The SFPQ (Splicing Factor Proline and Glutamine rich) protein, initially identified as a splicing factor, is a multifunctional nuclear protein involved in various cellular processes. Its main cellular partner is NONO (Non-POU domain-containing octamer-binding protein), with which SFPQ forms a heterodimer that is a crucial component of subnuclear structures called paraspeckles and located near nuclear speckles. However, SFPQ can also function independently in certain cellular processes and is essential for cell viability. There is substantial evidence of the involvement of SFPQ in the repair of double-strand DNA breaks (DSBs), but a definitive understanding of the mechanism of its participation in this critical cellular process is still lacking. In this review, we aim to summarize and systematize the existing data on the role of SFPQ and its complex with NONO in the repair of double-strand DNA breaks.

This study investigated permeability of the apical and basolateral membranes of rat corneal endothelial cells to water and urea. We demonstrated that the apparent water permeability of the basolateral membrane of endothelial cells (4.43E-05 ± 7.57E-07 cm/s) is more than three times higher than that of the apical membrane (1.21E-05 ± 1.03E-07 cm/s). Permeability of the basolateral membrane to urea (1.23E-04 ± 1.56E-06 cm/s) was statistically significantly higher than that of the apical membrane (9.52E-05 ± 1.02E-06 cm/s) by approximately 30%. We examined contribution of the phloretin-inhibited urea transport across the apical and basolateral membranes in these cells. Phloretin at concentration of 0.1 mM significantly reduced urea permeability by more than 20% through both the apical and basolateral membranes. The results suggest that the compositions of transporters involved in water transport in the apical and basolateral membranes differ significantly. It is hypothesized that high apparent water permeability of the basolateral membrane of endothelial cells is due to contribution of the concomitant water transport with ions involved in active transport processes. Presence of the phloretin-sensitive urea transporters in the plasma membrane of endothelial cells, likely involved in its transcellular transport, has been demonstrated. The results indicate potential significance of urea for corneal function.

Ferroptosis is an iron-dependent form of regulated cell death induced by hyperoxidation of polyunsaturated fatty acids (PUFAs) in cytoplasmic membrane phospholipids. Recent research has identified four key regulatory pathways of this process, with glutathione pathway (SLC7A11/SLC3A2)/GSH/GPX4 being the most central and well-studied. Functioning of all ferroptosis control systems is supported by the multilevel network of protein-coding and regulatory genes, whose dysregulated expression could trigger tumor cell transformation. Ferroptosis, alongside with other types of programmed cell death, plays a pivotal role in pathogenesis of many cancers, including non-small cell lung cancer (NSCLC). This review provides a comprehensive overview of the molecular mechanisms of ferroptosis and summarizes experimental evidence demonstrating involvement of the ferroptosis-associated non-coding RNAs (microRNAs and long non-coding RNAs) in the development and progression of NSCLC. Special emphasis is placed on the potential application of anti-ferroptotic and pro-ferroptotic non-coding RNAs in NSCLC therapy, focusing on targeted modulation of their expression to induce ferroptosis in tumor cells.

The process of signal transmission and transformation in the central nervous system requires active energy metabolism with high consumption of glucose and oxygen. Reactive oxygen species (ROS) produced as a result of these processes participate in intracellular signaling, but their overproduction leads to oxidative stress. Oxidative stress and α-synuclein aggregation are recognized as activators of neuronal death in Parkinson's disease. However, much less is known about the physiological role of monomeric synucleins. Using acute brain slices and primary co-cultures of cortical neurons and glial cells derived from transgenic animals with knockout of α-, β-, and γ-synuclein genes, we investigated the role of these proteins in ROS production and energy metabolism. We found that absence of synucleins leads to the reduced ROS production compared to the wild-type cells. The xanthine oxidase (XO) inhibitor led to the decrease in ROS production in the wild-type cells and the brain slices with β-synuclein knockout, whereas in the slices lacking α- or γ-synuclein, the XO inhibition was not observed, suggesting possible regulation of this enzyme by these proteins. Knockout of α- and γ-synucleins resulted in the decrease in mitochondrial membrane potential and reduction in energy capacity (in the form of ATP), which could be one of the mechanisms of XO regulation by synucleins.

Accurate quantification of extracellular vesicles (EVs) remains a significant challenge in biomedical research. Although various analytical methods have been developed, their reliability is often limited by the presence of non-vesicular nanoparticles and biological contaminants, particularly in biological fluids. Moreover, for some sources of EVs, such as uterine aspirates and gastric juice, quantitative evaluation of EVs has not been investigated. The aim of the study is to perform comparative analysis of three EV quantification methods: total protein content measurement, nanoparticle tracking analysis (NTA), and esterase activity assessment using commercial FluoroCet exosome quantitation kit in EVs isolated from various biological fluids: blood plasma, ascitic fluid, uterine aspirates, gastric juice, and medium conditioned by ovarian and non-small cell lung cancer cells. All three methods demonstrated strong correlation for the EV samples derived from the conditioned medium, supporting their validity for EV quantification in highly purified samples. In contrast, blood plasma, ascitic fluid, and uterine aspirates exhibited discrepancies between the methods, likely attributable to the presence of non-vesicular nanoparticles. Notably, the EVs from gastric juice demonstrated strong correlation between the protein content and esterase activity, indicating prevalence of the vesicle-associated proteins and, potentially, unique EV composition in this fluid. The findings underscore the necessity for multifactorial approach to EV quantification, taking into account factors such as sample origin and limitations inherent to the specific method employed. These results may serve as a basis for the development of standardized protocols for EV quantification, which is particularly relevant for clinical sample analysis.

The most critical problem in clinical oncology is the metastasis of malignant neoplasms. The survival and growth of metastases in a new microenvironment fundamentally depend on adaptations in the energy metabolism of metastasizing cells. However, these adaptations are far less studied compared to primary tumors. A promising method for assessing the metabolic status of cells is fluorescence lifetime imaging microscopy (FLIM), based on recording the decay parameters of cellular autofluorescence emitted by pyridine and flavin cofactors. This work aims to identify differences in the fluorescence decay kinetics of NAD(P)H between metastatic breast cancer cells and the primary tumor, as well as between metastatic cells and lymph node tissue in a 4T1 mouse model experiment. The study revealed a decrease in the relative fraction of the free form of NAD(P)H (a1, %), i.e., the form not bound to enzymes and associated with glycolysis, in metastases. This indicates a shift in the balance towards mitochondrial respiration. Furthermore, metastases were metabolically more heterogeneous at the cellular level than primary tumors, as evidenced by a higher dispersion of the mean NAD(P)H fluorescence lifetime τm. Additionally, it was found that metastatic cells have a higher contribution of the free NAD(P)H form a1 to the fluorescence decay and, consequently, a shorter mean lifetime τm compared to lymphoid tissue cells ( < 0.001). Thus, this study uses FLIM to demonstrate, for the first time, differences in the temporal characteristics of NAD(P)H autofluorescence between breast cancer metastases and the primary tumor, and between metastases and lymph node tissue. These findings align with existing concepts about the oxidative metabolism of breast cancer metastases. The obtained data are of interest for searching for therapeutic targets in the energy metabolism pathways of metastases and for developing new diagnostic approaches using autofluorescence.

Tumor-associated fibroblasts (TAFs) are a key cellular component of solid tumors, including gliomas. They support the growth of malignant cells, stimulate their invasion and metastasis, induce chemoresistance, and suppress the antitumor immune response. TAFs are formed from resident stromal cells under the influence of tumor cell secretome, including growth factors, chemokines, and extracellular vesicles. Communication between malignant cells and TAFs occurs through direct cell-cell contacts and exchange of secreted molecules and membrane vesicles. In this work, apoptotic bodies (apoBDs) were obtained from two types of glioma cells (T98g cell line and Gbl25 cells isolated from a glioblastoma biopsy) and characterized for surface markers. The surface of tumor apoBDs contained glioblastoma tumor-associated markers, such as GD2 ganglioside and A2B5 antigen. Glioma apoBDs contained lower levels of "don't eat me" molecules and higher levels of "eat me" molecules compared to the original intact glioma cells. On one hand, glioma apoBDs reduced the viability of normal dermal fibroblasts in a dose-dependent manner; on the other hand, they initiated their transformation into the inflammatory subtype of TAFs (iTAFs). iTAFs obtained in this way demonstrated upregulated transcription of genes encoding cytokines, chemokines, and growth factors (IL17A, IL18, IL33, IFN-γ, CCL3, CCL5, CXCL1, CXCL5, CXCL10, CXCL12, TGFB1, and TNF) responsible for maintaining both tumorigenesis itself and the ability of fibroblasts to support it. It was found that glioma apoBDs were able to transfer tumor-associated markers (GD2 ganglioside and A2B5 antigen) to normal fibroblasts. The assessment of the effects of anti-GD2 antibody-drug conjugates (ADCs) on TAFs suggests the possibility of development of targeted drugs effective not only against tumor cells but also against tumor stroma.

Butylated hydroxytoluene (BHT) is a well-known synthetic antioxidant and a commonly used synthetic food additive. It is prominently employed in pharmaceutical, rubber, oil, and petroleum industries. However, the evidence supporting its role in preventing skeletal muscle atrophy is lacking. In this study, the effect of BHT on the oxidative stress (100 µM HO)-induced atrophy was investigated in C2C12 myotubes. The antioxidative potential of BHT was compared to that of β-carotene. BHT demonstrated a superior free radical-scavenging ability compared to β-carotene in both DPPH and ABTS assays. Furthermore, pretreatment with 25 μg/ml BHT for 4 h preserved myotube morphology and membrane integrity and promoted creatine kinase activity in the oxidative stress-induced atrophy model. BHT also prevented the degradation of myosin heavy chain (a key structural protein) by downregulating the activity of calpain and suppressing expression of MuRF-1 mRNA (ubiquitin proteasome system), as well as reduced lipid peroxidation and ROS levels and increased lactate dehydrogenase activity, indicating improved cellular resilience. This study provides the first direct evidence of the protective effects of BHT against HO-induced atrophy in cultured myotubes and highlights a therapeutic potential of BHT in the mitigation of oxidative stress-related muscle atrophy.

The effect of the C-terminal tail on the bioactivity of mannose-binding protein (Abmb) was investigated. Based on the earlier obtained crystal structure of Abmb, it was suggested that the additional C-terminal tail can modulate the binding of sugars to the protein. According to glycan microarray, Abmb can bind β-Gal sugars, which contradicted the results of SPR analysis showing that Abmb only interacts with α-Man and not with α-Gal. Here, we used MCF-7 and MDA-MB-231 breast cancer cells to demonstrate that the presence of the C-terminal tail decreased the anti-proliferative activity of Abmb. Pre-incubating Abmb with α-Gal did not eliminate the anti-proliferative activity, while pre-incubation with α-Man attenuated it. At the same time, preincubation with a mixture of α-Gal and α-Man strongly promoted the anti-proliferative activity of Abmb. analysis using molecular docking suggested the presence of a second functional sugar-binding site for Gal, which had not been identified previously. The study provides new insights into the structure of lectins and their interaction with sugars.

Cysteine is an amino acid essential for normal functioning of living organisms. In bacteria and plants, the main mechanism of cysteine synthesis is the thiolation pathway, the second stage of which is catalyzed by either cysteine synthase A (CysK), if the substrate is inorganic sulfide, or cysteine synthase B (CysM), if the substrate is thiosulfate. The crucial role of these enzymes in cysteine synthesis makes them promising targets for antimicrobial agents and new herbicides, and well as possible components of industrial production of cysteine. In addition to their main functions, cysteine synthases show the antimicrobial and antibiofilm activities. The review discusses the physicochemical characteristics of CysK and CysM, their diversity, and potential applications in biotechnology and medicine.

Guanylins are intestinal natriuretic peptides that regulate water and electrolyte balance in the intestine and kidney. Their primary receptor is membrane guanylyl cyclase C (GC-C), while alternative functions related to feeding behavior and olfactory preferences are mediated by guanylyl cyclase D (GC-D) expressed exclusively in olfactory neurons. Evidence suggests existence of unidentified receptors activated by guanylin peptides in the absence of GC-C that affect sodium metabolism. Some of these receptors trigger the cGMP-dependent signaling pathways typical exclusively for guanylyl cyclases. This review provides a comparative analysis of the existing data on different membrane receptor guanylyl cyclases, including early discoveries and contemporary research, focusing on their potential as guanylin targets.

Skeletal muscle unloading results in muscle atrophy associated with the upregulation of proteolytic genes and suppression of protein synthesis, often accompanied by altered calcium signaling. Here, we used the inositol trisphosphate receptor (IP3R) inhibitor aminoethoxydiphenyl borate (2-APB) to explore the hypothesis that these changes are mediated by IP3Rs. Male Wistar rats were divided into 4 groups: (i) control, (ii) control with daily injections of 2-APB, (iii) 3 days of hind limb suspension, (iv) 3 days of hind limb suspension with daily administration of 2-APB. At the end-point, soleus muscles from the animals were analyzed by Western blotting for the markers of calcium, anabolic, and catabolic signaling. The 3-day hind limb unloading resulted in a decreased muscle weight index, upregulation of the anabolic suppressor pThr56-eEF2, downregulation of anabolic signaling via the mTOR pathway and rRNA expression, as well as the increase in the content of nuclear pThr286-CaMKII ( < 0.05) and cytosolic calcineurin A. While 2-APB did not affect the mTOR-governed changes in anabolism and catabolism, it significantly attenuated alterations in the calcium-dependent targets, such as CaMKII, calcineurin, and eEF2. By contrast, proteolytic signaling (expression of MuRF1, atrogin-1, Ulk1, and ubiquitin mRNAs) after 3-day hind limb unloading was equally upregulated in the control and 2-APB-treated animals. These results suggest that IP3Rs are involved in the unloading-induced muscle atrophy by controlling the nuclear content of calcium; however, they are dispensable for reduced mTOR activity and altered metabolism.

Reviewing published concepts on the chemical interactions between small molecules implicated in the origin of life suggests that their chemical properties have included not only those that might have been suitable for metabolic pathways. Some of the immanent "excessive" potencies of molecules make them able to form covalent adducts with proteins and nucleic acids. The accumulation of macromolecules damaged in this way could decrease the viability of protocells with increasing age. Thus, aging (senescence) could emerge concomitantly with life as its chemical heritage. Moreover, the exponential increase in mortality with age (the Gompertz law) could emerge when the kinetics of molecular disintegration according to the Arrhenius equation (disintegration rate depends exponentially on varying temperature at a constant activation barrier) was inherited by the kinetics of protocells dying out in their populations, the role of the independent variable passing from temperature, which was virtually constant on the Calvin scale, to viability. The cooperation of these two chemical heritages was enough to eliminate effectively old living objects and to make any evolved program of aging needless. Therefore, aging had not resulted from the biological evolution but rather has been and still is its independent factor. All this was possible without oxygen, which could only modify, rather than form , the primary chemical driving force of aging. With all that, the energy benefits of aerobic metabolism have provided for the advent of multicellular organisms, in particular, those featuring massive extracellular matter and unrenewable cell populations, including those comprising the brain. Their functions are incompatible with complete renewal. This makes the role of oxygen in aging not limited to being the source of reactive oxygen species. Oxygen had been indispensable for the advent of both accumulators of chemical damage and ability to recognize it. In a sense, it was not a problem for nature to develop aging in the course of evolution towards humans, for whom being aware of aging is a problem. Its satisfactory solution cannot be chemical, physical, pharmacological, or otherwise technical. It can only be mental.

Mitochondrial translation is a highly specialized process of synthesizing mitochondrially encoded proteins, mainly the components of the oxidative phosphorylation system. It involves four key stages: initiation, elongation, termination, and recycling of mitochondrial ribosomes. Each of these stages is regulated by a specific set of translation factors, most of which are encoded by the nuclear genome and imported into mitochondria. The termination of mitochondrial translation in yeast () is carried out by the MRF1 release factor. This nuclear-encoded factor is crucial for ensuring accurate protein synthesis within the organelle, as it recognizes stop codons and facilitates the release of completed polypeptide chains from the ribosome. In addition to this main function, MRF1 participates in maintaining mitochondrial genome stability. The aim of this study was to investigate the capacity of human homologues, hMTRF1, hMTRF1A, and mitoribosome rescue factors hMTRFR and hMRPL58, to compensate for the absence of the yeast mitochondrial translation termination factor MRF1 in cells. The results obtained suggest that human orthologues of MRF1, such as hMTRF1 and hMTRF1A, can contribute to maintaining the integrity of the yeast mitochondrial genome. However, they do not fully replace the function of MRF1, as they do not restore normal respiration of the mutant yeast strains.

In euryhaline fish species, including the three-spined stickleback, a key physiological response to freshwater adaptation aimed at maintaining osmotic homeostasis is enhancement of ion uptake from the environment and reduction of ion loss. Hormone prolactin, a central regulator of this process, primarily targets gills and intestine. Our previous work demonstrated that in the model of freshwater adaptation in sticklebacks prolactin expression and sensitivity of osmoregulatory tissues to prolactin differ between the males and females. In the present study, we measured expression levels of the genes encoding α1a and α3a subunits of Na/K-ATPase, as well as ion transporters NKCC1a, NKCC2, NCC, and NHE2, in the gill and intestinal tissues of the male and female three-spined sticklebacks ( L.) under conditions of acute (24 h) and chronic (72 h) freshwater adaptation, relative to the control conditions. During the freshwater adaptation, females, but not males, exhibited increased intestinal expression of and genes (as well as of the ratio of / expression), and the gene, along with the decreased expression of the gene. In contrast, only males showed increase in the gene expression in the intestine. In both sexes, exposure to fresh water led to the significant decrease in the gene expression in the gills. These findings support our hypothesis of sex-dependent plasticity in osmoregulatory function in sticklebacks, with females exhibiting a more pronounced response. This pattern further aligns with the previously reported stronger activation of the prolactin axis in the females under freshwater adaptation conditions.

Actin cytoskeleton is a key participant in numerous cellular processes, including organelle transport, motility, contractility, exocytosis, and endocytosis. It also plays a critical role in pathological processes such as malignant cancer cell invasion. The actin-binding proteins, particularly tropomyosins (Tpm) and cofilins, are involved in actin cytoskeleton remodeling. For this study, we selected the least studied isoforms of Tpm expressed from the gene - Tpm1.7, Tpm1.8, and Tpm1.9 - as well as the more well-known Tpm1.1 and Tpm1.6. We investigated mutual influence of these Tpm isoforms and cofilin-1 (cof-1) on actin filament dynamics. Using co-sedimentation assays, we demonstrated that Tpm1.7, Tpm1.8, and Tpm1.9 significantly inhibit cof-1 binding to the F-actin surface. Viscometry was employed to assess depolymerizing and severing effects of cof-1 on actin filaments. Tpm1.1, Tpm1.8, and Tpm1.6 effectively prevented depolymerizing/severing action of cof-1, while the protective effect of Tpm1.7 and Tpm1.9 was less pronounced. The rhodamine-phalloidin displacement assay was used to analyze the cof-1-induced conformational changes in F-actin. All studied Tpm isoforms effectively prevented effects of cof-1 on actin filaments. Our findings indicate that the gene products generally exert an inhibitory effect on cof-1 activity in relation to actin filament polymerization/depolymerization dynamics. Such properties of Tpm isoforms could be important for formation of specific intracellular populations of actin filaments.

17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for producing testosterone from phytosterol. Heterologous 17β-HSD from the fungus catalyzes NADPH-dependent reduction of the 17-oxo group of androstenedione/androstadienedione formed in mycolicibacterial cells as a result of the inherent polyenzymatic process of side chain oxidation of phytosterols, yielding testosterone/Δ-dehydrotestosterone, respectively. The object of this study was heterologous 17β-HSD from the fungus (17β-HSD) with a 6×His tag (6×His-17β-HSD), synthesized in the cells of actinobacteria . Isolation and purification of the recombinant enzyme were performed using affinity chromatography. The 6×His-17β-HSD enzyme preparation exhibited the highest activity toward androstenedione. Activity of the 6×His-17β-HSD depended on NADPH and was observed in the pH range from 6.0 to 9.0 with an optimum at pH 7.0. Analysis of kinetic characteristics showed that the properties of the heterologous enzyme 6×His-17β-HSD synthesized in cells are comparable with those reported for the 17β-HSD enzyme isolated from the fungus , as well as for the recombinant 17β-HSD enzymes synthesized in and cells. The results expand our knowledge on microbial 17β-HSDs and suggest potential for the use of the recombinant strains expressing a codon-optimized cDNA sequence encoding 17β-HSD from the fungus for producing testosterone from phytosterol.

The cytokine TRAIL is distinguished by its remarkable ability to preferentially induce apoptosis in transformed, but not in normal, cells. The recombinant TRAIL extracellular domain and other first-generation agonists of DR4 and DR5 death receptors (DRs) have shown very limited antitumor activity in clinical trials. To enhance the antitumor effect, we developed the multitarget recombinant fusion protein SRH-DR5-B-p48 based on the DR5-selective TRAIL variant DR5-B to simultaneously affect tumor cells (DR5-B-mediated apoptosis) and tumor microenvironment, in particular, to suppress angiogenesis. For this purpose, we modeled and produced the recombinant SRH-DR5-B-p48 fusion protein containing antagonistic synthetic peptides (SRH and p48) to VEGFR2 and FGFR1 receptors, respectively. Analysis of molecular trajectories using molecular dynamics methods showed that the SRH and p48 peptides form non-specific temporary contacts with the DR5-B domain. Using enzyme-linked immunosorbent assay, we showed that SRH-DR5-B-p48 was similar to DR5-B in its affinity for the death receptor DR5 and demonstrated a high affinity for VEGFR2 and FGFR1 with nanomolar dissociation constants. SRH-DR5-B-p48 killed tumor cells of various origin more efficiently than DR5-B and destroyed tumor-like structures in 3D cell models, as well as inhibited FGF2-mediated stimulation of fibroblast proliferation. Therefore, the SRH-DR5-B-p48 fusion protein can be considered as a promising agent for the therapy of solid tumors of various origin.

Argonaute proteins are an evolutionarily conserved family of proteins capable of recognizing and cleaving specific nucleic acid sequences using complementary guide molecules. Eukaryotic Argonautes play a key role in RNA interference by utilizing short RNAs of various classes to recognize target mRNAs. Prokaryotic Argonautes are much more diverse and most of them recognize DNA targets. The search for new Argonautes that would be active under varying conditions is important for both understanding their functions and developing new tools for genetic technologies. Many previously studied Argonautes exhibit low activity at low and moderate temperatures. To overcome this limitation, we isolated and studied two Argonaute proteins from psychrotolerant cyanobacteria, CstAgo from and CspAgo from  sp Both proteins use short DNA guides to recognize and cleave DNA targets. CstAgo displayed no specificity for the 5'-end structure of the guide, while CspAgo demonstrated a weak preference for the 5'-terminal nucleotide. CstAgo was highly active and capable of cleaving single-stranded DNA at temperatures from 10 to 50°C. CspAgo was more cold-sensitive but cleaved double-stranded plasmid DNA using specific guides. Therefore, the studied proteins can be potentially used for DNA manipulations under a wide range of conditions.

The article addresses a formal paradox related to the formation of molecular oxygen during photosynthesis. Following the studies of van Niel in the early 1930s, it has become clear that in the oxygenic photosynthesis, molecular oxygen originates from water rather than carbon dioxide. However, the overall equation of photosynthesis, CO + HO → (CHO) + O, suggests that the amount of oxygen produced exceeds what could be derived from the water molecules involved. This paradox can be resolved by analyzing the light and dark reactions of photosynthesis, which ultimately result in the incorporation of carbon from CO into carbohydrates and production of molecular oxygen. Despite its simplicity, the solution is not immediately obvious. One reason is that in the scientific and educational literature, the dark reactions of photosynthesis are often depicted schematically, without precise specification of all components involved. The author argues that analyzing this paradox and underlying physicochemical principles of photosynthesis can be valuable for students specializing in biochemistry.