Vascular organoids derived from human induced pluripotent stem cells (iPSCs) are promising models for studying vascular pathology, including neurodegenerative diseases. In this study, we investigated signs of mitochondrial dysfunction in the vascular organoids derived from the iPSCs of a healthy donor, as well as patients with Alzheimer's disease (AD) and Parkinson's disease (PD). In the conditioned medium of vascular organoids from the PD patient-derived cells, but not from the AD patient-derived cells, a trend toward a disrupted NAD+/NADH balance was observed, accompanied by the reduced expression of the connexin 43 (Cx43) protein. A sign of metabolic vulnerability of endothelial cells in the vascular organoids from the PD patient-derived cells, but not from normal or AD patient-derived organoids, manifested as reduced expression of c-Myc was observed. Changes in the membrane potential were detected in the vascular organoids from the AD and PD patient-derived, as well as increase in the mitochondrial superoxide anion production were observed, which may indicate development of oxidative stress in the microvessel cells during neurodegeneration.
The innate immunity of plants is a dynamic, multilevel system traditionally divided into pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Despite being activated by different types of receptors localized in different cell compartments, PTI and ETI are currently considered interdependent components of a single defense system. This view suggests that, due to various positive interactions between these two pathways, the innate immunity of plants is more than the sum of PTI and ETI. Available data indicate that PTI and ETI enhance each other synergistically, increasing the concentration of signaling molecules, such as components of kinase cascades, reactive oxygen species, calcium ions, and phytohormones. This leads to the activation of defense genes, providing a local response to pathogens and the development of systemic plant resistance.
Escherichia coli is one of the most common producers of recombinant proteins, including therapeutic antibody fragments. However, the outer membrane of E. coli contains high levels of lipopolysaccharide (LPS, also known as endotoxin), which can activate innate immune receptors, trigger immune responses, and induce systemic inflammation that may progress to septic shock. Ensuring extremely low endotoxin levels in preparations intended for in vivo applications is critically important. In this study, we investigated the endotoxin content in preparations of the bispecific mini-antibody MYSTI-2 produced in two E. coli strains: the Rosetta strain, which synthesizes conventional LPS, and the ClearColi strain, which synthesizes potentially non-toxic form of LPS. Our results demonstrate that near-complete removal of LPS can be achieved only through the use of a non-ionic detergent during purification, regardless of the bacterial strain used for protein production.
Proteins that bind components of bacterial cell wall play a key role in innate immunity and interactions between bacteria and host organisms. They participate in the control of peptidoglycan synthesis and degradation, determine the pathogenic specificity of bacteria, affect their ability to adhere and invade, and serve as important elements of molecular recognition. The review discusses proteins of diverse origins and their recombinant analogues, their structure and binding mechanisms, and prospects for application in the diagnostics of bacterial infections and functionalization of nanomaterials.
Chronic alcohol consumption in alcohol dependence (AD) is associated with various molecular and cellular dysfunctions. One of the key mechanisms involved is the extracellular release of HMGB1 (high-mobility group box 1), a nuclear protein that acts as a proinflammatory signaling molecule. However, the dynamics of peripheral HMGB1 levels across different stages of AD and its association with psychopathological symptoms remain insufficiently understood. To investigate peripheral HMGB1 concentrations in patients with AD during different disease stages - alcohol withdrawal syndrome (AWS), post-abstinence state (PAS), and remission - and to assess their associations with clinical characteristics of the disease. The study included 53 men aged 30-60 years diagnosed with AD and 19 conditionally healthy men who served as controls for biological analyses. Clinical and biological assessments were performed at three time points: (1) during AWS; (2) after completion of standard therapy (PAS); and (3) during remission following hospital discharge. The severity of psychopathological symptoms was evaluated using validated clinical rating scales. Serum HMGB1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Patients with AD demonstrated significantly elevated serum HMGB1 levels during AWS and PAS compared with controls, whereas HMGB1 concentrations decreased to control values during remission. Correlation and ROC analyses revealed that higher HMGB1 levels were associated with a less pronounced reduction in psychopathological symptoms following treatment. Peripheral HMGB1 may represent a promising biomarker for monitoring treatment response and predicting relapse risk in AD.
The primary role of sterols in the cell is to support plasma membrane function, and for this reason their concentration in this compartment is the highest among all cell membranes. In the yeast Saccharomyces cerevisiae, sterol transport between membranes is mediated by proteins of Osh and Lam families. The Lam1-Lam4 proteins are reported to transport sterols passively from plasmalemma to endoplasmic reticulum. The Lam5-Lam6 proteins transport sterols at the ER interface with vacuoles and mitochondria. Deletion of the LAM family genes does not impair cell growth under standard conditions, which makes their biological role unclear. We hypothesized that the Lam family proteins may play a role in yeast sporulation, as the spore plasma membrane is formed de novo from the ER-derived vesicles, which contain less sterol than the plasma membrane, necessitating sterol transport into the newly forming spore plasma membrane. To test this hypothesis, we generated diploid strains with the LAM1-LAM4 and LAM5-LAM6 deletions. We demonstrated that double deletion of the LAM5-LAM6 genes reduced both percentage of the sporulating cells and number of the spores per ascus. Conversely, deletion of the LAM1-LAM4 genes reduced proportion of the full asci but did not inhibit sporulation initiation. We demonstrated that deletion of the LAM1-LAM4 genes induces cell wall thickening and structural defects. Clusters of osmiophilic granules were detected at the cell wall surface of these spores. Spores with the deletions of the LAM family genes demonstrated reduced resistance to heat shock and alkali. Taken together, our data indirectly support our hypothesis and point out that sterol transport by the LAM family proteins is necessary for the sterol redistribution during sporulation.
This review focuses on current challenges associated with the study and standardization of chitosan, a promising biopolymer for medical applications. Its key properties, such as biodegradability, low toxicity, mucoadhesion, and antimicrobial effects, make it highly sought in various fields of medicine. Particular attention is given to the analysis of chitosan parameters, including molecular weight, degree of deacetylation, and pattern of acetylation, as well as compliance with pharmacopoeial requirements. The challenges associated with the reproducibility of chitosan properties and the absence of uniform analytical standards are addressed. The review also summarized the data on the relationship between the chitosan structural characteristics and biological activity (antimicrobial, antioxidant, immunomodulatory, etc.), which is important for predicting its behavior in biological systems. Regulatory considerations governing the medical use of chitosan and prospects for its introduction into medical practice are examined. This review will be useful for researchers engaged in the development, characterization, and standardization of chitosan-based biomaterials.
The α-ketoglutarate dehydrogenase complex (KGDHC) serves as a master regulator of cell's molecular machinery. Beyond its classical role as a rate-limiting enzyme in the tricarboxylic acid (TCA) cycle, KGDHC has emerged as a critical redox sensor that can act as both a source and a target of reactive oxygen species (ROS), thereby regulating cellular redox homeostasis. This review summarizes evidence from genetically modified animal models and cell culture studies demonstrating that compromised KGDHC activity affects neuronal metabolism, redox homeostasis, and cellular signaling. KGDHC dysfunction causes mitochondrial failure, resulting in reduced ATP synthesis and activation of AMP-activated protein kinase (AMPK). Although inhibition of KGDHC reduces mitochondrial ROS formation, it also disrupts physiological ROS-dependent signaling mechanisms. In KGDHC-deficient mice, impaired ROS signaling and energy deficit decrease brain adaptability, increase susceptibility to neurotoxins, and disrupt crucial pathways by downregulating PGC-1α and Nrf2. These alterations result in suppression of antioxidant defences and lead to neuronal death in the hippocampus and memory impairment. Moreover, KGDHC dysfunction induces mitochondrial fragmentation and is strongly linked to excitotoxicity, further accelerating neuronal dysfunction. As observed in heterozygous models, even partial KGDHC deficiency can exacerbate persisting cellular and mitochondrial defects, leading to the development of more severe pathological conditions.
In recent years, reconstructive surgery strategies have been supplemented with innovative approaches aimed at developing tissue-engineered structures using autologous tissues and biodegradable scaffolds and matrices, the purpose of which is to reduce the risk of postoperative complications, on the one hand, while ensuring accelerated restoration of the structure and functions of organs, on the other hand. The review systematizes modern scientific trends in the field of tissue engineering of the urethra, focused on creation of biocompatible and functionally active tissue-engineered structures using achievements of cellular technologies and materials science. Particular attention is paid to describing the mechanisms and ways of forming a complete and functional three-dimensional structure of the urethra, including the use of organoids. It is expected that this strategy will contribute to the development of personalized therapeutic approaches and improved clinical outcomes in the patients with urological diseases.
Previously, we demonstrated that administration of dexamethasone (Dex) at a dose of 1 mg/kg, 24 h before an ulcerogenic stimulus exerts a pro-ulcerogenic effect, accompanied by disturbances in carbohydrate metabolism. In the present study, we examined the influence of housing conditions - standard conditions (SC), social isolation (SI), and environmental enrichment (EE) conditions - on the Dex-induced changes in carbohydrate metabolism, as well as on hematological parameters. Experiments were conducted with male rats during the winter period. Starting from the age of 30 days, the animals were housed for 6 weeks under SC, SI, or EE conditions. Dex (1 mg/kg, intraperitoneal) or its vehicle (control) was administered 24 h prior to the glucose tolerance test (GTT), after which food was removed. Following the GTT, indomethacin (IM) was administered at an ulcerogenic dose; 4 h later, the rats were decapitated, and blood samples were collected to assess corticosterone levels and hematological parameters, including calculation of the neutrophil-to-lymphocyte ratio (NLR). Alongside the IM administration experiment, a control experiment including vehicle administration was performed according to the same protocol, in which the vehicle of IM was administered instead of IM itself. Administration of glucose during the GTT led to the increase in the blood glucose levels, reaching maximum (peak) at 30 min in all control, previously fasted animals (SC, SI, EE groups). Beginning at 60 min, the glucose levels gradually declined in all control groups, returning to the baseline only in the control rats from the EE group. In the rats maintained under SC conditions, pretreatment with Dex resulted in the reduction in the peak of the glycemic curve, accompanied by the corresponding decrease in the area under the curve (AUC) and reduced rate of decline in the blood glucose levels compared with the respective control group. In the rats housed under EE condition, resistance to the effects of Dex was observed, as evidenced by the absence of changes in the glycemic curve peak, AUC, or rate of decline in the blood glucose levels relative to the corresponding control group. The control rats from the SI group exhibited lower values of the glycemic curve peak, AUC, and rate of decline in the blood glucose levels than the rats from the SC and EE groups. Administration of Dex did not produce any further changes in these parameters. Dex administration induced a marked increase in the NLR in all groups (SC, SI, and EE), both in the rats treated with IM and in the animals receiving its vehicle. Taken together, these findings indicate that a single administration of Dex (1 mg/kg; 24 h after injection) to the rats from the SC group could alter glycemic response and increase NLR. Housing under EE conditions prevents the Dex-induced changes in the glycemic curve.
Macrophages are a heterogeneous cell population whose functional diversity is formed during their maturation and depends on factors of the microenvironment after their migration into the bloodstream or tissues. One such factor is the pro-inflammatory protein cyclophilin A (CypA, 18 kDa). Using a model of early human monocytic THP-1 cells, it was shown that recombinant human CypA (rhCypA) exerts a differentiating effect on these cells, inducing their maturation, adhesion, and spreading. Under the effect of rhCypA, the THP-1 cells developed an actin cytoskeleton characteristic of motile cells with numerous pseudopodia and podosomes, which ensure tight adhesion of the cells to the substrate and determine their migratory capabilities. Combination of low concentrations of rhCypA and other activators (phorbol myristate acetate) showed an additive effect and ensured effective monocyte differentiation. It was shown that rhCypA, along with other pro-inflammatory factors (IFNγ, TNFα), promotes cell fusion and induces formation of multinucleated macrophages, which are formed during osteoclast maturation under normal conditions as well as during granuloma formation in chronic inflammation (tuberculosis, Crohn's disease). Multinucleated giant cells have significantly higher functional activity (phagocytosis, bactericidal, and pro-inflammatory activity) compared to the mononuclear forms. The study showed that rhCypA enhances expression of the CD147 molecule, an integral functional regulator of CD29 and CD98 molecules involved in the processes of cell adhesion and fusion. Elevated doses of CypA cause deterioration in macrophages, inducing their apoptosis, which may play a role in regulation of the immune response. The findings of this study determined the mechanisms by which secreted CypA mediates monocyte differentiation and maturation, as well as it showed functional role of macrophages in the development of the immune response, which could facilitate further development of therapeutic approaches for the treatment of infectious, autoimmune, and other diseases.
Cancer-testis antigens are expressed in the germ cells of the testes, but can also be produced in some types of cancer cells, thus representing an important protein group in oncoimmunology. They include sperm-specific proteins of glycolysis, in particular, sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDHS). This isoform differs from somatic isoform in a number of properties. Normally expressed in spermatids, GAPDHS is also found in uveal and skin melanoma cells. Because GAPDHS is a glycolytic protein and glycolysis is a key component of energy metabolism in a growing tumor, the review summarizes the data on the functional and structural features of GAPDHS and its role in the regulation of glycolysis in melanoma cells.
Ultracentrifugation (UC) has long been considered the "gold standard" for extracellular vesicle (EV) isolation. However, due to its drawbacks such as high cost of an ultracentrifuge and rotors, time-consuming and labor-intensive protocol, low yield considering initial biofluid volume and low throughput, development of new EV isolation approaches is still ongoing. Here we compare three methods for isolating the most studied EV subtype, small extracellular vesicles (sEVs), from human plasma: ultracentrifugation (UC), express asymmetric depth filtration (ExADFi), and anti-CD9 immunoaffinity capture (AS-CD9) with focus on their Raman and proteomic profiles. For all three methods, purity and quality of the sEV isolation were assessed based on the level of contamination of the sEV fraction with major plasma proteins such as albumin and apolipoproteins (APOA1, APOH, APOA4, APOC2, APOC1, and APOC4). UC showed the highest ratio of protein to nanoparticle concentration. AS-CD9 and ExADFi provided comparable to UC purity and levels of non-vesicular contaminants with AS-CD9 requiring minimal time and labor. ExADFi showed characteristics including purity of the sEV samples, yield, and isolation time that is between the UC and AS-CD9 methods. Raman spectroscopy provided more details about characteristics of the isolated sEVs and confirmed differences observed in the proteomic profiles. The findings demonstrate that the AS-CD9 and ExADFi methods could be appropriate substitutes of the classical UC-based isolation method and be chosen depending on the final requirements and use of the purified sEVs such as further functional and biomarker studies.
Reductive stress caused by excessive accumulation of reducing equivalents (NADH, NADPH, glutathione), is increasingly recognized as a pathogenetic factor at the early stages of neurodegenerative diseases. However, its impact on the morphofunctional properties of astrocytes remains poorly understood. We performed the first comprehensive quantitative assessment of biochemical and morphological changes in cultured primary rat astrocytes under chronic reductive stress modeled using dithiothreitol (DTT, 500 μM, 24 h). To enable morphometric analysis of live, unstained cells, we developed and trained a semantic segmentation model based on the YOLOv11 architecture, which enables objective assessment of complex branched cell morphology in phase-contrast images while avoiding fixation-related artifacts. DTT-induced reductive stress caused a significant increase in the total pool of nicotinamide coenzymes (p < 0.01) and elevated mitochondrial superoxide production without compromising mitochondrial membrane potential. Morphometric analysis revealed a sustained enlargement of astrocyte soma area and increase in the number of astrocyte processes, along with a reduced branching complexity. These findings provide new insights into the role of redox imbalance in regulating glial function and may expand our understanding of the early mechanisms of neurodegenerative diseases.
Sex-specific interactions between neurosignaling systems, which generate, propagate, and terminate signals in nervous tissue, and metabolic pathways that support these processes may underlie sex differences in adaptation and therapeutic efficacy. This study aimed to characterize these interactions as systemic indicators of sex-specific adaptive responses in a rat model of metabolic stress induced by the inhibition of pyruvate dehydrogenase complex (PDC), which catalyzes the key reaction linking anaerobic glycolysis to aerobic glucose oxidation. To inhibit brain PDC, we used a single intranasal administration of methyl acetylphosphinate (MeAcP), a phosphinate analog of pyruvate, or dimethyl acetylphosphonate (AcPMe2), a membrane-permeable precursor of phosphonate pyruvate analogs. Effects were assessed 24 h post-administration by measuring biochemical and physiological parameters in the cerebral cortex, including glutamate levels, glutamine synthetase (GS) activity, and activities of enzymes in the tricarboxylic acid (TCA) cycle and affiliated pathways. Neurosignaling was evaluated using surrogate indicators: ECG (electrocardiogram) parameters and spontaneous behavior in the open field test. Relationships between measured parameters were analyzed using Spearman's rank correlation coefficients, with the correlation strength classified according to the Chaddock's scale. In control animals, no sex differences were observed in the mean values of biochemical or ECG parameters. However, behavioral parameters (e.g., grooming and locomotion) and the overall structure of correlations between the studied parameters exhibited marked sex dependence. In control females, strong correlations were detected between ECG parameters and GS activity, whereas in males, ECG parameters were strongly associated with malic enzyme (ME) activity. Male controls also showed strong correlations between locomotor/exploratory behavior and activities of ME, PDC, and 2-oxoglutarate dehydrogenase complex (OGDC). Administration of PDC inhibitors induced a sex-specific reorganization of relationships between neurosignaling indicators and glutamate metabolism, which eliminated pronounced sex differences in locomotor activity observed in controls, while revealing new sex-related differences in glutamate levels, glutamate dehydrogenase (GDH) and ME activities, grooming bout duration, and freezing time. The reduction in glutamate levels observed in females following PDC inhibition was consistent with the established decrease in de novo glutamate synthesis from glucose under conditions of impaired substrate flux through the TCA cycle. Overall, these findings demonstrate that the relationships among metabolic, behavioral, and ECG parameters are inherently sex-specific. Moreover, the homeostatic response of the cerebral cortex to PDC inhibition reshapes these relationships, thereby modifying sex-dependent biochemical and behavioral characteristics observed under control conditions.
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The mechanisms underlying the recently discovered catalase activity of immunoglobulin G (IgG) in patients with schizophrenia remain unclear. Using a series of rigorous criteria, it has been demonstrated that this activity is an intrinsic property of antibodies themselves. The present study shows that classical catalase inhibitors also suppress the catalase activity of IgG. Specifically, inhibitor analysis revealed a dose-dependent reduction in the IgG catalase activity following addition of sodium azide (IC50 = 140 μM) and 3-aminotriazole (IC50 = 16.06 μM), suggesting that the catalytic mechanism of IgG shares similarities with that of classical catalase and may be due to the ability of antibodies to bind metalloporphyrin complexes, including heme. IgG catalase activity in patients with schizophrenia during therapeutic remission was significantly reduced, being fourfold lower than in healthy controls (p = 0.0004) and twofold lower than in patients during disease exacerbation (p = 0.002). A moderate positive correlation was observed between the total Positive and Negative Syndrome Scale (PANSS) score and IgG catalase activity (R = 0.32, p = 0.01). Therefore, IgG catalase activity in patients with schizophrenia depends on the disease clinical state and may contribute to the regulation of reactive oxygen species (ROS) metabolism and the severity of oxidative stress in affected individuals.
The Escherichia coli bacterial expression system was the first platform developed for recombinant protein production and remains the fastest, simplest, and most cost-effective system for achieving high protein yields for fundamental research, as well as biotechnological and pharmaceutical applications. Bacterial surface display systems and secretion of target proteins have become widely used approaches. These strategies help prevent intracellular aggregation and proteolytic degradation of recombinant proteins, enabling the recovery of soluble, properly folded, and stable protein products. In the case of toxic proteins, secretion mitigates their inhibitory effects on essential host cell processes. Furthermore, secretion of target proteins and peptides significantly simplifies their purification. The review summarizes the data on E. coli secretion systems with a special focus on protein export and display strategies, and discusses their applications in scientific research, industrial biotechnology, and medicine.
To study biochemical mechanisms underlying the development of autism spectrum disorders (ASD), an experimental model based on early postnatal administration of valproic acid (VPA) to Wistar rats was used. Social behavior impairments characteristic of ASD was accompanied by cognitive impairments and were associated with the increased neutrophil elastase (NE) activity in the serum and cerebellum of the rats treated with VPA. Increased NE activity in the cerebellum may indicate development of neuroinflammation. Impaired antioxidant defense under the influence of VPA was manifested by the increased glutathione S-transferase (GST) activity in the serum and cerebellum of rats. Correlation analysis revealed a link between the NE activity and formation of social behavior in animals. Enzymes of glutamate metabolism (glutamate dehydrogenase and glutamine synthetase) and antioxidant defense enzymes that regulate oxidative stress (OS) levels correlated with formation and maintenance of the acquired skills in both control and experimental animals.
This special issue of Biochemistry (Moscow) "Interaction between Neural Signals and Metabolic Pathways: Role in the Functioning of a Healthy and Diseased Brain", includes studies on the mechanisms of close functional connections between the brain and other organs and tissues of the body. These mechanisms link brain metabolism with its signaling function under normal and pathological conditions. The metabolic signals that enable these connections are the focus of research in this field, which is crucial for an integrated understanding of how the body functions. An impairment in metabolic signaling leads to the development of various pathologies. Metabolites such as glucose, fatty acids, and amino acids act as primary signals that influence neural networks and brain chemistry. This connection between the body's metabolism and brain signaling is not merely a matter of fuel supply, but rather a complex information exchange process. The interaction between the brain and the body occurs within the framework of coordinated work of two main axes: the brain-to-body axis ("from top to bottom" or from center to periphery), and the body-to-brain axis (from periphery to center). This relationship between brain function and body metabolism forms a mechanical and logical connection between metabolic somatic diseases and brain disorders that may underlie their comorbidities. The close connection between brain function, metabolism, and the metabolism of peripheral organs and tissues forms the basis for treating "body-brain metabolic" disorders. Identifying the molecular and cellular mechanisms underlying this relationship allows identifying targets for treating and preventing comorbid somatic and brain conditions. The recent achievements, which prove the close relationship between metabolism and brain activity, have led to the emergence of a rapidly growing interdisciplinary field at the interface of neuroscience, philosophy of consciousness, and functional biochemistry of metabolism. This new synthetic field can be called "metabolic neurophilosophy". Its subject is to explore the integrity and inseparability of the body's metabolism (including both in the brain and peripheral organs and tissues) and the signaling and informational function of the brain. It also studies the dependence of all brain activity, including cognition, and mental states on energy processes and metabolic signaling throughout the body.