BACKGROUND. Ovarian adenocarcinoma is characterized by a high mortality rate due to late diagnosis and the development of resistance to standard chemotherapy. Despite the introduction of targeted therapies, the risk of recurrence remains high, highlighting the need for new therapeutic approaches. In this view, immunotherapy is a promising approach, but requires the identification of specific tumor antigens. AIM. To identify tumor antigens in ovarian adenocarcinoma that are promising for immunotherapy. METHODS. Immunopeptidome from cell lines and postoperative material of patients with ovarian adenocarcinoma were isolated using immunoaffinity chromatography followed by liquid chromatography-mass spectrometry analysis. RESULTS. In this study, we tested an affinity chromatography-based immunopeptidome isolation protocol, comparing various detergents (CHAPS, NP-40, SOD, and Triton X-100) for cell lysis, and observed no statistically significant differences in the number of identified peptides. Using NP-40, 5 peptides belonging to the proteins of cancer/testis antigens (CTA) were identified in the immunopeptidomes of postoperative material from patients with ovarian adenocarcinoma, 3 of which, according to the human protein atlas, are indeed not expressed in normal ovarian tissues. CONCLUSION. Each of the four tested detergents provides identification of unique sets of peptides. Immunopeptidome analysis allows the identification of peptides ofCTA proteins, but a larger sample of postoperative material from patients with ovarian adenocarcinoma and experimental testing of the immunogenicity of the identified peptides are needed for further research.
Chronic stress elicits a broad spectrum of molecular alterations affecting nearly all levels of metabolic regulation. The primary initiating mechanism involves activation of the hypothalamic-pituitary-adrenal axis by a stressor, resulting in sustained glucocorticoid release. This, in turn, drives a systemic reprogramming of energy metabolism, fosters low-grade chronic inflammation and oxidative stress, and instigates persistent epigenetic modifications. These pathological processes are characterized by a severe imbalance in pro- and antioxidant systems, significant shifts in adipose and muscle tissue metabolism including induced lipolysis and gluconeogenesis and impaired leptin and insulin signalling. Consequently, they elevate the risk for abdominal obesity, metabolic syndrome, type 2 diabetes, and related comorbidities. Of particular significance are stress-induced epigenetic changes, such as DNA methylation, which can serve to entrench a pathological metabolic phenotype over the long term. This review, therefore, aims to systematise and analyses current knowledge on the complex molecular mechanisms underpinning stress-induced dysfunction in metabolism and neuroendocrine regulation in mammals.
BACKGROUND: The number of studies investigating physical methods of cell targeting is steadily growing. Within the field of magnetic cell targeting, two main approaches have recently emerged: the use of microscale structures as magnetic carriers, e.g, porous spheroids, helices, and microrobots, that transport cells, and the use of nano- or microscale magnetic particles that directly label cells. AIM: The study aimed to review experimental studies on the use of magnetic particles for targeted delivery of mammalian cells in order to identify the main parameters of magnetic labeling and targeting systems. METHODS: Scientific data was searched in the PubMed, Cochrane Library, and eLIBRARY.RU databases for the period from January 2019 to September 2024 using the keywords magnetic cell targeting, magnetic cell delivery, magnetic cell localization, and magnetic cell guidance. Original experimental in vitro and in vivo studies were included if they involved labeling mammalian or human cells with magnetic nano- or microparticles for targeted delivery using magnetic fields. Data on study design, cell lines used, characteristics of magnetic particles, magnetic labeling conditions and efficiency, characteristics of magnetic trapping systems, efficiency of magnetic delivery, and clinical effects in in vivo disease models were extracted from the selected articles. RESULTS: A total of 62 articles were included in the analysis, of which 63% (39 studies) involved animal disease models, mainly affecting the nervous system, heart, eyes, urinary system, musculoskeletal system, and cancer. The most common labeled cells were multipotent mesenchymal stromal cells (27 studies), immune system cells (16 studies), and endothelial cells and their progenitors (7 studies). In most studies, superparamagnetic iron oxide nanoparticles were used for cell labeling (82% of studies) with more than 30 types of coatings, whereas neodymium magnets of various configurations at 0.005–1.450 T magnetic induction served as targeting systems. In 84% of studies, the optimal labeling concentration of magnetic particles ranged from 10–100 µg Fe/mL, with a labeling time of 4–24 h. A high degree of labeled cell magnetic controllability was demonstrated in vitro. In 19 animal studies, magnetic targeting resulted in a 1.16- to 20-fold increase in local cell concentration within the target area. In 85% of in vivo studies, magnetic targeting produced a more pronounced therapeutic effect compared with controls without targeting systems. Overall, the analysis confirms the high clinical potential of magnetic targeting in cell therapy. CONCLUSION: Magnetic targeting of cells using magnetic particles is a rapidly developing and promising technology in the field of regenerative medicine and cell therapy.
BACKGROUND: By the end of the first quarter of the 21st century, adeno-associated viruses (AAVs) have become a powerful tool for human gene therapy. A number of advantages have positioned these viruses as leaders among gene delivery vehicles for therapeutic purposes. One of the current challenges is the development of novel recombinant AAV variants with tropism for specific cell types, including tumor cells of various origins. This goal can be achieved by mutating the viral capsid using modern capsid engineering approaches. AIM: development of a recombinant AAV that selectively infects human melanoma cells. METHODS: Using a rational design approach for the viral protein shell, a capsid architecture was developed containing the insertion of an RGD peptide that preferentially binds to αvβ3 integrins on the surface of malignant melanoma cells. To generate a plasmid encoding the mutant capsid protein, the PIPE (polymerase incomplete primer extension) method was used. The transduction efficiency of the two AAV variants (one with the mutant capsid and one with the wild-type capsid) was assessed on the human MeWo and HaCaT cell lines. A competitive inhibition assay was used to analyze virus binding to cell surface receptors. RESULTS: A recombinant AAV carrying an insertion of the RGD15 peptide in the VR-VIII loop of the capsid protein (following asparagine 587 in the amino acid sequence) was generated. This mutant virus exhibits reduced tropism for normal skin keratinocytes while retaining the ability to efficiently infect human melanoma cells. Competitive inhibition assays demonstrated that the altered tropism of the AAV with the RGD peptide in the capsid is due to a shift in receptor specificity. CONCLUSION: The recombinant AAV with the proposed capsid design can be further used to deliver an expression cassette encoding one of the oncolytic proteins into human melanoma cells. Such an AAV would potentially exhibit dual specificity toward malignant cells, ensuring selectivity first at the stage of viral receptor binding and then at the transgene expression stage. The development of therapeutic agents with such a mechanism of action would help address the problem of cancer drug non-selectivity.
BACKGROUND:Replicative aging of human skin fibroblasts is closely linked to alterations in the mTOR signaling pathway, a critical regulator of cellular metabolism, proliferation, and autophagy. Despite the established role of mTOR in tissue aging, data on its component Raptor – a subunit of the mTORC1 complex responsible for substrate phosphorylation specificity – remain limited in the context of physiological skin aging. AIM:To study the dynamic changes in Raptor+ fibroblast content in human dermis across ontogenesis and aging. METHODS:A total of 134 human skin samples were analyzed, spanning an age range from 20 weeks of gestation to 85 years. Immunohistochemical (IHC) analysis was performed to evaluate Raptor+ fibroblast expression. Primary polyclonal rabbit antibodies targeting Raptor (GTX132303, GeneTex, USA, 1:50), vimentin (GTX100619, GeneTex, USA, 1:50), and PCNA (AHP1419, AbDSerotec, UK, 1:100) were applied. Morphometric quantification was conducted using anNikon Eclipse 200 light microscope (Japan) with NIS-Elements Br software, calculating the percentage of Raptor+, PCNA+, and vimentin+ fibroblasts relative to the total cell count per 1 mm² of dermal tissue. Statistical analysis included the parametric Student’s t -test for intergroup comparisons of mean values, nonparametric Spearman correlation to assess associations between marker expression and age, and one-way ANOVA to identify age-related trends. RESULTS:A statistically significant decline in Raptor+ fibroblast proportion was observed, decreasing from 92.8% at the embryonic stage (20 weeks gestation) to 87.2% by age 60 (p0.05). This negative trend correlated with reductions in PCNA+ and vimentin+ cell counts, highlighting a functional interplay between mTORC1 activity, fibroblast proliferative capacity, and metabolic status. CONCLUSION: Raptor emerges as a pivotal molecular regulator of replicative dermal aging, modulating the balance between anabolic and catabolic processes. These findings expand current knowledge on age-related mechanisms in human dermal fibroblasts and underscore its potential as a therapeutic target for modulating skin aging.
Synucleinopathies are a group of disorders associated with abnormal aggregation of synuclein family proteins. Aggregated alpha-synuclein serves as a histopathological hallmark of Parkinson disease and several other synucleinopathies, including dementia with Lewy bodies and multiple system atrophy. Under physiological conditions, alpha-, beta-, and gamma-synucleins exist in monomeric form and perform several functions, including regulation of synaptic transmission and intracellular transport. However, under the influence of various pathological factors, these proteins may undergo aggregation, acquire toxic properties, and thereby contribute to the development of neuropathological conditions. Nevertheless, synuclein aggregation–related disturbances are not confined to neurodegenerative disorders and may affect multiple organ systems. Considering that most of these diseases are currently incurable and lack reliable biomarkers for early diagnosis, investigation of synuclein function remains an important area of neuroscience research. In this review, we critically examine the impact of pathological synuclein behavior on the organism and their involvement in processes associated with seizures and epilepsy. In recent decades, accumulating evidence suggests that alpha-synuclein may play a substantial role in epileptogenesis. Pathological aggregation of this protein contributes to activation of neurotoxic mechanisms, including enhancement of oxidative stress, induction of neuroinflammation, and reduced efficiency of neurotransmitter release. Experimental and clinical studies indicate that alpha-synuclein dysfunction may promote the development of seizure activity. Of particular interest are studies demonstrating elevated concentrations of alpha-synuclein in the serum and cerebrospinal fluid of patients with pharmacoresistant epilepsy, which may reflect both its involvement in disease pathogenesis and its potential utility as a biomarker. There is also a rationale for investigating the role of other family members, beta- and gamma-synucleins, although currently available data is limited. Systematization and synthesis of accumulated evidence on the possible association between synucleins and epileptic processes may contribute to a more profound understanding of the molecular mechanisms of epileptogenesis, identification of novel therapeutic targets, and development of methods for early diagnosis.
BACKGROUND: antiseptic solutions and abrasive agents are widely used in dentistry to decontaminate surfaces, which can have a cytotoxic effect on progenitor cells. At the same time, there are no comparative studies evaluating the cytocompatible properties of such substances in cultures of multipotent human mesenchymal cells. AIM: to comprehensively evaluate the immediate and delayed cytotoxic effects of antiseptics and abrasives used in dentistry on MSCs of human adipose tissue (MSC AT) in vitro. METHODS: Human MSC AT was incubated with solutions of antiseptics (Miramistin 0.01%, Chlorhexidine 0.05%, Octenicept) and abrasive powders (sodium bicarbonate, glycine, erythritol) in initial concentrations and at dilutions of 2, 4 and 10 times. Cell viability was assessed immediately after exposure and after 24 hours using an MTT test and fluorescence microscopy. RESULTS: All substances demonstrated dose-dependent cytotoxicity. Among antiseptics, Miramistin turned out to be the least toxic. Chlorhexidine at baseline significantly reduced cell survival in both immediate and delayed assessment. Octenicept caused pronounced cell death and led to cell fixation. Among the abrasives, glycine and soda had acceptable cytocompatibility. Erythritol showed high cytotoxicity. CONCLUSION: For preserving the regenerative potential of MSC AT, Miramistin in diluted form and sodium bicarbonate are the most biocompatible agents used for decontamination. The use of Octenicept and erythritol in clinically significant concentrations is associated with irreversible damage to progenitor cells.
BACKGROUND: Olfactory dysfunction is a prodromal symptom of many neurodegenerative diseases. Neuroinflammatory and neurodegenerative processes in the olfactory bulbs may be initiated by infectious agents in the nasal cavity, particularly bacterial lipopolysaccharide (LPS). The molecular mechanisms mediating the effects of this endotoxin on the olfactory epithelium and the development of pathological processes in the olfactory bulbs remain insufficiently studied. AIM: This study aimed to investigate the effects of different doses of intranasally administered lipopolysaccharide on the intensity of immunopositive staining of the olfactory epithelium with antibodies against alpha-synuclein (α-syn) and the adaptor protein MyD88, as well as of olfactory bulbs with antibodies against α-syn and glial fibrillary acidic protein (GFAP). METHODS: The study included 18 male BALB/c mice weighing 20–33 g. Animals received unilateral intranasal injections of sterile saline 10 μL daily (control) or lipopolysaccharide at a high (0.1 μg/mL) or low (0.01 μg/mL) concentration. After 28 days, the brains and nasal structure complexes were excised and frozen using dry ice. Serial cryostat sections (14 μm) were prepared and stained with methylene blue and antibodies against α-syn, MyD88, and GFAP. The intensity of immunolabeling was quantitatively assessed using Image-Pro Insight 8.0 software. RESULTS: Unilateral intranasal administration of lipopolysaccharide solution to mice resulted in a dose-dependent increase in α-syn levels in receptor cells, olfactory nerve bundles, and olfactory bulb glomeruli, along with a dose-dependent increase in MyD88 in the receptor epithelium and GFAP in the glomerular layer of the olfactory bulb ipsilateral to endotoxin administration. CONCLUSION: The observed morphological and immunohistochemical changes in the olfactory bulbs following intranasal lipopolysaccharide administration indicate the development of dose-dependent neuroinflammation in the glomerular layer. Neuroinflammation in the olfactory bulb appears to be initiated by LPS-induced upregulation of α-syn expression in receptor neurons projecting to the bulbs, mediated by activation of MyD88, which participates in intracellular signaling downstream of Toll-like receptors and interleukin-1 receptor family members.
A hallmark of synucleinopathies, including Parkinson's disease, is the aggregation of alpha-synuclein, a regulatory protein that modulates presynaptic vesicle dynamics. Amyloid-type aggregates, whose structure is stabilized by intramolecular contacts of beta-strands from the N-terminal and central regions of alpha-synuclein, exhibit pronounced neurotoxicity. Peptides that exhibit affinity for these regions have been shown to impede the contacts that are characteristic of the pathogenic conformation of alpha-synuclein and thereby prevent its aggregation. These peptides have the potential to function as neuroprotectors, and thus may offer therapeutic benefits in the treatment of Parkinson's disease. In recent years, several dozen promising peptides that block alpha-synuclein aggregation have been identified. The protective effect of several leading peptides has been confirmed in animal models of Parkinson's disease; however, due to delivery challenges, the search for alternative penetrating peptides remains a pressing issue. This review examines key strategies for the rational design of peptide inhibitors of alpha-synuclein aggregation and approaches to their testing, and it summarises data on the mechanism of action of the most fully characterised effective inhibitors.
BACKGROUND: Probiotics are capable of modulating immune responses through interactions with the gut microbiota, potentially enhancing the efficacy of immunotherapy and reducing adverse effects of chemotherapy and radiotherapy. Certain probiotic strains have demonstrated the ability to suppress chronic inflammation and augment antitumor immunity; however, their clinical application requires further investigation. AIM: This work aimed to evaluate the effects of oral administration of the probiotic strains Lacticaseibacillus rhamnosus K32 and Bifidobacterium adolescentis 150 on tumor growth and gene expression in the B16-F10 melanoma model, as well as on gut microbiota composition in experimental animals. METHODS: The experiment was conducted in C57BL/6 mice bearing B16-F10 melanoma. Animals were divided into three groups: control (no intervention) and two experimental groups for oral administration of B. adolescentis 150 or L. rhamnosus K32, respectively. Changes in gut microbiota composition were analyzed by full-length 16S rRNA gene sequencing using Oxford Nanopore technology. The transcriptomic response of B16-F10 melanoma cells to probiotic administration was assessed by RNA sequencing. RESULTS: Substantial differences were observed in the effects of the studied probiotic strains on B16-F10 melanoma progression. B. adolescentis 150 significantly stimulated experimental tumor growth by 29% (padj. = 0.02 vs. control; padj. = 0.001 vs. L. rhamnosus K32; adj., Bonferroni correction applied). At the molecular level, this stimulation was associated with suppression of interferon signaling, activation of proliferative pathways (WNT/β-catenin, TGF-β), and reduced expression of immune cell markers in melanoma tissue. In contrast, L. rhamnosus K32 reduced tumor growth by 18% (not significant; padj. = 0.4) and was associated with increased expression of cytotoxic T lymphocyte and NK cell markers, as well as activation of interferon response pathways. Both probiotic strains induced marked alterations in gut microbiota composition, characterized by an increased relative abundance of Klebsiella spp., and were associated with activation of proinflammatory signaling pathways (NF-κB, IL-6/JAK/STAT3, IL-2/STAT5) in tumor tissue. Notably, administration of both probiotics was linked to activation of epithelial–mesenchymal transition and hypoxia in the tumor, potentially creating conditions favorable for tumor progression and metastasis. CONCLUSION: These findings highlight the complex and context-dependent effects of probiotics on tumor development and underscore the need for careful strain selection in the adjuvant therapy of melanoma and other malignancies.
This review discusses the mechanisms underlying the therapeutic effects and the clinical prospects of mesenchymal stromal cell (MSC) transplantation in dilated cardiomyopathy. The development of dilated cardiomyopathy is based on a complex interplay of multiple etiological factors and mechanisms. This condition is characterized by high prevalence, unfavorable prognosis, and substantial mortality, highlighting the need for innovative treatment methods. In dilated cardiomyopathy, mesenchymal stromal cells exert multifaceted effects on the injured myocardium, primarily through paracrine secretion of cytoprotective, immunomodulatory, proangiogenic, and antifibrotic factors; suppression of oxidative stress; and restoration of cardiomyocyte energy metabolism. Preclinical and clinical studies have demonstrated the ability of mesenchymal stromal cells to improve cardiac contractile function and patients’ quality of life. Sustained long-term benefits have been reported, with allogeneic cells showing greater therapeutic potential and a lower incidence of complications compared with autologous cells. To enhance the efficacy of MSC transplantation and facilitate its broader implementation in clinical practice, several challenges must be addressed, including optimization of cell sources, delivery methods, and strategies to improve cell survival within the hostile myocardial microenvironment. In this context, genetic modification of mesenchymal stromal cells aimed at enhancing their cardioprotective and regenerative properties represents a promising approach to improving outcomes in patients with dilated cardiomyopathy.
Regenerative medicine uses cells as therapeutic agents to heal tissues and organs. It is a rapidly evolving area of research worldwide. Cell-based therapy has emerged as a pivotal treatment approach for articular cartilage defects, recognizing the limited regenerative potential of cartilage inherent to its structural biology. Given the inherent challenges associated with the standardization of cell-based drugs compared to conventional pharmaceuticals, the evaluation of their safety and efficacy in preclinical or clinical trials incurs particular considerations. In the majority of cases, autologous chondrocytes and mesenchymal stem/stromal cells derived from various tissues become key components of cell-based therapies currently available for cartilage defects. The cell-based therapies that have been approved for clinical use vary in manufacturing methods, types of cells, and use of matrices as a cell carriers in the finished product. Furthermore, clinicians routinely use a range of surgical techniques to perform a biopsy procedure for the preparation and subsequent implantation of finished cell-based products. Each cell-based treatment option available for patients with cartilage diseases offers a particular indication, benefits, and limitations, underscoring the relevance of comparative analysis of the therapies currently used in clinical practice. This will facilitate clinicians in selecting the most suitable therapy, while researchers may potentially expand the range of diagnoses for such therapies or enhance their efficacy. This review will focus on certain cell-based therapies that have currently arrived at the stages of clinical investigation and have been approved for the treatment of cartilage defects.
BACKGROUND: Currently, there are only a few studies evaluating the role of perivascular mesenchymal stem cells in the pathogenesis of cardiovascular diseases. Heart defects are a broad category of conditions affecting people of all ages. Therefore, biological characteristics of perivascular mesenchymal stem cells may be relevant to this area of research. The expression profile of surface markers is a key characteristic of cells that represents their functional state. This work evaluated and compared immunophenotypic characteristics of perivascular mesenchymal stem cells obtained from patients of different ages with heart defects of various non-inflammatory origins. AIM: The study aimed to evaluate morphotypes and immunophenotypes of perivascular mesenchymal stem cells in pediatric and elderly patients with heart defects of various origins. METHODS: The study included 16 patients of various ages with heart defects. Mesenchymal stem cells were isolated from perivascular adipose tissue and cultured. The levels of the following surface markers expressed by these cells were evaluated using flow cytofluorimetry for passages 2–4: CD90, CD105, CD73, CD34, and HLA-DR. RESULTS: Only 47.97% of the cells in passage 2 in the pediatric group expressed specific surface markers. However, the number of cells showing a perivascular mesenchymal stem cell phenotype increased with each further passage (p = 0.0016). The subcultivation of perivascular mesenchymal stem cells obtained from older patients revealed that, in passage 2, 95.98% of the cells had specific surface markers, which decreased to 44.59% by passage 4 (p = 0.0016). CONCLUSION: The expression of the study surface markers (CD90, CD105, CD73, CD34, HLA-DR) was less significant in perivascular mesenchymal stem cells obtained from older patients with non-inflammatory heart defects than in cells obtained from children with similar defects.
Mesenchymal stem cells are a cell population with the ability to self-replicate and differentiate into various types of somatic cells. The present review focuses on the potential of mesenchymal stem cell cultures for cell therapy using transplantable cells or tissue-engineered constructs, and the paracrine factors secreted by mesenchymal stem cells. The methodological aspects of the use of these cells in various diseases both in clinical and preclinical trials have been demonstrated through examples of experimental therapy, with an overview of their primary mechanisms. In the context of cell therapy, mesenchymal stem cells are of significant interest because of their abundance and renewability. Although the differentiation pathways of mesenchymal stem cells are not yet fully elucidated, the cells themselves play a pivotal role in stem cell biology in view of their regulatory properties, including immunomodulatory, antiapoptotic, proliferation-promoting, and antifibrotic effects. It is important to emphasize that the paracrine functions of mesenchymal stem cells are the primary factor contributing to their enhanced integration into tissues, when compared to induced pluripotent stem cells, particularly in the context of cardiac tissue engineering. The review also highlights the role of exogenous factors, such as substrates, in modulating the efficacy of the paracrine effects of mesenchymal stem cells, which is crucial for identifying the optimal cellular microenvironment to enhance therapeutic outcomes without adverse effects.
BACKGROUND: Wilson disease is a rare autosomal recessive disorder involving mutations in the ATP7B gene, which encodes the copper-transporting ATPase. The dysfunctional protein disrupts biliary copper excretion, which results in copper accumulation in hepatocytes. The available range of cell models to investigate the molecular mechanisms of this disease and to identify novel therapeutic approaches is currently limited. AIM: To develop an in vitro induced human pluripotent stem cell-derived model for investigating the molecular mechanisms of Wilson disease and evaluating therapeutic strategies. METHODS: A 2D cell model has been developed using validated induced pluripotent definitive endoderm cells obtained from a healthy donor and differentiated by activin A and CHIR99021. The copper overload that is a hallmark of the pathogenesis of Wilson disease was simulated by introducing exogenous copper into the growth medium. Relative cell viability was measured by the Alamar Blue assay. RESULTS: The induced pluripotent stem cells demonstrate a normal karyotype. Their morphology is typical of embryonic stem cells. They express pluripotency markers (SOX2, OCT4, TRA-1-60, and SSEA-4) and form tissues of all three germ layers during spontaneous differentiation in embryoid bodies. The differentiation of these induced pluripotent stem cells using the suggested procedure produces definitive endoderm cells that exhibit the expected morphology and express the markers SOX17, FOXA2, and ATP7B. The obtained model demonstrates sensitivity to exogenous copper overload at IC50 197 μM. CONCLUSION: The developed platform of definitive endoderm cells obtained from healthy donor’s induced pluripotent stem cells can be used to model the copper overload in vitro, simulating a cellular metabolic dysfunction associated with Wilson disease. Therefore, the proposed model can be used for both fundamental research and the development of novel therapeutic approaches.
BACKGROUND: Embryonic stem cells are a unique type of cells derived from the pre-implantation epiblast of mammalian embryos. These cells demonstrate the ability to undergo indefinite division and maintain an undifferentiated state. The potential for differentiation into any cell type renders embryonic stem cells a significant tool for regenerative medicine. Furthermore, they may be used in investigating the mechanisms of embryogenesis and disease modeling. The maintenance of pluripotency and the directed differentiation of embryonic stem cells rely on strictly regulated cell processes, including ubiquitin-proteasome-mediated protein degradation. Aberrant functions of the ubiquitin-proteasome pathway are linked to loss of embryonic stem cell pluripotency and apoptosis initiation. These processes are driven, particularly under oxidative stress, by the regulatory complex PA28αβ, which consists of Psme1- and Psme2-encoded α- and β-subunits. The early stages of mouse embryonic stem cell differentiation are accompanied by an increase in the expression of PA28αβ, and the knockdown of the α-subunit results in the accumulation of carbonylated proteins. This suggests that PA28αβ plays a role in the early stages of embryonic stem cell differentiation. However, the functions of PA28αβ in maintaining embryonic stem cell pluripotency remain insufficiently studied. AIM: To determine the effects of knockout of the Psme1 gene, which encodes the α-subunit of the PA28αβ regulator, on mouse embryonic stem cell proliferation and accumulation of reactive oxygen species (ROS). METHODS: In the study, a Psme1 knockout mouse embryonic stem cell line was generated through the use of genome editing. Teratoma assay was used to assess the ability to differentiate in vivo. The expression of pluripotency markers in embryonic stem cells was determined by real-time polymerase chain reaction and western blotting. The experimental stage also included flow cytometry to assess cell proliferation and production of ROS. RESULTS: A Psme1 knockout mouse embryonic stem cell line was successfully obtained. The analysis of the expression of key pluripotency markers did not show statistically significant differences between the control and mutant embryonic stem cells. The teratoma assay confirmed the maintenance of pluripotency in Psme1 knockout embryonic stem cells, demonstrating their ability to differentiate into derivatives of all three germ layers (i.e., ectoderm, mesoderm, and endoderm). The mutant embryonic stem cells demonstrated a lower proliferation rate compared to the control cells, whereas the ROS level remained unchanged. CONCLUSION: Psme1 knockout mouse embryonic stem cells have been demonstrated to maintain pluripotency and the ability to differentiate into derivatives of all three germ layers in vivo. Although the ROS level remained unchanged, the knockout of the α-subunit of the PA28αβ regulator caused a reduction in the proliferation rate, thereby emphasizing the critical role of PA28αβ in maintaining the proliferative potential of embryonic stem cells.
BACKGROUND: Myeloid tissue is among the most radiosensitive tissues and represents one of the first structures to be affected by ionizing radiation exposure. Damage to myeloid tissue manifests as suppression of hematopoiesis, depletion of the hematopoietic stem cell pool, and impairment of bone marrow stromal components. One promising approach for hematopoietic recovery in acute radiation syndrome involves multipotent mesenchymal stromal cells (MMSCs). Their ability to form a “niche” for hematopoietic stem cells, secrete hematopoietic factors, and exert immunosuppressive effects allows MMSCs to be considered an effective tool of cellular therapy, particularly in allogeneic transplantation. However, the efficacy of MMSCs is limited by their low viability and functional activity post-transplantation. Therefore, enhancing the regenerative potential of MMSCs represents a key challenge. One potential approach to address this issue may involve modulation of autophagy in MMSCs. AIM: To investigate the effects of autophagy modulation in MMSCs on their functional activity and ability to stimulate myeloid tissue regeneration in a model of acute radiation syndrome. METHODS: MMSCs were isolated from the chorion of ICR (CD1) mouse placentas and cultured with autophagy activator sirolimus and autophagy inhibitor 3-methyladenine. In vitro assessments included cell viability, concentrations of autophagy proteins (Beclin-1 and LC3B), and secretion of hematopoietic growth factors: SCF (stem cell factor), G-CSF (granulocyte colony-stimulating factor), and FLT3 ligand (Fms-related tyrosine kinase 3 ligand). In vivo experiments involved modeling acute radiation syndrome followed by MMSCs transplantation into laboratory animals. Bone marrow analysis and blood parameters were evaluated on day 7 post-irradiation. RESULTS: Autophagy activation with sirolimus increased concentrations of hematopoietic growth factors: SCF by 70.5%, G-CSF by 59.6%, and FLT3 ligand by 62.3% (p ≤ 0.0001). Transplantation of MMSCs with activated autophagy promoted a more pronounced increase in cellularity within granulocytic (+4.5%, p = 0.026), lymphocytic (+18.8%, p 0.0001), and megakaryocytic (+30.5%, p = 0.011) lineages. Autophagy inhibition reduced growth factor secretion and abolished the therapeutic effect of MMSCs. CONCLUSION: Autophagy activation in MMSCs represents a promising approach for hematopoietic recovery in acute radiation syndrome models.
BACKGROUND: Lipopolysaccharide (LPS) administration in mice is a widely used model for studying inflammation-associated depression. However, the mechanisms underlying LPS-induced changes in the brain remain unclear. AIM: This study is aimed to investigate behavioral, cellular, and molecular changes induced by chronic-interval LPS treatment in two brain regions implicated in depression, the hippocampus and the prefrontal cortex. METHODS: The study involved adult wild-type male mice (2–3 months old, 25–35 g, n = 28) and glial cell primary cultures. The experimental design included a two-phase LPS administration protocol (1 mg/kg, 3 injections intraperitoneally) with a 7-day interval. During the first phase, behavioral assessments were performed; whereas in the second phase, tissue samples (prefrontal cortex and whole hippocampus) were collected for molecular and histological analyses. Behavioral assessment included the Open Field Test (general activity and anxiety-like behavior), the Tail Suspension Test, the Sucrose Preference Test (anhedonia), and the Y-Maze Test (spatial working memory). Glial cell primary cultures were incubated in the presence of LPS to induce neuroinflammation and fibroblast growth factor 2 (FGF2) to assess changes in the microglial phenotype. Molecular and cellular changes in vivo and in vitro were analyzed using real-time polymerase chain reaction and immunohistochemistry assays. RESULTS: LPS-treated mice exhibited depression-like behavior, including decreased interest in hedonic stimulus, increased immobility, reduced locomotor activity, and memory deficits. The inflammatory reaction was associated with the elevated expression of proinflammatory cytokines (TNF-α, IL-1β) in both the spleen and brain with distinct regional patterns of astrocytic and microglial activation. LPS increased the expression of tight junction protein 1 (TJP1), vascular endothelial growth factor A (VEGFA), and E-selectin, decreased the expression of claudin 3, occludin, FGF2, and significantly increased the number of mast cells. Microglial activation was observed in both regions with a shift towards the amoeboid phenotype. Glutamatergic signaling was altered with downregulation of glutamate transporters (GLT-1) and glutamine synthetase in the hippocampus, suggesting the impaired glutamate buffering. In vitro, LPS induced microglial activation, which was reversed by FGF2. CONCLUSION: LPS-induced neuroinflammation differentially affected the hippocampus and prefrontal cortex with the hippocampus appearing to be more vulnerable. FGF2 reversed LPS-induced microglial activation, indicating its potential as a therapeutic target for neuroinflammation-associated depression.
BACKGROUND: Tumor microsatellite instability/microsatellite stability (MSI/MSS) status is a crucial parameter determining both disease prognosis and potential response to immunotherapy. Patients are referred for MSI typing based on clinical indications; however, only a small proportion of patients meet these criteria. Consequently, most sequencing datasets available in repositories and collected locally do not inherently contain MSI information in their metadata. Project funding limitations and insufficient biomaterial quantities often preclude DNA isolation and subsequent MSI typing using polymerase chain reaction methods. RNA sequencing results from tumor tissue may also be used to determine MSI status, but this requires incorporating a bioinformatics tool for MSI assessment into the tumor whole transcriptome analysis protocol. AIM: To evaluate the availability and applicability of bioinformatics tools for determining MSI status in locally derived datasets of whole transcriptomes of colorectal cancer samples. METHODS: Publicly available bioinformatics tools designed for MSI status assessment using RNA sequencing data were selected for analysis. These tools were tested on a locally derived dataset of whole tumor transcriptomes from 13 patients following primary colorectal tumor resection. The number of somatic mutations was assessed as a surrogate marker of MSI/MSS status, along with MSI/MSS status based on the results of bioinformatics tool testing, and their correlation. RESULTS: We tested two bioinformatics tools, PreMSIm and MIRACLE, designed to determine MSI status using transcriptomic data. When using MIRACLE, microsatellite instability was detected in 3 out of 13 samples. The MSI status determined by MIRACLE correlated with tumor mutational burden (TMB) (mean, 2163 mutations in MSI samples vs. 122.9 in MSS samples) and reliably identified unstable samples. PreMSIm also detected MSI in 3 samples, but its results showed limited concordance with TMB. For two of the three samples with high TMB, we identified known pathogenic and likely pathogenic variants in MSH2 and MLH1 genes associated with Lynch syndrome, confirming MSI status. For one sample, we proposed sporadic MSI etiology due to MLH1 gene hypermethylation. CONCLUSION: PreMSIm and MIRACLE demonstrate different sensitivity and specificity profiles for MSI status determination when using TMB as a surrogate MSI/MSS marker in colorectal adenocarcinoma. The MIRACLE tool can be easily integrated into whole-transcriptome tumor analysis protocols and provides biologically plausible MSI/MSS assessments that correlate with transcriptome-derived TMB.
