Vol 65, No 4 (2023)

DNA in situ hybridization (DNA-ISH) is a widely used method in molecular cytogenetics that allows the localization of specific DNA sequences in particular regions of chromosomes. Implementation of DNA-ISH requires the use of DNA probes, which can be commercial or developed for specific research purposes as non-commercial (homemade) DNA probes. One of the significant drawbacks of non-commercial probes is the difficulty in obtaining a high signal intensity with a small DNA probe size. Therefore, developing approaches to enhance non-commercial DNA probes is an important task in modern molecular cytogenetics. To directly visualize small DNA sequences on a chromosome, the tyramide signal amplification (TSA) method is used. The TSA system is based on the formation of a covalent bond between electron-rich protein fragments in the sample and tyramide molecules linked to a hapten (in chromogenic in situ hybridization) or a fluorophore (in fluorescent in situ hybridization). This is achieved by converting tyramide molecules into free-radical intermediate compounds under the action of horseradish peroxidase (HRP), followed by deposition of precipitated molecules nearby. As a result, a low-intensity signal is amplified. Thus, TSA is a good complement to the DNA-ISH method, thanks to its high sensitivity and ability to detect small genomic imbalances, and can therefore become a valuable tool for diagnosing chromosomal rearrangements in clinical practice.

Deciphering the mechanisms of development of neurodegeneration at the presymptomatic stage is an urgent task. It’s solving allows optimizing the methods of early diagnostics and prevention of Alzheimer’s disease (AD). Goal of the study: to study the features of neurogenesis in brain neurogenic niches in experimental Alzheimer’s disease at the presymptomatic stage of neurodegeneration. Modeling of AD in vivo was carried out in experimental animals (male mice, C57BL/6, 8 months old) as follows: the control group, n = 30, animals were injected with 2 µl of a 0.9% NaCl solution in the CA1 field of the hippocampus; the experimental group, n = 30, animals were injected with a 1M solution of oligomerized beta-amyloid 25–35 (Aβ25–35) (2 μl bilaterally). Cognitive impairments have been assessed with the passive avoidance task (PAT). For immunohistochemical studies, we identified the subgranular zone of the hippocampus (SGZ) and the subventricular zone (SVZ) in frozen sections of the brain tissue. We have analyzed the expression of markers – Nestin, Pax6, NeuroD1, VEGFR2, as well as apoptosis (TUNEL protocol) in neurogenic niches. In the period preceding the manifestation of cognitive dysfunction (from 9 to 17 days after intrahippocampal administration of Aβ25–35), we registered multidirectional changes in the expression of markers of neurogenesis, neoangiogenesis and the severity of apoptosis in the SGZ of the hippocampus and in the SVZ. At 9th day since the beginning of development of Alzheimer’s type neurodegeneration, we found elevated expression of Pax6 and VEGFR2 in the SGZ and higher number of Nestin+ cells in the SVZ. Subsequent application of the PAT protocol with the presentation of an aversive stimulus (day 10) or the corresponding context (days 11 and 17) resulted in dynamic changes in the expression of cell markers at different stages of neurogenesis. In sum, аt the presymptomatic stage of the Alzheimer’s type neurodegeneration, SGZ and SVZ show signs of aberrant neurogenesis associated with a disruption in the pool of stem and progenitor cells and suppression of the production of neuroblasts/immature neurons in the period preceding the evident cognitive dysfunction.

Information about the effect of cryopreservation on functions and the genetic of cells of different genesis is not unambiguous and is in the process of accumulation. This work is aimed at studying the effect of long-term storage (7 years) of human endometrial mesenchymal stem cells (eMSCs) in the frozen state on the stability of their genome in vitro. The results showed destabilization of the karyotype structure in the descendants of cells after their thawing, namely, aneupolyploidization, increased fragility of chromosomes, resulting in a huge pool of aberrant chromosomes, and impaired condensation in homologues. Chromosomal breakds in centromeric regions offen accompanied by the preservation of genetic material in the form of independent chromosomes. Almost all chromosomes of the set were involved in the process of destabilization of the eMSCs cell genome. It has been shown that the procedure of long-term cryopreservation can become an inducer of premature cellular aging of eMSCs after their thawing. Comparison of the data obtained with the results of karyotyping of transformed Chinese hamster cells that underwent a similar procedure led to the conclusion that cryopreservation for biological systems can be a stress that induces genetic defects of various types at the karyotype level. The response of the genome of cells of different origin to the same conditions of cryopreservation may differ.

The ability of three model green proteins to covalently bind to microparticles (MP) based on poly(D,L-lactic acid) (PLA). Green fluorescent protein (sfGFP), recombinant human beta2-microglobulin-sfGFP fusion protein (β2M-sfGFP), and recombinant human amylin-sfGFP fusion protein (IAPP-sfGFP) were isolated by affinity chromatography. The double emulsion method was used to form PLA-MPs. The modification of PLA MPs by proteins was testified using laser scanning microscopy (LSM). Phagocytosis of PLA-MPs modified with different proteins and free model proteins by macrophages was also studied using LSM. Recombinant sfGFP has been shown to bind to particle surfaces at lower levels compared to β2M-sfGFP and IAPP-sfGFP. Presumably, this is due to the fact that amino groups that could potentially react with activated carboxyl groups on particle surfaces, are spatially unavailable for this reaction due to the structure of sfGFP. β2M and IAPP within the corresponding recombinant proteins are spacer structures between the surface of spherical particles and sfGFP. It was also found that increasing the protein/particle ratio by a factor of three did not lead to an increase in the amount of bound protein per unit mass of particles, which may indicate that the amount of protein that can be bound per unit mass of particles is limited by the capacity of the particles themselves. The study of phagocytosis of PLA-MPs modified with model proteins revealed that MPs bearing β2M-sfGFP and IAPP-sfGFP were captured by macrophages and, therefore, contribute to the activation of the cellular immune response, which is important in the fight against various viral infections. In addition, model proteins (β2M-sfGFP, IAPP-sfGFP) appeared to be also capable of phagocytosis. This may be due to the fact that both β2M and IAPP are amyloidogenic and aggregation prone proteins. Apparently, the aggregates of these proteins are also able to be absorbed by macrophages due to the increase in size compared to their monomeric forms.

The aim of this work was to analyze the morphology and morphometric parameters of peripheral blood eosinophils and eosinophilic granules in raccoon dogs Nyctereutes procyonoides (Grey, 1834). On blood smears stained by Pappenheim, the composition of the leukocyte formula was determined, the morphological features and morphometric parameters of eosinophils and their granules were evaluated. Cytochemical methods revealed the localization of peroxidase and cationic proteins in eosinophils. ANOVA was used to assess the effect of sex. As a result of the study, it was found that raccoon dogs are characterized by a high relative content of eosinophils (7–10%), as well as the presence of large secretory granules in them. Along with the common eosinophils, there were cells with abnormal granularity (low number of secretory granules, the presence of vacuole-like granules that did not stain with the dyes used). The influence of sex was expressed in a higher proportion of eosinophils with abnormal granularity in males compared to females, while the latter had higher morphometric parameters (the number and average area of granules in one cell, the ratio of the area occupied by granules to the area of the cell). Since the causes of abnormally granular eosinophils in raccoon dogs are not completely clear, there is a need for further research into this issue.

V. E. Yurinskaya, I. I. Marakhova, I. A. Gamaley, A. A. Rubashkin, A. V. Moshkov, A. N. Tomilin