Sperm have been fertilizing ovules for millions of years and yet we are only just beginning to understand the rudimentary fundaments involved. Over the millennia, the miracle of conception has occupied some of the most learned minds and, before the existence of the microscope, a vivid imagination was required to make sense out of the liquid men injected into women, which ultimately produced a new version of one of them. A baby boy or a baby girl. Hence the existence of what may seem preposterous theories to us now but which were perfectly sensible in the light of what was then known. For instance, two opposing schools of thought lasted for a very long time. There were those who believed that a complete – but very small version of a human – was contained within one sperm and, once in the woman’s womb, it grew to the size of a newborn. And then there were those who believed that a perfectly-shaped baby human was already floating in the ovule and that all it needed was the sperm to trigger off its growth.

Despite an apparent simplistic view of how Nature dealt with things, what had been understood is that both a sperm and an ovule are necessary for conception. Nothing happens in the absence of the other. And that remains a fact. What we know today is that both gametes can be present and, what is more very close to one another, but if there is something faulty on either one’s surface, nothing will happen. And this is frequently the cause of infertility – male or female. Gametes have a very specific and intricate makeup. There are molecules on their surface which have a protective role, molecules which recognise the other and molecules which allow them to adhere to one another. Zp3 – for zona pellucida 3 – is a protein receptor which is buried in the matrix which surrounds an ovule, known as the zona pellucida. This is the cushiony part of the cell, full of carbohydrate and protein in almost equal amounts, through which one sperm must find its way to initiate fertilization.

‘Tenderness’ by Vladas Velyvis

Zp3 is a cell surface receptor which is bound to the ovule’s membrane. Typically, the protein backbone of zp3 is covered in O-linked oligosaccharides which make up part of the zona pellucida cushion. These particular carbohydrates are recognised by a receptor found on the sperm’s surface. Despite this recognition, scientists believe that the binding of a sperm to an ovule also involves a host of other molecules. What is clear though is that zp3 and its oligosaccharides are needed to initiate the acrosome reaction – the reaction in which the sperm binds irreversibly to the ovule’s membrane and, in so doing, triggers off the chemical process which modifies the supra-molecular structure of the zona pellucida thus forming a barrier to the entry of a second sperm.

Besides discovering the beauty of how life starts on a very small scale, the finer our knowledge is on the molecular processes involved in the moments before and during fertilization, the better we can help men and women faced with infertility problems. Similarly, targeting essential molecules on the surface of sperm or ovules could lead to alternative methods of contraception. Research of the kind has been carried out in the hope of checking the exponential growth of animal populations. Protected from ivory poachers and squeezed into geographical spaces that are getting smaller all the time, elephants are overpopulating game reserves in Africa. Instead of shooting them, a means of birth control would be a more humane solution. Since the 1990s, scientists have been seeking ways of producing antibodies to zp3 for instance, which would create temporary infertility. Some have suggested engineering a virus with a copy of human zp3 and intentionally infecting an animal species whose growth has gone out of control. For instance, mice infected with the virus would produce zp3 antibodies, thereby causing infertility of their host because mouse zp3 is very similar to human zp3. Furthermore, one mouse would infect another mouse, and each infected mouse would in turn become infertile. The problem is: who says the virus won’t infect another animal population which is essential to the fauna’s equilibrium… It is a risk which cannot be taken.

Likewise, immunity of some kind against zp3 could be an elegant form of contraception for humans. It is common knowledge that, though very useful in many ways, the pill or intrauterine devices can be damaging to women – so any method which could prove to be absolutely harmless is welcome. Recently, some scientists suggested a gene-silencing technique whereby the zp3 gene is not translated. This is done by using a method known as RNA interference. In this case, short RNAi fragments bind to zp3 mRNA thus checking zp3 translation. Consequently, the zp3 receptor does not appear on the ovule’s surface and sperm are then unable to bind to an ovule – which is a sure means of avoiding conception. This kind of contraceptive could be administered through a skin patch or by means of a vaginal suppository. And scientists are confident that such treatment will only affect the zp3 receptors of maturing ovules – i.e. the ones that make an appearance once a month – and not the whole reserve in the ovary. It sounds promising and attractive. And far more appealing than a chastity belt.

Every single dog the human race has ever known not only belongs to the same species, Canis familiaris but is also descended from only one other species: the grey wolf, Canis lupus. The variation in size – which no other mammalian species has experienced – results from the selective breeding of dogs over the years and can be tracked all the way back to the beginnings of animal domestication. Medium-size dogs are probably more popular because they are easier to look after. Likewise, if a dog wants to be part of a household, it is better off medium-sized. Fossils, the size of Great Danes or of terriers, date back thousands of years. However, the huge variation we can testify to on a daily basis – from pocket dogs that hardly weigh a kilo to a hundred kilo Saint Bernard – is the result of strong selective breeding which has taken place within the past few hundred years only and has produced an array of 350 declared breeds.

Whatever it is that has made dogs small has been present for a long time. Scientists discovered an insulin-like growth factor 1 (IGF1) variant that is widespread in small dogs meaning – on evolutionary terms – that it was fixed in Canis familiaris many years ago. It is not present in the grey wolf’s genome and the early appearance of the IGF1 variant will have driven the rapid genesis of size diversity in the domestic dog. What is more, this particular variant is geographically widespread – most probably as a result of trading and human migration – thus supporting the fact that it appeared early on in time.

‘Skinny Vinny’ by Lisa Ballard

Dream Dog Paintings

Courtesy of the artist

A study was carried out on one particular breed of dog – the Portuguese water dog. The samples varied substantially in size due strong selective breeding. What the scientists discovered was that every single small-sized dog carried the IGF1 variant while the larger dogs did not. They then turned to other dog breeds to discover the same thing: all the small dogs carried the variant. Although most of the bigger dogs did not, a few did. This means that there is a ‘stronger’ mechanism which exists to dictate the size of larger dogs. It also means that size – like all other phenotypes – is defined by something far more complex than the mere action of just one protein.

insulin-like growth factor I promotes the growth of an organism by firing off a network of reactions – loosely known as the IGF pathway – which ultimately promote protein turnover, cell proliferation, tissue differentiation and even protection against cell apoptosis. Upstream, IGF1 action is triggered off by the pituitary growth hormone, or GH. IGF1 is at the heart of an organism’s maturity. It is synthesized in the liver from where it acts on a wide variety of tissues and mediates its action by binding to its receptor. This launches multiple IGF1 signals transduced by way of phosphorylation reactions to hosts of kinases downstream. IGF1 exists in two forms. It can either bind directly to its receptor or bind to IGF-binding proteins where it is buried in a protective envelope, thus delaying and modulating IGF1’s interaction with its receptor.

Besides finding a way to fit a dog into a matchbox, what is the point of such studies? Such a huge variation in a phenotype within one same species provides valuable information on two fronts. On the one hand, it can reveal how key elements are involved in intricate pathways and, on the other, it can give an idea as to how sets of genes interact to come up with a specific phenotype such as size, or even behaviour. The Canis familiaris model is also precious for the analysis of genetic polymorphism and the understanding of the differences between a healthy genome and a pathological one.

The IGF pathway has been under close scrutiny for some time. Not only is it involved in an organism’s maturity but, unsurprisingly perhaps, it also seems to play a part in an organism’s longevity. A faulty IGF, or indeed, a faulty receptor is likely to create some kind of havoc. One interesting study was carried out on a number of women with a faulty IGF pathway, who had not only lived to be a hundred years old but whose stature was slightly under the average, echoing the effect the IGF1 variant has on dog breeds… Do small dogs live longer than big dogs? The issue was not raised. Though there are no doubt many handbags out there that would enjoy the knowledge that they will get old with their closest companion.

Humans are particularly clever with their hands. One of the very first special events in our evolution was to get onto our hind limbs and free our hands for collecting food and making tools. A subsequent good move was to take away the burden of communication on our hands by developing our vocal instruments. Indeed speech, and its fine-tuned elaboration that only humans have managed to master so far, has given our hands great freedom which we have put to use in a multitude of ways. But none of this can explain why we are – for the great majority (90%) – right-handed. Hosts of other species also use their appendages for collecting food, eating or grooming but they don’t have a distinct preference for one hand over the other.

The passing of roles from hand to mind expresses a particular brain structure. In turn, the progressive use of speech has continued to mould our brain into a shape peculiar to the human species. But why would that make us right-handed? For speech to evolve, one part of our brain had to evolve differently too, and in so doing it made most humans right-handers. This is what is known as the ‘right-shift factor’. Consequently, our right-handedness is not the result of nature selecting right-handers over left-handers but rather of nature nudging our brain into a shape which encourages the act of speaking. As a result, over the millions of years, the human brain has been divided into two hemispheres. The right hemisphere is dedicated to the world of emotions and imagination, whilst the left hemisphere deals with talking and logical processes.

Two men engaged in conversation

Source unknown

LRRTM1, or leucine-rich repeat transmembrane neuronal protein 1, is a protein involved in brain development. It has a set of repetitive domains in its sequence which are known to be involved in protein-protein interactions, a vital activity in the light of brain structure and development. LRRTM1 may be one of the factors which bestows upon the brain its asymmetry. It is expressed very early in the development of forebrain structures and may function in neuronal differentiation and connectivity. It is also thought that it could have a role in intracellular trafficking in axons. Left-handedness, which is handed down by the father, may well be due to LRRTM1 dysfunction causing the original asymmetry to be flipped around, or reduced. However, it has been pointed out that chimpanzee LRRTM1 is 100% identical to human LRRTM1, yet no one has found a left-handed chimp, or an articulate one for that matter. Handedness and subtle brain asymmetry, as found in humans, are the result of much more than just one protein – and environmental factors are undoubtedly of great influence too.

With a role in brain development and possible neuronal connectivity, it is hardly surprising that LRRTM1 has been linked to neuronal diseases such as schizophrenia, autism and language impairment. Likewise, a logical step was to wonder whether left-handedness could not be taken as an indication to a predisposition for neuropsychiatric disorders. It so happens that in one study carried out on schizophrenic individuals many were left-handed. This kind of result has to be taken with caution though. It does not mean that every left-handed individual is prone to some form of psychosis. Many right-handers suffer from psychiatric impairment too. However, it does suggest that genetic components involved in the structure of our brain may be indicative of a predisposition to a neuronal illness, given the environment. Surprisingly, other studies have shown that left-handers are more prone to accidents than right-handers. No clear explanation has yet been given but it may just be because our society is really built for right-handers.

LRRTM1 is predicted to link to another protein – or proteins – where the bond would supposedly trigger off a reaction. With this in mind, if it can be shown that LRRTM1 does have a role in the development of neuropsychiatric diseases, it may well prove to be precious in the design of novel therapies to lessen such disorders. Once again though, no protein acts on its own. There are genes upstream and downstream of LRRTM1 involved in its expression. Furthermore, an individual’s environment is hugely important in triggering off a psychiatric disorder: drugs, alcohol, abuse, violence, stress etc. And, besides psychiatric disorders, what to think of someone who writes with their left hand and throws with their right? What is their brain structure? Is LRRTM1 also part of semi left-handedness? It is all very mysterious. But the fascinating part of the story is to realise that were it not for our words, we would not be able to carry out nearly as much as we do with our hands.

Why make a meal out of your kin when you have the option of surviving – albeit in a slow-motion form – as a spore? Sporulation occurs when the environment does not provide sufficient nutrients for micro-organisms such as Bacillus subtilis, for example, to survive. Though the aim of sporulation is to prevent cell death by enclosing it in a micro-environment in which the minimum is provided for a creature living at a minimum pace, the process is extremely energy-consuming. Sporulation calls on the activity of over five hundred genes, involved in complex molecular pathways. Once the first steps have been completed, the morphogenesis of sporulation is irreversible. When the environment becomes plentiful again, unraveling the doings of sporulation is just as energy-consuming before B.subtilis can settle down and feed. So if sporulation can be sidestepped, it is.

One way of doing it is to make sure B.subtilis has enough to live on. If it doesn’t, the next step is to delay sporulation as long as possible, just in case the conditions get better. Indeed, a non-sporulated bacterium will profit from bountiful surroundings much faster than a sporulated one. As a consequence, non-sporulated B.subtilis are far more favoured. In stark conditions, one gruesome way of putting off sporulation is by eating those closest to you. Though unicellular, B.subtilis is relatively sociable and frequently evolves in small colonies. When conditions become life-threatening, a number of the cells in the colony are thrust into the process of sporulation while the fate of the others – over two thirds of the whole colony! – is to be lysed and digested.

© Michael Leunig

Used with permission

Two proteins directly involved in this peculiar homicide are the sporulation killing factor, SkfA, and the sporulation delaying protein, SpdC. Cells which are not directed towards sporulation and whose fate is to become bait, produce neither SkfA nor SpdC. Both toxins are produced when B.subtilis enters the first steps of sporulation – steps which are not yet irreversible. Very little is known about the two proteins and their mode of action, besides the fact that without them, there would be no cell lysis, and hence no food supply.

Both proteins are toxic and secreted into the environment. SkfA is secreted by way of a pump which also only appears in sporulating cells. Being specifically pumped out of a cell may be one way of shunning suicide. Indeed, if SkfA can kill off identical cells, it can also kill the one which made it. It’s like turning your own weapon onto yourself. SpdC is also secreted. It is still unknown how but more importantly its existence seems to stimulate the production of an immunity protein known as SpdI which protects the sporulating cell from doing itself any damage.

How SkfA and SpdC lyse the sister cells remains a mystery. It may be that they are merely intermediate and that it is another molecule that is actually responsible for lysis. Perhaps they disrupt the cell membrane some way or another, either by lodging themselves inside it and causing molecular chaos. SpdC acts as a ligand and the attacked cell may have an SpdC receptor on its membrane. SpdI – which is only stimulated in sporulating cells – may act as an immunity molecule, either by binding directly to SpdC in the sporulating cell so that it doesn’t get the chance to bind to its receptor, or by binding to the SpdC receptor so that there’s no room for SpdC. Either way, the sporulating cell is saved from self-attack.

There are many unknowns in this peculiar bacterial cannibalism. Since B.subtilis assembles in colonies, the process can be seen as a form of programmed cell death where – in close proximity – a number of cells are sentenced to death in order to save others. It is also a singular case of chemical warfare – a very well documented event amongst distinct bacterial populations. In fact, SpdC and SkfA may enjoy a far broader toxicity than their own species. Especially if they can disrupt a cell just by squeezing into its membrane. Evidently, more time is needed to unravel the mystery of B.subtilis vs B.subtilis. And why see it as a way of avoiding sporulation? Perhaps feeding on sister cells is less to delay the process than simply to make sure that sporulation is completed – since it takes up so much energy. You wouldn’t want to find yourself half-sporulated with no nourishment to finish off the job… In which case, is it not more a case of altruistic martyrdom than cannibalism?

Keeping our balance is not such a simple task. You may think that it’s all a question of a little concentration and crafty positioning of the body. But it’s not. There is more to it than that. We have built-in devices which we need not only to keep an upright posture but also to sense movement. These devices are known as otoconia and are located in the darkest recesses of our inner ear. Otoconia are a mixture of organic and inorganic material. In humans, their size ranges from 3 to 30μm and they are bathed in an extracellular gelatinous matrix. The otoconia themselves have an organic protein core cloaked with calcium carbonate (CaCO3) crystals – a bit like the sweets our grandmothers gave us, which were hard on the outside and filled with a soft centre.

Otoconia synthesis is completed shortly after birth. Caught in an extracellular gelatinous matrix, itself packed full of protein, this peculiar biomineral cum protein mass forms a jelly-like membrane which rests on tiny hairs. These hairs are embedded in two small organs of the inner ear, known as the utricle and the saccule. Any movement of our head – with respect to acceleration or gravity – makes the otoconia move. And since they are resting on the hairs, their movement will, in turn, cause the hairs to move. The perception is then relayed to nerves which will make a point of telling the brain. The utricle is largely horizontal and will register any movement in the horizontal plane, such as acceleration. The saccule, on the other hand, lies in a vertical plane and registers movement due to the pull of gravity.

Ocean perch (Sebastes mentella) otoliths

Source: Wikipedia

Otoconia are formed during embryogenesis and their final design is completed shortly after birth. Otoconia seedlings are probably secreted into the extracellular matrix, and are most likely to involve a core protein matrix which is calcium-friendly. Calcium and carbonate concentration must also be heightened in the seedlings’ vicinity, so that the calcium carbonate crystal envelope can be shaped. Otopetrin 1 is part of the gelatinous extracellular matrix in which the otoconia float. When otopetrin 1 is deficient, the formation of otoconia is impaired. As a result, the perception of acceleration and gravity is confused, and mice carry tilted heads or zebrafish swim upside down.

Otopetrin 1 has all the characteristics of an integral membrane protein, yet it is found in the extracellular gelatinous matrix. This has led scientists to believe that the protein is most probably imbedded in the membranes of vesicles which bud off epithelium cells. In zebrafish, otopetrin 1 seems to be involved in protein transport, and in particular the trafficking and localization of a protein known as Starmaker. Starmaker is proving to be essential both for the fashioning of the fish biominerals – known as otoliths – and the crystal lattice on which they grow. Otoliths are of the same nature as otoconia, only far bigger. While we own many otoconia, fish only have three. But they are large, mainly because the deposit of calcium carbonate only stops when a fish doesn’t need a sense of balance anymore. Though a role in protein trafficking has not yet been witnessed in humans, otopetrin 1 does seem to be involved in calcium regulation in response to ATP. However, otopetrin 1 itself doesn’t sport domains that, until now, have been known to interact with ATP or calcium. Consequently, either otopetrin 1 activates proteins that do, or its sequence contains atypical domains that can! Whatever the outcome, otopetrin 1 is certainly essential for the formation of calcium carbonate crystals, and hence otoconia.

Otoconia – or the lack of them - can be the source of trouble. The passing of time can cause the structures to degenerate or change position. The sensation perceived is known as benign paroxysmal positional vertigo (BPPV) and, though harmless, those concerned suffer from dizziness or loss of balance. The elderly are prone to falling which frequently results in broken bones or even, sometimes, accidental death. Certain drugs can also be the cause of otoconia degeneration and patients suffer from the same disorders. Research on otoconia and otholiths is now focused on finding therapies which could improve their stability over time as well as enhance the biomineralization of remaining otoconia. Otoconia regeneration is also being considered. Furthermore, getting to know the mechanisms involved in otoconia synthesis could lead to a better understanding of how bone (CaCO4) is made – and without bone or otoconia, how could we fully enjoy a balanced life?

On the occasion of Amos Bairoch’s 50th Birthday

Life has its ways. We are given opportunities to make choices. We are even given opportunities to nudge life onto a path we wish. And yet, there seems to be an invisible force lurking beneath which leads you to the most unexpected places…an unexpected place which, in time, turns out to be the place where you should be. Call it destiny, perhaps. Today, fifty years after the day he was born, Amos is sitting in an office in Geneva at the head of a project which has travelled around the world and for which many people work. From a cramped attic to a large open space office, Swiss-Prot continues to grow both in work force and in use. Amos has won prizes for it. He has been praised for it. He has put much of his soul and his heart into it. And despite this, far from him was the desire of ever having really wanted it.

New path, Meg Harper

Courtesy of the artist

The story has been told many a time. Over twenty years ago, while working on his thesis, Amos felt the need to sort and perfect a protein sequence databank which already existed. And he did. Instead of listing the sequences of proteins, he felt it was of greater benefit to the scientific community to offer some information on the protein as well – such as its structure, its function and its possible involvement in illnesses. Having done so, he offered to those concerned what he saw as improvements made to the existing databank. No particular enthusiasm was shown for his effort, so Amos undertook to develop his concept, fully expecting to hand it over to someone else so he could get on with things he was more interested in, such as exobiology.

But that is not the way the wheel turned. Protein sequences kept on pouring in and, though the rate of submission was far slower than it is today, Amos took it on himself to maintain the rhythm. As a result, he continued not only to enter them manually into his embryo-databank but also to annotate them. There was little point in dropping something that was proving to be useful. That was the beginning of the…beginning. It was not long before he needed help to cope with the profusion of incoming data. And ever since, the number of scientists who strive to keep pace with automated sequencing and a competitive knowledgebase, has never ceased to increase. Today, Amos is surrounded by computer scientists, biologists of all walks of life, secretaries, receptionists, science writers and science communicators. And a lot of us are sitting in a big modern office in the basement of Geneva’s academic medical centre where it all started, whilst others are scattered in other parts of the world.

Hundreds of fingers boogie – or mooch – across keyboards every day, for the sake of proteins. Dozens of pairs of eyes scan computer screens every day, for the sake of proteins. Millions of neurons spark – or indeed fail – every day, for the sake of proteins. Numerous cups of coffee are absorbed, thousands of words are exchanged, hordes of paper are printed, hundreds of footsteps are made, hearts beat, lungs breath, sighs are lost and a few aspirin are swallowed – every day, and all for the sake of proteins.

And, in the hands of experts, these singular molecules of life are given a name, brought down to a sequence of letters, sorted into families, sculpted into space and predicted a role. They are fashioned into ribbons, moulded into globes and painted in the colours of a rainbow. They are explained by professors, sought after by researchers and discovered by students. Pharmaceutical companies commercialise them. Journalists write about them. Artists weave them, sculpt them and paint them. All of us, without exception, eat them. And, what is more, we make them.

Without proteins, life would not exist. And neither would any of us. Without proteins, Swiss-Prot would never have seen the light of day. Neither would Amos for that matter. 50 years ago and a little more, proteins were hard at work in that little spermatozoon which wriggled towards its mate. When it reached its other half, dozens of proteins made quite sure that no other would get a chance. A cell was born. The first. The very beginning of a human being. And from there, with the help of billions of proteins, millions and millions of divisions occurred. Proteins supplied energy and ferried chemicals, they triggered off pathways and monitored rates, transcribed DNA and translated RNA, built networks and provided heat. They let a heart beat, ears hear, eyes see, legs walk and they designed the mould from which a mind could grow. Undoubtedly, there is not much we would be without these remarkable molecules. And destiny? Could destiny also be in the hands of proteins? Hardly. But they certainly do have their say. ]]> Swiss-Prot cross references

The expression of a gene is dependent on so many different instances, from the presence of a certain ion to a specific moment in development. However, until recently, it had always been thought that transcription factors had the last word. They were the ones who decided whether a gene would be transcribed and ultimately translated. But it is not so. Tiny strands of RNA – known as microRNAs or miRNA – are beginning to raise their voices and show that they are just as good as any transcription factor. The concept is mind-blowingly simple: miRNAs recognise their complementary sequence encoded in a specific mRNA and bind to it. In so doing, they interfere with the proper translation of the mRNA sequence and no protein product is synthesized. It’s a bit like immobilising a locomotive on a railway, thereby hindering the passage of a train.

The knowledge that certain non-coding RNA molecules – the miRNAs – were actually part of protein expression regulation arose in the early 1990s whilst working on Caenorhabditis elegans. Since then, many different miRNAs have been discovered in animals and plants – and the viruses that infect them – and it is becoming obvious that their role in protein expression regulation is important. It is thought that the human genome may well house up to 1000 miRNA genes, which could regulate as many as one third of our protein-coding genes…which is not trivial.

3D structure of Dicer

Source: Wikipedia

Lending the silencing of genes to the sole existence of miRNA is, however, a little short-sighted. In truth, they need a battery of enzymes to meet their ends. Drosha and Dicer are two such enzymes, whose major role is to cleave RNA. The making of mature miRNA – which is the RNA strand that actually recognises its target – is part of a fascinating process. Drosha, like Dicer, carry two domains in their sequence known as ribonuclease III (or RIII) domains. Thanks to protein sequence folding, these two domains meet to form an intramolecular dimer which – in both cases – is the catalytic site for RNA processing.

Drosha is found in the cell’s nucleus and specifically recognises primary miRNA (pri-miRNA). Drosha – like Dicer – can only recognise double-stranded RNA so, conveniently, pri-miRNA folds into a hairpin conformation thus producing one double-stranded end. The two RIII domains form a cleft into which the RNA hairpin can lodge. Drosha then cleaves one end to produce what is known as pre-miRNA. Pre-miRNA is then shuttled out of the nucleus where it encounters Dicer. Dicer prepares the other end of the hairpin in much the same way as Drosha and releases the mature miRNA which is then ready to recognise its mRNA target and act upon it.

Plant miRNAs are specific: their sequences match their complementary RNA targets with great precision. Animal miRNAs are far more promiscuous. The match is far less demanding and, as a result, one miRNA will bind to as many as one hundred different target sequences. From Nature’s point of view, it may sound economical but it just makes things difficult in the laboratory. It is a relatively easy task to identify a specific miRNA within a specific pathway if the said miRNA only has one target. However, in reality, one particular miRNA regulates many different genes and is thus most probably involved in as many cellular pathways.

The fact that animal miRNAs are so dissolute makes it difficult to use them as diagnostic markers for disease or for exploiting them for therapeutic benefit. Nevertheless, much research is being carried out in this direction to make some sense out of the miRNA jungle. Since miRNA silencing in plants is far more straightforward, a number of applications have already been made. The very first was with on tomatoes where an engineered miRNA was used to prevent the expression of an enzyme which participated in the softening of the cell walls once ripe. Ripe tomatoes could then be harvested without falling apart. Such practises cannot be widely used though because of our whims of genetically modified crops.

An obvious question is why do miRNAs exist? Do they provide some special regulatory property that transcription factors do not? Is the silence Drosha and Dicer create of a different nature to that of the well known transcription factors? Time will tell. Nature continues to show that she has many facets and those she nurtures are usually there for a reason…

Antlers are not an uncommon sight these days. If you are lucky enough to live on the outskirts of a forest, there is a great chance that you will spy an antler or two, usually at dusk. Antlers are made out of bone. They grow from pedicles that form at puberty and which, in time, become permanent protuberances from where antlers bud and are cast seasonally. They can grow at the amazing rate of two centimetres a day and represent the only example of both irrigated and innervated cartilage in the animal kingdom.

Once antlers are cast, the next generation initiates immediately thanks to resident stem cells on the permanent pedicles. These cells differentiate first into chondroblasts and then into chondrocytes, which are associated with the formation of cartilage. Mineral deposition then occurs on the cartilage scaffolding, and bone is formed. This stage gradually gets rid of any blood supply which is made to the antlers. As a consequence, they stop growing and their metamorphosis to bone is completed. The final touch comes with the shedding of a thin film of velvet skin that coats the appendages, and the antlers are then ready for the rutting season.

Red Stag, from an original painting by Robert E. Fuller

Courtesy of the artist

Naturally, the regeneration of an organ demands the existence of a complex network of proteins. Annexin 2 is just one of these proteins but an essential one, since it seems to be directly involved in bone formation. More specifically, annexin 2 seems to be involved in the formation of calcium channels and mineralisation in the environment of chondrocytes and osteoblasts. Annexin 2 is also involved in many other mechanisms and it is now becoming obvious that it is a multifunctional protein. It assembles into a heterotetramer and belongs to the very large annexin family which is characterised by the existence, in their sequence, of an annexin domain – a 70 amino acid domain – of which each type of annexin has a defined number. This particular domain binds calcium – a feature characteristic to all annexins.

Annexins are distributed both in the animal and the plant kingdom; from humans to guinea pigs, frogs, flies, zebra fish, worms, moulds and mouse-ear cress. They are found in many different tissues and participate in a variety of physiological processes. All are calcium-dependent and bind to phospholipids, and are usually found in the periphery of the plasma membrane, either intracellular or extracellular. One form even seems to be not only extracellular but also soluble. This was a surprising discovery because annexins do not have a signal peptide, so they must follow a route other than the customary endoplasmic reticulum secretory pathway prior to secretion.

Besides antler formation, annexin 2 is involved in a host of other processes such as fibrinolysis, cell proliferation and differentiation as in bone formation, endo- and exocytosis, cell migration, cell shape and even immunity. As a consequence, it is expressed in many different tissue types such as the central nervous system, the cardiovascular system, bone marrow and the small intestine. In fact, annexin 2's tissue and function versatility is at the origin of an equally versatile nomenclature – as is frequently the case. And, with the years, it has been given a variety of names such as p39, calpactin I heavy chain, protein I, chromobindin 8 and lipocortin 2…

When a protein is multifunctional in this way, there is a big chance that it is involved in just as many diseases. So far, annexin 2 is known to be over-expressed in patients suffering from acute promyelocytic leukemia, which results in excessive fibrinolysis. On the brighter side of things, annexin 2’s involvement in fibrinolysis could be promising for the design of anti-coagulant drugs for those suffering from cardiovascular complications. Furthermore, though it is not clear why, annexin 2 also seems to have a suppressive effect on tumour malignancy – which is an excellent reason to get to know it better, although it is always a delicate thing to associate any protein with an anti-tumour effect when the said protein is blessed with so many functions.

Movement or not, some people seem to stay lean whatever their calorie intake. This could be because they are the carriers of a ‘fidget’ gene, which makes them shuffle and shift more than necessary – as far as energy balance goes. Bsx – or Brain specific homeobox – is the fidget gene. Mice that carry the wild type version were recently shown to be ten times more active than those who carry the mutated form. As a consequence, in an environment where food is plentiful, the laid back mice will tend to stock fat whereas their more active counterparts will keep trim. So far, nothing new. We are all aware that a lack of exercise is not ever going to help you lose weight. But if you take it one step further, what would be the point of a protein that is there to make you move with regard to energy homeostasis?

Such proteins are likely to be the product of a proposed complex gene network, known as the thrift gene network. The thrift gene theory made its first appearance in the 1960s. It hypothesizes that animals carry a set of genes where the act of overfeeding coincides with food abundance, and stocks are made with the future prospect of famine. In our day and age though, there is so little famine in some parts of the world that, in many instances, there is a lot of overeating. Subsequently, many people are overweight. The thrift gene theory is an elegant attempt to explain obesity which is spreading worldwide. However, like all theories, it has met with some distaste and a number of scientists have suggested that being overweight is less a question of fat collected in the event of future starvation, than humans who are less faced – if not at all – with problems linked to predation, which are not only the source of stress but also get you moving.

Old Woman Eating Peas, Jeannette Langmead

Courtesy of the author

It remains that Bsx certainly seems to be a protein involved both in locomotion and food intake. It is one of over a hundred homeoproteins isolated in mice. They are transcription factors and Bsx, in particular, is largely expressed in the hypothalamus, the part of the brain involved in many important activities, including appetite, or the lack of it. Furthermore, Bsx not only seems to be important for the correct development of the embryonic brain as well as its adult function, but is also involved in the formation and maintenance of the mammary glands.

The hypothalamus organises two major networks of neurons, one of which encloses the NPY/AgRP hypothalamic neurons which regulate feeding behaviour and body weight. Bsx is not only expressed in NPY/AgRP neurons but is also required for their expression. As a consequence, Bsx not only affects the hypothalamus itself but also distant target organs – such as the mammary glands, for instance. So far, Bsx has been found in vertebrates as diverse as humans, mice, chicken, zebrafish and frogs. Hence, it is a little surprising to discover that Bsx has a role in mammary gland formation. This implies that, over time, the protein has acquired an additional function in mammals. A function still linked to food, nevertheless…

Involved in the molecular regulation of not only feeding but also locomotion, Bsx protein proves to be at the heart of an exceptional behavioural response: one related to food and movement. Without it, calorie intake is upset, leading to weight problems. This could prove to be a drug target for future therapies that would help people who suffer from obesity, for example, which is the source of health problems that kill millions of people every year. Once again, Bsx cannot be held as the sole culprit. Weight imbalances are always due to labyrinthine molecular networks and, obviously, the environment plays a role too. Nevertheless, Bsx could prove to be a novel and precious drug target to help people shed fat.

Cover illustration for "journey into a tiny world"

© Vivienne Baillie Gerritsen, Sylvie Déthiollaz

Although this article celebrates the publication of the English version of “journey into a tiny world”, the original version – Globine et Poïétine sur la piste de la moelle rouge – was written in French five years ago. Once the idea of a story had been agreed upon, the next step was to decide what we were going to write about, and then write it. The tale had to involve proteins; that was our only dictum. The rest was up to us.

The idea of a journey into the recesses of a little girl’s body arose quite naturally. However, there had to be a reason for such a journey. One that made sense. It wasn’t long before we imagined that the little girl had to take medicine – a protein – that would lose its way inside her and, in so doing, get the chance to visit numerous parts of her body as well as meet other proteins. Our hope was that children would discover that an organism is full of different kinds of protein, which each have a specific job to do. Sylvie had just finished an article on EPO (erythropoietin), a protein which has the capacity to stimulate the production of red blood cells in the bone marrow. This became our starting point.

Little did we realise though that the choice of something so specific would put certain restrictions on our imagination… For one, EPO could not be swallowed but only taken by injection. Secondly, several weeks into the creation of our tale, Sylvie announced that the little girl was far more seriously ill than we had imagined! It didn’t change the course of our story though… And Sylvie and I moulded our characters, weaving them in and out of situations according to our fancy. It was a delightful experience.

You may wonder how two people can write together. There is no rule to how this can be done. But as far as the making of this tale goes, I wrote from my kitchen, while Sylvie wrote from the office. We wrote up the different chapters independently and then compared them. To our constant astonishment, our minds glided along the same paths and most of the time we were thinking along the same lines, which made things relatively easy. Sylvie then sifted and sorted the ideas and adventures of our molecules, keeping the best parts and discarding the poor ones. As a result, within a couple of months, we managed to produce the French version of the tale.

“Globine et Poïétine sur la piste de le moelle rouge” was the title. It is the story of Poietin who careers around a little girl’s body – Lily – with Globin, a friend she picks up on the way. Together, they experience great adventures discovering what it is to look through an eye, to sit on the edge of a wound or to watch a heart beat, while meeting other proteins like Actin, Myosin, Orexin or Insulin. Following a sequence of exploits, Poietin finally finds the bone marrow she needs, from where she will be able to stimulate blood cell production and save Lily’s life.

During the science fair, a modest set with two red armchairs, a carpet and a white canvas mounted on an easel – on which we projected the illustrations which are now part of the published book – was arranged in a far corner of one of the villa’s drawing rooms. We would have liked a local artist to do the illustrations for us. When we were told how much it would cost, it became obvious that we would have to rely on our own artistic talents. So Sylvie and I grabbed a pencil and a paintbrush and produced our interpretations of the various characters you can find strewn through the book. Despite a solid background in biology and, consequently, a sound understanding that proteins have no eyes, mouths, arms or feet, we decided that if we wanted to give them some kind of a literary life, this was one of the ways to go about it.

The tale was read several times over a weekend by two actors who managed to infuse life, beauty and magic into the story. And we will always remember the little girl who stayed to listen to more than one reading and who, by the end of the day, had learned some of the lines off by heart. Meanwhile, she had managed to drive her mother – whose only wish was to head back home – round the bend. The whole thing turned out to be a success, and a number of parents asked us whether we were thinking of making a book out of Globin’s and Poietin’s adventures. We thought it was a good idea, and the book was published in French in July 2003. And four years later, the time and energy was found to translate the tale into English.

Alois Alzheimer described the clinical and neuropathological traits of Alzheimer’s disease in November 1906, at the 37th meeting of the Society of Southwest German Psychiatrists. Five years earlier, Alzheimer had admitted a middle-aged woman into the Frankfurt hospital, who was suffering from memory loss, focal symptoms, delusions and hallucinations. She died in April 1906 and Alzheimer sent her brain to Munich where the novel technique of silver-staining highlighted the accumulation of protein plaques and tangles. Such deposits are still used today to define the neuropathological characteristics of the disease – although they are now known not to be particular to AD.

The accumulation of neuritic plaques and neurofibrillary tangles in our brain is a natural consequence of the passing of the years. However, in most cases, their accumulation is minimal and does not hinder normal neuronal function. The proteins at the heart of these plaques and tangles are amyloid-β and tau, respectively. In healthy organisms, both are soluble proteins. In hereditary AD, genes that are directly involved in the formation of these plaques and tangles are passed down through generations. Though sporadic AD – the form of AD that hits the great majority of those afflicted – presents the same neurophysiological traits, it is still not known what causes them. Why do some develop AD while others do not? ApoE could be an answer. Though just a whispered one.

Self-portraits (1996-2000) by artist William Utermohlen, suffering from Alzheimer's disease

Galerie Beckel-Odille-Boicos, Paris

Accompanied by hordes of other apolipoproteins, ApoE’s first function is to transport lipids, usually by cell-mediated endocytosis via an apoE receptor – of which there are many kinds. ApoE is thought to have variable structural conformations, depending not only on the size and shape of the lipids it binds but also on its receptor. The N-terminus docks to the apoE receptor while the C-terminus binds the lipid. Once a lipid is bound, the initial loose 3D structure of apoE is believed to adopt a hairpin conformation and then a belt-like configuration around a discoidal bilayer of phospholipids. Besides lipid transport, apoE seems to be involved in a number of other processes such as apoE fragmentation into toxic products, membrane disruption, neuronal sensitivity to injury and recovery. It is further known to have antioxidant properties and is associated with amyloid β-peptide and plaque formation.

All these mechanisms are the fruit of three common apoE isoforms: apoE2, apoE3 and apoE4. And where multiple functions are involved, rests a hive for multiple diseases. Indeed, together, the apoE isoforms affect the clinical outcome of a host of cardiovascular, neurodegenerative, and viral infectious diseases, including atherosclerosis, Alzheimer’s disease, hepatitis C and HIV. Each isoform structure behaves differently. For instance, apoE4 forms partially unfolded structures far more readily than its peers. Such a conformation not only facilitates lipid-binding but also makes apoE4 more sensitive to proteolysis. What is more, apoE4 is the apoE isoform which – when present – is proving to be an indicator of AD, or a sign of AD predisposition. How apoE4 is involved in AD remains a mystery but this particular allele does seem to have a role in neuronal plasticity and arborisation, as well as amyloid-β accumulation – traits which have a direct effect on the process of memory for instance.

No one gene will prove to be the definite Alzheimer gene. Besides the simple passing of time, AD is the result of a variety of genetic and environmental instances. To date, existing therapies relieve symptoms such as depression or insomnia, but there is still nothing that can halt – or even slow down – the process of neuron loss. Researchers seem to have put their finger on a probable genetic indicator – apoE4 – although almost half of the AD population does not carry the epoE4 allele… Concentrating their efforts on apoE4’s structure and biochemistry will surely lead to a greater understanding of its function, and in so doing perhaps give a way of challenging Alzheimer’s ugly face. Such an ugly face, that James Watson – the American component of the discovery of the helical structure of DNA – who has had his genome sequenced, announced that he didn’t wish to know whether he was the owner of the apoE4 isoform or not.

Over time and as a means of defense, Nature has devised the most diverse ways of hurting. Snakes spit venom. Nettles sting. Bacteria puncture. And dogs bite. However, deprived of the resources to sense pain caused by venom, or a nettle’s sting or a dog’s fangs, we wouldn’t understand the warning that goes with it. Likewise, pain which is caused by something inside us has to be detected so that our attention can be drawn to it. To this end, pain receptors line our body’s every nook and cranny, ready to send out a signal which will be relayed to our brain and translated into pain.

The Transient Receptor Potential (TRP) channels – or receptors – are pain receptors. Pain receptors are an essential part of the process which leads to the actual sensation of pain. The signal triggered off by a TRP receptor is sensed by nerve fibres which release neuropeptides that, in turn, inform the brain both of pain and inflammation. TRPA1 is one such receptor, known to be directly stimulated by the pungent components of mustard oil and garlic, which cause the familiar burning and pricking sensation we have all experienced. Besides mustard and garlic though, TRPA1 is also stimulated – though not directly – by other substances such as volatile irritants found in vehicle exhaust, tobacco products and tear gas, or components unleashed during chemotherapy treatment, or even environmental stimuli such as heat, cold. Why can TRPA1 relay so many signals of discomfort, while other TRP receptors deal with only one at a time?

"Hellbound", Matt Sesow

http://www.sesow.com

Like all TRP receptors, TRPA1 is membrane-bound and most likely acts as a heterodimeric voltage-gated channel. TRPA1 has a particular secondary structure: its N-terminus is lined with a large number of ankyrin repeats which are believed to form a spring-like edifice. Most receptors have intricate pockets which are specific to a certain kind of ligand, and the slightest alteration of either the pocket or the ligand has drastic effects. Since TRPA1 can respond to a variety of stimuli, it must have another system. Indeed, instead of presenting a pocket into which a ligand can lodge, the TRPA1 receptor forms covalently linked adducts with electrophilic compounds. The difference with other ‘pocket-binding’ TRP receptors is that TRPA1 ligand-binding persists for hours. The physiological response – i.e. pain in this instance – is greatly prolonged because the electrophile cannot readily dissociate from its receptor. Consequently, the receptor remains activated.

TRPA1 reacts to a variety of compounds, and does so in a variety of ways. It is directly stimulated by isothiocyanate and thiosulfinate compounds which give mustard oil and garlic their specific pungent qualities. This was discovered when mice, which didn’t carry the receptor, turned out to be insensitive to both. Volatile irritants such as those found in vehicle exhaust, tear gas and tobacco smoke, as well as heat and cold stimulate TRPA1 indirectly. How? TRPA1 is part of a sensory pathway – or a number of sensory pathways – and is most likely activated or even modulated downstream of other neurotransmitter or growth-factor receptors. Funnily enough, although TRPA1 is not specific to only one component, it is surprisingly fine-tuned. As an example, the receptor is stimulated by acrolein – a compound in tear gas – yet it is insensitive to acrolein’s corresponding saturated aldehyde: propanal.

Historically, mustard oil has been used extensively as a paradigm to study the mechanisms underlying inflammatory pain. Thanks to mustard oil, and a number of other substances, it has now been demonstrated that TRPA1 is capable of translating diverse signals of hostility into a singular sensation, i.e. pain. TRPA1 does seem to be on the crossroads of a number of sensory pathways. Consequently, it could prove to be an exceptional therapeutic target and may help to find ways of relieving those that suffer from chronic pain, or the secondary effects of chemotherapy and medication used in the treatment of arthritis for example. Likewise, engineering TRPA1 could help to counter the effects of noxious compounds in airways that induce not only inflammation but also chronic cough or asthma, which are a discomfort to many. Interestingly though, besides TRPA1’s multiple talents, one laboratory has shown that sexual dimorphism can be an essential factor in the modulation of pain, and how it is sensed. Yet another difference between man and woman.

The Dutch merchant Anton van Leeuwenhoek was the first to observe bacteria in the 17th century, under a microscope of his own design. He called the creatures he described, animalcules. Over 100 years later, the German naturalist Gottfried Ehrenberg renamed them ‘bacteria’ from the Greek ‘bacterion’ meaning ‘small staff’. It took a further century before the renowned French microbiologist Louis Pasteur and his contemporary, Robert Koch, demonstrated that although bacteria were essential in a number of vital daily processes, they could also be the carriers of fatal diseases.

The tiniest drop of water shelters over a million bacterial cells. A teaspoon of soil can be a home to 40 billion. Needless to say, there are a lot of bacteria around. They were amongst the first organisms to populate our planet, and have never stopped multiplying and evolving since. They live in earth’s every nook and cranny, in the deepest depths of oceans, and at temperatures none of us could stand. Minuscule though they may be, many of them carry out tasks we couldn’t do without, while others can ground us and leave us at death’s door.

Thousands of Myxococcus xanthus cells amassed into a fruiting body with spores, ready to glide...

Photo by Michel Vos, 2005

Source: Wikipedia

Bacteria have to move in all kinds of media, and they have thought up as many ways as they need to do so. With time, they have devised two ways of gliding across hard substrates: either on their own or with one – or more – of their kind. The two mechanisms have been named adventurous gliding motility and social gliding motility, respectively. Bacteria can use either mechanism, or indeed both. Myxococcus xanthus uses the two means of locomotion, and is the bacterium in which aglZ was identified. Researchers had been observing Myxococcus xanthus for years; when it wasn’t hitching a ride, it seemed to be moving on its own within a film of slime. Consequently, many believed that there was something in the slime that helped the micro-organism move forward. What they could not see were the tiny clumps that riddled the creature’s “belly” along its length, at regular intervals. These clumps were finally observed thanks to the use of fluorescence techniques, and aglZ turned out to be a major protein in their formation.

AglZ is a coiled-coil protein, reminiscent of myosin. In its absence, Myxococcus xanthus cannot adhere to substrate and, as a result, cannot move on its own. AglZ is part of a Lilliputian anchor that spans the bacterium’s membranes, and forms a gangway between its cytoskeleton and the surface along which it is gliding. If you could have a look at the bacterium’s underside, you would see a neat row of these anchors. While, to the naked eye, they appear to be immobile with regard to the surface, they are in fact moving along a helical runway in the opposite direction to that of the bacterium. This runway is in fact part of the organism’s cytoskeleton and its helical structure – combined with the ‘walking’ anchor – causes the bacterium to spin around its long axis. The whole mechanism seems very close to the myosin/actin motor system used in muscle contraction, for instance.

Because they are so ubiquitous, nowadays almost any discovery regarding bacteria is significant one way or another. Bacteria are used in the preparation of fermented foods and beverages such as cheese, bread, pickles, yoghurt, wine and beer, not to mention waste processing and bioremediation. They are used to replace pesticides and produce chemicals that are of pharmaceutical or agrichemical importance. Bio-engineered bacteria know how to synthesize therapeutic proteins, such as insulin, growth factors and antibodies, which have made a huge difference in our society in the past years. In short, without bacteria, research in molecular biology, genetics and biochemistry would not have taken such great leaps forward in the past decades. Yes, but their means of locomotion you’re thinking… The way – and the speed at which – bacteria move is paramount since their virulence depends on it. Consequently, aglZ is a great candidate to tinker when designing novel therapies which could counter adventurous gliding. And what better way to stop the troops from advancing than to tamper with their means of locomotion?

How an embryo becomes male or female has puzzled many a human for thousands of years. While menstrual blood was believed to be the material with which semen moulded the beginnings of a foetus, it was thought that heat favoured the development of little boys and cold shaped an embryo into a little girl. Semen that came from the right testicle gave boys; the left testicle harboured the wherewithal to make girls. Likewise, embryos that were positioned to the right of the womb became male, and those to the left, female. Once the notion of two germ cells and their meeting was acknowledged, the Ovists believed that a child was held within an egg and just needed sperm to trigger off its development, while the Spermists speculated that it was sperm that sheltered the embryo, and the egg was there to feed it. As time passed and techniques improved, scientists discovered not only chromosomes but also the fact that specific chromosomes defined sex. The X chromosome was the first to be discovered because of its size. Y was discovered shortly after.

The fate of the Y chromosome is bewildering. It all started 300 million years ago when Nature thought up sexual reproduction. To reproduce in this way, you need at least two entities of an opposite sex. It was a great way to promote biological diversity and a basis on which natural selection could work, but it meant compromising on one chromosome, i.e. the Y chromosome as we know it today. 300 million years ago, a large portion of an X chromosome was inverted. As a consequence, it was unable to pair with its sister chromosome. Left to fend for itself, it fast became prey to mutations and over the years it has lost hundreds of genes, gained a lot of rubbish and become very small. So much so that some scientists think that the Y chromosome may just wither away…and hence the male of the species? ‘Nonsense!’ say fellow researchers, other animals have lost their Y chromosome already and have simply found another way of promoting the male program. There is no reason why Homo sapiens wouldn’t do the same, and some of the future mutations on Y may even support its survival.

‘Seated nude male’, Pablo Picasso (1909)

© 2007, ProLitteris, Zurich

So as long as XY embryos carry Sry, their destiny is a male one. What does Sry do? It triggers off pathways that promote the development of masculine features which depend upon two types of cell: the Sertoli and the Leydig cells. Sertoli cells give rise to the testes and Leydig cells will ensure the production of androgens - the male hormones. How does Sry do this? Sry is a transcriptional factor and binds to the minor grooves of DNA. There – by way of bending the nucleotide structure at angles of 60 to 80° – it either activates or represses the transcription of genes. To date, no one is sure whether Sry activates ‘male’ genes or whether it represses ‘female’ genes – though there is growing evidence for the former. Naturally, Sry does not act on its own; without the help of molecules both upstream and downstream of Sry expression, there would be no baby boy at all – or one whose male features are hugely hindered.

Up until the 7th week of pregnancy, a human embryo’s phenotype is neither male nor female. If the embryo is XY, Sry is expressed – though only for a short time. In mice, for instance, Sry is active from the 10th day of development until the 12th day. And if it misses the bus, the embryo is heading for serious trouble. In fact, problems related to a person’s sex can be the cause of a dysfunctional Sry, either because it has not been expressed, because it is inactive or because it wasn’t expressed on time. Besides its role in sex differentiation, Sry is also found in male brains. Could it be involved in what could be described as ‘male’ behaviour? Possibly. It seems to have a role in the production of dopamine which is involved in motor control. The obvious question then is: are women handicapped in this respect? Of course not. In fact, the female hormone oestrogen also has a role in dopamine production and could replace that of Sry...

It takes a network of molecules to become a boy or a girl. Sex is not the work of only one protein though Sry is certainly sitting on a hinge. Integrated into an XX mouse embryo, it can switch the sex program and a female mouse becomes…male. Mice are not human though. Furthermore, the common belief that girls only become so because Sry is not expressed, is not accurate. Women are not passive in their making. One of Sry’s doings is to activate the production of a hormone known as AMH which ensures that any feminine features are impeded.

To cut a long story short, the chromosomal definition of a man is XY, and that of a woman XX. But it takes very little to confuse the nature of one or the other by providing our species with humans that carry both feminine and masculine attributes for example, or by ‘turning’ what ‘should’ have been males into females, or females into males. There are XY females where Sry is not expressed and XX males where Sry is expressed thanks to the presence of its gene on an X chromosome. And there are hermaphrodites whose phenotype is neither male nor female, but a bit of both. The making of a sex is not a trivial affair and we should bear in mind that it takes very little to tip the scales and confuse the program. It is a thin line between a man and a woman.