Biomedicine

In prof. C. Schmitt’s laboratory, molecular mechanisms of cell’s response on stress are studies. Stress-mediated responses are accompanied by the induction of apoptosis or cell aging that stops precancer state of the cell and afterwards kills (or isolates) the changed cell. However, when the cell undergoes oncogenic stimulation over a long period of time, it can overcome the barrier and irretrievably acquire oncogenic phenotype. In prof. C. Schmitt’s laboratory, mouse models of lymphoma and other tumors caused by known genetic disorders were created to study cell’s response on stress, which is induced by the anticancer therapy. On the basis of those models, antitumor role of Suv39h1 histone methylase was shown. Suv39h1 histone methylase inhibition in mice led to the development of the B-cell lymphoma directed by the constitutive expression of the embedded Ras proto-oncogene. Methylation of the H3R9 histone (it is a target for the Suv39h1 methylase) prevented formation of lymphomas and initiated the process of B-cell aging as protective mechanism against aging in the Suv39h1-deficient mice. It was also established that genetic disorders in the INK4a/ARF locus had modulatory role in sensitivity/resistance of different lymphoma types to anticancer therapy. Oncogenic signals activated stress response in normal cells. That stress response is a peculiar barrier on the way of cell transformation from the normal cell into the tumor one. So cell aging or apoptosis induced by antitumor therapy is a protective mechanism that prevents further expansion (metastasis) of tumor cells.

In the laboratory headed by K. Khrapko, particular interest is paid to the role of mtDNA somatic mutations in human aging. Scientists investigate the amount of mtDNA mutations in individual cells. Using such approach — evaluating the amount of mtDNA mutations in individual cells — it was found that the level of mtDNA deletions in human pigmented neurons was very high. Among other research guidelines of the laboratory, there are studies focused on mtDNA recombination and studies of mtDNA using microarrays. It is generally assumed that mtDNA mutations are created in the cells where those mutations are currently found. However, it was shown that cells with a particular mtDNA mutation tended to «cluster». Cells of those clusters are usually descendants of the single sell. Thus, mutations in mtDNA do not appear the cells of a cluster, but in progenitor cells, such as stem cells, or even earlier in the development. MtDNA mutatios in progenitor cells may be one of the major sources of mtDNA mutations in healthy aging tissue.

Many mitochondrial diseases are provoked by structural and functional changes in the complex I. Therefore, the major aim of the studies, T. Yagi’s team conducts, is to develop a therapy for compensation of the complex I defect. The most promising method is transfection with NADH gene of yeasts (Saccharomyces cerevisiae). That gene consist of one subunit (Ndi1) responsible for the NADH to ubiquinone-10 (UQ10) electron transfer in the mammalian mitochondria. Using complex I-deficient Chinese hamster CCL16-B2 mutant cell lines, the researchers from T. Yagi’s laboratory performed successful transfection of those cells’ mitochondria with the Ndi1 gene. The Ndi1-transfected cells with functionally active NADH dehydrogenase, as well as the healthy cells (control with the intact complex I gene) showed electron transfer when malate plus glutamate were employed as respiratory substrates. So, thansfection with Ndi1 gene opens new possibilities in the treatment of mitochondrial diseases with the use of gene therapy.

The main guideline of R. Freitas work is development of medical nanorobots for the diagnostics, the repair of damaged tissues, cells and organs (including those after cryopreservation), DNA analysis and correction, and elimination of bacteria and viruses. Using modern technologies, the manufacturing of nanorobots may start in the next 15–20 years. R. Freitas believes that the size of the device should not exceed 1×1×3 µm (without locomotor flagellums). Molecular manufacturing that uses nanotechnologies will enable to solve the problem of any organ treatment and restoration at the molecular level. Moreover, this technology will help to create artificial blood cells and cells with new functionality (for example, respirocytes that will help the human to breath underwater). In addition, it will help to stop the use of antibiotics in the treatment of any infectious diseases, cure cancer and heal any wounds, it will help to replace chromosomes and so, it will deliver people from genetic diseases. Nanorobots will enable to remove defects that have accumulated in the organism and cause aging. As the result, nanorobots may considerably prolong human youth and life span.

G. Forgacs is one of the leading scientists in the Organ Printing Project. Considerable part of his papers is devoted to study of physical mechanisms which underlie biological self-organization, particularly — self-assembly of cell structures. Concept of tissue fluidity firstly proposed in the Malcolm Steinberg’s differential adhesion hypothesis was experimentally validated in G. Forgacs’s studies and became the molecular base for the bioprinting technology. 3d printing of living tissues became possible due to existence of tissue surface tension and the ability of the same cells to stick together into spherical structures, The special «ink» is used in 3d bioprinting — it contains microspheres with 10 — 40 thousands of cells. It is established that, when such 3d bioprinting is used, each type cells migrate at the appropriate place and build up tissues and organs, which forms are defined by the arrangement of matrix particles («biopaper»). Biocompatible matrices, which are used in bioengineering, generally show positive results. However, they can cause a set of undesirable problems, too. For example, matrix immunogenicity, degradation velocity and toxicity of the products, formation of the fibrous tissue during degradation, interaction with adjacent tissues etc. can influence the late fate of transplantation and directly affect biological functions of the bioengineered tissue. Properties of extracellular matrix are extremely critical in the modelling of vascular tissues. Creation of artificial blood vessels with small diameter, which have mechanical strength comparable to those of the native vessels, is still one of the most difficult problems of tissue engineering. To solve that problem, a new approach, where agarous bars were used as building blocks for the form filled with tissue spheroids or cylinders, was developed. Layer-by-layer arrangement of agarous bars and standard multicellular structures (spheroids and cylinders) enable precise regulation of the internal diameter, the wall thickness and the pattern of vessel branching. All the process, which includes removing of the agarous bars, is automated and enables to obtain one-layered, as well as two-layered blood vessels. Such approach has many advantages, and it makes it possible to prevent a lot of problems linked to the presence of exogenous materials. Since constructions obtained are made of cells only, high cell density can be achieved. So the properties of such bioengineered vessels came close to the properties of the naive ones. Moreover, when multicellular cylinders are used as a «bioink», the maturing time decrease and forms of the final structures become more accurate. The important achievement of G. Forgacs and colleagues is the use of bioprinting for reconstruction of frameless blood vessels that enables to obtain vessels of various diameter and shape necessary for transplantation. The next stage of bioprinting must be creation of complicated branched macro- and microvascular systems with the internal diameter from 300 mkm and the wall thickness from 100 mkm, which will be available for clinical implantation.

The major research area of O. Srivastava’s laboratory is the study on the role of cross-links in crystallin protein aggregation during aging and cataractogenesis. Cristallin is the joint name for a family of proteins found in the eye’s crystalline lens and cornea of human and other mammals. Crystallin consists of several individual proteins: αА- and αВ- crystallins are the chaperone proteins maintaining the structure of crystalline lens proteins, and consequently, lens transparency; γ-crystallin is the structural protein found in the lens. The purpose of the study was to determine in vitro cross-linking of gamma D-crystallin fragments alone and with alpha-, beta- and gamma-crystallins. Moreover, the scientists tried to ascertain the existence of covalent multimers of the polypeptide in vivo, and post-translational modifications in the three isoforms of the polypeptide. The study has shown that crystallin fragments are covalently crosslinked via non-disulfide bonding. The polypeptide also exhibited crosslinking with individual alpha-, beta-, and gamma-crystallins. Tryptophan and methionine oxidation are posttranslational modifications of the crystallin polypeptide. So gamma D-crystallin fragment is able to crosslink another fragments and stimulates oxidation of tryptophan and methionine residues. The scientists have found two types of multimers which appear in youth and keep on accumulating during aging. The first type consists of 8 different crystallins (αА, αB etc.). The second type shows the presence of filensin and phakinin proteins in addition to crystallin fragments. Crystallin fragments undergo post-translational modifications. It is found that during cataractogenesis, multimers are accumulating more intensively than in heathy people. Insoluble βA3/A1 and βB1 crystallin fragments were found in the lens of patients with cataract. Further study on different types of crystallin proteins and its modifications found in the crystalline lens is necessary to develop a new treatment for cataract.

It has been known long ago that inhibition of TOR enlarges life span of invertebrates, particularly yeasts, nematodes and fruit flies. However, it has remained undecided for a long time whether inhibition of TOR enlarges life span of mammals. In the experiments carried out by D. Harrison’s team, TOR was inhibited by rapamycin. Three groups of genetically heterogeneous mice were used in the study. It was found that the life span considerably increased in the mice of all three groups: females showed 14 % increase, while males showed 9 % rise. As far as we now know, inhibition of TOR activates macroautophagy. That study has shown for the first time that inhibition of TOR enlarges life span of mammals, not only that of invertebrates.

The major research area of prof. A. Ryazanov is signaling molecules and protein synthesis, and their connection to cell growth, differentiation and aging. The scientist examines certified medicines only. If compounds having positive effect on life extension are found, the researcher won’t have to certify them again as they are already used in clinical practice. Finding of this study have not been published yet. In the future, this scientific project will probably be expanded. Clinical and histological analysis, as well as postmortem examination will be conducted in order to reveal how different compounds affect pathogenesis of cancer and age-related diseases. At the moment, A. Ryazanov leads the project on the large-scale screening of more than a thousand of medicines allowing to analyze their life extension effect in mice.

Prof. A. Hollander and his team have taken part in the creation of the world’s first bioengineered trachea as they have prepared autological chondrocytes. Monolayer culture of mesenchymal stem cell obtained from the patient’s bone marrow (BMSC), was grown in the chondrogenic medium where the cells differentiated into chondrocytes. Then the cells were plated at the decellularized donor framework using a bioreactor. A. Hollander and his colleagues have shown for the first time that chondrogenesis of adult stem cells can be induced by synthetic retinoic acid receptor inhibitor LE135, so pharmacological regulation of chondrogenesis is possible. According to A. Hollander’s hypothesis, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces in order for effective cartilage integration to take place. A chondrocyte/collagen-scaffold implant system has been developed as a method of delivering dividing cells at the interface between two cartilage surfaces. The cartilage-implant-cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.

A. Moskalev’s team carries out investigation in the field of radiation genetics and gerontology. A. Moskalev’s team has studied an adaptive response (changes in the life span) to low-dose radiation in Drosophila melanogaster. One of the wild type lines of D. melanogaster, together with mutant lines having mutations in the heat shock factor (Hsf) genes and in the heat shock protein genes, were used in the experiments. Scientists have shown that the radiation stress has no stimulatory effect on the life spans of the flies with mutated Hsp and Hsf genes, i.e. it does not cause the adaptive response. Research results indicate that induction of heat shock proteins is one of the primary protective mechanisms under stress conditions.