The aim of the study was to evaluate the biocompatibility of a hybrid material based on epoxy-treated bovine pericardium and cryostructured polyvinyl alcohol (PVA) after implantation into the rat aortic wall and subcutaneous tissue. Materials and Methods:The study evaluated hybrid material patches based on epoxy-treated bovine pericardium and cryostructured PVA; unmodified "KemPeriplasNeo" pericardial patches served as controls. The samples were implanted into the abdominal aortic wall and subcutaneously in male Wistar rats. The vascular implantation periods were 5 and 20 days, and the subcutaneous implantation periods were 60 and 120 days. The specimens were excised from the aorta and studied histologically using Russell-Movat staining and immunostaining for neutrophil myeloperoxidase (MPO) and the macrophage marker CD68. The calcium content in subcutaneously implanted samples was assessed by spectrophotometry and alizarin red S staining. The quantitative data were presented as the median, percentiles, minimal and maximal values (Me [25%-75%; min-max]). Results:After implantation into the abdominal aortic wall, the hybrid material samples showed a lower tendency toward thrombotic deposit formation on the surface compared to the pericardium. On day 5 of implantation, the thrombus thickness was 49.1 [34.7-64.6; 27.4-71.6] μm in the experimental group vs 170.3 [158.1-210.3; 124.4-217.0] μm in the controls (p<0.001). By day 20 of implantation, the macrophage density was lower in the peri-implant area of the modified samples compared to the controls: 219 [187-275; 112-362] vs 301 [244-338; 194-433] CD68+ cells per field of view (p<0.001), respectively. The neutrophil density at all experimental time points, as well as the macrophage density on day 5 of implantation did not differ significantly between the tested materials (p>0.17). No signs of calcification were detected in either group following the subcutaneous implantation over a period of 60 or 120 days. The control pericardium in the subcutaneous model showed the signs of cell-mediated degradation, whereas the hybrid samples were resistant to immune-cell infiltration and preserved their internal architecture. Conclusion:The modification of epoxy-treated bovine pericardium with cryostructured PVA cryogel improved its biocompatibility and enhanced resistance to biodegradation in a rat model after aortic-wall and subcutaneous implantation. The developed material may be used to create next-generation heart valve bioprostheses with potentially improved resistance to structural valve degeneration.
The aim of the study was to analyze the influence of various pipelines for preprocessing raw functional magnetic resonance imaging (fMRI) data on the accuracy of classification of subjects into schizophrenia patients and healthy controls using machine learning methods, and to give recommendations for optimizing the data preprocessing pipeline for this task. Materials and Methods. The study used fMRI data from 72 subjects acquired on a Siemens Magnetom Verio 3T MRI scanner (Siemens Healthineers, Germany) at the National Research Centre “Kurchatov Institute”. Seven different preprocessing pipelines were used on each dataset. Feature vectors for classification were constructed from each preprocessed dataset. We applied the following three algorithms for feature vector construction: ReHo, FCM, and FHR. Classification was performed using 15 machine learning methods from the scikit-learn package for each feature set and each preprocessing pipeline. The final accuracy was determined as the maximum accuracy among all machine learning methods. Results. No single optimal preprocessing pipeline for all feature vector construction algorithms was found. Smoothing increased accuracy for the voxel metrics ReHo and FHR, but not for the regional metric FCM. Spatial smoothing was the only preprocessing step that affected classification accuracy for the FHR method. The FHR feature set without smoothing demonstrated accuracy close to chance level. The steps of frequency filtering and median normalization substantially increased the accuracy for both the ReHo and FCM methods, both with and without smoothing. The steps of inhomogeneity correction and slice timing correction did not increase accuracy for the FCM feature set but did improve it by several percent for the ReHo feature set. Application of ICA filtering changed accuracy within a range of 5%, in either a positive or negative direction. Conclusion. Based on the obtained results, the following recommendations can be given for fMRI data preprocessing pipelines for binary classification of subjects into schizophrenia patients and healthy controls using machine learning methods and taking into account the feature vector construction algorithm. The most suitable for the ReHo metric pipeline includes motion correction, normalization, inhomogeneity correction, slice timing correction, frequency filtering, median normalization, and spatial smoothing. The most suitable for the FCM metric pipeline includes motion correction, normalization, inhomogeneity correction, slice timing correction, frequency filtering, and median normalization. Spatial smoothing is the most important factor for the FHR metric. The ICA filtering step does not provide a clear benefit and should therefore be applied with caution.
The aim of the study was to identify the regulatory potential of ascorbic acid and HOO• radicals in the formation of extracellular DNA networks and the internalization activity of mononuclear cells from rat blood and spleen. Materials and Methods. The isolated mononuclear cells from rat blood and spleen were exposed to ascorbic acid (10 and 50 μM), hydroperoxyl radicals (HOO•), and their combination. The following parameters were assessed: membrane toxicity (using the trypan blue exclusion test), internalization activity with latex particles (endocytosis), extracellular DNA networks formation, intracellular reactive oxygen species production kinetics (flow cytometry with the intracellular fluorescent probe dihydrorhodamine 123, DHR123), tryptophan fluorescence, glycated protein levels, and cellular redox balance (NAD+ and NADH). Results. Ascorbic acid at the concentrations of 10 and 50 μM suppressed the formation of extracellular DNA networks. At a concentration of 50 μM, it exerted a membrane-toxic effect, reduced tryptophan fluorescence, increased glycated protein levels, and shifted the redox balance toward oxidation (increased NAD+ and decreased NADH). HOO• radicals at concentrations ≥200 μM demonstrated concentration-dependent effects: stimulation of endocytosis, increased formation of extracellular DNA networks, and elevated intracellular production of reactive oxygen species. At a concentration of 600 μM, HOO• radicals reduced tryptophan fluorescence and depleted both NAD+ and NADH levels. The stimulatory effects on endocytosis and extracellular DNA network formation in response to HOO• radicals were the most pronounced in splenic mononuclear cells. The analysis of the combined action of HOO• radicals and ascorbic acid showed that ascorbic acid at a concentration of 10 μM protected cells from the membrane-toxic effects of HOO• radicals and reduced the formation of extracellular DNA networks induced by HOO• radicals. The ascorbic acid at a concentration of 50 μM enhanced the membrane-toxic effect of HOO• radicals and reduced the internalization activity of mononuclear cells. Conclusion. HOO• radicals at concentrations ≥200 μM stimulated the internalization activity of mononuclear cells and the intracellular generation of reactive oxygen species, whereas HOO• radicals at 600 μM caused a decrease in both oxidized and reduced coenzyme levels and increased the formation of extracellular DNA networks. Ascorbic acid at a concentration of 10 μM exhibited antioxidant properties and protected cells from the damaging effects of HOO• radicals, while at 50 μM it acted as a synergistic pro-oxidant, enhancing the membrane-toxic effect of the radicals and reducing the internalization activity of cells.
The aim of the study was to assess the efficiency of the mandibular bone defect regeneration by administering mesenchymal stem cells (MSC) from the Bichat's fat pad (B-MSC), as well as the osteoblasts differentiated from them, according to the morphological findings. Materials and Methods:The experimental study was carried out on male Wistar rats (n=120) aged 2 months, weighing 250-300 g. B-MSCs and the osteoblasts differentiated from them were used for the regeneration of rat mandibular bone defects formed by a round bur. In the control group, the defect was not filled, the defect was healing under the blood clot. The biopsy specimens were histologically examined on days 14, 30, and 90 after the injury formed. Results:The infiltration of the bone defect site by inflammatory cells was revealed for all study groups 14 days after the injury. The groups with cell administration showed the resorption of bone trabeculae (B-MSC and osteoblast groups) and a large number of capillaries (an osteoblast group). On day 30 after the injury, the preparations of the groups with cell administration demonstrated a large number of capillaries (B-MSC and osteoblast groups) and primitive bone trabeculae with osteoblasts (an osteoblast group). Active osteoclastic resorption and inflammatory infiltration were revealed in the control group. The groups with cell administration 90 days after the injury, in contrast to the control group, were found to have the formation of spongy mature bone tissue (B-MSC and osteoblast groups), the presence of bone mineralization and myeloid bone marrow (an osteoblast group) in the bone defect zone. It indicated high regenerative potential of the cells used when replacing a bone defect and the significant reduction of a regeneration period.
The aim of the present study was to improve the differential diagnosis accuracy of retinal diseases combining a transformer model to classify the biomarkers on OCT images and the large language model DeepSeek-V3. Materials and Methods:Two datasets were collected and annotated: a training set (3288 central retinal OCT images annotated for 8 biomarkers) and a validation set (50 clinical cases from octcases.com). For biomarker classification, we compared ResNet, DenseNet, EfficientNet, and Vision Transformer (ViT-Tiny-Patch16-224) architectures. The ViT-Tiny model demonstrated the highest performance, its F1 macro - 0.84±0.03. An integration algorithm was developed to combine predicted biomarker labels with clinical history data via the DeepSeek-V3 LLM API. Results:Combining OCT biomarkers and the medical history significantly improved the diagnostic accuracy: Top-1 Accuracy - 78%, Top-3 Accuracy - 94%, MRR - 84%, representing 10-44% improvement over using either data type alone. Conclusion:The suggested approach enabled to automate the detection of biomarkers on OCT images and enhance the differential diagnosis accuracy of eye diseases, reducing the image interpretation time and supporting clinical decision-making.
The aim of the study was to histologically validate the diagnostic potential of new systemic inflammation biomarkers (MLR, SIRI, CAR, dNLR, ALB/dNLR) in assessing the severity of rheumatoid arthritis (RA) induced in warm-blooded animals with genetically determined resistance to hypoxia. Materials and Methods:An autoimmune RA model (experimental groups) was induced by subcutaneous injection of complete Freund's adjuvant into the right hind limbs of 8-month-old male rats of strains with high (HR/SmY) and low (LR/SmY) resistance to hypoxia. Rats in the control groups received only the solvent. After 35 days, blood was collected from the hearts of all the rats. To calculate the new inflammation biomarkers - CAR, ALB/dNLR, SIRI, dNLR, NC/LC, and MLR - the following laboratory parameters were used: ALB (albumin), CRP (C-reactive protein), MC (monocytes), LC (lymphocytes), and NC (neutrophils). The effectiveness of disease severity assessment using these biomarkers was determined based on histological examination of plantar sections of the tarsal and metatarsophalangeal joints of control and experimental rats. Results:Genetically determined resistance to hypoxia is an important factor in the onset of the pathogenetic RA mechanisms. Low hypoxia resistance was associated with more severe connective tissue pathology compared to high resistance: LR/HR - 164 points/113 points, p≤0.05. The severity of pathomorphological RA changes (considering the organism's resistance to hypoxia), identified histologically (cartilage degeneration: LR/HR - 29 points/18 points, p≤0.01; general joint inflammation: LR/HR - 37 points/26 points, p≤0.01; osteolysis: LR/HR - 6 points/0 points, p≤0.01), was most effectively reflected by the following integral inflammation indices: CAR (LR/HR - 11.55·10-6 units/12.73·10-6 units), ALB/dNLR (LR/HR - 86.93 units/78.51 units), and SIRI (LR/HR - 0.14 units/ 0.19 units). Conclusion:The severity of pathomorphological RA signs depends on genetically determined resistance to hypoxia. For effective prediction of the disease, risk of complications, and treatment efficacy, it is advisable to use the new systemic inflammation biomarkers CAR, ALB/dNLR, and SIRI.
The aim of this study was to develop a quantitative method for assessing the ultrastructure of cardiomyocyte mitochondria in the right atrial appendage of patients with chronic heart failure (CHF). Materials and Methods. A single-center prospective study included 39 patients aged 67 [58; 71] years who underwent coronary artery bypass grafting. The inclusion criteria were as follows: heart failure with a left ventricular ejection fraction of <50%, atherosclerotic plaques in two or more large coronary arteries of 70% or greater, and the cardiac team's decision to perform coronary artery bypass grafting. To perform electron microscopy, biopsy samples were collected from the right atrial appendage during coronary artery bypass grafting. Two calculated parameters were used for the analysis: "total area of interfibrillar mitochondria" and "ratio of outer to inner membrane length". Based on these, the total index was calculated by the formula: the "total area of interfibrillar mitochondria" / the "ratio of outer to inner membrane length". Results. The median values of the calculated parameters characterizing the ultrastructure of cardiomyocyte mitochondria in the right atrial appendage were as follows: 43.7 [35.9; 54.3]% for the "total area of interfibrillar mitochondria", 31 [25; 37]% for the "ratio of outer to inner membrane length", and 1.4 [0.95; 2.00] for the total index accounting for both ultrastructural characteristics of mitochondria: the "total area of interfibrillar mitochondria" / the "ratio of outer to inner membrane length". To assess the clinical significance of the proposed total index, associations between the obtained values and atrial fibrillation (AF) were studied. Mitochondrial ultrastructural changes were more pronounced in patients with both AF and CHF. However, some parameters had no statistically significant differences: the total area of interfibrillar mitochondria was lower in patients with AF (42% vs 49%, p=0.224), and the ratio of outer to inner membrane length was higher in patients with AF (35% vs 31%, p=0.125). The total index was significantly lower in patients with AF (0.96 vs 1.75, p=0.021). ROC analysis was performed to identify a connection between total index and AF, and AUC was 0.773 (p=0.021). Conclusion. The proposed total index for analyzing cardiomyocyte micrographs, calculated as the "total area of interfibrillar mitochondria" / the "ratio of outer to inner membrane length", provides a comprehensive characterization of mitochondria that accounts not only for their number and size but also for their internal structure.
The aim of the study was to evaluate the efficiency of the KANU-Net 2D architecture based on U-Net in medical segmentation tasks of 2D brain MRI images on BraTS dataset with a limited number of training samples. Materials and Methods. The experiments were carried out using the subsamples containing 50, 100, and 150 images. The study described the data preprocessing steps, including normalization, gamma correction, cropping, and augmentation. A combination of Dice loss and BCE loss was used as a loss function. The network was optimized using AdamW. The network operation performance was evaluated using Accuracy and Dice coefficient for each region and its mean Dice. Results. KANU-Net 2D was experimentally demonstrated to achieve competitive performance comparable to current SOTA models of convolutional neural networks when trained on small samples. Specifically, the mean Dice coefficient reached 0.851 when using 100 training samples. Conclusion. The conducted studies showed KANU-Net 2D network to outperform the Med-DANet segmentation model both in terms of a mean value and region classes. The model effectiveness for different tumor regions highlighted the ability of the KAN-based (Kolmogorov-Arnold network) approach to adapt to various image characteristics in medical segmentation tasks. The obtained results demonstrated the undeniable promise of applying KAN for medical image segmentation in small samples and can lay the foundation for further research in this field.
The aim of the study was to evaluate the applicability of radioimmunoconjugates (RIC) of nanobodies against PD-L1 and HER2/ neu for the diagnosis and therapy of malignant tumors. Materials and Methods. The nanobodies to human biomarkers PD-L1 and HER2/neu were conjugated with radionuclides 68Ga and 177Lu using the chelating agent DOTA. RIC biodistribution was studied in experimental models on F1 (DBA/2xBALB/c) mice, which were inoculated with genetically modified CT26 murine carcinoma cells expressing human PD-L1 or HER2/neu. RICs containing the isotope 68Ga and intended for tumor detection were administered to animals intravenously at a dose of 1.0-1.2 MBq. In 0.5, 1.5, and 4 h the radioactivity accumulation in specific tumors carrying human biomarkers was assessed using direct dosimetry compared with control tumors. RICs containing 177Lu were administered for therapy at a dose of 0.8-1.6 MBq, and studied similarly for 96 h after administration. Results. After RICs containing 68Ga were administered to mice, the greatest difference between specific and control tumors was observed in 1.5 h. At the same time, there was a multiple excess of the specific tumor radioactivity over the blood and muscle tissue radioactivity, which should provide high contrast imaging. After administering RICs containing 177Lu to mice, the radioactivity in specific tumors persisted for 48 h producing a long-term effect of the radioisotope on the tumor. With the introduction of all RICs, there was a rapid elimination of radioactivity from the blood through urine, which was associated with the use of nanobodies with a molecular weight of 13 kDa and not containing sites of interaction with Fc receptors. There were found the significant accumulation and long-term retention of radioactivity in the kidneys. The work revealed a dependence of the radioactivity biodistribution on the isotope used: the time-normalized radioactivity of some organs and tissues, including tumors, after the administration of RICs containing 68Ga appeared to be higher than after administering the RICs of the same specificity, but containing 177Lu radioisotopes. Conclusion. The findings indicated the functional suitability of the pairs of RIC nanobodies with 68Ga and 177Lu radioisotopes for theranostics of malignant tumors expressing PD-L1 and HER2/neu biomarkers.
The aim of the study was to develop an optimal technique to predict the results of a bicycle ergometry test (BET) based on the parameters recorded during a six-minute walk test (6MWT) using machine learning methods. Materials and Methods. The study involved 56 patients who had experienced acute myocardial infarction and were undergoing the second stage of cardiac rehabilitation. The patients underwent a complete examination, including history taking, physical examination, anthropometric assessment, as well as a symptom-limited BET and 6MWT. During the 6MWT, we recorded the following: the distance covered, the heart rate, the blood pressure, the oxygen saturation, Borg rating of perceived exertion, the number of steps taken, and the electrocardiographic data. The algorithms for random forest, gradient boosting, k-nearest neighbors, and multiple linear regression were used to construct the machine learning models. The performance of the models was evaluated based on a determination coefficient, a mean absolute error, a mean square error, and a root mean square error. SHAP analysis was applied to interpret the findings. Results. The gradient boosting model provided the best prediction quality with a high determination coefficient (R2 being around 0.99) and low error values for both target metrics: the distance walked in the 6MWT and the metabolic equivalent achieved during the BET. The significance analysis of features revealed the heart rate, age, and the body mass index to have the greatest impact on predicting the 6MWT distance, while for predicting the metabolic equivalent, the distance covered, the number of steps, and the body mass index were the most significant. Conclusion. The developed gradient boosting-based machine learning model demonstrated its high efficiency in predicting the results of the 6MWT-based BET. The suggested method can serve as a valuable auxiliary tool to plan cardiac rehabilitation programs, particularly in cases when BET is difficult or impossible to perform. The use of SHAP analysis helped to understand the contribution of each feature to the prediction, increasing the confidence in the model results.
The aim of the study was to investigate the potential of using artificial intelligence technologies for age estimation in children from dental radiographs. Materials and Methods. Aretrospective study was conducted, analyzing orthopantomograms of 322 children (173 female, 149 male) aged 4-16 years. Fourteen permanent mandibular teeth were annotated on each radiograph. Neural network training was performed by splitting the data into training and test sets at a ratio of 80:20; 5-fold cross-validation was used. Age estimation was approached as a regression task. The neural network training and validation were conducted in Python using the PyTorch library. The accuracy of age prediction was assessed using the coefficient of determination (R2), mean squared error (MSE), and mean absolute error (MAE). Results. The study showed that the developed machine learning model was highly accurate in age estimation in children. The mean absolute error across cross-validation was 0.92 years, which was significantly lower than the error associated with traditional manual methods.
Chronic kidney disease (CKD), which often requires maintenance hemodialysis (MHD), represents a model of accelerated aging. Therefore, understanding the mechanisms underlying cerebral neurodegeneration (CND) and cognitive impairment (CI) in patients undergoing MHD is of particular importance. Brain damage in patients with end-stage CKD is considered a multicomponent process associated with the impact of cerebrovascular, neurodegenerative, and numerous dysmetabolic factors. Special attention is given to CND mechanisms mediated by the activation of microglia and astrocytes. The high prevalence of CI in the general population and especially among patients with end-stage CKD undergoing MHD determines the need to clarify CND risk factors and biomarkers. Furthermore, it is essential to explore therapeutic targets, as well as modifiable modern strategies and technologies aimed at slowing the CND progression. This review synthesizes data on the role of neuroglia and associated factors in the development of CND and CI in MHD patients. It presents the mechanisms and factors affecting neuroglia and involved in the pathogenesis of CND and CI. Particular focus is placed on chronic systemic inflammation, the effects of uremic toxins, and the activation of the cerebral renin-angiotensin-aldosterone system. The article studies such chronic systemic inflammatory factors associated with neuroglia as protein S100B, IL-1β, IL-6, TNF-α, and fibrinogen. Experimental and clinical data investigating the impact of uremic toxins (indoxyl sulfate, p-cresol sulfate, and imidazole propionate) on microglia and astrocytes, as well as the activation of the cerebral renin-angiotensin-aldosterone system, are analyzed. Potential further clinical research fields aimed at slowing the CND and CI progression in this patient population are highlighted.
The aim of this study was to study the role of transmission electron microscopy (TEM) in assessment of the phenotype of astrocytes obtained with the directed differentiation technique from induced pluripotent stem cells (iPSCs) from a healthy donor and from a patient with a hereditary form of Parkinson's disease (PD). Materials and Methods. Monolayer astrocyte cultures differentiated from iPSCs from a healthy donor and a PD patient having the G2019S mutation in the LRRK2 gene were used in the study. The obtained glial cultures were characterized using real-time PCR and immunocytochemical staining for glia-specific genes and proteins. TEM was used to examine astrocyte ultrastructure. Results. PCR analysis and immunocytochemical staining demonstrated that cell lines received from a healthy donor and a PD patient expressed the required pattern of glia-specific genes and synthesized astrocyte-specific proteins. However, some glia-specific genes were expressed at reduced levels by mutant cells. One of the most typical ultrastructural features of astrocytes received from iPSCs from a PD patient was destructive changes in mitochondria, including mitochondrial clearing, swelling, and cristae destruction. In many cells, mitochondria were completely absent after a long culturing. Another characteristic feature of cells with a mutation in the LRRK2 gene was the accumulation of vacuoles with contents of varied electron density. Distinct changes in the ultrastructure of nuclei, protein-synthesizing organelles, and cytoskeletal elements were also seen in cultured astrocytes with a PD-associated LRRK2 mutation. Here, the morphometric study did not reveal any differences in the average cell area, nuclear area, cytoplasm area, or nuclear-cytoplasmic ratio between astrocytes of the control line and the PD mutation line. Conclusion. Reprogramming and obtaining of astrocytes from iPSCs received from a donor with a PD-associated mutation in the LRRK2 gene allow to assess the nature and dynamics of pathological morphochemical and ultrastructural changes caused by the mutation during gliogenesis. The use of combined techniques (PCR, immunocytochemistry, TEM) to compare cell cultures differentiated from iPSCs allow to assess, on the one hand, general culture parameters, such as the dynamics of culture differentiation based on changes in the expression level of specific genes and immunocytochemical markers, and on the other hand, morphofunctional changes at the level of individual cells. TEM demonstrates significant potential for studying cell cultures differentiated from iPSCs. This technique is instrumental for phenotyping the resulting cells based on their ultrastructure, assessing the degree of their morphological maturity, and identifying minor ultrastructural changes in cells, both pathological and differentiation-associated. The results of this TEM-based study indicate a pronounced decrease in mitochondrial viability and other ultrastructural abnormalities, thus confirming the idea of a significant role of astroglia in the development of the neurodegenerative process in the LRRK2-associated PD; hence, astroglia can be a basis for development of new approaches as well as for searching pharmacological targets in the pathogenetic therapy of the disease.
The aim of the study was to develop a novel approach to treatment of non-healing wounds by using a portable Biogan bioprinter and an ink based on fibrin-gelatin hydrogel and spheroids derived from mesenchymal stromal cells (MSCs) from adipose tissue in a model of ischemic pig wound. Materials and Methods. To simulate the wound, titanium sealing rings were used, which mechanically compressed the skin to create a local ischemic wound. A day after, the rings were removed, and the epidermis of the skin was excised. The wound was treated one day and 2 weeks after the wound infliction. For this purpose, a combined ink was applied to the wound surface using a portable Biogan bioprinter (the prototype was developed by the authors). An adapted passive mixer allowed uniform mixing of the bioink based on a fibrin-gelatin hydrogel and spheroids derived from human adipose MSCs. Wound closure rates were assessed over 36 days, followed by histological analysis. Results. The use of inks based on fibrin-gelatin hydrogel and spheroids from adipose MSCs significantly accelerated healing, as evidenced by the reduction in the wound area compared to the control group and hydrogel-only group, as well as the complete restoration of all skin layers by day 36. The therapeutic effect of the developed approach was due to the spheroids in the bioinks and not to the hydrogel. The use of the developed mixer did not reduce the cell viability and ensured convenient ink application to the wound surface.
The aim of the study was to estimate the efficiency of identifying bacterial agents in native cardiac valves affected by infective endocarditis using 16S rRNA metabarcoding. Materials and Methods:The study material involved 20 native cardiac valve samples from 16 patients. Sequencing was carried out in the Center for Collective Use "Genomics" (Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Russia) on a sequenator MiSeq (Illumina, USA) using MiSeq Reagent Kit v3 (2×300 bp; Illumina, USA). Results:The study revealed major microorganisms (their portion in the sample under study was over 5% from all identified bacterial agents) belonging to Streptococcus (40% of all valves studied), Sphingomonas (35%), Pseudomonas (35%), Roseateles (25%), Phyllobacterium (25%), and Enterococcus (15%). Moreover, the studied valves were found to have rare cases of Ralstonia pickettii, as well as Bacillus and Klebsiella representatives. Conclusion:The findings demonstrated the efficiency of 16S rRNA metabarcoding in identifying bacterial agents compared to routine noninvasive diagnostic methods. Native cardiac valves isolated from the patients with infective endocarditis were characterized by numerous opportunistic pathogenic bacteria in negative blood cultures.
The aim of the study was to evaluate the possibilities and limitations of using the immunohistochemical detection of Iba-1 protein for morphofunctional analysis of microglia under different conditions. Materials and Methods. In the study we used brain samples from Wistar rats at different ages: 7 days of postnatal development (n=18), 14 days of postnatal development (n=18), 4-6 months (n=22); the brain samples from 3-6-month-old SHR (spontaneously hypertensive rats) male rats (n=4); human cerebral cortex samples (n=10). Rabbit polyclonal antibodies against calcium-binding protein Iba-1 (Biocare Medical, USA) were used to detect microglia immunohistochemically followed by light and confocal laser microscopy. A previously developed original technique was applied to simultaneously detect microglial cells and amyloid plaques. Results. Iba-1 protein in microglial cells was shown to be present in grey and white matter in all cerebral regions under study. Microglial cells in different regions were noted to be characterized by pronounced structural and functional features when stained for Iba-1. In addition to microglial cells, in the rat brain there were found other Iba-1-immunopositive macrophages, which were tissue cerebral macrophages differing from microgliocytes by specific morphology and localization. Iba-1 protein was demonstrated to be present in microglial cells in all investigated stages of postnatal development that makes Iba-1 an appropriate marker for comparative ontogenetical studies of microglial cells. The localization of Iba-1 in the bodies and processes of microgliocytes enables to more clearly identify the complex ramified cell morphology and make three-dimensional reconstructions. In the brain of spontaneously hypertensive rats (SHR line rats) the Iba-1-immunopositive microglia were shown to have a number of structural and functional features indicating the moderate activation of microglia. 48 h after ischemic injury, an anomalously great number of large Iba-1-immunopositive cells with amoeboid morphology were detected in the gray matter of the striatum in an ipsilateral hemisphere near the injured area in the rat brain. Microglial cells in human cerebral cortex were noted to be localized in the majority of the detected amyloid plaques and characterized by amoeboid morphology indicating their severe activation. Conclusion. The presented findings suggest Iba-1 protein to be a reliable and universal microglial marker. The immunohistochemical detection of the protein enables to identify and analyze qualitatively and quantitatively microglial cells in different brain regions in human and laboratory animals under normal and pathological conditions. The limitations of using Iba-1 as a marker of microglia include the inability to determine the microglial activation vector and the difficulty in distinguishing between microglia and infiltrating macrophages of the brain in pathology. In such cases, it is necessary to improve the technology for detecting microglia that can be based on using a multi-marker analysis.
Gene therapy has evolved into a sophisticated field encompassing diverse precision editing platforms and advanced delivery systems capable of addressing complex genetic disorders and age-related pathologies. This comprehensive review examines the current landscape of gene therapeutic technologies, including CRISPR-based genome editing, base editing systems, prime editing platforms, and emerging DNA polymerase-based editors alongside their corresponding delivery methodologies. The review encompasses viral vectors, including tissue-specific adeno-associated virus serotypes, non-viral delivery systems such as ionizable lipid nanoparticles and virus-like particles, and innovative platforms, including exosome-based delivery and the SEND system. We examine therapeutic applications spanning nuclear genome editing, mitochondrial genome modification, RNA editing, and epigenetic modulation, demonstrating the expanding scope of gene therapy beyond traditional monogenic disorders. Critical analysis reveals that while fundamental technological capabilities have been established, significant challenges remain in manufacturing scalability, long-term safety assessment, delivery across physiological barriers, and optimization of editing efficiency in post-mitotic tissues. The integration of artificial intelligence approaches for predictive analysis and rational vector design represents a promising avenue for addressing current limitations. This review concludes that successful clinical implementation requires systematic resolution of manufacturing, safety, and delivery challenges alongside the development of standardized protocols for patient stratification and robust regulatory frameworks that accommodate rapid technological innovation while ensuring patient safety.
Glial tumors are the most common neuroepithelial neoplasms of the brain. Consequently, investigating robust, non-invasive techniques for subtyping these tumors - specifically through advanced multimodal neuroimaging and radiomics - is warranted. The present systematic review of scientific literature, including meta-analysis, was conducted to specify the major challenges of radiomics and machine learning in diagnostics of glial tumors based on the MRI data as well as to assess the quality of such non-invasive diagnostics. We analyzed 42 publications utilizing radiomics and machine learning to predict molecular biomarker status in glial tumors based on MRI data. The analysis covered mutations in the IDH, ATRX, BRAF, and H3K27M genes, as well as TERT promoter mutations, 1p/19q codeletion, MGMT promoter methylation, and proliferative activity (Ki-67 labeling index). The overall accuracy of these techniques was high and equaled 0.86 [0.83; 0.89]. At the same time, the studies demonstrated significant methodological heterogeneity, in particular, related to the lack of uniform standards to select the location, size, and shape of the area of interest for obtaining radiomic features. This greatly hinders reproduction of the experimental results in clinical practice. Therefore, standardization of radiomics procedures remains relevant for further research of glial tumors.
Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor expressed in the central nervous system, the gastrointestinal tract, the pancreas, and the adrenal cortical tissue regulating their physiological functions. In addition to normal tissues, GRPR is overexpressed in many solid cancers. At present, several radiolabeled ligands targeting GRPR have been introduced into clinical practice for cancer diagnostics and radioligand therapy. However, there were found the high uptake of radiopharmaceuticals in normal organs and low bioavailability of the drugs caused by their stability. Therefore, GRPR radioantagonists with improved proteolytic stability and longer residence time in tumor foci with low uptake in non-target organs are currently being sought to improve the therapeutic efficacy. The aim of the study was to analyze the biodistribution of the BBN/C1-C2 molecule created on the basis of bombesin and knottin and its accumulation dynamics in the tumor in an in vivo model. Materials and Methods. The study analyzed the biodistribution of Cy7.5-labeled BBN/C1-C2 peptide based on bombesin and knottin U5-Sth1a and produced using solid-phase peptide synthesis. The investigation was carried out on a mouse model Nu/Nu with a prostate cancer solid tumor (PC-3 culture) transplanted into the right side, expressing GRPR, using real-time surface fluorescent imaging on days 2 and 5 after intravenous administration of the molecules under study. Results. The biodistribution analysis showed the selective binding of the BBN/C1-C2 molecule to a tumor, as well as its holding power on the tumor surface for up to 5 days without internalization into cells with low accumulation in normal organs and tissues. Conclusion. We managed to obtain a stable molecule; however, U5-Sth1a toxin tropic to ion channels was used as a scaffold, so the resulting molecule retained the domain responsible for attaching to the target channel and typical for knottin. In the future, when using the molecule as a ligand for radiotherapy, it can have a negative effect on the patient's heart due to the supplementary radiation load. In this regard, the BBN/C1-C2 molecule requires the extension study.
