119 Matching Annotations
  1. Aug 2021
  2. Jun 2021
  3. 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)

  4. 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)

  5. 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
  6. 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
    3. For detection of protein-nucleic acid interaction, an electrophoretic mobility shift assay (EMSA) was conducted as described by Hellman and Fried (2007). Breifly, 8-10 μg of nuclear extract protein was incubated with binding reaction-mixture containing either 32P end-labeled double-stranded oligonucleotides (NF-κB, AP-1, p53 or SP-1) or unlabeled oligonucleotide as shown in table 2.3. The reaction mixture was incubated at 37°C for 45-60 min. After completion of reaction, 6μl of 6X DNA-loading dye was added and mixed well by gentle tapping.Table 2.3: Binding conditions for DNA-protein complexes in EMSA
    4. Gel shift assay
    1. The desired S.cerevisiae strain was grown overnight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500x gin 15 mL polypropylene tubes. Yeast cells were washed with PBS, suspended in 500 μL lysis buffer (Section 2.1.6.2) and were transferred to a 1.5 mL microcentrifuge tube. Yeast cells were incubated for 15 min on a thermomixer set at 65 ̊C and 750 rpm.After incubation, a volume equivalent to 500 μLof glass beads (0.5 mm) were added and cells were lysedon a beating apparatus by mixing three times for 45 seconds each with intermittent cooling on ice to prevent overheating.Cell lysates were centrifuged at 12,000 x gfor 5 min and upper aqueous phase was transferred carefully to a new 1.5 mLmicrocentrifuge tube, to which 275 μL of 3M sodium acetate was added. To this solution, 500 μL of chloroform was added, mixed well, and centrifuged at 12,000 x gfor 5 min at 4°C (this step was repeated twice). Supernatant was transferred to a new 1.5 mLmicrocentrifuge tubeand500 μL of isopropanolwas added and mixed well by inverting the tube 3-4 times. To precipitate genomic DNA, the suspension was centrifuged at 12000 x gfor 15minat 4°C. Precipitated genomic DNA was washed with 70% ethanol and dried at room temperature. The genomic DNA pellet was dissolved either in 100μLof Sigma molecular biology grade water or TE buffer supplied with Qiagen plasmid mini prep kit, andadd 1 μL of RNase solution (30 mg/mL) was added to this and incubated at 37oC for 1 h. The extracted genomic DNA was checked for integrityon a 0.8%agarose gel by electrophoresis and stored at -20oC
    2. Genomic DNA isolation by glass bead lysis method
    1. 44KDa to 670 KDa; Ovalbumin (44KDa), Conalbumin (75KDa), Aldolase (158KDa), Ferritin (440KDa), Thyroglobulin (669KDa)] purchased fromGE Healthcare Life Science. Blue dextran was used for the void volume of the column. The molecular weight of the protein complex fractions was calculated from the plot. The different fractions that were collected as eluates from the column were concentrated,and the presence of WWP2 or WWP1 as a monomer or multimer was identified by western blotting using specific antibodies for WWP2 and WWP1
    2. The WWP2-WWP1 heterodimeric complex formation under normal conditions and upon cisplatin-induced stress conditions was studied by gel exclusion chromatography. HEK293T cells untreated or treated with cisplatin were harvested,and cell lysate was prepared by using the standard protocol. Sephacryl S-200 (GE Healthcare) columns were equilibrated with 1X NETN (without Triton X-100) at a flow rate of 1ml/min. 0.8ml of cell lysate (1mg/ml) was passed through the Sephacryl S-200 column,and different fractions (fraction size; 500μl) were collected using Bio-Rad 2110 fraction collectorat the same flowrate. To determine the molecular weight of the fractions, column was calibrated with high molecular weight markers [range
    3. Gel filtration
    1. flash (Thermoscientific). β-Glucuronidase activity for GUS was expressed as nanomoles of MU produced/minute/108 cells
    2. In planta siderophore gene expression was studied by measuring β-glucuronidase activity. GUS marked BXOR1 strain and wild-typeBXOR1 (control) were inoculated in the leaves of 14 day old susceptible Taichung Native 1 (TN-1) variety of rice. After 10 days of inoculation, leaves were crushed and dissolved in 1 ml of MUG extraction buffer without adding MUG substrate (4-methylumbelliferyl β-D-glucuronide). Subsequently, 250 μl extraction buffer containing MUG was added, and incubated at 37°C for appropriate time (Jefferson et al., 1987). Next, 75-μl aliquots were taken from each reaction mixture, and the reaction was terminated by the addition of 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone (MU; Sigma) as standard at excitation/emission wavelength of 365/455 nm, respectively in
    3. In plantaGUS expression assay for siderophore cluster
    1. shRNA were processed for invasion studies. A suspension of 1×105cells in 200μLof 1%FBS containing DMEM was prepared and plated as described in Section 2.2.16. Cells were incubated at 37ºC with 5% CO2for 24 h to allow invasion. Downstream processing of the samples i.e. removing non-invading cells, staining and imaging were followed as described in Section 2.2.16. The number of cells invadedto the lower surface was quantified by counting the total number of DAPI positive nuclei in at least 10 random fields. The total number of cells invaded was normalized to non-targeting control cells and expressed as percentage invasion
    2. Invasion assays were conducted as described previously (Raoet al., 2015).Invasion chambers with pre-coated matrigel (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning) were used to conduct the invasion assays. Packages containing invasion chambers were kept outside and allowed to come to room temperature. Inserts were equilibrated by adding with 500 μLwarm (37ºC) 1% FBS containing DMEM to the upper chamber of the insert and bottom well of the companion plate, and rehydrated for 2 h in a humidified tissue culture incubator at 37ºCwith 5% CO2. The rehydration media was removed carefully without disturbing the pre-coated matrigel matrix on the membrane. Meanwhile, HCT116 cells expressing either non-targeting or IP6K1 targeting
    3. Invasion assay
  7. Dec 2018
    1. foolish imaginations of his heart

      Constructs which mistake the head as superior to the heart or as an adequate starting place for imaginings will always lead to destruction since the head can not handle nor is it designed for the necessary embedding and recursion which is a seed bearing fruit in itself. But it is sure that, before the brain breaks down and eventually falls/fails, those who mistake it as the best foundation will inevitably turn to mocking them who follow through with flow of spirit through and back to the heart. (see Lehi's Dream 1 Nephi 8:26-27 and Nephi's visitation of the same dream in 1 Nephi 11:35-36)

      This phrase,"foolish imaginations" can and ought to be read as the "FULLish imaginations of his heart." In earlier verses we see that a FULL rendering of Lehi's heart brought about fulfillment on several levels already. This "fulfillment" even took the immediate form of a "filament" (pillar of light which struck a rock in front of Lehi just as the communication struck his heart with overpowering energy). The electrical charging effects of Full Imagining can be transmitted beyond the individual and others can be made to feel these effects, however, unless they are allowed to take hold in the heart where embedding and recursion can take place, then they are short lived and sometimes can have disastrous overall effect upon others who rely on second-hand spirit and external motivation. (see 1 Nephi 3:28-31 and 1 Nephi 17:53-54)

  8. Sep 2018
    1. MR. CORNELLIER—All the confederations which you have mentioned were or are republican, and had the common fate of republican institutions. You have not said a word about monarchical confederations. MR. JOLY—I have made no mention of monarchical confederations, because none have ever existed, and none can exist. The principle of a monarchy is that the power resides in one person; the principle of confederation is that it resides in all the members of the confederation. A confederation would, therefore, always be a republic, even if formed of several states subject to a monarchy; because the power would not be vested in one person, but in each of the several states, of which no one would acknowledge a head ; it would be a republic consisting of a very small number of members. Before I take leave of all the confederations, the names of which I have mentioned, I intend to say one word, at least, in their favor. We understand that states

      §§.9, 17, 91 and 92 of the Constitution Act, 1867.

  9. Aug 2018
    1. The seat of the Federal Government is to be at Ottawa, of course. The Governor General or other head of this magnificent future vice-royalty, or what not, will hold his court and parliament at Ottawa ; but a handsome sop is thrown to Quebec and Toronto, also. They, too, are each to have a provincial court and legislature and governmental departments.

      §.17 of the Constitution Act, 1867.

  10. Jul 2018
    1. 12

      Step 17:

      Place the desk surface on the studs previously inserted. The two holes should face away from the opening of drawers. Secure the surface by screwing the 1 100218 into the pre-drilled hole using the 100001 tool provided with your package. Consult the graph for proper alignment.

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  11. May 2018
    1. But Theresa May, I think to -- on the heels of the Skripal attack, of the Russian nerve poison on a former agent and for which she garnered considerable international support, riding that wave into this -- and that response was a surprise to her, I think, in many ways, that going into this without getting parliamentary support, which at best would have been ambiguous, given the level of divisiveness and the precariousness of her position in Parliament, meant that she went along with that, without consulting with lawmakers and the backlash has been not just from Jeremy Corbyn or the opposition but also within her party, where there's a lack of consensus on whether or not she should have gone into this with Donald Trump, with French president Emmanuel Macron without greater consultation with the international community.

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    1. So, you are talking about a soggy weekend, five, six, 700 upwards of 800 millimeters of rainfall in some of this regions across the northern tier of the island of Kawai.

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    2. The attack in Douma was horrific attack on civilians using chemical weapons part of a Civil War that has killed hundreds of thousands of people.

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    3. Again after public outrage grew and the two issues as you say, have come together, one is about the continuing concern about violence against women and people are asking more than five years after that awful rape, gang rape in Delhi in 2012.

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    1. He ordered a military strike on Syrian chemical weapons facilities but then he hesitated and then reversed direction on advice from his most senior advisers on taking sanctions against companies that helped facilitate that deadly chemical weapons infrastructure by Syria.

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    1. Well in everything any time that you're talking about Russia and the relationship between the United States and Russia, it's always in the context of this cloud that's over the president about whether or not he's got some kind of ulterior motive for his policies and looked the combination of James Comey going out there over the last several days saying I don't know if the Russians have something that they are using to blackmail the president, and then the sort of abrupt, like hey, I don't want to do sanctions on Russia, you know, yes, there were some sanctions but he was very much pushed in the direction by Capitol Hill and others.

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    1. $130,000, if that's viewed as a campaign contribution, that is $127,300 above the legal limit.

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    1. It's $131,000.

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    1. This was back in 2013 President Obama looked at that chemical attack, and at that point in time, his general was saying, hey, it's not a slam-dunk, because you can detect there were Syrian claims, you can detect Syrians dropped bombs, but it's sometimes hard to know if they dropped an existing vehicle of weapons, and Syria has been known to use chemical weapons, or if the Syrians did it.

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    2. I will do two things, balance the budget, balances over five years with a 1 percent across the board cut, but also expand health savings accounts larger than anyone has tried in the past.

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    3. A 1 percent cut each year is about $13 billion, actually balances the budget in five years.

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    4. I think when you win by three million votes but lose by less than 80,000 votes in three states that decided the election, there's a lot that should be factors, but I think this was a very significant factor.

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    1. They are originally from bacteria in fungi and they were designed to be much faster, 10 hundred times faster than the original within a period of five to 10 years.

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    1. But I'll tell you, some of these flexes that we're having here, one story I picked up that I've never told before or written, but William Webster, former head of the CIA, told me that -- it's one of his best stories, he's allowing me to tell it -- that they created a body double for George Herbert Walker Bush at CIA, meaning somebody who looked just like him and it was uncanny how much CIA had created a human that looked like 41.

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    1. Do you -- do you -- do you -- I'm sorry, do you disagree with the $4 million for school safety -- $4 billion for school safety, the $6 billion for opioids

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