Suspended animation II
Death on demand
Azamat Akkizov, "Biomolecule"
(see the first part of the article here)
In Hans Christian Andersen's fairy tale "The Snow Queen", in order to gain true freedom, it was necessary to put the word "eternity" out of ice cubes. Now scientists are thinking of achieving the same goal by turning the person himself into a piece of ice as the embodiment of immortality itself...
Echoes of the "primary broth" era
Why is dehydration associated with the inhibition of the "biochemical machine"? To answer this question, it is necessary to clearly understand what role water molecules play in the organization of the "brick" of all living things – a single cell.
Firstly, water is a unique medium for the self-organization of organic molecules into supramolecular structures such as the phospholipid framework of biomembranes or the tertiary structure of proteins. Secondly, water is a universal solvent in which, during the dissociation of salts, ions necessary for the generation of biopotentials are formed. "Correctly" assembled biomolecules together with a certain amount of electrolytes provide the spatiotemporal orderliness of metabolism that underlies the phenomenon of life . It's simple, isn't it?.. Too easy.
In fact, a lot has been written about the properties of water and its role in life processes. However, now we are only interested in the fact of the withdrawal of water molecules and their mysterious role in ensuring the reversibility of suspended animation. Although... it's not such a mystery.
Imagine a rich broth for making jelly. Scientifically speaking, we have a water–based gelatin emulsion in front of us. If the broth is cooled, it will turn into a jelly – gel, very different from its liquid state – sol. It is believed that life originated in the "primary broth", the droplets of which became the actual cells.
The contents of the cells periodically thicken, then liquefy, i.e. they are in a state of reversible phase transition "sol - gel", the equilibrium point in which is very unstable. However, during dehydration, the equilibrium shifts to one side: "sol → gel → amorphous state of the colloid → denaturation of the colloid". Based on this scheme, it is assumed that during suspended animation, the colloidal contents of cells are dehydrated only to a reversible amorphous state, without reaching irreversible (and therefore truly fatal) denaturation .
How much should the body be "dried" so that it plunges into suspended animation, and does not die from dehydration?
Observations have shown that this is possible if at least 10% of water remains in the body, which, however, is not enough even for a single-layer coating of protein molecules . This phenomenon of drying out to death, which is not uncommon in nature, is called xeroanabiosis. It contains spores of bacteria and fungi, mosses and lichens, as well as some groups of invertebrates (Fig. 1) .
Figure 1. In the plant kingdom, xeroanabiosis is characteristic of lower plants – mosses and lichens. Selaginella, or "resurrecting moss", is an example of this. However, it is extremely rare among higher plants, but there is an amazing ability to completely dry out and "resurrect" in a humid environment (for example, Ramonda Serbian, she is also "phoenix"). a – Selaginella lepidophylla (Selaginēlla lepidophylla). b – Ramonda Serbian (Ramonda serbica). Drawings from Wikipedia.
This is explained by the role played by xeroanabiosis: an emergency measure of survival in extreme conditions (Fig. 2). In some cases, this measure turned out to be so useful (for example, in the formation of pollen in higher plants or cysts in crustaceans) that it was evolutionarily fixed as an obligatory stage of ontogenesis.
Figure 2. Larvae of the African ringworm Polypedilum vanderplanki visited space within the framework of the Russian-Japanese experiment "Space Mosquito". It turns out that these relatively large organisms are able to withstand almost complete drying. In a state of xeroanabiosis, they can survive in extreme environmental conditions, which was tested in 2014 on board the ISS. Figure from .
The ability to reversibly lose part of the "bodily" water without harm to health is characteristic of all living things. However, the ability to extreme dehydration, as well as the duration of inhibition of metabolism, weaken with the structural and functional complexity of the body (Table. 1) .
But how to properly "dry" the body?
Obviously, part of the "body" water should be evaporated, let's say by heating... Stop! But a significant increase in body temperature is fatal, because already at 50 ° C irreversible destruction of protein molecules will occur! Nevertheless, back in the XIX century it was possible to do this. And more than once!
We are talking about the previously mentioned dispute "Dwyer vs. Pouchet" before the commission of the Paris Biological Society. Then the success of L. Dwyer's experiments consisted "in the gradual and perfect drying of animals. For these purposes, moss containing rotifers and slow-walkers was kept for 7 days under the bell of an air pump over sulfuric acid and only then placed in a water bath, where it was gradually heated to 100 ° C. " In order not to "skip" the stage of the amorphous state, the naturalist very "gently" heated the object in a rarefied atmosphere, and therefore the water slowly evaporated at a temperature less than 100 ° C, and the water vapor was immediately absorbed by sulfuric acid .
Today, vacuum drying of the body is recognized as the most effective. Its success depends on the gradual and uniform evaporation of water, which, in turn, depends on the volume of the biological object: the smaller the organism, the more successfully it will be dried. This explains the success of complete drying so far only microscopic animals (Table 1).
Following this logic, dehydration of small fragments of the body seemed very promising, which was proved by N.P. Kravkov back in 1922, in experiments with ... rabbit ears. And believe me, in the experiments of Nikolai Pavlovich, rabbit ears are far from the "nail of the program" (Fig. 3).
Figure 3. Nikolai Pavlovich Kravkov resorted to the method of isolated organs in his experiments. Along with rabbit ears, he mummified human fingers! Only after a couple of months they were soaked in saline solution. Surprisingly, the fingers recovered so much that the skin sweated on them and the nails grew back. Drawing from Wikipedia.
And yet, despite the phenomenal resistance of some creatures to complete drying, dehydration is stressful for the average cell! More precisely, hyperosmotic stress, which develops according to the following scenario.
First, dehydration of the periocellular space occurs with a regular increase in the concentration of dissolved substances, i.e. a hyperosmotic environment is formed. It draws water out of the cell, thereby dehydrating it. The cell loses volume, and its plasma membrane shrinks. At the same time, the intracellular concentration of electrolytes increases, which, by destroying ionic bonds, reduce the stability of protein molecules.
The evolution of microorganisms contrasted the process of dehydration with the accumulation of xeroprotectors – compounds that protect microbial cells from hyperosmotic stress (Table 2). These substances stabilize protein globules and biomembranes, replacing water molecules in them, and thereby facilitating the transition of colloids to an amorphous state [6, 7].
In the halls of the Snow Queen
Dehydrated cells become extremely resistant to extreme environmental conditions, and first of all – low temperatures (Fig. 4).
Figure 4. Spermatozoa (a), in accordance with their specific biological role, are adapted to survive in very harsh environmental conditions. They are practically devoid of the liquid part of the cytoplasm and therefore withstand complete freezing without loss of fertilizing ability. In order not to be accused of sexism, we note that the eggs of crustaceans, fish and amphibians can also do this (b). Drawing from Wikipedia.
There is a lot of evidence, but we will cite one thing: recently, scientists from the PNC RAS (Pushchino) managed to successfully revive a whole plant from the placental tissue of the fruits of the narrow-leaved resin (Silene stenophylla). It would seem that there is such a thing? However, the dry fruit-boxes have lain in the permafrost for about 30 thousand years !
Of course, here we are talking more about hypobiosis, because at a temperature of -7 °It could not have happened with complete freezing of tissues . But still, this study confirms the possibility of extremely long-term low-temperature storage of dehydrated cells.
And what if the body is frozen without pre-drying?
Theoretically, at temperatures below -80 °C, the Brownian motion of water molecules in tissues stops, and the body must plunge into a state of so-called "cryoanabiosis" [10, 11]. It is clear that this is impossible in natural conditions, and therefore cryoanabiosis is an unnatural condition, possible only in a laboratory.
The first systematic study of cryoanabiosis was conducted by Professor Porfiry Ivanovich Bakhmetyev of Sofia University. A series of his experiments on cooling various small animals to ultra - low temperatures (-90... -160 ° C) allowed us to conclude: the revival of a completely frozen organism is possible only if the water contained in it is preserved in a liquid supercooled state . If ice crystals have formed in the cells, then it is in vain to wait for its "resurrection" after the thawing of the body.
P.I. Bakhmetyev also found that the chance of survival after cryoanabiosis decreases with the structural and functional complexity of the test animal. It turns out that the features of xero- and cryoanabiosis are very similar and can be expressed by the thesis: the higher an organism stands on the evolutionary ladder, the more difficult it is to dry and/or freeze it without killing it.
The success of cryoanabiosis also depends on the volume of the biological object. If the body is not microscopic in size, then even at a very low temperature it freezes not instantly, but gradually, passing through a state of rigor mortis. In this state, when the soft tissues of the body seem to have stiffened (recall the forest frog from the first part), hidden life still continues to glow in its depths, i.e. low-temperature hypobiosis is observed. Complete freezing of the body usually leads to death due to the destructive effect of the ice formed in the cells. That is why it did not come to experiments with freezing a person, but his intentional immersion in low-temperature hypobiosis was carried out already in 1940!
Then American scientists Temple Faye and Lawrence Smith attempted to treat the last stage of cancer by hypothermia of the body. They repeatedly managed to immerse the patient, whose body temperature dropped to 28-30 ° C, into artificial hibernation for 5-8 days. The total duration of hibernation was 40 days ! Unfortunately, Faye and Smith did not achieve significant results in the treatment, but they definitely expanded their understanding of the capabilities of the human body.
And human capabilities are amazing. Judge for yourself by the following stories of amazing (if not miraculous) cases of "resurrection" of a person after an extremely long (!) period of clinical death that occurred as a result of hypothermia (Fig. 5). Are you ready?
Figure 5. Guests of the Ice Maiden... Survived! From left to right: Erika Nordby, Anna Bagenholm and Mitsutaki Yuchikoshi. Drawings from websites thestar.com , yle.fi and taringa.net .
The first case. In 1999, 29-year-old Austrian student Anna Bagenholm, skiing, fell through the ice. She stayed in the icy water for 80 minutes. Half of this time Anna was breathing thanks to an air bubble under the ice. When the body of the mountain skier was pulled to the surface, she was clinically dead: pupils dilated, breathing and heartbeat were absent, body temperature: 13.7 ° C. A stiffened corpse...
Anna came to life nine hours after, as it turned out, clinical death! After another 26 days, remaining completely paralyzed, she regained consciousness. Gradually, Anna's health was fully restored .
Case two. In 2001, 13-month-old Erika Nordby stayed naked for almost two hours in a 24-degree frost. When the girl was found, she was in a state of clinical death with body temperature: 16°C. Erica was successfully resuscitated, avoiding amputation of frostbitten limbs .
In both cases described, medical care was provided relatively quickly, which cannot be said about the third case, which occurred in 2006, when 35-year-old Japanese Mitsutaki Yuchikoshi received help only on the 25th day! All this time he was lying on Mount Rocco with fractures of the pelvic bones.
However, when Mitsutaki was found, his pulse was palpable, and his body temperature was 22 ° C. Therefore, there was probably not a state of hypobiosis, but, as in the experiments of Faye and Smith, hibernation .
All three cases are an example of a random coincidence of factors used in modern medicine (for example, in craniocerebral hypothermia) to immerse a person in a state of artificial hypobiosis, when complete freezing of the body does not occur. These cases are an example of low–temperature hypobiosis, but not human cryoanabiosis!
So is it possible to completely freeze a person reversibly?
Recipe for full freezing
The only way to immerse the body in cryoanabiosis is the so-called "shock freezing", when cooling occurs so quickly that the water instantly turns into an amorphous state before it has time to crystallize . Defrosting should also be fast, in order to avoid recrystallization of water.
So far, this trick can be carried out only with individual cells: the culture is sprayed in an environment with a temperature of liquid nitrogen (-196 ° C), where, thanks to its micro-volume, the cells instantly freeze . It is proved that almost all types of microorganisms can safely survive the "shock" freezing.
But the prospect of successful freezing of a multicellular organism is still vague, because it is associated with solving a complex problem: reducing the cooling rate of water with its subsequent transition to an amorphous, rather than crystalline, state [19, 20]. A certain hope here is placed on cryoprotectors – substances that give the body resistance to freezing. Some of the cryoprotectors are simultaneously xeroprotectors, which once again proves the similarity of the mechanisms of xero- and cryoanabiosis (Fig. 6).
Figure 6. The addition of trehalose (a) or glycerin (b) to the nutrient medium makes microbes resistant to freezing. These cryoprotectors are found in the hemolymph of frost-resistant insects, and "glycerin antifreeze" flows through the veins of polar fish [21-24]. Drawings from Wikipedia.
In fact, in all cases of suspended animation, one mechanism is found: liquid water changes its aggregate state, either evaporating (xeroanabiosis) or freezing (cryoanabiosis). Therefore, the ideal option for immersion in suspended animation is a combination of vacuum dehydration with freezing and storage of a biological object at ultra-low temperatures. In this state, the body is maximally resistant to many extreme environmental factors.
The endospores of bacteria are the most resistant to various extreme environmental factors . So, in 1995, microbiologists of the California Polytechnic University reported the revival of bacterial spores in Bacillus sphaericus extracted from the intestines of a bee "preserved" in a piece of amber 25-40 million years ago (Fig. 7)! And already in 2000 there was a message that the staff of West Chester University (Pennsylvania, USA) resurrected spores of an unknown bacterium that has been waiting in the wings in a salt crystal for about 250 million years . Part of the scientific community, of course, simply did not believe in the reliability of this information, considering that the combination of background radiation with the absence of DNA repair for such a long time must have led to the occurrence of a lethal mutation.
In second place in terms of resistance to extreme factors are more highly organized than bacteria, slow–moving organisms (Fig. 8). They are able to stay in suspended animation for more than a hundred years, while withstanding 100-degree heat and radiation, 1000 times higher than the lethal dose for humans. Slow walkers can withstand even a short stay in outer space [27-29]! By the way, the latter circumstance encourages modern supporters of the panspermia hypothesis .
Figure 7 (left): a prehistoric bee, "preserved" in a drop of amber. Drawing from the website altaj-inaki.com . Figure 8 (right):
a slow walker, or "water bear", in person. Drawing from Wikipedia.
The concepts of xero- and cryoanabiosis have been consciously used in biomedicine for a long time. For example, with the help of lyophilization (drying of a frozen biological object), microbial cells and spores, as well as human platelets, are preserved. And cryobanks with their very unusual "contributions": collections of microbial cultures, cell lines, seeds, gametes, embryos and tissues for transplantation are no surprise to anyone [18, 1, 31]. These are the realities of today.
What are the prospects for applying knowledge about suspended animation? And do we need it?
Of course you should! And here's why.
Mastering the technology of human immersion in suspended animation will allow a deeper understanding of the essence of life processes. Suspended animation as a fact of reversible stopping of these processes at the molecular level will be a real proof of the absence of a clear boundary between the concepts of "life" and "death". We are already on the verge of creating biomedical technologies that recently seemed fantastic...
"We suspend life, but we don't call it suspended animation, because it sounds like science fiction <...> we call it emergency preservation and resuscitation," says Samuel Tisherman, a leading surgeon of an experiment starting at the Presbyterian Hospital (Pittsburgh, USA), during which the patient will be connected to a heart–lung machine, after which his blood will be replaced with a special cooled liquid.
But why all this?
Let's let another participant in the experiment, surgeon Peter Rea, answer this question: "If a patient comes to us two hours after death, we will not be able to bring him back to life. But if he dies and we suspend his life processes, there is a chance to start them after the structural problems are fixed" (Fig. 9).
Figure 9. The experiment, which was conceived by a tandem of high-class Tisherman-Ri surgeons, literally freezes the blood in the veins, and this is not a metaphor. Left: Samuel Tisherman, right: Peter Rea. Photo from Wikipedia.
Of course, this technology of immersion in fact into a state of low-temperature hypobiosis has long been successfully tested on experimental subjects... pigs. In 2000, Peter Rea simply "froze" animals. Six years later, he had already "frozen" and then operated on mortally wounded havroni, achieving a 90% survival rate .
The first test of "emergency preservation and resuscitation" is planned to be carried out on 10 injured people with a pronounced cardiac arrest. The body temperature of the victims will be reduced for a couple of hours to 10 ° C. This time should be enough for surgeons to eliminate fatal wounds. The comparison group will be retrospectively evaluated ten sad cases of similar injuries with unsuccessful attempts of traditional resuscitation.
You don't have to die!
The mechanism of suspended animation was evolutionarily formed and acquired the biological meaning of an "emergency exit" in situations when life was on the verge of its irreversible disappearance – death. A well–known researcher of this phenomenon, Pyotr Yulievich Schmidt, expressed this idea more deeply and elegantly: "Life for its preservation creates the absence of life, as if temporary death!"
It is clear that extreme environmental factors can destroy any organism. But are the conditions of the outside world the only cause of death? Will a person, having created an ideal environment for his dwelling, avoid death? No, of course not. There is an internal process inherent in life itself that pushes it to disappear. This is an aging process that we can slow down, but not stop.
Aging is the situation when life is approaching the threshold of its final disappearance. So isn't it logical to use the "emergency exit" and temporarily "die", plunged into suspended animation? Of course, this will not stop aging, but it will stretch life for centuries! Perhaps even such a temporary "death" will help to live up to those glorious times (and maybe even to the Apocalypse – it's how lucky), when aging will be defeated, and all people will be young and happy. Then the person will look like Count Dracula, periodically rising from the grave... ugh – from suspended animation, in order to taste the fruits of the progress of the current century and again go into temporary oblivion...
Considering two circumstances: 1) the more water in the tissue, the better it will survive dehydration and 2) the development of an individual is accompanied by gradual dehydration of his tissues, – we can come to the following conclusion: a successful immersion in suspended animation is more likely in the early stages of individual development, in other words – long before old age. Otherwise, we should look for ways to slow down the "ontogenetic" dehydration, which is expressed in the compaction and wrinkling of aging tissues.
And finally the last question. Let's say we have learned to immerse a person in a real suspended animation. How will this affect his personality? How high is the probability of its partial or complete loss?
Considering that the substrate of the human psyche – brain tissues contain a large amount of water, which means they should survive dehydration well – one can hope for the successful preservation of the personality of a person in suspended animation.
Someday, the technology of human immersion in suspended animation will be developed. His life will be lengthened and his death delayed. It's hard to say how people will use this opportunity. We can only hope that by the time such a technology is created, a person will not forget his dream of flying to the edge of the universe – to distant stars!
Suspended animation and longevity, worms and genes
To live for many hundreds of years, periodically "rising from oblivion" and spending a significant part of the time in suspended animation, is not very interesting. It would be great to do without long periods of inactivity, living both long and active at the same time. It's the same as with sleep: if we didn't have to sleep, how many useful things we would have done! What would you have achieved! But, alas, it will not be possible to cancel the dream yet.
In a dream, at least you can entertain yourself by watching dreams, but in suspended animation you will not be able to do this. But is there a theoretical possibility to avoid long episodes of anabiotic "downtime" and live with it for 100 years or more?
Let's not underestimate our smaller brothers (in every sense) and turn to the help of well–known model organisms - roundworms Caenorhabditis elegans. Like many other invertebrates, these millimeter nematodes change their appearance several times during their life, in other words, they shed. Normally, C. elegans has four larval stages (L1–L4) and an adult stage.
If a fry always turns out to be a fish, and a tadpole always turns out to be a frog, then the development of Caenorhabditis elegans may have one interesting offshoot. If (right on Darwin!) there are too many relatives around, and there is too little food, this nematode at the L1 or L2 stage turns off the usual path "molt → molt → molt → mature individual" and falls into a state with a very low metabolic rate, without genitals and without visible increase in size. This condition is called dauer (from German "durable, long-lasting"; it is also called diapause or hypobiosis; Fig. 10). The same thing happens if the ambient temperature is too low or too high, and also if the worm's receptors pick up a dower-inducing pheromone. Actually, its concentration indicates the population density in the worm population.
Figure 10. Roundworm Caenorhabditis elegans. a – A sexually mature hermaphrodite worm lays eggs. The b – stage of the dower differs in structure from the adult and does not reproduce. Drawings from .
Simultaneously with the transition to the Dower stage, the daf-2 gene encoding the insulin receptor in nematodes and playing a role in the metabolic pathway of the insulin-like growth factor IGF-1 (insulin-like growth factor 1) begins to work actively in the C. elegans larva . (By the way, this metabolic pathway is arranged approximately the same in roundworms, fish with frogs, and us.) The result of reactions of the IGF-1 metabolic pathway is inhibition of the activity of the daf-16 gene. Meanwhile, daf-16 regulates the work of a whole hundred genes responsible for the production of heat shock proteins (molecules that help the cell to withstand elevated ambient temperatures and generally increase resistance to stressors) and antioxidants . Studies of the corresponding gene mutants have shown that both daf-2 and daf-16 are necessary for the onset of the dower stage, but these genes act in the opposite way.
From a dower worm can turn into a sexually mature individual if it is affected by a sufficient amount of steroid hormones, united by the common name dafachronic acids (Fig. 11). These acids, similar in structure to bile components, activate the daf-12 gene, which serves as a transcription factor for genes of several receptors. Among the latter are the vitamin D receptor, as well as Liver–X and Farnesoid-X (they regulate the metabolism of cholesterol and fatty acids). Daphachronic acids become plentiful in the presence of sufficient amounts of insulin and TGF-β (transforming growth factor beta, which restricts cell reproduction and does not control cancer cells). After reaching puberty, worms do not live long.
Figure 11. Diagram of interactions of the IGF-1 pathway with some signaling molecules under various environmental conditions. Drawing from the website www.age.mpg.de .
In the Dower state, nematodes can exist for up to four months. Compared to the three weeks of their life after becoming a sexually mature individual, this is a lot. But, of course, it would be much more interesting for science if C. elegans retained the ability to live for a long time without giving up reproduction and without going into the dower stage. It turned out that this is possible, only for this you need to be a mutant. Active and reproducible nematodes with a mutation in the daf-2 gene live twice as long as "normal" individuals who have not passed the dower stage . By the way, daf-2 mutations do not lead to the development of tumors (as sometimes happens in the case of excessively active work of probable "longevity genes"), but, on the contrary, suppress their development .
The role of daf-2 in the regulation of nematode lifespan was discovered in the early 1990s, after which many biochemical cascades were clarified, thanks to which it is possible to increase life expectancy and provide increased resistance to adverse environmental conditions (Fig. 12). By the way, most of the fateful signals "ordering" the worm to live longer come from the nervous system, and not, say, from muscle tissues and not from the intestine . It turns out that ultimately it is the brain that controls the longevity of nematodes...
Figure 12. Metabolic pathways, the influence on which provides a high life expectancy for worms of the species Caenorhabditis elegans. Arrows – activation, horizontal dashes at the end of the "arrows" – suppression. Figure from .
It would seem, what does human longevity have to do with it? The fact is that, firstly, all the listed nematode genes have their orthologs (correspondences) in the DNA of our species, and secondly, the metabolic pathway of insulin-like growth factor 1 in C. elegans is activated and suppressed by the same substances as in humans. However, it is not necessary to write off the fact that humans do not have larval stages, and even more so there is no dower. Therefore, the possibility of influencing human analogues of biochemical cascades that ensure the longevity of roundworms has yet to be comprehensively considered.
The author of the inset: Svetlana Yastrebova
biomolecule: "From the living to the inanimate and back";
Schmidt P.Y. Anabioz. M.: USSR Academy of Sciences, 1948. – 381 p.;
Clegg J.S. (2001). Cryptobiosis – a peculiar state of biological organization. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 128, 613–624;
Gechev T.S., Dinakar C., Benina M., Toneva V., Bartels D. (2012). Molecular mechanisms of desiccation tolerance in resurrection plants. Cell. Mol. Life Sci. 69, 3175–3186;
Zimina T. (2014). Mosquitoes have found a foothold in space. Science and Life;
Garcia A.H. (2011). Anhydrobiosis in bacteria: From physiology to applications. J. Biosci. 36, 1–12;
Crowe J.H. and Crowe L.M. (2000). Anhydrobiosis: A unique biological state. Amer. Zool. 40, 986;
Kosmachevskaya O.V. (2012). The ubiquitous Maillard reaction. Chemistry and Life. 2, 23–27;
Yashina S., Gubin S., Maksimovich S., Yashina A., Gakhova E., Gilichinsky D. (2012). Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost. Proc. Natl Acad. Sci. USA. 109, 4008–4013;
Zhmakin A.I. Fundamentals of Cryobiology. Physical phenomena and mathematical models. Springer-Verlag Berlin Heidelberg, 2009. – 292 p.;
Muldrew K. and McGann L.E. (1997). Cryobiology – a short course. University of Calgary;
Suspended animation. Medical Encyclopedia website;
Smith L.W. and Fay T. (1939). Temperature factor in cancer and embrional cell growth. J. Med. Am. Assoc. 113, 653–660;
Gilbert M., Busund R., Skagseth A., Nilsen P.A., Solbø J.P. (2000). Resuscitation from accidental hypothermia of 13.7 °C with circulatory arrest. Lancet. 355, 375–376;
Elements: "Suspended animation will prolong your life";
Terekh N. (2006). A 35-year-old Japanese man spent 24 days and nights in the mountains without food and water, while his body temperature dropped to 22 degrees! Website "Facts and comments";
biomolecule: "Vitrification is a controlled pause of development in a glass–like state";
Kirsop B.E. and Doyle A. Maintenance of microorganisms and cultured cells: a manual of laboratory methods. London: Academic Press, 1991. – 308 p.;
Fahy G.M., MacFarlane D.R., Angell C.A., Meryman H.T. (1984). Vitrification as an approach to cryopreservation. Cryobiology. 21, 407–426;
Fahy G.M., Wowk B., Wu J., Phan J., Rasch C., Chang A., Zendejas E. (2004). Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology. 48, 157–178;
Hubálek Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology. 46, 205–229;
biomolecule: "Is life possible without hemoglobin?";
Wharton D.A. Supercooling and freezing tolerant animals. In: Supercooling / ed. by Wilson P. InTech, 2012. P. 17–28;
Wharton D.A. Cold tolerance. In: Molecular and physiological basis of nematode survival / ed. by Perry R.N. and Wharton D.A. Wallingford: CABI Publishing, 2011. P. 182–204;
Setlow P. (2006). Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 101, 514–525;
Russell H., Rosenzweig W.D., Powers D.W. (2000). Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal. Nature. 407, 897–900;
Jönsson K.I., Rabbow E., Schill R.O., Harms-Ringdahl M., Rettberg P. (2008). Tardigrades survive exposure to space in low Earth orbit. Curr. Biol. 18, R729–R731;
Guidettia R., Rizzo A.M., Altiero T., Rebecchi L. (2012). What can we learn from the toughest animals of the Earth? Water bears (tardigrades) as multicellular model organisms in order to perform scientific preparations for lunar exploration. Planet. Space Sci. 74, 97–102;
Wełnicz W., Grohme M.A., Kaczmarek L, Schill R.O., Frohme M. (2011). Anhydrobiosis in tardigrades – the last decade. J. Insect Physiol. 57, 577–583;
Rozanov A. Yu. (2000). Bacterial-paleontological approach to the study of meteorites. Bulletin of the Russian Academy of Sciences. 70, 214–226;
Eiseman E. and Haga S.B. Handbook of human tissue sources: a national resource of human tissue samples. Santa Monica: RAND Corporation, 1999;
Syrov S. (2014). Suspended animation becomes a clinical practice. Website "XXII century – discoveries, expectations, threats";
Gami M. and Wolkow C. (2006). Studies of Caenorhabditis elegans DAF-2/insulin signaling reveal targets for pharmacological manipulation of lifespan. Aging Cell. 5, 31–37;
Gottlieb S. and Ruvkun G. (1994). daf-2, daf-16 and daf-23: genetically interacting genes controlling Dauer formation in Caenorhabditis elegans. Genetics. 137, 107–120;
Ogg S., Paradis S., Gottlieb S., Patterson G., Lee L., Tissenbaum H., Ruvkun G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature. 389, 994–999;
Kenyon C., Chang J., Gensch E., Rudner A., Tabtiang R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature. 366, 461–464;
Pinkston J., Garigan D., Hansen M., Kenyon C. (2006). Mutations that increase the life span of C. elegans inhibit tumor growth. Science. 313, 971–975;
Wolkow C., Kimura K., Lee M.-S., Ruvkun G. (2000). Regulation of C. elegans life-span by insulin-like signaling in the nervous system. Science. 290, 147–150.
Portal "Eternal youth" http://vechnayamolodost.ru