Stanislav Sergeevich Bachurin

Introduction. Adhesion to the intestinal mucosa is a key criterion for probiotic selection. A short-term Caco-2/HT-29 co-culture was used, combining enterocyte-like and HT-29 cells. Mucin production was not experimentally validated in this model. The aim was to assess the adhesion and cytotoxicity of the strains in this model, analyze SlpA interactions with MUC2 domains, and evaluate the feasibility of automated nuclei detection in the co-culture. Methods. The morphology of the co-culture and bacteria was examined by confocal microscopy. MTT-reduction activity of the bacteria-exposed co-culture was measured relative to untreated cellular controls, while cell membrane integrity was assessed using the trypan blue exclusion assay. Adhesion after 2 h was evaluated by cell counting and colony-forming unit (CFU) percentage. Caco-2/HT-29 nuclei were detected using Faster R-CNN and YOLO11 models. SlpA complexes with the D1 and D3 domains of MUC2 were analyzed by docking, molecular dynamics, and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Results. Monolayer integrity was preserved. Both nuclei detection models showed comparable performance (mAP@50 = 0.97; mAP 0.50–0.95 = 0.92 and 0.90). The relative MTT-reduction signal was higher after exposure to Bifidobacterium animalis subsp. lactis KB-54 (81.05 ± 33.21%), Bacillus subtilis KATMIRA 1933 (72.91 ± 31.26%), and Bacillus amyloliquefaciens B-1895 (59.47 ± 17.93%) and lower after exposure to B. animalis subsp. lactis KB-16 (13.53 ± 9.44%) and L. acidophilus KB-41 (7.35 ± 5.16%). A similar pattern was observed for cell viability. KATMIRA 1933 showed the highest number of adherent cells (12.72 ± 2.16); among the probiotic strains, higher values were observed for KB-41 and KB-16. The highest adhesion percentages were observed for KATMIRA 1933 (5.32%) and KB-16 (4.93%), whereas KB-54 and KB-41 showed the lowest values. In silico, the SlpA complex with the deglycosylated D1 domain of MUC2 showed the most favorable interaction energy (−10.63 ± 6.03 kJ/mol). Conclusions. The short-term Caco-2/HT-29 co-culture is suitable for the combined assessment of bacterial adhesion, cellular metabolic activity, and membrane integrity under the tested conditions, while nuclei can be quantified automatically. High adhesion was not always associated with preservation of cell status, and SlpA-MUC2 interactions depended on glycosylation.

Sleep maintains central nervous system homeostasis, but many molecular mechanisms of sleep–wake regulation remain poorly understood. Connexin-43 (Cx43), the astrocytic gap junction protein, supports neuroglial communication, yet its role in physiological sleep is little studied. Here, we analyzed Cx43 expression, gap junction organization and neuropil ultrastructure in the motor cortex of common noctule bats (Nyctalus noctula) during sleep and wakefulness using confocal microscopy, transmission electron microscopy, semi-automated image analysis, and molecular dynamics. Sleep was associated with higher Cx43 immunoreactivity and with more and larger astrocytic gap-junction plaques than wakefulness. Across neuropil fields, synaptic, process and mitochondrial counts and total active-zone length were higher during wakefulness, and mean mitochondrial profiles were larger during sleep; after averaging within animals, only total active-zone length remained significantly greater during wakefulness. More perisynaptic astrocytic process (PAP) profiles contacted synapses during wakefulness, whereas individual PAPs were larger during sleep; both contrasts remained significant after animal-level averaging. As a secondary, hypothesis-generating analysis, we simulated histidine protonation of the Cx43 hemichannel as a simplified model of acidic conditions. Cerebral pH was not measured, and the simulations do not explain the sleep-associated Cx43 increase. In silico, protonation reduced conformational flexibility and narrowed the central and extracellular pore regions.

Severe traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide. To date, there are no clinically effective neuroprotective agents. Biomaterials that combine structural support for damaged tissue with a depot for therapeutic agents may represent a key solution to this problem. To evaluate the neuroprotective potential of a collagen matrix derived from the jellyfish Rhizostoma pulmo (R. pulmo) and modified with sodium thiosulfate (Na₂S₂O₃) as an hydrogen sulfide (H₂S) donor in a bioengineered platform for the treatment of severe TBI. Comprehensive characterization of the collagen matrix (electrophoresis, fluorescence microscopy), its implantation in a mouse model of severe TBI, and subsequent morphological, histological, ultrastructural, and immunohistochemical analyses of connexin 43 (Cx43) and p53 protein (p53) were performed. In addition, molecular dynamics simulations of the interactions between sulfur-containing compounds and target proteins were conducted. The effects were compared with inhibition of endogenous H₂S synthesis using aminooxyacetic acid (AOAA). The collagen matrix retains the properties of type I collagen and forms a three-dimensional porous structure with high hydrophilicity and biocompatibility. Implantation ensures effective defect filling, reduces cystic degeneration, and preserves cortical structure. Modification with Na₂S₂O₃ results in a significant reduction in both nuclear and cytoplasmic accumulation of p53, prevention of Cx43 dysregulation, a decrease in the proportion of damaged neurons and inflammatory infiltration, and preservation of tissue ultrastructure. In contrast, inhibition of CBS with AOAA exacerbates pathological changes. Molecular modeling demonstrated that S₂O₃²⁻ is capable of forming stable electrostatic interactions with domains of p53 and Cx43 under conditions of acidosis and elevated Ca²⁺. A collagen matrix derived from R. pulmo and modified with Na₂S₂O₃ represents a promising biodegradable platform that combines structural support with local H₂S-dependent regulation of key mechanisms of secondary brain injury. This approach provides a multilevel neuroprotective effect and opens new opportunities for the development of therapeutic implants for severe TBI.

Traumatic brain injury (TBI) initiates a cascade of molecular and cellular reactions leading to long-term disturbances of neuronal and glial homeostasis. One of the key mechanisms of secondary injury is a pathological increase in intracellular Ca²⁺ concentration. Parvalbumin (PV) plays an important role in the regulation of Ca²⁺ homeostasis in neurons. In turn, connexin 43 (Cx43) is the principal protein of astrocytic gap junctions (GJs), which ensure neuroglial communication. The spatiotemporal changes in these proteins and the mechanisms of their interaction after TBI remain insufficiently studied. In the present study, a comprehensive analysis of the expression, localization, and spatial organization of PV and Cx43 in the cerebral cortex following TBI was performed. In intact tissue, PV was localized predominantly in neurons, whereas Cx43 formed typical punctate structures of astrocytic GJs. Twenty-four hours after TBI, a sharp activation of PV with pronounced nuclear translocation was observed against the background of a catastrophic decrease in Cx43 expression, accompanied by a reduction in the number of NeuN⁺ neurons and signs of apoptosis. However, after 7 days, a mirror-opposite effect was detected, characterized by decreased PV expression and increased Cx43 levels with its aggregation into cluster-like structures, as well as partial restoration of NeuN immunoreactivity. In addition, molecular dynamics simulations demonstrated that the stability of the PV–Cx43 complex is determined by the presence of Ca²⁺ and physiological pH, whereas acidosis and Ca²⁺ overload destabilize their interaction. Taken together, these results reveal a phase-dependent mirror-opposite pattern of PV and Cx43 expression and localization and emphasize the key role of Ca²⁺- and pH-dependent neuroglial interactions in TBI.

Hydrogen sulfide (H₂S) is a gasotransmitter that plays a crucial role in regulating pathological processes following injury to the central and peripheral nervous systems. This review systematizes current data on various classes of H₂S donors and inhibitors of its biosynthesis in neurotrauma and related experimental models. Inorganic donors (e.g., NaHS, Na₂S, and STS) rapidly suppress oxidative stress and inflammation, supporting the recovery of synaptic plasticity and cognitive function. Organic donors (e.g., GYY4137, ACS67, ACS84, SPRC, ADT-OH and its derivatives, S-memantine, and MTC) provide sustained H₂S release, stabilize the blood–brain barrier, and exhibit antiapoptotic activity. Natural donors (e.g., DADS, DATS, and SAMe) demonstrate high biocompatibility, inhibit pyroptosis, and enhance antioxidant defense mechanisms. Hybrid systems—including nanoparticles and hydrogels—enable targeted delivery and prolonged action, thereby stimulating regeneration and angiogenesis. Thiol-activated donors (e.g., COS/H₂S and AlaCOS) allow controlled H₂S release, offering broad opportunities for precise modulation of its concentration within target tissues. Inhibitors (e.g., AOAA, PAG, oxamic hydrazide 1, L-aspartic acid, benserazide, and NSC4056) of H₂S biosynthesis underscore the physiological importance of this gasotransmitter, as their administration enhances neuroinflammation and diminishes neuroprotection. The analysis reveals a general pattern: all classes of H₂S donors effectively modulate key pathological mechanisms, differing in their rate, duration, and specificity of action. These findings highlight the therapeutic promise of H₂S-based pharmacological agents in clinical neurotraumatology, while emphasizing the need for further research to optimize delivery systems, enhance efficacy, and minimize adverse effects.

Traumatic brain injury (TBI) is one of the most common forms of neurotrauma, accompanied by significant disruptions in neuronal homeostasis and intercellular communication. A key protein involved in these processes is connexin 43 (Cx43), which facilitates the formation of gap junctions in the astrocytic network. In this study, using confocal and immunofluorescence microscopy, ultrastructural analysis, and molecular modeling, we investigated the dynamics of Cx43 expression and structural changes in neuroglia during various post-traumatic periods following TBI. It was shown that in the acute phase, 24 h post-injury, there is a reduction in Cx43 expression, accompanied by apoptotic neuronal degradation, disruption of nuclear NeuN localization, and destruction of cellular ultrastructure. By 7 days post-injury, a significant increase in Cx43 levels was observed, along with the formation of protein aggregates associated with pronounced reactive astrogliosis. Peripheral blood analysis revealed persistent neutrophilia, lymphopenia, and reduced monocyte levels, reflecting a systemic inflammatory response and immunosuppression, which was corroborated by a custom-trained neural network-based computer vision model. Linear regression and correlation analyses further identified a strong positive association between normalized monocyte levels and Cx43 expression, a moderate negative correlation with lymphocytes, and no significant correlation with neutrophils. Using a custom-built computer vision model, we confirmed these hematological trends and detected subtle changes, such as early increases in platelet counts, that were not captured by manual evaluation. The model demonstrated strong performance in classifying common blood cell types and proved to be a valuable tool for monitoring dynamic post-traumatic shifts in blood. Molecular dynamics modeling of Cx43 identified a pH-dependent mechanism of conformational reorganization under post-traumatic acidosis, mediated by the interaction between protonated His142 and Glu103. This mechanism mimics the structural consequences of the pathogenic E103K mutation and may play a critical role in the neurotoxic effects of Cx43 in TBI. These findings highlight the complexity of Cx43 regulation under traumatic conditions and its potential significance as a target for neuroprotective therapy.

Transcription factors of the E2F1 family belong to those identified to be in the center of signaling cascade responsible for the propagation of apoptosis, including stroke-induced ischemia conditions in the brain. E2F1 activity is mediated by its binding to DP1 and retinoblastoma proteins and its phosphorylation and acetylation. We have studied the effects of E2F1 point acetylation at lysins 117 and 125 on its interaction with retinoblastoma and its role in penumbra cell fate after photothrombotic stroke. E2F1, in complex with retinoblastoma, is mainly unacetylated or acetylated at lysine 125 by PCAF. The formation of this complex is associated with the upregulation of E2F1 after stroke due to pRb protecting E2F1 from degradation vie ubiquitin-proteasome mechanism. Point acetylation of E2F1 at lysine 117 contributes to increased neuronal apoptosis induces by oxidative stress and is associated with inhibition of the “S”-site binding of retinoblastoma. Acetylation levels of E2F1 at K125 is regulated by HDAC1. The identification of selective deacetylase for E2F1 at lysine 117 would provide new means for the modulation of oxidative stress-induced neuronal apoptosis.

This study focuses on understanding the role of c-Myc, a cancer-associated transcription factor, in the penumbra following ischemic stroke. While its involvement in cell death and survival is recognized, its post-translational modifications, particularly acetylation, remain understudied in ischemia models. Investigating these modifications could have significant clinical implications for controlling c-Myc activity in the central nervous system. Although previous studies on c-Myc acetylation have been limited to non-neuronal cells, our research examines its expression in perifocal cells during stroke recovery to explore regulatory mechanisms via acetylation. We found that in peri-infarct neurons, c-Myc is upregulated with acetylation at K148 but not K323 during the acute phase of stroke, with SIRT2 deacetylase primarily affecting K148 acetylation. Molecular dynamics simulations suggest that lysine 148 plays a crucial role in stabilizing c-Myc spatial structure. Increased acetylation at K148 reduces c-Myc compaction, potentially limiting its nuclear penetration, promoting calpain-mediated cleavage, and decreasing nuclear localization. Additionally, cytoplasmic acetylation at K148 may alter c-Myc's interaction with unidentified proteins, potentially influencing its pro-apoptotic effects and promoting cytoplasmic accumulation. Targeting SIRT2 with selective inhibitors could be a promising avenue for future stroke therapy strategies.

CRISPR/Cas systems are used for genome editing, both in basic science and in biotechnology. However, CRISPR/Cas editors have several limitations, including insufficient specificity leading to “off-targets” and the dependence of activity on chromatin state. A number of highly specific Cas9 variants have now been obtained, but most of them are characterized by reduced activity on eukaryotic chromatin. We identified a spatial cluster of amino acid residues in the PAM-recognizing domain of Streptococcus pyogenes Cas9, whose mutations restore the activity of one of the highly specific forms of SpyCas9 without reducing its activity in Saccharomyces cerevisiae. In addition, one of these new mutations also increases the efficiency of SpyCas9-mediated editing of a site localized on the stable nucleosome. The improved Cas9 variants we obtained, which are capable of editing hard-to-reach regions of the yeast genome, may help in both basic research and yeast biotechnological applications.