In the Buddhist world we even in, in a way you can say you're always dying. You're already dying. So just thinking about it in those terms: what's the cultural impact of thinking about life as death, actually—as a process that maybe never ends?

I'll talk about some cases in a moment— there's very sudden decomposition when the "tukdam" ends. You can have people who have been in tukdam for 27 days and then on the— as one of the cases that we're gonna be publishing on soon— and on the 28th day there's like very dramatic decomposition. Just boom! It seems to happen.

And this is one of the big problems right now— [pointing at the slide] tukdam regularly occurs in non-experts, right? You find, you know, people who are not great trained tantric practitioners who know all the commentaries and, you know, who aren't even monks or nuns— who are just ordinary lay people—and they go into "tukdam."

I've encountered several people in the Kagyu and Nyingma traditions who say, "Oh, we, you know 'tukdam,' yeah, people go in 'tukdam,' "but it's like, you know, not that big a deal. It's, we don't care that much." Part of the reason they don't care that much is that the idea that you need to go into this completely, kind of, a state where there's no phenomenal content— that's just a pure clear light mind— actually is something that many of the contemporary practitioners and teachers in those lineages don't agree with.

it's said that you can get there by doing like philosophical analysis, but this is using basically physiological techniques to get to the same place phenomenologically. So that's what "tukdam" is theoretically

So the concept here is that you're actually no longer even capable of thinking, you're no longer capable of seeing, you're no longer capable of hearing, and so on. All that's left is just this kind of sheer consciousness itself, which doesn't even have a subject-object structure. So for the Gelugpas that lack of subject-object structure is not really relevant. For the other traditions it's extremely relevant, because it's said that if you're going to understand the nature of the mind, the fundamental distortion in the mind is precisely that subject-object structure. So you have to cultivate a non-dual awareness,

in the Perfection Stage— what's called the Perfection Stage— one is going to actually begin to bring the winds into the central channel. And when one is able to do so and bring them into the heart cakra.

he made this three-dimensional, so this is the maṇḍala.

And how one is gonna do that, one is gonna become not you. You're gonna become somebody else—specifically, you're gonna become a fully enlightened tantric deity, right? And you, with a sense of what's called dignity or pride, right, the, the... "lha’i nga rgyal," the "pride of being the deity."

Unexcelled Yoga Tantra

So what's the first thing to do? It's to stop being ordinary. So they say, "tha mal gyi rtog shes spang ba," "abandon ordinary thoughts and ordinary attitudes," ordinary experience.

these winds, right— these energies—are already flowing, of course, and they flow in very deep patterns that basically constitute one's own ordinary identity. And so quite literally one's own ordinary identity is, is the patterning of these winds.

There are different forms of energy, five primary forms and five secondary forms of energy, and they flow in channels in the body. And at the time of death, there, there's a certain kind of configuration of those energies that occur and you can actually, you can, in a sense, force those energies— maybe that's not the right term, but some people would agree with that metaphor— you can force those energies to enter into that configuration through various forms of yogic practices.

The simulation, however, requires a high degree of control over the winds— "rlung" in Tibetan or "vāyuḥ" in Sanskrit, not "prāṇa," but "vāyuḥ" in Sanskrit—that are involved in the death process.

To rehearse that moment, essentially what one does, is you induce a simulated version of this clear light mind.

We wanna get down in a sense to the foundational state of mind, a most fundamental form of mind, and that occurs at death.

if we're gonna really understand the ultimate nature of reality, it means to understand the ultimate nature of the mind

Gelugpa tradition eventually rejects, really, almost everything about Yogācāra. But tantra itself really emerges out of that perspective, which is essentially that the only thing we have access to is our own experience.

The only way you can become a buddha is to see the nature of ultimate reality with the motivation of relieving the suffering of sentient beings. And in order to do that, you have to cultivate this wisdom.

avidyā in Sanskrit or "ma rig pa" in Tibetan,

from the standpoint of Mahāyāna theory that a buddha is special because a buddha can teach in this incredibly effective way.

More bee

Elon Goitein

One large study by Ben D. Woodand Frank N. Freeman in 1932 paved theway for acceptance in elementary schools.The study included 14,947 children ofelementary-school age in an experimenton the effect of the typewriter on class-room performance (3). The children whohad typing instruction actually spent onlyan hour or two a week at the typewriter,yet at the end of the first year they out-performed the nontyping pupils in read-ing.

In 1938, President Franklin D. Roosevelt signed the Fair Labor Standards Act, which created a right to overtime pay for those who worked more than 40 hours a week.

Edward Teach (alternatively spelled Edward Thatch, c. 1680 – 22 November 1718), better known as Blackbeard, was an English pirate who operated around the West Indies and the eastern coast of Britain's North American colonies.

Hubert Czerniak TV - Fakty i mity o witaminie D3! Lepiej usiądźcie -

Witamina D3 + K2 MK-7 - 5.000 IU + 200mcg - 120szt.

Leczenie doustne: dorośli z niedoborem (<20 ng/ml 25(OH)D3): 40 000 IU/tydzień przez 7 tyg. lub 50 000 IU/tydzień przez 6 tyg.[4

clicking

Дикий шок первого дня.Безумная неделя в коридоре, крики с крыш «Точно свои? Сейчас гранату кинем!», закрытые магазины и бесплатный хлеб.Выезд снежным утром, пустой город и бесконечная очередь машин, надписи «Дети» и брошенные на обочинах Теслы.Бесконечно холодные март и апрель за Днепром. Попытки собрать свой мир обратно из маленьких кусков.Возвращение в пустой и грохочущий Харьков. Безлюдные проспекты, надписи «Закрыто, ничего нет», следы от прилетов.Белгородский экспресс в 10, возвращение девочек из Германии, привычку к горящему горизонту и взрывам за окном.Сентябрь 2022, когда фронт отодвинули от Харькова, непривычную тишину и улыбки на лицах.Момент, когда мы отмечаем компанией снятие осады — и после тяжелого взрыва за окном гаснет свет. На сутки. Полгода блекаутов и жизни с батарейками.«Дальше будет только лучше, войне скоро конец!» Бравые записи в каналах и новостях, намеки от властей и командующих. Вирусные открыточки и мемчики.Разочарование лета 2023. Ничего не закончилось.

A typewriter repair technician by trade from Michigan, Carl Elmer Anderson started the Anderson Typewriter Company in Pasadena in 1912 after falling in love with the City as a vacationer.

every person who got apnea got to be on vitamin d vitamin d makes a tremendous change for so many different 00:24:20 chemical changes in our entire body uh it affects you not only our immune system because you know we look at covet they you know they found like eight over eighty percent of every covert case had deficiency of vitamin d so you think about if your immune system 00:24:33 is weakened that means other systems are weakened which what does the immune system do it keeps away inflammation you see it's all tied together it's so important so now you say why do i need vitamin d well how much are you in the 00:24:46 sun as we get older we're just not in the sun all that much and oh i eat healthy you're not getting it from you're not from your food you need to supplement

Data and analytics engineers for Gentrack Logical Data Model

In 1950 Dwight Eisenhower wrote a letter to a U.S. diplomat in which he ascribed a military-oriented version of the saying to an anonymous soldier. Emphasis added to excerpts by QI:[1]1984, The Papers of Dwight David Eisenhower, Volume XI: Columbia University, Editor Louis Galambos et al, Letter from: Dwight Eisenhower, Letter to: Hamilton Fish Armstrong, Date: December 31, 1950, … Continue reading . . . I always remember the observation of a very successful soldier who said, “Peace-time plans are of no particular value, but peace-time planning is indispensable.”

Dr. Martin Luther King, Jr.

my peers, stared, their faces as unconvinced as blank sheets of paper.

«В Японии бывало много религиозных споров и, между прочим однажды о том, какого цвету диавол: одна секта утверждала, что он черный как уголь, другая, напротив, что у него кожа снежной белизны; но тут возникли еще две партии и, увлекаясь духом противоречия, стали утверждать, одна что он красный, другая, что зеленый. Распря разгоралась, умы воспламенялись, и чуть не дошло до междоусобной войны. Наконец дело представлено было на разрешение императора, и сын неба помирил всех, объявив, что все правы. С тех пор японский ад населился чертями черными, белыми, красными и зелеными, народ перестал спорить и с равным удовольствием смотрит на тех и других, любуясь их пестротою и разнообразием».

As socialist realism was imposed on Soviet writers, one form of permissible resistance, of finding an inner freedom, was to read translations of foreign writers. No private library was complete without Hemingway, Faulkner, London, Fitzgerald, Steinbeck, Salinger—all officially permitted as “progressive writers” exposing the “ulcers of the capitalist world.”

Or, did you ever see a dog with a marrowbone in his mouth,—the beast of all other, says Plato, lib. 2, de Republica, the most philosophical? If you have seen him, you might have remarked with what devotion and circumspectness he wards and watcheth it: with what care he keeps it: how fervently he holds it: how prudently he gobbets it: with what affection he breaks it: and with what diligence he sucks it. To what end all this? What moveth him to take all these pains? What are the hopes of his labour? What doth he expect to reap thereby? Nothing but a little marrow

Cohen was reluctant to provide reference values for his standardized effect size measures. Although he stated that d = 0.2, 0.5 and 0.8 correspond to small, medium and large effects, he specified that these values provide a conventional frame of reference which is recommended when no other basis is available.

Justice Louis D.Brandeis instructedlawyers that “there is nosuch thing as good writ-ing. There is only goodrewriting.”23

Additionally, Henry Giroux claims that it was originally "military–industrial–academic complex".[16]

Одна из лучших возможностей нашего времени, кроме стоматологии с обезболиванием — возможность всегда с кем-то поговорить о чем угодно

Set frequency to 145.670

У нас в стране происходит самая тяжелая гуманитарная катастрофа последних 80 лет, во всех смыслах, и что же мы видим, или вернее не видим?Я встречал гуманитарные склады и кухни в спортзалах, подвалах офисов, школьных столовых, конюшнях и моллах.Но никогда не видел и не слышал, чтобы они были в церквях или монастырях.Центры эвакуации? Бесплатная раздача еды? Центры психологической поддержки? Координаторы волонтеров? Организация поставок через границу, черт возьми?Кто угодно, только не товарищи в рясах.Такое ощущение, что их нет, и все эти огромные халабуды стоят брошенными именно в тот момент, когда они понадобились.Честно говоря, я не представляю, как такое может быть, но факт остается фактом. Церковь эту войну проиграла начисто, независимо от того, кому она подчиняется.- я вижу не так мало священников, которые пытаются что-то делать и часто успешно. Но какой-то организации, за ними стоящей, я не вижу: все разрозенно и держится на подвиге отдельных людей.Так что церкви как организации проигрывают несмотря на то что кто-то определенно будет прославлен в лике святых, явно или неявно.

Те, с кем я общался после начала войны, потухли. Буквально. Все, что они знали и любили, весь старый Харьков потерял смысл.Как можно после февраля рассказывать о том, как по приказу из Москвы строили Госпром? Или про то, как наши проходчики потом поехали делать метро в тогдашнюю столицу? Как делали Т-34 в прошлую войну?Вся, вся старая история бессмысленна, ее теперь невозможно рассказывать. Это не запрещено — просто невозможно. Она отошла, осталась где-то там, в нулевых. И ее знатоки вместе с ней.Прошлое время прошло.Гамлет, Розенфельд, Комаровский. Бушвокер.А новая история только пишется и новый город только возникает.И никто пока не может сказать какой он будет.Точно другой....И это еще не начали ломать, еще не упал Пушкин в начале бывшей Немецкой.

D*nald Trump

Oh, never, surely, was there knight So served by hand of dame, As served was he, Don Quixote hight, When from his town he came; With maidens waiting on himself, Princesses on his hack—

Ce programme nommé « I can word it too », disponible en hébreu et arabe, a été spécialement créé pour cette étude. Il reproduit les activités quotidiennes (jouer à des jeux, prendre les repas, faire sa toilette…) et demande à l’enfant ce à quoi il veut jouer, en lui présentant un choix de jeux sur l’écran

Make Use of Frames FacebookLinkedInTwitterPinterestEmailPrint Using frames or templates is a great way to scaffold instruction and build learners’ confidence in writing, particularly in writing tasks and genres with which they have little prior experience. A writing frame consists of a skeleton outline given to learners to scaffold their writing. By providing a few sentence starters and some rhetorical phrases common to the task or genre, frames give learners a structure that allows them to focus on expressing their thoughts

Depuis longtemps, je suis d’avis que la rigueur d’un cours ne se mesure pas à la quantité de connaissances dont l’enseignant fait étalage, mais aux apprentissages que les étudiants font.

PTH

Miscible with alcohol

(+)-Limonene is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide. The solubility of (+)-Limonene in these solvents is approximately 20 mg/ml.

les directeurs d'école et les chefs d'établissement doivent communiquer aux associations de parents d'élèves qui en font la demande la liste des parents d'élèves de l'école ou de l'établissement scolaire mentionnant leurs noms et adresses postale et électronique

108,574

The more coachable someone is, the more they can grow, and the more quickly they can grow. Someone with low coachability can find it so hard to do anything outside of their expertise that it is understandable when managers focus their energy on the people they can help and grow instead.

Empower managers to facilitate effective learning transfer As Fergal explains, managers have a key role to play in facilitating effective learning transfer. “Research shows that managers play the most critical role in learning transfer - especially in the post-training environment. Every learner needs a manager who understands them, and how they want to learn and grow. They need to have the right coaching style, and they need the right resources.”In most organizations, instructional design focuses on the needs of the learner. But as Fergal explains, focusing on the needs of your managers can pay dividends. “Ideally, you’d have the manager attend the same training as the learner. The problem is, managers are always stretched. So, what you can do instead is develop specific guidance for your managers.” Provide a script for managers to support their team’s learning

Your employee experience action plan Delivering a more positive experience for employees begins with diagnosis – listening to the voice of your employees frequently and consistently through the power of tools such as pulse surveys. It continues by identifying the culturally relevant workplace practices that you can build on and improve. Once that’s done, it’s time to take action:  Enable managers to design experiences consistent with your organization’s core values

Why do 87% of data science projects never make it into production?

Following are strategies for facilitating SDL. The teacher can help the learner to Conduct a self-assessment of skill levels and needs to determine appropriate learning objectives. Identify the starting point for a learning project. Match appropriate resources (books, articles, content experts) and methods (Internet searches, lectures, electronic discussion groups) to the learning goal. Negotiate a learning contract that sets learning goals, strategies, and evaluation criteria. Acquire strategies for decision-making and self-evaluation of work. Develop positive attitudes and independence relative to self-directed learning. Reflect on what he/she is learning.

What Are the Differences Between Facilitation, Presentation, and Training? Trainers help others improve their performance by teaching, instructing, or facilitating learning. As such, facilitation and presentation are both tools in a trainer’s toolkit. In most cases, effective and engaging trainers will spend less time presenting content through lectures or lecturettes and more time facilitating learning around that content. Presentation vs. Facilitation

Learning is not linear, and meaningful learning resists being quantified. Our assessment approaches should create space for learning not arbitrarily delimit it.

The best course of action is to be intentional and systematic from the get-go. That is, rather than seeing the development, communication, and management of knowledge as “nice-to-haves” within your organization, start building these processes into your organization’s standard routine: Include knowledge management in your project descriptions and timelines. Set a “shelf life” for your knowledge documents (the maximum amount of time that can pass before revisiting said documents in some way). Perform scheduled maintenance to your knowledge documentation on a monthly, quarterly, and yearly basis.

MediaWi

A crucial difference between representations of relative error inthese equations compared withEquations 6and7 for the single-facet designs is that three sources of measurement error varianceare separately represented, withpt2ntequaling specific-factor error,po2noequaling transient error, andpto,e2ntnoequaling random-responseerror. Effects fortasks, occasions, and their interaction are includedin the denominator for the D-coefficient but not the G-coefficientbecause those effects can change the absolutemagnitude of scoresbut not their relative differences.

Most C++ template idioms will carry over to D without alteration, but D adds some additional functionality

Create an /etc/apt/preferences.d/debian-chromium

Learners’ practices, for example, should develop from low-level procedures such as sharing initial ideas to mid-level comparing and evaluatingsequences and eventually toward high-level challenging/debating and synthesizingroutines.

identify invariant (i.e., constant) characteristics of subsequent transfer domains

The freedesktop.org project also developed a free and open-source software library called libdbus, as a reference implementation of the specification. This library should not be confused with D-Bus itself, as other implementations of the D-Bus specification also exist

relief

Cholesterol

article D. 111-9 du code de l’éducation

À cette fin, comme le prévoit l'article D. 111-8 du Code de l'éducation, les directeurs d'école et les chefs d'établissement doivent communiquer aux associations de parents d'élèves qui en font la demande la liste des parents d'élèves de l'école ou de l'établissement scolaire mentionnant leurs noms, adresses postale et électronique, à la condition que ceux-ci aient donné leur accord exprès à cette communication.

Reverse engineering a bronze cannon from theLaBelleshipwreck

More girls

tamper-proof qubit

Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.

Primary culture of E. coli was grown in LB medium containing either ampicillin (Amp) and/or kanamycin (Kan) to final concentration of 100 j..tg/ml and 25 J..tg/ml respectively. Depending on the vector construct, antibiotics were used for expression of different proteins as described in Table 3.1. Medium was inoculated with 1 ml glycerol stock of E. coli and incubated overnight at 37 oc at 200 rpm.

The DFD between an atom pair is the normalized frequency distribution of the interatomic distances sampled from equal time snapshots taken from the MD simulations. DFDs of corresponding atoms taken from the different simulations were used to qualitatively compare the effect of mutational perturbations on the HbS fiber. Considering that the fiber simulations were carried out only for a relatively short time scale of 1.2ns, the calculated DFDs might suffer from errors due to limited sampling. Hence a quantitative comparison of the different DFDs were not attempted, however given that the global parameters monitored during the simulation had already become reasonably stable after 0.2ns (Chapter!, Table 5), it is expected that the gross features of the DFDs would remain unaltered even in much longer simulations.

The fluctuation maps (Fu) were calculated from the MD trajectories of the 0-chains as described earlier (Hery et al, 1997). The fluctuation value Fy is given by the equation, where dy(t) is the distance between a pair of designated atoms ( ca atoms as used here) at time t and the angle brackets represent time averages. The Fy values are the standard deviation of interatomic distance. The fluctuation maps in Figure 6 has a black dot wherever the Fu value is less than or equal to 0.5A. Thus dark regions of the map indicate those parts of the molecule which undergo strongly coupled movements.

chains were generated in the same way as for the isolated a-chains. Thus the model consisted of eight polypeptide chains ( 4 a-chains and 4 13-chains) with the central two a and two 13 chains making up the axial contact interface. The simulations of the above fiber models were carried out without explicit solvent using the GROMOS96 vacuum force field as implemented within the GROMACS suite of programs. Models of HbS mutants were generated using the program SCWRL and the mutant fiber models were generated as for the native HbS model. SCWRL replaces only the side chains of desired residues with the best possible rotamer of the mutated amino acids. Thus the initial backbone conformation of the isolated a-chains and the fiber models remained identical in the native HbS and the mutants. The MD simulation protocol for the fiber models consisted of an initial steepest descent energy minimization for 1000 steps followed by full MD simulation for 1.2ns at 300 K. Essential parameters of the simulation like the radius of gyration, root mean squared deviation from the initial structure as well as the kinetic and potential energies are summarized in Chapter I, Table 4. It was observed that all the global indicators of the simulation stabilized to their average values within 0.2ns. Hence data from 0.2ns till the end of the simulations were used for all subsequent analysis

MD simulations on the a-chain of HbS and the mutants were carried out using the GROMACS suite of programs (Lindahl eta!, 2001). The initial structure of the native protein was taken from the high-resolution x-ray crystal· structure (Harrington et a!, 1997) of HbS (PDB entry: 2HBS). The structures of the mutants were generated interactively using INSIGHT-II. The initial model structures were placed in a simulation box of size 42.8 x 31.5 x 42.7 A. The closest distance from any protein atom to the walls of the box was not less than 9 A. The system was then solvated by adding a bath of SPC (Berendson eta!, 1981) waters in such a way that the density of the system was as close to 1 as possible. The overall charge of the system was neutralized by placing suitable counter ions wherever necessary (Chapter 1, Table 4). The resulting system was then energy minimized for 1000 steps using the steepest descent algorithm. This was followed by 0.3ns of position restrained MD during which the solvent and counter ions were allowed to move freely but the protein atoms were harmonically restrained to their initial positions. Finally, normal MD was run for 3ns using the default GROMACS force field. Bond lengths were restrained to their equilibrium values using the LINCS (Hess et a!, 1997) algorithm and a cut-off radius of 0.9nm was used for non-bonded interaction calculation. The temperature of the system was maintained close to 300K by weak coupling to an external temperature bath with a coupling constant of 0.1 ps. The integration time step used throughout the simulation was 1 fs. MD simulations of single mutant a-chains (K 16Q, E23Q and H20Q) as well as the double mutants (K16Q/H20Q, K16Q/E23Q and H20Q/E23Q) were carried out in a similar fashion for 3ns. In order to directly analyze the effects of the mutations on the contact interface, we also carried out MD simulations on a miniature model of the sickle hemoglobin fiber consisting of a complex of two hemoglobin tetramers. In the low salt crystal structure of deoxyhemoglobin S (Harrington et a!, 1997) the asymmetric unit consists of two HbS tetramers that pack as two strands of HbS molecules running parallel to the crystallographic a axis. Axial contacts occur between two tetramers of the same strand and lateral contacts occur between tetramers of different strands. A canonical fiber model was generated by taking one of the two tetramers in the asymmetric unit together with its neighbor translated along the crystallographic a axis [Figure 7 (A, B)]. Fiber models incorporating mutant a

Electrostatic potentials were calculated by the Finite Difference Poisson-Boltzmann (FDPB) method using the program MEAD running within the PCE web server (http://bioserv.rpbs.jussieu.fr/PCE) (Miteva et al, 2005; Bashford et al, 1992). Additions of hydrogen atoms as well as assigning of atomic radii and charges were performed automatically within the server. MEAD numerically solves the Poisson-Boltzmann equation to yield the distribution of electrostatic potential on the protein surface. Calculations were performed on one of the native a-chains of the 2HbS crystal structure (Harrington et al, 1997) as well as its SCWRL (Dunbrack et al, 1993) generated mutants. All calculations were performed by setting the internal protein dielectric constant to 4 and the external solvent dielectric constant to 80. The ionic strength parameter was held at 0.1

The time kinetics of deoxyhemoglobin polymerization were studied in 1.8M, 1.5 and 1 M potassium phosphate buffer (pH 7.25) respectively as described by Adachi and Asakura (1979a, b) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. Deoxygenation of the hemoglobin sample was ensured by passing moist gaseous nitrogen over the sample in an airtight cuvette and by addition of sodium dithionite. The polymerization of the resultant deoxyhemoglobin samples was initiated by a temperature jump from 4 to 30 oc within 10 sec and the progress of the reaction was followed by monitoring turbidity changes at 700 nm. The delay time was calculated from the kinetic traces

with the plunger of a Hamilton syringe. The tube was centrifuged at room temperature at 14,000 rpm for 30 min. The above process of gel-disruption and centrifugation was repeated twice subsequent to which the oil layer was aspirated and suitable aliquots from the supernatant were taken for estimation of Csat by Drabkin' s reagent (Goldberg eta!, 1977).

he gelation concentrations of HbS constructs were determined by the dextran-Csat method of Bookchin et a! (Bookchin et a!, 1999). This method allows measurement of Csat under near-physiological conditions and at much lower concentration of HbS (about 5-fold or less) than that required in standard Csat assays, but essentially provides the same information. Briefly, a suitable aliquot of a concentrated solution of hemoglobin in potassium phosphate buffer (0.05 M, pH 7.50) was taken in a 1.5 ml micro-centrifuge tube. A concentrated dextran (70 kDa) solution prepared in the same buffer was added to it and mixed well. This mixture was overlaid with 0.5 ml of mineral oil, chilled on ice bath and deoxygenated with an anaerobically prepared dithionite solution through an airtight Hamilton syringe. The final concentrations of dextran and dithionite in the mixture were 120 mg/ml and 0.05 M respectively. The deoxygenated sample above was allowed to polymerize at 37°C for 30 min after which the gel under the oil layer was disrupted

All experiments were carried out on a Beckman XL-A analytical ultracentrifuge, equipped with absorbance optics, and an An60-Ti rotor, at 20 °C. Sedimentation velocity experiments were performed at 40,000 rpm. Data were collected at 540 nm and at a spacing of 0.005 em with three averages in a continuous scan mode. The protein concentration varied in the range 4-40 IJ.M (heme) in 50 mM phosphate buffer, pH 7.2

acid. The respective buffer baselines were subtracted from the sample CD data. The ellipticity of the protein samples is reported as mean residue ellipticity (MRE) in deg/cm2/dmol units. The first derivative UV spectra of the oxy and deoxy-HbS were recorded on a Lambda Bio20 spectrophotometer (Perkin Elmer Life ScieAces). The hemoglobin concentration used for the spectral measurements was approximately 50 )!M on heme basis.The spectra of unliganded proteins was recorded subsequent to deoxygenating the hemoglobin samples by passing moist gaseous nitrogen extensively over the sample in an airtight cuvette. Completion of deoxygenation was ascertained by recording the visible spectrum of the deoxygenated Hb sample.

Circular dichroism (CD) spectra were recorded on a J71 0 Spectropolarimeter (Jasco, Japan) fitted with a Peltier type constant temperature cell holder (PTC-348W). The calibration of the equipment was done with (+)-! 0-camphorsulfonic

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.

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.

concentrators (Amicon), and subjected to reduction with 0.0.5 M sodium dithionite. For this an appropriate amount of anaerobically prepared dithionite solution was added to the reconstituted Hb and the reaction mixture was quickly passed through a Sephadex G25 gel filtration column (30 em x 1.5 em) equilibrated with 0.05 M Tris HCI (pH 7.4), in order to minimize the duration of contact of dithionite with the protein. The reduced Hb was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and loaded onto a CM52 column (1 Ocm x 1.5cm) equilibrated with the same buffer. A linear gradient of 150 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) was employed to elute the protein from the column

Construction of mutant a globins

The semisynthetic a globin was purified from the mixture by CM52 urea chromatography as explained below. The lyophilized sample was dissolved in 0.005 M phosphate buffer (pH 6.9) containing 8 M urea and 0.05 M 2-mercaptoethanol at a concentration of I 0-15 mg/ml and loaded onto a CM52 column (16 em x 1.5 em) equilibrated with the same buffer. After an initial wash with the same buffer, two linear gradients of(a) 100 ml each of0.005 M to 0.03 M and (b) 100 ml each of0.02 M to 0.05 M phosphate buffer (all buffers contained 8 M urea and 0.05 M 2-mercaptoethanol, and were adjusted to pH 6.9) were employed at a flow rate of 45 ml/h to elute the semisynthetic a globin. The column was finally washed with 0.05 M buffer to elute unreacted a31-141 fragment from the column. The elution profile was monitored at 280 nm. The fractions for semi-synthetic a globin were pooled, extensively dialyzed against 0.1% TF A and lyophilized. The semisynthetic yield of the protein varied between 35% to 45%

V8 protease-mediated semisynthesis of a globin was carried out at 4°C in 0.05 M ammonium acetate buffer (pH 6) containing 30% 1-propanol. For this, the lyophilized samples of natural or synthetic analogs of a 1-30 and respective a31-141 were individually prepared in water. Suitable volumes of the complementary fragments were mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized material was dissolved in appropriate amount of ammonium acetate buffer (pH 6). To this solution, a suitable volume of 1-propanol was added to a final concentration of 30% 1-propanol and 20 mg/ml substrate. The mixture was cooled on ice subsequent to which suitable volume of V8 protease solution prepared in water ( 1% w/w of substrate) was added. The ligation reaction mixture was incubated at 4°C for 24 hours. The extent of synthesis was monitored on RPHPLC by loading an aliquot of the reaction mixture on an analytical reverse phase column. The reaction was stopped by addition of chilled 5% TF A solution (0.2 fold v/v) and lyophilized

Peptides were synthesized by standard solid phase synthesis protocols using Fmoc chemistry on a semi-automated peptide synthesizer (Model 90, Advanced Chemtech). For this, Wang resin pre-loaded with N-a-Fmoc-Glu was used as the starting material. The stepwise coupling of Fmoc amino acids was performed with DIPCDIIHOBT activation procedure. The coupling of each step was monitored by Kaiser test for free amine and wherever necessary, a double coupling was used to increase the yield. Before each coupling step and on completion of the synthesis, the N-terminal Fmoc group was removed using 20% piperidine (v/v in DMF). The peptides were cleaved from the resin and the side chains deprotected with appropriate volume of a mixture containing TF A, ethanedithiol, phenol, thioanisole and water (80:5:5:5:5, v/v). The resin was removed by filtration and the crude cleaved peptides were precipitated using cold diethyl ether and extracted in water. The peptides were purified by RPHPLC and their chemical identity was checked by mass spectrometry

The complementary segments of a globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (Sivaram eta/, 2001). The a globin was dissolved in 0.01 M ammonium acetate buffer (pH 4) at a concentration of 1.0 mg/ml and digested at 37°C with V8 protease (1: 200, w/w) for 3 hours. The completion of digestion was ascertained by RPHPLC, after which the reaction was quenched by addition of neat TFA to a final concentration of 0.1 %. The complementary segments, al-30 and a31-141, from the digestion mixture were isolated in pure form by size-exclusion chromatography on a Sephadex G50 column (98cm x 2.8cm). The column was equilibrated and run in 0.1% TFA. The lyophilized sample of the digest was dissolved in the above solvent and loaded on to the column. The column was run at a flow rate of 30 mllhour and the elution profile monitored at 280 nm. The individual chromatographic profile of a globin digest showed only two peaks, a31-141 and a1-30 respectively, as expected from a single cleavage at the 30-31 peptide bond. The peak fractions were pooled separately and lyophilized

The a-PMB chain was subjected to acid-acetone treatment to separate the heme from the a globin. Briefly, a solution of concentrated a-PMB chain (5 ml; 30 mg/ml) was added dropwise to I 00 ml of thoroughly chilled acid-acetone solution (0.5% v/v HCI in acetone) with constant shaking, and then incubated at -20°C for 30 min to allow complete precipitation of the globin. The precipitated globin was isolated by centrifugation at 7000 rpm (4°C) for 15 min and the supernatant containing soluble heme was discarded

The [3-PMB and a-PMB chains were eluted with a linear gradient of 500 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) at a flow rate of 50 ml/hour. The chains were separately concentrated using Centriprep concentrators (Amicon) and stored in liquid nitrogen till further use

The heme bound a and ~ subunits were obtained as described by Bucci (1981 ). Briefly, hemoglobin was reacted with PMB in an eight fold molar excess (8 moles of PMB per mole of hemoglobin). The reaction mixture was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and then loaded onto a CM52 column (30cm x !Scm) that was pre-equilibrated with the same buffer.

Blood was drawn from appropriate source into heparinised tubes. The blood sample was centrifuged at 4000 rpm for 15 min ( 4 °C). The supernatant was discarded, and the erythrocytes (pellet) were subsequently washed thrice with chilled isotonic buffer [0.01 M PBS (pH 7.4)] by centrifugation at 4000 rpm and 4°C for 15 min. The washed erythrocytes were lysed in water. The resultant red cell lysate was then dialyzed extensively against PBS (pH 7.4) at 4°C to obtain stripped hemoglobin (hemoglobin devoid of bound allosteric modulators like BPG). The stripped hemoglobin was then loaded onto a pre-equilibrated DE52 column (30cm x 15cm) after extensive dialysis against 0.05 M tris acetate buffer (pH 8.5). The protein was eluted from the column employing a linear gradient of 500 ml each of 0.05 M tris acetate (pH 8.5) and 0.05 M tris acetate (pH 7) at a flow rate of 50 ml/hour. The purified hemoglobin was estimated spectrophotometrically at 540 nm (molar extinction coefficient= 53236 cm-1/M) and stored at -70°C till further use.

Fmoc protected amino acids and other chemicals used in peptide synthesis were obtained from Novabiochem (Switzerland). V8 protease and TF A were procured from Pierce Chemical Company (USA), while }-propanol, PMB," Hemin, Dithiothreitol, EDT A were obtained from Sigma Chemical Company (USA). DE52 and CM52 ion exchange resins were purchased from Whatman (UK). Sodium diothinite was procured from Fluka (Switzerland) and Catalase from Boehringer Mannheim (Germany). Carboxypeptidase was obtained from Worthington Biochemical Corporation (USA).

Cells growing in culture medium were harvested by trypsinization and washed twice with ice cold PBS. Cells were fixed by adding ice cold 70% ethanol and stored at 4°C. Before harvesting cells were washed twice with PBS and re-suspended in adequate amount of PBS containing Propidium Iodide (PI) to a final concentration of 50μg/ml and RNase to a final concentration of 10μg/ml. Thereby the cell suspension was incubated at 37°C for 30 minutes in dark. Analysis was done by running the samples in BD FACS Vantage System according to the standard procedures after calibration of instrument with Calibrite beads

The plates were kept in incubator gently and the colony formation was monitored every week. Media (500μl) was added to the plates every 4th-5th day to avoid drying. Colonies formed in soft agar photographed were taken without staining, under a microscope in light field

Agar solution was prepared in a sterile 50ml Schott Duran Bottle and boiled in microwave until fully dissolved and kept at 55°C to 65°C. Master Mix with the rest of the components of bottom agar was made in a sterile corning 50ml tube prewarmed at 55°C and agar solution was added. The solution was once vortex briefly and then added (2ml) carefully to each well avoiding air bubbles. The plates were left undisturbed in laminar flow hood until the agar set fully. Two days before final assay, the bottom agar plates were kept in tissue culture incubator for equilibration. On the day of assay the following mix was prepared for Top Agar 4 dishes 5 dishes1.media with FBS, L-glutamine and Pen-Strep 4.8 ml 6 ml 2.fetal bovine serum 1.8 ml 2.5 ml 3.sterile water 1.8 ml 2.5 ml 4.agar 1.8% (1.8 g/100mLs) 1.8 ml 2.5 ml 5. cell suspension 1.0 X 105/ dish 100 to 350 μl 100 to 350 μl 6. Total 10.2 ml 13.5 ml Top agar mix without cells was first prepared and kept at 42°C. The cells were then trypsinized and re-suspended after counting in final volume of 100μl to 200 μl. Cells were then mixed with top agar and solution was quickly poured over the bottom agar.

For soft agar assays 2x104, (A549) or 1x105 cells (E-10) were used in 1.5ml top agar. For preparing bottom agar plates (0.64% final con. of agar), a following mix was prepared for five dishes. 1.2X media with FBS, L-glutamine and Pen-Strep 10 ml 2.fetal bovine serum 5 ml 3.sterile water 1 ml 4.noble agar 1.8% (1.8 g/100mLs) 9 ml 5.Total 25 m

For clonogenic assays, 1x103 (A549) or 2x103 (E-10) cells were seeded per well of a six well tissue culture plate and grown for 15 days. For identification of signaling pathways various inhibitors were used viz, PI3K inhibitor LY294002 (10μM), MEK inhibitor PD98059 (10μM) or p38 inhibitor SB203580 (10μM). Cells were grown in the presence of inhibitor for seven days following which fresh medium was added. For staining, cells were washed twice with PBS and fixed in 10% formalin for 10 minutes, washed extensively with water and stained with 0.25% crystal violet prepared in 25% methanol for 4hrs at 4°C. Plates were then washed with milli Q water and dried before scanning

and fixed with 100μl of fixative solution per well, for 10 minutes at room temperature. The cells were then washed twice with PBS and 100μl of staining solution was added to each well. The plate was kept at 37° C, until the color development.

4x103-5x103 cells were plated in 96 well plate, well. Cells were transfected with reporter plasmid 18 -24 hrs after plating. After 48 hrs, cells were washed once with PBS

1X PBS diluted in distilled water 1X fixative solution diluted in distilled water 2.4.12.3 Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF

20 mg/ml X-gal in dimethylformamide Solution A as 40 mM potassium ferricyanide. Solution B as 40 mM potassium ferrocyanide. Solution C as 200mM magnesium chloride. 10X fixative (20% formaldehyde; 2% glutaraldehyde in 10X PBS) 10X PBS as 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7

This protocol is for the detection of β-gal expression in fixed cells. It was performed on 96-well plates for initial screening of tTA transfected clone, and is a modification of Sanes et al., 1986

β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well

Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice

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

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

The PCR parameters were as follows

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).

appropriate secondary antibody (conjugated with horse-radish peroxidase) diluted in 5% fat free milk solution (in PBST) and incubated for 45 minutes at room temperature. After incubation the membrane was washed and processed for the detection of protein bands using ECL-plus detection reagent (Amersham Biosciences) followed by detection of signal on X-ray film (Hyperfilm-ECL, Amersham Biosciences)

The proteins were resolved using denaturing SDS-PAGE gel and after completion of the run, the gel was over laid on a nitrocellulose paper cut to the size of gel and kept in the blotting cassette in the presence of blotting buffer. Finally the cassette was put in the mini transblot apparatus (Bio Rad) and blotting was done for 4 hours at a constant voltage of 60 V. Then the membrane was taken out and rinsed in PBS containing 0.1% Tween - 20 (PBST) for 5 minutes by gentle shaking. Later the membrane was immersed in 5% non-fat milk solution in PBST with gentle shaking for 1 hour at 37°C. The membrane was washed off from the traces of the fat free milk with PBST and the membrane was over laid with primary antibody diluted in PBST for 3 hours at 4°C with shaking. After incubation the membrane was washed with PBST and layered with

Immunobloting

For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel

Lysate Preparation for Immuno- blotting

DTT. Then contents were then mixed and 1μl (200 units) of M-MLV was added. The mixture was then incubated at 37°C for 50 minutes. The reaction was stopped by incubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then used as a template for PCR. The expression of the investigated genes was determined by normalizing their expression against the expression of actin or GAPDH gene

Semi-quantitative RT-PCR

μg of total RNA was reverse-transcribed using poly-T oligonucleotide and M-MLV reverse transcriptase (Invitrogen) according to manufacturer’s protocol. Briefly, a 20μl reaction volume was made for 1μg of RNA. In a microcentrifuge tube, 1μl oligo (dT)(500μg/ml) , 1μg total RNA, 1μl 10mM dNTP mix and sterile water was added to afinal volume of 13μl. The mixture was then incubated at 65°C for 5 minutes ad quickly chilled on ice. To this mixture were added 4μl of 5X first strand buffer and 2μl of 0.1M

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed, from 35mm dish and 1ml to TRIzol was added directly to the dish and kept at room temperature for 5 minutes. The cells were harvested by pipetting up and down three four times and transferred to a 1.5ml microfuge tube. For each 1mlTRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed for 10 minutes. The upper aqueous phase was transferred into a fresh microcentrifuge tube and 500 μl of isopropanol was added and this was incubated at -20°C for 1 hour. The RNA was pelleted by centrifugation at maximum speed for 30 minutes at 4°C. The supernatant was decanted and the pellet washed with 1ml of 70% ethanol followed by a second wash with 1ml of 90% ethanol and centrifugation at maximum speed for 10 minutes. The supernatant was removed and the pellet air-dried for about 5 minutes and re-solubilized in 30-50 μl RNase free deionized (DEPC-treated Milli-Q) water and aliquots were stored at -70°C

Transient transfection of plasmid DNA in culture cells was performed using Lipofectamine 2000 according to manufacturer’s protocol. Briefly, forty million cells were seeded in a 35mm tissue culture dish, one day before transfection. Transfection was performed 18-24 hrs after seeding the cells. 4μg DNA was mixed in 50μl of Opti-MEM in one eppendorf tube. In another tube, 5μl of Lipofectamine 2000 was diluted in 50μl Opti-MEM and incubated at room temperature for 5 minutes. After five minutes, DNA and Lipofectamine 2000 were mixed together and complexes, incubated for 30 minutes at room temperature. Meanwhile, the adherent cells were washed twice with PBS and 1ml of Opti-MEM was added. 100μl of complexes were then added to each dish containing cells and medium. After 6hrs, the medium containing complexes was removed and complete medium was added and transgene expression was accessed 24-48 hrs after transfection

Transient transfections in adherent cells