If you have ever worked in a molecular or medical biology research laboratory, chances are one of the first things you learnt was tissue culture (or the microbiology equivalents). Even if you know nothing about biology, you’ve probably heard mentions of “cell culture” on the news, or at the very least heard about the results of studies in tissue culture. If you hear about “cell lines”, you’ve got culture. If you hear about a “laboratory study” showing that your favourite chemical is carcinogenic, you’ve probably got culture. If you hear about new trials on a miracle cancer cure that has been shown to be effective in “preliminary laboratory tests”, you’ve probably got culture. Everything from zombie epidemics to £10,000 animal-free beef is cell culture. Knowing a little bit about what tissue culture is, and what its uses and limitations are, is therefore important when answering such questions as “is my baby’s bottle poisonous?”, “is stem-cell research ethical?” and “is vitamin C an effective cure for colds/cancer/HIV?”

So. What is tissue culture (TC)? It’s when you take specific cells from a multi-cellular animal and grow them in a dish full of nutrients (a mimic of your blood serum). The point of doing this is to create a system on which to experiment which does not require growing and killing lots of individuals — something that is, for some reason, considered unethical. Especially when it’s humans you propose using. Typically, human or other mammalian tissues are used — especially “model organisms” such as mice. You can use healthy or diseased cell lines from all sorts of different organs. Once you’ve grown up a nice batch of cells in your dish, you can see how they respond to your cancer drug, environmental contaminant, or new junk food ingredient. You can see exactly how the behaviour of your cells changes over the minutes, hours and days of exposure; how they recover after the chemical has been flushed away; how your cancer drug works in dozens of different tumours; how your junk food ingredient works in the old and young, male and female, fit and fat; and how your environmental contaminant interacts with other environmental contaminants. It’s great. If you work hard enough, you can know everything you want to know about your chemical within a week. Wipe out cancer and save the world by next Monday. At least, that’s what the animal-rights movement would have you believe. And the tabloid press fall for it daily.

Trouble is, it’s very easy to get superficially interesting answers using TC. Which makes it very easy to convince a journalist that you have important results, but very difficult to convince a scientist. That’s not to say that TC is not important. But everything that we measure in TC is an estimate of what happens in real life situations. It’s a model that uses surrogate measures from which we can develop hypotheses about what happens in reality. A bad analogy is in order, I think. Suppose you are building a car. You want to protect your future drivers from side-on impacts. Very early on in the design process, you have an engineer conduct strength tests on different materials and designs for doors. From this, you can narrow down the field of designs, and make hypotheses about which designs will perform best on the road. But you can not be sure that the strongest material will provide the best protection against injury and death. You would want play with the crash test dummies, before putting the car on the road. And once the car is on the market, you would analyse incidents. Because when the door is attached to the car and put on the road, a huge number of other variables comes into play. And so it is with, er… what was the topic again? Tissue culture.

Cells did not evolve for growth in a dish. They evolved in the context of cooperation with a vast number of other specialist cells in a body. They are not fine-tuned for survival in the absence of skin, an immune system, a digestive system, liver and kidneys. They are not supposed to live like barnacles on plastic. But if you’ve worked with research quality cell lines, you’ll know that it’s surprisingly easy to make them grow in a dish. Feed them every couple of days, and they’ll happily live for many months. Well go and say that to the post-docs and technicians who made it that way. They were up until midnight processing disgusting lumps of freshly excised tumour. They spent months trying out different combinations of nutrients and fungicides in an attempt to make the cells survive longer than a week. They may be easy to grow now, but don’t think there wasn’t any effort involved. Billions of cells died in the process of making those few grow. Under these circumstances, you can hardly expect the cells not have evolved a little. You are introducing them to a vast number of novel mutagens by taking them away from the protection of skin. And putting anything into a new environment is going to mean new selection pressures. When you finally manage to immortalise your cell line, is it because you’ve perfectly adapted the conditions to the cells, or because the cells have adapted to the conditions?

So. There are all sorts of reasons why TC can not be anything more than an approximation of what is happening in real life. A useful approximation, but unreliable in the absence confirmatory evidence from in vivo and population studies. But these are only the intrinsic limitations of TC. When judging the merits of TC based research, you must also take into the account the fact that TC is easily misused and misrepresented, and that charlatans are doing it all the time. TC is a favourite of cargo-cult healers and nutritionists — those who like to keep up a superficial appearance of having a scientific basis for their quackery. Take, for example, the shamen who pedal vitamin C as an HIV/AIDS drug (Patrick Holford, for example) or as a cancer therapy. They will tell you that in TC, vitamin C has been shown to kill tumour cells, or those cells that are infected with HIV. Therefore, the reasoning goes, we should abandon proven therapies, in favour of taking some vitamin supplements. Trouble is, you can chuck a big lump of any chemical in a dish of cells and the cells will die. I could pour a bag of vitamin C into a dish of healthy cells. They will die. Conclusion: those vitamin supplements are deadly poisonous. Except that your cells will never be exposed to a bag of vitamin C, because you have skin, a digestive system, and kidneys. And because people just don’t go around pouring bags of vitamin C down their throats. I could spit in a dish of cells and tell you that spit is a killer. It’s not.

But it’s not just charlatans that abuse TC. Many legitimate scientists bend the rules a little. They may not even be aware that they are doing it. Take the case of Bisphenol A (BPA), something I did a little work on a couple of years ago. BPA is a component of some plastics, notably bottles. It is known to very slowly leach out of the bottles and into your drink. There is a little bit of evidence (mostly from rats) to show that consuming BPA may be harmful. And there are a lot of TC experiments on the chemical. BPA is a xenoestrogen, meaning that it mimics the activity of estrogens. Estrogen, of course, regulates prolactin release, and cell division (particularly in the breasts). We know that BPA mimics estrogens because when we put some in our dish of tumour cells, we see that within seconds the estrogen receptors have been activated, and all the other effects of estrogen follow. There are loads of results to confirm this because there are a lot of experiments into the effect of estrogen (there’s plenty of money in breast cancer research). If you’re doing the experiment anyway, it’s hardly any more effort to look at BPA. And you can pretend that your research has another potential medical application. Since it’s not the primary aim of your research, the journal’s reviewers won’t notice that you’re using it at a thousand times the concentration that you would find it in the body. So even if enough BPA does leach out of your bottle, and even if BPA does do interesting things in the body, a large proportion of the TC studies will be irrelevant to understanding how it does those things, because they look at inappropriately large concentrations and inappropriately small timescales.

So, next time you are flicking through the health pages of the Daily Mail — which I know all of you like to do — engage healthy skepticism when they update the list of miracle cures and carcinogens. Like statistics, tissue culture is incredibly useful — whether you’re searching for the truth, or a convincing lie.

Here’s an interesting one: Genesis chapter 30. If you think Darwin got inheritance wrong, try the Bible.

30:28 And he [Laban] said, Appoint me thy wages, and I will give it.

30:29 And he said unto him, Thou knowest how I have served thee, and how thy cattle was with me.

30:30 For it was little which thou hadst before I came, and it is now increased unto a multitude; and the LORD hath blessed thee since my coming: and now when shall I provide for mine own house also?

30:31 And he said, What shall I give thee? And Jacob said, Thou shalt not give me any thing: if thou wilt do this thing for me, I will again feed and keep thy flock.

30:32 I will pass through all thy flock to day, removing from thence all the speckled and spotted cattle, and all the brown cattle among the sheep, and the spotted and speckled among the goats: and of such shall be my hire.

30:33 So shall my righteousness answer for me in time to come, when it shall come for my hire before thy face: every one that is not speckled and spotted among the goats, and brown among the sheep, that shall be counted stolen with me.

30:34 And Laban said, Behold, I would it might be according to thy word.

30:35 And he removed that day the he goats that were ringstraked and spotted, and all the she goats that were speckled and spotted, and every one that had some white in it, and all the brown among the sheep, and gave them into the hand of his sons.

30:36 And he set three days’ journey betwixt himself and Jacob: and Jacob fed the rest of Laban’s flocks.

30:37 And Jacob took him rods of green poplar, and of the hazel and chesnut tree; and pilled white strakes in them, and made the white appear which was in the rods.

30:38 And he set the rods which he had pilled before the flocks in the gutters in the watering troughs when the flocks came to drink, that they should conceive when they came to drink.

30:39 And the flocks conceived before the rods, and brought forth cattle ringstraked, speckled, and spotted.

30:40 And Jacob did separate the lambs, and set the faces of the flocks toward the ringstraked, and all the brown in the flock of Laban; and he put his own flocks by themselves, and put them not unto Laban’s cattle.

30:41 And it came to pass, whensoever the stronger cattle did conceive, that Jacob laid the rods before the eyes of the cattle in the gutters, that they might conceive among the rods.

30:42 But when the cattle were feeble, he put them not in: so the feebler were Laban’s, and the stronger Jacob’s.

30:43 And the man increased exceedingly, and had much cattle, and maidservants, and menservants, and camels, and asses.

So, I think the deal here is that Jacob makes some pact where he gets to take all of the stripey, speckled, and spotted cows, sheep, and goats, from this other dude Laban’s stock. He does this, leaving Laban with homogeneous flocks of plain individuals. He then attempts fraud by making Laban’s plain individuals mate while looking at stripey things, so that the offspring will be stripey, and Jacob can claim they are his own. And, hey, guys, it totally worked.

It’s not actually at all surprising that the offspring of two plain individuals turn out stripey, speckled, or spotty. That sort of thing is pretty normal. Traits can skip generations and reappear later for a variety of reasons. It could be that one or the other trait is linked to a dominant/recessive gene system; or that they are influenced by complicated combinations of genes, which are shuffled in each generation; or that they are capable of being thrown either way by developmental switches. Indeed, it’s possible even to speculate on reasons why the “feebleness” of cattle might be linked to the tendency to breed true for more traits.

So, assuming that the story has some basis in reality (lets pretend, anyway), Jacob probably just developed a superstition. A convenient myth to explain a mysterious natural phenomenon, while allowing him to believe that he had some influence over that phenomenon. Perhaps he noticed some stripey pattern in the landscape one day, noticed that there was mating going on in the vicinity of the stripes, and then noticed the stripey lambs being born. A meaningless correlation would then appear, superficially, to be a principle of inheritance. From there, the superstition would develop as the believer started counting hits, forgetting misses, and discovering his “ability” to select the stronger more desirable individuals as parents (or post-hoc reasoning that because it has sired a stripey calf, it must be a strong bull).

Alternatively, of course, Goddidit.

Continue reading at At War With The Motorist…

Sorry, I just can’t get over these extraordinary pages on the BMA’s website. Here’s a very quick run through some of the Bad Science Bingo points that leaped out.

There were the canards, fallacies, and methods of misdirection:

Continue reading at At War With The Motorist…

Continue reading at At War With The Motorist…

The BMJ is the journal of the British Medical Association, the professional association and trade union of British doctors. Part of the BMA’s remit it to lobby the government on issues that its members believe are important, and it has some clout in this area. These policies are decided by a representative democracy — a group of members elected by region and by field. In recent years, this body has decided that it is BMA policy to support legislation that would make helmets compulsory for cyclists.

Continue reading at At War With The Motorist…

We also made careful note of the fact that a reduction in the rate of head injury in the event of a crash is a different finding to a reduction in the rate of injury and death of bicyclists. We briefly began the exploration of what this means by considering the fact that helmets are not much defence against a motor vehicle.

How could a reduction in head injury in cyclists who crash not mean a reduction in injury and death in bicyclists?

Continue reading at At War With The Motorist…

Continue reading at At War With The Motorist…

The central dogma of molecular biology, first described by Francis Crick in 1958, describes the flow of information between DNA, RNA, and proteins.^[1] The central dogma is interesting, but I believe that its use in teaching is somewhat misleading and gives it undue importance. If you’ve come across the central dogma before, it was probably in an undergraduate or perhaps high-school lecture, where it was casually mentioned when explaining that gene expression involves the flow of information from DNA sequence to messenger RNA, and from there to protein sequence and structure. Because we think of gene expression in terms of the information carrying molecule, introductory biology teaches gene expression in those terms: we think of it as a two step process of transcription (DNA to RNA) and translation (RNA to protein).

Gene expression is not a two step process, and of all the steps involved, transcription and translation are not necessarily the most interesting. This week’s Thursday paper is “Pre-mRNA Secondary Structures Influence Exon Recognition”,^[2] by Hiller, Zhang, Backofen, and Stamm, and it looks at a particular aspect of one of the lesser known steps: alternative splicing. The story as told by introductory biology is that DNA is transcribed into messenger RNA (mRNA): a carbon copy of the information in DNA whose sole purpose is to convey the information from the precious DNA archive, which is kept safely in the nucleus, out to the sites where the proteins are produced. In fact, the result of transcription is “pre-mRNA” (or “primary transcript”), which undergoes a series of modifications before it is ready for translation. One such modification is splicing.

When researchers started examining the human genome, they were surprised at how many genes they found — eventually coming down from an estimate of hundreds of thousands, to something around 25,000. But they were sure there were far more proteins than that. The reason proteins outnumber genes is that evolution has stumbled upon an efficient way organising things: make several proteins with a single gene. Thus most genes (in “higher” organisms, anyway) are split into many “exons”, each specifying a different section of the protein sequence, and “introns” (non-coding sequences which contain metadata). Thus the protein coding sequence may be split into sections A, B, and C, and the gene may have, say, three alternative versions of each; the protein can be constructed with A1, B1, and C1, or A1, B2, and C3, or A1 and C1 alone, and so on. Splicing is the process that organises the exons. WhatHiller at al are asking is: how does the gene expression machinery know which exons to pick for the protein desired?

It has already been shown that certain sequences within the pre-mRNA, and even features on the DNA, act as signals for the splicing machinery, by altering how the RNA and splicing machinery interact. At the DNA level, alternative promoter sequences situated at different positions upstream of the gene allow the production of a variety of different primary transcripts. Then there are “enhancer” and “silencer” sites, which occur both in introns and in exons (the four are ESEs, ESSs, ISEs and ISSs), and are collectively known as splicing regulatory motifs.^[3] We tend to talk about the information contained in nucleic acids in terms of nucleotide sequence. However, unlike DNA, which forms the famous double stranded helical structure, RNAs are (usually) single stranded, but can fold into a variety of 3-dimensional structures by forming double stranded sections with distant regions on the RNA strand. Often, this 3-D structure is what determines howRNAs interact with other biological molecules. So, the first question that Hiller et al asked was: what is the relationship between splicing regulatory motifs and 3-D structure?

This question is rather difficult to answer. Very few mRNA structures have been empirically determined, and the methods for determining them remain expensive and time consuming. However, enough is known about how these structures form to allow the creation of computer programs to predict likely 3-D structures. Variables affecting structure include the locations at which protein co-factors bind to the transcript, the length of the double stranded region that is formed by folding, the proximity of the sections which come together to form double stranded regions, and most importantly, energy minimisation. Using this knowledge, each nucleotide in the pre-mRNA is assigned a probability of being unpaired in the folded structure. While structure has to be estimated, the location of the regulatory motifs is on firmer ground: the AEdb database contains gene sequences for which the locations of motifs have previously been determined experimentally. Using the predicted structures of these sequences, it was found that splicing regulatory motifs are far more likely to be single stranded than the average sequence.

Indicative of something interesting, but it’s not very convincing on its own. So Hiller et al set out to show that alternative splicing is affected by the single- versus double-stranded structure of splicing regulatory motifs. They looked at the SXN-minigene, a gene whose splice variants are already well understood, and in which the effect of motif sequences on splicing has already been characterised. They engineered versions of theSXN gene which had either silencers or enhancers (or, as a control, random sequences of equal length) added either within a single stranded section or within a double-stranded section. They predicted that regulatory motifs would be less effective when hidden in double-stranded structures, and this is what they found. Enhancers, whose job is to make sure that the exon is kept, and silencers, whose job is to make sure that the exon is removed, only worked efficiently when located in single-stranded structures.

The conclusion, therefore, is that mRNA structure is part of what they call the “splicing code” (an analogy to the genetic code, which maps nucleotides to amino acids). This conclusion is nothing particularly surprising — it has long been known that many DNA and RNA interacting proteins directly interact with unpaired nucleotides.^[4] But it leads me to make a hypothesis that I’d like to put to the splicing experts — I don’t have enough background in this field, and have not yet had time to read up on whether it’s a plausible hypothesis, or even a novel one. My hypothesis is this: alternative promoters cause a frame-shift of the transcript in terms of the regulatory motifs. Different promoters will therefore be associated with a different set of regulatory motifs because the transcript that they produce has a different 3-D structure. Is such a simple solution possible, and has anybody else considered it?

References

1. Crick, F. (1970). Central Dogma of Molecular Biology. Nature 227, 561-563
2. Hiller, M., Zhang, Z., Backofen, R., Stamm, S. (2007). Pre-mRNA Secondary Structures Influence Exon Recognition. PLoS Genetics, 3(11), e204. DOI: 10.1371/journal.pgen.0030204
3. Blencowe BJ (2000) Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci 25:106-110. (Cited in Hiller et al 2007.)
4. e.g. S.D.Auweter, F.C. Oberstrass & F.H. Allain (2006) Sequence-specific binding of single-stranded RNA: is there a code for recognition? Nucleic Acids Res 34:4943-4959.

I wrote the majority of this post a couple of years ago, when I had the intention to do a regular “creationist claim” feature, but for some reason never got around to polishing and posting it at the time.

When children wish to wind each other up in an argument, they need look no further than the chant “I know you are, you said you are, but what am I?” It’s a phrase to suit all occasions, and it sounds the death knell for the ailing argument. This week’s “creationist claim” is an example of how creationists (and other pseudoscientists) adopt this chant. The following are from WellingtonGrey.net:

Creationists do not follow the scientific method. The Answers in Genesis “Creation Museum” in Kentucky presents the visitor with a pair of paleontologists: a “creationist” and an “evolutionist”, and tell you that they accept the same data, but simply reach different conclusions based upon it. Paul Taylor of Answers in Genesis UK, in his talk at Skeptics in the Pub a few months ago, told us that the difference between creationism and evolutionism is only that the two set out with different but equally valid assumptions — the evolutionist’s is just that the universe is natural and material; the creationist’s is just that the bible is the literal and infallible word of God. Two equally valid assumptions, I’m sure you’ll agree.

But the idea that creationists are looking at the data — any data — is simply not true. Sometimes good data is discarded; sometimes data is invented; sometimes a piece of data only fits their conclusions because other facts are ignored. Most of the time, however, data is simply irrelevant: creationists are ignorant of the vast majority of the relevant science and evidence, and that doesn’t hinder them in their cause in the slightest. Creationism, it an exercise in spin; the science is irrelevant.

The theory of evolution was discovered in the mid-19th century, when Christianity still permeated to every corner of society, politics and science. Though we had begun the task of filling in those gaps in our knowledge in which God resided — with Copernicus, Newton, et al — God was still the unchallenged creator of everything, especially life, and a literal Genesis was common, even among scientists. Indeed, Darwin was middle-aged before the term “science” became widespread; he would have been more familiar with “natural philosophy” and “natural history”: defined then as the study of God’s handiwork. Men that we now call scientists saw themselves as investigating God’s creation, and many thought it was a simple case of filling in the details of a story summarised in Genesis. When scientists in the 19th century were convinced of the old earth and of evolution, it was despite their prejudices, and not because of them. Upon closing the Origin, Huxley kicked himself for not thinking of such an obvious idea himself: the power of and evidence for the theory was great even then, but the intellectual climate had prevented others from discovering it.

When creationists accuse scientists of bending observations to fit their theories, it suggests that they have an incomplete understanding of the scientific method. Creationists take the data produced by scientists (for they have produced none of their own), and see if they can use it to construct an argument that will convince non-scientists that creationism is scientific. They assume, therefore, that scientists are doing something similar: taking the data they find, shoehorning it into their own explanation of the world, and dressing it up to parade for the public and seek their acceptance.

Needless to say, this is not how science works. Observation is an important part of science, but it is not unfocused observation. It is observation guided by questions specifically designed to test the truth of theories. Scientists do not say “lets go observe the world”, but ask “if I observe this system, what would I expect to find, based on my theory?”, and “what kinds of observations would I not expect to find if my theory is true?” Upon seeking those observations, we discover something about the validity of the theory. Note also that these questions are not “what evidence should I gather to prove my theory right?”, but, “what questions should I ask whose answers could potentially prove my theory wrong?”

The creationist community is relatively homogeneous, consisting primarily of the American evangelical Christianity branch, and the Islamic branch (there are others, but they are not part of the same phenomenon). Their preconceptions are the same, based ultimately upon the same story, invented by Middle-Eastern nomadic herdsmen several thousand years ago; and their methods are the same, based upon faith, authority and revelation. The scientific community is not homogeneous: it includes liberals and conservatives, people of all nations, races and cultures, people with non-religious upbringings, and religious upbringings including Christianity and Islam, but also Hinduism, Buddhism, and many more besides; and its methods are based on questioning, skepticism, and competition to make the big new discoveries. Scientists have their prejudices, but it’s hard to believe that in such a large, diverse and inquisitive group, any particular prejudice could have such a pervasive and long-lasting effect.

Creationist argument is sham science, put on as a PR exercise. It is reminiscent of the cargo cult science of alternative medicine. It is supposed to look like science to those who do not look too closely. There are data used and numbers cited, and these put in the context of an explanation of how and why the world works. But when you look closer, you find that the data are carefully chosen, and the numbers a diversionary tactic. And in place of the scientist’s toolkit — empiricism, rationalism, skepticism, and logic — there is a creationist toolkit: faith, revelation, and spin. And they are so lacking in imagination that whenever this is exposed, they can only chant “I know you are. You said you are. But what am I?”