[Modern approaches to studying the molecular mechanisms of lung functioning in normal and pathological conditions]. / Sovremennye podkhody k izucheniyu molekulyarnykh mekhanizmov funktsionirovaniya legkikh v norme i pri patologii.

Problems with breathing and lung function are caused by the development of various lung diseases associated with lifestyle, harmful environmental factors and genetic predisposition. Knowledge of the molecular mechanisms of the development of the pathological process will allow on time identification of the disease or the development of targeted therapy. The article provides an overview of modern methods that make it possible to most accurately reproduce the structural, functional and mechanical properties of the lung (organ-on-a-chip), to perform non-invasive molecular studies of biomarkers of bronchopulmonary pathology using saliva diagnostics, as well as using DNA and RNA aptamers, verify tumor markers in biological samples of human tissue. Analysis of alterations in the pattern of protein glycosylation using glycodiagnostic methods makes it possible to detect lung cancer in the early stages.

[Caldesmon and tumor growth: prospects for optimizing diagnosis and targeted therapy]. / Kal'desmon i opukholevyi rost: perspektivy optimizatsii diagnostiki i targetnoi terapii.

Tumor invasion plays a key role in the progression of tumors. This process is regulated by the interactions of cells and tissues, in which physical, cellular and molecular determinants undergo changes throughout the entire period of progression of tumor growth. Tumor invasion is triggered and maintained by specialized signal cascades that control the dynamic state of the cytoskeleton in tumor cells, the processes of rearrangement of cell-matrix and intercellular connections, followed by cell migration to neighboring tissues. Studying the mechanisms of regulation of cell motor activity and determining its main regulators is an important task for understanding the pathophysiology of tumor growth. Caldesmon is an actin, myosin and calmodulin binding protein. It is involved in the regulation of smooth muscle contraction by inhibiting actin and myosin binding, in the formation of actin stress fibers, and in the transport of intracellular granules. Currently, caldesmon is considered as a potential biomarker of tumor cell invasion, migration, and metastasis. The study of signaling molecules involved in tumor progression, such as caldesmon, is necessary to predict response to chemotherapy and radiotherapy. This review highlights the main functions of caldesmon and analyzes its role in oncological pathology.

[Slowdown of replicative aging of fibroblasts by hyaluronan complexes with gold nanoparticles.]

Determination the activity of the genes of sirtuin-1, hyaluronidase, TGF-ß cytokine, calreticulin in the process of replicative aging of human fibroblasts in vitro and the effect of hyaluronan preparations with gold nanoparticles on the activity of replicative cell aging. Compared the expression of proteins of the studied genes using specific markers at 7 and 14 passages of cultivation of fibroblasts isolated from human skin, without drugs and in the presence of drugs in the growth medium. This work shows a decrease in the activity of the sirtuin 1 gene and an increase in the expression of hyaluronidase in the process of replicative aging of human fibroblasts. Found a means of slowing down replicative aging by activating the SIRT-1 gene and reducing the activity of hyaluronidase in action in the growth medium of hyaluronan preparations with gold nanoparticles. The discussed variants of cell transitions to the pathological state, caused by replicative aging and the mechanisms of slowing down the replicative aging of human fibroblasts.

[Secretory phenotype of the human endothelial cells associated with replicative and induced aging.]

The aging-associated secretory phenotype (SASP) is a heterogeneous phenotype of cells secreting pro-inflammatory cytokines, growth factors, apoptosis' regulatory molecules, and proteases. SASP is one of the three main hallmarks of senescent cells. Dysregulation of the synthesis of SASP-forming molecules leads to the development of age-associated diseases, including cardiovascular pathology. The aim of this study is to characterize the SASP of human endotheliocytes during replicative and induced aging. Isolated human umbilical vein endothelial cells HUVEC were used to model replicative and inflammation-induced aging. It has been established that the molecules that form SASP during replicative and inflammation-induced aging of HUVEC are molecules that control apoptosis (p16, p21, p53), adhesion (E-selectin, VCAM-1) and some cytokines (IL-1ß, IL-6). With replicative aging of endotheliocytes, the synthesis of apoptosis' regulatory molecules increases to a greater extent. Inflammation-induced aging of HUVEC is characterized by a multiple increase in the synthesis of adhesion molecules and pro-inflammatory cytokines.

[The influence of AEDG and KE peptides on mitochondries stain and L7A ribosomes protein expression during human pineal gland and thymus cell senescence in vitro.]

It was verified new molecular targets of geroprotective activity of AEDG (epitalon) and KE (vilon) peptides by the method of confocal laser scanning microscopy. It was shown that the MitoTracker Red mitochondries staining decreased and L7A ribosomal protein synthesis compensatory increased during pineal and thymic cell senescence in vitro. AEDG peptide increases in 1,5 times the square of MitoTracker Red mitochondries staining and decreases on 22% the expression of ribosomal protein L7A in cultures of human pineal gland cells during its senescence. KE peptide increases in 1,5 times the square of MitoTracker Red mitochondries staining and decreases on 15% the expression of ribosomal protein L7A in cultures of human thymic cells during its senescence. The square of MitoTracker Red mitochondries staining decreases and the expression of L7A ribosomal protein compensatory increases during pineal gland and thymic cells senescence. We can suppose that AEDG and KE peptides have a tissue-specific effect that normalizes the functions of mitochondria and ribosomes of pinealocytes and thymocytes.

Thymalin: Activation of Differentiation of Human Hematopoietic Stem Cells.

Thymalin is a polypeptide complex isolated from the thymus and regulating the functions of the immune system. Thymalin is effective in therapy of acute respiratory syndrome, chronic obstructive bronchitis, and other immunopathology. Thymalin increases functional activity of T lymphocytes, but the targeted molecular mechanism of its biological activity requires further study. We studied the influence of thymalin on differentiation of human hematopoietic stem cells (HSC) and expression of CD28 molecule involved in the implementation of antiviral immunity in COVID-19 infection. It was found that thymalin reduced the expression of CD44 (stem cell marker) and CD117 (molecule of the intermediate stage of HSC differentiation) by 2-3 times and increased the expression of CD28 (marker of mature T lymphocytes) by 6.8 times. This indirectly indicates that thymalin stimulated differentiation of CD117+ cells into mature CD28+T lymphocytes. It is known that in patients with severe COVID-19, the number of CD28+, CD4+, CD8+T lymphocytes in the blood decreased, which attested to a pronounced suppression of immunity. It is possible that the antiviral effect of thymalin consists in compensatory stimulation of HSC differentiation into CD28+T lymphocytes at the stage of immunity suppression in unfavorable course of viral infection. Thymalin can be considered as an immunoprotective peptide drug for the prevention of COVID-19.

Sirtuins as Possible Predictors of Aging and Alzheimer's Disease Development: Verification in the Hippocampus and Saliva.

Verification of signaling molecules in the saliva is a non-invasive method of diagnosis and evaluation of treatment effectiveness in different pathologies. Sirtuins (SIRT), proteins from NAD-dependent histone deacetylases, are supposed to be involved in the pathogenesis of Alzheimerэs disease. Age-related decrease in sirtuins expression induces many pathophysiological processes that could lead to neurodegeneration. We studied the expression of SIRT1, SIRT3, SIRT5, and SIRT6 in the hippocampus and saliva of humans without neurological pathologies and in patients with Alzheimer's disease of elderly and senile age. In elderly and senile patients, the expression of SIRT1, SIRT3, and SIRT6 in the hippocampus and saliva was 1.5-4.9-fold reduced in comparison with healthy individuals of the corresponding age. In healthy senile persons, the expression of SIRT6 in the hippocampus and saliva was 2.5-4.5-fold lower than in healthy elderly individuals. Measurement of SIRT1, SIRT3, and SIRT6 concentration in the saliva can be used as an additional method for intravital non-invasive diagnosis of Alzheimer's disease in patients of advanced age. SIRT6 concentration in the saliva can be recommended as a marker for assessment of the rate of aging.

Placental protein expression of kisspeptin-1 (KISS1) and the kisspeptin-1 receptor (KISS1R) in pregnancy complicated by diabetes mellitus or preeclampsia.

PURPOSE: Kisspeptins regulate the trophoblast invasion. The disturbance of this process might lead to the development of preeclampsia (PE). Diabetes mellitus (DM) is associated with the high rate of this complication. The main hypothesis was to investigate the placental protein expression of kisspeptin-1 (KISS1) and its receptor (KISS1R) in diabetic, preeclamptic, and healthy pregnancies. METHODS: Placentae (n = 65) were divided into the following groups: the control group (n = 20), either PE or non-PE type-1 diabetes mellitus (T1DM) (n = 10), either PE or non-PE type-2 diabetes mellitus (T2DM) (n = 10), either PE or non-PE gestational diabetes mellitus (GDM) (n = 10) and preeclampsia without diabetes (PE) (n = 15). Immunohistochemistry analysis was used for demonstrating the presence and location of KISS1/KISS1R in placental tissue and to measure the area of immunopositive expression. Correlation analyses were performed to detect the links between protein expression of these biomarkers and the main obstetric outcomes. RESULTS: The highest placental protein expressions of KISS1 were detected in the PE (35.4%) and GDM (33.2%) groups. In case of DM, levels of KISS1 expression depended on the presence of PE and were higher compared with DM no PE and control groups: (30.6%) in T1DM + PE and (30.1%) in T2DM + PE group. The lowest expression was detected in the control group (14.1%). The expression of KISS1R was higher in DM and PE compared to the control group. We detected the strong direct link between PE and placental expression of KISS1 (r = 0.81) and KISS1R (r = 0.56), and inverse correlation link between KISS1 and preterm birth weight (r = - 0.73). The low correlation links were found between KISS1 and IUGR (r = 0.29), and preterm birth (r = 0.24). The same trend was detected for KISS1R. We did not find any significant correlations between placental expressions of KISS/KISS1R and placental weight or HbA1c levels. CONCLUSION: Increased expression levels of KISS1 and KISS1R in case of diabetes mellitus may play a role in the altered placentation process and lead to the development of preeclampsia.

[Kisspeptins in human ovarian tissue during ontogenesis.]

The article presents the results of studies of the expression hormones-kisspeptins and their receptors in human ovarian tissues during ontogenesis of this organ. Kisspeptins regulate the hypothalamic-pituitary-gonadal axis, the most important function of which is to launch the mechanism of puberty. Verification of kisspeptins and their receptors in ovarian tissue, suggests that they promote ovulation, as well as controls the expression of matrix metalloproteinases involved in tissue remodeling. The KISS1/KISS1R system begins be active in the period of prenatal development, so that already at week 22 a positive reaction with antibodies to kisspeptin was recorded in the ovarian tissue. It has been established that, at reproductive age, the expression of kisspeptins remains at a consistently high level, whereas during menopause, the expression of kisspeptins in the ovaries has its peak, which may be due to a compensatory mechanism for reducing the synthesis of ovarian estrogens. In postmenopausal period defined minimum values. Further studies of the metabolism of kisspeptins during menopause will contribute to the expansion of knowledge about their mechanism and the possibility of using them as targeted therapeutic agents.

[Investigation of signal molecules in saliva: prospects of application for diagnostics of myocardial infarction and the aging rate of different age people.]

Among the diseases of the cardiovascular system in elderly people, ischemic heart disease and myocardial infarction (MI) occupy the first place in the structure of mortality. One of the main causes of disability and death from MI is late diagnosis. In this regard, the search for new, highly informative and non-invasive methods for diagnosing MI is an important task of molecular gerontology. An enzyme immunoassay showed that the concentration of TNF-α, IL-8 cytokines and p16 aging marker in saliva in elderly people without cardiovascular pathologies (CP) increases in 2,1-4,8 times as compared with middle-aged people. At the same time, in elderly people without CP the concentration in the saliva of the hormone irisin (FNDC5) decreases by 1,8 times as compared with middle-aged people. In middle-aged patients with MI the concentration of IL-8, TNF-α, MMP8, MMP9 in saliva increases 4,3-15,3 times, and FNDC5 decreases 1,8 times compared with those parameters without CP in this age group. In elderly people with MI the concentration of IL-8, TNF-α, MMP8 and MMP9 in saliva increases 4,3-7,1 times as compared with elderly people without CP. Thus, the study of the concentration of signaling molecules IL-8, TNF-α, MMP8, MMP9 in saliva can be used as a non-invasive method for diagnosing MI in people of middle and elderly age. To assess the rate of aging of the organism in middle-aged and elderly people without CP, a study of the concentration of p16 and FNDC5 molecules in saliva is recommended.

[The sirtuin family of proteins: the role in the aging processes and regulation of cell metabolism.]

In recent years, the key role of sirtuins in the regulation of the most important cellular processes has been established. Sirtuins are involved in histone deacetylation, regulation of fat and glucose metabolism. Violations of their synthesis and secretion can induce carcinogenesis, aging and cell death. The wide range of processes in which sirtuins are involved indicates their possible role in the pathogenesis of many diseases including metabolic syndrome, neurodegenerative diseases, inflammation associated diseases and tumor growth. All of these diseases are associated with aging. In this article, we analyze the existing data on sirtuins and their role in the pathogenesis of aging, as well as their possible verification as markers for the diagnosis of age-related diseases and as targets of geriatric therapy and prevention.

[Molecular markers of Alzheimer disease early diagnostic: investigation perspectives of peripheral tissues.]

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of elderly and old age people. For intravital diagnosis of the expression of signaling molecules - AD markers, cerebrospinal fluid (CSF) and peripheral tissues are used: lymphocytes and blood platelets, buccal and olfactory epithelium, skin fibroblasts. There are several changes in the production of hyper phosphorylated form of τ-protein, BACE1 and peptide Аß42 in CSF in case of AD, but CSF taking may have a number of side effects. Less traumatic taking of sampling tissues for the diagnosis of AD is in use of epithelium biopsy and blood portion. An increase in the expression of the hyper phosphorylated form of τ-protein is shown in blood lymphocytes of AD patients. An increase in the content of high molecular weight forms of phosphorylated t-protein and amyloid precursor protein-APP was also revealed in blood platelets of AD patients. Changes in the amount of 2 miRNA families - miR-132 family and miR-134 family were revealed in blood cells 1-5 years before the manifestation of clinical signs of AD. An increase in the concentration of bound calcium, synthesis of peptides Aß40 and Aß42, τ protein was observed in AD skin fibroblasts. In the olfactory and buccal epithelium an increase in the expression of hyper phosphorylated form of τ-protein and Aß peptide was detected in patients with AD. Verification of AD markers in peripheral tissues for biopsy have the important significant for life diagnostics, prevention and and target AD treatment.

[MELATONIN: THE POSSIBILITY TO ANALYSE THE MARKER OF AGE-RELATED PATHOLOGY IN THE BUCCAL EPITHELIUM AND URINE].

Extrapineal and pineal melatonin is the marker of the aging rate of organism making it possible to characterize functional condition of the neuro-immuno-endocrine system. In this article we have used the new method for non-invasive diagnostics of melatonin expression in buccal epithelium and determination of the main melatonin metabolite 6-hydroxymelatonin sulfate (6-HMS) in urine of elderly people. Normal, impaired and enhanced melatonin expression was documented in 20.5%, 43.2% and 36.30% of the patients respectively. Such comprehensive melatonin and 6-COMT studies can be recommended for elderly patients with oncological, neurodegenerative, cardiovascular diseases, and ageing macular dystrophy. Moreover, melatonin expression analysis in buccal cells can be used for integral investigation of biorhythms in elderly people.

[Effect of combined application of ozone therapy and gravitational therapy on the remote results of complex treatment of geriatric patients].

Skin aging is one of the topical issues in modern gerontocosmetology. Application of cosmetic products with short peptides is a promising measure for retardation of skin aging. This research is aimed at investigation of KE (Lys-Glu, Vilon) dipeptide influence on the expression of markers of aging in human skin fibroblasts in vitro. Collagen type I and sirtuin-6 expression in «young¼ and «old¼ skin cell fibroblasts cultures was studied using immunofluorescence confocal microscopy method. The areas of expression of collagen type I and sirtuin-6 are known to decrease in skin fibroblasts with aging by 3,5 and 3,6 times accordingly. KE dipeptide increases collagen type I expression area in «old¼ skin fibroblasts cultures by 83%. KE dipeptide increases expression area of sirtuin-6 in «young¼ and «old¼ skin fibroblasts cultures by 1,6 and 2,6 times correspondingly. Thus, KE dipeptide promotes functional activity of skin fibroblasts and inhibits their aging.

Peptide Regulation of Skin Fibroblast Functions during Their Aging In Vitro.

The effect peptides KE, KED, AED and AEDG on proliferation (Ki-67), regeneration and aging (CD98hc), apoptosis (caspase-3), and extracellular matrix remodeling (MMP-9) in skin fibroblasts during their aging in culture were studied by immunofluorescent confocal microscopy. All studied peptides inhibited MMP-9 synthesis that increases during aging of skin fibroblasts and enhanced the expression of Ki-67 and CD98hc that are less intensively synthesized during cell aging. Peptides AED and AEDG suppressed caspase-dependent apoptosis that increases during aging of cell cultures.

[DIFFUSE NEUROENDOCRINE SYSTEM OF THE STOMACH AND CHARACTERISTICS OF PROLIFERATION IN REALIZATION OF CORREA'S CASCADE IN DISEASES ASSOCIATED WITH HELICOBACTER PYLORI].

AIM: To study the role of the vascular endothelial growth factor (VEGF), Ki-67, BCL-2, and endocrine cells (EC) of gastric mucosa producing somatostatin (SS), glucagon (GL), and pancreatic polypeptide (PP) in diseases associated with Helicobacter pylori. To use the data obtained to develop early diagnostic criteria for the progress of structural changes in gastric mucosa of the patients with stomach ulcer disease [SUD), chronic atrophic gastritis (CAG), gastric adenomatous polyps (GAP), and gastric cancer (GC) before and after surgical intervention and eradication of H. pylori. MATERIALS AND METHODS: We examined 104 patients with gastric pathology associated with Helicobacter pylori including 30 with SUD, 30 with CAG, 20 with GAP and CAG, 24 with stage II noncardia GC. The effectiveness of the treatment was evaluated 2 months after alleviation of inflammation in the stomachfollowing polypectomy in case of GAP or gastrectomy in case of GC. Material for immunohistochemical studies was taken from the fundus. Monoclonal antibodies against VEGF SS, GL, PP Ki-67, BCL-2 (1:100, Novocastra) were used. Histobacterioscopy was conducted using antral mucosal smears stained by the Romanowsky-Giemsa procedure. RESULTS: Exacerbation of SUD was accompanied by a decrease in the number of epithelial cells and EC producing VEGF glucagon (GL), and pancreatic polypeptide (PP) with the increase in the number of SS-secreting EC along with intensification of proliferative processes determined from the number of Ki-67 and BCL-2 immunopositive epithelial cells. CAG and GC were associated with persistence of H. pylori, hyperplasia of EC producing VEGF glucagon (GL), pancreatic polypeptide (PP) and hypoplasia of SS-secreting EC along with high proliferative activity of epitheliocytes expressed via Ki-67 and BCL-2. CONCLUSION: Endothelial growth factor (VEGF), somatostatin (SS), glucagon (GL), and pancreatic polypeptide (PP) are of importance for the prognostication of development and clinical course of diseases associated with Helicobacter pylori since their pathological properties are realized either directly or indirectly through H. pylori, Ki-67 and BCL-2. Eradication of H. pylori does not result in the disappearance of intestinal metaplasia.

Peptide regulation of gene expression and protein synthesis in bronchial epithelium.

INTRODUCTION: Some studies have shown that peptides have high treatment potential due to their biological activity, harmlessness, and tissue-specific action. Tetrapeptide Ala-Asp-Glu-Leu (ADEL) was effective on models of acute bacterial lung inflammation, fibrosis, and toxic lung damage in several studies. METHODS: We measured Ki67, Mcl-1, p53, CD79, and NOS-3 protein levels in the 1st, 7th, and 14th passages of bronchoepithelial human embryonic cell cultures. Gene expression of NKX2-1, SCGB1A1, SCGB3A2, FOXA1, FOXA2, MUC4, MUC5AC, and SFTPA1 was measured by real-time polymerase chain reaction. Using the methods of spectrophotometry, viscometry, and circular dichroism, we studied the ADEL-DNA interaction in vitro. RESULTS: Peptide ADEL regulates the levels of Ki67, Mcl-1, p53, CD79, and NOS-3 proteins in cell cultures of human bronchial epithelium in various passages. The strongest activating effect of peptide ADEL on bronchial epithelial cell proliferation through Ki67 and Mcl-1 was observed in "old" cell cultures. ADEL regulates the expression of genes involved in bronchial epithelium differentiation: NKX2-1, SCGB1A1, SCGB3A2, FOXA1, and FOXA2. ADEL also activates several genes, which reduced expression correlated with pathological lung development: MUC4, MUC5AC, and SFTPA1. Spectrophotometry, viscometry, and circular dichroism showed ADEL-DNA interaction, with a binding region in the major groove (N7 guanine). CONCLUSIONS: ADEL can bind to specific DNA regions and regulate gene expression and synthesis of proteins involved in the differentiation and maintenance of functional activity of the bronchial epithelium. Through activation of some specific gene expression, peptide ADEL may protect the bronchial epithelium from pulmonary pathology. ADEL also may have a geroprotective effect on bronchial tissue.

Effects of pancragen on the differentiation of pancreatic cells during their ageing.

Expression of differentiation markers was found to be reduced during ageing of pancreatic cells. Tetrapeptide pancragen stimulates the expression of differentiation factors of acinar (Pdx1, Ptfla) and islet of Langerhans (Pdx1, Pax6, Pax4, Foxa2, NKx2.2) cells in "young" and " aged" cultures. Differentiation of acinar and islet pancreatic cells induced by pancragen can be a mechanism underlying its anti-diabetic and anti-inflammatory effects. Thus, transcription factors that regulate differentiation of pancreatic cells are a pharmacological target for pancragen, which allows considering it as an effective tool in the treatment of diabetes mellitus and pancreatitis.

Immunomodulating effects of Vilon and its analogue in the culture of human and animal thymus cells.

We studied molecular mechanisms of immunoprotective effects of two dipeptides, AB-O and R-1, on cultured human and rat thymic cells. Both dipeptides were shown to increase the expression of lymphocyte differentiation marker CD5 in thymic cells. Dipeptide AB-O induced T-cells precursor differentiation towards CD4(+)T-helpers and its effect was weaker than that of dipeptide R-1. Dipeptide R-1 stimulates differentiation of CD5(+) cells to mature T-helpers and cytotoxic CD8(+) T cells and hence can be considered as a bioactive substance possessing immunomodulator and antiallergic activity.

A method of creation a cell monolayer based on organotypic culture for screening of physiologically active substances.

We developed a method of culturing and phenotyping of a monolayer of cells of the retinal tissue, thymus and spleen on the basis of organotypic culture. All characteristic types of neurons and fibroblasts were found in their microenvironment in the retinal cell monolayer. Lymphocytes, macrophages, and fibroblasts were verified in the monolayer of thymus and spleen cells. Histological staining, immunocytochemistry, and electron microscopy demonstrated the possibility of assessing the differentiation degree and functional activity of the cell monolayer. The developed technique preserves cell-cell interactions and a variety of cell types characteristic of the examined organ in the monolayer. This opens up new prospects for its application in basic research and in screening of different physiologically active substances.