Wednesday, October 28, 2015

It’s All in the Numbers - Sizes in Nature

If all the animal species are broken up into groups, the light 
blue section includes insects, and the rest of the 
circle colors represent every other animal on Earth!
Comparisons help to make very big or very small numbers meaningful, and biology is chock full of big and small numbers. For instance, there are more insects in the world than there are humans. By more, I mean ALOT MORE, something like 1.5 x 1018 insects. But what does that number mean? Consider looking at it this way; the world population hit 7 billion last year and that's a big number, but even if we were to double our population again in the next ten minutes, there would still be 100 million insects for every human on earth. This certainly makes an impression, but it seems small when comparing the most numerous organisms, bacteria, to humans.

Bacteria outnumber us by orders of magnitude more than insects do; they live everywhere, in every environment. They have been found in 0.5 million year-old permafrost as well as 40 miles up in the atmosphere. There are approximately 100 million to 1 billion bacteria in every teaspoon of dirt, so in total there are currently 5 x1030 bacteria carrying out their daily routines. That means there are about 5 x 1019 living bacteria (that is 50,000,000,000,000,000,000) for every person who has EVER LIVED. Another way of visualizing this might be to imagine that each bacterium is a penny being stacked. The column would be a trillion light years high. That’s about five times the diameter of the observable universe.

Nanobacteria are still controversial, the 0.2 µm diameter is 
close to the smallest size that could still hold DNA. 
For comparison, the white line in panel A is 1 µm long, 
and in Panel C the line is just 0.1 µm.
While the redwoods might be slightly taller than the sequoias, 
the mass of the sequoias is much greater because the 
trunks have such a large diameter.
Even using comparisons and analogies, these numbers are almost too big to comprehend. It isn’t much easier when talking about sizes. The scale of life is amazing, from the smallest bacteria (called nanobacteria), just 0.2 µm in size (1/5,000,000 of a meter), to the biggest living thing on Earth, a Giant Sequoia called General Sherman. This behemoth of a tree is more than 83 meters (272 ft.) in height and 1,225,000 kilograms (2,701,000 lb.) in mass. This means that from smallest to largest, life spans more than eight orders of magnitude. In terms of biomass, the difference between the smallest bacterium and General Sherman is even greater, about 1 x 1023, about the same as difference in mass as one human compared to seven Earths.

On a smaller scale, the difference in size between bacteria and nucleated cells (eukaryotic cells) is still pretty stunning. A single macrophage cell of your immune system can ingest more than 100 bacteria without flinching, and macrophages are nowhere near the biggest eukaryotic cells. These different sizes demand some distinctions in how cells conduct their business; for example, how they move molecules into and within themselves.

A macrophage reaching out and ingesting bacteria.
The bacteria are the small, connected rods.
Eukaryotic cells, unlike prokaryotic cells (bacteria and Archaea), have specialized systems, like actin filaments, cytoskeleton, and microtubules. These apparatus are designed to act like conveyor belts; they carry different molecules through the cell to their needed destinations. Eukaryotes also have specific receptors for bringing in specific molecules. These are fast systems of uptake and movement, and can work against a concentration gradient.

The cytoskeleton of the eukaryotic cell stretch out like fibers.
They help it move, can convey molecules from place to place,
and holds the cells shape.

Unfortunately, bacteria only have diffusion to move molecules around their insides. This makes things doubly hard on them because bacteria have limited access to resources; most often they meet up with few molecules that are important to them (being a small cell in a big environment). Therefore, they need to get as many of these resources into their cell as possible and move throughout their entire volume quickly.

Diffusion is the movement of molecules from places where there a lot of them toward places here there are fewer of them (from high concentration to low concentration). Think of a crowd pouring out onto the football field after a big win. You start with many people in the stands and very few on the field, but end up with about an equal number of people in all parts of the stadium. Bacteria count on consuming their nutrients this way. Important molecules diffuse into the cell, and then get metabolized for energy or other building blocks. This breaking down and reassembly of molecules helps ensure that the concentration of important molecules is always lower inside the cell, so diffusion into the cell can continue. Importantly, as the width or length of a cell doubles, the volume increases by a factor of eight; therefore, prokaryotic cells remain small so that they can get molecules everywhere they need them quickly. It is the only way for diffusion to remain profitable for them.

Diffusion is the movement of from where there are 
many to where there are few. If it is water 
molecules that are moving, then call it osmosis.
Diffusion is not quite as simple as people pouring out the stands. There are several aspects of this process that are important to bacteria. The first of these is the diffusion rate, which is based on a diffusion coefficient for each different molecule, and the liquid it is moving through. For oxygen moving through water, the diffusion rate is about 1 mm/hr. This means that for an average sized bacteria it only takes 1 millisecond (1/1000th of a second) for an oxygen molecule to travel across the entire cell.

There is also the mixing rate; this refers to the time it takes for a molecule that enters the cell to have an equal probability of being found in any part of the cell. A 1µm (1/1,000,000 of a meter) bacterium has a mixing time of roughly 1 millisecond. But since the volume increases by a factor of eight as the size doubles, it would not take much growth for the mixing time to become problematic if a cell was to rely on diffusion alone.

Finally, there is the issue of traffic time. Every reaction that takes place in a cell involves two or more molecules finding one another and then interacting. In both prokaryotic and eukaryotic cells there are some systems designed to help bring molecules together, but in the end, it is basically luck – they have to run into one another. The number of molecules can affect this time; say you want molecule A to meet molecule B. If the cell contained only one of each molecule, this could take a while, but if there are 1000A’s and 1000B’s, then the traffic time will be decreased considerably. For average sized bacteria, traffic times exist in the range of 1 second, but again, if they are much bigger, the chances of molecules meeting their partners goes down dramatically.

If the bacterium grows too big, the diffusion rate, mixing time, and traffic time can become too long to permit survival. Therefore, size limitations seem to be set for bacteria. However, some bacteria just have to be rule breakers. There are two excellent examples of bacteria that have evolved ways to overcome the diffusion problems associated with increased size, and we'll start to look at them next week.

Schulz, H., & Jørgensen, B. (2001). Big Bacteria Annual Review of Microbiology, 55 (1), 105-137 DOI: 10.1146/annurev.micro.55.1.105

For more information on numbers in nature, diffusion, and cytoskeleton, as well as web-based activities and experiments, go to:

Cell size and volume:

scaling in nature:



Wednesday, October 21, 2015

Mostly Dead Is Slightly Alive

Halloween has morphed into a holiday where people see how much it takes to scare them. Horror movies, haunted houses, dangerous pranks; people like to be scared.

Miracle Max had his own methods for determining if someone was all dead 
or just mostly dead. They involved a bellows and Carol Kane’s 
voice.  But the point is made, for centuries, people were just guessing 
if others werereally dead. There were few experts, and they were
probably just comedians in make-up.
What scares you the most– spiders, public speaking, death? These three are high on every list of common fears, but it wasn’t so long ago that another fear was in first place – taphophobia. Never heard of it? I bet that its mere definition will be enough to send a chill up your spine.

Technically, taphophobia means “fear of graves” (taphos = tomb, and phobia = fear of), but its common use is “fear of being buried alive.” Premature burial is not an urban legend, incidents have been documented in nearly every society – and not all of them were just in the movies or books.

In the 1800’s and earlier, being dead was a lot like being a duck….. you know, if it looks like a duck, walks like a duck, and quacks like a duck….. The appearance of death was often enough to make a diagnosis and start going through their pockets.

As a good example of the wisdom of the age, George Washington had these last words, "Have me decently buried, but do not let my body be put into a vault in less than three days after I am dead…….., tis well." He wanted a sufficient amount of time to pass to ensure that he was in fact dead.

The Irish wake probably originated in the leaving of the
tomb unsealed for several days, just in case the dead
person might wake. Later, stories came about concerning
the lead in pewter tankards from which the Irish would
drink. Lead poisoning could induce a state that resembled
death. Sometimes, a wake is just another reason to raise
a glass of ale.
Many cultures built time delays into their death rites to make sure someone was truly dead. Greeks washed the dead….. and some would wake up. In more difficult cases, they would cut off fingers or dunk the bodies in warm baths. The custom of the Irish wake began with the Celts watching the body for signs of life. But mistakes were made, often in times of epidemic.

The hopes of preventing the spread of infection often lead to burying the dead before they were quite dead. I give you plague victim Eric Idle in Monty Python’s Search for the Holy Grail – “But I’m not dead yet…. I’m feeling much better.”

Even without epidemic, most people in the 18th, 19th, and early 20th centuries died at home, having never seen a doctor. If someone couldn’t hear a heartbeat or feel a pulse, then the patient was dead. But these were lay people, did they know how to feel for a pulse? Maybe they relied on another indicator of death - rigor mortis (rigor = stiffness, and mort = death).

In humans, rigor mortis begins 2-6 hours after death. Rigor is caused by a loss of ATP production. Follow me here--- no breathing, no oxygen; no oxygen, no ATP production. With no ATP, the muscle  can’t relax. This may seem strange, since it takes ATP to contract a muscle in the first place.

As described in the text, the thick filament (myosin) pulls
itself along the thin filament (actin) by grabbing and releasing
actin monomers. A single sarcomere (contractive subunit,
~100,000 in a muscle cell) has millions of myosin heads. They
grab actin fibers that run on all sides of the myosin fiber.
The picture at the side should help with this explanation, but I won’t give you all the gory details. Your muscle cells have systems that look like ratchets, using to proteins called myosin and actin which pull past one another to shorten (contract) the muscle fiber. The myosin is bound by ATP, which then hydrolyses to form ADP + P. When ADP + P is bound to myosin, it can reach out and bind to the actin.

The ADP + P is released from the myosin and it flexes the head of the protein, which pulls it along the actin. When a new ATP is bound, the myosin lets go from the actin, and the cycle is repeated.  Each muscle fiber in each cell has millions of myosin heads resulting in a contracted muscle.

In rigor, there is no more ATP, so the myosin doesn’t let go of the actin, therefore, no relaxation can take place. The muscles remain the length they were at death. After about 72 hours, the muscle proteins start to break down, rigor will lessens and the body will become limp again. But as we will see below, some conditions can mimic the signs of rigor, increasing the chances of premature burial.

In an effort to see how bad the situation was, the English reformer, William Tebb, in 1905 made a study of accidental premature burial. Tebb was quite the joiner; the weirder the society, the more he wanted to join or lead it. He worked with the Vegetarian Society, the anti-vivisection movement, the national Canine Defense League, and formed National Anti-Vaccination League in 1896.

William Tebb’s book on premature burial was a best seller.
You’d think he had a product to sell given the way he
described some of the incidents. In one, Madame Blunden
was buried in a crypt under a boys school. The next day, the
students heard noises from below. They opened the tomb
and coffin just in time to see her die from lack of oxygen.
In his book, Premature burial, and how it may be prevented, with special reference to trance catalepsy, and other forms of suspended animation, Tebb professed that he had found 219 cases of near premature burial and 149 live burials. He had some stunning stories of scratches on the lids of coffins and noises from newly filled graves.

In her 1996 book, The Corpse: A History, Christine Quigley documents many instances of premature burial and near-premature burial (I LOVE the title). Skeletons were outside their coffins, sitting up in the corner of their vault after being opened years later. Others were found turned over in their caskets, with tufts of their own hair in their hands.

How might this happen? What conditions might make it look so much like you were dead that even your loved ones would let them plant you in the ground? The list is long and varied, but here are some of the more common things that can make you look dead:

Asphyxiation – anything that cuts off your supply of air can make you look dead once you fall unconscious – continuation of this condition leads to actual death. You look dead enough and won’t respond to external stimuli, so people assume you are dead. Close the coffin lid, and soon you really will be dead of asphyxia.

Catalepsy – Many things can bring on this catatonic state in which the muscles are rigid (like rigor mortis after death) and no pain is enough make you respond, one example is epilepsy. Hypnotists call their trances catalepsy (Greek for to grab and take down), but true catalepsy is much more severe and can last hours to days. Severe emotional trauma can also bring it on, so you can certainly be scared enough to look like you are dead.

Catalepsy is denoted by muscle rigidity, so it can look like
rigor mortis. But there is also waxy flexibility in some cases.
The dead-looking not dead people can be posed, and they
will hold the pose indefinitely. What little girl wouldn’t love
a cataleptic doll for Christmas!
Coma – In medicine, a coma is unconsciousness that lasts more than six hours and from which a person cannot be roused and will not respond to stimuli. Injury or inflammation of the cerebral cortex and/ or the reticular activating system in the brain stem can lead to coma. The things that can injure these structures are myriad, from traumatic injury, to drug overdose, to stroke or hyperthermia, etc.

To show how medicine has changed, there is now a battery of assessments called the Glasgow coma scale (GCS) that are carried out on coma victims to assess their state and prognosis. In centuries past, you might look at them, hold a mirror under their nose, maybe lift and drop an arm….. bury them.

The GCS has traditionally been used in the hospital environment, but new evidence shows that a prehospital GCS (assessment at scene or in route) can be just as accurate and may benefit treatment choice in pediatric traumatic brain injury patients. The study compared prehospital and emergency department GCS scores and showed that they were similar. They also compared outcomes with prehospital scores and showed a positive correlation. If assessment and treatment can be begun earlier, outcomes should improve.

Apoplexy – this not a very accurate term any longer, and has meant different things at different times. It can refer to bleeding within an organ or bleeding during a stroke. A stroke is very likely to leave survivors that look like they are dead, and are unresponsive. Nevertheless, there are stroke victims who regain consciousness.

Due to the above conditions, many people in the 1700’s and 1800’s made a hunk of change by promoting safety coffins and vaults. These might be as simple as attaching a rope to the hand of the deceased, and running this rope to the surface where it was attached to a bell.

In other coffins the alterations were more elaborate. There might be glass plates to view the face of the dead or a periscope to keep an eye on the corpse. Some thirty designs were patented just in Germany in the second half of the 19th century, including some that contained vibration sensors, and later… a telephone line.

Waiting mortuaries were built in the 1800’s, mostly in
Germany. Since the best sign of death was the beginning
of the rotting process, these mortuaries were basically
holding cells for bodies while nature took its course. If they
didn’t start to smell, they had to look for fangs or a way to
arouse them.
To be successful, those folks above ground must have been very alert. A coffin has only about 20-40 minutes of air, so a person could go from dead to live to dead without the change being noted. To counteract this small window of time, Germany also built waiting mortuaries, where dead bodies could be held for longer periods of time. It came to be accepted that the only reliable sign death was putrefaction --- waiting mortuaries did not smell like flowers or fresh baked bread.

Modern EEG and EKG have reduced the chance of premature burial or cremation, but mistakes do get made. In 2007, a Venezuelan man awoke during his own autopsy, and Quigley also writes of several modern instances of near-premature burial. Furthermore, the need for quick burial during epidemics has been replaced by the need for timely organ harvests – maybe they aren’t done with that kidney yet!

Next week we will take Halloween and death one-step further – could Halloween, or anything else for that matter, literally scare you to death?

Wednesday, October 14, 2015

Blood --- Not Just For Vampires Anymore

“Nosferatu” was the first film (1922, directed by F.W. Murnau)
made about the blood sucking undead. It followed the Stoker
novel so closely that his estate sued and a court ordered all the
copies destroyed. Only five survived, and were used to restore
the film in 1994. One area where did deviate from the novel
was in the way the vampire dies. Murnau introduced the idea
of sun sensitivity, which caught on and was accepted as part
of the myth.
It may not be surprising, but there’s a lot of pathology in Halloween. Since the holiday is coming up soon, let's take a look at some of the gory details.

Pathology (pathos = disease, and ology = study of) is the study of disease, and being dead is the worst disease - O.K., maybe being undead is worse. Between life and death is where the vampires live, so maybe this is a good place to start.

One prerequisite for being a vampire is that you have a taste for blood, but if that was the only rule, then almost everyone would be a vampire. Hematophagy (hemo = blood, and phagy = eat) is as common as bad Dracula impressions. Almost every culture consumes blood.

Many people eat cooked blood. The Poles eat blood soup (czernina), and the Brits love their blood pudding as much as the Chinese gobble their fried blood tofu. The next time you go to a French restaurant for the coq au vin, remember that the sauce is made with rooster blood!

There are also those cultures that drink blood. The inuit peoples drink fresh seal blood, and the Maasi in Africa rely on a mixture of cow’s milk and cow’s blood as a staple of their diet. And why not, blood is a decent source of nutrition.

Blood has a lot of protein and is a good source of lipids. Of course it is iron rich, and is a source of fluid and salt if you happen to be caught in the desert. If a vampire happens to pick out an uncontrolled diabetic, a drink of blood could also be a good source of carbohydrates.

These are Finnish blood pancakes. You have to wonder about 
a recipe whose first ingredient is 40 ml of blood. But 
the lingonberry jam on top is a nice touch; you would 
hardly remember that you are eating blood.
Many animals practice hematophagy. Female mosquitoes consume blood; both sexes of the Cimicidae family (bed bugs) survive solely on blood, as do arachnids of the Ixodida order (ticks). Some of the 700 species of leeches feed on blood only, but most eat small invertebrates as well. There is even a vampire finch on the Galapagos Islands that bites the rumps of other birds and licks off the blood. And then there are the vampire bats.

As members of the Chiroptera order (chira = hand, and ptera = wing), vampire bats are members of a grand biologic exception. Bats are the only mammals that truly fly. True flying requires lift, being able to sustain a rise in altitude by mechanical means. Closest to this is soaring, which is the use of upwelling air currents to gain altitude. But gliding is the most common type of aerial motion in reptiles, amphibians, mammals. Gliding is really controlled falling; it means moving at less than a 45˚ angle to the ground.

Bats are so finely evolved for flying that they have lost most of their ability to walk, but vampire bats are an exception even in the world of bats. They often approach their victims by walking or running up to them from behind. Vampire bats were quite the biologic discovery.

The vampire bat wasn’t named as such until 1774, but vampire legends (4000 BCE) and the word vampire (circa 1734) had been around much longer. Therefore, the bat was named after the undead, blood-drinking person, not the other way around.

Three species of bat, ranging from Mexico to Chile, subsist exclusively on blood. Each has evolved tricks to help them secure the blood they need. Their noses house special thermoreceptors to help them find areas of flesh where blood vessels lay close to the surface. The way their brain perceives and interprets this information (see this post) is very similar to the way pit viper snakes sense live prey (see this post).

Common vampire bats like to bite and lick blood from around 
the hooves of cattle and such. They are so sneaky, they run 
up to the animals from behind instead of flying. Their wings 
are stronger than most bats, so they can help support their 
body weight when they run or hop.
Two species (Diphylla ecaudata, Diaemus youngi) feed on the blood of birds, while the other (Desmodus rotundus, a.k.a. common vampire bat) feeds on mammals, including humans, but they all feed exclusively at night. This may have helped to link the bats to the monsters, as vampires are supposedly harmed by sunlight.

The common vampire bat will shave away the hair away with its teeth and then plunges its incisors in about 7-8 mm to bring blood, as its incisors are conical and are designed for cutting. Vampire bats are an exception in that they are the only bat species that do not have enamel on their incisors.

Enamel is very strong in compression and wear, but is brittle and rounds off the points of the teeth. Vampire bats need very sharp incisors, so they have forgone the enamel. Broken enamel would blunt their teeth, a lethal problem for a bloodsucker (although they don't suck).

The Swiss Federal Institute for Technology at Lausanne has
developed a drone that can walk and fly, based on the movement
of the vampire bat. When it goes terrestrial, it pulls in the the
middle section of wing and the rotates the wing tips
to propel itself (2015).
Importantly, vampire bat salvia contains anticoagulants to keep the blood flowing and vessel relaxants to keep the local blood vessels from constricting.  A new study has shown that bat saliva may have potential in human medicine. The common vampire bat is the source of a new clot-dissolving compound called desmoteplase; it activates an enzyme called plasminogen, which breaks down early clot formation.

Desmoteplase is structurally similar to a currently used clot buster called tPA (tissue plasminogen activator), but has some differences that make it more selective for fibrin. Importantly, it doesn’t cause nearly as much neuronal apoptosis or breakdown of the blood-brain barrier as does tPA. Desmoteplase is in phase III clinical trials for use in ischemic stroke patients (a brain blood vessel is blocked by clot). I wonder if human vampires have such useful saliva.

Ischemic stroke occurs when a blood vessel in the brain
is occluded so oxygen rich blood can’t reach the brain
tissue beyond the occlusion. The middle cerebral artery is
a common site for these cerebrovascular accidents. 
Desmoteplase appears to be effective against occlusions
caused by blood clots, but there can be other occlusions,
name scar tissue from infection or atherosclerotic plaques.
Vampire bats usually slice open a small vessel with their incisors, and then lick the 20-25 ml of blood that flows out. This is very different from the idea of vampires sucking out all the blood from a human; something not consistent with long life. But could losing blood ever be considered a good thing? You know there has to be an exception.

In certain diseases, removing excess blood is beneficial. We talked earlier about excess iron in hereditary hemochromatosis, for which bloodletting is an appropriate treatment, but there are others.  Polycythemia vera is a genetic disease in which too many red blood cells are produced, leading to high blood volume and pressure, excess bleeding and clotting. To bring the volume closer to normal, a pint of blood may be removed once a week.

Finally, in chronic hepatitis C infection there is damage to the liver, a major storehouse of iron. This releases iron into the blood, and causes a secondary hemochromatosis. Small amounts of blood can be removed to help lessen the iron overload. Maybe old-timey medicine didn’t have everything wrong.

These same old cultures had myths about the undead that would feed on human flesh, but our current vampire myths date from early 1700’s Southern Europe. There are diseases that could be mistaken for some or all of the aspects of vampirism, but are they the chicken or the egg? In many cases, myths and folklore have some basis in fact, but in these cases hindsight is hardly ever 20/20.

Tuberculosis and rabies have a few aspects that are similar to the common tales of vampires. TB leaves its victims emaciated; they end up pale with swollen eyes that make them sensitive to light. They might cough up blood, and the first victim often gave the disease to other members of the house, so it have might appeared that the first was draining the others.

Similarly, people with rabies may exhibit a bloody froth from the mouth because lesions on the throat make it very painful to swallow. They may also be driven to bite people due to the encephalitis (encephalo = brain, and itis = inflammation) that the rabies virus causes. Other behaviors associated with rabies are sleeplessness (night time activity) and fear of looking at one’s own reflection.

Rabies spreads through the nerves, and the brain is the main
organ affected by the infection. Without vaccination or
treatment rabies is 100% fatal. Animals with the infection lose
fear of man, and become very aggressive, and then so do people
who contract the virus. Two cases of human bit rabies have been
confirmed (both in Ethiopia in the 1990’s).
Vampire bats are carriers of rabies, and this may contribute to their use in vampire lore, but recent evidence says bat rabies may not be such a bad thing. A 2012 CDC study shows that many Peruvian natives have a natural immunity to rabies, a disease that kills 55,000 people each year. The vampire bat maybe helping drive this immunity. It’s bite can deliver a sub-pathogenic dose of virus, enough to convey immunity, but not enough to cause disease. A case of vaccination by bite!

Another disease that mimics some vampire characteristics is xeroderma pigmentosum (XP). XP leads to an extreme sensitivity of the skin to the radiation of the sun. XP was first described in the scientific literature in 1874, just a couple of years before the first tales of sun sensitivity in vampires. There are several different types of XP, but all are autosomal recessive genetic diseases. Most involve mutation and inactivation of nuclear excision repair enzymes.

Sunlight contains UV radiation that causes DNA mutation. Excision repair enzymes usually fix the DNA damage. Without them, afflicted individuals manifest hundreds of skin cancers, and acquire others that are lethal (malignant melanoma). The patients’ eyes are very sensitive to light; they sunburn almost instantly, and must be kept out of sunlight. The children from the 2001 film, “The Others” had XP (while they were alive).

Congenital Erythropoietic Porphyria (CEP) is by far the disease most often associated with vampirism. Exceedingly rare, this autosomal recessive genetic disease has only been diagnosed in about 200 people, but there are many variants of porphyria that carry some or most of the same symptomology as CEP.

Porphyria can lead to deposits of porhyrins in the enamel
of developing teeth. The word porphyrin comes from the
Greek word for purple, so the discoloration is often darker
than what is shown here. Interestingly, tetracycline use in
pregnant women and children can lead to a similar
deposition, but for very different reasons.
The mutation common to the porphyrias is in the gene for an enzyme called uroporphyrinogen cosynthetase. Involved in heme synthesis, the loss of this enzyme leads to the buildup of heme intermediates called porphyrins. The porphyrins accumulate in the skin and organs and act as a sun-activated toxin.

The symptoms of the porphyrias do make you think of vampires: sun sensitivity with extreme burning, white skin, bloodshot eyes, sensitive eyes, anemia (low number and therefore a need for red blood cells), reddish tears, reddish urine, red pigment in the enamel of the teeth (erythrodontia).

The red teeth really bring to mind feeding on flesh or blood, and porphyrias also bring increased body and facial hair (hirsutism), so they may contribute to the werewolf legend as well. This is interesting because Medieval Europeans would burn the corpses of people who were thought to be werewolves, so as to prevent them from returning as vampires - better safe than sorry! 

Next week we will continue our look at Halloween by investigating death – how likely is that you might be buried alive?

For more information or classroom activities, see:

Hematophagy –

Vampire bats –

Xeroderma pigmentosum –

Congenital Erythropoietic Porphyria –

Medcalf RL (2012). Desmoteplase: discovery, insights and opportunities for ischaemic stroke. Br J Pharmacol. DOI: 10.1111/j.1476-5381.2011.01514.x

Amy T. Gilbert, Brett W. Petersen, Sergio Recuenco, Michael Niezgoda, Jorge Gómez, V. Alberto Laguna-Torres and Charles Rupprecht (2012). Evidence of Rabies Virus Exposure among Humans in the Peruvian Amazon Am J Trop Med Hyg DOI: 10.4269/ajtmh.2012.11-0689


Wednesday, October 7, 2015

Twin Sons Of Different Mothers…… Or Fathers

Biology concepts – twins, superfecundity, superfetation, hormones, reproduction

Best In Show is one of the great movies; it also gives you
a good idea how crazy some people get about their dogs.
I applaud that they love them that much, but they expect
you to love them that much as well. I like old movies too,
but I don’t need to see dogs recreating the iconic scenes.
When it comes to their pets, some people can really come unglued. People have had them stuffed after death so they can pet them forever. There has even been paternity suits concerning the offspring of said pets.

True, in some cases a lot of money might be involved in stud fees and in selling purebred pups, but that just goes to show how crazy things can get when pets are involved. One case concerned a female Shih Tzu was bred to two different males in the same estrus cycle – why they did that I have no idea. What was dumber, the two male dogs, a Shih Tzu and a Coton de Tulear, look very similar.

When the pups were born, each owner claimed that they were the pups of his male dog. A DNA fingerprinting method called barcoding was new at the time, and was used to determine that one pup came from each male.

Giving birth at one time to offspring fathered by more than one male is called superfecundation (super = beyond, and fecund= fruitful). Dogs, cats, and many other mammals that have litters are capable of superfecundity; although it is usually seen in stray animals that may mate several time in a single day. Raccoons have had superfecundation litters, and I’m sure it has happened with other animals as well, but whose watching.

With superfecundity in dogs as an example, let's ask our last two questions concerning the definition of twins. We saw in the last two posts that twins can be born months apart and don’t even need to be of the same "race." Now let’s ask – do twins have to be conceived at the same time, or even by the same father?

A litter of puppies where one looks a lot different than the
others. Different breeds mating will give something in the
middle, and even pure breds can have puppies of different
colors, but I don’t think a mom and dad hound will ever
produce a huskie (right) on their own. This is definitely
a superfecundation litter.
The definition of superfecundity is two or more eggs released in the same estrus cycle fertilized by one or more males and implanted and developed in the uterus. Superfecundity can be seen in two different situations. The first is homopaternal superfecundation, when the eggs are fertilized by the same male, but at different times of the same cycle.

The question that immediately popped into my mind when read about homopaternal superfecundation was – how would you know? Run of the mill dizygotic twins would be from the same cycle, the same father, delivered at the same time (usually). How would homopaternal superfecundation twins look any different? How could you tell the two situations apart? And if you can’t tell them apart, how do you know if they can happen in humans?

Consider the following possibility: a husband and wife undergo in vitro fertilization because they have not been able to have a baby in several attempts. Her eggs and his male gametes are used. Two embryos are transferred and a several weeks later they do ultrasound to see if they implanted and are developing. Low and behold – there are five fetuses in there!

Is this proof of superfecundation? No, but the couple did attempt to make a baby the natural way again after the embryos were transplanted – no, that’s not proof. Maybe the two transferred embryos split into a set of twins and a set of triplets. We know that it is possible from our discussions of monozygotic twinning rates during assisted reproductive therapies (see this post).

If you isolate and amplify DNA from several different areas
of the genome (loci, each is a locus), then mom will have
two alleles and dad will have two alleles. For the child, every
allele should match one from mom and/or dad. If not, you
may have the wrong guy.
The only way to tell would be to test the DNA of the parents and all five kids. If the results say that the husband is the father of all of them – is that evidence of superfecundity? No – they could still be monozygotic twins and triplets. But what if the genetic profiles of all the kids are different; what would this mean?

Dizygotic twins and trizygotic triplets don’t have the same chromosome profiles, just like regular siblings do not. Random assortment in the production of gametes means that the odds of two fetuses having exactly the chromosomes is millions to one (see this post). Monozygotic twins or triplets will have the same genetic profile – that’s sort of the definition of monozygotic multiples (unless they are chimerics, see this post and this post).

So, if all the kids have the same mother and father and none are monozygotic multiples, then where did the other three come from? Yep, a fertilization by the father separate from the IVF procedure. This is proof of homopaternal superfecundity. Do you think this is just a hypothetical case and the odds are too long to ever have it ever happen? Well it did, not once, but twice in the literature of the last 15 years, once in 2001 and once in 2011.

Remember that IVF isn’t just an egg harvest and return. Lots of hormones have to be given to the woman to make her ovulate several eggs and to prepare the uterus for implantation after the embryo transfer. This makes it possible for her to ovulate again, and makes it more probable that any fertilized eggs released later might implant and develop as well. And there you have it.

FSH rises just before ovulation and is the reason women
may ovulate more than one egg. Progesterone is produced by
the follicle left from the egg (corpus luteum) and keeps FSH,
LH and estrogen lower, so no other eggs will be released. If the
embryo implants (right side of chart) then progesterone would
be produced by the placenta and stay high – this would prevent
cycling until the pregnancy is over.
A study in 1993 suggested that superfecundity might be responsible for up to 0.5% of dizygotic twins, but that would be a hard thing to prove. The cases that come to light are usually when a question of paternity arises, and that arises much too often.

The second possibility is that eggs can be fertilized by gametes of two different males; the term is heteropaternal superfecundation. There are several cases where this has been proven by genetic testing (1997 and 2000), and in ancient Greek accounts, it has been cited as the reason for any set of twins – they were an untrusting, and apparently, philandering lot.

But you can see how one might assume that there are two dads – remember our discussion of different "race" twins a few weeks back – one paper states that in the case of black and white twins, heteropaternal superfecundity has to be ruled out before a case of different race twins can be proposed. And why does it matter – paternity suits show that heteropaternal superfecundation is present in 2.4 % of the cases that come to court concerning multiples.

Romulus and Remus were raised by wolves – their dad
(Zeus) had sent someone to kill them, but they banished
them to the wild instead. They were twins, and twins in
Rome and Greece were admired and feared. They might be
offspring of the gods, and they might be a sign of infidelity.
If your definition of twins includes a condition that they have to be conceived at one time or by one father – then your definition just got shot down. But don’t feel bad, I’m getting the idea that it’s impossible to pin down just what the true definition of twinning might be. Case in point – we haven’t even gotten to the weird exception yet.

Have you ever heard of superfetation? It is like superfecundity, but stretched out through time. To define it sounds like a riddle – can a pregnant woman get pregnant? Believe it or not, the answer is yes.

After an egg is released from the ovary, the follicle becomes the corpus luteum (see this post). This structure releases progesterone hormone which acts on the hypothalamus, so does the progesterone released from the placenta. In both cases, the progesterone down-regulates the action of the hypothalamaus on the pituitary, so the pituitary releases less hormone that stimulates ovulation in the ovary.

Basically, the reproductive system is telling the brain, "Wait a minute, we may have an implanted embryo, don’t release any eggs in the next estrus cycle." This is why women who are pregnant more often have eggs and will ovulate longer in their life; they go nine months without releasing any eggs every time they are pregnant.

But if there is an over abundance of a hormone called human chorionic gonadotropin (hCG), the signal from the brain may get overridden. HCG is the only hormone known to stimulate ovulation in a woman who might be is releasing progesterone from the placenta and follicle.

Several types of fish, like this black molly, are the first
animals to produce a placenta and give birth to live offspring.
They lost and regained the placenta many times, so some
fish have it and some don’t. Even in the same family, so
species will give birth to live young (viviparity), and some
will scatter eggs to be fertilized outside.
If the first embryo isn’t taking up too much room, the egg released the next month is capable of being fertilized and implanting in the wall of the uterus. Now you have two developing fetuses that may differ in gestational age by as much as five weeks! You can imagine that this might be mistaken for growth discordance (see last week’s post) or growth discordance might be mistaken for superfetation.

Superfetation occurs in other mammals. Placental fish – yes, some fish have placentas and give birth to live young instead of releasing eggs into an underwater nest – are notorious for having immature eggs fertilized while they are carrying a brood.

In brown hares (Lepus europaeus), the same thing occurs; the hypothesis is that it is a way to increase reproductive success in one breeding season for animals that don’t know how long they have before something big might eat them. Still, it has been hard to document superfetation in humans.

In 2007 there was a case of growth discordant twins, but it was the smaller one that was the right size for the gestational age – sounds promising. And in 1999 there was a case where the ultrasound early in the first trimester shows very different sized embryos. Usually growth discordance wouldn’t be seen until much later. The predicted gestational ages for the two embryos differed by four weeks. hmmmm

One big sign that our species may survive – reality shows that
announced paternity testing results used to be a big deal, but
we moved past them. Finally, humans show some good taste.
I wonder if any ever tackled the problem of superfecundity or
superfetation? I think the Jerry Springer’s head would
explode when he contemplated the ratings.
I ask you one last time – how do you define dizygotic twins? Same father, same conception time, same delivery, same gestational age, same size, same "race"? It seems none of them apply concretely. The next time someone tells you that they are a twin, you’ll have a lot to talk about.

And just to blow your mind a bit more – consider a case of heteropaternal superfecundation where the two embryos merge either totally or partially to create chimeric(s). Could you actually end up with a person who has two biological fathers?

In the next weeks, we go way back into the vault to look at a couple of posts about Halloween. It turns out that nearly everyone is a vampire - and then we'll see all the different ways one might end up being buried alive. 

Pollux, B., Meredith, R., Springer, M., Garland, T., & Reznick, D. (2014). The evolution of the placenta drives a shift in sexual selection in livebearing fish Nature, 513 (7517), 233-236 DOI: 10.1038/nature13451

Peigné, M., Andrieux, J., Deruelle, P., Vuillaume, I., & Leroy, M. (2011). Quintuplets after a transfer of two embryos following in vitro fertilization: a proved superfecundation Fertility and Sterility, 95 (6), 2147483647-2147483647 DOI: 10.1016/j.fertnstert.2011.01.029

James WH (1993). The incidence of superfecundation and of double paternity in the general population. Acta geneticae medicae et gemellologiae, 42 (3-4), 257-62 PMID: 7871943

Baijal N, Sahni M, Verma N, Kumar A, Parkhe N, & Puliyel JM (2007). Discordant twins with the smaller baby appropriate for gestational age--unusual manifestation of superfoetation: a case report. BMC pediatrics, 7 PMID: 17239246

Claas, M., Timmermans, A., & Bruinse, H. (2010). Case report: a black and white twin Journal of Perinatology, 30 (6), 434-436 DOI: 10.1038/jp.2009.156

For more information or classroom activities, see:

Superfecundity –

Superfetation –