Similar things happened earlier when we played the game with “birds” as the category.  In this case I thought of colors, which brought me: blue jay, red cardinal, yellow canary, flamingo, parrot, macaw, and so on.

Why is this interesting, you ask?  Consider the following background information for context:

(1) During my visit at Carnegie Mellon University in early March this year (I was there as a prospective BME grad student), I spoke with a professor who tells me it is possible to use EEG to demonstrate the mechanism of optical illusions.  i.e. we are tricked by optical illusions, but by mapping synaptic activity in the brain of someone looking at an optical illusion, it should be possible to explain how illusion happens, which brain regions are responsible, and in what way.

(2) There are studies showing that when faced with solving a cognitively demanding problem, smarter people’s brains show less activity than average brains.*  It is hypothesized that this diminished activity is due to smarter people using their brains more efficiently and therefore needing to go through fewer or shorter neuron pathways.

In this context, I got to thinking about how my friend and I dealt with what is essentially a vocabulary game.  My search algorithms were better in this case.  Might it be that I was using my brain more efficiently? We both have about the same knowledge of types of birds and rodents, we are both of similar intelligence, though we have different strengths (he’s a Math PhD at Columbia and beats me at Gipf all the time).  It might be interesting to use EEG to look at how people play games like what I played today, to see how they use their brains to search for words and see if common conclusions can be drawn about increasing the efficiency of searches through declarative memory.

If useful conclusions can be drawn, it might be possible for people seeking to train themselves to think more efficiently to do so using biofeedback machines that let them see how they are using their brains when searching for information.

Maybe I’ll get into this kind of research one day, after I’ve satisfied my tissue engineering research thirst.  I’m going into an MD-PhD program this fall.. I’m excited!

*Neubauer and Fink. Intelligence and neural efficiency: Measures of brain activation versus measures of functional connectivity in the brain.

In other words, suppose research group X discovers something about colon cancer cells, specifically colon cancer cell line Y.  They write up a paper with their data results and try to submit to Journal A.    Journal A demands them to prove  that the cells they’ve been working on are actually Y, and not some other colon cancer cell line expressing totally different morphology/proteins/receptors etc.  To prove that Y is Y, research group X would need to get the DNA of their putative Y and verify that it matches the canonic DNA for the Y cell line.

So you ask, Why wouldn’t they already have the DNA fingerprint?  Why would there be any doubt?  Didn’t they engineer these cells themselves?  Probably not.   What happens when you work on a colon cancer cell line is that usually you get the cells from a biotech company like ATCC, or you get a donation of cells from another lab.  To prove to the  journal that these cells you’ve been working on really are the cell line you say they are, you need to go get the DNA fingerprint.

Boring so far.  But now let’s ask why journals are beginning to require DNA fingerprints.

Case 1:  MD Anderson Cancer Center, one of the biggest kahunas of cancer research in the United States, once conducted a series of breakthrough studies on thyroid cancer, using thyroid cancer cell lines.  Here’s one from 2001.  Here’s another one from the same year.

In 2008, a bunch of party-pooper scientists led by Rebecca Schweppe proved that these thyroid cancer cell lines were not of thyroid origin.  The one MD Anderson used?  Actually a colon cancer cell line.

[http://jcem.endojournals.org/cgi/content/abstract/93/11/4331]

Too bad for MD Anderson.  Too bad for these guys in Japan, who published a 2006 review in Endocrine-Related Cancer involving the Kat50 “thyroid” cancer cell line.  Oh well, everyone’s naive at least once.

Case 2:  Lewis Lung Carcinoma

LLC was long considered a relatively uncommon form of lung cancer, and many research groups poured impressive amounts of time and energy into studying the carcinoma cell line.

It was later discovered to be a sarcoma.

Case 3:  Once popular breast cancer cell line MDA-MB-435 was also the subject of many scientific papers.  The MD Anderson Breast Cancer Cell Line Database includes an extensive list of its protein expression profile.

It was later proven to be a melanoma.

[http://www.ncbi.nlm.nih.gov/pubmed/17004106]

The list could go on much further, but I think I’ve amused myself enough today.

Overview

Over time, there have been a lot of different ideas about what dark energy is, how it behaves over space-time, how strong it is, and how it works. Common to all the main theories on dark energy is the premise that dark energy is a repulsive force, and is causing an acceleration in the expansion of the known universe. It is necessary to consider the existence of such a force because compelling empirical observations show we do live in a Universe that is expanding at an accelerating rate. By Newton’s equation of Force = mass * acceleration, we expect that where there is acceleration, there must be some force at work. Some force is causing the observed accelerating expansion. We do not know exactly what the force is, because it does not interact with matter and space-time in the way we are used to; we are “in the dark” about this force. So we provisionally call this force, “dark energy.”

Although the existence of this repulsive force could explain why we see accelerating expansion, we have replaced one puzzle with another: even if dark energy is the explanation for accelerated expansion, what is the explanation for dark energy? Early 20th century cosmologist physicists saw dark energy as a convenient way of explaining data or shoring up TOEs. Later physicists faced the thorny problem of devising models of dark energy that would plausibly explain why dark energy should take on the convenient values required by the data of their predecessors.

The competing theoretical models of this non-electromagnetic repulsive force are varied and often confusing to the casual physics reader. At various points in history the “repulsive force” has been called “the cosmological constant”, “vacuum energy,” “quintessence,” or simply “dark energy,” depending on expectations about the force’s behavior through space-time. To add further confusion, the different terms denoting different conceptions of dark energy are often used interchangeably. The particular model of dark energy in question must often be gleaned from context. In this paper I will try to use “dark energy” to refer generally to the alleged non-electromagnetic repulsive force accelerating the universe’s expansion.

To begin from the beginning, dark energy first entered into mainstream theoretical physics in the early 20th century. After publishing his theory of general relativity in 1915, Einstein proposed the existence of a “cosmological constant”. This mysterious space-time-invariant factor would oppose the tendency of gravitational attraction to cause the Universe to contract. For various reasons, Einstein did not want his theories to predict a contracting (or expanding) universe, hence the addition of the cosmological constant. Later, the field of quantum mechanics generated the concept of “vacuum energy,” which theoretically produces a general repulsive effect causing hugely accelerated expansion of the Universe. This idea brings extra problems, as the paper will discuss. Our itinerary for this paper is to tour through these historic incarnations of dark energy, giving theoretical and empirical context along the way, before we move on to current popular models of dark energy.

Part I, Early 20th Century: We Should Be Contracting (or decelerating expansion).

The cosmological constant as a bid for a static universe with gravity

There are four accepted fundamental forces in modern physics: gravity and the three forces of the Standard Model (electromagnetism, weak force, and strong nuclear force). Together, these four forces are supposed to completely describe all observable particle interactions in the universe, except maybe the accelerated expansion of the universe. But in 1915, we have not discovered this expansion problem yet, nor even learned much about the strong or weak force. These latter two forces can only act at ranges of significantly less than a nanometer anyway, so they do not matter when considering the general expansion or contraction of the universe. Nor does electromagnetism play a significant role here, since it depends on relative differences of charge and the Universe is remarkably homogeneous and neutral in charge as objects reach larger scales. Only gravity, an attraction felt between massive particles, exerts significant effect across vast expanses of space.

If one imagines a volume of space (the Universe) containing some number of massive particles (galaxies, dust, etc.), it is easy to see that every particle will exert a net attractive force on every other particle. If the particles begin in a static state (neither expanding nor contracting), then unless the particles are arranged in a perfectly symmetrical lattice, they will tend to move closer to their closest neighbors, converging into particle clouds that further compact themselves under the influence of gravity, until all matter is compacted into a small dense point. Observations of nearby stars and galaxies show the universe’s matter is not arranged in a perfectly symmetrical lattice that is identical everywhere even at small scales. According to the rules of gravity established under Einstein’s theory of General Relativity, even if a universe were static at some point, it cannot remain static; a static universe should be contracting in on itself. It isn’t.

Einstein did not know the universe was not contracting when he proposed a cosmological constant to oppose the contraction caused by gravitation. He proposed the cosmological constant so his theory could lead to a prediction of a static universe, neither contracting nor expanding. There were two main reasons to cling to the idea of a static universe: (1) Slipher’s empirical observations of red shifts suggesting universe-expansion were not well-known at the time, nor was there any other effectively publicized evidence of an expanding or contracting universe, so the static universe seemed to be a null hypothesis requiring the least number of unsupported assumptions; (2) the static universe idea was extremely popular and well-entrenched amid the scientific and general community, and Einstein did not feel like bucking yet another traditional idea of the universe. The cosmological constant in Einstein’s formulation behaved as an unchanging force exerted evenly throughout space-time, without variations in strength (i.e., constant). This constant would conveniently have a repelling strength value exactly canceling the contractive tendencies resulting from gravitational attraction. Einstein did not really try to explain why the constant was there, he just included it in his theory to force a prediction of a universe he wanted, i.e. a static one.

Empirical evidence against a static universe

In 1912, Vesto Slipher observed significant red-shifts in spiral nebulae. Red-shift is a phenomenon observed of light that is coming from a source moving away from the viewer. These spiral nebulae seemed to be moving away from the viewer (i.e. Slipher, on Earth) in all directions. Assuming we are not specially able to repel spiral nebulae, this means that spiral nebulae (and matter-clusters in general) are moving away from each other and other matter besides moving away from Earth. This points to an expanding universe, not a static one. Interestingly, an expanding universe is one possible solution to Friedmann’s equations, a set of cosmological equations derived from Einstein’s gravitational field equations by Alexander Friedmann. Friedmann omitted use of the cosmological constant in his calculations and published a paper in 1924 suggesting that (without a cosmological constant) the universe must be dynamic: either contracting at an accelerating rate or expanding at a decelerating rate. The dynamic universe seen in Slipher’s data would seem to refute Einstein’s cosmological constant. Further observations on the nature of the universe’s expansion refuted Friedmann’s world theories as well.

Hubble brings more evidence for expansion

Edwin Hubble used state of the art telescopes to measure brightnesses of relatively nearby Cepheid variable stars. Although variable in brightness, these stars tended to follow certain regular rules of luminous behavior, allowing Hubble to more accurately estimate their distances from Earth. Data on their distance and associated brightnesses helped Hubble to calibrate distance estimates of spiral nebulae (which were often farther away and more difficult to gauge in terms of distance). For similar reasons, Hubble was able to map distances in the observable universe using data from Type 1a supernovas, which tend to have fairly uniform intrinsic brightness and are therefore easier to gauge, distance-wise, given a measurement of perceived brightness coupled with parallax considerations. He discovered that the farther a spiral nebula was from Earth, the more red-shifted it would appear. More redshift implies the light source is moving away from the viewer faster than a source with less redshift. Even without considering different amounts of redshift, it was possible to notice that faraway galaxies and supernovas were giving off light of dimmer brightness than would be expected if they were moving away from us at the speed of more nearby light sources. This would suggest that the faraway light emitted was encountering a lot more distance to cover on its way to us than one would expect if its sources were moving away at the same rate as nearby light sources (Ferreira). Therefore, the faraway light sources must be moving away faster, producing more distance for the emitted light to cover on its way toward us.

These empirical observations made for compelling evidence that the universe was expanding. More or less evenly distributed expansion everywhere in a generally homogeneous Universe would account for objects farther away from us moving away faster than nearer objects are moving away. In the 1990s, scientists further discovered that this expansion was accelerating. Of course, no one yet had developed an idea of why the universe should be expanding at an accelerated rate. All theoretical models seemed to suggest the world should be contracting, or at least expanding at a decelerating rate due to gravity. No one had a theoretical model involving accelerating expansion, until 1948.

Part II, Mid 20th Century: We Should Be Expanding Much Faster Than We Are.

Heisenberg’s Uncertainty Principle

Heisenberg discovered that there are limits to how precisely one can make measurements of a particle’s position and velocity. If one gets the position exact, the velocity is unknown. If one gets the velocity exact, the position is unknown. If one gets the position with a position that is not exact but within a small range of possibilities, one can get the velocity as well, also within a finite range of possibilities. The corollary to this irreducible uncertainty in measurement is that given a finite amount of time and a large space, it is impossible to have exactly zero energy throughout that space for the entire time. The more certain you are about time-space, the less certain you can be about energy. A consequence in quantum mechanical theory is that there exists a minimum nonzero energy even in a vacuum. This energy can spontaneously transform through quantum fluctuations into virtual matter-anti-matter pairs which then annihilate each other to give back the original energy. This “vacuum energy” has interesting consequences.

Casimir plates

Ferreira gives a decent description of the Casimir effect in his book, The State of the Universe: A Primer in Modern Cosmology, and I will paraphrase. Imagine a fixed volume containing a huge vacuum and two identical, neutral, conducting plates, like in the diagram below.

The distance between the inner sides of the plates (a) is much smaller than the expanse on either side of the outer sides of the plates. Any sort of energy or force particle existing between these plates has to conform to a certain set of wavelengths that fit exactly to the space between the two plates. [Otherwise the particle will accidentally skip past one or both of the plates instead of interacting with the plate(s).] Since the space between the plates is relatively small, there are a relatively small number of energy or force particles that can fit between the plates. By contrast, there are many more possible wavelengths allowed for particles existing in the external vacuum, because the distance to the destination plate can be so much bigger and accommodate more possibilities. Also, by the uncertainty principle, more virtual particles are likelier in a larger space (i.e. the space on external sides of the two plates) while less virtual particles are likely to pop into a smaller space, such as that between the plates. Force and energy particles in the vacuum tend to exert an “energy pressure”, which pushes on the plates. Because there are more of these virtual particles (and more energy) pushing on the external sides of the plates than on the inner sides, the Casimir plates will tend to move together. This is the Casimir effect.

A consequence of this effect is that as one reduces volume of a space, the energy pressure due to vacuum energy decreases. This is unlike what happens in matter-filled space, as reducing a mass-filled volume tends to increase energy pressure. Another way to explain it is to say that vacuum energy has negative pressure. A negative pressure under the influence of gravity would tend to repel things instead of attracting them.* If positive pressure corresponds to a depression in space-time, becoming deeper as more pressure is added, translating into a curvature corresponding to high gravity, then negative pressure would translate to a “curvature” corresponding to “negative” gravity. Thus, vacuum energy due to quantum fluctuations would account for an accelerating expansion of the universe as objects increasingly repel each other as more space (and vacuum energy) comes between them. The vacuum energy neatly does not interfere with models of the early, small Universe, because the vacuum energy shrinks to negligible amounts when confined to small volumes such as that of the very early universe. Unfortunately, the repelling force predicted by quantum mechanical vacuum energy is about 10^120 times stronger than what we actually observe (Ferreira).

Physicists who had tried so hard to explain the puzzle of why the universe is not contracting now had a different problem: why isn’t it expanding faster? By the predictions of vacuum energy theory, particles should be repelling each other so fast that light should never be able to reach our eyes at all, from anywhere. Also, we shouldn’t be able to have eyes. In short, the expansion predicted by vacuum energy would not explain the Hubble data. Modern physicists try to deal with the problem by breaking it down into two parts.

(1) Somehow get rid of the vacuum energy issue completely by appealing to symmetric theories of the universe that might somehow cancel out some or all of the repelling energy.

(2) Find something else to explain the expansion of the universe, preferably something very small in strength, so that it would not interfere much with everyday experiences on Earth nor with theories of the early Universe.

Supersymmetry

Supersymmetry theory postulates that for every fermion there exists a boson that can be paired with it and vice versa. Because there seem to be an unequal number of fermions and bosons in the observed universe, supersymmetry theory would claim that something happened in the past (symmetry-breaking) to make the two equal groups of particles unequal. For reasons involving some extremely technical knowledge, it is possible to exploit this boson-fermion asymmetry to reduce the amount of repelling force predicted by vacuum energy theory (Ferreira). Nevertheless, the reduction is not nearly enough and the repulsion predicted is still at least 60 orders of magnitude greater than that observed.

In similarly vague fashion, Ferreira mentions that Supergravity theory gives rise to fields (particles) having properties similar to those ascribed to the ideal dark energy (i.e., one that drives accelerated expansion at the rate we actually observe). Furthermore, such dark energy fields or particles would not have to be uniformly distributed throughout the Universe under supergravity theory. This frees us from having to explain why some permeating repulsive force throughout the universe should happen to be exactly the value most convenient for the evolution of planets and life. Of course, it still does not get rid of the problem of canceling out that pesky vacuum energy, but sometimes it is easier to address one puzzle at a time. For now, we will pretend the vacuum energy is canceled by some amazing symmetry theory, and deal with the most popular models of dark energy. This is a key bit of intentional ignorance, because the most popular modern models of dark energy tend to pretend that vacuum energy was just a bad dream we are not going to acknowledge.

Part III, later 20th Century: Four Dark Energy Models

By the late 20th century, requirements for ideal dark energy are getting strict. Dark energy must

• Be weak in the early universe so as to have had negligible effect until recently.

• Be strong enough to have effects we observe

• Not be so strong that it smooths out all inhomogeneities as it stretches the universe spatially through time (galaxies represent locally inhomogeneous parts of the universe)

• Not be affected by distance the way other forces are (the force of gravity decreases as distance increases; if that happened with dark energy, sustained acceleration of expansion would seem unlikely)

For a while, the working model of dark energy simply designated the convenient value fulfilling these requirements as the strength of dark energy throughout all of space-time. This seemed too neat for many skeptical physicists, even when incorporated with the trusty Anthropic Principle, so another condition joined the existing restrictions on dark energy.

• The behavior of dark energy should not be too neat.

Quintessence

In brief, the Quintessence theory of dark energy postulates the existence of a repulsive (fifth) force, quintessence, which does not interact strongly with the four fundamental forces of gravity plus the Standard Model. Just after the Big Bang, the universe was small and particles all had most of their energy bound up in their rapid motion under high temperature and pressure. Thus, all particles moved essentially like massless particles, whether they had mass or not. They moved like radiation particles, such as photons. During such an era, the energy pressure exerted by these particles would have been enormous. Ferreira notes that the contractive tendency of clouds of matter under gravity is even stronger when that matter is in radiation form, because gravity is proportional to energy density plus energy pressure (heavy slow particles have the former but very little of the latter, whereas radiation type particles have more equal amounts of both). In such a situation, the intrinsic repelling force of quintessence would be barely felt. As the universe expanded, the amount of particles per unit volume would have decreased, and so would the strength of their interactions via the other four forces. Only when the universe had reached a size wherein the average energy density was just above absolute zero (i.e., today), would the tiny force of quintessence cause stronger “interactions” overall than those caused by the other four forces.

Quintessence theory deals with the neatness problem by giving quintessence the unusual property of taking on the strengths of whichever force is dominating the universe at the moment. So at the beginning of the universe, before it reached Planck lengths, supergravity would have dominated, and quintessence would have taken on supergravity’s strength levels, driving very rapid expansion (the inflation period). As the universe got older, quintessence would take on the strength of the electroweak force once gravity crystallized out of the unified forces. As objects moved even farther from each other and the universe evolved into a gravity-dominated era, quintessence would take on the strength of gravity. As expansion continued, gravity’s strength over huge expanses of space would wane even more, until the gravity field would be dwarfed by the quintessence field, which would then take on its own intrinsic value. Since the other four forces decay comparatively rapidly as space between particles expands, it is possible for quintessence, which decays much much more slowly over distance, to eventually dominate the universe. In some ways, I think quintessence theory is even neater than the cosmological constant-style view of invariant dark energy. However, quintessence allows dark energy to take on a larger range of values, which is more believable for some cosmologists. Quintessence theory predicts that the universe will continue to expand until the other galaxies are moving away from us faster than the speed of light, at which point we shall have no more information about them and only be able to see our own galaxy. No other part of the universe will be visible to us.

Big Rip

A more extreme version of the dark energy theory goes further than the isolation of our galaxy. The Big Rip theory postulates that as the universe continues to expand, even very local forces will no longer be able to hold atoms and molecules together, and everything will be ripped apart into individual hadrons and leptons and bosons. Then those particles will themselves be ripped apart into something even smaller, forever ripping until/unless they reach something too small to rip.

Cyclic Universe, or Big Bang-Big Crunch

The cyclic universe theory of dark energy took Paul Steinhardt and Neil Turok and entire book (Endless Universe: Beyond the Big Bang) to explain, so I will have to skip many details in order to give the gist in my limited space. In the cyclic universe model, dark energy behaves like a coiled spring. At the Big Bang, it was compressed very tightly and then released, whereupon it behaved essentially like it does in Quintessence theory, stretching outward as the universe expanded, eventually driving some of that expansion itself as it became the dominant energy in the universe. According to the cyclic model, the dark energy spring will eventually reach an end to its stretch and then begin to snap back toward compression again, like a real spring. This will drive the universe to contract into a Big Crunch, which will conveniently put the universe in a state of high density and pressure sufficient to fulfill initial conditions of the Big Bang. A Big Bang will occur, and the cycle will begin again as the universe stretches outward. The cyclic model has the advantage of explaining the general homogeneity of the universe plus the existence of small heterogeneous areas. The homogeneity is explained by dark energy smoothing space-time during previous expansions, and the heterogeneity is explained by M-theory membranes discussed later. Previous noncyclical models are hard pressed to explain how the Universe could expand so rapidly and uniformly that it is remarkably homogeneous on a “global” scale, and yet maintains heterogeneous galaxies at the local scale.

M-Theory, membranes

Steinhardt and Turok explain the membrane part of their theory by asking us to picture two flat, flexible sheets, or membranes, fixed in space and positioned in parallel so that there is less than a centimeter separating their planar surfaces. Each membrane represents a three-dimensional world, and they are separated by a short distance along the axis of a fourth spatial dimension. If a wind blows against these sheets, or if something in the sheets causes a mild perturbation or ripple, parts of the membranes may collide, releasing energy on impact. This energy could exert gravitational attraction on nearby particles within each sheet, producing a locally inhomogeneous area as particles clump around the bit of “seed energy.” Many collisions produced by random vibrations or billowing would produce an essentially even distribution of inhomogeneous clusters of particles distributed across two globally homogeneous membranes. Steinhardt and Turok ask us to consider a theory wherein our universe resides in one of those membranes. As the membranes stretch while they move toward each other and away after collision, wrinkles (local inhomogeneities) in the membranes might be smoothed out, as happens when the fabric of the universe expands.

Closing statements

Current models of dark energy are myriad, creative, and often technically dense. Many seem confused or glaringly incomplete. Some models of the universe insist dark energy does not exist as a separate force at all, but is actually some weird aspect of gravity’s behavior on large scales. Because of all the confusing ideas out there, it is sometimes difficult for the layman to make head or tail of what people mean by “dark energy.” The word itself brings to mind images of sinister super villains, or new age terminology. This does not help to lend credibility to the physicists trying to put forth their theories with seriousness and rigor. Nor is it desirable that something with such popular name-recognition in the general population should be simultaneously so little understood beyond the name and association with physics. For this reason I hope to have provided a decent tour of dark energy, challenging but not overwhelming in breadth and depth.

Informal Works Cited:

Friedmann. On the possibility of a world with constant negative curvature of space. 1924

Ferreira, Pedro. The State of the Universe – A Primer in Modern Cosmology.

Paul Steinhardt and Neil Turok. Endless Universe: Beyond the Big Bang.

Let’s say instead of one pair of plates you have two pairs of plates, and each pair is much farther from the other pair than the space between plates in a pair.. Sort of like this (ignore the dashes, I put them in as spacers):

|—|—————————————–|—|

Call the plates a, b, c, and d from left to right.

Now pretend for a moment that a and d are fixed in space. Then, because there’s more space between b and c compared to space between ab and cd, b and c are going to tend to move away from each other and move toward their partners, because there’s more energy pressure (and space) between b and c than there is between ab and cd.. So you get this:

|-|———————————————|-|

In a way, b and c seem to have “repelled” each other and the plate pairs are now a little bit farther apart.

If you pretend the plate pairs were really galaxies or galaxy clusters, then the ab galaxy just moved away from the cd galaxy and vice versa.

Now, in reality there is no reason why a and d are going to be fixed in space, so you might think that the centers of each galaxy are still going to stay the same distance apart, but consider a couple ideas:

(1) What if ab is on one “edge” of the universe and cd is on the opposite “edge” of the universe? If the vacuum counts as part of the universe then there isn’t even vacuum space on the external sides of a and d:

-|—|—————————————–|—|-
^not much here———————-not much here either^

So a is not going to be pushed toward b as much as b is pushed toward a, since there’s less vacuum energy on the other side of a to do any net pushing of the plate pairs toward each other.
(if a dot represents half a dash) You get
.|–|———————————————|–|.

instead of
–|-|——————————————-|-|–
(^here the center point between plate pairs hasn’t moved even though individual plates moved. b and c are farther apart but a and d are closer together than they were before)

Now, consider another idea..

(2) What if you have a whole bunch of plate pairs, and they’re not arranged in neat little rows anymore? What if you have something like this:

Pair 1——————— Pair 2

———Pair 3—————–

Pairs 1 and 3 are relatively closer to each other than they are to pair 2. The bigger space between them and Pair 2 will tend to push pair 2 farther away from 1 and 3 compared to anything trying to push 1 and 3 away from each other. Furthermore, pair 2 is going to want to move away from 1 and 3 (relatively speaking) at an angle, which may put it in a position to “interact” with some other plate pair not depicted. If you have a lot of galaxies distributed randomly like this, over time the farther galaxies will seem to be repelling each other.

Again it could be that the repulsion effect doesn’t actually have an intuitive mechanism like what I’ve just described–Ferreira is unclear about that. But it helped me immensely with the concept to go through the above thought experiments.

Example Procedure:

Choose a DFHR -/- cells and electroporate with mouse/human chimeric anti-CD20 IgGI (immunoglobulin G1, an antibody) expression vector and select in media lacking hypoxanthine and thymidine

Example Procedure:

Human B-lymphoma Raji-cell microsomal fraction is coated onto 96-well immunoplates and incubated overnight (4°C).

Introduce various antibodies into wells, incubate 2 h at room temp.

Detect antibody binding to CD20 (on lymphomas) with 1/3000 dilution goat anti-human IgG1 peroxidase-conjugated polyclonal antibody.

Develop with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS).

• initial mRNA preparation may have DNA contaminants
  • therefore design primers using sequences from adjacent exons
  • OR treat with RNase-free DNase

Key terms: gene-specific primer (GSP), universal primer (at either end of DNA molec.), mRNA/cDNA heteroduplex

terminal deoxynucleotidyl transferase (TdT) – non-specifically adds deoxynucleotides to 3′ end of a single DNA strand (i.e. NOT RNA)

Rapid Amplification of cDNA Ends (RACE)

3′ RACE – amplifies cDNA corresponding to 3′ end of eukaryotic mRNA having poly-adenine tail; First strand initiated by oligo-dT universal primer, second strand initiated by GSP.

**optional additional anchor sequence on oligo-dT to provide more effective site for subsequent amplification + restriction site for subseq. cloning

———————————————————-
Prep: Cell lysis=>RNA extraction=>Oligo dT cellulose column chromatography

=>(1) purify RNA solution, +Oligo-dT universal primer, + reverse transcriptase, +nucleosides

mRNA 5′ ————————————-AAAAA………….. 3′

1st strand cDNA 3′ …………<——————-[TTTT|Anchor] 5′

*[]s denote primers

…….. s are just spacers

=>(2) Denature=>RNase, purify 1st strand cDNA with centrifuge or phenol => +GSP, reanneal +DNA polymerase +nucleosides

1st strand cDNA 3′ ———————————————[TTTT|Anchor]………….. 5′

2nd strand 5′ [GSP]——————————–AAAA|Anchor_match] 3′

=>(3) Amplify by usual PCR: Denature, anneal primers (GSP+oligo-dT or GSP+anchor), extend with DNA polymerase, repeat

*Specificity can be increased using nested PCR in which further amplification uses second nested primer instead of GSP.

———————————————————-

5′ RACE – amplifies cDNA corresponding to 5′ end of eukaryotic mRNA having a poly-adenine tail; First strand initiated by random primers, second strand initiated by oligo-G (if you’re using deoxycytosines for your TdT stage).

Prep as with 3′ RACE

=>(1) purify RNA solution, +random primers (more likely to produce 1st strand corresponding to 5′ end than oligo-dT), + reverse transcriptase, +nucleosides

mRNA 5′ ———————————————————————-AAAAA 3′

1st strand cDNA 3′ <—————-[Random primer] 5′

=>(2) Denature=> + terminal deoxynucleotidyl transferase (TdT) +deoxycytosines (dCTPs) => RNase and denature, purify 1st strand(s) cDNA with centrifuge or phenol => +anneal with oligo-dG primer & anchor +extend with DNA polymerase +nucleosides

1st strand cDNA 3′ …………….CCCC————————–[Random primer] 5′

2nd strand cDNA [Anchor|GGGG]————> 5′

=>(3) Amplify by usual PCR: Denature, anneal primers (GSP+anchor), extend with DNA polymerase, repeat

*Nested PCR again possible, replacing GSP with second specific primer.

Sources: Dr. Daniel Kalderon’s lectures; Dale and Schantz. From Genes to Genomes.

1. Lyse cells using lysozyme (breaks bacterial cell walls), EDTA (destabilizes outer memb. in bacteria, also inhibits DNases) and detergent (ex. sodium dodecyl sulphate; SDS) to solubilize membrane lipids.

——————————————————————–

2. Denaturing and Extraction methods:

D) Enzyme treatment

+RNase (pre-heat RNase to denature any contaminant DNase)

+proteolytic enzyme (ex. proteinase K)

-

D+E) phenol-chloroform extraction [removes proteins]

*Use (i) for DNA extraction, (ii) for RNA extraction

i) cell lysate+phenol (equilibrated with neutral or alkaline buffer such as water) =>agitate vigorously =>proteins denature and ppt at interphase, DNA+RNA in aqueous layer=>RNase

ii) cell lysate+ acidic phenol=>agitate vigorously =>proteins denature and ppt at interphase, RNA in aqueous layer, DNA in organic/phenol layer

-

E) alcohol precipitation. To the RNA or DNA solution,

add isopropanol or ethanol with monovalent cations (Na+, K+, or NH4+) to ppt nucleic acids

centrifuge + remove supernatant, remove salt by washing ppt with 70% ethanol

-

E) FOR PLASMIDS, alkaline denaturation:

Raise pH to 12, denaturing DNA in your DNA solution. Linear DNA will separate and tangle; plasmids can’t separate because of supercoiling.

Lower pH again. Everything (linear DNA, cell wall debris, proteins…) clumps to bottom except plasmids, which stay in solution. This method purifies enough for restriction digestion.

-

E) faster and simpler than alcohol precipitation is size-selection chromatography:

this column purification method uses a matrix of small porous beads which trap smaller molecules and let long ones through

-

E) affinity chromatography uses resins on the beads that bind what you want to purify and let everything else go through.

Advantages of using CHO cells:

• high growth rate
• high productivity
• ease of genetic manipulation
• good proliferation in large-scale suspension culture
• adaptability to protein-free media
• one of most universal hosts in biopharm production (this is only an advantage because you’re more likely to get extensive literature/applicable accessories for CHO cells than some more obscure cell line)

Problems with gene-targeting:

• Though you need somatic cells (rather than ES cells) if you want to make antibodies, homologous recombination in somatic cells (ex. CHO cells) occurs at > 100-fold lower frequency than in ES cells (Arbones et al., 1994; Hanson and Sedivy, 1995).
• For CHO cells
  • targeting vector frequently copies target sequence on CHO genome (Adair et al., 1989; Nairn et al., 1993; Penningon and Wilson, 1991) and the truncated end of the vector sequence is extended for several kb beyond target homologous region on the vector (Aratani et al., 1992; Scheerer and Adair, 1994), and randomly integrated elsewhere on the genome. This gives rise to many pseudo-homologous recombinants with the same sequence on nontarget and target loci. They can’t be distinguished from true homologous recombinants by genomic PCR so you will need to introduce a probe on your construct and/or target neighborhood for Southern blot analysis so you can distinguish what you want.
  • chromosomal abnormalities that affect copynumber and chromosome location of target loci accumulate in CHO cells (Siciliano et al., 1985; Warner, 1999). Most cultured somatic cell lines are aneuploid and go through chromosomal rearrangement during long cultivation.

Example staining procedure:

1a) Suspend 2 x 10^5 cells in 50 uL PBS (phosphate-buffered saline) containing 1% bovine serum albumin and 2 ug/mL fluorescein isothiocyanate (FITC)-labeled LCA.

1b) For the control, do the same thing above, replacing the FITC-labeled LCA with a 1/100 dilution of FITC-labeled streptavidin (which lacks glycosylation)

2) Incubate both mixtures at 4°C for 30 min, then analyze stained cells by FACScalibur.

*There are two types of oligosaccharides found attached to proteins by glycosylation: the N-linked oligos are attached to the amino group of asparagine side chains, and the O-linked oligos are attached to the hydroxyl of serine and threonine side chains.

Tour: A large breadth of related material covered haphazardly by (1) choosing a few arbitrary points of interest to expound upon and (2) doing uneven justice to the rest. Allows for more speculation and questions than summary.

Exploration: A single point of interest pursued to relatively great depth, with occasional forays into tangentially relevant supporting points.