225 Matching Annotations
  1. Jun 2022
    1. User participation in any online internet community generally follows the 90-9-1 rule:90% of community members are lurkers who read or observe, but don’t contribute9% of community members edit or respond to content but don’t create content of their own1% of community members create new content
  2. Mar 2022
    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.

  3. Feb 2022
  4. Dec 2021
    1. These designers value expression over style

      It's about expressing oneself or creates distortion rather than valuing or achieving a certain look that they should convey.

    2. syntactical

      in a way that relates to the grammatical arrangement of words

      in a sentence: a syntactically complicated language


    3. Shattering the constraints of minimalism was exhilarating and far more fun than the antiseptic discipline of the classical Swiss school.

      That's understandable because doing the same type of style over and over again can be boring sometimes.

  5. Nov 2021
    1. As a result, the Texas law is a permissible regulation of speech.

      incorrect, they ruled that Johnson's actions did not threaten to disturb the peace or that the state's interests in preserving the flag as a symbol.... justify his criminal conviction for engaging in political expression

    1. Only certain religious groups are free to participate.

      I don't think this is apart of the opinion, I cant find where it says that only certain groups are free to participate.

  6. Oct 2021
  7. 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.

  8. Jun 2021
    1. For example, saver's credits make certain retirement savings tax-exempt, meaning savers get to keep more of the interest they earn on savings investments.

      This example is pulled from Giacomo's slides. Possible replacement: education fund accounts? (Same concept of tax-exemption, less common)

    2. In the United States and many other countries, the government taxes gains from private investment. Low capital gains taxes encourage investment and so also economic growth.

      This sentence is from OS

  9. May 2021
  10. Mar 2021
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 5.2

      AssayResultAssertion: Normal

      StandardErrorMean: 0.39

    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.1189A>T p.(Thr397Ser)


      AssayResult: 92.68

      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.242A>G p.(Lys81Arg)

    1. Source Data

      AssayResult: 95.74

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 14.87

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

    2. Source Data

      AssayResult: 62.31

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 11.49

      StandardErrorMean: 8.13

      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.13C>T p.(P5S)

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

      AssayResult: 32

      AssayResultAssertion: Abnormal

      ReplicateCount: 31

      StandardErrorMean: 5

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

    2. we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

      HGVS: NM_198056.2:c.1186G>C p.(Val396Leu)

  11. 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: 45

      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.216dup p.(Val73Argfs*76)

  12. Oct 2020
    1. Figure 9 shows the decreasing optical transmit-tance at the visible region with increasingfilm thickness(increasing immersion time)

      La figura 9 muestra el decrecimiento de las transmitancia óptica en la región visible con un incremento del grosor de la película (incrementando el tiempo de inmersión).



    1. Men who worship me,thinking solely of me,always disciplined,win the reward I secure.

      it state in this part that the man who worship me and think about me will be reward at the end by lord.

    2. I am the universal father,mother, granter of all, grandfather,object of knowledge, purifier,holy syllable OM, threefold sacred lore.

      Does Krishna showing his power to the world by calling himself as a universal father?

  13. Sep 2020
    1. 71 1. The Standard Version, Tablet IX [He] clad himself in their skins, he ate their flesh. Gilgamesh [dug] wells that never existed before, [he] drank the water, as he chased the winds. Sham ash grew worried, and bending down, he spoke to Gilgamesh: '0 Gilgamesh, where are you wandering? The life that you seek you never will find.' Said Gilgamesh to him, to the hero Shamash: 'After roaming, wandering all through the wild, when I enter the Netherworld will rest be scarce? I shall lie there sleeping all down the years! 'Let my eyes see the sun and be sated with light! The darkness is hidden, how much light is there left? When may the dead see the rays of the sun?'

      Gilgamesh seemed sad after the death of his friend Enkidu.

    2. The life that you seek you never will find.'

      Shamash was worried about Gilgamesh the death of Enkidu.

  14. Jul 2020
  15. Jun 2020
  16. Mar 2020
  17. Nov 2019
    1. Christian Reclamation Center

      christain reclamation center potential workplace

    2. a drive-in restaurant

      drive in near funeral home, reached by car

    3. THE BUDDHIST church up on the hill next to a playground

      buddhist church were mama's funeral was held

    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.

  18. Jul 2019
    1. Analysis of internal transcribed spacer region
    2. Effect of temperature on xylanase activity
    3. Effect of pH on xylanase activity
    4. Effect of various carbon sourceson xylanase activity
    5. Assayof xylanase activity
    6. Dinitrosalicylate reagent (DNS)(per liter)
    7. Citrate phosphate buffer
    8. Reagents
    9. Xylanase activity
    10. Harvesting of cultures
    11. Enzyme production (EP) medium
    12. Inoculum preparation
    13. Sporulation medium used for Trichodermasp
    14. Maintenance of Trichodermasp. culture
    15. Sterilization
    16. Materials for xylanase induction
    17. Induction of xylanase from Trichodermasp
  19. Jun 2019
    1. Purified HbS was digested with carboxypeptidase B (200 mg hemoglobin to 1 mg of the enzyme) for 3 hours in freshly prepared 0.05 M Tris-acetate buffer pH 7.1) at 25°C , followed by passage through a cation-exchange column using Whatman CM52.
    2. Preparation of HbS{des arg 141a
  20. May 2019
    1. On December 14, 2017, FCC commissioners revoked Network Neutrality rules by a 3–2 vote. As a result, ISPs can now legally offer “tiered service” favoring some websites, services, and applications with faster connections, blocking others, or charging some conNet Neutrality Why It Matters to School Librarians Feature ARTICLE tent providers greater fees to connect to their customers (Fung, 2017). This is the “fast lane” and “slow lane” concept

    1. Protein concentrations were detennined using BCA protein estimation kit (Pierce, .. USA). The assay was perfonned according to the instructions provided by the manufacturer. Various dilutions of the sample or BSA were made in appropriate buffer and 200 J.ll of supplied reagent mix (1 :50 ratio) was added to each well in a 96 well plate. The plate was incubated at 37°C for 1 h and the absorbance was measured at 540 nrn.
    2. Protein Estimation
    1. antibody at an appropriate dilution. The immunoreactivity was detected by enhanced chemiluminescence using an ECL detection kit (Amersham Biosciences) and were recorded on X-ray films after appropriate exposure and development. It is important to note that the blots for probing phosphorylated proteins were performed using 1% BSA as blocking agent instead ofblotto
    2. Whole cell extracts were prepared by treating cells with lysis buffer (0.125M Tris, 4% SDS, 20% glycerol, and 10% ~-mercaptoethanol), and protein estimation was performed using CB-X protein assay kit as per manufacturer's protocol. Lysates were resolved on 12% SDS-PAGE gel, following which Western transfer was performed onto nitrocellular membranes using a BioRad Western transfer apparatus. The blots were incubated with 5% blotto (non-fat dry skimmed milk) in 0.05% PBS-Tween 20 for 1 h to block non-specific binding sites following which they were incubated for 1 h with primary antibody at an appropriate dilution prepared in 1% blotto in 0.05% PBS-Tween-20. The blots were washed 3x with 0.05% PBS-Tween-20 at 5 min intervals following which they were incubated for 1 h with secondary
    3. SDS-PAGE and Western blot
    1. Sequencing of cloned inserts was done by Sanger's dideoxy chain termination method (Sanger et al., J 977) using Sequenase version 2.0 kit from USB. 5 J.ll of J mg/ml suspension of plasmid DNA was incubated in denaturation buffer at 37 °C for 30 min. in a reaction volume of 50 J.ll, and then the precipitation was carried out in the presence of 5.5 J.ll of 3M sodium acetate and 4 volumes of chilled ethanol at -70 °C for 30 min. The pellet, obtained by centrifugation at 10,000 g, for 20 min., at 4 °C, was washed with 70% ethanol and resuspended in 7 J.ll of sterile water. J pmole of sequencing primer in J J.ll water and 2J.ll of 5X sequenase reaction buffer were added to denatured DNA and the reaction mix was incubated at 65 °C for 5 min. for primer annealing. The reaction mixture was cooled slowly to about 35 °C, by putting the heat block at room temperature. For labeling, J J.ll of 0. J M DTT, J J.ll radioactivity containinig J 0 J.lCi of 35S dATP, 2 Jlllabeling mix diluted 5-fold in steriJe water and 2 Jll sequenase enzyme diluted 8-fold in sequenase dilution buffer were added to the· primer annealed DNA. Incubated the reaction mixture at room temperature for 2-5 min. and added 3.5 J.ll to each of the 4 different tubes containing 2.5 J.ll dideoxy nucleotides ddATP, ddTTP, ddCTP, and ddGTP separately. The mixture was incubated at 37 °C for 5 min. and finally, the reaction was stopped by adding 4 J.ll of estop solution to each tube. Reaction products were separated on a 6% polyacrylamide sequencing gel made in TBE buffer containing 7.5 M urea. The samples were heated at 75 °C for 2 min. and immediately loaded on the gel. The gel was run at a constant power of 60 watts maintaining the temperature of gel between 50-55 °C, dried and exposed to an X-ray film.
    2. DNA Sequencing
    1. r-bZP3 was arsanilated using a modification of the procedure of Nisnoff (1967). Briefly, arsanilic acid (100 mg) was dissolved in 5 ml of I M HCI. A IO ml stock of NaN02 (10 mg/ml) was also prepared fresh and added dropwise to the arsanilic acid solution while vortexing. Activation of arsanilic acid was checked on starch-KI paper. The ice cold activated arsanilic acid solution was added dropwise to the protein solution (5 mg of r-bZP3 in 100 mM PB, pH 7.4) stirring constantly in an ice water bath, while pH was maintained between 9.0 and 9.5 with 10 N NaOH. The protein solution was dialyzed extensively against I 00 mM PB having 4 M urea. Arsanilation of r-bZP3 was checked by ELISA using ars-r-bZP3 for coating ( 1 flg/well) and using a 1:100 dilution of murine anti-ars MAb, R 16.7 (Durdik et al., 1 989). Bound Ab was revealed using anti-mouse HRPO conjugate (I :5000). Three monkeys previously immunized with r-bZP3-DT conjugate (MRA-375, MRA-640 and MRA-672) and 2 naive monkeys (MRA-446 and MRA-670) were immunized at 2 intramuscular sites with 250 flg of ars-bZP3 conjugate using Squalene:Arlacel A ( 4: 1) as an adjuvant. Boosters were administered at intervals of 20 days and bleeds were collected I 0 days post immunization. Bleeds were analyzed by ELISA using r-bZP3 and ars-BSA for coating to determine anti-bZP3 and anti-ars Ab titres as described earlier.
    2. Arsanilation of r-bZP3
    3. Cells and supernatant collected 72 h pi were analyzed on a 0.1% SDS-I 0% PAGE and Western blot using polyclonal Abs generated in rabbit against peptide-DT conjugates using (i) 23-45 aa residue N-terminal peptide with an extra lysine at the N-terminus (KQPFWLLQGGASRAETSVQPVLVE), (ii) 300-322 aa residue C-terminal peptide (CSFSKSSNSWFPVEGPADICQCC) corresponding to the bZP3 sequence (iii) MA-451 and (iv) goat anti-GST Ab. The C-terminal anti-peptide Ab was used for determining whether or not the full length bZP3 was being expressed by the cells infected with the different viruses. Anti-mouse (I :500), anti-rabbit ( 1 :500) or anti-goat ( 1 :500) Abs conjugated to HRPO were used for revealing bound Ab.
    4. Analysis ofr-bZP3 in VI, V2, V3 and V4 Infected Cells and Supernatant
    1. From an 0/N grown culture, 1 ml cells were pelleted in a 1.5 rnl eppendorf tube. The cells wer~ washed once with 100 ul of solution I ( 50 rnM glucose in 25 rnM Tris. HCl ,· pH 8. 0 ) . The cells were pelleted again and resuspended in 70 ul of solution I. To this, 20 ul of a freshly prepared solution of lysozyme 10 rng 1 ml in distilled water was added. The tube was vortexed to mix the contents and incubated in ice for 5 minutes. Next, 10 ul of 0.1 M EDTA, pH 8.0, was added, vortexed and the tube incubated in ice for 5 minutes. Next, 200 ul of solution IV ( 0.2 N NaOH + 1 % SDS was added, the contents vortexed quickly but briefly to mix and incubated in ice for 5 minutes. Finally, 150 ul of 5 M potassium acetate, pH 4. 8 was added and the tube incubated in ice. After 60 minutes, the tube was centrifuged for 10 minutes at 10,000 rpm, at 4°C. 450 ul of the supernate was removed to another tube and DNA precipitated with two volumes of ethanol at -7 0°C for 15 minutes. The DNA pellet was collected by centrifugation and after draining off the supernate, the pellet was washed with 80 % ethanol. The pellet was dried briefly under vacuum and finally resuspended in 150 ul TE. From this, a 10 ul aliquot was used for checking on gel or for•setting up digestions with restriction endon~cleases.
    2. Plasmid DNA minipreps.
    3. Nitrocellulose membranes ( BA85 ) were obtained from Schleicher and Schuell, Germany. GeneScreen and GeneScreen Plus membranes were from DuPont, USA. Millipore membranes ( 0.45 um ) were from Millipore Corporation, USA. 3 MM and 1 MM chromatography filter papers were from Whatman Ltd, U.K.
    4. other Materials.
    1. Selected transformants were grown in 250 ml of LBamp 0/N. The cells from the 0/N cultures were harvested by centrifugation (4°C) at 4000 X g for 30 min. The cell pellet was resuspended in 5 ml of TEG solution containing lysozyme (2.0 mg/ml in 10 mM Tris-HCl, pH 8.0) and incubated at RT for 15 min. Alkaline-SDS (10 ml) was added to the mixture and again incubated at R T for 10 min after mixing the contents gently by inverting the tube. Post-incubation, chilled sodium acetate solution (7.5 ml) was added and the contents were incubated on ice for 15 min. After incubation, the mixture was centrifuged at 10,000 X g at 4°C and processed in the similar fashion as described above upto addition of isopropanol. The DNA pellet was resuspended in 500 Jll TE containing 20 Jlg/ml RNase and incubated for 1 h at 37°C. Plasmid DNA was then extracted as described above. The DNA pellet was air-dried and finally dissolved in 200 Jll ofTE
    2. Large scale plasmid DNA isolation
    1. EROD
    2. TheO2consumptionofthetissueswasmeasuredbymanometrictechniquesinaWarburgconstantvolumerespirometer(Gallenkemp,England)aspertheproceduresgivenbyUmbreitetal.(1959).Thecontrolandeffluentexposedfisheswerekilledandthebrain,gill,muscle,liver,heart,kidneyandair-breathingorganswereisolated.ThetissueswereslicedandplacedinWarburgflasks(60-80mgtissuesflask)containing2.5mloffishringersolutionwithphosphatebufferatpH7.5asthesuspensionmediumforthetissuesand0.2ml15%KOH.Temperaturewas28°CduringO2uptakedetermination.Inordertocomparedatafromthedifferentseriesoftreatment,arespiratoryindexwascalculatedusingthefollowingformula:KO2treated-KO2controlr=100-------------------------------------x100KO2controlWhere,K=O2consumptioninpi/100mgwettissue/hrThisindexindicatespercentrespirationoftreatedtissuesrelatedtothecontrolvalues ofthesameseries.
    3. TissueRespiration
    1. incubator until the cells were 60% confluent. For each transfection, 1-2pg of DNA was diluted in 100 pi serum free media. Also, lOpl of lipofectin reagent · was diluted in 100 pi of serum free media and allowed to stand at room temperature for 30-45 minutes. The two solutions were combined, mixed gently and incubated at room temperature for 15 minutes. The cells were washed once with 2ml of serum free medium. For each transfection, 0.8 ml of serum free medium was added to each tube containing lipofectin-DNA complexes. The complex was mixed gently and overlaid onto cells. The plate was incubated for 4-6 hrs in a CDl incubator. The medium in each well was replaced with serum containing medium and the cells were further incubated for varying periods of time at 370C. The concentration of lipofectamine 2000 was used in the ratio 1:2 or 1:3 with DNA. The Rzs and Dzs were either co-transfected with the plasmid DNA of interest or when required to be transfected alone then pBSK+/-was used as carrier plasmid for better transfection efficiency. In order to ensure uniform transfection efficiency a reporter plasmid DNA (pSV -~ gal, Promega) was used
    2. Transfection of cell lines used was carried out u5ing lipofectin reagent (Invitrogen, U.S.A.). In a six well plate 10 s cells/ well were seeded in 2m1 medium supplemented with serum. The cells were incubated in a CD2
    3. Materials & Methods dried. These were counted directly to determine the total counts. In duplicate tubes, 1pl of the diluted probe was added to 100pg of carrier nucleic acid (tRNA or Herring Sperm DNA) in a total volume of 100pl. To this 500pl of ice-cold 5% TCA was added, mixed thoroughly and incubated on ice for 15-20 min. Glass fiber filters were wet (in duplicate) properly with 5% TCA and then these samples were applied on to them under vacuum. The filters were washed twice with 5ml of chilled 5% TCA and then air dried after rinsing with 2m1 of acetone. All the dry filters were inserted into scintillation vials containing scintillation fluid and the counts were taken in a liquid scintillation a-counter (LKB Wallac, 1219 Rackbeta, Sweden). The percentage incorporation, specific activity and the total amount of RNA made was then calculated according to the standard procedures. % incorporation =Incorporated cpm X100 Totalcpm Total RNA made (ng) = % incorporation X 338 Specific activity of probe = Total cpm incorporated p.g of RNA synthesized Cell culture media and cell lines: All the cell lines were grown and maintained in Dulbecco' s modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (penicillin, streptromycin and amphotericin B). The cells were maintained at 37<>C with 5% C02 in a humidified CD2 incubator (Nuaire-IR Autoflow CD2 Water-Jacketed incubator). Transient transfection
    1. After PCR, 1 μl of Dpn1 enzyme (10U/μl) was added to the amplification mix and incubated at 37°C for 6hours. After that, 10ml of the amplification mix was taken to transform Dh5a cells. Positive clones were selected after confirming the sequence of plasmid DNA
    2. The PCR parameters were as follows
    3. The reaction mix included 2ml of PSKll(39+) (50ng) containing wild type K-Ras cDNA , 5ml 10x buffer, 20pmoles of primers , 1ml of 10mM dNTP mix and 1ml of deep vent polymerase (NEB).
    4. The following K-Ras-ras mutants were generated by site directed mutagenesis according to the protocol described in QuickChange site directed mutagenesis kit (Stratagene). The primers used are shown in the following table
    5. Site Directed Mutagenesis
    1. Denaturation 95°C 1 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C
    2. The PCRs were normally performed using a PCR amplification kit from Fermentas/Sigma (USA), following the company's protocols. Approximately, 10 ng of chromosomal or 1-2 ng of plasmid DNA was used as template in a 50 μl reaction volume containing 0.2 mMM of each dNTP, 20 picomoles each of forward and reverse primer and 0.5 units of Taq DNA polymerase. In some cases, freshly streaked E.coli cells from a plate were resuspended in 50 μl of sterile Milli Q water to get a cell suspension (~ 109 cells/ml) and 10 μl from this was used as the source of DNA template. The samples were subjected to 30 cycles of amplification and the typical conditions of PCR were as follows (although there were slight modifications from one set of template/primers to another): The initial denaturation was done at 95°C for 3 min and the cycle conditions were as given below. Annealing 55°C 1 min Elongation 72°C (1 min/kb of DNA template to be amplified)
    3. Polymerase chain reaction (PCR)
    4. Most chemicals were obtained from commercial sources. The sources for some of the fine chemicals used in this study are given below. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were routinely from Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4 DNA ligase, DNA polymerases for PCR amplification and DNA size markers were obtained from companies including New England Biolabs and Fermentas. Quiagen or HiPura Kits used for plasmid isolation, purification of DNA fragments. The oligonucleotide primers used in this study were mainly synthesized on order by Ocimum Biosolutions or MWG Biotech Pvt. Ltd
    5. Chemicals
    1. 1mL medium were stained with 1p.L of the dye (1ng/mL) and incubated at 37°C for 10 min in the dark. The unbound dye was washed off with either PBS or medium and cells analysed by flow cytometry or microscopy. Nonylacridine Orange (NAO): NAO (Molecular probes) is a probe which interacts specifically with non-oxidized cardiolipin, a lipid that is exclusively localized in . the inner mitochondrial membrane (Petit et al., 1992). A stock solution of 100p.M was prepared in DMSO and 1p.l (1nM) was used to stain 107 cells in 1mL medium for 10 min at 37°C. The excess dye washed off with PBS and cells were fixed with 4% formaldehyde for 3 min. Subsequently they were analysed with flow cytometry or microscopy
    2. Mito Tracker Green®: Mito Tracker® Green (Invitrogen, Carlsbad, CA) is an agent that interacts with mitochondrial lipids and is essentially non-fluorescent in aqueous solution, only becoming fluorescent once it accumulates in the lipid environment of the mitochondria. Since the entry of the dye is not dependent on the mitochondrial membrane potential, it can be used to measure/ compare the mitochondrial mass of cells both in live as well as fixed states. MitoTracker Green® stock solution was prepared in DMSO (1pg/pL) and stored at -20°C. To stain cells
    3. Assay for measuring mitochondrial mass
    4. The PCR products generated using protocol mentioned above were purified using QIAquick PCR purification kit from Qiagen (Hilden, Germany) as per manufacturer's protocol. Briefly, S volumes of buffer PB was added to 1 volume of PCR sample. This was applied to QIAquick spin column and centrifuged at 10,000 x g for 30-60s. The flow-through was discarded and the column washed with 0.7SmL of buffer PE. After discarding the flow-through again, the column was dried by a quick spin. The DNA was eluted using 30-SO J..lL of elution buffer (Buffer EB (10mM Tris-Cl pH 8.S)) or alternatively in 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 facto
    5. Purification of PCR produc
    1. were maintained in log-phase by continuous passaging in fresh YNB medium every 4 h
    2. For phosphate starvation,yeastcells grown to log-phase in YNB medium were harvested,washed with water,transferred to either regular YNBor YNB medium lackingphosphate and were grown for 16 h at 30 ̊C. Cells cultured in YNB medium
    3. Phosphate starvationof yeast cells
    4. .coliBW23473 electro-competent cell aliquots werethawed on ice and mixed with 1-2 lof plasmid DNA. Mixture was pulsed with the Gene Pulser®electroporation apparatus(Bio-Rad) at 1800 Volts with 25 μF and 200 Ωcurrentin a chilled0.1 cm electroporation cuvette(Bio-Rad). Immediately after successful pulsing, 1 ml LB medium was added to the cuvetteand suspension was transferred toa 1.5 ml sterile centrifuge tube. Cells wereincubated at 37°C for 1 hwith shaking and further plated onLB plates containing kanamycin(30μg/ml). Positive colonies were inoculated in LBliquid medium containing kanamycin(30μg/ml)for plasmid isolation


    5. E.coliBW23473transformation by electroporationE
    6. C. glabratayeast cells were grown overnight in 5 ml YPD medium at 30 ̊C. An aliquot from the overnight culture was inoculated in 10 ml fresh YPD medium to an initial OD of 0.1. Cells were incubated at 30 ̊C till the cultureOD600was between 0.4 and 0.6. Cells were harvested in a sterile 50 ml centrifuge tube and washed twice with sterile Milli-Q(MQ)water. Washed cells were suspended in 100 μl of 100 mM LiOAc, mixed thoroughly and transferred to a sterile 1.5 ml microcentrifuge tube. A transformation mix containing 240 μlpolyethylene glycol(PEG) (50% (w/v)), 36 μl LiOAc(1 M), 25μl ultrapure single-stranded salmon sperm DNA (2 mg/ml) (Clonetech) was added to 50 μl cell suspension. 50 μltransforming DNA (1μg circular plasmid DNA) was added to the above suspension. Whole mixture was vortexed gently and incubated at 30 ̊C for 45 min. 43 μl DMSO was added to the tubeand incubated at 42 ̊C for 15 min. Cells were collected after centrifugation at 5,000 rpm for 1 min and suspended in minimal medium containing 0.6% Bacto-Casamino acid. Transformation mixture was plated on CAA plates and transformants were selected for uracil prototrophy
    7. Yeast transformation usinglithium acetate (LiOAc) strategy
    1. Nucleosomal-associated DNA was extracted from RPMI-grownand macrophage-internalized C. glabratacells using EZ NucleosomalDNA prep kit (ZYMO Research),treated withmicrococcal nuclease digestionfor 2.5, 5, 7.5 and 10 min at 25ºC andwasresolved on 2% agarose gel
    2. Micrococcal nuclease digestion assay
    3. centrifugation at 5,000 rpm for 4 minat room temperature. Harvested cells werewashed with PBS and treated with different compoundse.g.H2O2. After treatment,cells were harvested and further processed according to the type of experiments performed
    4. For several experiments, log-phase C. glabratacells were harvested and treated with different compounds. For this, single colony of aC. glabratastrain was inoculated in YPD-liquid medium and grown for 14-16 h at 30ºC withcontinuous shaking at 200 rpm. Overnight cultures were reinoculated in YPD medium to an initial OD600of 0.1 andgrown for another 4 h. These log-phase cells were harvested by
    5. Harvesting of and treatment to logarithmic phase C. glabratacells
    6. To collectmacrophage-internalized yeast cellsfor RNA and protein extraction, 107THP-1 monocytes were seeded in 100 mm cell culture dishes and treated with PMA. PMA-differentiated THP-1 macrophages were infected with appropriateC. glabratastrainsto a MOIof 1:1. Equal numberof C. glabratacells wasinoculated inRPMI medium as control. Two hourspost infection,non-phagocytosed yeast cells were removed by washing THP-1 macrophages thrice with PBS. At different time points, culture dishes were washed twice with chilled PBS and 2 mlchilled sterile water was added toeach dish to lyse the macrophages. Corresponding cultures grown in RPMI medium were transferred to50 ml polypropylene tubesand transferred on ice. Lysates were collected by scrapping the macrophage monolayer and transferred to50 ml polypropylene tubes.RPMI-grown and macrophage-internalized C. glabratacells were harvested by centrifugation at 2,500g for 8 min. Macrophage cell debris were removed frommacrophage-internalized cells by repeated washing with chilled sterile water. Harvested C. glabratacells were stored at -20ºC till further use
    7. Harvesting of macrophage-internalized C. glabratacellsfor RNA and protein extraction
    1. nvolved use of GFP based vector system, the expression of the transgene was visualized under fluorescent microscope with excitation filter of 485+20 nm
    2. Transient transfection of plasmid DNA in cellswas performed usingLipofectamine 2000transfection reagentaccording to manufacturer’s protocol. Briefly, 0.5 to 1million cellswere seeded in a 35mm tissue culture dish one day prior totransfection. For each 35mm dish, 4μg DNA was mixed in 250μl of Opti-MEMin one polypropylene tube. In another tube 10μl of Lipofectamine 2000 was diluted in250μl Opti-MEM and incubated at room temperature for 5 minutes. DNA and Lipofectamine 2000 were mixed together and allowed to form complexes for 30minutes at room temperature. Meanwhile, the adherent cells were washed twicewithPBS and 1ml of Opti-MEM was added. 500μl of complexes were then added to each dishcontaining cells and medium. After 6-8 hrs, the medium containing complexes wasremoved and complete medium was added and transgene expression was evaluated 24-48 hrs after transfection. Since most of the experiments
    3. Transient transfection in adherent cells
    4. Inoue buffer
    5. For preparation of Ultra competent cells
    6. Aprotinin
    1. Co-Immunoprecipitation assays were performed essentially as described by Lee (Lee, 2007). For a typical immunoprecipitation assay, cells were washed with ice-cold PBS and scapped in ice-cold microfuge tube. Then, cells were lysed with NETN buffer (containing 1 μg/ml each of leupeptin, aprotinin, 10mMeach ofNaF and phenylmethylsulfonyl fluoride (PMSF))on shaking rotator in cold room for 30 min. After centrifugation, the whole cell lysate (500 μg-1 mg) obtained was incubated with 1 μg of antibody of interest(orwithisotype control)on shaking rotator in cold room for 3 h, followed by addition of 10-20 μl of ProteinSepharose A/G beads (Santa Cruz) for 1 h. The immuno-complexes bound to beads were then pelleted atlow speed centrifugation (2500 rpm for 3 min) and washed three times with NETN buffer. The proteins bound to beadswere resolved by SDS-PAGE and immunoblotting was performed accordingto standard protocol described earlie


    2. Co-Immunoprecipitation (Co-IP)
    3. Stripping buffer
    1. After the HPLC run, vials numbered from 50 to 65 were surveyed using a Geiger counter, 4 vials with high counts were pooled into one vial and 5 μL of this 4 mL solution was measured in a liquid scintillation counter (Perkin Elmer). To the remaining solution, 800 μL of 50% ammonia solution was added to neutralise the pH and the tube was kept on ice. In a 50 mL conical tube, 45 mL of chilled water was taken, to which the neutralised IP7solution was added and kept on ice. A Sep-Pak cartridge [Waters, WAT020545] was equlibrated with 10 mL of ice cold deionized water using a 10 mL syringe. The Sep-Pak cartridge was attached to a 60 mL syringe and 50 mL of diluted IP7 solution was passed slowly through ot so that IP7would bind to the Sep-Pak column. The cartridge was washed with 8 mL of chilled water, followed by chilled 8 mL 0.2 M triethylammonium bicarbonate solution pH 8.5 (4 mL of 1.5 M triethylammonium bicarbonate, pH 8.5 + 26 mL chilled water). The bound IP7 was eluted in 4 mL of 1.5 M triethylammonium bicarbonate solution, pH 8.5, into three 1.5 mL microfuge tubes. The eluted IP7 was concentrated in a vacuum concentrator (Scanvac) at 2000 rpm, 25°C, to obtain 30-50 μL of a 1-2 μCi/μL solution of radiolabelled IP7
    2. A mixture of 40 mL of DEPC treated water and 0.6 gof agarose was meltedby boiling. After cooling down the temperature to 55°C, 8.4 mL of formaldehyde (final concentration 2.2 M)and 5 mL of 10X MOPS were added, mixed well and poured into a boat to cast a gel
    3. Preparation of formaldehyde agarose gel(50 mL) and RNA sample
    1. protein agarose beads for 1 hour at 4 c and then washed three times with 1X NETN. The proteins bound to S-protein agarose beads were resolved by SDS-PAGE and visualized by Coomassie staining. Proteinspresent in the gels were analyzed by Mass Spectroscopy
    2. HEK293T cells were transfected with S-protein/FLAG/SBP (streptavidin binding protein)-triple tagged WWP2/Dvl2 and then three weeks later puromycin-resistant colonies were selected and screened for WWP2/Dvl2 expression. The positive stable cells were then maintained in RPMI1640 supplemented with 10% FBS and 2 g/ml puromycin. The stable cells(harvested cells from ~30 tissue culture plates of 10cm size)were collected in 1X PBS by scraping them off the plates, and were lysedwith NETN buffer (20 mM Tris-HCl,pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 % Nonidet P-40) containing 50 mM -Glycerophosphate, 10 mm NaF, 0.5 mM PMSF, 1 g/ml of each Pepstatin and Aprotinin on ice for 20 minutes. After removal of cell debris by spinning, cell lysates were incubated with streptavidin sepharose beads for 1 hour at 4C. The bound proteins were washed three times with 1X NETN and then eluted twice with 2 mg/ml Biotin for 60 minutes at 4 C. The eluateswere incubated with S-
    3. Tandem affinity purification
    1. culture dishes and dishes were incubated at 28ºC. OD600 was measured after 16 and 42 h of incubation, and percentage inhibition of growth was determined with respect to the growth in the corresponding control cultures containing PS media without streptonigrin
    2. For streptonigrin sensitivity assay, different strains of Xanthomonas oryzaepv. oryzicolawere grown overnight with appropriate antibiotics as described earlier. 0.2% of primary inoculum was added into fresh PS medium and grown for 24 h till the OD600reached 0.6. Serial dilution of bacterial cultures were performed as mentioned earlier, and 5μl diluted cultures were spotted on PSA plates containing different concentration of streptonigrin (0.05 μg/ml, 0.1 μg/ml and 0.15 μg/ml). Plates were incubated at 28ºC for 72 h and plate images were captured and analyzed for comparative growth inhibitionin different strains caused by streptonigrin. Further, streptonigrin sensitivity assay in liquid broth was performed by growing different strains as described previously (Wilson et al., 1998).Briefly, Xanthomonas oryzae pv. oryzicolastrains were grown to an OD of 1 in PS medium with appropriate antibiotics. Cells were pelleted down, and resuspended in fresh PS medium at an OD600of 0.6. Next, 100 μl culture was inoculated in 4 ml PS medium with or without streptonigrin. Streptonigrin was added to a final concentration of 0.1μg/ml into
    3. Streptonigrin sensitivtity assay
    4. insert of 1:3 for sticky end ligations. Ligation mix was incubated either at 22°C for 30 min or 16°C for 14-16 h. After incubation, T4DNA ligase was inactivated at 65°C for 20 min
    5. After restriction enzyme digestion, digested products were resolved on agarose gels, and desired DNA fragments were extracted from the gel. Otherwise digested DNA fragments were precipitated by Phenol-choloroform-isoamyl alcohol method. Concentration of gel extracted or precipitated fragments were determined using spectrophotometer and ligation reactions were set up using a molar ratio
    6. Ligation
    1. The reaction mixture contained 10 μl of culture filtrate, 490 μl of double distilled water (DDW) and 300 μl of methanolic rhodanine solution. This mixture was incubated for 5 min at 30°C in a water bath. The reaction was stopped by adding 0.3 ml of methanolic rhodanine solution (0.667 %), which resulted in the formation of complex between gallate and rhodanine. This was followed by the addition of 0.2 ml of KOH solution (0.5N) and the tubes were further incubated at 30°C for 5 min. The total reaction mixture in each tube was diluted with 4.0 ml of distilled water. Tubes were further incubated at 30°C for 10 min. The absorbance was measured at 520 nm against a control having distilled water in place of culture filtrate. The absorbance thus obtained was used to calculate the amount of gallic acid present in the culture filtrate, from the standard gallic acid curve prepared in the range of 100-1000 μg/ml.
    2. The procedure of Sharma et al. (2000) was used to estimate the gallic acid in the culture filtrate. Reagents: Methanolic rhodanine solution (0.667% w/v): Prepared by dissolving 0.667 g of rhodanine in 100 ml of methanol.Potassium hydroxide (0.5 N): 2.8 gpotassium hydroxide dissolved in100 ml of distilled water.
    3. Gallic acid estimation (Sharma et al., 2000)
    1. For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction
    2. Yeast colony PCR
    3. For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction
    4. Yeast colony PCR
    1. dropped on clean prechilled slides held at a 45°angle from a height of 18 inches, dried at 55°Con a hot plate and stained with 3% geimsa in phosphate buffer (pH 6.8) for 10 min. Slides were washed three times and dried, individual metaphase spreads were analyzed using bright field microscopy (Zeiss Axio Scope), and chromosomal aberrations were scored manually from each spread
    2. MEFsplated in 6 cmdishes were treated with MMC (300 nM) for 12 h. Post damage cells were treated with colcemid (0.1 μg/mL) for 4 hto arrest cells in metaphase. After 4 h cells were washed twice with PBS to remove traces of colcemid and harvested by trypsinisation, pelleted down at 100 x g for 5 min in 15 mL conical tubes. The supernatant was removed by leaving 0.5 mL and resuspended thoroughlyby pippeting. Cells were subjected to hypotonic swelling in pre-warmed 0.075 M KCl for 15 min at 37°C[Note: key to this step is to add hypotonic solution drop by drop slowly otherwise cell clumps will form which are impossible to disperse. Add a few drops first then pipette the cells up and down to mix them thoroughly and add few more drops. Invert tubes 2-3 times during the incubation]. After 15 min cells were fixed by adding few drops of chilled fresh fixative (methanol: glacial acetic acid; 3:1) kept in the deep freezer. Cells were pelleted down at 100 x g for 8 min, the supernatant was removed, 0.5 mL of fixative was left behind and the pellet was resuspended very gently. Slowly fresh fixative was added in the same manner as the KCl solution and cells were pelleted down at 100 x g for 8 min; this step was repeated twice. The supernatant was removed by leaving 0.5 mL fixative and the cells were resuspended and mixed well before preparing metaphase spreads. Cell suspensions were
    3. Cytogenetic analysis
  21. Apr 2019