Making Movies in DNA

Last time we reminded ourselves of one of the ways in which cancer is odd but, of course, underpinning not just cancers but all the peculiarities of life is DNA. The enduring wonder is how something so basically simple – just four slightly different chemical groups (OK, they are bases!) – can form the genetic material (the instruction book, if you like) for all life on earth. The answer, as almost everyone knows these days, is that there’s an awful lot of it in every cell – meaning that the four bases (A, C, G & T) have an essentially infinite coding capacity.

That doesn’t make it any the less wonderful but it does carry a huge implication: if something you can squeeze into a single cell can carry limitless information it must be the most powerful of all storage systems.

A picture’s worth a thousand words

We looked at the storage power of DNA a few months ago (in “How Does DNA Do It?”) and noted that its storage density is 1000 times that of flash memories, that it’s fairly easy to scan text and transform the pixels into genetic code and that, as an example, someone has already put Shakespeare’s sonnets into DNA form.

Now Seth Shipman, George Church and colleagues at Harvard have taken the field several steps forward by capturing black and white images and a short movie in DNA. Moreover they’ve managed to get these ‘DNA recordings’ taken up by living cells from which they could subsequently recover the images.

Crumbs! How did they do it?

First they used essentially the text method to encode images of a human hand: assign the four bases (A, C, G & T) to four pixel colours (this gives a grayscale image: colours can be acquired by using groups of bases for each pixel). These DNA sequences were then introduced into bacteria (specifically E. coli) by electroporation (an electrical pulse briefly opens pores in the cell membrane).

The cells treat this foreign DNA as though it was from an invading virus and switch on their CRISPR system (summarized in “Re-writing the Manual of Life”). This takes short pieces of viral DNA and inserts them into the cell’s own genome in the form of ‘spacers’ (the point being that the stored sequences confer ‘adaptive immunity’: the cell has an immunological memory so it is primed to respond effectively if it’s infected again by that viral pathogen).

In this case, however, the cells have been fooled: the ‘spacers’ generated carry encoded pictures, rather than viral signatures.

Because spacers are short it’s obvious that you’ll need lots of them to carry the information in a photo. To keep track when it comes to reassembling the picture, each DNA fragment was tagged with a barcode (and fortunately we explained cellular barcoding in “A Word From The Nerds”).

Once incorporated in the bugs the information was maintained over many bacterial generations (48 in fact) and is recoverable by high-throughput sequencing and reconstruction of the patterns using the barcodes.

And the movie bit?

Simple. In principle they used the same methods to encode sequential frames.

Pictures in DNA.

Top: Using triplets of bases to encode 21 pixel colours. Images of a human hand (top) and a horse (bottom) were captured. For the movie they used freeze frames taken in 1872 by the English photographer Eadweard Muybridge. These showed that, for a fraction of a second, a galloping horse lifts all four hooves off the ground. Seemingly this won a return for the sometime California governor, Leland Stanford (he of university-founding fame) who had put a wager on geegees doing just that. From Shipman et al., 2017. You can see the movie here.

Getting the picture clear

To recap, in case you’re wondering if this is some scientific April Fools’ prank. What Church & Co. did is scan pictures and transform pixel density into the genetic code (i.e. sequences of the four bases A, C, G & T). They then made DNA carrying these sequences, persuaded bacteria to take up the DNA and incorporate it into their own genomes and, after growing many generations of the bugs, extracted their DNA, sequenced it and reconstructed the original images. By scanning sequential frames this can be extended to movies.

It’s not science fiction – but it is pretty amazing. With a droll turn of phrase Seth Shipman said “We want to turn cells into historians” and the work does have significant implications in showing something of the scope of biological memory systems.

Won’t be long before the trendy, instead of birthday presents of electronic family photo albums, are giving small tubes of bugs!

References

Shipman, S.L., Nivala, J., Macklis, J.D. & Church, G.M. (2017). CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349.

The Shocking Effect of Boiled Bugs

There’s never a dull moment in science – well, not many – and at the moment no field is fizzing more than immunotherapy. Just the other day in Outsourcing the Immune Response we talked about the astonishing finding that cells from healthy people could be used to boost the immune response – a variant on the idea of taking from patients cells that attack cancers, growing them in the lab and using genetic engineering to increase potency (generally called adoptive cell therapy).

A general prod

Just when you thought that was as smart as it could get, along comes Angus Dalgleish and chums from various centres in the UK and Spain with yet another way to give the immune system a shock. They used microorganisms (i.e. bugs) as a tweaker. The idea is that bacteria (that have been heat-killed) are injected, they interact with the host’s immune system and, by altering the proteins expressed on immune cells (macrophages, natural killer cells and T cells) can boost the immune response. That in turn can act to kill tumour cells. It’s a general ‘immunomodulatory’ effect. Dalgleish describes it as “rather like depth-charging the immune system which has been sent to sleep”. Well, giving it a prod at least.

bugs-pic

Inactivating bugs (bacteria) and waking up the immune system.

And a promising effect

The Anglo-Spanish effort used IMM-101 (a heat-killed suspension of a bacterium called Mycobacterium obuense) injected under the skin, which has no significant side-effects. The trial was carried out in patients with advanced pancreatic cancer, a disease with dismal prognosis, and IMM-101 immunotherapy was combined with the standard chemotherapy drug (gemcitabine). IMM-101increased survival from a median of 4.4 months to 7 months with some patients living for more than a year and one for nearly three years.

Although the trial numbers are small as yet, this is a very exciting advance because it looks as though immunotherapy may be able to control one of the most serious of cancers in which its incidence nearly matches its mortality.

References

Dalgleish, A. et al. (2016). Randomised, open-label, phase II study of gemcitabine with and without IMM-101 for advanced pancreatic cancer. British Journal of Cancer doi: 10.1038/bjc.2016.271.

 

Fast Food Fix Focuses on Fibre

If you’re like me you’re probably more bored than absorbed by the seemingly continuous stream of ‘studies’ telling us what we should and shouldn’t eat. No one’s going to argue it’s unimportant but gee, I wish they’d make their minds up. Of course the study of diet and its effects is tricky – as we noted in Betrayed by Nature – not least because you generally need enormous numbers of people to tease out significant effects.

Fortunately authoritative sources like The American Heart Association offer generally sane and simple advice: “eat a balanced diet and do enough exercise to match the number of calories you take in.”

A balanced diet includes fibre, sometimes called roughage, the stuff we eat but can’t digest that assists in taking up water and generally keeping our insides working. There’s much evidence that eating plenty of fibre helps to prevent bowel cancer – usually accumulated from vast numbers (e.g., the European Prospective Investigation into Cancer and Nutrition study involved over half a million people from ten European countries). But even for fibre, when you might just be thinking the answer’s clear-cut, there are other studies showing no protective effect.

So hooray for Stephen O’Keefe and friends from the University of Pittsburgh and Imperial College London for coming up with a dead simple experiment – and some pretty astonishing results (though to prevent panic we should reveal at the outset that they confirm that a high fibre diet can substantially reduce the risk of colon cancer).

Doing the obvious

The experiment compared what happened to two groups of 20, one African Americans, the other from rural South Africa, when they swapped diets for two weeks. So, in principle ‘dead simple’ but to describe it thus does a great injustice to the huge amount of effort involved – for a start they had to find two lots of 20 volunteers willing to have a colonoscopy examination before and after the diet swap. The Western diet was, of course, high protein, high fat, low fibre, whereas the typical African diet was high in fibre and low in fat and protein. Just to be clear, the American diet included beef sausage and pancakes for breakfast, burger and chips for lunch, etc. The traditional African diet comprises corn based products, vegetables, fruit and pulses, e.g., corn fritters, spinach and red pepper for breakfast.

B'fast jpegCompare and contrast.

A rural South African diet (corn fritters for breakfast) and the American diet (Getty images)

Shock – and horror

Almost incredibly, within the two weeks of these experiments there were significant, reciprocal changes in both markers for cancer development and in the bug army – the microbiota – inhabiting the digestive tracts of the volunteers. That is, the dreaded colonoscopy revealed polyps (tumour precursors) in nine Americans (that were removed) but none in the Africans. Cells sampled from bowel linings had significantly higher proliferation rates (a biomarker of cancer risk) in African Americans than in Africans. After the diet switch the proliferation rates flipped, decreasing in African Americans whilst the Africans now had rates even higher than in the starting African American group. These changes were paralled by an influx of inflammation-associated cells (lymphocytes and macrophages) in the now high-fat diet Africans whilst these decreased in the Americans on their new, high-fibre diet.

Equally amazing, these reciprocal shifts were also associated with corresponding changes in specific microbes and their metabolites. You may recall meeting our microbiota (in The Best Laid Plans of Mice and Men and It’s a Small World) – the 1000 or so assorted species of bacteria that have made you their home, mostly in your digestive tract, of which there are two major sub-families, Bacteroidetes and Firmicutes (Bs & Fs). We saw that artificial sweeteners in the form of saccharin shifts our bug balance: Fs down, Bs up. Here feeding Americans high-fibre diet was associated with a shift from Bs To Fs. As we noted before, the composition of the bug army is important because of the chemicals (metabolites) they produce – in this case the diet switch resulted in more short chain fatty acids (e.g., butyrate) in the American group and a reciprocal drop therein for the Africans.

The bottom line

It really is quite remarkable that these indicators of cancer risk manifest themselves so rapidly following a change to a typical Western diet. Of course ‘markers’ are one thing, cancer is another. As one of the authors, Jeremy Nicholson of Imperial College London, said: “We can’t definitively tell from these measurements that the change in their diet would have led to more cancer in the African group or less in the American group, but there is good evidence from other studies that the changes we observed are signs of cancer risk.”

Put less scientifically, “a nod’s as good as a wink to a blind horse.”

Reference

O’Keefe, S.J.D. et al. (2015). Fat, fibre and cancer risk in African Americans and rural Africans. Nature Communications 6, Article number: 6342 doi:10.1038/ncomms7342

Our Inner Self

Richard Gettner is the anti-hero of Christopher Fry’s wonderful play The Dark is Light Enough, set in the Austro-Hungarian war of 1848. Viewing himself as a failed author, failed husband and all-round disaster, he’s just absented himself from the Austrian Army on the basis of not being too nifty at soldiering either. Their minions are hot on his heels, intent on meting out the retribution that the military traditionally reserve for deserters, and he’s taken refuge in the family home of his former wife. In a tête á tête with her she rebukes him for his knack of self-destruction and points out that his book was actually quite well received and wasn’t really a failure. All Gettner’s frustration then bursts forth in a tirade of brutal philosphising:

‘Is there another

Word in the language so unnecessary

As ‘fail’ or ‘failure’?

No one has ever failed to fail in the end;

And for the very evident reason

That we’re made in no fit proportion

To the universal occasion; which, as all

Children, poets and myth-makers know,

Was made to be inhabited

By giants, fiends, and angels of such size

The whole volume of human generations

Could be cupped in their hands;

And very ludicrous it is to see us,

With no more than enough spirit to pray with,

If as much, swarming under gigantic

Stars and spaces.’

Fry deserves to be remembered as one of the great poetic wordsmiths of the English language, if only for The Dark is Light Enough but, had he known that nine out of ten cells in our bodies are bugs, he might have added a final blast to his demolition of the human condition:

Our failings should not surprise as we are but a sinister symbiosis,

More bacterial than human,

Helpfully poised such that when our hour is done

The microbial hordes surge forth to reduce us to our component parts.

bacteria and virus cartoon

The range of the hordes

Our rising preoccupation with the bug army (see it’s a small world & The Best Laid Plans In Mice and Men …) has been promoted by several recent studies that have propelled our ‘inner organism’ from the bowels of biology into the limelight. The story is somewhat fragmented but it’s a good time to see if we can make sense of the current threads.

We’ve known for many years that a motley collection of microorganisms are happy residents in most of our nooks and crannies, ranging from tummy buttons and through the skin, to saliva and our guts. They include bacteria and fungi, they’ve become known as the human microbiome (or microbiota), are said to outnumber human cells 10 to 1 and, all-told, can be viewed as a co-evolved ‘super-organism’ that has many benefits, including making our metabolism more efficient and hence improving nutrition. However, as with everything else in biology, this close relationship is a balancing act, the disturbance of which carries risks for disease development.

It’s critical to note that this vast microbial army, toiling away on our behalf in the dungeon of our innards, mostly dwelling in our gut, is a really mixed lot. It’s estimated to include about 700 different species of bacteria, of which perhaps thirty or forty species make up the bulk. It’s a bit like a mini Great Barrier Reef, well known as the world’s largest coral reef system and extraordinary in that, although it’s made up of billions of tiny organisms, the thing can behave in an integrated way, most dramatically illustrated by mass spawning.

Within the gut there are two major sub-families of microorganisms (Bacteroidetes (Bs) and Firmicutes (Fs)). Although more close-knit genetically speaking, each of these still includes many different classes of microbe. So, they’re a bit of a rabble but, by and large, not only are they harmless, they actually play a vital part in keeping us healthy.

Bacterial army manoeuvres

The power of DNA sequencing means that we can now interrogate our inner armies as to their make up under different conditions, because each type of microbe has a distinctive genome. The first thing to emerge is a dramatic shift in the balance between the major sub-families in obese individuals, be they mice or humans. That is, obese animals have about half the number of Bs and double that of Fs, compared to normal. And the link here is that the bug switch alters the pool of genes available, the upshot being increased energy harvest from nutrients consumed. In other words the switch helps animals get fatter.

It’s possible to breed mice that do not have any gut bugs and ask what happens when you transfer a colony from another animal. Bacteria-free mice on receipt of a normal gut army promptly double body fat: microbiota transferred from obese mice makes ’em twice as fat and, remarkably, human gut microbes from someone who’s obese also makes mice obese, if fed a high-fat rather than a normal diet.

Chemical warfare

Because we use antibiotics on a massive scale to control infections, we might ask whether they cause the good guys to suffer what the military call collateral damage – the point being that antibiotics don’t target bacteria on the basis of whether they’re good for us or potentially fatal. Inevitably, it turns out that ‘good guys’ do get hit by some antibiotics, and when this happens mice gain weight and build up fat. Unsurprisingly, a high-fat diet makes things worse. The sequence is that the drug changes the balance in microbiota before mice become obese and – a real shock – one course of antibiotic treatment imprints these effects on the animal permanently: it acts for life.

To clever for our own good

In our panic to avoid obesity and still pander to our sweet tooth, mankind has taken to using artificial sweeteners on a massive scale in the mistaken belief that these low-calorie agents do no harm. Only recently has this come to light as yet another example of the old adage about there being no such thing as a free lunch. It’s remarkable: saccharin, the most commonly used artificial sweetener, causes big shifts in the proportions of different types of gut bacteria – some increasing whilst others go down – the overall effect again being much more efficient energy harvesting from food. This is a direct effect of saccharin on the bugs, blocked by commonly used antibiotics.

The story so far

The regiments from which our foot soldiers are drawn (i.e. the species that form the microbiota) affect our metabolism and in particular can influence obesity – and that’s inextricably linked with type 2 diabetes and heart disease. With that in mind, it seems obvious that upsetting them with drugs is a risky business. What’s more, seemingly harmless food supplements can also be fraught with danger.

Marching to a beat

Yet another amazing feature of our inner army is that it keeps time. That is, the abundance of different sub-types fluctuates in synchrony with the day/night cycle. Put another way, it marches to a circadian rhythm along with many other physical, mental and behavioral changes that respond mainly to light – and hence roughly follow a 24-hour cycle. These can be big changes in composition: a particular type of bug can double in amount in 6 hours and return to its initial level by 6 hours later. One of the most familiar examples of the importance of biological rhythms comes from upsetting them by flying long distances on an east–west axis. Sure enough, mice have the same problem and, just like us, their clock is disturbed by jet lag (rather than shuttling them business class across the Atlantic you can simulate the effect simply by shifting the light-dark cycle under which they live forwards or backwards by 8 hours every three days). This largely blocks microbiota rhythmicity, the overall effect being to reduce the total number of bacteria. This in turn raises blood sugar level and the mice become obese. These events are absolutely dependent on what has happened to the microbiota because they are replicated in germ-free mice after transfer of jet-lagged faeces.

That’s more astonishing than might appear at first glance because it places the daily variation in gut bug populations alongside the basic circadian rhythms of the sleep-wake cycle, body temperature and other important functions. Circadian rhythms are driven by a ‘master clock’ in the brain that coordinates all the body clocks so that they are in synch. Four proteins are at the heart of the clock (CLOCK and BMAL1, highly expressed during the light phase, and cryptochromes (CRYs) and period proteins (PERs) expressed in the dark phase). These regulate the expression of many genes, thereby controlling the overall response (see Twenty More Winks). The implication is, therefore, that far from being a kind of add-on that occasionally gets upset, our microbiota play central role in a healthy body.

A recent example of it doing just that comes from another mouse model showing our ‘inner organism’ acting to protect against bacteria from the outside world. In response to infection, cells that line the small intestine switch on the production of a particular sugar (fucose): that is then released from the cells and consumed by members of the microbiota – this novel energy source seemingly helping the host to survive the onslaught of infectious microorganisms.

And finally …

All this stuff about germs being our best friends is riveting but what about the important question? Well, there appears to be a complex interaction between diet, microbial metabolism and colorectal cancer, with bacteria able to make some agents that protect against cancer and some others that drive carcinogenesis. There’s evidence that a wide range of tumours can be promoted by transferring microbiota to germ-free mice and, on the other hand, that depleting intestinal bacteria reduces the development of liver and colon cancers.

Space invaders

Personal space is, apparently, a big thing for many of us these days. So big that ‘scientists’ have had a go at measuring it – they never miss an opportunity do they? Actually, boffins being boffins, they measured something called the defensive peripersonal space (DPPS) – a ‘vital safety margin surrounding the body’ – by sticking a pair of electrodes to the wrists of volunteers who held their hands different distances from their faces whilst receiving bursts of current through the electrodes. That made them blink (!) and the nearer the hand to the face the more they blinked, as the shock was perceived to be a greater threat to their face. There is, seemingly, a sharp boundary: up to somewhere between 20 cm and 40 cm is a high-risk area where we get very aerated: beyond that we don’t much care – with large personal variations depending on how twitchy you are. Debrett’s, which styles itself as the arbiter of society etiquette, has a simpler test, its distilled wisdom revealing that if you can feel the warmth of someone’s anxious breath upon your face, then you’re standing too close.

With all this neurosis it’s probably a good job no one mentioned our inner army: a ten-to-one cellular takeover (albeit that bugs are much smaller) is not so much a bit of heavy breathing as a blitzkrieg. Even so, it’s a delicately poised occupation upon which we depend for survival – and it’s one that we disturb at our peril.

References

Sambo, C.F. and Iannetti, G.D. (2013). Better Safe Than Sorry? The Safety Margin Surrounding the Body Is Increased by Anxiety. The Journal of Neuroscience 33, 14225-14230; doi: 10.1523/JNEUROSCI.0706-13.2013.

The Best Laid Plans In Mice and Men …

I never thought I’d find myself indebted to one R. Burns, said to be Scotland’s national poet, but as a title for today’s piece it’s hard to avoid a mild bit of adaptive plagiarism. And after all, if John Steinbeck could do it …

Artificial sweeteners are wonderful things …

The next thing to do is to pass up all pretence at suspense and give the upshot of a remarkable new bit of work first. The story is of artificial sweeteners (non-caloric artificial sweeteners: NAS for short – most commonly saccharin) – among the most widely used food additives worldwide. Introduced over a century ago, they’ve long been considered great as they pander to our sweet teeth yet are low on calories – what can possibly go wrong?

Saccharin  StructureSweet'N Low

Well, according to Jotham Suez and his pals in The Weizmann Institute, Israel, quite a lot, once you get round to looking in the right places. They found that artificial sweeteners, particularly saccharin, make normal folk glucose intolerant (i.e. cause metabolic conditions – including diabetes – in which blood glucose levels are raised, aka hyperglycemia). Moreover, they do so by changing the make up of the bacteria in our gut (our intestinal microbiota – we’ve already met these guys in it’s a small world). The effects of NAS are reversed by antibiotics which, as we described in it’s a small world, can have drastic, permanent effects on our insides.

It’s a real shocker because, put another way, it shows NAS can dirDiet Coke etcectly drive the very outcomes we’re trying to avoid – diabetes and obesity.

How do they do it?

Suez & Co first showed that saccharin increases blood glucose in mice (glucose intolerance). Treatment with commonly used antibiotics (e.g., ciprofloxacin) blocks this effect. Sequencing DNA extracted from faeces revealed big shifts in the proportions of different types bacteria (taxa) – with some increasing whilst others went down. The overall effect is that the intestinal bugs (microbiota) as a whole became much more efficient at energy harvesting from food (e.g., producing more short-chain fatty acids) – an effect known to be associated with obesity in both mice and humans.

Obese miceDirect or indirect?

To show whether saccharin does this by directly acting on gut bugs they grew samples of faeces in the lab with and without added saccharin and – you’ve guessed it – the bug balance changed: Firmicutes down, Bacteroidetes up (from 89 to 79% and 6 to 22%, respectively). Transferring the saccharin-treated microbiota to germ-free (normal) mice made them glucose intolerant.

Lolli the Saccharin by Trinity FateRe-think required

The upshot of all this is that NAS may be doing the very thing we’re trying to avoid. Suez et al. note that the cult of NAS use has coincided with the epidemics of diabetes and obesity – but their results suggest very strongly that, far from being coincidence, it is yet another example of optimism and our hunger for easy solutions diverting our attention from our ignorance of the underlying science.

Grim reaperSo the message is there isn’t a short-cut to dealing with our sugar craving – if we aren’t to go on making ourselves very ill on a big scale we just have to show more self-discipline.

Reference

Suez , J. et al. (2014). Artificial sweeteners induce glucose intolerance by altering the gut microbiota. Nature 514, 181-186.

Heir of the Dog

I’ve probably in the past owned up to causing generations of students to do that raised eyebrow thing, familiar to all parents of teenagers, that, far more pointedly than words, says ‘The old boy’s finally lost it.’ Indeed I may well have a bit of a causative repertoire but one that unfailingly works is revealing that, even after a life in science, I still get ‘Wow’ moments every couple of months or so when I read or hear of some new discovery, method or insight that brings home yet again the wonder of Nature – or has you asking ‘Why didn’t I think of that?’ (The response to that one’s easy, by the way, so please don’t write in).

A common question

The most recent of these jaw-dropping events relates to a question often asked about cancer: ‘Can you catch it from someone else?’ In other words, can cancers be passed from one person to another by infection, much as happens with ’flu? The answer’s ‘No’ but, as usual in this field, even the firmest statement can do with a little explanation. The first point is that the ‘No’ is true even for 20% or so of cancers that are actually started by microbial infection – what you might call ‘bugs’ – bacteria, fungi, and viruses. One such, the bacterium Helicobacter pylori, can cause stomach ulcers that may lead to cancer. Those even smaller bugbears, viruses (typically one one-hundredth the size of a bacterium), are responsible for much of the cervical and liver cancer burden world-wide. Oh, and there’s a little, single-cell parasite (Trichomonas vaginalis), the most common non-viral, sexually transmitted infection in the world that, in men, can cause prostate cancer. But these infections are not cancers even though they may be an underlying cause – bacteria through prolonged inflammation and effects on the immune system and viruses by making proteins that affect how cells behave. Only when these perturbations cause genetic damage – i.e. DNA mutations – do you have a cancer. Which is why the answer to the original question is ‘No.’

There’s always one

Well, two in this case – and, given that we’re talking about cancer, you won’t be surprised that there are some oddities. They’re not exceptions to the ‘No’ answer because they occur in other animals – not in humans – but, in each, tumour cells are directly transferred from one creature to another – so it is cancer by infection. One such contagious tumour occurs in the Tasmanian devil. It’s transmitted by biting, an activity popular with these little chaps, and it gives rise to a particularly virulent facial tumor, eventually fatal because it prevents eating. To counter the probability that Tasmanian devils will become extinct in their native habitat, a number of Australian sanctuaries have breeding programmes aimed at setting up a disease-free colony on Kangaroo Island, South Australia.

TDs

Tas D

 

 

 

 

 

Tasmanian devils – cancer-free – Lone Pine Koala Sanctuary, Brisbane

A very similar condition in dogs known as canine transmissible venereal tumour (CTVT: also called Sticker’s sarcoma), mainly affects the external genitalia. First spotted in the nineteenth century by a Russian vet, it too is spread either by licking or biting and also through coitus. Dogs with CTVT can now be found on five continents and, from DNA analysis, we’ve known for some time that – remarkably – all their cancers are descended from a single, original tumour cell that appeared many years ago. They’re like one of those cell lines grown in labs all over the world, except they’ve been going far longer than any lab – with man’s best friend doing the cultivating.

So what is new?

Elizabeth Murchison and colleagues at The Wellcome Trust Sanger Institute, Cambridge have just produced the first whole-genome sequences of two of these tumours – from Australia and Brazil (an Aboriginal camp dog and a purebred American cocker spaniel). These confirmed that all CTVTs descend from a single ancestor who, they estimated, was trotting around about 11,000 years ago. The last common relative of the two dogs whose tumours were sequenced lived about 500 years ago, before his descendants went walkies to different continents.

And the ‘Wow’?

We already had a pretty good idea of how CTVTs have been handed down. In this paper the really amazing bit came in the detail. The authors estimated roughly how many mutations were present in each tumour. Answer: a staggering 1.9 million. And it’s staggering partly because it’s only slightly less than a change every 1,000 units (bases) in dog DNA but it’s truly awesome when you note that it’s several hundred times more than you find in most human cancers. We’re getting used to the idea of thousands or tens of thousands of mutations turning up in human cancer cells with associated gross disruptions of individual chromosomes. But these canine cancers display genetic mayhem on a massive scale – perhaps best visualized by comparing their chromosomes with those of a normal dog using a method that labels each with a different colour. A glance at the two pictures tells the story: all the cancer chromosomes from one of the tumour-bearing dogs (on the right) have been shuffled as if in some molecular card game. The full range of colours can still be seen, but of the normal pattern of 39 pairs of identical segments of DNA (left) there is no sign.

Two dogs chromos

Dog chromosomes. Left: normal; right: CTVT

(from Murchison, E.P. et al. (2014) Science 343, 437-440)

It seems incredible that cells can survive such a shattering of their genetic material – a state called ‘genetic instability’ because, once DNA damage sets in, mutations usually continue to accumulate. These cancers are uniquely bizarre, however, because although their genomes have been blown to smithereens, not only do the cells survive but they’ve continued suspended in this surreal state for centuries. They’re genetically stable – it really is the cellular equivalent of balancing an elephant on a pin.

‘Wow’ Indeed – but so what?

So like me you’ve been blown away by these discoveries but you may be asking, apart from the excitement, what’s in it for us humans? Well, there’s one other very strange thing about these dog cancers. Infected animals do indeed develop the most unpleasant, large tumours – but most of them are eventually rejected by the host dog. That is, its immune system gets to work to eliminate them – and after that the dog is immune to further infection. We are only just beginning to find ways of boosting the human immune system so that it can attack cancers and maybe, just maybe, we can extract from the stable chaos of the CTVT genome the secret of how they provoke rejection – and maybe that will guide human treatments.

Reference

Murchison, E.P. et al. (2014). Transmissible Dog Cancer Genome Reveals the Origin and History of an Ancient Cell Lineage. Science 343, 437-440.

it’s a small world

Once upon a time I went to Disneyland. My excuse is that it was a long time ago. So long, in fact, that I don’t need to specify where — it was before theme park cloning got going. Goodness knows why I went — given that if I was inclined to sticking pins in things, Mickey Mouse would be a prime target — though, logically, a model of Walt would come first. But one memory of that visit recurs unbidden to this day: the song ‘it’s a small world (after all).’ I know. I shouldn’t blame Disney as it was the Sherman Brothers greatest hit — and what with also writing the scores for Mary Poppins and Chitty Chitty Bang Bang, they’ve got a lot to answer for. Nevertheless and irritating though the jingle may be, it contains a rather profound line:

There’s so much that we share that it’s time we’re aware, It’s a small world after all’.

And that will do very well as our theme for the day.

SmallWorldFront83_wbYour inner self

A sobering thought about being human is that we’re mostly bugs – that’s to say on a cell to cell basis the microbes in our bodies outnumber us by ten to one. Ten to one: time for lunch, to recycle the old Goon Show gag, but first perhaps you should survey your microbiota – the 1000 or so assorted species of bacteria that have made you their home. Most of them (99%) reside in your digestive tract and we don’t notice them, of course, because they’re so much smaller than the cells of our body (they make up less than 3% of our mass). Sometimes called gut flora, they’re important in squeezing the last ounce of energy from what we eat by helping to digest sugars and they also make some vitamins that we need. You could, then, think of this unseen army of tiny cells as an organ in their own right. Unnoticed they may be but you upset them at your peril, as everyone knows who’s taken a course of antibiotics (e.g., penicillin) to get rid of unwanted bugs.

Bugs tummy

This vast force of bacteria, toiling away on our behalf in the dungeon of our innards, includes two major sub-families, Bacteroidetes and Firmicutes. Don’t worry about pronunciation: think of them as B & F. What’s important is that obese animals (including humans) have about half the number of Bs and double that of Fs, compared to normal. That’s a startling shift – the sort of result that gets scientists thinking: something fishy going on here. But what really gets their antennae twitching is the follow-up result. Each bug has its own genetic material (DNA) carrying a set of genes — different for each species. From faecal samples (i.e. stools) the total number of microbial genes can be estimated and — astonishingly — it turns out that there are several hundred times the number of our own genes. We have about 20,000, the bugs muster several million. But the really provocative result is that this total of microbial genes in our gut drops if we become obese:

Fewer genes = more body fat

More genes (a more diverse microbiome) = healthy status.

Cause or effect?

A good question — that can be answered by man’s best friend. Yes, I’m afraid it’s Mickey again. Mice born under aseptic conditions by Caesarean section don’t have any gut microbes — they’re ‘germ-free’ mice — and they grow up with less body fat than normal mice. However, give them the gut army from a normal mouse and they more than double their body fat in a couple of weeks. The microbiota from an obese mouse makes them gain twice as much fat. What happens if you colonise germ-free mice with human gut microbes? If they’re from someone who’s obese the mice also become obese, if fed a high-fat rather than a normal diet.

Because obesity is all about the balance between energy extracted from food and that expended, all this suggests that obesity-associated microbiomes increase the efficiency of extraction.

But if that’s the case maybe there are some slackers in the bug world – types that are pretty hopeless at food processing. Might they offset obesity? Well, at least one (by the name of Akkermansia muciniphila) does just that — again in mice — and its numbers are much reduced in obese people but go up after gastric bypass surgery that reduces the absorption of nutrients from food. This offers the seductive notion that some types of bug might help to reduce obesity.

Debugging

You may have spotted a bit of a cause for concern: if the make up of our gut bugs can affect how our bodies work — and especially whether we put on weight — what happens when we zap ourselves with antibiotics? The problem is, of course, that these drugs target a range of bacteria — they’re not particularly choosy — which is why you get diarrhœa when you take penicillin for a throat infection. And it’s not just you. In the UK we consume 30 million antibiotic prescriptions a year: Americans get through over 250 million and their children get an average of 15 courses of antibiotics in their early years.

The problem here is not about antibiotics being wonderful and saving millions of lives but the possibility that they might have long-term effects. Evidence for this has come from Martin Blaser’s group at New York University who showed that some antibiotics make mice put on weight and build up fat. What’s more, a high-fat diet adds to this effect. Remarkably, changes in the mice microbiota occur before they become obese — and the effects are for life. It seems extraordinary that a short drug pulse, such as we might give a child to cure an ear infection, can have permanent effects. The explanation may be that some gut bacteria are better at surviving the drug treatment resulting in a shift of microbiota balance to give more efficient digestion — i.e. greater energy provision.

It may not be coincidence that the escalation in antibiotic use since the 1940s has paralleled the obesity explosion. In 1989 no USA state had an obesity level above 14%; by 2010 none was below 20% — and the national average is now 30%.

Bugs and cancer: drivers or mirrors?

Those who follow this blog will know that where obesity lurks cancer looms. Indeed transferring microbiota to germ-free mice has been shown to promote a wide range of tumours and, conversely, depleting intestinal bacteria reduces the development of liver and colon cancers. It’s also worth noting that bowel cancer occurs more frequently in the large intestine than in the small — which may reflect the much higher microbial density.

Is it a small world after all?

All these findings suggest that our bug contingent can influence the onset of obesity and various cancers and that even brief drug treatments can have permanent effects on its make up. We have only the vaguest idea how this happens and most of the evidence so far comes from Mickey’s rellos. Even so, maybe in time we will be able to manipulate our personal gut micro-worlds to augment our defences against these potent foes.

Reference

Martin J. Blaser: Missing microbes, Henry Holt & Company 2014

http://www.amazon.com/Missing-Microbes-Overuse-Antibiotics-Fueling/dp/0805098100/ref=sr_1_1_ha?s=books&ie=UTF8&qid=1400478180&sr=1-1&keywords=missing+microbes

Dyslexic DNA

Writing in code

Did you notice a few months back that some boffins had written a book in DNA? No, that’s not a typo: what they did was to transcribe a 53,000 word book – plus pictures – into a synthetic DNA sequence. In essence, they re-wrote the book in binary by taking the four bases that make the genetic code of life and setting A and C to equal zero whilst G or T represented one. The result wasn’t without its typos: in the just over five million bits needed there were ten mistakes. So rather better than my touch-typing then. But there was a real commercial point behind this exercise, aside from showing, yet again, the astonishing coding capacity of our genetic material. One gram of DNA (you’ve got 500 grams) can store more than 100 billion DVDs, so not merely is it the ultimate in compacted data but it’s amazingly tough stuff – think of sequencing the woolly mammoth, in the freezer for thousands of years – by comparison with the latest software updates for my computer which usually mean I can’t read files 10 years old. And if I dig out my 20 year old 35 mm slides from the attic, chances are they’ll adorned by fungal growths.

Genetic switches

So DNA’s great for long-term information storage but this was by no means the first attempt to use biological molecules in ways we normally associate with electronic devices. When the code of DNA is ‘read’ to make an intermediate (RNA) from which, in turn, proteins can be made it’s acting as a biological transistor: a switch and amplifier that responds to an input signal. The DNA code ‘reader’ is a molecular machine called RNA polymerase (RNA pol) that moves step-wise along a strand of DNA, adding units one at a time to a growing molecule of RNA, complementary in sequence to the DNA template. This process is called ‘transcription’. In its wake another molecular machine can ‘translate’ the RNA codes into protein. RNA pol therefore ‘flows’ along a strand of DNA rather like a current of electrons through a transistor and, because RNA can makes lots of copies of a protein, the system has built-in amplification. Input control is via proteins that stick to segments of DNA called promoters and ‘switch on’ RNA pol (i.e., an analog input). After that the sequence of DNA itself can, in effect, say either ‘go’ or ‘stop’: short sequence motifs can wave RNA pol through or make it stall. The output signal is the protein made – and if you make green fluorescent protein (GFP) you can shine light on it and measure how much you’ve got from the fluorescence emitted.

Over the last few years a number of such gadgets have been made and inserted into bacterial cells to work as simple digital logic gates. In electronic-speak these have included DNA AND gates (giving a high output only if two inputs are high) and OR gates (a high output if one or both the inputs to the gate are high). They’re genetic transistors, processing signals like the logic gates built from transistors that, in combinations of billions, are the basis of computer memory and microprocessors.

Throwing a DNA switch

Throwing a DNA switch

So what’s new?

For biological gates the problem has been that each needs its own construct (a DNA plasmid) and to make more complicated bits {e.g., EXCLUSIVE OR (XOR) gates (high output only if the inputs are different) or EXCLUSIVE NOR (XNOR) gates (output high only if inputs equal)} lots of constructs are required, each having to be persuaded to enter bacteria and to work in a stable fashion.

Step forward Drew Endy and colleagues from Stanford who, by dint of some very clever molecular biology, have combined multiple logic elements into a single construct – which they call a ‘transcriptor’. The switching capacity of their devices comes from integrases – enzymes made by viruses that infect bacteria – that can invert (flip) short stretches of DNA. These can be designed as switchable ‘go’ or ‘stop’ signals for RNA pol. Back in the 1940s Barbara McClintock, working on maize, discovered that stretches of DNA can be shifted around within the genome – they’re called ‘transposons’ – and integrases do the same thing as the enzymes that switch transposons around. McClintock remains, incidentally, the only lady to win a Nobel Prize for Medicine on her own. The great thing about integrases is that they can be turned on simply by adding the appropriate activator to the medium surrounding the cells.

This remarkable advance means that essentially any kind of gate can be built into a single, synthetically made genetic transistor, regulated by a range of integrases. The potential is somewhat mind-boggling but includes being able to monitor in real time the effects of drugs on the behavior of individual cells.

When John Bardeen, Walter Brattain and William Shockley (a Brit by origin but really another Stanford man) invented the transistor (they got the 1956 Nobel Prize in Physics) they can have had little idea of the impact it would have on mankind. But they really would have been staggered to know that, 60 years on, their successors would be shaping our genetic material to act as semiconductors in living cells.

Anything else?

So, as far as I can see, Drew Endy and his chums have done pretty well everything except build an EOR gate that responds to any input with “Don’t blame me”. But they’re such smart guys I bet they’ve got one of those in the fridge too – it was just that the journal editor lacked a sense of humour and wouldn’t publish it. Science editors have form in this department – recall the tale of Albert Szent-Gyorgyi who, whilst a member of my department back in the 1920s, isolated ascorbic acid (the vitamin that stops you getting scurvy) and, convinced it was a sugar (so it should have the suffix -ose – it’s actually made from glucose by oxidation) but not knowing the exact structure, sent his results to the Biochemical Journal calling it ‘ignose’. When the editor said ignose was silly Albert suggested ‘godnose’, getting a predictable response!

References

http://www.guardian.co.uk/science/2012/aug/16/book-written-dna-codehttp://www.huffingtonpost.com/2013/03/29/biological-computer_n_2981753.html

Bonnet, J., Yin, P., Ortiz, M.E., Subsoontorn, P. and Endy, D. (2013). Amplifying Genetic Logic Gates. Science 28 March 2013 / Page 1/ 10.1126/science.1232758http://www.sciencemag.org/content/early/recent