List of Nobel Prize winners in physiology. Prize in Physiology or Medicine. The secret becomes clear
In 2016, the Nobel Committee awarded the Physiology or Medicine Prize to Japanese scientist Yoshinori Ohsumi for discovering autophagy and deciphering its molecular mechanism. Autophagy is a process of recycling spent organelles and protein complexes; it is important not only for the economical management of the cellular economy, but also for the renewal of the cellular structure. Deciphering the biochemistry of this process and its genetic basis suggests the possibility of controlling and managing the entire process and its individual stages. And this gives researchers obvious fundamental and applied perspectives.
Science rushes forward at such an incredible pace that the non-specialist does not have time to realize the importance of the discovery, and the Nobel Prize is already awarded for it. In the 80s of the last century, in biology textbooks, in the section on the structure of the cell, one could learn, among other organelles, about lysosomes - membrane vesicles filled with enzymes inside. These enzymes are aimed at splitting various large biological molecules into smaller units (it should be noted that at that time our biology teacher did not yet know why lysosomes were needed). They were discovered by Christian de Duve, for which he was awarded the Nobel Prize in Physiology or Medicine in 1974.
Christian de Duve and colleagues separated lysosomes and peroxisomes from other cellular organelles using a then new method - centrifugation, which allows particles to be sorted by mass. Lysosomes are now widely used in medicine. For example, targeted drug delivery to damaged cells and tissues is based on their properties: a molecular drug is placed inside the lysosome due to the difference in acidity inside and outside it, and then the lysosome, equipped with specific labels, is sent to the affected tissues.
Lysosomes are illegible by the nature of their activity - they break up any molecules and molecular complexes into their constituent parts. Narrower "specialists" are proteasomes, which are aimed only at the breakdown of proteins (see:, "Elements", 11/05/2010). Their role in the cellular economy can hardly be overestimated: they monitor the enzymes that have served their time and destroy them as needed. This period, as we know, is defined very precisely - exactly as much time as the cell performs a specific task. If the enzymes were not destroyed upon its completion, then the ongoing synthesis would be difficult to stop in time.
Proteasomes are present in all cells without exception, even in those where there are no lysosomes. The role of proteasomes and the biochemical mechanism of their work was investigated by Aaron Ciechanover, Avram Hershko and Irwin Rose in the late 1970s and early 1980s. They discovered that the proteasome recognizes and destroys those proteins that are labeled with the protein ubiquitin. The binding reaction with ubiquitin comes at the expense of ATP. In 2004, these three scientists received the Nobel Prize in Chemistry for their research on ubiquitin-dependent protein degradation. In 2010, while looking through a school curriculum for gifted English children, I saw a row of black dots in a picture of the structure of a cell, which were labeled as proteasomes. However, the school teacher at that school could not explain to the students what it is and what these mysterious proteasomes are for. With lysosomes in that picture, no questions arose.
Even at the beginning of the study of lysosomes, it was noticed that parts of cell organelles are enclosed inside some of them. This means that in lysosomes, not only large molecules are disassembled, but also parts of the cell itself. The process of digesting one's own cellular structures is called autophagy - that is, "eating oneself." How do parts of cell organelles get into the lysosome containing hydrolases? Back in the 80s, he began to deal with this issue, who studied the structure and functions of lysosomes and autophagosomes in mammalian cells. He and his colleagues showed that autophagosomes appear in mass in cells if they are grown on a nutrient-poor medium. In this regard, a hypothesis has arisen that autophagosomes are formed when a reserve source of nutrition is needed - proteins and fats that are part of extra organelles. How are these autophagosomes formed, are they needed as a source of additional nutrition or for other cellular purposes, how do lysosomes find them for digestion? All these questions in the early 1990s had no answers.
Taking on independent research, Osumi focused his efforts on the study of yeast autophagosomes. He reasoned that autophagy should be a conserved cellular mechanism, hence, it is more convenient to study it on simple (relatively) and convenient laboratory objects.
In yeast, autophagosomes are located inside vacuoles and then disintegrate there. Various proteinase enzymes are engaged in their utilization. If the proteinases in the cell are defective, then autophagosomes accumulate inside the vacuoles and do not dissolve. Osumi took advantage of this property to obtain a culture of yeast with an increased number of autophagosomes. He grew cultures of yeast on poor media - in this case, autophagosomes appear in abundance, delivering a food reserve to the starving cell. But his cultures used mutant cells with inactive proteinases. So, as a result, cells quickly accumulated a mass of autophagosomes in vacuoles.
Autophagosomes, as follows from his observations, are surrounded by single-layer membranes, which can contain a wide variety of contents: ribosomes, mitochondria, lipid and glycogen granules. By adding or removing protease inhibitors to wild cell cultures, one can increase or decrease the number of autophagosomes. So in these experiments it was demonstrated that these cell bodies are digested with the help of proteinase enzymes.
Very quickly, in just a year, using the random mutation method, Osumi identified 13–15 genes (APG1–15) and the corresponding protein products involved in the formation of autophagosomes (M. Tsukada, Y. Ohsumi, 1993. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae). Among colonies of cells with defective proteinase activity, he selected under a microscope those in which there were no autophagosomes. Then, cultivating them separately, he found out which genes they had corrupted. It took his group another five years to decipher, as a first approximation, the molecular mechanism of these genes.
It was possible to find out how this cascade works, in what order and how these proteins bind to each other, so that the result is an autophagosome. By 2000, the picture of membrane formation around damaged organelles to be processed became clearer. The single lipid membrane begins to stretch around these organelles, gradually surrounding them until the ends of the membrane approach each other and fuse to form the double membrane of the autophagosome. This vesicle is then transported to the lysosome and fuses with it.
APG proteins are involved in the process of membrane formation, analogs of which Yoshinori Ohsumi and colleagues found in mammals.
Thanks to the work of Osumi, we have seen the whole process of autophagy in dynamics. The starting point of Osumi's research was the simple fact of the presence of mysterious small bodies in the cells. Now researchers have the opportunity, albeit hypothetical, to control the entire process of autophagy.
Autophagy is necessary for the normal functioning of the cell, since the cell must be able not only to renew its biochemical and architectural economy, but also to utilize the unnecessary. There are thousands of worn-out ribosomes and mitochondria, membrane proteins, spent molecular complexes in the cell - all of them need to be economically processed and put back into circulation. This is a kind of cellular recycling. This process not only provides a certain economy, but also prevents the rapid aging of the cell. Disruption of cellular autophagy in humans leads to the development of Parkinson's disease, type II diabetes, cancer, and some disorders associated with old age. Controlling the process of cellular autophagy obviously has great prospects, both in fundamental and applied terms.
Anastasia Ksenofontova
The Nobel Committee named the 2018 Physiology or Medicine Prize Laureates. This year the award will go to James Ellison of the Cancer Center. M.D. Anderson of the University of Texas and Tasuku Honjo of Kyoto University for their "discovery in the field of inhibition of the immune system to more effectively attack cancer cells." Scientists have figured out how a cancerous tumor "cheats" the immune system. This made it possible to create an effective anti-cancer therapy. Read more about the opening in the material RT.
- 2018 Nobel Laureates in Physiology or Medicine James Ellison and Tasuku Honjo
- TT News Agency/Fredrik Sandberg via REUTERS
The Nobel Committee of the Karolinska Institute in Stockholm on Monday, October 1, announced the winners of the 2018 Prize. The award will be presented to the American James Ellison from the Cancer Center. M.D. Anderson of the University of Texas and Tasuku Honjo of Japan from Kyoto University for "the discovery in the field of inhibition of the immune system to more effectively attack cancer cells." Scientists have figured out how a cancerous tumor "cheats" the immune system. This made it possible to create an effective anti-cancer therapy.
Cell Wars
Among the traditional methods of cancer treatment, chemotherapy and radiation therapy are the most common. However, there are also “natural” methods of treating malignant tumors, including immunotherapy. One of its promising areas is the use of inhibitors of "immunity checkpoints" located on the surface of lymphocytes (cells of the immune system).
The fact is that the activation of “immunity checkpoints” suppresses the development of the immune response. Such a "checkpoint" is, in particular, the CTLA4 protein, which Ellison has been studying for many years.
In the coming days, winners of awards in other categories will be announced. On Tuesday, October 2, the committee will announce the laureate in physics. On October 3, the name of the winner of the Nobel Prize in Chemistry will be announced. On October 5, the Nobel Peace Prize will be awarded in Oslo, and on October 8, the winner in the field of economics will be determined.
The winner of the Literature Prize will not be named this year - it will be announced only in 2019. This decision was made by the Swedish Academy due to the fact that the number of its members has decreased, and a scandal erupted around the organization. 18 women have accused the husband of the poetess Katharina Frostenson, who was elected to the academy in 1992, of sexual harassment. As a result, seven people left the Swedish Academy, including Frostenson herself.
In recent years, we have almost forgotten how to understand why they receive the Nobel Prize in Medicine. So complex and incomprehensible to the ordinary mind are the studies of the laureates, so ornate are the formulations explaining the reasons for its award. At first glance, the situation is similar here. How do we understand what "suppression of negative immune regulation" means? But in fact, everything is much simpler, and we will prove it to you.
First, the results of the laureates' research have already been introduced into medicine: thanks to them, a new class of drugs for the treatment of cancer has been created. And they have already saved the life of many patients or significantly extended it. The drug ipilimumab, made thanks to research James Ellison, was officially registered in the US by the Food and Drug Administration in 2011. Now there are several such drugs. All of them affect the key links in the interaction of malignant cells with our immune system. Cancer is a great deceiver and knows how to deceive our immunity. And these drugs help him to restore his working capacity.
The secret becomes clear
Here is what the oncologist, MD, professor, head of the scientific laboratory for cancer chemoprevention and oncopharmacology of the National Medical Research Center of Oncology named after N.N. N. N. Petrova Vladimir Bespalov:
- Nobel laureates have been conducting their research since the eighties, and thanks to them, a new direction in the treatment of cancer was then created: immunotherapy with the help of monoclonal antibodies. In 2014, it was recognized as the most promising in oncology. Thanks to the research of J. Ellison and T. Honjo Several new effective drugs have been developed for the treatment of cancer. These are high-precision tools aimed at specific targets that play a key role in the development of malignant cells. For example, the drugs nivolumab and pembrolizumab block the interaction of specific proteins PD-L-1 and PD-1 with their receptors. These proteins, produced by malignant cells, help them "hide" from the immune system. As a result, tumor cells become as if invisible to our immune system and it cannot resist them. New drugs make them visible again, and thanks to this, the immune system begins to destroy the tumor. The first drug created thanks to the Nobel laureates was ipilimumab. It has been used to treat metastatic melanoma but has had serious side effects. New generation drugs are safer, they treat not only melanoma, but also non-small cell lung cancer, bladder cancer and other malignant tumors. Today, there are already several such drugs, and they continue to be actively investigated. Now they are being tested in some other types of cancer, and perhaps the range of their application will be wider. Such drugs are registered in Russia, but, unfortunately, they are very expensive. A single course of administration costs more than a million rubles, and they need to be repeated later. But they are more effective than chemotherapy. For example, up to a quarter of patients with advanced melanoma are completely cured. This result cannot be achieved by any other drugs.
Monoclones
All these drugs are monoclonal antibodies, absolutely similar to human ones. Only our immune system does not make them. Preparations are obtained using genetic engineering technologies. Like conventional antibodies, they block antigens. The latter are active regulatory molecules. For example, the first drug, ipilimumab, blocked the regulatory molecule CTLA-4, which plays a critical role in protecting cancer cells from the immune system. It is this mechanism that was discovered by one of the current laureates, J. Elisson.
Monoclonal antibodies are the mainstream in modern medicine. Based on them, many new drugs for serious diseases are being created. For example, such drugs have recently appeared for the treatment of high cholesterol. They specifically bind to regulatory proteins that regulate cholesterol synthesis in the liver. By turning them off, they effectively inhibit its production, and cholesterol is reduced. Moreover, they act specifically on the synthesis of bad cholesterol (LDL), without affecting the production of good cholesterol (HDL). These are very expensive drugs, but the price of them is rapidly and sharply declining due to the fact that they are used more and more often. That's how it used to be with statins. So over time, they (and new cancer treatments, hopefully, too) will become more accessible.
In 2018, the Nobel Prize in Physiology or Medicine was awarded to two scientists from different parts of the world - James Ellison from the USA and Tasuku Honjo from Japan - who independently discovered and studied the same phenomenon. They found two different checkpoints - the mechanisms by which the body suppresses the activity of T-lymphocytes, immune killer cells. If these mechanisms are blocked, then T-lymphocytes "go free" and go to battle with cancer cells. This is called cancer immunotherapy, and it has been used in clinics for several years.
The Nobel Committee loves immunologists: at least one in ten awards in physiology or medicine is given for theoretical immunological work. This year we are talking about practical achievements. The Nobel laureates of 2018 are recognized not so much for theoretical discoveries as for the consequences of these discoveries, which have been helping cancer patients fight tumors for six years now.
The general principle of the interaction of the immune system with tumors is as follows. As a result of mutations in tumor cells, proteins are formed that differ from the “normal” ones that the body is used to. Therefore, T cells react to them as if they were foreign objects. In this they are helped by dendritic cells - spy cells that crawl through the tissues of the body (for their discovery, by the way, they were awarded the Nobel Prize in 2011). They absorb all the proteins passing by, break them down and expose the resulting pieces to their surface as part of the MHC II protein complex (major histocompatibility complex, see for more details: Mares determine whether or not to become pregnant by the major histocompatibility complex ... neighbor, "Elements" , 01/15/2018). With this baggage, dendritic cells travel to the nearest lymph node, where they show (present) these pieces of trapped proteins to T-lymphocytes. If a T-killer (cytotoxic lymphocyte, or killer lymphocyte) recognizes these antigen proteins with its receptor, then it is activated - it begins to multiply, forming clones. Then the cells of the clone scatter throughout the body in search of target cells. On the surface of every cell in the body there are MHC I protein complexes, in which pieces of intracellular proteins hang. The killer T is looking for an MHC I molecule with a target antigen that it can recognize with its receptor. And as soon as recognition has occurred, the T-killer kills the target cell, making holes in its membrane and triggering apoptosis (death program) in it.
But this mechanism does not always work effectively. A tumor is a heterogeneous system of cells that use a variety of ways to elude the immune system (read about one of the recently discovered such ways in the news Cancer cells increase their diversity by merging with immune cells, "Elements", 09/14/2018). Some tumor cells hide MHC proteins from their surface, others destroy defective proteins, and still others secrete substances that suppress the immune system. And the "angrier" the tumor, the less likely the immune system is to cope with it.
Classical methods of fighting a tumor involve different ways of killing its cells. But how to distinguish tumor cells from healthy ones? Usually, the criteria are “active division” (cancer cells divide much more intensively than most healthy cells in the body, and radiation therapy is aimed at this, damaging DNA and preventing division) or “resistance to apoptosis” (chemotherapy helps fight this). With such treatment, many healthy cells, such as stem cells, suffer, and inactive cancer cells, such as dormant cells, are not affected (see:, "Elements", 06/10/2016). Therefore, now they often rely on immunotherapy, that is, the activation of the patient's own immunity, since the immune system distinguishes a tumor cell from a healthy one better than external drugs. The immune system can be activated in a variety of ways. For example, you can take a piece of a tumor, develop antibodies to its proteins and inject them into the body so that the immune system “sees” the tumor better. Or pick up immune cells and train them to recognize specific proteins. But this year's Nobel Prize is awarded for a completely different mechanism - for removing the blockage from killer T cells.
When this story was just beginning, no one thought about immunotherapy. Scientists tried to unravel the principle of interaction between T cells and dendritic cells. Upon closer examination, it turns out that not only MHC II with the antigen protein and the T cell receptor are involved in their “communication”. Next to them on the surface of the cells are other molecules that also participate in the interaction. This whole structure - a set of proteins on membranes that connect to each other when two cells meet - is called an immune synapse (see Immunological synapse). The composition of this synapse includes, for example, costimulatory molecules (see Co-stimulation) - the very ones that send a signal to T-killers to activate and go in search of the enemy. They were the first to be discovered: this is the CD28 receptor on the surface of the T cell and its ligand B7 (CD80) on the surface of the dendritic cell (Fig. 4).
James Ellison and Tasuku Honjo independently discovered two more possible components of the immune synapse - two inhibitory molecules. Ellison worked on the CTLA-4 molecule discovered in 1987 (cytotoxic T-lymphocyte antigen-4, see: J.-F. Brunet et al., 1987. A new member of the immunoglobulin superfamily - CTLA-4). It was originally thought to be another co-stimulator because it only appeared on activated T cells. Ellison's merit is that he suggested that the opposite is true: CTLA-4 appears on activated cells specifically so that they can be stopped! (M. F. Krummel, J. P. Allison, 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation). Further, it turned out that CTLA-4 is similar in structure to CD28 and can also bind to B7 on the surface of dendritic cells, even more strongly than CD28. That is, on every activated T cell, there is an inhibitory molecule that competes with an activating molecule to receive a signal. And since there are many molecules in the immune synapse, the result is determined by the ratio of signals - how many CD28 and CTLA-4 molecules could bind to B7. Depending on this, the T-cell either continues to work, or freezes and cannot attack anyone.
Tasuku Honjo discovered another molecule on the surface of T cells - PD-1 (its name is short for programmed death), which binds to the PD-L1 ligand on the surface of dendritic cells (Y. Ishida et al., 1992. Induced expression of PD- 1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death). It turned out that PD-1 knockout mice (deprived of the corresponding protein) develop something similar to systemic lupus erythematosus. It is an autoimmune disease, which is a condition where immune cells attack normal molecules in the body. Therefore, Honjo concluded that PD-1 also works as a blocker, holding back autoimmune aggression (Fig. 5). This is another manifestation of an important biological principle: every time any physiological process is triggered, the opposite one (for example, blood coagulation and anticoagulation systems) is launched in parallel in order to avoid “overfulfillment of the plan”, which can be detrimental to the body.
Both blocking molecules - CTLA-4 and PD-1 - and their corresponding signaling pathways were called immune checkpoints (from the English. checkpoint- checkpoint, see Immune checkpoint). Apparently, this is an analogy with cell cycle checkpoints (see Cell cycle checkpoint) - the moments at which the cell "makes a decision" whether it can continue to divide further or some of its components are significantly damaged.
But the story didn't end there. Both scientists decided to find a use for the newly discovered molecules. Their idea was that immune cells could be activated by blocking blockers. True, autoimmune reactions will inevitably be a side effect (as is happening now in patients who are treated with checkpoint inhibitors), but this will help defeat the tumor. Scientists proposed blocking blockers with the help of antibodies: by binding to CTLA-4 and PD-1, they mechanically close them and prevent them from interacting with B7 and PD-L1, while the T cell does not receive inhibitory signals (Fig. 6).
At least 15 years have passed between the discoveries of checkpoints and the approval of drugs based on their inhibitors. Currently, six such drugs are used: one CTLA-4 blocker and five PD-1 blockers. Why did PD-1 blockers work better? The fact is that the cells of many tumors also carry PD-L1 on their surface in order to block the activity of T-cells. Thus, CTLA-4 activates killer T cells in general, while PD-L1 has a more specific effect on the tumor. And complications in the case of PD-1 blockers occur somewhat less.
Unfortunately, modern methods of immunotherapy are not yet a panacea. First, checkpoint inhibitors still do not provide 100% patient survival. Secondly, they do not act on all tumors. Thirdly, their effectiveness depends on the genotype of the patient: the more diverse its MHC molecules, the higher the chance of success (on the diversity of MHC proteins, see: Diversity of histocompatibility proteins increases reproductive success in male reed warblers and reduces in females, "Elements", 29.08 .2018). Nevertheless, it turned out to be a beautiful story about how a theoretical discovery first changes our understanding of the interaction of immune cells, and then gives rise to drugs that can be used in the clinic.
And the Nobel laureates have something to work on further. The exact mechanisms by which checkpoint inhibitors work are still not fully understood. For example, in the case of CTLA-4, it is not clear which cells the drug-blocker interacts with: with T-killers themselves, or with dendritic cells, or in general with T-regulatory cells - a population of T-lymphocytes responsible for suppressing the immune response. . So this story is actually far from over.
Polina Loseva
The Nobel Committee has announced the winners of the 2017 Physiology or Medicine Prize today. This year the award will once again travel to the US, with Michael Young of the Rockefeller University in New York, Michael Rosbash of Brandeis University and Geoffrey Hall of the University of Maine sharing the award. According to the decision of the Nobel Committee, these researchers were awarded "for their discoveries of the molecular mechanisms that control circadian rhythms."
It must be said that in the entire 117-year history of the Nobel Prize, this is perhaps the first prize for the study of the sleep-wake cycle, as well as for anything related to sleep in general. The famous somnologist Nathaniel Kleitman did not receive the award, and Eugene Azerinsky, who made the most outstanding discovery in this area, who discovered REM sleep (REM - rapid eye movement, rapid sleep phase), generally received only a PhD degree for his achievement. It is not surprising that in numerous forecasts (we wrote about them in our article) there were any names and any research topics, but not those that attracted the attention of the Nobel Committee.
What was the award for?
So, what are circadian rhythms and what exactly did the laureates discover, who, according to the secretary of the Nobel Committee, greeted the news of the award with the words “Are you kidding me?”.
Geoffrey Hall, Michael Rosbash, Michael Young
Circa diem translated from Latin as "around the day". It so happened that we live on planet Earth, where day is replaced by night. And in the course of adapting to different conditions of day and night, organisms developed an internal biological clock - the rhythms of the biochemical and physiological activity of the organism. It was only in the 1980s that it was possible to show that these rhythms had an exclusively internal nature by sending mushrooms into orbit. Neurospora crassa. Then it became clear that circadian rhythms do not depend on external light or other geophysical signals.
The genetic mechanism of circadian rhythms was discovered in the 1960–1970s by Seymour Benzer and Ronald Konopka, who studied mutant lines of fruit flies with different circadian rhythms: in wild-type flies, circadian rhythm fluctuations had a period of 24 hours, in some mutants - 19 hours, in others - 29 hours, and the third had no rhythm at all. It turned out that rhythms are regulated by the gene PER - period. The next step, which helped to understand how such fluctuations in the circadian rhythm are created and maintained, was taken by the current laureates.
Self-adjusting clockwork
Geoffrey Hall and Michael Rosbash suggested that the gene encoded period PER protein blocks the work of its own gene, and such a feedback loop allows the protein to prevent its own synthesis and cyclically, continuously regulate its level in cells.
The picture shows the sequence of events over 24 hours of fluctuation. When the gene is active, PER mRNA is produced. It exits the nucleus into the cytoplasm, becoming a template for the production of the PER protein. The PER protein accumulates in the cell nucleus when the activity of the period gene is blocked. This closes the feedback loop.
The model was very attractive, but a few pieces of the puzzle were missing to complete the picture. To block the activity of a gene, the protein needs to get into the nucleus of the cell, where the genetic material is stored. Jeffrey Hall and Michael Rosbash showed that the PER protein accumulates overnight in the nucleus, but did not understand how it managed to get there. In 1994, Michael Young discovered the second circadian rhythm gene, timeless(English "timeless"). It codes for the TIM protein, which is essential for our internal clock to function properly. In his elegant experiment, Young demonstrated that only by binding to each other, TIM and PER paired can enter the cell nucleus, where they block the gene period.
Simplified illustration of the molecular components of circadian rhythms
This feedback mechanism explained the reason for the appearance of oscillations, but it was not clear what controls their frequency. Michael Young found another gene double time. It contains the DBT protein, which can delay the accumulation of the PER protein. This is how fluctuations are “debugged” so that they coincide with the daily cycle. These discoveries revolutionized our understanding of the key mechanisms of the human biological clock. Over the following years, other proteins were found that influence this mechanism and maintain its stable operation.
Now the prize in physiology or medicine is traditionally awarded at the very beginning of the Nobel week, on the first Monday in October. It was first awarded in 1901 to Emil von Behring for the development of a serum therapy for diphtheria. In total, the prize has been awarded 108 times throughout history, in nine cases: in 1915, 1916, 1917, 1918, 1921, 1925, 1940, 1941 and 1942, the prize was not awarded.
Between 1901 and 2017, the prize was awarded to 214 scientists, a dozen of whom are women. So far, there has not been a case of someone receiving a prize in medicine twice, although there have been cases when an already acting laureate was nominated (for example, our Ivan Pavlov). Excluding the 2017 award, the average age of the laureate was 58 years. The youngest Nobel laureate in the field of physiology and medicine was the 1923 laureate Frederick Banting (award for the discovery of insulin, age 32), the oldest was the 1966 laureate Peyton Rose (award for the discovery of oncogenic viruses, age 87).
