iipofectamine complex was added dropwise to the cells and the plate incubated for 8 h at 3 7°C in humidified atmosphere of 5% C02• Subsequently, 1 ml of DMEM containing 10% FCS was added per well and cells allowed to grow for 48 h. After incubation, cells were processed for visualization of r-bmZPl by indirect immunofluorescence assay. Cells were washed twice with phosphate buffer saline (PBS; 50 mM Phosphate and 150 mM NaCI, pH 7.4), fixed in chilled methanol (-20°C) for 3 min and blocked with 3% bovine serum
    10. Detection of the expressed recombinant protein following i11 vitro transfection of mammalian cells with the plasmid DNA.
    11. 6000 X g for 15 min at 4°C. Plasmid DNA was purified from the pellet using QIAGEN DNA purification kit according to the manufacturer's instructions. The purified plasmid DNA (1.5 -2.0 mg/ml) was dissolved in autoclaved double distilled water and stored in aliquots (500 f.!l each) at -20°C until further use.
    12. A single colony of the respective clones was picked up from a freshly streaked LB + Kan (50 f.!g/ml) plate, inoculated into 5 ml of LB + Kan medium and incubated for 8 hat 37°C with vigorous shaking (-250 rpm). Subsequently, 500 fll of this primary culture was inoculated into 500 ml LB+ Kan and grown at 37°C 0/N. The culture was centrifuged at
    13. Purification of plasmid DNA in large amount
    14. N-VITRO TRANSFECTION OF MAMMALIAN CELLS WITH V ARlO US PLASMID DNA CONSTRUCTS AND CHARACTERIZATION OF EXPRESSED RECOMBINANT PROTEINS
    1. After transfection the cells were harvested and RNA was isolated from the celllysates using Trizol reagent (Invitrogen) and purified according to the manufacturer's directions. Briefly, the cells were lysed directly in the culture dish by adding 1ml of Trizol reagent to each well. The homogenized sample was incubated at room temperature for 5 min to permit complete dissociation of the nucleoprotein complexes. For purifying the RNA, 200pl of chloroform was added, the tubes were shaken vigorously for 15 seconds and incubated at room temperature for 2-3 min. Tubes were centrifuged at 12,000 ref for 15 min at 40C. The aqueous phase was collected, mixed with 500pl isopropanol and incubated at room temperature for 10 min. Centrifugation was carried out at 12,000 ref for 10 min at 40C. The supernatant was carefully removed and the RNA pellet was washed with 1ml of 70% ethanol by vortexing and then centrifuging at 7500 ref for 5 min. The pellet was air dried and dissolved in 20pl of NFW.
    2. RNA isolation from celllysates
    1. Procedure:
    2. β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well
    3. Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water
    4. Solutions:
    5. β-gal assay in transfected cells
    1. Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions by either CDFD or an outsourced sequencing facility
    2. DNA sequencing
    3. To 2 ml of the fresh overnight culture of the recipient strain grown in Z-broth, 108 pfu of P1 lysate was added and incubated at 37°C without shaking for 15 min to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 4000 rpm for 5 min and the pellet of bacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated at 37°C for 30 min with slow shaking to allow for phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspended in 0.3 ml of citrate buffer. 100 μl aliquots were plated on appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without the addition of the P1 lysate, was processed in the similar way as described above. In case of selection for nutritional requirements, the infection mixture was centrifuged, washed once in 5 ml of citrate buffer and plated without phenotypic expression
    4. P1 transduction
    1. hydrolysis of the non-fluorescent derivative dichlorodihydrofluorescein. In the presence of an appropriate oxidant, dichlorodihydrofluorescein is oxidised to the highly fluorescent 2, 7 -dichlorofluorescein. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were resuspended in fresh phenol red-free DMEM containing 10% FBS to get a cell density of 107 cells per mL. The cells were loaded with the dye (Stock solution prepared in DMSO to a final concentration of 1pg/pL) by incubating every 107 cells with 2pL of stock solution for 15 to 20 min on an end to end shaker at RT and then washed with medium. The cells were incubated for another 15 min to allow de-esterification to occur. 200pL was aliquotted into each well of a black plate and a basal reading taken at 485nm/ 520nm. Subsequently stained cells were exposed to appropriate treatments and fluorescence monitored at appropriate intervals of time. For each experiment, measurements were prepared in quadruplets and expressed as arbitrary f1uorescence intensity units (AFU)
    2. CM-H2DCFDA (5-(and-6)-chloromethyl -21,7'-dichlorodihydro fluorescein diacetate, acetyl ester) has been used as a detector of ROS as described previously (Mukherjee et al., 2002).This probe is a non-polar, non-fluorescent dye that diffuses readily into cells, where it is trapped by
    3. Assay for measuring intracellular ROS
    4. Ultra competent cells were prepared by using the method described by Inoue (Inoue et al., 1990). Briefly, the DH5-a cells were grown in the SOB culture medium (20gm/L Tryptone, 5 gm/L Yeast extract, 0.5 gm/L Sodium chloride, 2.5 mM Potassium chloride and 10mM Magnesium chloride, pH 7.0) at 18°C till the O.D.6oo of 0.55 was attained. The flasks were then shifted to ice-water bath for 10 min. The cells were harvested by centrifugation at 3220 x g, all media was discarded and the cell pellet was resuspended in Inoue transformation buffer (55mM Manganese chloride, 15mM Calcium chloride, 250mM Potassium chloride, 10mM PIPES, pH 6. 7). The suspension was centrifuged at 3220 x g, the buffer discarded and cell pellet was resuspended in fresh Inoue transformation buffer. DMSO (1.5mL/20mL of buffer) was added and the cells were frozen at -70°C. Cells were checked for transformation efficiency and were used if transformation efficiency was above 5 X 108 transformed colonies / Jlg of DNA
    5. Preparation of ultra-competent cells of E. coli DHS-a
    1. Total cellular polyphosphates were quantified viapolyacrylamide-Tris borate gel electrophoresis (PAGE-TBE).Briefly, PAGEwas performed with Tris-borate buffer (pH 8.3) todeterminethe quantityand the typeof polyphosphatesextracted fromyeaststrains. Equal amount of total RNA (20 to 100 μg) was loadedon 34%PAGE-TBE gel (22cm long, 16 cm wide and 0.8 mm thick) and electrophoresedat 500Voltsfor 20-24 hin cold-roomtill the marker dye bromophenol blue(BPB)had migrated 15-16 cm awayfrom the well.After electrophoresis, total polyphosphates werevisualizedby staining the gel with 0.05%toluidine blue staining solutionfollowed by destaining. Polyphosphates wereobserved both as asmearin the top most portion of the gel as well asdiscreet bands of long chain polyphosphatesand shortchain polyphosphatesin middle andbottom half of the gel,respectively.Polyphosphateband intensity in the gelwas quantified using ImageJ software (http://rsbweb.nih.gov/ij/)and relative amountsof long chain and short chain polyphosphatesin C. glabratacells werecalculated
    2. Quantitative analysisof polyphosphates
    1. Complementary-DNA synthesis was doneusing reverse transcriptase enzyme and oligo-dT primers. For this, 1 μg good quality RNA was treated with1μl(1 unit) DNase I for 15 min to remove DNA contamination. Next, SuperScript III First-Strand Synthesis System kit (Invitrogen) was used to synthesize cDNA according to the manufacturer’s instructions. cDNA synthesized was stored at -20 ̊C till further use
    2. Synthesis of complementary DNA (cDNA)
    3. A microtipful of cellsfor each yeaststrainfrom appropriate mediumwassuspended in 10μlzymolyase cocktailandincubated at 37ºC for 90 min. 2 μlof zymolyase-treated cell suspension was used as template in 25 μlPCR reaction
    4. Yeast colony PCR
    1. CHX pulse chase assay was performed as essentially described by Zhou (2004). Cycloheximide (CHX), a protein biosynthesis inhibitor was used to determine the half-life and stability of protein of interest. CHX blocks translation elongation step, thereby halting the synthesis of new proteins and therefore, time course degradation of protein can be studied. Briefly, parental and profilin-stable cells were seeded in 35 mm culture dishes and treated with CHX (50 μg/ml) the following day. Cells were harvested at different time points and level of protein was determined by immunoblotting
    2. Cycloheximide (CHX) chase assay
    1. Overnight grown yeast were sub-cultured at 0.2 OD600and growntill 0.8 OD600. Cells equivalent to 1 OD600were harvested and washed twice with SC-Uramedium to remove any residual uracil from the cells. Cells were incubated in SC-Ura medium containing 3 μCi/mL [14C]uracil for 1, 5, 10 and 20 min, and RNA was extracted as described in Section 2.2.8. Equal total RNA was resolved on a formaldehyde agarosegel and transferred to an N+Hybond membrane (GE Life Sciences). Radiolabeled rRNA was detected using a phosphorimager scanner (Fujifilm FLA-9000)
    2. [14C]uracillabelling of total RNA
    1. Triton X-100, 0.5 mg/ml RNase A, 40 μg/ml propidium iodide). Cell nuclei were then incubated for 30 min. at 30°C and were subsequently analyzed by FACS. The hypodiploid nuclei in the histograms were considered and represented aspercentage of apoptotic cells
    2. Apoptosis was measured by Nicoletti method (Nicoletti et al., 1991). Cells treated with appropriate apoptotic stimuli for the indicated times were harvested by centrifugation at 800g for 5 minutes at room temperature. Cells were washed once with PBS, and then resuspended in hypotonic PI lysis buffer (1% sodium citrate, 0.1%
    3. Apoptosis
    1. media containing 50 μM 2’2’-dipyridyland grown for 24 h at 28°C with continuous shaking at 200 rpm. Cells were harvested by centrifugation at 7000 g for 10 min at 4 °C, washed twice with 50 mM phosphate buffer (pH-7.4), and finally resuspended in phosphate buffer. The bacterial suspension was then diluted with chelex-100 treated PS to get a final OD600of 1.0 and incubated at 28°C for 5 min. Iron transport assay was initated by adding 55FeCl3(American radiolabeled chemicals, Inc., St. Louis, USA,specific activity 10.18 mci/mg) to a final concentration of 0.4 μM into the bacterial suspension. The radiolabelled stock solution was diluted with water and 1M sodium ascorbate for 55Fe3+uptake and 55Fe2+uptake studies, respectively. For uptake of FeCl3bound vibrioferrin, both vibrioferrin (7.6 mM stock) and 55FeCl3were incubated in 1:1 ratio by diluting it appropriately with water and uptake was initiated with a final concentartion of 0.4 μM. To stop the uptake, 200 μl of bacterial cell suspension was layered and immediately centifuged (13000 g; 1 min) through 300 μl of di-butylphthalate and di-octyl phthalate (1:1) mixture. The upper aqueous layer and organic solvent was aspirated, and pellet was resuspended in 100 μl Triton-X-100. The suspension was added to 5 ml scintillation cocktail, and radioactivity count was determined in the 3H channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). As control, both Fe2+and Fe3+uptake assays were performed in presence of proton motive force uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP; 50 μM), to distinguish between non-specific uptake of readiolabelled Fe by the bacterial cells. However, no significant increase in the incorporation of Fe2+and Fe3+ was observed in presence of FCCP, which indicated that iron uptake by these strains is energy-dependent process
    2. In vitro transport assay was performed by using radiolabelled iron to measure the capacity of Xanthomonas oryzaepv. oryzicola strains to transport 55Fe(II) and 55Fe(III) forms of iron as described previously with slight modifications (Ardon et al., 1997; Velayudhan et al., 2000). For iron uptake asssay, Xocwild-type BXOR1 strain, ∆rpfF mutant and the complemented strain harboring full length rpfFgenewere grown overnight in PS medium. 0.2% of the overnight grown culture was inoculated in fresh P
    3. 55Fe uptake assay