153 Matching Annotations
  1. Jul 2021
    1. Zebrafish: Tg(kdrl:GFP)s843

      DOI: 10.1016/j.celrep.2019.05.052

      Resource: (ZFIN Cat# ZDB-ALT-050916-14,RRID:ZFIN_ZDB-ALT-050916-14)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-050916-14


      What is this?

    1. ZDB-ALT-070427-14

      DOI: 10.1523/ENEURO.0286-16.2017

      Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14


      What is this?

    2. ZDB-ALT-070427-14

      DOI: 10.1523/ENEURO.0286-16.2017

      Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14

      Curator comments: Allele Name: Hu2098 Danio Rerio ZFIN Cat# ZDB-ALT-070427-14


      What is this?

    1. ZDB-ALT-070427-14

      DOI: 10.1016/j.neuron.2017.06.001

      Resource: (ZFIN Cat# ZDB-ALT-070427-14,RRID:ZFIN_ZDB-ALT-070427-14)

      Curator: @Zeljana_Babic

      SciCrunch record: RRID:ZFIN_ZDB-ALT-070427-14


      What is this?

    2. ZDB-ALT-170522-14

      DOI: 10.1016/j.neuron.2017.06.001

      Resource: (ZFIN Cat# ZDB-ALT-170522-14,RRID:ZFIN_ZDB-ALT-170522-14)

      Curator: @Zeljana_Babic

      SciCrunch record: RRID:ZFIN_ZDB-ALT-170522-14


      What is this?

  2. Jun 2021
  3. Apr 2021
    1. La composition ainsi que les missions et modalités de pilotage des comités d'éducation à la santé et à la citoyenneté sont détaillées dans la circulaire n°2016-114 du 10 août 2016.
  4. 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: 7.2

      AssayResultAssertion: Normal

      StandardErrorMean: 0.01

    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.1250C>A p.(Ser417Tyr)

    1. SUPPLEMENTARY DATA

      AssayResult: 41

      AssayResultAssertion: Indeterminate

      PValue: < 0.0001

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

    2. SUPPLEMENTARY DATA

      AssayResult: -31

      AssayResultAssertion: Abnormal

      PValue: < 0.0001

    3. SUPPLEMENTARY DATA

      AssayResult: 82.22

      AssayResultAssertion: Indeterminate

      PValue: 0.004

      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.2840T>C p.(Leu947Ser)

    1. Source Data

      AssayResult: 117.58

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 0.81

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

    2. Source Data

      AssayResult: 95.02

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 0.08

      StandardErrorMean: 0.06

      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.1676A>G p.(Q559R)

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

      AssayResult: 0.9

      AssayResultAssertion: Abnormal

      ReplicateCount: 12

      StandardErrorMean: 0.6

      Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (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.2204C>A p.(Ala735Glu)

  5. Feb 2021
    1. RRID:ZDB-ALT-131122-14

      DOI: 10.7554/eLife.54491

      Resource: (ZFIN Cat# ZDB-ALT-131122-14,RRID:ZFIN_ZDB-ALT-131122-14)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-131122-14


      What is this?

    1. Supplemental material

      AssayResult: 91

      AssayResultAssertion: Normal

      Comment: See Table S2 for details

    2. Supplemental material

      AssayResult: 4.9

      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.323G>A p.(Gly108Asp)

  6. Oct 2020
    1. The graphs of ground state confinement energy againstsize (radius) for zinc sulfide nanoparticles in Figure 14 showthe dependence of confinement on the size of quantum dots.The result shows that ground state confinement energy is

      Las graficas de la energía de confinamiento en su estado fundamental en contra del tamaño (radio) por nanopartículas de sulfato de zinc en la Figura 14 muestran la dependencia de confinamiento en el tamaño de los puntos quánticos. El resultado muestra que el estado fundamental de energía en confinamiento es inversamente proporciona al tamaño (radio). Por lo tanto, cuando uno incrementa su radio (tamaño) la energía de confinamiento decrece pero nunca llega a cero. Eso es, el energía mas baja posible para el punto quántica de muestra no es cero. El confinamiento comienza cuando el radio del punto cuántico de muestra es comparable o del orden del radio exciton de Bohr.

  7. Jul 2019
  8. Jun 2019
    1. The time kinetics of deoxyhemoglobin polymerization were studied in 1.8M, 1.5 and 1 M potassium phosphate buffer (pH 7.25) respectively as described by Adachi and Asakura (1979a, b) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. Deoxygenation of the hemoglobin sample was ensured by passing moist gaseous nitrogen over the sample in an airtight cuvette and by addition of sodium dithionite. The polymerization of the resultant deoxyhemoglobin samples was initiated by a temperature jump from 4 to 30 oc within 10 sec and the progress of the reaction was followed by monitoring turbidity changes at 700 nm. The delay time was calculated from the kinetic traces
    2. Kinetics of polymerization
  9. May 2019
    1. A synthetic peptide (KMMTSKDNLNIDIPS) based on the PfCDPK4 sequence was custom synthesized (Peptron Inc.) and conjugated to keyhole limpet hemocyanin via an additional N terminus cysteine residue. It was used to raise polyclonal antisera against PfCDPK4 in rabbit. First immunization was performed using 1 00 ~g of peptide diluted in PBS and mixed 1: 1 v/v with Complete Freund's Adjuvant (CF A). Subsequently, three booster doses of 50 ~g each were given on the 14th, 28t\ 42nd day post first immunization. Blood was collected from animais on 7th, 21 S\ 35th, 49th day. Antibody titers were checked by ELISA using recombinant proteins or ovalbumin conjugated peptides as an antigen. In all cases, pre immune sera from the same rabbit were used as control
    2. Generation of anti-PfCDPK4 antisera
    1. were represented as arbitrary fluorescence units and comparisons were made against the untrea!ed control samples. Exogenous addition of hydrogen peroxide to cells was used as a positive control for the assay
    2. he generation of reactive oxygen species in macrophages was detected by fluorimetry using the fluorescent dye CM-H2DCFDA, which can detect hydrogen peroxide, hydroxyl radical, peroxyl radical, and peroxynitrite anion (5, 6). To perform the assay, THP-1 macrophages were washed and resuspended in serum and phenol-red free RPMI-1640 medium and incubated at room temperature for 30 min in the presence of CM-H2DCFDA at a final concentration of 1 JiM. Subsequently the cells were washed once with fresh media to remove the excess probe and fluorescence measurements were commenced on a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and an emission of 520 nm. Appropriate treatments were initiated and time-kinetic measurements were carried out and the values obtained
    3. Detection of intracellular reactive oxygen species generation
    1. 1.2% acetylphenylhydrazine (APH) solution to render it anemic. The APH solution wa5 prepared in sterile water and pH was neutralized to 7.0 with 1 M HEPES buffer (pH 7.5). The rabbit was allowed to recover for 5 days, after which it was bled. The blood was collected in a sterile tube, containing an equal volume of prechilled salt solution and the mixture was filtered through a cheese cJoth. Filtrate was centrifuged at 2000 g for 10 min. at 4 °C. Supernatant was discarded, the pellet was washed twice with salt solution without heparin and finally, resuspended in equal volume of chilled sterile water. It was kept on ice for a minute and centrifuged at 20,000 g for 20 min. at 4 °C. The supernatant, containing the lysate, was immediately stored in liquid Nitrogen in 0.5 ml aliquots.
    2. The rabbit reticulocyte lysate was prepared as described by Sambrook et al ( 1989). A young male NZW rabbit weighing 2-2.5 Kg was subcutaneously injected for five consecutive days respectively with 2 ml, 1.6 ml, 1.2 ml, 1.6 ml and 2.0 ml of
    3. Preparation of rabbit reticulocyte lysate
    1. with glutamine. Rat-2 and FWIL cells were cultured in IMDM supplemented with glutamine. All media were supplemented with 10 % fetal calf serum~ The cells were maintained in a 5 % co2 atmosphere and were split after 72 hours in culture, at a ratio of 1 : 15 approximately. For splitting the adherent cells, the cells were washed once with HBSS and 0.1 % trypsin in PBS added to the cells. The flask was shaken briefly to ensure a uniform distribution of trypsin over the cells. The cells were incubated in trypsin for 1 - 2 minutes after which 2 ml of FCS was added to the cells to inactivate the trypsin. The trypsin was carefully aspirated from the flask and fresh culture medium was added into the flask. The cells were dislodged from the bottom of the flask by gently tapping the flask against the working bench. Alternately, the cells were resuspended by vigorous pipetting up and down of the medium. The cells were centrifuged at 1500g for 5 minutes at room temperature and the supernate was discarded aseptically. The cells were resuspended in a known volume of fresh culture medium, an aliquot counted on a haemocytometer, and then accordingly seeded at the desired density in a fresh flask. For long term storage, the cells were frozen in a mixture of 95 ~ 0 culture medium and 5 ~ 0 DMSO in liquid nitrogen.
    2. eHO-Kl cells were cultured in Ham s F-12 med1a. NIH3T3, mouse LMtk-and HeLa cells were cultured in DMEM supplemented
    3. Growth and maintenance of cell lines.
    1. weight) as a general anesthetic and ovaries were snap frozen in liquid nitrogen. Ovarian sections of 5 J.lm thickness were cut in a cryostat at -20°C and fixed in chilled methanol for 15 min at RT. Sections passing through follicles were selected, washed with 50 mM PBS and blocked with 3% normal goat serum (NGS) in PBS (v/v) at RT for 1 h. Sections were then washed two times with PBS and incubated with 1: 1 0 dilution of immune serum samples. Ovarian sections incubated with 1:10 dilution of mouse preimmune or immune sera from mice immunized with VR1020 vector served as negative controls. After incubation, the sections were washed three times with PBS and incubated with 1:800 dilution of goat anti-mouse IgG conjugated to FITC (Sigma) for 1 hat RT. The slides were washed three times with PBS, mounted in glycerol : PBS (9 : 1 ), and examined under fluorescence microscope (Optiphot, Nikon, Japan).
    2. Ability of mouse polyclonal antibodies, generated subsequent to immunization with VRbmZP1 and VRdZP3 plasmid DNA, to recognize native ZP was evaluated by indirect immunofluorescence assay. A normal cycling female bonnet monkey and a female dog were ovariectomized after administration of ketamine hydrochloride (5 mg/kg body
    3. REACTIVITY WITH NATIVE ZP OF THE IMMUNE SERUM SAMPLES OBTAINED FROM MICE IMMUNIZED WITH VRbmZPl AND VRdZP3 PLASMID DNA
    1. Bloodsampleswereobtainedbycuttingthecaudalpeduncleandanalysedforlacticacidusinganenzymatictechnique(SigmaCo,1974)MeasurementsweremadeinQuartzcuvettesat340nmwithaBeckmanAVSpectrophotometer.Standardcurvesweremadeonthedaythebloodlacticaciddeterminationsweremade.
    2. Bloodlacticacid
    1. ubjected to three washes with PBST and two washes with PBS. The blot was developed using the substrate DAB (Sigma, U.S.A.) or with ECL (Amersham Biosciences, U.K.)
    2. he protein samples were diluted with 4X sample buffer which is essentially SDS-reducing buffer (O.SM Tris-Cl, pH 6.8, Glycerol, 10% (w /v) SDS, 2-J3-mercaptoethanol, 0.05% (w /v) bromophenol blue). The samples were denatured at 1000C for 10 min and the proteins were resolved on 12-15% SDS-polyacrylamide gel at 25-30mA. For detection, the proteins were transferred on to nitrocellulose (NC) membrane (Hybond-C extra, Amersham, U.K.) at 200mA, for either 1 hr or at 12 rnA, 40C for overnight. After the transfer was over, the NC membrane was washed thrice with PBST (1X PBS with 0.1% Tween 20) and blocked with 2% BSA for 2hrs (in PBST) at room temperature. Primary antibody to HBx/Vif/ APOBEC3G-NT raised in rabbit and were diluted to 1:1,000 in PBST. One hour incubation with the primary antibody was followed by three washes with PBST (10 min each) and then 1 hr incubation with 1:1,000 dilution of the secondary antibody (Anti-rabbit IgG (Fe) HRP conjugate) was carried out. The blot was further
    3. Westem blot analysis
    1. The plates were kept in incubator gently and the colony formation was monitored every week. Media (500μl) was added to the plates every 4th-5th day to avoid drying. Colonies formed in soft agar photographed were taken without staining, under a microscope in light field
    2. Agar solution was prepared in a sterile 50ml Schott Duran Bottle and boiled in microwave until fully dissolved and kept at 55°C to 65°C. Master Mix with the rest of the components of bottom agar was made in a sterile corning 50ml tube prewarmed at 55°C and agar solution was added. The solution was once vortex briefly and then added (2ml) carefully to each well avoiding air bubbles. The plates were left undisturbed in laminar flow hood until the agar set fully. Two days before final assay, the bottom agar plates were kept in tissue culture incubator for equilibration. On the day of assay the following mix was prepared for Top Agar 4 dishes 5 dishes1.media with FBS, L-glutamine and Pen-Strep 4.8 ml 6 ml 2.fetal bovine serum 1.8 ml 2.5 ml 3.sterile water 1.8 ml 2.5 ml 4.agar 1.8% (1.8 g/100mLs) 1.8 ml 2.5 ml 5. cell suspension 1.0 X 105/ dish 100 to 350 μl 100 to 350 μl 6. Total 10.2 ml 13.5 ml Top agar mix without cells was first prepared and kept at 42°C. The cells were then trypsinized and re-suspended after counting in final volume of 100μl to 200 μl. Cells were then mixed with top agar and solution was quickly poured over the bottom agar.
    3. For soft agar assays 2x104, (A549) or 1x105 cells (E-10) were used in 1.5ml top agar. For preparing bottom agar plates (0.64% final con. of agar), a following mix was prepared for five dishes. 1.2X media with FBS, L-glutamine and Pen-Strep 10 ml 2.fetal bovine serum 5 ml 3.sterile water 1 ml 4.noble agar 1.8% (1.8 g/100mLs) 9 ml 5.Total 25 m
    4. Soft Agar Assay
    1. f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine
    2. a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.
    3. Activation of CM10 (weak cation exchange ) array
    4. d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine
    5. One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once
    6. Activation of LSAX (strong anion exchange ) array
    7. Activation of H50 protein chip array
    8. b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine
    9. 5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once
    10. 5 μl of 0.1% TFA was applied to the spots on the SEND array and removed after 30 sec using Whatman paper (care was taken not to touch the spot surface). b. 5 μl of cell lysate sample was spotted on the SEND array and incubated in a humid chamber for 10 min. Removed after 30 min. c. 5 μl of 0.1% TFA was then added and removed after 30 sec. d. 2 μl of 25% ACN in 0.1% TFA was added to the spots and allowed to dry. e. The chip was then placed in the SELDI machine
    11. Activation of SEND arrays for peptide analysis
    12. Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA
    13. Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised
    14. Destaining of gel, trypsinization and peptide extraction
    15. 12% resolving gel (for 25 ml)Water = 8.2 ml 30% Acrylamide = 10.0 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.01 ml 5% stacking gel (for 10 ml)Water = 6.8 ml 30% Acrylamide = 1.7 ml 1.5 mM Tris (pH 6.8) = 1.25 ml 10% SDS = 0.1 ml 10% APS = 0.1 ml TEMED = 0.01 ml
    16. A double cylinder gradient former was used with 12% poly acrylamide gel mix in the inner cylinder and a 3% polyacrylamide gel mix in the outer cylinder that was stirred using a magnetic bead on a magnetic stirrer. A pump was connected to the flow tube and the flow rate adjusted at 5-8 to cast a 12-3% gradient gel. A 5% stacking gel was used. After the protein samples were run on the gradient gel, it was stained in instant blue over night under shaking. 3% resolving gel (for 25 ml)Water = 15.68 ml 30% Acrylamide = 2.5 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.02 ml
    17. Casting a gradient SDS-polyacrylamide gel
    18. 1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl
    19. Protein estimation by Bradford method
    20. microfuge tubes and snap frozen in liquid nitrogen and were stored at ─80 ̊C. Protein estimation was performed simultaneously with one of these aliquots
    21. The strains were grown to stationary phase in 500 ml LB supplemented with ampicillin (100 μg/ml) overnight. Cells were pelleted at 2100g for 30 min at 4 ̊C and dissolved in 5 ml of 1X PBS with 2X protease inhibitor and 3 mM DTT. Cells were lysed using French Press at 1500 psi for three cycles. The lysed cells were pelleted at 20,000g for 45 min at 4 ̊C. Clear supernatant was collected in sterile 2 ml
    22. Preparation of cell extracts
    23. These experiments were undertaken in the laboratories of Dr. Sylvie Rimsky and Dr. Malcolm Buckle at the Ecole Normale Superioure, Cachan, Paris (France)
    24. Methods for SELDI (Surface Enhanced Laser Desorption/Ionization)
    25. A single plaque of λ contains approximately 105-106 pfu/ml. The method of propagation of λ from a single plaque was as follows. The contents of a single isolated plaque were drawn into a 1-ml pipette tip and dispensed into 0.2 ml of LB broth. After addition of a drop of chloroform, the contents were vortexed and centrifuged. The clear supernatant was mixed with 50 μl of λ-sensitive cells and incubated for 20 min at room temperature for adsorption. 10 ml of Z-broth supplemented with 5 mM MgSO4 was then added to the infection mixture, and incubated at 37°C with shaking until lysis. The lysate thus obtained usually contained 109pfu/ml
    26. Preparation of λ lysate by propagation from a single isolated plaque
    27. The method used was essentially the same as that described for preparation of P1 lysate, except that the λ-sensitive C600 cells used for infection were grown in LB broth containing 0.4% maltose and 10 mM MgSO4. The lysate thus prepared was checked for supE+revertants by plaquing on both supE (C600) and supE+strains (MG1655) using various dilutions of the lysate. To be used for experimental purposes, a phage titre of the order of 1010 to 1011 on the supE strain and around four orders of magnitude lower on the supE+strain, indicating very less frequency of supE+ revertants in the lysate is ideal
    28. Preparation of lambda (λ) lysates
    1. 30nm corresponds only to the intermediate. Since equal number of cells was taken for estimation, the height of the peak was taken as a measure of the ergosterol content
    2. Ergosterol content was measured as described by Arthington-Skaggs et al. (1999) with slight modifications. Briefly, equal number of cells were harvested and washed with PBS to remove media and FBS. They were resuspended in 3mL of 25% alcoholic KOH (25g KOH, 35mL sterile distilled water brought to lOOmL with ethanol) and vortexed for one minute. The cell suspension was transferred to a glass vial and incubated at 85°C for 1 hr. The vial was cooled to room temperature followed by the addition of 1mL sterile distilled water and 3mL of n-heptane. The vial was vortexed for 3 mins after which the heptane layer was collected. It was diluted 5 times in absolute ethanol and transferred to a cuvette. A spectrum was recorded between 220nm and 300nm. The peak at 281.5nm corresponds to ergosterol along with some intermediates.
    3. Spectrophotometric estimation of ergosterol
    4. Plasmid DNA was isolated at small scale using QIAprep Miniprep kit according to manufacturer's protocol. Briefly, 5mL of overnight E. coli culture was pelleted and resuspended in 250J..LL Buffer P1(50mM Tris-Cl, pH8.0; 10mM EDTA and 100p.g/mL RNase A). To this, 250 J..LL of Buffer P2(200mM NaOH and 1 %w /v NaOH) was added and mixed thoroughly by inverting the tube 4-6 times. 350 IlL of Buffer N3 (proprietary) was added and mixed immediately and thoroughly by inverting the tube 4-6 times. This was followed by centrifugation at 13000 x g for 10min at RT. The supernatant was applied to a QIAspin column and centrifuges at 13000 x g for 30-60s. The column was washed with 0.5mL Buffer PB followed by 0.75 mL Buffer PE. Residual wash buffer was removed by centrifugation for an additional 60s. The plasmid DNA bound to the column was eluted using the elution buffer, 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
    5. Miniprep to isolate plasmids
    1. cellswere collected and washed with chilled sterile water.1 OD600cells were resuspendedin 20 μl chilled10%TCA solution containing 8 mM EDTA (pH 8.0) and incubated at room temperature for 15-20 min.Followingincubation, cellsuspension was centrifuged at 12,000 rpm for 5 minat 4 ̊Cand supernatant was transferred to a fresh 1.5 ml microcentrifuge tube. 10 μl of this supernatant fraction was diluted 75-foldwith ATPassay mix dilution buffer provided with the kit. 50 μl of diluted suspension was added to anequal volume of ATPassay mix (Sigma # FLAAM) which containedfirefly luciferase and luciferin with MgSO4, EDTA, DTT and BSA inTricine buffer.Luminescence was measured inluminometer (Varioskan flash-3001,Thermo Scientific). Total ATP was quantified usingpurified ATP as the standardand expressed in moles/OD cells
    2. ATPconcentrationin yeast cells was measuredby luminometricluciferase-luciferinbased assayusingATPbioluminescent kit(Sigma # FLAA).Briefly, log-phase yeast
    3. Determination of intracellular ATPlevels
    4. Estimation of total glycogen in cells was performed asdescribed previously (Parrou et al., 1997) with slightmodifications.Briefly, YPD medium-grown C. glabratacells were harvested, washed once with 1 ml ice-cold waterandresuspendedin 250 μl sodium carbonate(0.25 M)solution. After incubation at95 ̊C for 4 hin water bath with occasional stirring, cell suspension was cooled and pH of the suspension was adjusted to 5.2 by adding 150 μl 1 M acetic acid. Tothis suspension,600 μl 0.2M sodium acetatewas added and cell suspension was incubated with 1-2 U/ml of α-amyloglucosidase from A.niger(Sigma #A7420)at 57 ̊C for overnight with constant agitation.Resultant glucose liberated by α-amyloglucosidase digestion was collected in the supernatant fraction and quantifiedby phenol-sulphuric acid methodof carbohydratedetermination.For quantification, commercially available purified glucose was used as a standard and total glycogen incells was expressed as μg/2 x 107cells tonormalizeagainstcell density
    5. Estimation of glycogenlevels
    6. Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells
    7. Estimation of trehalosecontent
    8. Estimation oftrehalose, glycogen and ATPlevels
    1. QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA sample was resolved on 1-1.2% agarose gel and gel piece containing desired fragment was cut ona UV-transilluminator. DNA fragment was purified as per the kit manufacturer’s instructions
    2. Gel extraction of DNA
    3. To prepareelectrocompetent cells, a single colony of E. coli BW23473 strain from a freshly-streaked LB agar plate was inoculated in 50 ml LB liquid medium. Culture was incubated at 37°C for 14 h with continuous shaking at 200 rpm. 25 mlovernight-grown E. coliBW23473 culture was transferred to 500 ml LB liquid medium and incubated at 37°C till the OD600 reached to 0.4. Post incubation, cultures were transferredto ice and centrifuged at 1,000g for 15 minat 4°C. Cells were washed twice with 500 ml ice-cold sterile water, three times with 250 ml ice-cold 10% glycerol solution and resuspended in 1 ml 10% glycerol solution. After absorbance measurement, cell suspension was normalized to final cell density of 3X1010cells/ml and dispensed in 50 μl aliquots to sterile ice-cold 1.5 ml microcentrifuge tubes. Aliquots were snap frozen in liquid nitrogen and stored at -70ºC
    4. Preparation of E. coliBW23473electrocompetent cells
    1. Secretory alkaline phosphatase (SEAP) assay: For SEAP assay, the culture supernatant was analyzed for SEAP activity essentially as per the Clontech kit protocol (Palo Alto, CA). Briefly, cells were transiently co-transfected with Lipofectamine 2000 transfection reagent, 0.5 μg of required plasmid DNA(s) with the protein of interest or empty vector, 0.5 μg of reporter plasmid containing NF-κB binding site cloned upstream of heat-stable SEAP (designated asNF-κB-SEAP)and 0.5 μg of green fluorescence protein (GFP) expression plasmid (Clonetech) in Opti-MEM media.After 6 h of transfection, cells were washed and cultured for 12 h in complete media, followed by treatment with different inducers. GFP positive cells were then counted to ensure similar transfection efficiency. At the end of treatment, cell culture-conditioned medium was harvested and 25 μl of medium was mixed with 20 μl of 5X buffer (0.5 M Tris, pH 9 and 0.5% bovine serum albumin) in a total volume of 100 μl in a 96-well plate followed by incubation at 65°C for 30 min. The plate was chilled on ice for 2 min and 50 μl of 1 mM 4-methylumbelliferylphosphate (MUP, substrate) was added to each well and incubated at 37 °C for 2 h. The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan, Lab Systems, MA) with excitation set at 360 nm and emission at 460 nm. The average number (± SD) of relative fluorescent light units for each transfection was then determined and reported as fold activation with respect to empty SEAP-transfected cells.Luciferase (Luc) assay:The cell pellet was lysed and extract was analysed as per Promega kit protocol.Briefly, cells wereco-transfected with Lipofectamine with 0.5 μg of reporter plasmid containing p53 binding site cloned upstream of luciferase (designated as p53-luciferase) and 0.5 μg of GFP constructs. After 6 h of transfection, cells were washed and cultured for 12 h, followed by treatment with different inducers of apoptosis. GFP positive cells were then counted.Cellswere pelleted down and lysed using the lysis buffer. The samples were freeze-thawed twice by storing them at -70oC to ensure total lysis. The supernatant,obtained by centrifuging the same at 11,000 rpm for 2 min was transferred to a fresh tube. About 100 μl of the substrate (Firefly luciferin, Promega) was added to the supernatant and light emission wasmeasured in luminometer by using a delay time of 2 sec andread time of 10 sec.The values were calculated as fold of activation over vector-transfected value
    2. Reporter gene transcription assays
    1. apparatus.Membranes were neutralised in 2X SSC, and denatured plasmids were cross-linked to Hybond-N+membranes usingaUV cross linker at 2000 J/inch square energy for 2 min
    2. +610),end 5’ETS (+611 to +952),25S (+5270 to +5630) and NTS2(gifted byDr. Susan J Baserga) (Gallagheret al., 2004); ACT1 cDNA (+175 to +701) cloned into TOPO vector; pUC12 plasmid containing 5S rDNA construct (giftedby Dr. Purnima Bhargava). Empty TOPO plasmid and genomic DNA extracted from wild type yeast were used as controls. Plasmids and gDNA were extracted, quantitated and denatured in alkaline denaturing solution. 10 μg of each plasmid and gDNAin replicates weredenaturedin alkali, blotted on a Hybond N+membrane using a 96-well Dot Blot
    3. The following plasmids (Table 2.2)were used as probes to detect the transcribed RNA (Fig. 2.1). The TOPOplasmids containing rDNA start (+1 to +177), 5’ETS (+351 to
    4. Dot blot membrane preparation
    1. Cells were transfectedwith various combinations of plasmids and treated with cycloheximide (50ug/ml) 24hrs.post-transfection. Cells were harvested at different time points, and the protein levels were determined by using the standard protocol for western blotting/immunoblotting
    2. Cycloheximide-chase assay
    1. concentarion in the samples were determined based on their peak area against standard oxalic acid plot.For GC-MS analysis, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization was performed with the dried HPLC fraction of samples as well as standards as described previously (Šťávová et al., 2011). Briefly, 200 μl BSTFA, and 100 μl hexane were added to the sample, and incubated at 50 °C for 70 min. GC analyses were performed using a Shimazdu GP 2010 plus instrument equipped with an autosampler, and a split injector.Separations were accomplished using a 30-m long DB-5 capillary column, 0.25 mm internal diameter (I.D.) at a constant helium flow rate of 1.5 mL/min. Samples (10 μL) were injected with a split ratio of 10 into the column at 100 °C. The final column temperature program started at 100 °C and attained final temperature 280°C with a gradient increase of 5 °C/min. The MS data (total ion chromatogram,TIC) was acquired in the full scan mode (m/z of 50–500) at a scan rate of 1000 amu using the electron ionization (EI) with an electron energy of 70 eV. The acquired spectrum was searched against standard NIST-05 library
    2. Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium supplemented with appropriate antibiotics. 0.2 % of the overnight grown culture was reinoculated in 250 ml of fresh PS medium supplemented with 50 μM 2,2’-dipyridyl, and allowed to grow till OD600reached 1. Cultures were centrifuged to obtain cell free culture supernatant, concentrated on vaccum evaporator, and freeze dried at regular time intervals to remove the water completely. Oxalic acid was estimated from the dried supernatant by using Agilent 1100 series HPLC system as described previously with slight modifications (Ding et al., 2006). In brief, dried supernatant fractions of different cultures were dissolved in mobile phase of pH 2.7, and allowed to stand for 3 h for the precipitation of humic substances. These samples were filtered through membrane filter (porosity, 0.45 μm), and 20 μl volume of the filtrate was injected into the Agilent C18 (4.6 mm× 250 mm× 5 μm) column. The mobile phase used was 10 mM KH2PO4-CH3OH (95:5, pH 2.7), and the samples were separated by isocratic elution at 0.8 mL/min at 26°C temperature. Standard oxalic acid was detected in similar way in mobile phase (pH 2.7 at 210 nm) with retention time (RT) of 6.7 min. Likewise, oxalic acid in the test samples were also detected at 210 nm with RT 6.7
    3. Oxalic acidestimation from culture supernatant of different strains of X. oryzaepv. oryzicola by HPLC and GCMS analysis
    1. fluorophore-conjugated phalloidin (Alexa Fluor 488 or rhodamine) for 45 min, followed by DAPI staining for 2 min. Cover slips were mounted onto glass slides using Fluoromount G (Southern Biotech) or Vectashield (VectorLabs),and imaged using an LSM 710 laser confocal-anisotropymicroscope (Zeiss, Zen acquisition software, 40x 1.3 N.A. objective) for 15 min spreading,or LSM 510laser confocal microscope (Zeiss, LSM acquisition software, 63x 1.4 N.A. objective) for 24 hspreading.To measure the cell spread area for serum depleted cells (0.2% serum), images captured at identical zoom settings were analyzed using the Image J software (NIH)as follows:thresholdvalues were set to define the cell edge and amaskwas then createdfor each cellto get the totalcellarea(with arbitrary units)within the mask.For 15 min spreading (10% serum) and 24 h spreading, the exact spread area was calculated based on pixel dimensions during image acquisition
    2. Cell spreading assays were done as described previously (Balasubramanianet al., 2010).Briefly, cells were cultured in complete medium (10% FBS) or subjected to serum starvation (0.2% FBS for 12-14 h), trypsinized, counted using a hemocytometer, and held in suspension in the same medium containing 1% methylcellulose at 37°Cand 5% CO2in a 50 mL tube in a slanted position. After 90 min, cells werepelleted down at 1350 rpm for 8 min at 4°Cand the supernatant was discarded leaving 5 mL at the bottom. Cells were dislodged or displaced gently without touching the walls of the tubes, which might lead to activation of adhesion dependent signalling. Now, cells were washed once with excess of cold media (4°C)and one more time with warm media (37°C). A day before the experiments coverslips were coated with fibronectin (2 μg/mL)overnight at 4°Cand washed once with PBS before plating the cells. Cell suspension equivalent to 105cells was plated onto each fibronectin (2 μg/mL) coated coverslip and allowed to spread for 15 min or24 hin serum depleted (0.2% FBS) or complete medium. At each time point, unadhered cells were washed off with PBS and the coverslips were fixedwith 3.5-4% paraformaldehyde for 20 minat room temperature. After fixation coverslips are washed thrice with PBS and cells were permeabilized with 0.1% Triton X-100 for 5 min,followed by blocking with 5% BSAfor 30 min at room temperature. Fixed cells were stained with
    3. Cell spreading
  10. Apr 2019
  11. Dec 2018
    1. A geographical line has been drawn across the Union, and all the States north of that line have united in the election of a man to the high office of President of the United States, whose opinions and purposes are hostile to slavery. He is to be entrusted with the administration of the common Government, because he has declared that that “Government cannot endure permanently half slave, half free,” and that the public mind must rest in the belief that slavery is in the course of ultimate extinction.

      14 + 16 - The author expresses disdain towards the election of a Republican president, not elected in the South, is considered a hostile to slavery, and plans on abolishing it. These concerns imply that he should not be the president of the South.

  12. Sep 2018
    1. The whole of the clauses which refer to the latter are as complete as the most ardent supporters of union could desire, tempered by the lew exceptions by means of which the provinces have wished to shelter their local institutions from attack.

      §§.92(14) and 101 of the Constitution Act, 1867. of the Constitution Act, 1867.

    2. The 34th paragraph of the 29th clause of the scheme reads thus: ” The establishment of a General Court of Appeal for the Federated Provinces.” What is the object—what will be the character of the tribunal?

      §§.92(14) and 101 of the Constitution Act, 1867. of the Constitution Act, 1867.

    1. [Page 248]
    2. There is a provision that the nomination of the judges of the superior courts shall be vested in the General Government, but it would seem that the constitution of the courts is to be left to the local governments ; and I put the question, What does this mean ? Do you mean that the local governments are to establish as many courts as they please, declare of how many judges they will be composed, and that the General Government will have to pay for them ? Is a local government to say, here is a court with three judges ; we want five, and those five must be appointed and paid by the General Government ?

      §§.92(14), 96, and 100 of the Constitution Act, 1867.

    1. There is one currency here, another in Newfoundland, another in Prince Edward Island, and so on. The shilling and pound of this province are different from the shilling and pound of Newfoundland and those of the other Maritime Provinces. But, with Confederation, all these matters would be placed under the control of our central legislature; the currency would become uniform throughout, and capital might be everywhere invested without obstacle. So also it will be with respect to the rights of authors, patents for mechanical inventions, &c.

      §§.91(14) and 91(23) of the Constitution Act, 1867.

  13. Jul 2018
    1. 10

      Step 13:

      Attach the side panels to the back panel by using 4 101345 studs. Consult the graph for proper alignment.

      Step 14:

      Attach the previously assembled piece into the underside of the desk using 4 101345 studs. Consult the graph for proper alignment.

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    Annotators

  14. Mar 2018
    1. The control of property and civil rights, the administration of justice, including the constitution, maintenance, and organization of the courts of civil jurisdiction, and the procedure in civil matters, were also left to the local legislatures. From the peculiar position of Lower Canada it was felt impossible to confide the matter of civil law to the General Legislature. The principles upon which the civil law of Lower Canada were founded differed entirely from those of the English law. Under it property was secured, and civil rights of every kind maintained, and the people had no particular wish to see it changed, especially at this moment, when the work of codifying and simplifying it was about completed, and when they knew that within the next three or four months they would have it put into their hands in one volume. He thought it was undesirable to do away with that law, which had been beneficial to the country and under which it had prospered. It was necessary to have it left to the local Legislature, because all in Lower Canada were unwilling to have substituted another law with which they were unacquainted.

      §§.92(13)(14) of the Constitution Act, 1867.

    2. It was desirable the General Government should have the control of the medium through which the trade and commerce of the country was carried on, and that in the establishment of banks, the issue of paper money and in offering to the public the paper representative of their labor, in whatever part of the country, there should be the same legislative security for the people

      §§.91(2)(14)(15)(16) of the Constitution Act, 1867.

  15. May 2017
    1. We want to end gender inequality, and to do this, we need everyone involved

      This is also ethos because she uses the word 'we' again to show us that there is more than one person working on this problem.

    2. We want to try and make sure that it’s tangible.

      Emma wants you to trust her with this issue, so she is using words such as 'we' and 'make sure' to gain her audience's trust that she'll help this issue.

  16. Mar 2017
  17. Feb 2017
  18. Dec 2016