The amniotic variant of glycodelin (Gd) has pronounced immunomodulatory properties and is involved in the formation of immune tolerance during pregnancy. The role of recombinant Gd at physiological (0.2 and 2 μg/ml) and superphysiological (10 μg/ml) concentrations in regulating the differentiation and functional activity of human myeloid-derived suppressor cells (MDSCs) was investigated in vitro. MDSCs were generated from CD11b+ peripheral blood cells of healthy donors by two-step induction (IL-1β + GM-CSF and then lipopolysaccharide (LPS). The effect of Gd on the content of polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC), intracellular expression of indoleamine 2.3-dioxygenase (IDO), arginase-1 (Arg1, and cytokine profile in cell cultures was investigated. In general, the transformation of CD11b+ cells into MDSCs exhibits the following characteristics: as a result of cytokine induction, predominantly M-MDSCs but no PMN-MDSCs are formed and Arg1 expression is virtually undetected. Gd was found to increase the number of M-MDSCs at concentrations of 2 and 10 μg/ml. Gd was found not to affect Arg1 expression but increased the percentage of MDSCs expressing IDO (10 μg/ml). Gd also modulated the cytokine profile of CD11b+ cells by suppressing the production of IL-19, IL-26 and TWEAK/TNFsF12 at a physiological concentration of 2 μg/ml and the production of IFN-α2 and IL-26 at a supraphysiological concentration. Thus, the role of Gd in the conversion of CD11b+ cells to MDSCs was examined under conditions of cytokine induction in vitro.
Amitriptyline is a tricyclic antidepressant widely used in clinical practice for the treatment of anxiety, depression and chronic pain. These drugs have a multifaceted effect on cellular processes. One of their targets is sigma-1 receptors. Sigma-1 receptors are molecular chaperones located in endoplasmic reticulum membrane; they are characterized by a unique structure and pharmacological profile. Sigma-1 receptors regulate many cellular processes in health and disease, including Ca2+ signaling. Using Fura-2AM microfluorimetry, it was shown for the first time that sigma-1 receptor agonist, antidepressant amitriptyline, significantly suppresses both Ca2+ mobilization from intracellular Ca2+-stores and subsequent store-dependent Ca2+ entry into cells, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, as well as disulfide-containing immunomodulators glutoxim and molixan, in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in a complex signaling cascade induced by glutoxim or molixan, leading to an increase of intracellular Ca2+ concentration in macrophages. The results also indicate the participation of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.
Voltage-gated cardiac sodium channels Nav1.5 are responsible for the initiation and propagation of action potentials in cardiomyocytes. Dysfunction of Nav1.5 can be caused both by pathogenic variants in the SCN5A gene itself, which encodes Nav1.5, and by genetic variants in the genes of other proteins, regulating channel activity and trafficking. The change of different phases of the action potential is determined by the strict temporal organization of activation and inactivation of various ion channels. Transitions between channel functional states (for example, to slow inactivated state) can be influenced by various factors and proteins interacting with the channel. Despite the fact that the process of slow inactivation of the channel has been known for several decades, its role in the mechanism of development of hereditary heart pathology remains unclear. In this work, using the patch clamp method in whole-cell leads, we studied changes in the process of slow Nav1.5 inactivation under the influence of various mutations in structural genes (DSP-H1684R, LMNA-R249Q, FLNC-R1267Q, FLNC-V2264M) associated with a genetically determined myocardial pathology leading to dysfunction of cardiomyocytes. The study used a model of cardiomyocytes differentiated from induced pluripotent stem cells (СM-iPSCs). We have demonstrated an increase in slow inactivation in the model of CM-iPSCs obtained from patients with a phenotype of cardiomyopathy combined with ventricular arrhythmias. Thus, this work contributes to understanding the role of the slow inactivation process in the mechanism of the development of heart pathology.
Objectives: The aim of this work was to study the effect of nanocluster polyoxometalate Mo72Fe30 on macrophages. In vitro studies of polyoxometalate’s effect on cultured macrophages allowed us to evaluate the reaction of immune cells (in particular, peritoneal and alveolar macrophages) to the nanoparticles. Results and discussion: The analysis of the obtained data allowed to establish that Mo72Fe30 is not toxic for peritoneal and alveolar macrophages, has no significant effect on the morphology of cells, as well as on the activity of α-naphthyl acetate esterase. Conclusion: However, polyoxometalate significantly reduces phagocytic activity, which may indicate possible polarization of macrophages. The obtained results confirm the possibility of using Mo72Fe30 in the field of biomedicine.
A new non-immortalized fibroblast-like cell line, named MSCWJ-3, was generated and characterized. Characteristics during long-term cultivation (6–24 passages) confirm the status of MSCs. It is shown: 1) a gradual increase in the proportion of senescent cells during long-term cultivation; 2) a significant decrease in the proliferation index by the 24th passage; 3) preservation of the normal diploid karyotype of the man (46, XY) during the entire period of cultivation, trisomy for different autosomes in single cells, absence of structural chromosomal rearrangements; 4) a high proportion of cells carrying surface antigens characteristic of MSCs: CD44, CD73, CD90, CD105, HLA-ABC and a low proportion with antigens CD34, CD45 and HLA-DR over 24 passages. Cells of the MSCWJ-3 line are capable of differentiation in the osteogenic and adipogenic directions at early and late passages; differentiation in the chondrogenic direction is absent. In general, there are some differences with previously obtained lines isolated from the same source and are associated mainly with the degree of expression of a number of status characteristics.
In this study, the viability and adhesive features of BRO and SK-MEL-2 cell lines were evaluated using a melanoma model. It was revealed that in BRO cells, the development of apoptosis after exposure to dacarbazine was combined with the transition of the proportion of cells to the G0 phase of the cell cycle, that corresponded to previously obtained results. The absence of apoptosis in 3D spheroids and the absence of exit from the cell cycle were observed in SK-MEL-2 melanoma cells. It was also revealed that in the control spheroids (cells without exposure) of the BRO and SK-MEL-2 melanoma lines, adhesion to fibronectin was higher compared with the cells of the control monolayer, which is explained by the three-dimensional structure requiring cell communication with the extracellular matrix. In spheroids formed by SK-MEL-2 cells, dacarbazine induced a decrease in adhesion to fibronectin, which may be associated with the development of drug resistance. An increase in the expression of integrins AV and β8 in BRO and SK-MEL-2, as well as integrin β5 in SK-MEL-2, was determined in cells after exposure to dacarbazine, which may indicate the involvement of aforementioned molecules in the exit from proliferative stage of tumor cells.
Primary microcephaly is a brain growth disorder of which the main phenotypic hallmarks is a reduction of brain size with varying degrees of intellectual disability. MCPH1 is the first gene reported to cause primary microcephaly. Microcephalin (MCPH1), the encoded protein product, has been implicated in various cellular processes deregulation of which can negatively affects neurogenesis. In our review we will discuss the clinical cases of MCPH1 primary microcephaly and summarize the knowledge about the functions of MCPH1 employing animal models with mutations in various domains of MCPH1. We also pay special attention to the role of MCPH1 in in the evolution of the human brain.
Objective: Scientists need cell models from human tissues to develop methods of gene therapy and genome editing for monogenic diseases. It is preferable to use minimally invasive methods to obtain samples; these tissues can be applied for further screening to select the most effective approach to restore the synthesis of the target protein. Methods: We used the CRISPR/Cas9-SAM transcriptional activation system, which ensures expression of the DYSF gene in HEK293Т cells, as well as in fibroblasts from patients with dysferlinopathy (c.2779delG (Ala927LeufsX21)). Results and Discussion: After targeted activation of DYSF, it was possible to detect the main gene products: mRNA and protein (HEK293Т_ТА) and mRNA (fibroblasts). Conclusions: Transcriptionally activated dysferlin-deficient fibroblasts and HEK293 cells can be used to evaluate the in vitro efficacy of gene therapy for dysferlinopathies.
Objectives: Mesenchymal stem/stromal cells (MSC) isolated from equine adipose tissue (AT) represent a promising material for bioveterinary products for the prevention and treatment of many diseases. The production of these cells for clinical use requires improved serum-free culture conditions. The microenvironment can influence the properties of MSC. It is believed that the requirements for culture conditions without animal blood serum are species-specific. The purpose of this study was to evaluate the commercially available serum-free medium (SFM) MesenCult (STEMCELL Technologies, USA), created for human MSC, for the cultivation of equine MSC(AT). Materials and methods: One part of the cells was propagated for 10 passages in the standard DMEM medium with low glucose content (1 g/L) and 10
The BOB1 protein (OBF1, OCA-B) is a transcriptional coactivator of two POU domain proteins — OCT1, expressed in all cells, and lymphoid-specific OCT2. The interaction of BOB1 with OCT1/2 plays an important role in the regulation of immune responses in both physiological and pathological contexts. BOB1 is known to form a ternary complex with OCT1/2 bound to DNA in monomeric and certain dimeric configurations, changing the sequence specificity of the binding. To analyze DNA sequences from these complexes, in this work we proposed the EMSA-SELEX-seq method, based on the separation of OCT/BOB1 complexes of various compositions in a non-denaturing polyacrylamide gel (EMSA) followed by the isolation and amplification of the oligonucleotides that they contain (SELEX). Based on several rounds of the enrichment followed by the NGS sequencing and bioinformatics analysis, the DNA sequences were determined and the relevance of this approach was confirmed. Thus, the proposed EMSA-SELEX-seq method allows the analysis of DNA sequences in DNA-protein complexes with varying dimensions of its protein components.
Objective: Over the past decades, gene therapy based on the adenoviral E1A has proven its benefit against several tumor diseases, both in animal models and in clinical studies. It has been shown that in addition to its antiproliferative activity, E1A also can enhance the cytotoxic effect of some anticancer drugs. The use of E1A in combination therapy can solve several problems in clinical oncology, among which the most pressing is the problem of drug-resistance of tumor cells. Methods: This work describes the establishment of a cell model based on human colorectal cancer cells HCT116 and cisplatin-resistant HCT116/C cells with doxycycline-inducible expression of adenoviral E1A. Results and Discussion: We have shown the concentration-dependent and time-dependent dynamics of E1A expression upon doxycycline treatment and shown the antiproliferative effect of adenoviral E1A in the HCT116-E1A and HCT116/C-E1A cells both in vitro using MTT and clonogenic activity assays and in vivo using xenograft mouse models. Thus, as a result of our work, a model was created to explore the antiproliferative and sensitizing properties of E1A in platinum-sensitive and platinum-resistant colorectal cancer cells and to search for new approaches to anticancer therapy both in vitro and in vivo. Conclusions: The resulting cell line is a convenient model for selecting the most promising combinations of cytostatic drugs with E1A-based gene therapy.
An important task of topical application of medicines in the treatment of eyes is to achieve a compromise between their effectiveness and safety. The development of new multifunctional local ophthalmic drug delivery systems and in vitro screening of potential medicinal eye products are key areas in solving this problem. In this study, primary in vitro screening of the effect of echinochrome (Ech), the carrageenan complex of echinochrome (CRG/Ech) and its liposomal form (CRG/Ech-Lip) was performed on cultured epithelial cells of the outer shell of the eyeball: conjunctival epithelial cells (Chang Conjunctiva, Clone 1-5c-4) and corneal epithelium human (HCE). The cell viability was assessed by their morphology and metabolic activity using light microscopy and MTT test methods. The direct dependence of the intensity of the cytotoxic effect of Ech on its concentration in the nutrient medium, the form of use, the cellular test system and the incubation time of cells was revealed. Ech in the form of an alcoholic solution in its final concentration of 0.1 mg/ml of the nutrient medium exhibits pronounced cytoxicity against both cellular test systems. The same final concentration of Ech in the nutrient medium, but already as part of the carrageenan complex of echinochrome (CRG/Ech), turned out to be critical only for the viability of corneal epithelial cells, the survival rate of conjunctival cells under these conditions was about 50 %. A high biocompatibility of the liposomal form of the carrageenan complex of echinochrome (CRG/Ech-Lip) with cells of both test systems and a stimulating cytoprotective effect against the cells of the conjunctiva epithelium was revealed.
This mini-reviewsystematizes information on methods for quantitative assessment of intracellular hydrogenperoxide concentration based on the use of a genetically encodedperoxide sensor HyPer. Two approaches are being considered: 1) calibrationof the biosensor using exogenous hydrogen peroxide, based on assessingthe rate of peroxide penetration into cells and intracellular peroxidaseactivity; 2) direct determination of the intracellular peroxide content, basedon measuring the level of oxidation of the biosensor, theoxidation reaction constant and the reduction reaction constant of HyPerin the cells. The use of these methods makes itpossible to solve a wide range of tasks in cellularredox biology—to determine the range of physiological anddamaging concentrations of hydrogen peroxide in cells, to evaluate theeffectiveness of the antioxidant defense system in various cellular compartmentsunder conditions of oxidative stress, to determine the contribution ofvarious enzymatic systems to the peroxidase activity of cells, andto characterize antioxidant defense systems in various biological contexts (inthe process of cellular senescence, differentiation, reprogramming, during the developmentof pathologies). The described methods can be adapted for othergenetically encoded hydrogen peroxide biosensors.
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) using certain factors. The low efficiency of the reprogramming, as well as the heterogeneity of iPSCs, limits the potential application for iPSCs in cell therapy. Here, we show that lithium chloride (LiCl), a known activator of the Wnt signaling pathway, reduces or enhances the efficiency of iPSC generation from mouse embryonic fibroblasts (MEFs) depending on the timing of its addition during the reprogramming. Our results not only demonstrate a method to improve the efficiency of iPSC formation by LiCL, but also indicate its dual role in this process.
The effect of N-acyl derivatives of 2-amino-4,6-di-tert-butylphenolon the functions of neutrophils was studied. It has been established that these derivatives with a free hydroxyl group in the benzene ring, in contrast to O-methylated ones, modify the properties of cells, which is expressed in a decrease in hypochlorous acid generation during the “respiratory burst” formation. These compounds are scavengers of HOCl/OCl– generated by activated neutrophils and reduce the secretion of myeloperoxidase (MPO) from cells. N-(3,5-di-tert-butyl-2-hydroxyphenyl)acetamide has been shown to be the most effective hypochlorous acid scavenger. This substance significantly suppresses the secretory degranulation of neutrophils and has a cytoprotective effect under conditions of halogenating stress.
Preterm birth can contribute to the development of diseases of circulatory system in adulthood due to the incompleteness of the morphogenesis of the blood vessels wall. Smooth muscle cells are the leading cell population in the middle shell of the aortic wall and are plastic in nature, i. e. they are able to change their phenotype depending on the conditions of their environment. The presence of synthetically active smooth muscle cells in the aortic wall of an adult individual is a predictor of the formation of a wide range of cardiovascular diseases. The aim of our study is to identify the morphofunctional features of molecular phenotype and ultrastructure of smooth muscle cells of ascending aorta wall in rats born 12 and 24 hours prematurely. The paper presents the results of immunohistochemical and morphometric, as well as ultrastructural analysis of ascending aorta wall in Wistar rats born 12 and 24 hours prematurely. It has been shown that preterm birth leads to a later change in the phenotype of smooth muscle cells from synthetic to contractile, which can negatively affect the morphofunctional state of the cardiovascular system.
A cell is a complex three-dimensional system, which possesses a number of highly dynamic structures with extended, rugged, and uneven morphology. The actin cytoskeleton consists of fibrillar and globular actin, as well as auxiliary proteins that regulate organization. The shape and the rearrangements of actin cytoskeleton are closely related to functioning of the cell. The ability to characterize these changes allows scientists to confirm or refute any hypotheses in the research. Obtaining a numerical equivalent of the actin cytoskeleton organization could help compare actin structures in biological experiments (example: exposure to biologically active substances). The review summarizes methods for analyzing images of intracellular actin structures labeled with phalloidin using ImageJ. The methods considered make it possible to obtain a quantitative characteristic of the organization of actin structures for further evaluation and comparison of experimental results.
Tissue damage activates programs aimed at the survivalof the body and restoration of its integrity, but fora long time the molecular and cellular mechanisms of theirimplementation remained undeciphered. In recent years, a number of importantinformation has been obtained about the regulation of regeneration processes,which significantly clarify a number of ideas about the regulationof the response to damage and deserve attention in termsof searching for new targets for controlling this process. Thisreview briefly summarizes the basic regulatory mechanisms associated with theearly stages of the human tissue response to injury. Italso provides current data on the mechanisms of damage receptionand the role of stromal cells in the formation ofprimary connective tissue as a structure that determines the outcome.
Objective: The purpose of this study was to analyze the patterns of cytoplasmic granulation of GV oocytes that differ in their ability to resume and complete meiotic maturation in vitro. Methods: Although in vitro fertilization (IVF) programs usually use mature (metaphase II, MII) oocytes, it is also possible to use diplotene oocytes at the germinal vesicle (GV) stage after their in vitro maturation (IVM). Results and Discussion: Morphological characteristics of native GV oocytes are promising predictors of their capacity for spontaneous maturation in stimulated cycles. Conclusions: It has been shown that the central granulation pattern negatively correlates with the ability of GV oocytes to spontaneously mature in vitro.
Amitriptyline is a tricyclic antidepressant widely used in clinical practice for the treatment of anxiety, depression and chronic pain. These drugs have a multifaceted effect on cellular processes. One of their targets is sigma-1 receptors. Sigma-1 receptors are molecular chaperones located in endoplasmic reticulum membrane; they are characterized by a unique structure and pharmacological profile. Sigma-1 receptors regulate many cellular processes in health and disease, including Ca2+ signaling. Using Fura-2AM microfluorimetry, it was shown for the first time that sigma-1 receptor agonist, antidepressant amitriptyline, significantly suppresses both Ca2+ mobilization from intracellular Ca2+-stores and subsequent store-dependent Ca2+ entry into cells, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, as well as disulfide-containing immunomodulators glutoxim and molixan, in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in a complex signaling cascade induced by glutoxim or molixan, leading to an increase of intracellular Ca2+ concentration in macrophages. The results also indicate the participation of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.
