262 Matching Annotations
  1. Last 7 days
    1. “If now, in addition to all these things, you have properly reflected upon the odd disorder of the chamber, we have gone so far as to combine the ideas of {dd}an agility astounding, a strength superhuman,{dd} a ferocity brutal, a butchery without motive, a grotesquerie{e} in horror absolutely alien from humanity, and a voice foreign in tone to the ears of men{f} of many nations, and devoid of all distinct or intelligible syllabification. What result, then, has ensued? What impression have I made upon your fancy?”

      I love how Dupin uses "Fancy" at the end of the sentence. Since it emphasize again the case is not normal, so you have to fancy it and see it with your imagination.

    2. In a word, why did he abandon four thousand francs in gold to encumber himself with a bundle of linen? The gold was abandoned. Nearly the whole sum mentioned by Monsieur Mignaud, the banker, was discovered, in bags, upon the floor. I wish you, therefore, to discard from your thoughts the blundering idea of motive{m} engendered in the brains of the police by that portion of the evidence which speaks of money delivered at the door of the house.

      The motivation is not out of money but something peculiar.

    3. This relieves us of all doubt upon [page 549:] the question whether the old lady{c} could have first destroyed the daughter, and afterward{d} have committed suicide. I speak of this point chiefly for the sake of method; for the strength of Madame L’Espanaye would have been utterly unequal to the task of thrusting her daughter's corpse up the chimney as it was found; and the nature of the wounds upon her own person entirely preclude the idea of self-destruction. Murder, then, has been committed by some third party; and the voices of this third party were those heard in contention. Let me now advert — not to the whole testimony respecting these voices — but to what was peculiar{e} in that testimony. Did you observe any thing peculiar about it?” I remarked that, while all the witnesses agreed in supposing the gruff voice to be that of a Frenchman, there was much disagreement in regard to the shrill, or, as one individual termed it, the harsh voice.

      They know that the testimonies might already lead the observation in the wrong way, which is earlier to be accepted. However, it limits the imagination of thinking out of the box.

    4. There was something in his manner of emphasizing the word “peculiar,” which caused me to shudder, without knowing why. “No, nothing peculiar,” I said; “nothing more, at least, than we both saw stated in the paper.”

      We see that the attitudes of the narrator and Dupin toward the case are so different. And the narrator's perspective is more like usual but this case is obviously strange and peculiar as it is mentioned. Therefore, this two sentences highlight again and alert the reader - the case should be thought the other sad around.

    5. Upon reaching the first landing, heard two voices in loud and angry contention — the one a gruff voice, the other much shriller — a very strange voice. Could distinguish some words of the former, which was that of a Frenchman. Was positive that it was not a woman's voice. Could distinguish the words ‘sacré’{s} and ‘diable.’ The shrill voice was that of a foreigner. Could not be sure whether it was the voice of a man or of a woman. Could not make out what was said, but believed the language to be Spanish.{t}

      This is the first testimony mentioned the voices of the murderer, which to the point of the core characteristics of the murderer. Thus, the readers might be easily misled that the murderer is human.

    6. Did not see any person in the street at the time. It is a bye-street{f} — very lonely.

      From this testimony, we can see that it implies that no other people know the old lady had huge amount of money at home.

    7. “Several witnesses, recalled, here testified that the chimneys of all the rooms on the fourth story were too narrow to admit the passage of a human being.

      This is the first hint to ask reader think out of the box, maybe the murderer is not a regular murderer, maybe the murderer is not even a human being.

    8. You will say that it might have been the voice of an Asiatic — of an African. Neither Asiatics nor Africans abound in Paris; but, without{l} denying the inference, I will{m} now merely call your attention to three points.{n} The voice is termed by one witness ‘harsh rather than shrill.’ It is represented by two others to have been ‘quick and unequal.’ No words — no sounds{o} resembling words — were{p} by any witness mentioned as distinguishable.

      From this point, we can see that Dupin see this case from the other side. He did not take all the testimonies as the proof or observation but try to link them together to see the whole picture of the murderer.

    1. “I am now awaiting,” continued he, looking toward{z} the door of our apartment — “I am now awaiting a person who, although perhaps not the perpetrator of these butcheries, must have been{a} in some measure implicated in their perpetration. Of the worst portion of the crimes committed, it is probable that he is innocent.

      Dupin is speculating about who the person he might be and arguing that he is innocent. Dupin is right.

  2. Sep 2023
    1. whisker

      Whisker is any of the long, stiff hairs growing on the face of a cat, mouse, or other mammal.


    2. agility

      Agility is the ability to move your body quickly and easily.


    3. Jardin des Plantes

      Jardin des Plantes is French for "Garden of the Plants."

    1. Because adding research to your essays requires the use of others’ writing, and because many assignment require you to write about others’ writing, it is possible to plagiarize accidentally through incorrect citation.

      When we’re writing an essay, we often quote others writing in our paper, that might cause some copyright issues.

    2. Using ideas from elsewhere requires correct citation, and using ideas from elsewhere without citation is plagiarism.

      If you need to use ideas from elsewhere, you must require correct citations. Don’t use other’s creation!

    1. “The Purloined Letter,”

      It is another detective short story written by Allan Poe.

    2. I do not mean to say they are not ingenious — but people think they are more ingenious than they are — on account of their method and air of method

      The author was slightly annoyed at the attention which was paid to the Dupin stories.

  3. Jun 2023
    1. kons-9 is that it combines the power of a software development IDE with the visual tools of 3D graphics authoring system. It does this by being implemented in Common Lisp, an object-oriented dynamic language which provides powerful facilities for exploratory development and rapid prototyping within a live interactive software environment

      IDE + 3D + Lisp = Unique features:: * software development IDE with visual toold of 3d graphics authoring system * unlimited extensibility (no distinction between developers and end users)


    2. REPL-based

      Read-Eval-Print Loop and is a software programming environment that allows developers to interactively execute their code

  4. Apr 2023
    1. I figure that most of the learning students lost in Zoom school is learning they would have lost by early adulthood even if schools had remained open.

      Here we have a correlation between zoom school and the feeling of missing out on the school experience. Is this true? Well he states "I figure" so this doesn't typically mean there is evidence backing up the claim and there also is no article linked to this part of the essay. One way to do this is to survey children in Zoom school and collect data from the numbers.

    1. Most grown-ups believe that school is only for learning back-to-back. They don’t understand the parts that build character for us teens.

      Grown ups, sometimes, don't understand what school is like now compared to when they were in school. What I disagree with is the fact that grown ups don't understand that it builds character. That is one thing that hasn't changed over the years. Making life long friends and finding yourself. The statement leaves out these important factors. We have to consider the statement from every angle.

    1. 2.7-9 Theorem (Continuity and boundedness)

      Let T be a linear mapping between 2 normed space, then:

      1. T is continous if and only if it's bounded.
      2. T is continous at a single point then it's continous.
    2. 2.6-9 Theorem (Range and null space).
      1. Range is a vector space.
      2. If the dim of the range is less than infinitely, then the dim of the range is \(\le\) dim of the domain.



  5. Feb 2023
    1. rocessing of special categories of personal data 45. Subject to compliance with the Data Protection Regulation and any other relevant enactment or rule of law, the processing of special categories of personal data shall be lawful to the extent the processing is— (a) authorised by section 41 and sections 46 to 54 , or (b) otherwise authorised by Article 9.

      scd #specialcategoriesdata

    1. collecting and checking the content of declarations of private interests, of personal data that are liable to disclose indirectly the political opinions, trade union membership or sexual orientation of a natural person constitutes processing of special categories of personal data, for the purpose of those provisions.

      Second question: If you collect it, can you infer from it?

    2. those provisions cannot be interpreted as meaning that the processing of personal data that are liable indirectly to reveal sensitive information concerning a natural person is excluded from the strengthened protection regime prescribed by those provisions, if the effectiveness of that regime and the protection of the fundamental rights and freedoms of natural persons that it is intended to ensure are not to be compromised.

      And here's the key element for indirect/inferred data. In order for Article 9 to matter, it must also include data that infers SCD.

  6. Jan 2023
    1. surveillance capitalism, invented by Google in 2001, benefitted from a couple of important historical windfalls. One is that it arose in the era of a neoliberal consensus around the superiority of self-regulating companies and markets. State-imposed regulation was considered a drag on free enterprise. A second historical windfall is that surveillance capitalism was invented in 2001, the year of 9/11. In the days leading up to that tragedy, there were new legislative initiatives being discussed in Congress around privacy, some of which might well have outlawed practices that became routine operations of surveillance capitalism. Just hours after the World Trade Center towers were hit, the conversation in Washington changed from a concern about privacy to a preoccupation with “total information awareness.” In this new environment, the intelligence agencies and other powerful forces in Washington and other Western governments were more disposed to incubate and nurture the surveillance capabilities coming out of the commercial sector.

      !- summary : surveillance capitalism incentives - popularity of neoliberal norm of minimizing government regulation - 9/11 accelerated popularity of a global surveillance state

  7. Dec 2022
  8. Oct 2022
    1. For the second time Goutor mentions using different size cards for different note types, but doesn't specifically advise for it or provide a reason. Perhaps his advice for consistency and card size applies only to cards of particular types? (p28)

      link to: https://hypothes.is/a/XPphjkNZEe2s3i9VV4qt1g

      Incidentally he also specifically mentions 7x9" cards here too. How frequently used were these as a standard?

  9. Aug 2022
  10. 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
  11. 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.

  12. 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.

  13. 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.

  14. Oct 2021
  15. 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.

  16. 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

  17. May 2021
  18. 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)

  19. 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)

  20. 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?

  21. 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.

  22. Jul 2020
  23. Jun 2020
  24. Mar 2020
  25. 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.

  26. 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
  27. 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
  28. 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