John Sulston: Biologist, Geneticist and Guardian of our Heritage

 

Sir John Sulston died on 6 March 2018, an event reported world-wide by the press, radio and television. Having studied in Cambridge and then worked at the Salk Institute in La Jolla, California, he joined the Laboratory of Molecular Biology in Cambridge to investigate how genes control development and behaviour, using as a ‘model organism’ the roundworm Caenorhabditis elegans. This tiny creature, 1 mm long, was appealing because it is transparent and most adult worms are made up of precisely 959 cells. Simple it may be but this worm has all the bits required for to live, feed and reproduce (i.e. a gut, a nervous system, gonads, intestine, etc.). For his incredibly painstaking efforts in mapping from fertilized egg to mature animal how one cell becomes two, two becomes four and so on to complete the first ‘cell-lineage tree’ of a multicellular organism, Sulston shared the 2002 Nobel Prize in Physiology or Medicine with Bob Horvitz and Sydney Brenner.

Sir John Sulston

It became clear to Sulston that the picture of how genes control development could not be complete without the corresponding sequence of DNA, the genetic material. The worm genome is made up of 100 million base-pairs and in 1983 Sulston set out to sequence the whole thing, in collaboration with Robert Waterston, then at the University of Washington in St. Louis. This was a huge task with the technology available but their success indicated that the much greater prize of sequencing of the human genome — ten times as much DNA as in the worm — might be attainable.

In 1992 Sulston became head of a new sequencing facility, the Sanger Centre (now the Sanger Institute), in Hinxton, Cambridgeshire that was the British component of the Human Genome Project, one of the largest international scientific operations ever undertaken. Astonishingly, the complete human genome sequence, finished to a standard of 99.99% accuracy, was published in Nature in October 2004.

As the Human Genome Project gained momentum it found itself in competition with a private venture aimed at securing the sequence of human DNA for commercial profit — i.e., the research community would be charged for access to the data. Sulston was adamant that our genome belonged to us all and with Francis Collins — then head of the US National Human Genome Research Institute — he played a key role in establishing the principle of open access to such data, preventing the patenting of genes and ensuring that the human genome was placed in the public domain.

One clear statement of this intent was that, on entering the Sanger Centre, you were met by a continuously scrolling read-out of human DNA sequence as it emerged from the sequencers.

In collaboration with Georgina Ferry, Sulston wrote The Common Thread, a compelling account of an extraordinary project that has, arguably, had a greater impact than any other scientific endeavour.

For me and my family John’s death was a heavy blow. My wife, Jane, had worked closely with him since inception of the Sanger Centre and not only had his scientific influence been immense but he had also become a staunch friend and source of wisdom. At the invitation of John’s wife Daphne, a group of friends and relatives gathered at their house after the funeral. As darkness fell we went into the garden and once again it rang to the sound of chatter and laughter from young and old as we enjoyed one of John’s favourite party pastimes — making hot-air lanterns and launching them to drift, flickering to oblivion, across the Cambridgeshire countryside. John would have loved it and it was a perfect way to remember him.

Then …

When John Sulston set out to ‘map the worm’ the tools he used could not have been more basic: a microscope — with pencil and paper to sketch what he saw as the animal developed. His hundreds of drawings tracked the choreography of the worm to its final 959 cells and showed that, along the way, 131 cells die in a precisely orchestrated programme of cell death. The photomontage and sketch below are from his 1977 paper with Bob Horvitz and give some idea of the effort involved.

Photomontage of a microscope image (top) and (lower) sketch of the worm Caenorhabditis elegans showing cell nuclei. From Sulston and Horvitz, 1977.

 … and forty years on

It so happened that within a few days of John’s death Achim Trubiroha and colleagues at the Université Libre de Bruxelles published a remarkable piece of work that is really a descendant of his pioneering studies. They mapped the development of cells from egg fertilization to maturity in a much bigger animal than John’s worms — the zebrafish. They focused on one group of cells in the early embryo (the endoderm) that develop into various organs including the thyroid. Specificially they tracked the formation of the thyroid gland that sits at the front of the neck wrapped around part of the larynx and the windpipe (trachea). The thyroid can be affected by several diseases, e.g., hyperthyroidism, and in about 5% of people the thyroid enlarges to form a goitre — usually caused by iodine deficiency. It’s essential to determine the genes and signalling pathways that control thyroid development if we are to control these conditions.

For this mapping Trubiroha’s group used the CRISPR method of gene editing to mutate or knock out specific targets and to tag cells with fluorescent labels — that we described in Re-writing the Manual of Life.

A flavor of their results is given by the two sets of fluorescent images below. These show in real time the formation of the thyroid after egg fertilization and the effect of a drug that causes thyroid enlargement.

Live imaging of transgenic zebrafish to follow thyroid development in real-time (left). Arrows mark chord-like cell clusters that form hormone-secreting follicles (arrowheads) during normal development. The right hand three images show normal development (-) and goiter formation (+) induced by a drug. From Trubiroha et al. 2018.

John would have been thrilled by this wonderful work and, with a chuckle, I suspect he’d have said something like “Gosh! If we’d had gene editing back in the 70s we’d have mapped the worm in a couple of weeks!”

References

International Human Genome Sequencing Consortium Nature 431, 931–945; 2004.

John Sulston and Georgina Ferry The Common Thread: A Story of Science, Politics, Ethics and the Human Genome (Bantam Press, 2002).

Sulston, J.E. and Horvitz, H.R. (1977). Post-embryonic Cell Lineages of the Nematode, Caenorhabitis elegans. Development Biology 56, 110-156.

Trubiroha, A. et al. (2018). A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function. Scientific Reports 8, Article number: 5647.

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Bonkers Really … but …

 

This is just in case you spotted the headline in January 2018: ‘Scientists Counted All The Protein Molecules in a Cell And The Answer Really Is 42. This is so perfect.’ 

Them scientists eh! The things they get up to!! The scallywags in this case were Brandon Ho & chums from the University of Toronto and Signe Dean, the journalist who came up with the headline, was referring, of course, to Douglas Adams’s “Answer to the Ultimate Question of Life …” in The Hitchhiker’s Guide to the Galaxy — though it may be noted that Ho’s paper includes neither the number 42 nor mention of Douglas Adams.

The cult that has evolved around this number is both amusing and bizarre, not least because Adams himself explained that he dreamed 42 up out of the blue. In a different context a while ago (talking about how the way you get to work might affect your life expectancy) I recounted happy evenings spent carousing in The Baron (well, having a quiet jar or two) with Douglas Adams and friends from which it was clear that he was not into abstruse mathematics, astrology or the occult. He just had a vivid imagination.

Anything for a catchy headline but

Aside from the whimsy, is there anything interesting in this paper? Well, yes. Ho & Co studied a type of yeast (Saccharomyces cerevisiae) that is mighty important because it’s been a foundation for brewing and baking since ancient times. So no merry sessions in The Baron of Beef without it! Its cells are about the same size as red blood cells (5–10 microns in diameter) but you can actually see them sometimes as films on the skin of fruit. It’s played a huge role in biology as a ‘model organism’ for studying how we work because the proteins it makes that are essential for life are pretty well identical to those in human cells — so much so that you can swap those that control cell growth and division between the two. Yeast proteins work just fine in human cells and vice versa.

 

Yeast on the skin of a grape. Photo: Barbara W. Beacham

 

The question Ho & Co asked was ‘how many protein molecules are there in one cell?’ In the age when you can sequence the DNA of practically anything at the drop of a hat, you might think we’d know the answer already but in fact it’s not been at all clear. Accordingly, what these authors did was to pull together all the relevant studies that have been done to come up with an absolute figure. The answer that emerged was that the number of protein molecules per yeast cell is 4.2 x 107 — which, of course, can also be written as 42 million. Eureka! We have our headline!! Albeit, as the authors noted, with a two-fold error range.

Does anyone care?

Now you’re just being awkward. You should be grateful to be made to picture for a moment tens of millions of proteins jiggling around in little sacs so small you could get tens of thousands of these cells on the head of a pin. And somehow, in that heaving molecular city, each protein manages to carry out its own task so that the cell works. It is quite staggering.

Mention of tasks leads to the other question Ho et al looked at: how many copies are there of the different types of protein? We know from its DNA sequence that this yeast has about 6,000 genes (Saccharomyces Genome Database). So that’s at least 6,000 different proteins. Not surprisingly, it turns out that about two thirds of them are in the middle in terms of abundance — i.e. there’s between 1,000 and 10,000 molecules of each sort per cell. The rest are either low abundance (up to about 800 molecules per cell) or at the high end — 140,000 to 750,000, i.e. somewhere in the region of half a million copies of each type of protein.

Does this distribution make sense in terms of what these proteins do?

You know the answer because if it didn’t the Toronto team wouldn’t have got their work published but, indeed, proteins present in large numbers are, for example, part of the machinery that makes new proteins (so they’re slaving away all the time) whereas, those present in small numbers do things like repair and replicate DNA and drive cells to divide — important jobs but ones that are only intermittently needed.

These results aren’t going to turn science on its head but it is awe-inspiring when a piece of work really brings us face-to-face with stunning complexity of biology. And if it takes a bonkers headline to catch our eye, so be it!

Reference

Ho, B. et al. (2018). Unification of Protein Abundance Datasets Yields a Quantitative Saccharomyces cerevisiae Proteome. Cell Systems. Published online: January 23, 2018.

Hitchhiker Or Driver?

 

It’s a little while since we talked about what you might call our hidden self — the vast army of bugs that colonises our nooks and crannies, especially our intestines, and that is essential to our survival.

In Our Inner Self we noted that these little guys outnumber the human cells that make up the body by about ten to one. Actually that estimate has recently been revised — downwards you might be relieved to hear — to about 1.3 bacterial cells per human cell but it doesn’t really matter. They are a major part of what’s called the microbiome — a vast army of microorganisms that call our bodies home but on which we also depend for our very survival.

In our personal army there’s something like 700 different species of bacteria, with thirty or forty making up the majority. We upset them at our peril. Artificial sweeteners, widely used as food additives, can change the proportions of types of gut bacteria. Some antibiotics that kill off bacteria can make mice obese — and they probably do the same to us. Obese humans do indeed have reduced numbers of bugs and obesity itself is associated with increased cancer risk.

In it’s a small world we met two major bacterial sub-families, Bacteroidetes and Firmicutes, and noted that their levels appear to affect the development of liver and bowel cancers. Well, the Bs & Fs are still around you’ll be glad to know but in a recent piece of work the limelight has been taken by another bunch of Fs — a sub-group (i.e. related to the Bs & Fs) called Fusobacterium.

It’s been known for a few years that human colon cancers carry enriched levels of these bugs compared to non-cancerous colon tissues — suggesting, though not proving, that Fusobacteria may be pro-tumorigenic. In the latest, pretty amazing, installment Susan Bullman and colleagues from Harvard, Yale and Barcelona have shown that not merely is Fusobacterium part of the microbiome that colonises human colon cancers but that when these growths spread to distant sites (i.e. metastasise) the little Fs tag along for the ride! 

Bacteria in a primary human bowel tumour.  The arrows show tumour cells infected with Fusobacteria (red dots).

Bacteria in a liver metastasis of the same bowel tumour.  Though more difficult to see, the  red dot (arrow) marks the presence of bacteria from the original tumour. From Bullman et al., 2017.

In other words, when metastasis kicks in it’s not just the tumour cells that escape from the primary site but a whole community of host cells and bugs that sets sail on the high seas of the circulatory system.

But doesn’t that suggest that these bugs might be doing something to help the growth and spread of these tumours? And if so might that suggest that … of course it does and Bullman & Co did the experiment. They tried an antibiotic that kills Fusobacteria (metronidazole) to see if it had any effect on F–carrying tumours. Sure enough it reduced the number of bugs and slowed the growth of human tumour cells in mice.

Growth of human tumour cells in mice. The antibiotic metronidazole slows the growth of these tumour by about 30%. From Bullman et al., 2017.

We’re still a long way from a human therapy but it is quite a startling thought that antibiotics might one day find a place in the cancer drug cabinet.

Reference

Bullman, S. et al. (2017). Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science  358, 1443-1448. DOI: 10.1126/science.aal5240

RoboClot

 

It was the Chinese, inevitably, who invented paper – during the Eastern Han period around 200 CE (or AD as I’d put it). Presumably by 201 AD some of the lads at the back of the class had discovered that this new stuff could be folded and launched to land on the desk of the local Confucius, generating much hilarity and presumably a few whacks with a bamboo cane.

Folding molecules

Not to be outdone some 21st century scholars have shown that you can do molecular origami with DNA. The idea is fairly simple: take a long strand of DNA (several thousand bases) and persuade it to fold into specific shapes by adding ‘staples’ — short bits of DNA (oligonucleotides). When you mix them together the staples and scaffold strands self-assemble in a single step. It’s pretty amazing but it’s driven by the simple concept of Watson-Crick base pairing (adenine (A) binds to thymine (T): guanine (G) to cytosine (C)).

These things are, of course, almost incomprehensibly small — they are biological molecules remember — each being a few nanometers long. Which means that you can plonk a billion on the head of a pin.

Working on this scale has given rise to the science of nanorobotics ­— making gadgets on a nanometre scale (10−9 meters or one thousandth of a millionth of a metre) and the gizmos themselves are nanorobots — nanobots to their friends.

Making parcels of DNA must be great fun but it’s not much use until you include the fact that you can stick protein molecules to your DNA carrier. If you choose a protein that has a known target, for example, something on the surface of a cell, you can now mail the parcels to an address within the body simply by injecting them into the circulation.

Molecular origami: Making a DNA parcel with a targeting protein. A bacteriophage is a virus that infects and replicates in bacteria, used here to make single strands of DNA. Short DNA ‘staples’ are designed to fold the scaffold DNA into specific shapes. Adding an aptamer (e.g., a protein that binds to a specific target molecule on a cell (an antigen)) permits targeting of the nanobot. When it sticks to a cell the package opens and the molecular payload is released (from Fu and Yan, 2012).

Open with care

Hao Yan and colleagues from Arizona State University have now taken nanobots a step further by adding a second protein to their targeted vehicle. For their targeting protein they used something that sticks to a protein present on the surface of cells that line the walls of blood vessels when they are proliferating (the target protein’s called nucleolin). Generally these (endothelial) cells aren’t proliferating so they don’t make nucleolin — and the nanobots pass them by. But growing tumours need to make their own blood supply. To do that they stimulate new vessels to sprout into the tumour (called angiogenesis) and this is what Hao Yan’s nanobots target.

As an anti-cancer tactic the nanobots carried a second protein: thrombin. This is a critical part of the process of coagulation by which damaged blood vessels set about repairing themselves. Thrombin’s role is to convert fibrinogen (circulating in blood) to fibrin strands, hence building up a blood clot to plug the hole. In effect the nanobots cause thrombosis, inducing a blood clot to block the supply line to the tumour.

Blood clotting (coagulation). Platelets form a plug strengthened by fibrin produced by the action of thrombin.

Does it work?

These DNA nanorobots showed no adverse effects either in mice or in Bama miniature pigs, which exhibit high similarity to humans in anatomy and physiology.

Fluorescently labeled nanobots did indeed target tumour blood vessels: the DNA wrapping opens when they attach to cells and the thrombin is released …

Fluorescent nanobots targetting tumour blood vessels (Li et al. 2018). The nanorobots have stuck to cells lining blood vessels (endothelial cells: green membrane) by attaching to nucleolin. After 8 hours the nanorobots (red) have been taken up by the cells and can be seen next to the nucleus (blue).

Most critically these little travellers did have effects on tumour growth. The localized thrombosis caused by the released thrombin resulted in significant tumour cell death and marked increase in the survival of treated mice.

Robotic DNA machines are now being referred to as ‘intelligent vehicles’ — a designation I’m not that keen on. Nevertheless, this is a cunning strategy, not least because, although much effort has gone into anti-angiogenic therapies for cancer, they have not been notably successful. Simply administering thrombin would presumably be fatal but, well wrapped up and correctly addressed, it seems to deliver.

Reference

Fu, J. and Yan, H. (2012). Controlled drug release by a nanorobot. Nature Biotechnology 30, 407-408.

Suping Li et al. (2018). A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nature Biotechnology doi:10.1038/nbt.4071

One More Small Step

 

Back in the nineteenth century a chap called Augustus De Morgan came up with a set of laws that, when explained in English, sound like the lyrics of a Flanders & Swann song. Opaque to non-maths nerds they may be but they helped to build the mathematics of logic, so next time you meet AND / OR gates in electronics, spare him a thought.

In fact Augustus is rare — maybe unique — among mathematicians in that he’s not completely forgotten, for it was he who penned the lines:

Big fleas have little fleas upon their backs to bite ’em,
And little fleas have lesser fleas, and so, 
ad infinitum.

Given that we now know there’s over 2,500 species of fleas ranging in size from tiny to nearly one centimeter long, it may be literally true. But here, for once, the truth doesn’t matter. It’s a silly rhyme but nonsense verse it is not for it could well serve as a motto for biology because it really captures the essential truth of life: the exquisite choreography of living systems by which incomprehensible numbers of interactions come together to make them work.

Human fleas. Don’t worry: you’ll know if you have them.

Unbidden, De Morgan’s ditty came into my head as I was reading the latest research paper from David Lyden’s group, which he very kindly sent me ahead of publication this week. Avid readers will know the name for we have devoted several episodes (Keeping Cancer Catatonic, Scattering the Bad Seed and Holiday Reading (4) – Can We Make Resistance Futile) to the discoveries of his group in tackling one of the key questions in cancer — namely, how do tumour cells find their targets when they spread around the body? Key because it is this process of ‘metastasis’ that causes most (over 90%) of cancer deaths and if we knew how it worked maybe we could block it.

A succinct summary of those already condensed episodes would be: (1) cells in primary tumours release ‘messengers’ into the circulation that ‘tag’ metastatic sites before any cells actually leave the tumour, (2) the messengers that do the site-tagging are small sacs — mini cells — called exosomes, and (3) they find specific addresses by carrying protein labels (integrins) that home in to different organs — we represented that in the form of a tube train map in Lethal ZIP codes that pulled the whole story together.

The next small step

Now what the folks from Weill Cornell Medicine, New York, Sloan Kettering and a host of other places have done is adapt a flow system to look more closely at exosomes.

Separating small bodies. Particles are injected into a flowing liquid (left) and cross flow at right angles through a membrane (bottom) permits separation on the basis of effective size (called asymmetrical flow field-flow fractionation).

They found that a wide variety of tumour cell types secrete two distinct populations of exosomes — small (60-80 nanometres diameter) and large (90-120 nm). What’s more they found a third type of nanoparticle, smaller than exosomes (less than 50 nm) and without a membrane — so it’s a kind of blob of lipids and proteins (a micelle would be a more scientific term) — that they christened exomeres.

Is it real?

A perpetual problem in biology is reproducibility — that is, whether a new finding can be replicated independently by someone else. Or, put more crudely, do I believe this? This is such an important matter that it’s worth a separate blog but for the moment we’re OK because the results in this paper speak for themselves. First, by using electron microscopy, Lyden et al could actually look at what they’d isolated and indeed discerned three distinct nano-populations — which is how they were able to put the size limits on them.

Electron microscopy of (left) the input mixture (pre-fractionation) and separated fractions: exomere, small exosomes and large exosomes released by tumour cells.. Arrows indicate exomeres (red), small exosomes (blue) and large exosomes (green), from Zhang et al. 2018.

But what’s most exciting in terms of the potential of these results is what’s in the packets. Looking at the fats (lipids), proteins and nucleic acids (DNA and RNA) they contained it’s clear that these are three distinct entities — which makes it very likely they have different effects.

Given their previous finding it must have been a great relief when Lyden & Co identified integrin address proteins in the two exosome sub-populations. But what’s really astonishing is the range of proteins born by these little chaps: something like 400 in exomeres, about 1000 in small exosomes and a similar number in the big ones — and the fact that each contained unique sets of proteins. The new guys — exomeres — carry among other proteins, metabolic enzymes so it’s possible that when they deliver their cargo it might be able to change the metabolic profile of its target. That could be important as we know such changes happen in cancer.

It’s a bewildering picture and working out even the basics of what these little guys do and how it influences cancer is, as we say, challenging. But I think I know a good man for the job!

Augustus De Morgan looking down.

Mathematicians have a bit of a tendency to look down on us experimentalists thrashing around in the undergrowth and I suspect that up in the celestial library, as old Augustus De Morgan thumbed through this latest paper, a slight smile might have come over his face and he could have been heard to murmur: “See, I told you.”

References

Zhang, H. et al. (2018). Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nature Cell Biology 20, 332–343. doi:10.1038/s41556-018-0040-4

You Couldn’t Make It Up … But They Do!!

 

Having just posted a somewhat critical commentary on a recent, much-headlined, study looking at the effect of ‘ultra-processed’ food on cancer risk that was based on what folk said they ate, who should come galloping into the fray this morning but the Office for National Statistics (ONS).

They’ve analysed a National Diet and Nutrition survey and, surprise surprise, found that the adults surveyed (4,500 of them) said they ate 50% fewer calories than they were actually tucking away!

So much for relying on people telling the truth!!

How do they know?

Well, they persuaded 200 punters to drink doubly labelled water as part of their diet (the water is made of chemical variants of hydrogen and oxygen, deuterium and oxygen-18) and pee the truth into a bottle (from the proportions of deuterium and 18O  in urine you can work out calorie consumption).

The upshot of all this is that, whilst a rough average figure for desirable calorie intake is 2,500 for a man and 2,000 for a woman, the 4,500 were eating the equivalent of an extra Big Mac a day, with men consuming 3,119 calories rather than the 2,065 they claimed. Women consumed 2,393 calories instead of 1,570.

Actually, this didn’t come as a great surprise to the ONS guys because they’d spotted that 1 in 3 (34%) of the 4,500 claimed a calorie consumption figure that wouldn’t keep them alive! And, guess what, overweight people and men (of course) are most likely to tell dietary fibs.

Oh dear, I told the French folk in Please … Not Another Helping they shouldn’t believe a word people said. Of course, they will be quick to point out that the ONS is a British outfit reporting on Brits who are notorious cads and bounders. That’s OK then: we can confidently believe what the French tell us about their eating habits — just as we accept that they are the best lovers and have the most sex.

Please … Not Another Helping

 

You may have seen the headlines of the: “Processed food, sugary cereals and sliced bread may contribute to cancer risk” ilk, as this recently published study (February 2018) was extensively covered in the media — the Times of London had a front page spread no less.

So I feel obliged to follow suit — albeit with a heavy heart: it’s one of those depressing exercises in which you’re sure you know the answer before you start.

Who dunnit?

It’s a mainly French study (well, it is about food) led by Thibault Fiolet, Mathilde Touvier and colleagues from the Sorbonne in Paris. It’s what’s called a prospective cohort study, meaning that a group of individuals, who in this case differed in what they ate, were followed over time to see if diet affected their risk of getting cancers and in particular whether it had any impact on breast, prostate or colorectal cancer. They started acquiring participants about 20 years ago and their report in the British Medical Journal summarized how nearly 105 thousand French adults got on consuming 3,300 (!) different food items between them, based on each person keeping 24 hour dietary records designed to record their usual consumption.

Foods were grouped according to degree of processing. The stuff under the spotlight is ‘ultra-processed’ — meaning that it has been chemically tinkered with to get rid of bugs, give it a long shelf-life, make it convenient to use, look good and taste palatable.

What makes a food ‘ultra-processed’ is worked out by something called the NOVA classification. I’ve included their categories at the end.

Relative contribution of each food group to ultra-processed food consumption in diet (from Fiolet et al. 2018).

And the result?

The first thing to be said is that this study is a massive labour of love. You need the huge number of over 100,000 cases even to begin to squeeze out statistically significant effects — so the team has put in a terrific amount of work.

After all the squeezing there emerged a marginal increase in risk of getting cancer in the ultra-processed food eaters and a similar slight increase specifically for breast cancer (the hazard ratios were 1.12 and 1.11 respectively). There was no significant link to prostate and colorectal cancers.

Which may mean something. But it’s hard to get excited, not merely because the effects described are small but more so because such studies are desperately fraught and the upshot familiar.

One problem is that they rely on individuals keeping accurate records. Another problem here is that the classification of ‘ultra-processed’ is somewhat arbitrary — and it’s also very broad — leaving one asking what the underlying cause might be: ‘is it sugar, fat or what?’ Furthermore, although the authors tried manfully to allow for factors like smoking and obesity, it’s impossible to do this with complete certainty. The authors themselves noted that, for example, they couldn’t allow for the effects of oral contraception.

The authors are quite right to point out that it is important to disentangle the facets of food processing that bear on our long-term health and that further studies are needed.

I would only add ‘rather you than me.’

Perforce in these pages we have gone on about diets good and bad so there is no need to regurgitate. Suffice to say that my advice on what to eat is the same as that of any other sane person and summarized in Dennis’s Pet Menace — and it’s not been remotely affected by this new research which, in effect, says ‘junk food is probably bad for you in the long run.’ But let’s leave the last word to Tom Sanders of King’s College London: “What people eat is an expression of their life-style in general, and may not be causatively linked to the risk of cancer.” 

Reference

Fiolet, T. et al. (2018). Consumption of ultra-processed foods and cancer risk: results from NutriNet-Santé prospective cohort. BMJ 2018;360:k322 http://dx.doi.org/10.1136/bmj.k322

NOVA classification:

The ultra-processed food group is defined by opposition to the other NOVA groups: “unprocessed or minimally processed foods” (fresh, dried, ground, chilled, frozen, pasteurised, or fermented staple foods such as fruits, vegetables, pulses, rice, pasta, eggs, meat, fish, or milk), “processed culinary ingredients” (salt, vegetable oils, butter, sugar, and other substances extracted from foods and used in kitchens to transform unprocessed or minimally processed foods into culinary preparations), and “processed foods” (canned vegetables with added salt, sugar coated dried fruits, meat products preserved only by salting, cheeses, freshly made unpackaged breads, and other products manufactured with the addition of salt, sugar, or other substances of the “processed culinary ingredients” group).

Sweet Love …

 

Sweet love, renew thy force; be it not said

Thy edge should blunter be than appetite,

Which but to-day by feeding is allay’d,

To-morrow sharpen’d in his former might:

No prize for knowing I didn’t write those lines — or even that they’re down to The Bard of Avon. What he was on about here is the distinction between genuine (sweet) love and lust (appetite), the problem being that the latter may be assuaged today but will surely return tomorrow. Had we, by some Star Trek-like device, been able to secure his services for this piece, Shakespeare, master of the double-entendre, would quickly have spotted an opportunity in his new role as pop-sci scribe. For sweet read sugar: for appetite addiction.

Gary Taubes considers sugar to be the root of most western illnesses. Photograph: Alamy

The combination can be toxic, as the estimable US journalist Gary Taubes has argued over the last 15 years. His latest book The Case Against Sugar has just come out and I’m keen to give it a plug. In so doing I should point out that we’ve also done our best in these pages to make the same case — particularly in relation to cancer. However, it’s a little while since we wrote specifically on sugar, diet and cancer, mainly because nothing really new has caught my eye. Reading again the most relevant of our blog stories I thought they did a pretty good job (as Shakespeare might have said, being a chap not known for modesty). Three I thought worth looking at again are:

Biting the Bitter Bullet: how obesity and cancer quite often come hand-in-hand and how it is that we’re seduced into eating more and more of something that can help us get fat and ill.

A Small Helping For Australia: makes the point that this is a global problem (even though Australia’s wonderful).

The Best Laid Plans in Mice and Men..: artificial sweeteners aren’t the solution – just another problem.

Actually, there is one recent result we might mention — from Ken Peeters, Johan Thevelein & colleagues at the University of Leuven. Bearing in mind the long-established ‘Warburg effect’ by which cancer cells switch the energy supply system that breaks down glucose from respiration (using oxygen) to fermentation (making lactate), they looked at yeast cells that grow fastest when they ferment — much as cancer cells grow quicker than normal cells. Rather remarkably, they discovered a hitherto unknown way in which fermentation links to a key pathway controlling cell proliferation. That pathway centres around a protein called RAS that we met in Mission Impossible.

This finding does not show that eating lots of sugar gives you cancer but what it does show is a way by which, if yeast cells ‘eat’ more sugar, they grow faster. It seems quite possible that the underlying mechanism might work in human cells (the human version of the protein that links sugar metabolism to RAS, called SOS1, works in yeast) — giving an explanation for the well-known fact that the more sugar you eat the fatter you are likely to become. And what we do know is that obesity does raise cancer risk.

I dare say Gary might reckon this result worth a footnote in the second edition of: The Case Against Sugar by Gary Taubes is published by Portobello Books (£14.99).

Reference

Peeters, K. et al., (2017). Fructose-1,6-bisphosphate couples glycolytic flux to activation of Ras. Nature Communications 8, Article number: 922 doi:10.1038/s41467-017-01019-z.

Desperately SEEKing …

These days few can be unaware that cancers kill one in three of us. That proportion has crept up over time as life expectancy has gone up — cancers are (mainly) diseases of old age. Even so, they plagued the ancients as Egyptian scrolls dating from 1600 BC record and as their mummified bodies bear witness. Understandably, progress in getting to grips with the problem was slow. It took until the nineteenth century before two great French physicians, Laënnec and Récamier, first noted that tumours could spread from their initial site to other locations where they could grow as ‘secondary tumours’. Munich-born Karl Thiersch showed that ‘metastasis’ occurs when cells leave the primary site and spread through the body. That was in 1865 and it gradually led to the realisation that metastasis was a key problem: many tumours could be dealt with by surgery, if carried out before secondary tumours had formed, but once metastasis had taken hold … With this in mind the gifted American surgeon William Halsted applied ever more radical surgery to breast cancers, removing tissues to which these tumors often spread, with the aim of preventing secondary tumour formation.

Early warning systems

Photos of Halsted’s handiwork are too grim to show here but his logic could not be faulted for metastasis remains the cause of over 90% of cancer deaths. Mercifully, rather than removing more and more tissue targets, the emphasis today has shifted to tumour detection. How can they be picked up before they have spread?

To this end several methods have become familiar — X-rays, PET (positron emission tomography, etc) — but, useful though these are in clinical practice, they suffer from being unable to ‘see’ small tumours (less that 1 cm diameter). For early detection something completely different was needed.

The New World

The first full sequence of human DNA (the genome), completed in 2003, opened a new era and, arguably, the burgeoning science of genomics has already made a greater impact on biology than any previous advance.

Tumour detection is a brilliant example for it is now possible to pull tumour cell DNA out of the gemisch that is circulating blood. All you need is a teaspoonful (of blood) and the right bit of kit (silicon chip technology and short bits of artificial DNA as bait) to get your hands on the DNA which can then be sequenced. We described how this ‘liquid biopsy’ can be used to track responses to cancer treatment in a quick and non–invasive way in Seeing the Invisible: A Cancer Early Warning System?

If it’s brilliant why the question mark?

Two problems really: (1) Some cancers have proved difficult to pick up in liquid biopsies and (2) the method didn’t tell you where the tumour was (i.e. in which tissue).

The next step, in 2017, added epigenetics to DNA sequencing. That is, a programme called CancerLocator profiled the chemical tags (methyl groups) attached to DNA in a set of lung, liver and breast tumours. In Cancer GPS? we described this as a big step forward, not least because it detected 80% of early stage cancers.

There’s still a pesky question mark?

Rather than shrugging their shoulders and saying “that’s science for you” Joshua Cohen and colleagues at Johns Hopkins University School of Medicine in Baltimore and a host of others rolled their sleeves up and made another step forward in the shape of CancerSEEK, described in the January 18 (2018) issue of Science.

This added two new tweaks: (1) for DNA sequencing they selected a panel of 16 known ‘cancer genes’ and screened just those for specific mutations and (2) they included proteins in their analysis by measuring the circulating levels of 10 established biomarkers. Of these perhaps the most familiar is cancer antigen 125 (CA-125) which has been used as an indicator of ovarian cancer.

Sensitivity of CancerSEEK by tumour type. Error bars represent 95% confidence intervals (from Cohen et al., 2018).

The figure shows a detection rate of about 70% for eight cancer types in 1005 patients whose tumours had not spread. CancerSEEK performed best for five types (ovary, liver, stomach, pancreas and esophagus) that are difficult to detect early.

Is there still a question mark?

Of course there is! It’s biology — and cancer biology at that. The sensitivity is quite low for some of the cancers and it remains to be seen how high the false positive rate goes in larger populations than 1005 of this preliminary study.

So let’s leave the last cautious word to my colleague Paul Pharoah: “I do not think that this new test has really moved the field of early detection very far forward … It remains a promising, but yet to be proven technology.”

Reference

D. Cohen et al. (2018). Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 10.1126/science.aar3247.

Lorenzo’s Oil for Nervous Breakdowns

 

A Happy New Year to all our readers – and indeed to anyone who isn’t a member of that merry band!

What better way to start than with a salute to the miracles of modern science by talking about how the lives of a group of young boys have been saved by one such miracle.

However, as is almost always the way in science, this miraculous moment is merely the latest step in a long journey. In retracing those steps we first meet a wonderful Belgian – so, when ‘name a famous Belgian’ comes up in your next pub quiz, you can triumphantly produce him as a variant on dear old Eddy Merckx (of bicycle fame) and César Franck (albeit born before Belgium was invented). As it happened, our star was born in Thames Ditton (in 1917: his parents were among the one quarter of a million Belgians who fled to Britain at the beginning of the First World War) but he grew up in Antwerp and the start of World War II found him on the point of becoming qualified as a doctor at the Catholic University of Leuven. Nonetheless, he joined the Belgian Army, was captured by the Germans, escaped, helped by his language skills, and completed his medical degree.

Not entirely down to luck

This set him off on a long scientific career in which he worked in major institutes in both Europe and America. He began by studying insulin (he was the first to suggest that insulin lowered blood sugar levels by prompting the liver to take up glucose), which led him to the wider problems of how cells are organized to carry out the myriad tasks of molecular breaking and making that keep us alive.

The notion of the cell as a kind of sac with an outer membrane that protects the inside from the world dates from Robert Hooke’s efforts with a microscope in the 1660s. By the end of the nineteenth century it had become clear that there were cells-within-cells: sub-compartments, also enclosed by membranes, where special events took place. Notably these included the nucleus (containing DNA of course) and mitochondria (sites of cellular respiration where the final stages of nutrient breakdown occurs and the energy released is transformed into adenosine triphosphate (ATP) with the consumption of oxygen).

In the light of that history it might seem a bit surprising that two more sub-compartments (‘organelles’) remained hidden until the 1950s. However, if you’re thinking that such a delay could only be down to boffins taking massive coffee breaks and long vacations, you’ve never tried purifying cell components and getting them to work in test-tubes. It’s a process called ‘cell fractionation’ and, even with today’s methods, it’s a nightmare (sub-text: if you have to do it, give it to a Ph.D. student!).

By this point our famous Belgian had gathered a research group around him and they were trying to dissect how insulin worked in liver cells. To this end they (the Ph.D. students?!) were using cell fractionation and measuring the activity of an enzyme called acid phosphatase. Finding a very low level of activity one Friday afternoon, they stuck the samples in the fridge and went home. A few days later some dedicated soul pulled them out and re-measured the activity discovering, doubtless to their amazement, that it was now much higher!

In science you get odd results all the time – the thing is: can you repeat them? In this case they found the effect to be absolutely reproducible. Leave the samples a few days and you get more activity. Explanation: most of the enzyme they were measuring was contained within a membrane-like barrier that prevented the substrate (the chemical that the enzyme reacts with) getting to the enzyme. Over a few days the enzyme leaked through the barrier and, lo and behold, now when you measured activity there was more of it!

Thus was discovered the ‘lysosome’ – a cell-within-a cell that we now know is home to an array of some 40-odd enzymes that break down a range of biomolecules (proteinsnucleic acidssugars and lipids). Our self-effacing hero said it was down to ‘chance’ but in science, as in other fields of life, you make your own luck – often, as in this case, by spotting something abnormal, nailing it down and then coming up with an explanation.

In the last few years lysosomes have emerged as a major player in cancer because they help cells to escape death pathways. Furthermore, they can take up anti-cancer drugs, thereby reducing potency. For these reasons they are the focus of great interest as a therapeutic target.

Lysosomes in cells revealed by immunofluorescence.

Antibody molecules that stick to specific proteins are tagged with fluorescent labels. In these two cells protein filaments of F-actin that outline cell shape are labelled red. The green dots are lysosomes (picked out by an antibody that sticks to a lysosome protein, RAB9). Nuclei are blue (image: ThermoFisher Scientific).

Play it again Prof!

In something of a re-run of the lysosome story, the research team then found itself struggling with several other enzymes that also seemed to be shielded from the bulk of the cell – but the organelle these lived in wasn’t a lysosome – nor were they in mitochondria or anything else then known. Some 10 years after the lysosome the answer emerged as the ‘peroxisome’ – so called because some of their enzymes produce hydrogen peroxide. They’re also known as ‘microbodies’ – little sacs, present in virtually all cells, containing enzymatic goodies that break down molecules into smaller units. In short, they’re a variation on the lysosome theme and among their targets for catabolism are very long-chain fatty acids (for mitochondriacs the reaction is β-oxidation but by a different pathway to that in mitochondria).

Peroxisomes revealed by immunofluorescence.

As in the lysosome image, F-actin is red. The green spots here are from an antibody that binds to a peroxisome protein (PMP70). Nuclei are blue (image: Novus Biologicals)

Cell biology fans will by now have worked out that our first hero in this saga of heroes is Christian de Duve who shared the 1974 Nobel Prize in Physiology or Medicine with Albert Claude and George Palade.

A wonderful Belgian. Christian de Duve: physician and Nobel laureate.

Hooray!

Fascinating and important stuff – but nonetheless background to our main story which, as they used to say in The Goon Show, really starts here. It’s so exciting that, in 1992, they made a film about it! Who’d have believed it?! A movie about a fatty acid!! Cinema buffs may recall that in Lorenzo’s Oil Susan Sarandon and Nick Nolte played the parents of a little boy who’d been born with a desperate disease called adrenoleukodystrophy (ALD). There are several forms of ALD but in the childhood disease there is progression to a vegetative state and death occurs within 10 years. The severity of ALD arises from the destruction of myelin, the protective sheath that surrounds nerve fibres and is essential for transmission of messages between brain cells and the rest of the body. It occurs in about 1 in 20,000 people.

Electrical impulses (called action potentials) are transmitted along nerve and muscle fibres. Action potentials travel much faster (about 200 times) in myelinated nerve cells (right) than in (left) unmyelinated neurons (because of Saltatory conduction). Neurons (or nerve cells) transmit information using electrical and chemical signals.

The film traces the extraordinary effort and devotion of Lorenzo’s parents in seeking some form of treatment for their little boy and how, eventually, they lighted on a fatty acid found in lots of green plants – particularly in the oils from rapeseed and olives. It’s one of the dreaded omega mono-unsaturated fatty acids (if you’re interested, it can be denoted as 22:1ω9, meaning a chain of 22 carbon atoms with one double bond 9 carbons from the end – so it’s ‘unsaturated’). In a dietary combination with oleic acid  (another unsaturated fatty acid: 18:1ω9) it normalizes the accumulation of very long chain fatty acids in the brain and slows the progression of ALD. It did not reverse the neurological damage that had already been done to Lorenzo’s brain but, even so, he lived to the age of 30, some 22 years longer than predicted when he was diagnosed.

What’s going on?

It’s pretty obvious from the story of Lorenzo’s Oil that ALD is a genetic disease and you will have guessed that we wouldn’t have summarized the wonderful career of Christian de Duve had it not turned out that the fault lies in peroxisomes.

The culprit is a gene (called ABCD1) on the X chromosome (so ALD is an X-linked genetic disease). ABCD1 encodes part of the protein channel that carries very long chain fatty acids into peroxisomes. Mutations in ABCD1 (over 500 have been found) cause defective import of fatty acids, resulting in the accumulation of very long chain fatty acids in various tissues. This can lead to irreversible brain damage. In children the myelin sheath of neurons is damaged, causing neurological defects including impaired vision and speech disorders.

And the miracle?

It’s gene therapy of course and, helpfully, we’ve already seen it in action. Self Help – Part 2 described how novel genes can be inserted into the DNA of cells taken from a blood sample. The genetically modified cells (T lymphocytes) are grown in the laboratory and then infused into the patient – in that example the engineered cells carried an artificial T cell receptor that enabled them to target a leukemia.

In Gosh! Wonderful GOSH we saw how the folk at Great Ormond Street Hospital adapted that approach to treat a leukemia in a little girl.

Now David Williams, Florian Eichler, and colleagues from Harvard and many other centres around the world, including GOSH, have adapted these methods to tackle ALD. Again, from a blood sample they selected one type of cell (stem cells that give rise to all blood cell types) and then used genetic engineering to insert a complete, normal copy of the DNA that encodes ABCD1. These cells were then infused into patients. As in the earlier studies, they used a virus (or rather part of a viral genome) to get the new genetic material into cells. They choose a lentivirus for the job – these are a family of retroviruses (i.e. they have RNA genomes) that includes HIV. Specifically they used a commercial vector called Lenti-D. During the life cycle of RNA viruses their genomes are converted to DNA that becomes a permanent part of the host DNA. What’s more, lentiviruses can infect both non-dividing and actively dividing cells, so they’re ideal for the job.

In the first phase of this ongoing, multi-centre trial a total of 17 boys with ALD received Lenti-D gene therapy. After about 30 months, in results reported in October 2017, 15 of the 17 patients were alive and free of major functional disability, with minimal clinical symptoms. Two of the boys with advanced symptoms had died. The achievement of such high remission rates is a real triumph, albeit in a study that will continue for many years.

In tracing this extraordinary galaxy, one further hero merits special mention for he played a critical role in the story. In 1999 Jesse Gelsinger, a teenager, became the first person to receive viral gene therapy. This was for a metabolic defect and modified adenovirus was used as the gene carrier. Despite this method having been extensively tested in a range of animals (and the fact that most humans, without knowing it, are infected with some form of adenovirus), Gelsinger died after his body mounted a massive immune response to the viral vector that caused multiple organ failure and brain death.

This was, of course, a huge set-back for gene therapy. Despite this, the field has advanced significantly in the new century, both in methods of gene delivery (including over 400 adenovirus-based gene therapy trials) and in understanding how to deal with unexpected immune reactions. Even so, to this day the Jesse Gelsinger disaster weighs heavily with those involved in gene therapy for it reminds us all that the field is still in its infancy and that each new step is a venture into the unknown requiring skill, perseverance and bravery from all involved – scientists, doctors and patients. But what better encouragement could there be than the ALD story of young lives restored.

It’s taken us a while to piece together the main threads of this wonderful tale but it’s emerged as a brilliant example of how science proceeds: in tiny steps, usually with no sense of direction. And yet, despite setbacks, over much time, fragments of knowledge come together to find a place in the grand jigsaw of life.

In setting out to probe the recesses of metabolism, Christian de Duve cannot have had any inkling that he would build a foundation on which twenty-first century technology could devise a means of saving youngsters from a truly terrible fate but, my goodness, what a legacy!!!

References

Eichler, F. et al. (2017). Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. The New England Journal of Medicine 377, 1630-1638.