Vol 14, No 3 (2022)


Visualization of G-Quadruplexes, i-Motifs and Their Associates

Dubrovin E.V., Barinov N.A., Klinov D.V.


The non-canonical structures formed by G- or C-rich DNA regions, such as quadruplexes and i-motifs, as well as their associates, have recently been attracting increasing attention both because of the arguments in favor of their existence in vivo and their potential application in nanobiotechnology. When studying the structure and properties of non-canonical forms of DNA, as well as when controlling the artificially created architectures based on them, visualization plays an important role. This review analyzes the methods used to visualize quadruplexes, i-motifs, and their associates with high spatial resolution: fluorescence microscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). The key approaches to preparing specimens for the visualization of this type of structures are presented. Examples of visualization of non-canonical DNA structures having various morphologies, such as G-wires, G-loops, as well as individual quadruplexes, i-motifs and their associates, are considered. The potential for using AFM for visualizing non-canonical DNA structures is demonstrated.

Acta Naturae. 2022;14(3):4-18
pages 4-18 views

The Role of a Pathological Interaction between β-amyloid and Mitochondria in the Occurrence and Development of Alzheimer’s Disease

Nikolaeva N.S., Yandulova E.Y., Aleksandrova Y.R., Starikov A.S., Neganova M.E.


Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases in existence. It is characterized by an impaired cognitive function that is due to a progressive loss of neurons in the brain. Extracellular β-amyloid (Aβ) plaques are the main pathological features of the disease. In addition to abnormal protein aggregation, increased mitochondrial fragmentation, altered expression of the genes involved in mitochondrial biogenesis, disruptions in the ER–mitochondria interaction, and mitophagy are observed. Reactive oxygen species are known to affect Aβ expression and aggregation. In turn, oligomeric and aggregated Aβ cause mitochondrial disorders. In this review, we summarize available knowledge about the pathological effects of Aβ on mitochondria and the potential molecular targets associated with proteinopathy and mitochondrial dysfunction for the pharmacological treatment of Alzheimer’s disease.

Acta Naturae. 2022;14(3):19-34
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Human Artificial Chromosomes and Their Transfer to Target Cells

Ponomartsev S.V., Sinenko S.A., Tomilin A.N.


Human artificial chromosomes (HACs) have been developed as genetic vectors with the capacity to carry large transgenic constructs or entire gene loci. HACs represent either truncated native chromosomes or de novo synthesized genetic constructs. The important features of HACs are their ultra-high capacity and ability to self-maintain as independent genetic elements, without integrating into host chromosomes. In this review, we discuss the development and construction methods, structural and functional features, as well as the areas of application of the main HAC types. Also, we address one of the most technically challenging and time-consuming steps in this technology – the transfer of HACs from donor to recipient cells.

Acta Naturae. 2022;14(3):35-45
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Reporter Transgenes for Monitoring the Antitumor Efficacy of Recombinant Oncolytic Viruses

Semenova A.V., Sivolobova G.F., Grazhdantseva A.A., Agafonov A.P., Kochneva G.V.


Accurate measurement of tumor size and margins is crucial for successful oncotherapy. In the last decade, non-invasive imaging modalities, including optical imaging using non-radioactive substrates, deep-tissue imaging with radioactive substrates, and magnetic resonance imaging have been developed. Reporter genes play the most important role among visualization tools; their expression in tumors and metastases makes it possible to track changes in the tumor growth and gauge therapy effectiveness. Oncolytic viruses are often chosen as a vector for delivering reporter genes into tumor cells, since oncolytic viruses are tumor-specific, meaning that they infect and lyse tumor cells without damaging normal cells. The choice of reporter transgenes for genetic modification of oncolytic viruses depends on the study objectives and imaging methods used. Optical imaging techniques are suitable for in vitro studies and small animal models, while deep-tissue imaging techniques are used to evaluate virotherapy in large animals and humans. For optical imaging, transgenes of fluorescent proteins, luciferases, and tyrosinases are used; for deep-tissue imaging, the most promising transgene is the sodium/iodide symporter (NIS), which ensures an accumulation of radioactive isotopes in virus-infected tumor cells. Currently, NIS is the only reporter transgene that has been shown to be effective in monitoring tumor virotherapy not only in preclinical but also in clinical studies.

Acta Naturae. 2022;14(3):46-56
pages 46-56 views

Type 2 Diabetes Mellitus: Pathogenic Features and Experimental Models in Rodents

Gvazava I.G., Karimova M.V., Vasiliev A.V., Vorotelyak E.A.


Type 2 diabetes mellitus (T2DM) is the most common endocrine disorder (90%) in the world; it has numerous clinical, immunological, and genetic differences from type 1 diabetes mellitus. The pathogenesis of T2DM is complex and not fully clear. To date, animal models remain the main tool by which to study the pathophysiology and therapy of T2DM. Rodents are considered the best choice among animal models, because they are characterized by a small size, short induction period, easy diabetes induction, and economic efficiency. This review summarizes data on experimental models of T2DM that are currently used, evaluates their advantages and disadvantages vis-a-vis research, and describes in detail the factors that should be taken into account when using these models. Selection of a suitable model for tackling a particular issue is not always trivial; it affects study results and their interpretation.

Acta Naturae. 2022;14(3):57-68
pages 57-68 views

Research Articles

Mechanisms of P-Glycoprotein Regulation Under Exogenous and Endogenous Oxidative Stress In Vitro

Abalenikhina Y.V., Shchulkin A.V., Mylnikov P.Y., Rokunov E.D., Yakusheva E.N.


We investigated the mechanisms of P-glycoprotein (P-gp) transporter regulation in Caco-2 cells under exogenous and endogenous oxidative stress (OS). Exogenous OS was modeled by exposure of the growth medium to hydrogen peroxide at concentrations of 0.1, 0.5, and 1 µM for 24 h or 10 µM for 72 h. Endogenous OS was modeled by incubating cells with DL-buthionine sulfoximine (BSO, gamma-glutamylcysteine synthetase inhibitor) at a concentration of 10, 50, and 100 μM for 24 h. The levels of intracellular reactive oxygen species (ROS) were assessed using MitoTracker Red CM-H2XRos fluorescent probes. Relative P-gp contents were analyzed using Western blot. Exogenous and endogenous OS was shown to increase relative to P-gp contents. An important role played by the Nrf2-Keap1 signaling pathway in increasing the P-gp contents under H2O2-induced exogenous OS was revealed using specific inhibitors. The transcription factor HIF1 is involved in the regulation of the P-gp levels under 24-hour exogenous OS, and the transcription factor CAR is involved in the regulation of transporter levels under 72-hour OS. All tested transcription factors and signaling pathways are involved in P-gp induction under endogenous OS. Most likely, this is associated with the bimodal effect of BSO on Pgp. On the one hand, BSO induces the development of OS; on the other, BSO, as a xenobiotic, is able to stimulate PXR and CAR, which, in turn, increase the P-gp contents.

Acta Naturae. 2022;14(3):69-78
pages 69-78 views

MicroRNA Expression Profile Changes in the Leukocytes of Parkinson’s Disease Patients

Ardashirova N.S., Abramycheva N.Y., Fedotova E.Y., Illarioshkin S.N.


Parkinson’s disease (PD) is one of the most common movement disorders. It is primarily diagnosed clinically. A correct diagnosis of PD in its early stages is important for the development of a pathogenic treatment, which necessitates a search for potential biomarkers of the disease. We evaluated the diagnostic value of several microRNAs and their relationship with the clinical characteristics of PD. The study included 70 PD patients and 40 healthy volunteers. We analyzed the expression of 15 microRNAs in blood leukocytes, which were selected based on literature data and modern concepts of molecular PD pathogenesis. All patients were evaluated using the Hoehn and Yahr scale, UPDRS, NMSQ, and PDQ-39. The data analysis revealed a statistically significant increase in the expression of miR-7-5p, miR-29c-3p, and miR-185-5p and a statistically significant decrease in the expression of miR-29a-3p and miR-30c-1-5p in leukocytes in PD. However, the altered microRNA profile was shown to have a moderate diagnostic value for PD diagnosis. MicroRNA expression changes were associated with the motor and non-motor phenotypic features of PD and administration of anti-Parkinson’s drugs. Also, a relationship between some of the microRNAs studied and the duration and severity of PD was found, which may potentially be used to monitor disease progression.

Acta Naturae. 2022;14(3):79-84
pages 79-84 views

Suppression of the Testis-Specific Transcription of the ZBTB32 and ZNF473 Genes in Germ Cell Tumors

Bulanenkova S.S., Filyukova O.B., Snezhkov E.V., Akopov S.B., Nikolaev L.G.


The family of genes containing C2H2 zinc finger domains, which has more than 700 members, is one of the largest in the genome. Of particular interest are C2H2 genes with potential tissue-specific transcription, which determine the functional properties of individual cell types, including those associated with pathological processes. The aim of this work was to identify C2H2 family genes with tissue-specific transcription and analyze changes in their activity during tumor progression. To search for these genes, we used four databases containing data on gene transcription in human tissues obtained by RNA-Seq analysis. The analysis showed that, although the major part of the C2H2 family genes is transcribed in virtually all tissues, a group of genes has tissue-specific transcription, with most of the transcripts being found in the testis. After having compared all four databases, we identified nine such genes. The testis-specific transcription was confirmed for two of them, namely ZBTB32 and ZNF473, using quantitative PCR of cDNA samples from different organs. A decrease in ZBTB32 and ZNF473 transcription levels was demonstrated in germ cell tumors. The studied genes can serve as candidate markers in germ cell tumors.

Acta Naturae. 2022;14(3):85-94
pages 85-94 views

Extracellular Vesicles Derived from Metastatic Melanoma Cells Transfer α7-nAChR mRNA, Thus Increasing the Surface Expression of the Receptor and Stimulating the Growth of Normal Keratinocytes

Bychkov M.L., Kirichenko A.V., Mikhaylova I.N., Paramonov A.S., Kirpichnikov M.P., Shulepko M.A., Lyukmanova E.N.


We have previously shown that extracellular vesicles secreted by metastatic melanoma cells stimulate the growth, migration, and stemness of normal keratinocytes. This study showed for the first time that extracellular vesicles secreted by the metastatic melanoma cell lines mel H, mel Kor, and mel P contain, both at the mRNA and protein levels, the α7-type nicotinic acetylcholine receptor (α7-nAChR), which is involved in the regulation of the oncogenic signaling pathways in epithelial cells. Incubation with the vesicles secreted by mel H cells and containing the highest amount of mRNA coding α7-nAChR increased the surface expression of α7-nAChR in normal Het-1A keratinocytes and stimulated their growth. Meanwhile, both of these effects disappeared in the presence of α-bungarotoxin, an α7-nAChR inhibitor. A bioinformatic analysis revealed a correlation between the increased expression of the CHRNA7 gene coding α7-nAChR in patients with metastatic melanoma and a poor survival prognosis. Therefore, extracellular vesicles derived from metastatic melanoma cells can transfer mRNA coding α7-nAChR, thus enhancing the surface expression of this receptor and stimulating the growth of normal keratinocytes. Targeting of α7-nAChR may become a new strategy for controlling the malignant transformation of keratinocytes.

Acta Naturae. 2022;14(3):95-99
pages 95-99 views

Morphological Characterization of Astrocytes in a Xenograft of Human iPSC-Derived Neural Precursor Cells

Voronkov D.N., Stavrovskaya A.V., Guschina A.S., Olshansky A.S., Lebedeva O.S., Eremeev A.V., Lagarkova M.A.


Transplantation of a mixed astrocyte and neuron culture is of interest in the development of cell therapies for neurodegenerative diseases. In this case, an assessment of engraftment requires a detailed morphological characterization, in particular an analysis of the neuronal and glial populations. In the experiment performed, human iPSC-derived neural progenitors transplanted into a rat striatum produced a mixed neuron and astrocyte population in vivo by the sixth month after transplantation. The morphological characteristics and neurochemical profile of the xenografted astrocytes were similar to those of mature human astroglia. Unlike neurons, astrocytes migrated to the surrounding structures and the density and pattern of their distribution in the striatum and cerebral cortex differed, which indicates that the microenvironment affects human glia integration. The graft was characterized by the zonal features of glial cell morphology, which was a reflection of cell maturation in the central area, glial shaft formation around the transplanted neurons, and migration to the surrounding structures.

Acta Naturae. 2022;14(3):100-108
pages 100-108 views

An ELISA Platform for the Quantitative Analysis of SARS-CoV-2 RBD-neutralizing Antibodies As an Alternative to Monitoring of the Virus-Neutralizing Activity

Kostin N.N., Bobik T.V., Skryabin G.A., Simonova M.A., Knorre V.D., Abrikosova V.A., Mokrushina Y.A., Smirnov I.V., Aleshenko N.L., Kruglova N.A., Mazurov D.V., Nikitin A.E., Gabibov A.G.


Monitoring of the level of the virus-neutralizing activity of serum immunoglobulins ensures that one can reliably assess the effectiveness of any protection against the SARS-CoV-2 infection. For SARS-CoV-2, the RBD-ACE2 neutralizing activity of sera is almost equivalent to the virus-neutralizing activity of their antibodies and can be used to assess the level of SARS-CoV-2 neutralizing antibodies. We are proposing an ELISA platform for performing a quantitative analysis of SARS-CoV-2 RBD-neutralizing antibodies, as an alternative to the monitoring of the virus-neutralizing activity using pseudovirus or “live” virus assays. The advantage of the developed platform is that it can be adapted to newly emerging virus variants in a very short time (1–2 weeks) and, thereby, provide quantitative data on the activity of SARS-CoV-2 RBD-neutralizing antibodies. The developed platform can be used to (1) study herd immunity to SARS-CoV-2, (2) monitor the effectiveness of the vaccination drive (revaccination) in a population, and (3) select potential donors of immune plasma. The protective properties of the humoral immune response in hospitalized patients and outpatients, as well as after prophylaxis with the two most popular SARS-CoV-2 vaccines in Russia, were studied in detail using this platform. The highest RBD-neutralizing activity was observed in the group of hospitalized patients. The protective effect in the group of individuals vaccinated with Gam-COVID-Vac vaccine was 25% higher than that in outpatients and almost four times higher than that in individuals vaccinated with the CoviVac vaccine.

Acta Naturae. 2022;14(3):109-119
pages 109-119 views

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