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Vol 19, No 3 (2024)

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Reviews

Methods of karyoplast cell cycle synchronization for increasing the efficiency of somatic cloning of farm animals

Zhukova A.S.

Abstract

Somatic cloning is a method of obtaining genetically identical offspring, which for some reason have extremely low efficiency. Methods to increase the effectiveness of this procedure aimed at optimizing each of its stages, one of which is karyoplast preparation. In most experiments, to obtain cloned offspring, oocytes are used as recipient cells at the metaphase of the second meiotic division without prior activation, which determines the choice of somatic cells as karyoplasts at the G0/G1 stage, the most optimal for subsequent nuclear reprogramming by oocyte cytoplasmic factors. Serum starvation and/or contact inhibition are the most commonly used methods for arresting cells in this phase, which allows the arrest of up to 90% of cells at the G0/G1 stage. Despite the effectiveness of these methods, they have some significant limitations; therefore, the addition of components to the culture medium of somatic cells that prevent the progression of cells through the cell cycle stages is becoming widespread. Some chemical inhibitors have a protective effect on somatic cells, preventing the induction of apoptotic changes. Although the efficacy of butyrolactone I, mimosine, and aphidicolin application is controversial, several studies have attested the possibility of using these drugs to synchronize the karyoplasts. Thus, several methods can be employed for effective synchronization of the cell cycle of karyoplasts. When choosing the optimal method, the type of cells, species of animals from which they were obtained, and permissible duration of cultivation under given conditions must be considered to minimize the negative effect of conditions on karyoplast viability.

Genes & Cells. 2024;19(3):319-333
pages 319-333 views 68

Molecular signaling of key neurotrophic factors in the brain while the occurrence of affective disorders

Karabanov S.Y., Kibitkina A.A., Vasilevskaya E.R., Fedulova L.V.

Abstract

The COVID-19 pandemic and the increasingly tense political situation worldwide have led to an increase in the incidence of mood disorders. The occurrence of affective disorders is usually associated with neurotransmitters such as serotonin and dopamine; however, modern trends aim at studying the involvement of neurotrophic factors in the mechanisms of mood disorders.

This review aimed to summarize and systematize knowledge about key neurotrophic factors and the molecular mechanisms of their relationships.

The key metabolic mechanisms of proteins such as brain-derived neurotrophic factor, vascular endothelial growth factor, insulin-like growth factor-1, basic fibroblast growth factor-2, nerve growth factor, and glial cell-line derived neurotrophic factor are considered. Molecular pathways were analyzed, and a complex diagram of a multiple cascade with interconnected reactions was compiled, including each factor. Key molecular targets chosen included nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) and cAMP response element-binding protein. The review also presented candidates for the role of limiting factors for these molecular targets. For the NF-kB cascade, neurotrophin receptor p75(NTR) was proposed as a limiting factor, and those for the CREB cascade were intracellular phospholipase C (PLC-γ), binary molecular switches (RAS-GTP), and protein kinase B (AKT).

Genes & Cells. 2024;19(3):334-347
pages 334-347 views 53

Use of atomic force microscopy to assess the biomechanical properties of 3D tumor cell models

Puchkova K.S., Lopareva V.R., Shepeleva E.V., Magomedova O.A., Ivanova D.V., Zamskaya Y.V.

Abstract

Multicellular spheroids are a unique object model for toxicological studies. Cells in a three-dimensional (3D) cluster contain a microenvironment and intercellular communication, which allows spheroids to be used as a more realistic model than traditional cell cultures. Tumor microregions consist of heterogeneous populations of cancer cells, in which cell growth and response to antitumor drugs depend on their 3D architecture, intercellular contacts, and interaction with the microenvironment. Tumor growth and progression are also strongly influenced by mechanical cues. Currently, 3D cell culture models are powerful tools for studying the toxicity of drug compounds and nanomaterials of different compositions and morphologies.

This review presents data on the use of various techniques, particularly atomic force microscopy, to investigate changes in the mechanical properties of cells in spheroids. Specifically, the use of atomic force microscopy as a tool to reveal physicochemical parameters of cells during pathophysiological processes or drug exposure is considered. The relevance of this review is attributed to the increasing interest in the role of biomechanical properties of tissues, cells, and subcellular structures as markers of pathophysiological conditions.

Genes & Cells. 2024;19(3):348-358
pages 348-358 views

Effect of insulin on the phosphoproteome of skeletal muscles in normal conditions and with insulin resistance

Yakupova E.I., Popov D.V.

Abstract

The skeletal muscles are the main site of insulin-dependent glucose uptake, and the development of insulin resistance in skeletal muscles is the main factor in the progression of insulin resistance in the whole organism. This disorder is associated with defects in the canonical insulin cascade regulating glucose uptake; however, the specific molecular mechanisms are still debatable. Global mass spectrometry-based phosphoproteomic analysis appears to be an optimal approach to studying complex signaling networks.

The review summarizes data from phosphoproteomic studies investigating changes in intracellular signaling in skeletal muscles upon insulin stimulation under normal conditions and insulin resistance. In vitro and in vivo studies have shown that insulin stimulation/food intake causes large-scale changes in the phosphoproteome (hundreds of phosphosites). These changes affect not only the canonical insulin cascades but also other signaling pathways and proteins with different functions (enzymes of carbohydrate and fat metabolism, sarcomeric and mitochondrial proteins, transcription factors, chaperones, etc.) and cause transcriptomic changes. Insulin resistance impairs the phosphoproteomic response to insulin; however, these changes only slightly affect the canonical insulin cascade regulating glucose uptake. The causes of impairments in insulin-dependent glucose uptake are hypothesized to be related primarily by a combination of multiple defects in various signaling molecules that regulate glucose uptake directly or indirectly; however, they are not associated with the canonical insulin cascade.

Genes & Cells. 2024;19(3):359-371
pages 359-371 views

Original Study Articles

Evaluation of chondrogenic potential of human dermal fibroblasts after modification with differentiation media and cytokine TGF-β3

Bozhokin M.S., Marchenko D.M., Mikhaylova E.R., Rakhimov B.R., Bozhkova S.A., Korneva Y.S., Aleksandrova S.A., Khotin M.G.

Abstract

BACKGROUND: Hyaline cartilage is an avascular tissue that envelopes the surface of major joints. The regenerative capacity of this tissue is restricted due to its high content of extracellular matrix proteins and its modest number of cells. Articular cartilage recovery remains relevant and yet unresolved to date. Utilizing biomedical products that contain modified cells is a promising approach.

AIM: To examine the effects of various culture media on the chondrogenic modification of cells and to compare the results.

MATERIALS AND METHODS: Human dermal fibroblasts and rabbit multipotent mesenchymal stromal cells were modified using the chondrogenic differentiation medium StemPro or recombinant TGF-β3 protein. Alterations in the expression of genes involved in chondrogenesis (Acan, Tgfβ3, Col2α1, Comp) were assessed using the real-time polymerase chain reaction method ΔΔCt.

RESULTS: It was demonstrated that human dermal fibroblasts can induce chondrogenic modification when used in protein media. This cell type is easy to harvest, does not necessitate special cultivation conditions, is readily scalable, and is suitable for allogeneic transplantation.

CONCLUSION: The obtained data can be employed to develop tissue engineering products for the regeneration of hyaline cartilage using allogeneic dermal fibroblasts.

Genes & Cells. 2024;19(3):372-386
pages 372-386 views

Comparison the impact of mesenchymal stromal cells, their microvesicles and plasma enriched with soluble platelet factors on survival and apoptosis of rat spleen lymphocytes in vitro

Klimenkova O.V., Potapnev M.P., Goncharova N.V., Kudelich O.A., Kondratenko G.G.

Abstract

BACKGROUND: The trophic function is one of the important but insufficiently studied features of mesenchymal stromal cells (MSCs), their microvesicles (MVs), and plasma enriched with soluble platelet factors (PRPs), which ensure the viability of target cells.

AIM: To analyze the capability of MSCs, MVs, and PRP to affect the viability and spontaneous and activation-induced apoptosis of rat spleen lymphocytes during culturing in vitro.

MATERIALS AND METHODS: MSCs were isolated from the mononuclear cell fraction of the femoral bone marrow of Wistar rats using the plastic adhesion method. MVs were obtained from the conditioned medium of MSCs by centrifugation at 14,500 g. PRP was prepared by freezing/thawing rat peripheral blood platelet concentrate. Rat spleen lymphocytes were isolated on a density gradient of 1.077 g/cm3. The viability and degree of lymphocyte apoptosis in vitro were assessed by flow cytometry for 7-AAD incorporation and binding to annexin V.

RESULTS: The presence of MSCs at 10 and 20% concentrations caused an increase in the number of living intact and PMA-activated lymphocytes by the end of 3-day in vitro cultivation. Compared with controls, the amount of necrotic cells decreased 8.3–13.5 times, and the number of apoptotic cells decreased 2.3–4.0 times, mainly due to lymphocytes at the late stage of apoptosis. MVs of MSCs at the indicated concentrations did not significantly affect the viability of lymphocytes cultured in vitro but reduced the level of apoptosis of intact and PMA-activated lymphocytes by 3.6 (р=0.03) and 4.8–5.2 (р=0.048; р=0.03) times respectively. Studies have shown that 1.25% rat PRP has a growth-stimulatory activity against MSCs but not against lymphocytes cultured in vitro. In the culture of intact lymphocytes, PRPs did not significantly affect cell viability with a slight decrease (2.7–2.9 fold, p >0.05) in the number of necrotic cells. In the culture of PMA-activated lymphocytes, 1.25–2.50% PRP increased the number of living cells by 1.6–2.2 times (р=0.002; р=0.01) and the number of necrotic cells by two times (р=0.02).

CONCLUSION: MSCs, MVs of MSCs, and PRP, in decreasing order, enhanced the viability of rat spleen lymphocytes and suppressed late apoptosis during in vitro cultivation. Lymphocytes activated by phorbol myristate acetate are more sensitive to their vital action compared with resting cells.

Genes & Cells. 2024;19(3):387-399
pages 387-399 views

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