Natella Iosifovna Enukashvili

Heat shock activates heat shock factor 1 (HSF1), which induces classical heat shock genes and promotes transcription of pericentromeric HSAT3 repeats that contribute to nuclear stress body (nSB) formation. Although well characterized in epithelial cells, this response remains poorly understood in myeloid cells. We compared heat-induced HSAT3 transcription and nSB assembly in epithelial and monocytic cell lines, using A549 and THP-1 as models. pHSF1 (Ser326) levels, HSPA1A and HSAT3 expression, and nSB formation were assessed by Western blotting, qPCR, immunofluorescence, RNA-FISH, and 5-ethynyluridine labeling. Heat shock increased pHSF1 (Ser326) levels and HSPA1A expression in both models. A549 cells showed increased HSAT3 transcript levels and prominent nSB formation, whereas undifferentiated THP-1 cells showed little immediate change in HSAT3 transcript levels and did not form prominent nSBs. 5-EU labeling revealed discrete nascent RNA foci in heat-shocked A549, but not THP-1, cells. These foci overlapped with HSF1 and were associated with HSAT3-related hybridization signals. However, click chemistry increased accessibility of genomic satellite sequences, limiting RNA-specific interpretation of the HSAT3 probe signal. PMA-induced differentiation enabled an increase in HSAT3 transcript levels during heat shock and nSB assembly. These findings identify cellular state as a determinant of heat shock-induced satellite transcription and nSB assembly.

Movement of genetic material between non-parental organisms, called horizontal gene transfer (HGT), is well recognized in prokaryotes but represents an underestimated force for the acquisition of novel traits in eukaryotes. The mechanisms of cross-domain HGT remain poorly understood despite numerous reports of its occurrence. Thus, progress in the field remains limited by the lack of targeted approaches for detecting HGT events. Using BLASTp search and sequence identity, we describe an HGT-derived protein from the chytridiomycete fungus Neocallimastix californiae . This protein has two cysteine-rich repeats (CysRReps) and other functional domains that are highly similar to those of prokaryotes. Based on the alignment of several CysRReps, we identified 859 additional eukaryotic proteins spanning 43 protein families, each with at least one match to bacterial, archaeal, or viral proteins. HGT-derived proteins with CysRReps belong to multiple eukaryotic taxa, some of which are well-known HGT models. Bacterial, archaeal, and viral proteins comprise the prokaryotic counterparts. Bacteria belong to 15 phyla, spanning a diverse array of physiologies, habitats, and lifestyles. Viral diversity is largely restricted to Caudoviricetes, suggesting a virus-mediated mechanism of DNA integration underlying these transfers. Although the function of CysRReps remains unclear, their association with HGT events across diverse eukaryotic and prokaryotic taxa suggests the existence of a universal molecular mechanism that facilitates gene transfer across phylogenetic boundaries.

Regenerative endodontics is a strategy aimed at the regeneration of dental pulp or at least the formation of pulp-like tissue, ideally the pulp-dentin complex; regeneration of damaged coronal dentin, resorbed root, cervical or apical dentin. Regenerative endodontic strategies include stem cell transplantation and regenerative endodontic procedures (REP). REP methods are currently used in the endodontic treatment of open-apice teeth in patients aged 7–10 years. The most likely outcome of this treatment is repair (“maturation”) of the root, rather than regeneration of its tissues. Aim of the study : to develop the method and evaluate the efficacy of dental pulp stem cells in combination with fibrin gel for regenerative endodontic treatment of mature permanent teeth in a large animal model. Materials and methods . The study was conducted on minipigs. While under general anesthesia (xylazine and tiletamine-zolazepam), the animals underwent an X-ray examination of their oral cavities. This examination was performed to select a tooth that most closely resembled a human tooth in terms of its position in the jaw arch, as well as the number and shape of its roots and canals. The first left two-rooted molar (P2) was selected. The crown pulp was amputated, and the root pulp was extirpated. The canals were then filled with fibrin gel containing allogeneic pig dental stem cells. The gel was mixed with fibrinogen polymerization catalysts ( thrombin and calcium chloride) immediately before being injected into the canals for polymerization. Thus, the gel (3 μL, containing 15,000–18,000 cells per canal) took the shape of the canal during polymerization. Next, the gel was isolated with mineral trioxide aggregate, and the crown was filled with chemically cured, reinforced, packable glass ionomer cement. Four months after the endodontic treatment, the tooth was removed, fixed, decalcified, and used for a standard histological examination. Stepwise serial sections, 3–5 μm thick, were prepared and stained with hematoxylin and eosin. For quantitative assessment, seven sections were selected at different levels with a step of 10 μm, the images were scanned, and morphometric calculations were performed. Absolute and relative values of lengths and areas were assessed, as well as the degree of vascularization. Results . The length, shape and volume of the root canals of the first left premolar (P2) are similar to those in humans, which allowed us to use approaches and equipment developed for the endodontic treatment of human teeth, thus increasing the relevance of the model. Histological sections in the apical part of the canal showed the formation of vascularized pulp-like stromal tissue, and in the middle and apical thirds of the root, areas with a palisade layer of cylindrical cells — active odontoblasts producing dentin — were detected next to basophilic masses. There were no inflammatory processes in the periapical area of the endodontically treated tooth. No changes in the animals’ well-being or eating behavior were observed. Conclusion . The results of the pilot study indicate the prospects for the development of regenerative endodontics methods for the treatment of mature permanent teeth with formed roots. Scaffolds based on fibrin gel and dental pulp stem cells are generally suitable for the purposes of regenerative endodontics, but the method needs to be further improved to form pulp architectonics.

BACKGROUND: In modern regenerative medicine, the development of personalized biomedical cellular products for the replacement of bone tissue defects is crucial. Such products consist of a cellular component, a scaffold designed to retain cells and provide mechanical support, and auxiliary components. In vitro experiments do not adequately assess the effects of these constructs on bone regeneration or on the systemic response of the recipient organism. AIM: This study aimed to determine the outcomes of implanting scaffolds based on polycaprolactone, hydroxyapatite, and dental pulp stem cells into jaw bone defects in rabbits. METHODS: The study was conducted in Soviet (Russian) Chinchilla rabbits (n = 10) weighing 3.5–4.5 kg and aged 1–1.5 years bred under vivarium conditions. Four teeth were extracted from each animal (40 teeth in total), then the animals were divided into 5 groups. The regenerative outcomes were assessed after 4 months. To evaluate differences in the extent and rate of defect healing, histopathological examination of the implantation sites was performed. The degree of fibrosis, inflammation, and bone tissue remodeling was assessed in hematoxylin and eosin–stained sections, whereas the distribution pattern of implanted cells labeled with iron oxide nanoparticles was evaluated using Perls’ staining. RESULTS: At the implantation sites, scaffolds (both cell-seeded and cell-free) accelerated bone defect remodeling and fibrosis without the formation of coarse scar tissue. The most favorable combination of outcomes—namely minimal inflammation, the most advanced stage of bone tissue remodeling, and mature connective tissue formation at the implantation site—was observed in the group receiving copper-coated polycaprolactone scaffolds seeded with dental pulp stem cells. CONCLUSION: The results confirm the promise of this approach for the development of bioengineered constructs for the reconstruction of bone defects.

Objective: Single embryo transfer is a strategy used in assisted reproductive technologies that can reduce the incidence of multiple pregnancies. In this approach, the importance of methods for assessing embryo quality increases substantially, as it is necessary to select the embryo with the highest implantation potential from among several embryos. One way to improve embryo quality assessment is through non-invasive methods of diagnosing oocyte competence. The aim of the study was to evaluate the potential use of the expression levels of long non-coding RNAs (lncRNAs) NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, VIM-AS1, HSAT2/3, LTR12c-375, HERVS71-int9, MER52c-175 as predictors of oocyte quality. Methods: Nine donors and 19 patients with various IVF outcomes participated in the study. The groups of donors and patients did not differ significantly in age, body mass index, antimüllerian hormone level, duration of stimulation, or daily dose of hormones. RNA was isolated from the cumulus cells and used for cDNA synthesis and subsequent amplification by qPCR. The data were analyzed using statistical methods to determine the correlation between the level of the lncRNAs expression and IVF procedure outcome. Results and discussion: The expression levels of the lncRNAs were analyzed in relation to the IVF outcome and the embryologic status of patients and donors (total number, number of mature oocytes, and number of day 5 blastocysts). Decreased expression of NEAT1 and increased expression of VIM-AS1 were the predictors most strongly associated with successful IVF outcomes, i.e., birth (correlation coefficient r (–0.7) and 0.6 respectively). Logistic regression parameters were determined for NEAT1 to describe the relationship between IVF outcomes and non-coding RNA transcription levels (b0 = 3.852; b1 = –1.418). There was no correlation between the NEAT1 transcription level and the number of oocytes or blastocysts. This suggests that NEAT1-dependent factors that reduce oocyte competence play a role in post-implantation stages. VIM-AS1 expression showed a weak correlation with the total number of oocytes (r = 0.3). Transcript 2 isoform of the VIM-AS1 correlated also with the number of mature oocytes and day 5 blastocysts. Conclusions: NEAT1 and VIM-AS1 non-coding RNAs of cumulus cells are involved in follicle maturation processes and can be considered as potential biomarkers for assessing oocyte quality and predicting the outcome of IVF procedure.

The c-ErbB receptor family is a fundamental cell signaling system that regulates cell proliferation, motility, apoptosis, differentiation, and other key cellular functions. Overexpressed and mutated in some tumors, c-ErbB receptors play a pivotal role in their progression but are also present in many non-malignant cells, including those that are promising from the point of view of regenerative medicine, such as mesenchymal stromal cells (MSCs). The role of c-ErbB receptors in these cells is not clearly understood, and the data on their expression are sporadic. Therefore, the systemic characterization of c-ErbB receptor family expression in MSCs from a wide range of tissues is of high priority. Here, using RT-qPCR and Western blotting analysis, we evaluated the c-ErbB receptors expression pattern at the mRNA and protein levels in human MSCs isolated from six different tissues. We found that MSCs possess considerable EGFR and HER2 mRNA levels comparable to those in some malignant cells while showing trace HER3 and HER4 expression. However, EGFR but not HER2 was detected in MSCs at the protein level. We also show that the absence of HER2 protein is not associated with its rapid lysosomal degradation. We conclude that c-ErbB signaling in human MSCs is exclusively mediated by EGFR.

Mesenchymal stromal cells of the tumor microenvironment (TME) play a significant role in the progression of multiple myeloma (MM). The cells of the TME demonstrate resistance to treatment, thereby creating a favorable environment for disease relapse. The status of the TME during remission is poorly understood. An association between treatment response and TME status (including signaling pathways) has been suggested. One of the key players in the establishment of the MM TME is WNT signaling. In this study, we evaluated the expression of WNT family proteins in the TME and MM cells to assess their potential as TME markers and predictors of treatment response. A bioinformatic analysis of normal and malignant plasma cells, combined with an analysis of published data, revealed the following differentially expressed WNT genes: WNT5A, WNT10B, CTNNB1, and WNT3A. Immunohistochemical staining with the antibodies against the proteins encoded by the genes was conducted on trephine biopsy samples of bone marrow from healthy donors and patients with different responses to therapy. A quantitative analysis of the immunohistochemical data revealed differences in the amounts of WNT3A, WNT5A, WNT10B, and β-catenin proteins in the bone marrow before treatment depending on the subsequent responses of the patients to therapy. Multiplex fluorescent immunohistochemical staining with tyramide signal amplification revealed that WNT3A was predominantly present in mesenchymal stromal cells, whereas WNT5A and WNT10B were primarily observed in plasma cells. β-catenin was detected in both cell types. We analyzed the mRNA levels of the WNT gene family and CTNNB1 in MSC cultures from healthy donors and patients using qPCR. These genes were differentially expressed in MSC cultures derived from patients and healthy donors, as well as between patients grouped according to their response to therapy. Therefore, WNT proteins and β-catenin can be considered potential markers to assess the state of the tumor niche.

Objective: Cumulus cells respond to the effects of hormones and signalling molecules synthesised by the oocyte by changing their expression profile. In turn, cumulus cells control the growth and maturation of the oocyte. Therefore, analysis of the transcriptional profile of cumulus cells is likely to be one of the approaches for non-invasive prediction of oocyte quality in assisted reproductive technology programs. To evaluate the expression of selected genes in cumulus cells in women with primary and secondary types of infertility with positive and negative results of assisted reproductive technologies. Methods: 9 healthy donors and 19 patients undergoing infertility treatment with ART methods participated in the study. RNA was isolated from cumulus cells obtained during oocyte preparation for fertilisation, and cDNA was synthesised and used as a matrix for real-time PCR with primers for AREG, SCD4, PTGS, SCD5, HAS2, VCAN, STAR and two lncRNA genes (ANXA2P2, MALAT1). Results and Discussion: The genes of interest expression did not depend on the type of infertility but rather on the IVF attempt outcome. The panel of mRNA biomarkers (SDC4^upAREG^upMALAT1^{not changed}ANXA2P2^{not changed} ) was associated with poorer oocyte competence prognosis and SDC4^{not changed}AREG^downMALAT1^downANXA2P2^down set of biomarkers was associated with better quality of oocytes. Conclusions: This non-invasive method can be used to access oocyte quality in patients with primary and secondary infertility.