Techniques in Molecular Biology: Monoclonal Antibody Production

Antibodies are perhaps one of the most important tools in the arsenal of molecular biologists. They have a wide variety of applications, from targeting macromolecules with fluorescent dyes or other indicators to aid in visualization, or as a component of other molecular techniques, such as immunopercipitation of proteins. Antibodies that specifically target a researcher's molecule of interest, though, have not always been in the biologist's toolbox.

Before researchers had this ability, antibodies were produced using a simple technique: inject your antigen of choice into a mouse (or goat, or rabbit), and the mouse will produce antibodies targeted against the antigen. After taking a blood sample from the mouse and collecting the serum, the antibodies could be purified. Antibodies produced in this manner were said to be polyclonal: that is, they were derived from multiple antibody producing B-cells in the spleen. Each antibody producing cell produces a different antibody¹, and antibodies produced in this way will be a mixture of different antibodies from different cells.

Monoclonal antibodies, in contrast to the polyclonal variety, are derived from a single antibody producing cell. Antibodies from a single cell will all be specific for the same antigen epitope. This confers several advantages over polyclonal antibodies. Perhaps the biggest advantage is that they allow researchers to target specific epitopes on an antigen. Let's say you wanted to mark a specific residue on a protein using antibodies that are labelled with a fluorescent dye. Using polyclonal antibodies, your protein would end up labelled all over, since the polyclonal antibodies would bind of a variety of epitopes on the protein's surface.  Monoclonal antibodies specific to the residue of interest would get rid of all the problematic non-specific binding.

The production of monoclonal antibodies, however, is somewhat different, and pretty cool. The technique starts off just as it would if you were making polyclonal antibodies: inoculate a mouse using your antigen of interest. The antibody producing B-cells in the mouse spleen will begin to make antibodies that target your antigen. Where Köhler, and Milstein's technique differs is in what is done with those B-cells. Normally, the antibody-producing cells only start making antibodies near the end of their life. Isolating individual cells (thus isolating only one type of antibody) and growing them in culture would work to produce monoclonal antibodies, but only for a short amount of time. Milstein and co. got the idea of fusing the B-cells with immortalized mylenoma cells. Cancer cells can, for a variety of reasons, become immortalized², and continue replicating - indeed, this is what makes cancer a problem! By creating hybrids between B-Cells and mylenoma cells - called hybridomas - they were able to created antibody-producing cells that live forever and keep on producing antibodies. Culturing these cells and purifying the antibodies now became a more viable option. Separate cell lines were isolated and cultured, so that each culture contained only cells from an individual lineage, and consequentially, produced only one kind of antibody. These cells are first grown on plates to establish a lineage, but are eventually transferred to large tissue culture flasks. This allows for tons of monoclonal antibodies to be produced, isolated, and used by researchers the world over.

This technique pioneered by  Milstein and Köhler revolutionized the way research is done in molecular biology.  It was important enough that it won Georges Köhler, and César Milstein, the Nobel Prize in Medicine in 1984³. Since then, antibodies that target any antigen imaginable have been developed, and can easily be ordered from companies that specialize in production of monoclonal antibodies. It is unlikely, I think, that monoclonal antibodies will be ever displaced as a staple tool for molecular biology research.

FURTHER READING:

I you're interested in reading more about the history of antibodies, their discovery and the story of Milstein and Köhler's work, I suggest reading through "A Brief History of the Antibody" posted at the Proteintech Group blog [Part I Part II Part III]. Milstein's Nobel lecture also contains some interesting insights into antibodies, antibody research and the development of Hybridoma technology, and can be found here.


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1. The different antibodies produced are specific for the same antigen, but are directed towards different epitopes on the antigen.

2. This is to say that they are not subject to the Hayflick Limit. The Hayflick Limit, named after American researcher Leonard Hayflick, is the number of times a given cell line can divide before stopping. Originally, cells were thought to replicate indefinitely, and failure to keep cell lines alive was thought to be due to ignorance of optimal techniques. Hayflick and Paul Moorhead, working at the Wistar Institute in Philadelphia in 1961, showed experimentally that cell lines impose a limit on the number of times they can divide. The limit differs between cell types, but for humans it is around 52 divisions.

3. The prize was shared with Neils Jerne, who was awarded the prize for his work on the development and control of the immune system.

Rick Perry and Galileo: BFFs?

With the impending doom that is the Republican presidential nomination looming ominously on the horizon, Wednesday was the night of the latest debate between the Republican candidates. On the list of talking points was the inevitable question about global warming. Denying global warming is de rigueur in the Republican party these days, so it wasn't surprising that, with the exception of Huntsman, the idea of anthropogenic climate change was scoffed at across the board. It was Rick Perry, though, that did manage to raise some eyebrows when he delivered this gem:

"The science is not settled on this. The idea that we would put Americans' economy at jeopardy based on scientific theory that's not settled yet to me is just nonsense...just because you have a group of scientists that have stood up and said here is the fact, Galileo got outvoted for a spell."

Oh, Rick, you silly man. How art thou wrong? Let me count the ways:

1) The science is settled on the issue. Anthropogenic climate change is a reality, one that is accepted by the vast majority of scientists. Some of the exact particulars of the issue are currently under debate - long-term climate projections for the future, the extent to which particular pollutants have contributed, etc - but these are not the things that Perry claims are unsettled. It is the mere existence of anthropogenic climate change that Perry denies, and as far as science is concerned, that issue most certainly is settled.

2) Galileo was not "outvoted". Perhaps this was just a bad choice of words on Perry's behalf, but I fear that it indicates a deeper misunderstanding of how science works. Scientific debates are not settled by a vote. Scientists are not polled for their opinions and the ideas of the majority put forward as the scientific reality. The "truth" is not determined by popular vote, but by careful and critical consideration of the evidence. I'd like to think that Perry realizes this, but given the anti-science attitude that he and many others in the Republican party display, I'm not so sure that he does.

3) Does Perry not realize that Galileo's ideas were not suppressed by the scientific community, but rather, by the Church? Perhaps, as a religious conservative himself, Perry has chosen to overlook this fact. Galileo's case does not parallel the criticisms against climate change denialists. Galileo did not meet resistance from the scientific community, for one. The heliocentric model was supported by a large number of Galileo's contemporaries, including Copernicus (who was the father of modern heliocentrism!), Johannes Kepler, and to some extent Tycho Brahe (who had posited his own heliocentric model of the solar system). The condemnation of heliocentrism came from outside the scientific community. It raised the ire of the religious community (much like the concept of global warming does today!). Compare this to climate change. Climate change "skeptics" do not form a large portion of the current scientific community and are largely found on the fringes of science and often in disciplines unrelated to climate change. They are criticized by the scientific community itself, and this in no way parallels the persecution experienced by Galileo.

Researchers discover "7th and 8th bases of DNA"? Hardly. Here's your daily dose of science journalism fail.

Making the rounds on the blogosphere and the news sites today is the announcement that researchers have discovered the "7th and 8th bases of DNA". This announcement comes from a paper published online on Science's pre-print server¹, Science Express by researchers at the University of North Carolina School of Medicine, and most of the news reports seem to be based on an article posted to Science Daily. The article reads:

"For decades, scientists have known that DNA consists of four basic units -- adenine, guanine, thymine and cytosine. Those four bases have been taught in science textbooks and have formed the basis of the growing knowledge regarding how genes code for life. Yet in recent history, scientists have expanded that list from four to six. Now, with a finding published online in the July 21, 2011, issue of the journal Science, researchers from the UNC School of Medicine have discovered the seventh and eighth bases of DNA."

Oooh! Exciting! What are these bases, exactly?

"These last two bases -- called 5-formylcytosine and 5 carboxylcytosine -- are actually versions of cytosine that have been modified by Tet proteins, molecular entities thought to play a role in DNA demethylation and stem cell reprogramming."

So, wait a second. These "new" bases are only modified forms of cytosine? So what? This is no big deal at all. There are well over a dozen known modified bases. Here, let me list a few:

• 5-hydroxymethylcytosine
• 5-hydroxymethyluracil
• N⁴-methylcytosine
• 7-methylguanine
• N⁶-methylcytosine
• β-D-hydroxymethyluracil

Need I go on? If we're counting modified bases, then there are perhaps two dozen or more known bases. Why do 5-formylcytosine and 5-carboxylcytosine get the elevated status as the '7th and 8th' bases, when there are so many more modified bases that seem to have gone ignored (and who, for that matter, gave 5-methylcytosine and 5-hydroxymethylcytosine the distinction of being the 5th and 6th)?

Is the discovery of 5-formylcytosine and 5 carboxylcytosine interesting and exciting? Yes, most definitely. Are they the "7th and 8th" bases of DNA? Nope, not at all.

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Ito, S., Shen, L., Dai, Q., Wu, S.C., Collins, L.B., Swenberg, J.A., He, C., and Zhang, Y.  Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine. 2011.  Science Published Online 21 July 2011 doi:10.1126/science.1210597

Of Hens Teeth and IDiots.

The literature published by the Discovery Institute often confuses me. I'm never quite sure if it should frustrate me or amuse me. Their constant mangling of science combined with their propensity for telling half-truths and distorting reality both makes me laugh (hah! They really think they have science on their side?) and makes me embittered (How dare they twist science to deceive and miseducate?). I guess this recent article by Discovery Institute crony Casey Luskin should be of no surprise, then. In the piece, titled Of Hen's Teeth and Neutral Mutations, Luskin attempts to dismantle a claim made by Stephen Jay Gould about hen's teeth (or the lack thereof):

"Evolutionists often cite an experiment which purportedly induced tooth growth in chickens, supposedly confirming that birds have genes for teeth and are descended from toothed reptilian ancestors. For example, in his book Hen's Teeth and Horse's Toes, Stephen Jay Gould discusses this experiment...But there's a problem with Gould's argument: as Sean Carroll explains, neo-Darwinism has a 'use-it-or-lose-it' rule. According to neo-Darwinism, if a trait is not used then the DNA which encodes it will accumulate neutral mutations, and eventually the trait will be lost forever. If supposed chicken genes for producing teeth haven't been used for 60+ million years, then that would strongly suggest that neutral mutations should have long-since destroyed their ability to function."

For those of you who might be unfamiliar with the experiment in question - and with Gould's discussion of it - it would be worth the while to go into detail.  In 1980, Gould published the book Hen's Teeth and Horse's Toes, a collection of articles he had written for various magazines (primarily for Natural History). Included was an article of the same name where he discussed atavisms - apparent reversions in individuals to an ancestral phenotype. Gould claimed that atavisms are a shining example of the evolutionary past of a species coming to the surface. He illustrated his point with two examples: polydactyl horses and chickens with teeth. It is this second example towards which Luskin has aimed his bow and launched forth a volley of ignorance.

On p.182¹, Gould explains a curious experiment performed by E.J. Kollar and C. Fisher: they devised a way to prompt chickens to develop teeth. If it's been a while since you took a good look in the mouth of your local avian friends, then it might interest you to know that birds don't have teeth. The most recent known fossil of toothed birds dates to around 80 million years ago, so somewhere in the intervening time, birds lost the ability to produce teeth. Odontogenesis in vertebrates is a complex process (then again, developmental programs always are!). It requires two different tissue types to occur: epithelial tissue and mesenchyme. The outer enamel layer of a tooth is formed by the epithelial tissue, while the inside dentin later of your tooth grows from the mesenchyme. But there's a catch: the mesenchyme cannot produce dentin by itself, it needs to be in contact with epithelium for dentin production to begin - that is to say, epithelium induces the production of dentin. This dentin, in turn, induces the production of enamel in the epithelium. Birds don't produce dentin, nor, consequently, enamel, so birds are born toothless. Kollar and Fisher's idea was brilliant but simple: what happens if you graft chick epithelium with murine (mouse) mesenchyne? Mice most definitely have teeth, so we know their mesenchyne is capable for producing dentin if prompted by epithelial tissue. What they found was astounding: when mouse mesenchyne was grafted to chick epithelium, teeth (dentin and all) were produced. This meant that avian epithelial tissue - despite the fact that birds have no teeth, and have not had teeth for as long as 80 million years - is still able to induce dentin production in the appropriate mesenchyne. Gould mused that this experimental result displayed the evolutionary history of birds. Why else would avian epithelial tissue have the latent ability to induce dentin production unless they had descended from toothed ancestors?

Luskin, however, thinks that Gould was completely wrong. Luskin argues that, if birds lost the ability to produce teeth 80 million years ago, then the genes for tooth production would have accumulated so many mutations that it would be impossible to revert back to the original toothed phenotype. The tooth production genes, he claims, would have since been destroyed beyond the ability to function. He bases this argument in something called Dollo's Law. Dollo's Law, put simply, states that evolution cannot reverse itself, and that genes which escape selection pressure will degrade fast enough that reverting to the original phenotype is tantamount to impossible. According to Luskin, the example of toothed hens is not the resurrection of a lost developmental pathway but the result of an experimental mistake.

Luskin cites a paper from Marshall, Raff and Raff² that seemingly supports his argument. In the paper, the authors devise an equation that determines the probability of a silenced gene's reversion as a function of time passed. They concluded that, for a gene that has been silenced for 10 million years, there is a near-zero probability for reactivation. How do they account for Kollar and Fisher's results? They state that "the classic example of the resurrection of "hen's teeth" is most likely an experimental artifact". Well, that settles it, right?

Well, no. Marshall, Raff and Raff's paper was published in 1994, and despite what Luskin might think, science has progressed in the two decades since. Perhaps if he had read through more recent literature he would have realized some problems with his argument and with Marshall et al's conclusion.

First, let's tackle the "experimental artifact" claim. When Kollar and Fisher's original paper was published, there was some skepticism about their results. There was controversy over whether or not the mouse mesenchyne they used was contaminated with mouse epithelial tissue. If this was the case, then their results would be invalid: it would be impossible to tell whether or not the formation of dentin was prompted by the chick epithelium or the mouse epithelium. Despite the experiment being repeated by other researchers, the possibility of contamination meant that many people wrote off their result as an "experimental artifact". This debate was put to rest, however, by an paper published by Cai et al in 2009³. In their paper, the team repeated the tissue graft experiment using mesenchyne from transgenic mice expressing the LacZ gene (LacZ is used in molecular biology as a reporter gene, because it produces a dark blue pigment when supplied the proper substrate). Like Kollar and Fisher, Cai et al's results showed the induction of dentin by chick epithelium. To prove that there was no contamination by mouse epithelium, they took cross sections of the graft and stained them. The transgenic mouse tissue, expressing the LacZ gene, stained a dark blue while the chick tissue remained unstained. What they found was that the entire epidermal tissue remained unstained, while only the mesenchyne stained blue, ruling out the possibility of contamination. Kollar and Fisher's original results, then, are still valid.

So if Kollar and Fisher were correct all along, then don't their findings go against Dollo's Law? Shouldn't the genes for tooth production, being free from selective pressures, have accumulated many mutations that would prevent the pathway from functioning at all? The answer, again, is no. Perhaps if Luskin had read the Marshall et al paper more closely (if, indeed, he had read it at all, since he only quotes the abstract) he would have gotten a hint. The authors mention in their discussion that "[r]eversals of long-lost structures do occur but evidently result from the cooption of genes that continue to survive in other roles". In other words, genes involved in traits no longer expressed can avoid the fate of accumulating mutations if they have other roles in development. The genes for tooth production most certainly fit this description. Work by West et al in 1998⁴ found that many of the genes required for odontogenesis are still expressed in the developing chick embryo, indicating that they still play important roles. BMP4, for example, plays important roles in muscle development and bone development as well as in the development of teeth. Members of the  Hedgehog family of proteins are involved in a whole slew of developmental processes, only one of which is odontogenesis. Toyosawa et al⁵, in 1999, looked at one protein in particular, Dentin Matrix Protein 1, or DMP1. Since birds don't produce dentin, what use would they have for such a gene? Toyosawa et al not only found that birds have this gene but found it was being expressed in the jaws of chickens. The case of hen's teeth escapes Dollo's Law because many of them are not silenced, and many of them have other functions in the developing embryo.

If you think about it, this really should come as no surprise. Dollo's Law describes what happens to single genes that control single phenotypes when they become silenced. Dollow's Law makes no claims about what happens to genes involved in complex developmental pathways. In order for Luskin to be correct, then it would require all the genes in a developmental pathway to have become silenced. Given the interconnected nature of developmental pathways, this simply is not a reality. One or two genes in the pathway may be lost, but the rest remain due to their involvement in other roles. If the missing genes are supplied, then the original, ancestral pathway is reconstructed and the ancestral phenotype is "resurrected". This is precisely what is going on in the example of hen's teeth. The tooth development pathway remains largely intact since many of the genes are involved in other roles. The genes in chick mesenchyne that respond to signals from the epithelial tissue have been lost, which is why birds do not develop teeth. But if you supply these genes in the form of mesenchyne from mice, then the lost pathway is reconstructed and teeth develop. This in no way violates Dollo's Law.

As for Dollo's Law itself, there is mounting evidence that would indicate apparent exceptions to Dollo's Law might be the rule. In the last ten years, many examples of exceptions to Dollo's Law have been noted, including the evolution of  wings in stick insects⁶, the larval stage in salamanders⁷, digit loss in some lizards⁸, egg laying in sand boas⁹, teeth in frogs¹⁰ (which, by the way, have been toothless for 200 million years, more than twice as long as birds), shell coiling in limpets¹¹, and even the re-evolution of sexuality in Oribatid mites¹². As noted by Collin and Miglietta¹³:

"with the growing number of phylogenetic studies showing patterns consistent with re-evolution of characters, and genetic data showing that developmental pathways can be maintained for tens of millions of years, is it time to give up Dollo’s Law? Perhaps."

So what remains of Luskin's argument but smouldering rubble? Kollar and Fisher's experimental results were not due to experimental error, their results don't violate Dollo's Law, and Dollo's Law itself is on shaky ground. Gould was perfectly correct in referring to hen's teeth as an atavism hearkening back to a bygone day of toothed birds.

Once again, an argument put forth by the ID crowd has failed. Are they incapable of delivering a good argument? It sure seems hard to find one that is the least bit compelling. You might even say they're as scarce as hen's teeth.

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1. Stephen Jay Gould . Of Hen's Teeth and Horse's Toes. 1980

2  C. Marshall, E. Raff and R. Raff . Dollo's law and the death and resurrection of genes. Proceedings of the National Academy of Sciences. 1994. 91:12283-12287

3. Cai J, Cho S-W, Ishiyama M, Mikami M, Hosoya A, Kozawa Y, Ohshima H, Jung H-S. Chick tooth induction revisited. 2009. J. Exp. Zool. (Mol. Dev.Evol.) 312B:465–472.

4. Philippa Francis-West, Raj Ladher, Amanda Barlow, Ann Graveson, Signalling interactions during facial development. 1998. Mechanisms of Development. 75(1-2):3-28, DOI: 10.1016/S0925-4773(98)00082-3.

5. Satoru Toyosawa, Akie Sato, Colm O'hUigin, Herbert Tichy and Jan Klein. Expression of the Dentin Matrix Protein 1 Gene in Birds. 1999. Journal of Molecular Evolution. 50(1), 31-38, DOI: 10.1007/s002399910004

6. Whiting MF, Bradler S, and Maxwell T. Loss and recovery of wings in stick insects. Nature. 2003 421(6920):264-7.

7. Chippindale PT, Bonett RM, Baldwin AS, and Wiens JJ. Phylogenetic evidence for a major reversal of life-history evolution in plethodontid salamanders. 2004.  Evolution. 58(12):2809-22.

8. Kohlsdorf T, Wagner GP. Evidence for the reversibility of digit loss: a phylogenetic study of limb evolution in Bachia (Gymnophthalmidae: Squamata). 2006. Evolution. 60(9):1896-912

9.V. Lynch and G. Wagner. Did egg-laying boas break Dollo's Law? Phylogenetic evidence for reversal to oviparity in sand boas. 2010. Evolution. 64(1):207-216

10.Wiens JJ. Re-evolution of lost mandibular teeth in frogs after more than 200 million years, and re-evaluating Dollo's law. 2011 . Evolution. 65(5):1283-96.  doi: 10.1111/j.1558-5646.2011.01221.x

11. Collin R, and Cipriani R. Dollo's law and the re-evolution of shell coiling. 2002. Proceedings of the National Academy of Sciences. 270(1533):2551-5

12. Domes K, Norton RA, Maraun M, and Scheu S. Reevolution of sexuality breaks Dollo's law. 2007 . PNAS . 104(17):7139-44

13. Collin R, and Miglietta MP. Reversing opinions on Dollo's Law. 2008. Trends Ecol Evol. 23(11):602-9

$100 worth of science fail

So the Canadian government has decided to give our bills a makeover. Gone are the days of money made out of cotton! A new era of synthetic polymer bank notes has arrived. They look pretty cool, which is good. Supposedly, these notes are harder to counterfeit, which is even better. And the $100 bill is a celebration of science, which is even more awesome. The reverse side of the bill shows a bottle of insulin, a lady working at a microscope, and a strand of DNA, as shown below.

 

 Wait a second. Something looks wrong here. Let's take a closer look...

 That helix is left-handed! DNA is a right-handed helix, not a left-handed helix. I applaud the Government for making the bill science-centric, but really, how hard would it have been to get the art accurate? The left-handed helix mistake is incredibly common, but that's really no excuse.

Wait...what?

Where do I even begin with this one?



You know what? I'm just going to let this picture do the talking.

OM NOM NOM

From the journal Entomological Science (published online May 18th) comes this interesting predator-prey role reversal¹:

 

Yes, that is a giant water beetle eating a turtle.

The photo was snapped by Shin-ya Ohba outside of Hyogo, Japan, while collecting samples for his research. While giant water beetles are known to chow down on smaller insects and small fish, finding one dining on a baby turtle is quite bizarre. Ohba writes:

"The bug inserted his proboscis into the neck of the turtle....Although I could not confirm whether the bug caught the turtle by himself in this observation, the dead body of the turtle was fresh. Probably, he had just captured the turtle. This is a first report of a Lethocerinae eating a turtle."

Pretty neat. And also pretty scary.

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1. S. Ohba . "Field observation of predation on a turtle by a giant water bug" . 2011 . Entomological Science . doi:10.1111/j.1479-8298.2011.00450.x

This Week in Science (June 5, 2011)

Just a pair of papers for this week's TWIS. I haven't had much time to dedicate to reading new research this week, but I'll have more next week!

To start off, I'll point you in the direction of the infamous "arsenic bacteria" paper, which has finally been published in Science after languishing on Science's pre-print server for months. This paper, if you're unfamiliar with it, claimed to show the discovery of a new species of bacteria that utilized arsenic in its DNA rather than phosphorus. It was torn to shreds by critics due to problems in the authors' methodology and doubts about the results - and for good reason. Science's response to all the negative criticism that the paper has recieved has been to publish a list of criticisms (and the authors' responses) along with the paper. Jerry Coyne has a good rundown of the paper's publication on his blog.

Continuing on:

• DNA computing and square roots  - Published this week in Science is a paper by L. Qian and E. Winfree detailing an interesting advancement in DNA computing. The authors were able to design and string together DNA logic gates to create a simple molecular computer able to calculate square roots. Creating a DNA logic gate is actually quite simple, and there are multiple ways it can be done. The authors did it by using a "seesaw gate"design: the gate consists of a short stretch of DNA that can pair with multiple different sequences. One such sequence is added as an "input", which competes and replaces a second sequence that is already bound to the gate. This replaced sequence becomes the "output", which is then free to act as an input for a second gate. Gates can be strung together to create more complicated systems. The authors devised a way to string such gates together to create a system that could calculate square roots. As cool as this is, though, this early biological calculator takes a while to complete calculations - up to eight hours. Nevertheless, this is a big step towards creating bigger and more powerful DNA computers. A more detailed summary of the paper has been posted on Wired, which I highly recommend, as they do a better job explaining logic gates than me!

(L. Qian and E. Winfree . "Scaling up digital circuit computation with DNA strand displacement cascades" . Science . 2011 . 332(6034): 1196-1201, doi: 10.1126/science.1200520)

• Proteins successfully extracted from mammoth bones - A paper published this week¹ in the journal Geochimica et Cosmochimica Acta describes the successful extraction of collagen protein from 600,000 year old mammoth bones. The authors were investigating the idea that peptide mass spec. could be used to identify fossils; that is, if you could determine the sequence of proteins in the fossil, you could compare the sequences to a database of known proteins. Finding proteins which are a close match would narrow down the identity of the fossils in question. To test this idea, the authors used bones from two mammoth fossils and one mastodon fossil. After grinding samples of the fossils into powder, they performed a series of chemical extractions and washes, then prepared them for mass spec. This allowed them to sequence the extracted protein, which was confirmed as collagen. The sequence data was good enough that the collagen - and consequently, the fossils - were correctly identified as being related to elephants. The authors were even able to use this data to distinguish the samples as elephantid (mammoth) or mammutid (mastodon). These findings are important for two reasons: first, they show that proteins can successfully be recovered from fossils as old as 600,000 years; and secondly, they demonstrate that protein mass spec. can be used to correctly identify ancient fossils (or at the very least, identify the closest living relative). Exciting indeed!

(M. Buckley, N. Larkin, and M. Collins . "Mammoth and Mastodon collagen sequences; survival and utility" . Geochemica et Cosmochimica Acta . 2011 . 75(7): 2007-2016

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1. This paper actually wasn't published this week, it was published in April. For whatever reason, it looks like it only made the news now.

Fir-Tex: Bullshit in vest form.

Move over Q-ray, there's a new nonsense product in town.

Everyone is familiar with Q-Ray bracelets. Little more than two ball bearings attached with a piece of steel cable, Q-Ray bracelets are marketed as some sort of mystical device that will revitalize your "bio-energy" (whatever that means). Despite being a known fraud, the Q-Ray bracelet remains popular. In the face of mounting evidence that it's a load of baloney, we've seen the nonsense "bio-energy" fad expand in the last year. Products like the phony Power Balance bracelets have hit the market (and like the Q-Ray before it, have been debunked a thousand times over by the skeptic community). But if you've ever said to yourself "I really wish I had a similar product that covered my entire torso", then you've been out of luck.

Until now.

Introducing "Fir-Tex", the latest in wallet-draining pseudoscientific technology. Fir-Tex claims to be the first "energized textile", that can be fashioned into clothing. Products such as Wear Wings - brought to you by Red Bull - are being marketed as the world's first in "energy fashion". Having a slow day? Feeling drained at work? Don't worry, just send in a big cheque, pull a Fir-Tex vest on over your body and your motor will be revving like a chihuahua on speed.

But how does it work? Or better yet, does it work?

Bold Claims
Before we get into the "science" behind Fir-Tex, let's take a look at just what the product has been claimed to do. A banner at the bottom of the Fir-Tex splash page tells us that Fir-Tex clothing will "improve energy and wellbeing", allow you to "jump 10% higher", "improve performance", "boost power by 10%" and "optimize balance and microcirculation". These claims are pretty generic as far as "energized" products go. You'll see similar claims made for Power Balance and Q-Ray bracelets. Also note that claims like these are incredibly ambiguous. What exactly does "improved wellbeing" mean? How do you "optimize" microcirculation? How do you "boost" power, and for that matter, how do you measure power in order to determine that it's been boosted? These claims are little more than technobabble; they're utterly meaningless strings of sciency-sounding words. Just how ridiculous the Fir-Tex gobbledygook is can easily be illustrated. Of the following three items, one is related to Fir-Tex and the other two are taken from Star Trek. Can you tell which is which?

• decyon field fluctuations
• biogenic energy in the 40 to 50 micron range
• aggregate field of plain polarization

Fir-Tex stops short of making  any sort of medical claims, however. They're probably aware of how Q-Ray's medical claims worked out (very poorly), and to avoid lawsuits, they've added a disclaimer to their site:

"So by entering this website you must first acknowledge that FIR-TEX has not been designed for and does not make any claims whatsoever to be any kind of medical cure. Because it isn’t! The aim of FIR-TEX is to bring technological fabrics on the market and their products will help some subjects to simply perform better or feel better while others won't experience any difference or could even feel discomfort."

So despite their claims of "increased performance", they acknowledge that "others won't experience any difference". How convenient. If Fir-Tex doesn't work for you, it doesn't mean the product is a fraud, it just means that you're different.

But enough about bogus product claims, let's get down to some science.

Im in ur body vibratin ur waterz
So how does Fir-Tex work? Luckily for us, the Fir-Tex site has a science section. According to their site, Fir-Tex works on the principle of Far Infrared Rays. These are energetic rays that lay just outside the visible spectrum. They have wavelengths greater than that of visible light, but shorter than that of microwaves. What do far infrared rays do? Well, I'll let the Fir-Tex folks explain it to you:

"The human body is a reservoir of all kinds of bio-toxins which cannot be expelled immediately and become stored in the body, thereby triggering illness. When toxic gases such as sulfur dioxide and carbon dioxide, or potentially fatal heavy metal toxins such as mercury, lead and chlorine, meet large water molecules, they are encapsulated by clusters of water and trapped in the body. Where these toxins are accumulated, blood circulation is blocked and cellular energy is impaired. However, when a 7 to 11 micron FIR wave is applied to these large water molecules, the water begins to vibrate. This vibration reduces the ion bonds of the atoms which are holding together the molecules of water. As the water evaporates, the encapsulated gas and toxins can be released and this is exactly what FIR technologies can do for the human body."

Wait....what? It took me a few times to read that paragraph to understand what they're trying to say. From what I understand, it's this: "bio-toxins" accumulate in your body by being "encapsulated" by water, and causing illness. FIR "vibrates" the water, "reducing" the ion bonds, causing the water in your body to evaporate and setting the toxins free so they can be expelled from the body.



This paragraph also represents a massive chemistry fail. When toxins enter your body, they do not become "encapsulated" by water molecules. This is another instance of technobabble - water cannot "encapsulate" anything, let alone toxins¹. Perhaps they mean that toxins will bind with water molecules, but this won't happen either. Water molecules won't bind with gasses, nor will water bind with mercury.

The paragraph continues to tell us that applying far infrared rays to water, the molecules will vibrate, and this vibration "reduces the ion bonds". They're correct in stating that applying far infrared rays will cause the water molecules to vibrate; this is because applying ANY kind of energy will make water molecules vibrate. Think back to junior high chemistry class. When you apply energy to molecules, what happens? They speed up, move around faster, begin to vibrate more. If you put a glass of water in the microwave, what will happen? It boils. The microwave radiation increases the energy of the water molecules and they start moving faster and faster. Applying infrared radiation is no different - it increases the energy of the water molecules and they will move around faster. This is the whole idea behind the infrared heat lamps you see keeping the burgers warm at McDonald's. So while infrared rays will make water vibrate, they aren't special in their ability to do this.

Furthermore, even though infrared heat can get water vibrating faster, it won't "reduce the ion bonds of the atoms which are holding together the molecules of water" because water does not contain ionic bonds. All bonds are not the same. Some bonds have more energy than others. Some bonds are made by sharing electrons between atoms, while some are made by the transfer of an electron from one atom to another. The different types of chemical bonds are given different names to distinguish them from one another. Ionic bonds are once such type of bond. They're formed by the electrostatic attraction of two differently charged ions. Sodium ions and chloride ions will forms ionic bonds and produce salt, for example. Water does NOT form ionic bonds. Water forms hydrogen bonds. These are a very different type of bond.

But perhaps that's just semantics. The point Fir-Tex makes is that applying far infrared rays will make the water molecules in your body evapourate, and allow toxins to be removed. Now, if you don't immediately see how that incredibly terrible that is, then read it again. Let's ignore the fact that it would take an extraordinary amount of radiation to make the water molecules evapourate. Considering how important water is for the proper functioning of your body, does evapourating the water from your very tissues sound like a good idea? And, if the water is supposedly "encapsulating" toxins, wouldn't freeing them from the ensnaring water molecules be even worse? Then they would have free reign to move about your body. Take mercury as an example, since it is one of the toxins mentioned by Fir-Tex above. Mercury does its damage by accumulating in neurons and inhibiting the formation of myelin. At high concentrations, it can cause severe impairment of mental faculties and can potentially be fatal. If mercury were "encapsulated" by water molecules in the blood, it would actually prevent mercury from reaching your central nervous system and doing damage. Fir-Tex products would actually be doing more harm than good!

It's also interesting to note that Fir-Tex is not the only "far-infrared technology" on the market. However, Fir-Tex does claim to be "different" from all those other products. How is it different? Well, Fir-Tex doesn't seem to want us to know:

"During our meetings with our customers we explain the difference between our product/technology and any other product using FIR technologies. The technical information is not public for the moment for obvious reasons which one would of course understand."

No, actually, I don't understand.

So the principle by which far-infrared technology is supposed to rely on is nonsensical at best and utterly batshit-stupid at worst. Does Fir-Tex have any evidence that it works?


Science, schmience. We use Chinese medicine!
The Fir-Tex website's science section has a page called "Live Blood Analysis", which they present as scientific evidence that their product works. I'll get to that in the following section. What I want to go through first is this "scientific presentation" that can be found on their website.

The presentation begins with a summary of infrared rays. They claim that infrared rays are "most beneficial" for "the living beings". In what ways they are "most beneficial" is not explained. They do, however, give three properties of infrared rays. First, they "can generate heat by direct irradiation but localized objects can also reflect them". Well, so can microwaves, but sitting next to a microwave tower is a bad idea. In fact, that's WHY it's a bad idea. Secondly, they "deeply penetrate the living tissues". Well, so can X-rays, but again, basking in X-rays isn't a good idea either. And finally - and this one's a doozy - they "activate (water) molecules, increasing overall temperature of the system". Increasing the overall temperature of the system is the same thing as generating heat, mentioned in the first point. But what does it mean to "activate" water molecules. Are all the water molecules in your body normally "inactive"? How do they become "active" in the presence of infrared rays? This is yet more technobabble (sensing a theme here?). These three properties, Fir-Tex says, are why far infrared rays are called “bio-genetic rays” (another made-up word).

Continuing on, Fir-Tex evokes Wein's displacement law as some sort of justification for their claims. Wein's Law takes the form of λ=K/T, where T is temperature in degrees Kelvin, λ is wavelength in nm, and K is a constant, equal to about 2.896x10^-3m.K. Using this equation, Fir-Tex calculated the "frequency of emitted radiation" at 35°C as 9.4μm. If you whip out your calculator, you'll see that their math checks out. Human bodies (at 37°C, not 35°C, but whatever) do emit infrared heat at about 10μm, and this is what IR cameras are calibrated to measure. Armed with this number, what does Fir-Tex do? They conclude that "this explains why human body [sic] easily absorbs far infrared rays between 4 and 16 microns". Holy non sequitor, Batman! Using Wein's law shows what wavelength the human body emits, but says nothing about what wavelength it absorbs. And even if it did show that the body absorbs light of a certain wavelength, how does that show it is the "most easily" absorbed wavelength? What would that even mean?

What does any of this have to do with Fir-Tex's brand of far infrared technology? Well, as the presentation points out, our bodies are composed of large amounts of water. Infrared radiation can make water warmer. Fir-Tex

"works like an active mirror; it captures/receives the thermal radiations from the body heat. Then it reacts and uses these thermal/Far Infrared rays (rays of life) to send energy back into the body with multiple beneficial consequences on cells and tissues."

In other words, Fir-Tex is the world's most expensive blanket! All it purports to do is trap the heat radiated by your body. The same effect can be achieved by a wool blanket. $30 at Bed Bath and Beyond and you can be soaking in all the Rays of Life (whatever that means) that you want.

But Fir-Tex is firm in their belief that infrared radiation is beneficial. Afterall, it's what Qigong masters use!

"Energy medicine is very old, at least as old as the first Qigong masters and other ancient practitioners of healing touch therapies. These healers all had in common the ability to emit energy through their hands, and so do many modern day healers, such as Dolores Krieger, Ph.D., R.N., who began teaching healing touch techniques in the U.S. in the 1970s. Contemporary researchers have now proved that these forms of energy medicine use wavelengths in the infrared range."

Qigong "healers'" hands emit radiation in the infrared range? Does this come as a surprise to them? That's the range at which everyone's body emits radiation. That's what the Wein's equation above showed!

But none of this actually shows that Fir-Tex works at all. Fir-Tex's supposed evidence can be found in the Live Blood Analysis section of their Science page.

Live Blood Analysis, or How Not to do a Scientific Study
Prior to this, I had not heard of Live Blood Analysis (LBA). I'm more than familiar with haemotological techniques, but Life Blood Analysis was not a technique that I knew. A few minutes on Google, and I discovered that LBA is a technique pioneered by a company called Sevenpointfive. Who's Sevenpointfive, you ask? They're a South African naturophathic company that pushes their brand of "natural supplements". They present the same, tired "modern medicine is wrong, buy our products instead" line as every other "natural supplement" company. Their schtick, though, seems to be their LBA technique. What they do is simple. They take a drop of your blood, and look at it under a microscope for "imbalances" and "deficiencies". Whether or not Sevenpointfive's "technicians" can accurately diagnose physiological conditions using LBA is rather dubious to me. As far as I can tell, LBA is not a proper technique used by the medical community.

It's this exact method that Fir-Tex used to test their product. They took blood samples of individuals, then gave them all Fir-Tex vests to wear for 10 minutes, and took another sample. Then they compared the two. That is as detailed as their test gets. There's a huge problem here: they didn't use a  negative control subject (or if they did, they never mentioned it or showed the results)! This is a gigantic experimental flaw, and any results from their tests are worthless because of it. They also make no mention of what kind of activities the subjects were doing when they were using the Fir-Tex vests? Were they sitting still for ten minutes? Were they exercising? Was this controlled at all? Their methodology is severely lacking. Such a study would never get published in any reputable medical journal, which is probably why it exists only on the Fir-Tex website.

What were the results of their "tests"? Well, I haven't the slightest clue. Take a look at what they present as evidence:

 

Maybe it's because I don't have sufficient training to understand what I'm looking at, but I don't see a significant difference in the two treatments. All I see are alot of erythrocytes with some platelets here and there, in both cases. Nevertheless, Fir-Tex claims results like these indicate that their product protects cells from damage from free radicals (free radicals, according to Fir-Tex, are "atoms that are missing ions", a laughably stupid statement), reduces cholesterol, and boosts the immune system. None of their LBA pictures indicate any of these things. It is interesting to note that Fit-Tex admits "that each individual reacts differently to the same treatment, the results are thus never the same", but they don't accept this as evidence that their product does nothing. Quite to the contrary, they take this as evidence that Fir-Tex does work, just in different ways in different people. These tests definitely do not bolster their earlier claims of increasing your jump height by 10%, or "optimizing your balance". And even if they DID show such results, we have no way of knowing any of this is attributable to the Fir-Tex vests because of their shoddy methodology!

No doctor worth their medical degree would produce a medical study as awful as this. So who is responsible? Fir-Tex informs us that the tests were performed by "Dr Annelise Bunce, certified Clinical Metal Toxicologist and certified Dark Field and Multi Phase Microscopy and expert in Live Blood Analysis". Here is her CV. Masters in homeopathy? Oooh. That explains it.

More money than sense
So just what will one of these babies set you back? Between €325 and €475 (that's $453CAN to $663CAN). Hell, while you're at it, €525 will get you a saddle for your horse. This is an unbelievably colossal waste of money. Fir-Tex does nothing more than make you warm. The "science" behind it is complete and utter nonsense, none of their claims can be validated and it simply does not work. Just like the Power Balance and the Q-Ray before it, Fir-Tex is a scam that will have its manufacturers laughing all the way to the bank.


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1. It's been brought to my attention that this point is not entirely correct. Water molecules can, in fact, "encapsulate" some ions. This is called a "solvation shell". It occurs when a positively charged ion is attracted to the electrostatically negative oxygen of water molecules. Water molecules will effectively form a shell around the ion. I have my doubts about whether water can form a solvation shell around toxins, which are rather large compounds. Perhaps a more chemistry-minded reader could clarify this.

Central Dogma not so challenged after all?

In last week's This Week In Science, I summed up a paper published in Scicence by Li et al.¹ which was hyped up as being a challenge to the Central Dogma of molecular biology. The authors compared the RNA sequences from numerous individuals to the original DNA sequences they were derived from. What they found were a multitude of sites where the RNA sequences differed from that which would be expected given the original DNA sequence. Moreover, these variations were shared across individuals, indicating that they were not likely due to random mutation. Furthermore, the team found proteins that matched the varied RNA sequences and not the DNA sequences. These results suggested that there exists some yet unknown editing step during transcription that alters individual nucleotides in the resulting RNA transcripts.

However, declaring the Central Dogma to be toppled may have been a bit premature. According to Lior Pachter at the University of California, Berkley, the variations discovered by the researchers could be artifacts caused by their sequencing equipment. Many of the sites that were found to contain altered nucleotides lie in regions which are known to often cause RNA sequencing errors. In other words, the variations that the team observed, in many cases, might just be sequencing mistakes. 

Further skepticism has been shown by Joe Pickrell at the blog Genomes Unzipped (which I highly suggest reading, as he goes into quite a bit more detail than what I've presented below). He points out that the differences in RNA and DNA that the authors discovered might be false positives created by attributing a particular RNA sequence to the incorrect DNA sequence. For any given DNA sequence, there are bound to be other sequences very similar - even almost identical - to it². If you are given an RNA sequence, then, how do you determine which of the very similar DNA sequences it is derived from? Unless one takes steps to remove the incorrect sequences, it is very likely that you will end up with a false-positive. It would appear that Li et al. did not take such steps.

Pickrell points out another problem with sequencing and mapping through RNA splice sites. Mammalian genes are frequently alternatively spliced, and a cDNA library like the ones Li et al. used will have multiple isoforms of a gene. When mapping such transcripts back to the genome, you have to keep in mind that the genomic sequence will still contain the introns that have been excised in the mature mRNA transcripts. If you compare the shorter, edited mRNA to the longer, unedited DNA, you're likely to find many differences between the two. Mapping a particular sequence read to the wrong isoform will generate false-positives. Pickrell shows that Li et al. did just this on at least one occasion.

So widespread RNA editing in humans might not be a reality. It's possible that it is, but problems with the procedure used by Li et al. raise many doubts. I'm looking forward to reading the follow-up research. Until then, as perhaps Mark Twain would say, reports of the death of the Central Dogma have been exaggerated.

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1. M. Li et al. "Widespread RNA and DNA sequence differences in the human transcriptome". 2011. Science. doi:10.1126/science.1207018

2. It should be obvious that the larger the given sequence, the fewer (near)identical sites there will be.