233 Matching Annotations
  1. Nov 2022
  2. Oct 2022
    1. After his retirement in 1947 the Carnegie Corporation of New York persuadedhim to write a history of American state universities, which came to interest him in-tensely. At the end of a year he had written half of the book and sorted his lastnotes. He notified the university administration that he was unable to complete thejob before leaving his study for the hospital where he died, October 24, 1948. Thevolume is being completed by one of his students, Professor Walton E. Bean of theUniversity of California.

      Even following his retirement in 1947, Paxson continued to take notes specifically on education for a project which he never got to finish before he died in hospital on October 24, 1948.

    1. Sometimes a Scrum Team is hired and assembled fully by an organization without any input from the Scrum Team members, and there's nothing in Scrum preventing that. Likewise, there is nothing in Scrum that prevents a group of people from forming their own Scrum Team and organizing themselves into a structure that gives them their best shot at achieving the organization's goals.

      the Scrum Team may be both assembled or self-assembled

    2. In Scrum, the Product Owner manages the Product Backlog, Scrum Masters manage Scrum's effectiveness, and Developers manage how Increments are created.

      this is important, who is accountable for the management of what

    1. who

      is it really about who (customer/user) we're working for? or can we say that it's about what (goal/value/outcome /impact) we're working for?

    1. Artykuł przedstawia wnioski z badań poświęconych zastosowaniu haseł przedmiotowych i słowników kontrolowanych w katalogach bibliotecznych. Badania wykazują, że zastosowanie ich jest zasadne i potrzebne.

  3. Sep 2022
    1. The song, known as the Black national anthem, was played more than 20 minutes before “The Star-Spangled Banner,” which was written by slaveholder Francis Scott Key and includes lyrics about the capture of escaped slaves who fought for the British during the War of 1812.

      Do you believe that we should countinue with the current national anthem "The Star-Spangled Banner" or should we change it to "Lift Every Voice" ? Is it wrong that we are singing a song written by a slaveholder towards our country ?

  4. Aug 2022
    1. One year ago this month, the final order of library catalog cards was printed by the Online Computer Library Center (OCLC) in Dublin, Ohio. On October 2, 2015, The Columbus Dispatch wrote, “Shortly before 3 p.m. Thursday, an era ended. About a dozen people gathered in a basement workroom to watch as a machine printed the final sheets of library catalog cards to be made …”
  5. May 2022
    1. Target Questions: 1. Why is there a second modernity? 2. What is second modernity? 3. How does the second modernity relate to Medellin?

      Outline: Your own heading, subheadings, lists of examples, evidence, your own thinking

      1-4 Second Modernity [Time for a Second Modernity] [Defining Second Modernity]

      “…they found that the ‘repture’ theorized in the literature of postmodernism was more a response to the systemetic and sometimes dramatic failures of ‘high modernism’ …” (1)

      What are the failures of post-modernism and high modernism? It seems that architecture proposed temporary solutions that did not consider long-term issues.

      “… there is a growing recognition that widespread disillusionment with ‘the modern project’— the strategies for carrying out the ambitions of modernism – stops short of displacing the underlying ambitions for a more universal human progress.” (2)

      The ‘modern project’ or ‘modernity’ addresses immediate issues rather than foreseeing, resolving, adapting to long-term issues in architecture and the world.

      5-9 Second Modernity [Reflexive Modernism]

      “The common sense understanding of reflexivity comes from its root ‘reflex’ meaning an action performed in response to a stimulus without conscious thought.” (5)

      “… their design and construction should be subjected to intense scrutiny and their ongoing operation should be subject to reflexive mechanism of course correction including systems of checks and balances.” (5)

      “…research at the intersection of architecture, reflexivity, and informality has so far proven more effective at generating such questions than providing satisfactory responses.” (7)

      What is reflexivity? Reflexive architecture is architecture that perhaps responds to previous “modernities” and issues (political, economic, global etc.)

      9-13 Medellin, Colombia [Library Parks]

      “To make Medellin a city of ‘the most educated’ citizens in the nation, Fajardo’s team invited the demobilized soldiers to enroll in short-term informal educational and business development programs based in a series of community centers.” (9)

      “In contrast, Fajardo and his architect’s have earned the right to speak of design as a ‘vehicle’ for conveying basic human dignity or as a ‘catalyst’ for deep social transformation.” (12)

      Library Parks were created in response to the education issues after the war. Library Parks can be classified as second modernity or reflexive modernity as Library Parks take issues in Medellin, such as education, and responds to them by creating possible solutions.

      Takeaway:

      High modernity and post-modernism provided temporary solutions that did not allow for adaptability and resiliency. Reflexive modernism and second modernity are a proposition to create long-term solutions and adaptability to old issues left behind from high modernity and post-modernism.

  6. Apr 2022
    1. The top 10 percent of households have plenty of other potential for slashing emissions.

      The key is to educate and track.

    2. The top 10 percent is a broad category that includes more than the jet-fliers and yacht-owners. According to the IPCC, the top 10 percent includes households that spend more than $23 (in US dollars, compared using a metric called purchasing power parity) per person a day. The bottom 50 percent spend less than $3 per person per day.

      This is a key demographics to target.

  7. Feb 2022
    1. கரு சிறுகதையிலும், சிவம் சிறுகதையிலும் ஒரு யுகமுடிவென்பது அதீத வளர்ச்சியினால் வரும் என்ற ஒரு கருத்தும், விஷ்ணுபுரம் என்னும் புனைவு நகரத்தின் அழிவும்,வெண்முரசின் இறுதிப் போரும் கூட அத்தகைய முடிவுகள் தான். பின்னும் நம் இந்திய வரலாற்றில் மிகப் பெரும் சாம்ராஜ்ஜியங்கள் அழிந்ததும் இத்தகைய அதீத சமமின்மையால் தான். குறளினிது உரையில் நீங்கள் சொல்வது போல “அழுதாற்றித் தொழுத கண்ணீரால்” அழிந்த சோவியத்தையும் இந்தப் பட்டியலில் பொருத்தலாம்.

      Theory of Everything - Wind Pressure

  8. Dec 2021
    1. 3. Using the game bar Another way to capture the screenshot is by the game bar if you want to capture some scene from the game you are playing. Windows 10 offer the flexibility to use the game bar for a screenshot. Start the game either by Xbox for video games 2 or start the menu For expressing game bar overlay during the game, press the Windows + G. After this, click the camera icon to take the screenshot. Another way to capture screenshot is the keyboard shortcut (windows + alt + print screen) That screenshot you will find in videos with the name of captures.
  9. Nov 2021
    1. 10 Best SaaS Startups in 2022 for Your InspirationDmitryCEOStartupSaaSHomeBlogEntrepreneurship10 Best SaaS Startups in 2022 for Your InspirationPublishedJul 29, 2020UpdatedNov 5, 202111 min readToday, the SaaS industry is gaining momentum. According to research, 80% of businesses already use at least one SaaS application. Hence, building a SaaS company is currently a skyrocketing business idea. To help you find inspiration and launch the best SaaS startup ever, in this article you will find 10 great examples of SaaS startups you can learn from. All of them produce valuable and fast-growing products for now. Likewise, Growthlist and AngelList marked them as promising SaaS startups of 2021-2022. Without further ado, let’s take a closer look at them.

      Today, the SaaS industry is gaining momentum. According to research, 80% of businesses already use at least one SaaS application. Hence, building a SaaS company is currently a skyrocketing business idea.

      To help you find inspiration and launch the best SaaS startup ever, in this article you will find 10 great examples of SaaS startups you can learn from. All of them produce valuable and fast-growing products for now. Likewise, Growthlist and AngelList marked them as promising SaaS startups of 2021-2022.

      Without further ado, let’s take a closer look at them.

    1. the program most likely violates the establishment clause.

      Shouldn't it not violate if they are neutral? I think this is wrong

      book says "The program permits the participation of all schools within the district, religious or nonreligious"

    1. Here, the program is not one of true private choice. It is not neutral in all respects toward religion and is part of an attempt to channel funds to wealthy citizens who send their children to religious schools. Only certain religious groups are free to participate.

      This entire statement is false; The program was found to be one of true private choice, with no obvious religious preferences nor discrimination against economic status/class.

  10. Oct 2021
  11. bafybeiery76ov25qa7hpadaiziuwhebaefhpxzzx6t6rchn7b37krzgroi.ipfs.dweb.link bafybeiery76ov25qa7hpadaiziuwhebaefhpxzzx6t6rchn7b37krzgroi.ipfs.dweb.link
    1. Recent research suggests that globally, the wealthiest 10% have been responsible foras much as half of the cumulative emissions since 1990 and the richest 1% for more than twicethe emissions of the poorest 50% (2).

      this suggests that perhaps the failure of the COP meetings may be partially due to focusing at the wrong level and demographics. the top 1 and 10 % live in every country. A focus on the wealthy class is not a focus area of COP negotiations perse. Interventions targeting this demographic may be better suited at the scale of individuals or civil society.

      Many studies show there are no extra gains in happiness beyond a certain point of material wealth, and point to the harmful impacts of wealth accumulation, known as affluenza, and show many health effects: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950124/, https://theswaddle.com/how-money-affects-rich-people/, https://www.marketwatch.com/story/the-dark-reasons-so-many-rich-people-are-miserable-human-beings-2018-02-22, https://www.nbcnews.com/better/pop-culture/why-wealthy-people-may-be-less-successful-love-ncna837306, https://www.apa.org/research/action/speaking-of-psychology/affluence,

      A Human Inner Transformation approach based on an open source praxis called Deep Humanity is one example of helping to transform affluenza and leveraging it accelerate transition.

    2. 10% per annum

      Anderson has contextualized the scale of such an impact in his other presentations but not here. A recent example is the temporary emission decreases due to covid 19. A 6.6% global decrease was determined from this study: https://www.nature.com/articles/d41586-021-00090-3#:~:text=After%20rising%20steadily%20for%20decades,on%20daily%20fossil%20fuel%20emissions. with the US contributing 13% due to lockdown impacts on vehicular travel (both air and ground). After the pandemic ends, experts expect a strong rebound effect.

  12. Sep 2021
    1. Regular, sustained practice.• This may be one of the most important of all anti-dotes. I have seen some very impressive friends and colleagues fall into traps of stagnation, depression, and addiction. One of the common factors seemed to be that they had given up their daily spiritual practice. Yet continuity of practice is one of the most recurrent refrains across spiritual traditions, as for example, in the advice of the Koran, “Be constant in prayer”

      Wise words ...

    2. This is a key requirement for anyone who aspires to truly understand and communicate the integral vision.

      Wow! Spot on. Correctly and bluntly stated.

  13. Aug 2021
  14. Jul 2021
  15. Jun 2021
    1. .

      In the lecture notes, Giacomo says "The logic behind decreasing returns is that, if K increases while holding L fixed machines per worker and worker productivity falls." I don't have great clarity on what "worker productivity falls" truly means and don't want to put something incorrect--fill in once lecture videos are out

  16. Mar 2021
    1. Il peut opposer un refus à la tenue d'une réunion ou à la participation de personnalités extérieures lorsque celles-ci sont de nature à porter atteinte au fonctionnement normal de l'établissement ou à contrevenir aux principes du service public de l'enseignement.
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 4.9

      AssayResultAssertion: Normal

      StandardErrorMean: 0.32

    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.1190C>T p.(Thr397Ile)

    1. SUPPLEMENTARY DATA

      AssayResult: 90

      AssayResultAssertion: Normal

      PValue: Not reported

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

    2. SUPPLEMENTARY DATA

      AssayResult: -10

      AssayResultAssertion: Normal

      PValue: Not reported

    3. SUPPLEMENTARY DATA

      AssayResult: 101.6

      AssayResultAssertion: Normal

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    4. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.2590C>T p.(Pro864Ser)

    1. Source Data

      AssayResult: 74.18

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 6.49

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

    2. Source Data

      AssayResult: 98.55

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 5.74

      StandardErrorMean: 4.06

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

    3. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.1492G>T p.(D498Y)

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

      AssayResult: 113

      AssayResultAssertion: Normal

      ReplicateCount: 17

      StandardErrorMean: 28.6

      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.1859G>A p.(Arg620His)

    1. ReconfigBehSci on Twitter: ‘1 week to the SciBeh workshop “Building an online information environment for policy relevant science” Join us, register now! Topics: Crisis open science, interfacing to policy, online discourse, tools for research curation talks, panels, hackathons https://t.co/Gsr66BRGcJ https://t.co/uRrhSb9t05’ / Twitter. (n.d.). Retrieved 2 March 2021, from https://twitter.com/SciBeh/status/1323207455283826690

  17. Feb 2021
    1. Supplemental material

      AssayResult: 82

      AssayResultAssertion: Normal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 2.9

      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.314G>A p.(Gly105Asp)

  18. Nov 2020
  19. Oct 2020
    1. n order to determine optical bandgap, the Tauc relation-ship is used as follows [24]:ahv=Ahv−Egn,3whereais absorption coefficienta=−107lnT/t,Aisa constant,his Planck’s constant,vis photon frequency,Egis optical bandgap, andnis 1/2 for allowed direct semi-conductor bandgap. The bandgap energy of ZnS thinfilmwas estimated by plottingahv2againsthvas in Figure 10;

      En orden de determinar band gap óptico, la relación de Tauc fue usado como sigue:

    Tags

    Annotators

    1. I am the self abidingin the heart of all creatures;I am their beginning,their middle, and their end.

      Lord Krishna mentioned his power to the world that his power is endless.

  20. Sep 2020
    1. [ my friend, whom I loved so dear,] [who with me went through every danger,] [my friend Enkidu, whom I loved so dear,]

      This is how Gilgamesh express their friendship.

  21. Jun 2020
  22. May 2020
  23. Apr 2020
    1. The big thing about the Web isn’t the technology, it’s that it’s the first-ever platform without a vendor (credit for first pointing this out goes to Dave Winer). From that follows almost everything that matters, and it matters a lot now, to a huge number of people. It’s the only kind of platform I want to help build.

      First, and nearly last atm.

    1. Environmental policies much like the Sustainable Development discourse have become part of these established regimes and have primarily served to optimize these regimes making them a bit less unsustainable. I thus come to conclude that Sustainable Development itself has become part of the problem. The currently dominant regimes based on the foundations of modernity, are systemically unsustainable in a fundamental way.

      Yes! It is an elastoplast on the gaping wound. The painkiller that let's you get by your dislocated shoulder so you don't do anything about it.

    2. The two cases are to me clear examples of the ́sustainability lock-in’: we seem to be caught in a vicious cycle of optimizing and inherently unsustainable system which closes down the space for the development of inherently better alternatives
  24. Jan 2020
    1. I like that the Lambda Architecture emphasizes retaining the input data unchanged. I think the discipline of modeling data transformation as a series of materialized stages from an original input has a lot of merit. This is one of the things that makes large MapReduce workflows tractable, as it enables you to debug each stage independently. I think this lesson translates well to the stream processing domain. I’ve written some of my thoughts about capturing and transforming immutable data streams here.

      Great point 👍

      Something i've thought about and emphasized for doing FDF - ability to debug per step or re-run after a given step.

    1. RRID:ZFIN_ZDB-GENO-140612-10

      DOI: 10.1016/j.cell.2019.01.019

      Resource: (ZFIN Cat# ZDB-GENO-140612-10,RRID:ZFIN_ZDB-GENO-140612-10)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-GENO-140612-10


      What is this?

  25. Dec 2019
    1. I think this has the added benefit of making data warehousing ETL much more organizationally scalable. The classic problem of the data warehouse team is that they are responsible for collecting and cleaning all the data generated by every other team in the organization. The incentives are not aligned: data producers are often not very aware of the use of the data in the data warehouse and end up creating data that is hard to extract or requires heavy, hard to scale transformation to get into usable form. Of course, the central team never quite manages to scale to match the pace of the rest of the organization, so data coverage is always spotty, data flow is fragile, and changes are slow.
  26. Nov 2019
    1. Top 10 Global DevOps Consulting Companies

      Are you wishing to develop the websites at global level? Here you can find top 10 Devops Consulting Companies that aim to deliver first class solutions forever. In addition to this, the expert developers able to provide the friendly services and thus get attention on familiar sources forever.

  27. Sep 2019
    1. Americans today are some of the unhealthiest people on Earth

      That is correct, due to the high fatty food, lack of exercises,smoking and other related segments make them unhealthy. So the heart related problems are looming. According to the studies America is on the 10th place

  28. Aug 2019
    1. the set fee per transaction of 10%

      delete

    2. charged 10%

      replace with "a small fee"

    3. 10 percent

      Delete "10 percent" and replace with "a"

    4. Negotiate with the shipper with 10% fee in mind.

      delete

  29. Jul 2019
    1. Myth: Refugees are all Muslim.

      Do people actually think that? That is ridiculous and so ignorant. People shouldn't stereotype like that. Does the general public really believe that all refugees are from the middle east and are Muslim? I wonder if they know that there are thousands of Christians in the middle east."Christians now make up approximately 5% of the Middle Eastern population, down from 20% in the early 20th century" That's part of the problem. It's a war on freedom. Religious freedom, basic human rights, and personal desires. Sheesh!

    1. Production of β-galactosidase (ortho-Nitrophenyl β-D-galactopyranoside (ONPG)) test
    2. Production of β-galactosidase (ortho-Nitrophenyl β-D-galactopyranoside (ONPG)) test
  30. Jun 2019
    1. Expression of costimulatory molecules on splenocytes harvested from immunized and control animals
    1. The synthetic peptides were purified by RPHPLC on an Aquapore RP300 column (250 x 7 mm) using a 4-72% linear gradient of solvent B (acetonitrile containing 0.1% TFA) in 130 min at a flow rate of 2 mllmin. Globin chains from respective hemoglobins were separated on a similar column of a smaller dimension (250mm x 4.6 mm) under identical conditions but at a flow rate of0.7 ml/min. Electro spray mass spectrometric analysis was carried out on a VG Platform (Fisons) mass spectrometer. The instrument was usually calibrated with standard horse heart myoglobin or gramicidin S solution. Appropriate amount of each sample was taken in 50% acetonitrile containing I% formic acid and analyzed under the positive ion mode.
    2. nalytical procedures
  31. May 2019
    1. The results of the study indicate that education of the future business leaders calls for a contextualized approach to business ethical dilemmas.

      The main goal of this article is to understand how the perspective about business ethics between business student and non-business students differ, and the results show that we indeed do need contextualized approach to business ethical dilemmas

    1. An easy solution would be for the school to change to an ISP that would agree not to filter traffic. Unfortunately, in many rural areas, there are often few choices for ISPs, creating a lack of competition. An FCC report from June 2017 found that about 75% of U.S. census block regions have zero choice in terms of high speed Internet/ broadband access (Brodkin, 2017). The FCC has claimed that market competition will provide a check on potential ISP abuse. “Given the extent of competition in Internet access supply,” the FCC’s (2017) new order states, “the protections regulating ISPs are not necessary” (p. 144). Despite the frequent claims of competition throughout the document, the statistics included by the FCC show that competition is not as widespread as it would like to claim.

    1. The protein samples were resolved by SDS PAGE using the Laernrnli buffer system (Laernrnli, 1970). The protein sample was denatured by boiling at 100°C for 10 min in Laernrnli's buffer (List I). Resolving gel (10%) was prepared in a minigel (Bio-Rad, USA) system alongwith 3% stacking gel and the electrophoresis was carried out at 120 volts for 125 min. The gel was stained with 0.25% Coomassie blue R staining solution for Ih followed by destaining with successive washes of de staining solution. Staining was avoided when. gel was used for irnrnunoblotting. Details of reagents used for SDS-PAGE are given in List 1.
    2. SDS PAGE
    1. Sub-cellular fractionation of THP-1 macrophages was performed after lysis with hypotonic buffer. THP-1 macrophages after appropriate treatment were allowed to swell for 10 min in hypotonic buffer (1 0 mM NaCl, 1.5 mM MgCh, 10 mM Tris-HCl, pH 7 .5) followed by homogenization with 50 strokes using a Dounce homogenizer. More than 90% cellular lysis was ensured by visualizing under a light microscope, and immediately after lysis, the mitochondrial membranes were stabilized by addition of 2.5x mitochondrial stabilization buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.5) to a final concentration of 1x. The homogenate was centrifuged at 1300 x g for 15 min to isolate the nuclear fraction. The post-nuclear fraction was further centrifuged at 17,000 x g for 15 min in an ultracentrifuge (Beckman Optima XL-1 OOK ultracentrifuge) to isolate the mitochondria. The post-mitochondrial supernatant was centrifuged at 100,000 x g for 1 h to obtain the membranous fraction as a pellet and the supernatant obtained was the cytosol. The homogeneity of the obtained fractions was determined by probing for fraction specific proteins by Western blotting
    2. Sub-cellular fractionation
    1. Heterologus expression of various proteins was done in BL21 (A.DE3) strain of E. coli. Bacterial cells were transformed with the desired construct and grown in Super broth (pH 7.2) containing 100 J.lg/ml ampicillin, at 37 °C with continous shaking in a gyratory shaker at 225 rpm. The cultures were induced, at ~oo of 2.0, with I mM IPTG, and harvested two hours later by centrifugation at 4000 g, at 4 °C, for 20 min .. The recombinant proteins were purified from the inclusion bodies using the procedure described by Buchner et al ( 1992). The total cell pellet from a liter of culture was homogenized in 180 ml of inclusion bodies washing buffer containing 8 ml of freshly prepared lysozyme solution (5 mg/ml). The suspension was incubated at room temperature for 1 hr with intermittent shaking. Added 20 ml each of 5M NaCI and 25% Triton X-100 were added to the suspension and incubated at room temperature for 30 min. with vigorous shaking. The suspension was centrifuged at 13,000 g at 4 °C, for 50 min. and the pellet was resuspended, in the washing buffer containing 1% Triton X-1 00, using a polytron homogenizer and centrifuged at 13,000 g for 50 min. The pellet was washed four times with washing buffer without Triton . X-100. The pellet containing inclusion bodies was solubilized in 6 M guanidine hydrochloride by incubating for 2 hours at room temperature. The solubilized protein was centrifuged at 50,000 g, at 4 °C, for 30 min. and the protein concem.ration was adjusted to 10 mg/ml in the supernatant with 6 M guanidine hydrochloride. The denatured protein thus obtained was reduced by adding 65 mM dithioerythritol and incubated at room temperature for 2 h. To renature, the protein was diluted 1 00-fold in the refolding buffer and incubated at 10 °C for 48 h without stirring or shaking. Renatured material, after dialysis in 20 mM MES buffer, pH 5.0 containing 100 mM urea, was loaded on a S-Sepharose column, and the protein bound to the column was eluted with a 0-1 M NaCI gradient in 20 mM MES on an FPLC system (Pharmacia). The fractions containing the desired protein were pooled and concentrated, and the protein was further purified to homogeneity by gel filtration chromatography on a TSK 3000 column (LKB) in PBS, pH 7.4.
    2. Isolation and Purification of Proteins from the Inclusion Bodies
    1. Sf9 cells infected with AcNPV, VI, V2, V3, or V4 were harvested 72 h pi, washed twice with 10 mM PBS, air dried on a slide and fixed in chilled methanol for 15 min. Cells were incubated with MA-451 culture supernatant and N-terminal anti-peptide serum ( 1 :500) at 37°C for 1 h, washed with PBS and further incubated at 37oc for 1 h with I :50 dilution of anti-mouse FITC or I :2000 anti-rabbit FITC. Slides were washed extensively, mounted in 90% glycerol in PBS (50 mM, pH 7.4) and examined under Optiphot fluorescent microscope (Nikon, Tokyo, Japan).
    2. Immunocytochemical Localization of Recombinant Proteins in Infected Cells
    1. lysed directly in 1. 5 ml of solution D ( 4 M guanidium thiocyanate, 25 rnM sodium citrate, pH 7.0, 0.5 % sarcosyl and 0.1 M 2-mercaptoethanol ) . For every 2 ml of the lysate, 0.2 ml of chloroform was added, followed by vigorous mixing for 15 seconds, and incubation on ice for 15 minutes. The lysate was spun at 12, OOOg, at 4 °c for 15 mins. , and the aqueous phase transferred to another tube. RNA was precipitated with an equal volume of isopropanol and incubation at -2o0c for 45 mins. The samples were then spun at 12,000g for 15 mins. at 4°c, and the supernate discarded. The RNA pellet was washed twice with 75 % ethanol. Finally, the pellet was dried briefly under vacuum for 10 -15 mins. and dissolved in 0.5 % SDS. All chemicals and glassware used for handling RNA were treated with diethylpyrocarbonate ( DEPC ) .
    2. Total RNA was isolated from cultured mammalian cells by the method of Chornczynski and Sacchi ( 1987 ), with slight modifications. Briefly, cells from a 3.5 ern petri-dish were
    3. Isolation of RNA
    4. All computer software facilities were provided by the NII computer centre.
    5. Computer software.
    1. stranded DNA. The reaction was carried out at 37°C for 1 h. The reaction mixture contained 100 ng Bgl II digested VR1020 vector, SAP (0.5 U) and 1 fll lOX SAP buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCh) in 10 f.!l oftotal reaction volume. The reaction was stopped by inactivating the enzyme at 65°C for 15 min. The digested bmZP1 eDNA was ligated with SAP treated VR1 020 at vector : insert ratio of 1:10 in a 10 fll reaction volume for 16 h at l6°C. The reaction mixture contained 10 ng VR1020 vector, 26 ng bmZPl insert, 1 fll lOX ligase quffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCh, 10 mM DTT and 1 mM ATP), lfll T4 DNA ligase (20 U) in a total reaction volume of 10 fll. The ligation product was used for transformation of DH5a competent cells as described previously. Transformants were selected on LB plates containing 50 f.!g/ml Kanamycin (Kan). Similarly, the inserts corresponding to dZP3, rG and dZP3-rG fusion were digested with Bgl II restriction enzyme, gel purified and cloned in VR1020 vector, except that the ligation product of dZP3-rG fusion with VR1020 was transformed into JM109 competent cells
    2. The insert corresponding to bmZP1 was released from the pPCR-Script-bmZPl clone by Bgl II restriction and purified on the agarose gel. VR1020 vector was similarly digested and gel purified. To prevent self-ligation, the digested vector was treated with Shrimp Alkaline Phosphatase (SAP), which removes 5'-phosphate from the termini of double
    3. Cloning in VRl 020 mammalian expression vector
    1. Meancorpuscularhaemoglobinconcentration(MCHC)istheaverageHbconcentrationperunitvolume(100)ofpackedredcells(W/V).Henceitisexpresseding/1whichisthesameaspercent(%).ItiscalculatedbythefollowingformulaHbMCHC=—......x100(g/dl)PCV
    2. MCHC
    3. MeancellVolume(MCV).Itisexpressedinfentolitres(1fentolitreorflisequivalentto10'151)andcalculatedby thefollowingformula:PCVMCV=.....................x10(fl)RBC8.10.6.2.MCHMeancellhaemoglobin(MCH)=AverageweightofHbinanerythrocyte.Itisexpressedinpicograms(pg)whichisequivalentto10"12g.Itiscalculatedbythefollowingformula:HbMCH=-----------------x10(ppg)RBC
    4. MCV
    5. RedBloodcellsindices
    6. micro-haematocrittubewasfilledto100mmwithanticoagulatedblood.Oneendofthetubewassealedwithsealingwaxandthetubewasthenkeptinaverticalpositioninaglassbeakerstuffedwithcotton.Afteronehour,lengthoftheplasmacolumnwasmeasuredwitha rulergraduatedin0.5mm.
    7. ESRwasdeterminedbythemicromethodbecausethequantityofbloodavailablefromindividualfishwasinsufficienttoadoptanymacromethod.Anon-heparinised
    8. ErythrocytesedimentationRate(ESR)
    9. BloodwascollectedfromtheheartbycardiacpunctureusinganRBCpipette.ItwasdilutedwithHayem’sfluidintheratioof1:200.Thecontentswereshakenwell.AdropofthedilutedbloodwasplacedinaNeubauerdoublehaemocytometer(Germany)countingchamberandtheredbloodcellcountpercubicmmwascalculated
    10. Redbloodcellcount(RBC
    11. Thepackedcellvolumeorhaematocritisthevolumeoccupiedbythepackedredcells,afteravolumeofanticoagulatedvenousbloodisfullycentrifuged.Thevolumeofpackedcellisexpressedasapercentageoftheoriginalvolumeoftheblood.ThePCVisusedtoestimatehaematologicalindices,includingthemeancellhaemoglobinconcentration(MCHC)andmeancorpuscularvolume(MCV).PCVdetermination followedthemethodsofBlaxhallandDaisley(1973).Thehaematocritvaluewasdeterminedbycentrifuging(3000rpm)aknownvolume ofincoagulantbloodkeptinWintrobe’stubes
    12. PackedCellVolume(PCV)orHaematocrit(Ht)
    13. HaemoglobinwasdeterminedbySahlimethod.HaemoglobinisconvertedtoacidhaematinbytheactionofHC1.Theacidhaematinsolutionisfurtherdilutedwiththeaciduntilitscolormatchesexactlythatofthepermanentstandardofthecomparatorblock.TheHbconcentrationisreaddirectlyfromthecalibrationcurve.
    14. Haemoglobin(Hb)determination
    15. BloodwastakenbyheartpunctureusingMS222astheanaesthetic.Nofishwasusedmorethanonce.
    16. CollectionofBlood
    17. HaematologicalAnalysis
    1. The cells were assayed for Luciferase gene expression using Luciferase Assay kit (Promega, U.S.A.). After transfection, the cells were washed twice with PBS and then lysed by adding reporter lysis buffer provided in the kit. The cell lysate was collected from individual wells in eppendorf tubes, the cells were twice freeze-thawed in liquid N2 and then centrifuged at 13,000 rpm for 10 min at 40C. The supernatant was transferred to a fresh tube. 20¢ of cell extract was mixed with lOOp! of luciferase assay reagent that was kept at room temparature. The activity was determined using a luminometer (Packard lumicount, U.S.A.
    2. Luciferase assay
    1. normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice
    2. Growth curves were prepared for various cell lines using the modified method adopted by Serrano et al, 1997. Briefly, 10, 000 cells were seeded in a 24 well plate in quadruples. At the indicated times, cells were washed once with PBS and fixed in 10% formalin for 20 minutes and rinsed with distilled water. Cells were stained with 0.05% crystal violet for 30 minutes, rinsed extensively and dried. Cell associated dye was extracted with 1.0ml acetic acid. Aliquots were diluted 1:4 with water and transferred to 96 well microtitre plates and the optical density at 590nm determined. Values were
    3. Growth Curves:
    1. Inverse PCR is a technique to amplify unknown regions flanking the site of transposon insertion using the primers designed from the known sequence from one end of the transposon element. Genomic DNA was digested with a 4-base recognition restriction enzyme, Sau3A1 followed by intramolecular ligation set up at high dilutions. These ligated molecules were then used as templates for the PCR performed with a pair of divergently-oriented primers designed from one end of the transposon named AH1-AH2. The PCR product thus obtained was sequenced with the same set of primers to identify the junction sequence at the site of transposon insertion and hence the identity of the gene disrupted in each case. Typical PCR conditions used were as follows:- Annealing 55°C 2 min Elongation 72°C (1 min/kb of DNA template to be amplified) Denaturation 95°C 2 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C
    2. Inverse PCR
    3. To quantitate the P1 phage lysate preparations, titration was done using a P1-sensitive indicator strain such as MG1655. 100 μl each of serial dilutions of the phage (typically 10−5, 10−6) were mixed with 0.1 ml of the fresh culture grown in Z-broth. After 15 min adsorption at 37°C without shaking, each mixture was added in a soft agar overlay to Z agar plates, and incubated overnight at 37°C. The phage titre was calculated from the number of plaques obtained on the plates
    4. To 0.3 ml of infection mixture, 10 ml of Z-broth was added and incubated at 37°C with slow shaking until growth followed by the visible lysis of the culture occurred (in ~ 4-6 h). The lysate was treated with 1 ml of chloroform, centrifuged and the clear lysate was stored at 4°C with chloroform
    5. Broth method
    6. 0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107 plaque forming units (pfu) of a stock P1 lysate prepared on strain MG1655. Adsorption was allowed to occur at 37°C for 15 min and the lysate was prepared in the following ways
    7. Phage P1 lysate preparation
    1. Rhod-2 acetoxymethyl ester is a fluorescent long wavelength calcium indicator, where the AM ester forms are cationic, resulting in a potential driven uptake into the mitochondria making them selective detectors of mitochondrial calcium. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were washed twice with Kreb' s buffer (118mM Sodium chloride, 5.4mM Potassium chloride, 1.2mM Magnesium chloride, 1.2mM Potassium dihydrogen phosphate, 25mM Sodium hydrogen phosphate, llmM glucose, 1.5mM Calcium chloride, pH 7.4) by centrifugation at 1258 x g for 5 min to wash off all traces of medium and FBS. Washed cells were loaded with 1:1 mixture of Rhod -2 AM (1p.g/p.L stock solution prepared in DMSO) and 20%w/v Pluronic F127 for 1h at RT in the dark with shaking. Excess dye was removed by one wash with Kreb' s buffer followed by incubation of cells at RT for a further 30min for complete hydrolysis of the dye trapped in the mitochondria. Fluorescence intensities of the stained cells were measured fluorimetrically at excitation of 530nm and emission of 576nm or alternatively acquired by flow cytometer.
    2. Assay for measuring mitochondrial calcium