Quinazolinone derivatives are well-known anticancer agents; anticancer properties are also part of the broad spectrum of biological activity of coumarins. Conjugates containing quinazolin-4(3H)-one and coumarin fragments linked by polymethylene bridges of varying lengths were designed to improve properties of both parental compounds and create new anticancer or antibacterial agents. 3-{3-[(4-Methyl-2-oxo-2H-chromen-7-yl)oxy]propyl}quinazolin-4(3H)-one was synthesized as the base compound. It demonstrated moderate cytotoxicity against leukemia (K562 and HL60) and neuroblastoma (SH-SY5Y) cells in vitro, combined with relatively low acute, subacute, and chronic toxicity in vivo. Conjugates with various substituents and linkers were then synthesized to evaluate the structure-activity relationship. A study of the synthesized compounds on cell cultures showed that the introduction of a methyl substituent into the benzene ring of the coumarin fragment led to both an increase in cytotoxicity and expansion of its spectrum of action. Testing of the hybrids against Gram-positive and Gram-negative bacteria revealed that the introduction of halogens into the quinazoline fragment in the compounds or the elongation of the linker led to the emergence of pronounced antibacterial properties, which were most clearly manifested against Acinetobacter baumanii. The possibility of directing activity of quinazoline-4(3H)-one-coumarin hybrids by varying the substituents and the length of the linker was shown.

Alzheimer's disease (AD) is a multifactorial systemic disease that is triggered, at least in part, by the accumulation of β-amyloid (Aβ) peptides in the brain, but it also depends on immune system-mediated regulation. Recent studies suggest that B cells may play a role in AD development and point to the accumulation of clonally expanded B cells in AD patients. However, the specificity of the clonally expanded B cells is unknown, and the contribution of Aβ-specific B cells to AD pathology development is unclear. In this study, we have developed a novel method to identify Aβ-specific B cells by flow cytometry using fluorescent tetramers. The suggested method also enables the identification of B-cell clones specific to a more pathology-provoking form of Aβ with an isomerized Asp7 residue (Iso-D7-Aβ) that accumulates in elderly people and in AD patients. The method has been verified using mice immunized with antigens containing the isomerized or non-isomerized Aβ N-terminus peptides. In addition, we describe a new method for the detection of Iso-D7-Aβ-specific antibodies, which was tested on mouse serum. These methods are of potential importance in research aimed at studying AD and may be also utilized for diagnostic and therapeutic purposes.

Biosynthesis of ribose-5-phosphate (R5P) underlies all biosynthetic processes associated with biomass growth. Actively dividing cells continuously require building blocks for genome replication, synthesis of ribosomes and other derivatives containing R5P as a carbohydrate backbone. The main source of R5P in the cell is the pentose phosphate pathway (PPP), which is an anabolic sensor designed to coordinate the level of pentose phosphates and reduced NADPH required for anabolic processes. This review is devoted to a comparative analysis of R5P biosynthesis pathways among different domains of microorganisms, the features of PPP regulation in bacterial cells depending on physiological conditions, as well as genetic modifications of PPP and their effect on cell viability. We emphasize that ribose metabolism is a factor in the consolidation of cellular homeostasis under conditions of intensive biomass growth and the discrepancy between the processes of ribose synthesis and consumption is marked by spontaneous cell death.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, characterized by the formation of neurotoxic beta-amyloid (Aβ) oligomers in the central nervous system. One of the earliest pathological effects of Aβ is the induction of oxidative stress in brain tissue, mediated by NADPH oxidase 2 (NOX2). This study aimed to determine whether short-term inhibition of NOX2 could disrupt the pathological cascade and prevent the development of Aβ-induced pathology. We demonstrated that suppressing NOX2 activity by GSK2795039 during the first three days after intracerebral Aβ administration prevented the development of the pathological process in mice. Two weeks after the induction of Aβ pathology, animals treated with GSK2795039 showed no neuropsychiatric-like behavioral changes, which correlated with the absence of chronic oxidative damage in brain tissue. Moreover, GSK2795039 prevented microglial activation and reduced microglia-associated neuroinflammation. These findings indicate that short-term NOX2 inhibition effectively suppresses the development of Aβ-induced pathology, suggesting that NOX2 is a potential target for treatment and prevention of AD pathology.

Endogenous cardiotonic steroids (CTS), which are specific ligands of Na,K-ATPase, have been detected not only in blood plasma but also in cerebrospinal fluid and in the brain tissue. Consequently, the role of CTS in the central nervous system has gained increasing relevance. Na,K-ATPase serves not only as a receptor for CTS but also as a target for beta-amyloid (Aβ42). Previously, we demonstrated that ouabain binding to Na,K-ATPase prevents Aβ42-induced activation of Src kinase and the subsequent change in the level of the amyloid precursor protein (APP) in human neuroblastoma SH-SY5Y cells. In this study, we characterized the effects of other CTS—marinobufagenin, bufalin, and digoxin, on APP level in these cells. We found that, unlike ouabain, bufalin and digoxin increased APP levels in cells independently of Aβ42. Marinobufagenin amplified the increase of the general APP level caused by Aβ42. In contrast, the addition of Aβ42 in the presence of bufalin or digoxin does not further elevate APP levels. Src kinase activation is observed only with marinobufagenin. This suggests that unlike Aβ42, which activates Src kinase, the CTS-induced rise in APP occurred through a Src-independent pathway. CTS does not lead to the accumulation of APP in neurites. Furthermore, ouabain, marinobufagenin, and digoxin reduce the rise in APP that Aβ42 induces in neurites. Molecular modeling data indicate that CTS binding to Na,K-ATPase alters the number of contacts formed between the enzyme and subsequently bound Aβ42. It means that CTS binding to Na,K-ATPase alters its interaction with Aβ42. Taken together, these results show that endogenous CTS are important regulators that can maintain the balance between APP and beta-amyloid in the brain.

Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of beta-amyloid peptide (Aβ) leading to formation of soluble neurotoxic Aβ oligomers and insoluble amyloid plaques in various parts of the brain. Aβ undergoes post-translational modifications that alter its pathogenic properties. Aβ is produced not only in brain, but also in the peripheral tissues. Such Aβ, including its post-translationally modified forms, can enter the brain from circulation by binding to RAGE and contribute to the pathology of AD. However, the transport of modified forms of Aβ across the blood–brain barrier (BBB) has not been investigated. Here, we used a transwell BBB model as a controlled environment for permeability studies. We found that Aβ42 containing isomerized Asp7 residue (iso-Aβ42) and Aβ42 containing phosphorylated Ser8 residue (pS8-Aβ42) crossed the BBB better than unmodified Aβ42, which correlated with different contribution of endocytosis mechanisms to the transport of these isoforms. Using microscale thermophoresis, we observed that RAGE binds to iso-Aβ42 an order of magnitude weaker than to Aβ42. Thus, post-translational modifications of Aβ increase the rate of its transport across the BBB and modify the mechanisms of the transport, which may be important for AD pathology and treatment.

Alzheimer’s disease (AD) is associated with the accumulation of β-amyloids (Aβs) and the formation of Aβ plaques in the brain. Various structural forms and isoforms of Aβs that have variable propensities for oligomerization and toxicity and may differentially affect the development of AD have been identified. In addition, there is evidence that β-amyloids are engaged in complex interactions with the innate and adaptive immune systems, both of which may also play a role in the regulation of AD onset and progression. In this review, we discuss what is currently known about the intricate interplay between β-amyloids and the immune response to Aβs with a more in-depth focus on the possible roles of B cells in the pathogenesis of AD.

Abstract—Solving the problem of multidrug resistance currently requires the development of nonstandard approaches, since the potential for creating new antibiotics is almost exhausted. Controlling the metabolism of a pathogen in order to increase its susceptibility to antibacterial therapy is considered the most promising area of research for the creation of new combination drugs. In recent years, the number of studies devoted to investigation the role of the biosynthesis of the cell wall component ADP-heptose in the sensitivity of bacteria to antibiotics, as well as in the pathogenesis of bacterial infection, has increased. This review examines the main directions of scientific research in the field of use of ADP-heptose and its analogues in the treatment of bacterial infections. The exclusive role of ADP-heptose in the induction of an immune response is known through the activation of the NF-κB signaling pathway and the synthesis of pro-inflammatory cytokines. Our latest work has shown that disruption of the synthesis of ADP-heptose and the efflux of sedoheptulose-7-phosphate from the pentose phosphate pathway induces a redox imbalance and completely disorganizes the metabolism of low molecular weight thiols such as hydrogen sulfide, cysteine, and glutathione, which makes the bacterial cell extremely vulnerable to the action of antibiotics. We demonstrate that the hypersensitivity of ADP-heptose mutants to a wide range of antibiotics is explained by a new metabolic status rather than by changes in cell wall permeability. Thus, potential inhibitors of ADP-heptose biosynthesis can combine several positive qualities: an immunomodulatory effect and a powerful potentiating effect in combination with antibiotic therapy.

This study aims to investigate the potential role of antioxidants in oxidative stress and its consequent impact on the mechanical properties of neuronal cells, particularly the stress induced by amyloid-beta (1-42) (Aβ42) aggregates. A key aspect of our research involved using scanning ion-conductance microscopy (SICM) to assess the mechanical properties (Young's modulus) of neuronal cells under oxidative stress. Reactive oxygen species (ROS) level was measured in single-cell using the electrochemical method by low-invasive Pt nanoelectrode. We investigated the effects of the low molecular weight antioxidant N-acetylcysteine (NAC) and the antioxidant enzyme superoxide dismutase 1 (SOD1) on the physiological and mechanical properties of neuronal cells using SICM. Using electrochemical method and SICM, NAC effectively reduces oxidative stress and restores Young's Modulus in SH-SY5Y cells exposed to hydrogen peroxide and Aβ42 oligomers. Our study first examined the influence of SOD1 on intracellular ROS levels in the presence of Aβ oligomers. The investigation into the effects of SOD1 and its nanoparticle form SOD1 on SH-SY5Y cells reveals impacts on mechanical properties and oxidative stress. The combined use of SICM and electrochemical measurements provided a comprehensive understanding of how oxidative stress, including that triggered by the Aβ oligomers affects the mechanical properties of cells.

Alzheimer's disease (AD) is associated with the accumulation of β-amyloids (Aβs) and the formation of Aβ plaques in the brain. Various structural forms and isoforms of Aβs that have variable propensities for oligomerization and toxicity and may differentially affect the development of AD have been identified. In addition, there is evidence that β-amyloids are engaged in complex interactions with the innate and adaptive immune systems, both of which may also play a role in the regulation of AD onset and progression. In this review, we discuss what is currently known about the intricate interplay between β-amyloids and the immune response to Aβs with a more in-depth focus on the possible roles of B cells in the pathogenesis of AD.

Disruption of both branches of the canonical pentose phosphate pathway (PPP) in E. coli by combined inactivation of the zwf and talAB genes provokes the restoration of the ancient anabolic variant of PPP (aPPP). In the aPPP, pentose-5-phosphates are synthesized unidirectionally from fructose-6-phosphate and glyceraldehyde-3-phosphate by transketolase B, aldolase A, and phosphatase GlpX, converting sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate. Unexpectedly, the double zwf talAB mutant exhibits decreased survival after treatment by diverse classes of antibiotics with little effect on the minimal inhibitory concentration. Simultaneously, we found that killing effect of antimicrobials on the zwf talAB mutant could be reversed by the inactivation of either purR or deoB genes, both responsible for ribose-5-phosphate content in the mutant strain. Enhanced biosynthesis of the cell wall component ADP-heptose from sedoheptulose-7-phosphate also suppressed killing effect of antibiotics on the zwf talAB mutant. Furthermore, the inactivation of the Entner-Doudoroff pathway ( Δedd ) or shifting the metabolic equilibrium by the addition of exogenous phosphogluconate reverts aPPP to glycolysis, preventing the accumulation of excess pentose phosphates and the occurrence of the futile cycle in zwf talAB cells, thus desensitizing them to antibiotics. Our findings show that ribose-5-phosphate metabolism plays a crucial role in bacterial tolerance to a wide range of bactericidal antibiotics. We propose that targeting PPP could be a promising strategy for developing new therapeutic agents aimed at potentiating clinically significant antimicrobials. IMPORTANCE Recent studies have revealed the crucial role of bacterial cell’s metabolic status in its susceptibility to the lethal action of antibacterial drugs. However, there is still no clear understanding of which key metabolic nodes are optimal targets to improve the effectiveness of bacterial infection treatment. Our study establishes that the disruption of the canonical pentose phosphate pathway induces one-way anabolic synthesis of pentose phosphates (aPPP) in E. coli cells, significantly increasing the killing efficiency of various antibiotics. It is also demonstrated that the activation of ribose-5-phosphate utilization processes restores bacterial tolerance to antibiotics. We consider the synthesis of ribose-5-phosphate to be one of the determining factors of bacterial cell stress resistance. Understanding bacterial metabolic pathways, particularly the aPPP’s role in antibiotic sensitivity, offers insights for developing novel adjuvant therapeutic strategies to enhance antibiotic potency. ### Competing Interest Statement The authors have declared no competing interest.

Intracellular tripeptide glutathione is an important agent of cell survival under hypoxia. Glutathione covalently binds to SH groups of hemoglobin cysteine residues, protecting them from irreversible oxidation, and changes its affinity to oxygen. Reduced glutathione (GSH) can also form a noncovalent complex with hemoglobin. Previously, we showed that hemoglobin tetramer has four noncovalent binding sites of glutathione GSH molecules inside, two of which are released during hemoglobin transition to deoxy form. In this study, we characterized the conserved cysteine residues and residues of noncovalent glutathione binding sites in the sequences of a number of hypoxia-tolerant and hypoxia-sensitive mammals. The solvent accessibility of all HbA and HbB residues in oxy and deoxy forms was analyzed. The alpha subunit of all species considered was shown to have no conserved cysteines, whereas the beta subunit contains Cys93 residue, which is conserved across species and whose glutathionylation changes the affinity of hemoglobin for oxygen 5–6-fold. It was found that the key residues of noncovalent glutathione binding sites in both alpha and beta subunits are absolutely conserved in all species considered, suggesting a common mechanism of hemoglobin redox regulation for both hypoxia-sensitive and hypoxia-tolerant mammals.

Src kinase is one of the key regulators of cellular metabolism and is dysregulated in numerous diseases, including cancer, neurodegenerative diseases, and particularly Alzheimer’s disease. Despite its therapeutic importance, its full-length structure has never been obtained before, as it contains an intrinsically disordered regulatory region, SH4UD. The SH4UD region is crucial for Src activation, functional dimerization, and regulation by other kinases. In this study, we used the replica exchange molecular dynamics approach with a hybrid temperature and Hamiltonian tempering to obtain the conformational ensemble of full-length Src kinase in its non-phosphorylated state and in the presence of its two key regulatory phosphorylations: pY419 and pY530. The representative structures and simulation trajectories of non-phosphorylated pY419 and pY530 Src are available in open access. We demonstrate that pY419 phosphorylation, which is associated with Src activation, enhances its motility, whereas inhibited pY530 Src preserves relatively compact conformation. This study also provides insights into how SH4UD contributes to Src substrate binding, dimerization, and autophosphorylation, highlighting the putative role of 14-RRR-16 in this process.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance. Ovarian cancers frequently develop resistance to therapy. Here, using transcriptomics, proteomics, and preclinical models to analyse paired ascitic fluids before and after chemotherapy in ovarian cancer patients, the authors discover that extracellular secretion and spliceosomal components contribute to therapy resistance, enhancing the DNA damage response in recipient cancer cells.

The blood-brain barrier (BBB) is typically compromised in Alzheimer’s disease (AD): its permeability and intracellular parameters of the endothelial cells are affected. AD-related amyloid isoforms –Aβ42, isoD7-Aβ42 and pS8-Aβ42, inducing amyloidogenesis in a different mode – may have peripheral origin and affect the BBB cells. This study aimed to assess this effect. Mouse brain endothelial cells bEnd.3 underwent incubation with 10 µM of Aβ42, isoD7-Aβ42 and pS8-Aβ42 for 4, 24 and 48 hours to assess nitric oxide (NO), glutathione (GSH), reactive oxygen species (ROS), mitochondrial potential, Ca 2+ , cell viability by flow cytometry and mitochondrial functioning by Seahorse technology. To analyze the integrity of the cellular monolayer, the expression level of tight junction proteins and permeability of bEnd.3 monolayer to fluorescent tracers were assessed by Western Blotting and transwell-modeling, correspondingly . pS8-Aβ42 and isoD7-Aβ42 effects on the redox status of the cells and mitochondria functioning differ from the effects of Aβ42. Even at 4 hours, Aβ42 and isoD7-Aβ42 induce immense changes in mitochondrial potential and NO level but pS8-Aβ42 does not. Prolonged incubation with three isoforms leads to an increase in ROS, and greater increase is observed after incubation with isoD7-Aβ42. pS8-Aβ42 induces ROS increase later than other isoforms. Above all, incubation with pS8-Aβ42 results in a less pronounced NO и GSH growth compared with Aβ42 и isoD7-Aβ42 which induce activation of mitochondrial respiration and increase in mitochondrial potential, compared with pS8-Aβ42. IsoD7-Aβ42 is mostly toxic to bEnd.3 cells. Although Aβ42 and isoD7-Aβ42 significantly affect redox parameters of bEnd.3 cells, their effects on BBB permeability and tight junction protein expression are less pronounced. Our data demonstrate that inhibition of NO synthases and NMDA receptors induces changes in BBB cells’ response to Aβ peptides. Amyloid isoforms differently affect redox parameters of BBB cells significantly modulating mitochondria functioning. Iso-Aβ 42 induces higher cytotoxicity, thus, being a more pathogenic form. Post-translational modifications of Aβ 42 alter the amyloid effect on the BBB cells. It may result in different changes in BBB functioning and its permeability under amyloid isoforms. This research was funded by Russian Science Foundation (Grant No. #19-74-30007).

Ovarian cancer is known to be the most lethal malignancy among all gynecological cancers affecting a large number of women worldwide. The treatment of ovarian cancer is challenging due to the high recurrence rate of the disease and is further complicated by acquired chemoresistance. Most ovarian cancer deaths are the result of the metastatic spread of drug-resistant cells. The theory of cancer stem cells (CSC) suggests that both tumor initiation and progression are driven by a population of undifferentiated capable of self-renewal, tumor initiation and development of chemoresistance. The CD117 mast/stem cell growth factor receptor (KIT) is the most commonly used marker for ovarian CSCs. Here, we analyze the correlation between CD117 expression and histological tumor type in ovarian cancer cell lines (SK-OV-3 and MES-OV) and in small/medium extracellular vesicles (EVs) isolated from the urine of ovarian cancer patients. We have demonstrated that the abundance of CD117 on cells and EVs is correlated with tumor grade and therapy resistance status. Moreover, using small EVs isolated from ovarian cancer ascites, it was shown that recurrent disease is characterized by a much higher abundance of CD117 on EVs than primary tumor.

: Intracellular tripeptide glutathione is an important agent of cell survival in hypoxia. Glutathione covalently binds to SH-groups of hemoglobin cysteine residues, protecting them from irreversible oxidation and changes its affinity to oxygen. Reduced glutathione (GSH) can also form a non-covalent complex with hemoglobin. Previously, we showed that hemoglobin tetramer has four noncovalent binding sites of glutathione GSH molecules inside, two of which are released during hemoglobin transition to deoxy form. In this study, we characterized the conserved cysteine residues and residues of non-covalent glutathione binding sites in the sequences of a number of hypoxia tolerant and hypoxia-sensitive mammals. The alpha subunit of all species considered was found to have no conserved cysteines, whereas the beta subunit contains Cys94 residue, which is conserved across species and whose glutathionylation changes the affinity of hemoglobin for oxygen 5-6-fold. In addition, the key residues of noncovalent glutathione binding sites in both alpha and beta subunits are absolutely conserved in all species considered, suggesting a common mechanism of hemoglobin redox regulation for both hypoxia-sensitive and hypoxia-tolerant mammals. The solvent accessibility of all HbA and HbB residues in oxy- and deoxy- forms was analyzed. The key residue betaCys94 is solvent accessible only in the deoxy form.

Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein’s thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells.