Acta Naturae最新文献

The hippocampus is a key component of the brain that is associated with the formation of longterm memory, the energy metabolism of neurons playing a pivotal role in its mechanisms. The P2X3 receptor in the hippocampus is considered an attractive target when searching for novel biologically active substances that could work to reduce anxiety, epileptic conditions, and improve cognitive functions. In this work, the intensity of mitochondrial respiration, the glycolytic capacity, and the energy phenotype of hippocampal neurons were studied in p2rx3 knockout mice. The p2rx3 knockout mice were engineered by genome editing using the CRISPR/Cas9 system. The primary mixed culture of hippocampal neurons was derived from two-day-old newborn mice with the p2rx3^-/- and p2rx3^+/- genotypes. Mitochondrial respiration was measured on a Seahorse Bioscience HS mini Cell Metabolism Analyzer (Agilent, USA) using the appropriate kits for the Mitostress test, glycotest, and energy phenotype assessment test. The transgenic mice with the p2rx3^-/- genotype were characterized by an aerobic type of mitochondrial respiration, an increase in ATP production by 84.4% (p < 0.05), an increase in maximum respiration by 72.3% (p < 0.05), and a 36% (p < 0.05) increase in the respiratory reserve. Meanwhile, the spare respiratory capacity of mitochondria, the rate of glycolysis, and the glycolytic capacity in these mice were reduced by 36.6, 75.7 and 78.6% (p < 0.05), respectively. Our findings indicate that mitochondria work at close to maximum energy capacity. The p2rx3 knockout animals are a unique model for the search for pharmacological targets that can help correct the energy metabolism of brain cells and eliminate cognitive dysfunctions.

High-grade gliomas are among the most aggressive malignant pathologies of the brain. The high invasive potential of tumor cells causes relapses of the disease even after radical resection of the tumor. The signatures of the genes associated with the invasion of glioma cells have now been identified. The expression products of these genes are involved in various signaling pathways, such as cellular protein catabolism, the p53 signaling pathway, transcription dysregulation, and the JAK-STAT signaling pathway. Therefore, they can indirectly modulate the invasive potential of tumor cells. Using RNA interference technology, it is possible to change the expression level of the detected genes and reduce the invasive and proliferative potentials of cancer cells. This review focuses on the use of this technology to influence various links in signaling pathways and, accordingly, the cellular processes associated with the invasion of glioblastoma cells. Furthermore, the review discusses the problems associated with delivering interfering RNAs into cells and ways to solve them.

This paper introduces a test system for the investigation of biotin transport following inactivation of the SLC5A6 gene, which encodes the sodium-dependent multivitamin transporter SLC5A6. The aim was to develop a method for assessing the efficiency of biotin penetration across the cell membrane following inactivation of the SLC5A6 gene and to explore the feasibility of delivering biotin derivatives into cells independent of SLC5A6. The test system is built upon modified HEK293 cell lines with overexpression of the BirA* biotin ligase, with the first line comprising a functional SLC5A6 gene and the second one involving an inactivated version of this gene mimicking impaired biotin transport. This test system was used to investigate the transport of biotin and its two derivatives, namely the biotin conjugate with p-aminophenylalanine (Bio-1) and biotin methyl ester (Bio-2), through the cell membrane. It has been determined that biotin and its methyl ester (Bio-2) can enter cells independently of the SLC5A6 transporter, which points to the presence of alternative transport pathways. The biotin derivative Bio-1, which contains p-aminophenylalanine, is internalized into cells solely through the hSMVT transporter. The novel test system will serve as a tool for investigating the pathways involved in vitamin entry into cells and for developing therapeutic strategies for individuals with mutations in the SLC5A6 gene, as well as other transport-related genes.

Widespread environmental contamination with polychlorinated biphenyls (PCBs) leads to serious health problems for humans and animals. Our main focus should be on studying the negative effects of exposure to medium- and highly chlorinated PCBs in the human body. There is limited information on the impact of low-chlorinated biphenyls containing 1-2 substituents per molecule on the functions of mammalian organs and systems. Under natural conditions, PCBs can undergo bacterial degradation; the resulting compounds belong to a group of secondary pollutants and are considered hazardous to the environment. Because of limited research, the question regarding the impact of mono-substituted chlorobiphenyl congeners, as well as the products of their biotransformation, remains open. In the presented work, the effects of ortho- and meta- substituted monochlorinated biphenyls on the functions of immune system cells and the morphofunctional state of the liver of mammals in vivo are revealed for the first time. PCB 1 and PCB 2 were found to suppress humoral immunity and induce a productive inflammatory response, as well as widespread protein dystrophy with necrotic foci in the liver. The products of a aerobic bacterial transformation of PCB 1 and PCB 2 were shown to not have a negative effect on the mammalian immune system but proved toxic to hepatocytes, although to a lesser extent than the original chlorobiphenyls.

Neuroblastoma is a malignant solid tumor caused by the transformation of neural crest cells. Neuroblastoma predominantly occurs in children and is associated with a poor prognosis. In this regard, the development of novel approaches to neuroblastoma treatment, including combination therapy, is relevant. DNA hypermethylation of neuroblastoma cells indicates that it is possible to use hypomethylating agents in a combination therapy of the disease. In order to identify effective combinations of antitumor drugs, we analyzed the transcriptomic changes that take place in neuroblastoma SH-SY5Y cells after treatment with the hypomethylating agent 5-azacitidine and then experimentally tested the effectiveness of these combinations. Mithramycin A and lonafarnib were the two drugs that, in combination with 5-azacitidine, appeared to exert a synergistic effect on SH-SY5Y cell death. These drugs inhibit the signaling pathway associated with the transcription factor Sp1 and RAS-MAPK signaling pathway, which are activated by 5-azacitidine. An analysis of the signaling pathways also revealed an activation of the signaling pathways associated with neuroblastoma cell differentiation, as well as apoptosis induction, as confirmed by multiplex and confocal microscopy. Hence, by analyzing the changes in the signaling pathways, the mechanisms of cell death and cell adaptation to hypomethylating agents can be understood, and this can be further used to develop novel therapeutic approaches to neuroblastoma therapy.

In eukaryotic cells, the nonsense-mediated decay (NMD) pathway degrades mRNAs with premature stop codons. The coupling between NMD and alternative splicing (AS) generates NMD-sensitive transcripts (NMD targets, NMDTs) that play an important role in the gene expression regulation via the unproductive splicing mechanism. Understanding this mechanism requires proper identification of NMDT-generating AS events. Here, we developed NMDj, a tool for the identification, classification and quantification of NMDTgenerating AS events which does not rely on the best matching transcript partner principle employed by the existing methods. Instead, NMDj uses a set of characteristic introns that discriminate NMDTs from all protein-coding transcripts. The benchmark on simulated RNA-Seq data demonstrated that NMDj allows to quantify NMDT-generating AS events with better precision compared to other existing methods. NMDj represents a generic method suitable for the accurate classification of arbitrarily complex AS events that generate NMDTs. The NMDj pipeline is available through the repository https://github.com/zavilev/NMDj/.

Extracellular vesicles (EVs) are secreted by nearly all mammalian cells and play a major role in intercellular communication via the transport of various active biomolecules. In cancer, pathological EVs contribute to tumor progression by participating in metastasis, angiogenesis, and immune evasion. Recent advancements in EV research have revealed their potential as noninvasive biomarkers. This review addresses the latest advancements in EV isolation and characterization techniques, elucidates the molecular mechanisms underlying EV biogenesis, and examines their functional roles in cancer progression. Furthermore, we discuss emerging strategies that leverage EV profiling and molecular composition analysis, in conjunction with liquid biopsy technologies, offering possible breakthroughs in early cancer diagnosis and treatment monitoring. By synthesizing these insights, this review emphasizes the growing significance of EVs as versatile and powerful diagnostic tools in oncology.

Neurodegenerative disorders classified as synucleinopathies (Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy) are characterized by the accumulation of aberrant α-synuclein aggregates in neurons and glial cells. These diseases manifest clinically several years after the initial formation of pathological protein aggregates in the brain, making early and accurate diagnosis challenging. In recent years, a new method, which is based on real-time quaking-induced conversion (RT-QuIC) of α-synuclein, has been developed and validated. This technology holds great promise as a powerful diagnostic tool for the early and precise identification of synucleinopathies, potentially opening new horizons in the study of neurodegenerative diseases. RT-QuIC detects misfolded α-synuclein aggregates in human physiological fluids by introducing an excess of recombinant α-synuclein, which undergoes conformational conversion in an exponential, prion-like manner. The production of high-quality recombinant α-synuclein is a critical step in the effective application of this method, as protein purity significantly affects the sensitivity and specificity of the assay - key factors in its diagnostic utility. Using a three-step chromatographic purification protocol, we produced recombinant monomeric α-synuclein with a purity exceeding 97% from the periplasmic fraction of bacterial cells. While higher purity increases the assay duration, it also reduces the background signal and permits extended incubation times, which are essential for reliably detecting synucleinopathies with weak RT-QuIC responses, such as the cerebellar subtype of multiple-system atrophy. The data presented support the conclusion that optimized components of the RT-QuIC system will enable an accurate diagnosis of neurodegenerative synucleinopathies.

Red fluorescent proteins (RFPs) are often probes of choice for living tissue microscopy and whole-body imaging. When choosing a specific RFP variant, the priority may be given to the fluorescence brightness, maturation rate, monomericity, excitation/emission wavelengths, and low toxicity, which are rarely combined in an optimal way in a single protein. If additional requirements such as prolonged fluorescence lifetime and/or blinking ability are applied, the available repertoire of probes could dramatically narrow. Since the entire diversity of conventional single-component RFPs belongs to just a few phylogenetic lines (DsRed-, eqFP578- and eqFP611-derived being the major ones), it is not unexpected that their advantageous properties are split between close homologs. In such cases, a systematic mutagenetic analysis focusing on variant-specific amino acid residues can shed light on the origins of the distinctness between related RFPs and may aid in consolidating their strengths in new RFP variants. For instance, the protein FusionRed, despite being efficient in fluorescence labeling thanks to its good monomericity and low cytotoxicity, has undergone considerable loss in fluorescence brightness/lifetime compared to the parental mKate2. In this contribution, we describe a fast-maturing monomeric RFP designed semi-rationally based on the mKate2 and FusionRed templates that outperforms both its parents in terms of molecular brightness, has extended fluorescence lifetime, and displays a spontaneous blinking pattern that is promising for nanoscopy use.

The Pou5f1 gene encodes the Oct4 protein, one of the key transcription factors required for maintaining the pluripotent state of epiblast cells and the viability of germ cells. However, functional genetics provides convincing evidence that Pou5f1 has a broader range of functions in mouse ontogeny, including suppression of atherosclerotic processes. Related studies have primarily focused on the functions of the Oct4 protein, while the regulatory sequences within the Pou5f1 gene have not been considered. In this study, we have developed a genetic model which is based on mouse embryonic stem cells (ESCs) for assessing the roles of the Pou5f1 gene promoter in the transcriptional regulation of neighboring genes within the major histocompatibility complex (MHC) locus. We have demonstrated that deletion of this promoter affects the expression of selected genes within this locus neither in ESCs nor in the trophoblast derivatives of these cells. A notable exception is the Tcf19 gene, which is upregulated upon Pou5f1 promoter deletion and might be associated with the atherosclerosis pathology due to its pro-inflammatory activity. The developed genetic model will pave the way for future studies into the functional contribution of the cis-regulatory association of Pou5f1, Tcf19, and, possibly, other genes with the atherosclerotic phenotype previously reported for mice carrying the Pou5f1 promoter deletion in vascular endothelial and smooth muscle cells.