Vol 14, No 3 (2022)

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.

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.

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.

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.

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.

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.