bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiated approach to the pharmacotherapy of autism spectrum disorders: biochemical aspects
Authors: Boksha I. S , Prokhorova T. A , Savushkina O. K , Tereshkina E. B , Burbaeva G. Sh
Published: April 2024

Autism Spectrum Disorders (ASD) are highly heterogeneous neurodevelopmental disorders caused by a complex interaction of numerous genetic and environmental factors and leading to deviations in the nervous system formation at very early developmental stages. Currently, there are no accepted pharmacological treatments for so-called core symptoms of ASD, such as social communication disorders and restricted and repetitive behavior patterns. The lack of knowledge about biological basis of ASD, the absence of clinically significant biochemical parameters reflecting abnormalities in signaling molecular cascades controlling the nervous system development and functioning, and the lack of methods for selection of clinically and biologically homogeneous subgroups are considered among the causes for the failure of clinical trials of ASD pharmacotherapy. This review considers the possibilities of applying a differentiated clinical and biological approach to the targeted search for ASD pharmacotherapy with an emphasis on biochemical markers associated with ASD and attempts to stratify patients by biochemical parameters. The use of approach such as “target-oriented therapy and assessment of the status of the target before and during the treatment to identify patients with a positive response to treatment” is discussed using the published results of clinical trials as examples. It is concluded that the identification of biochemical parameters for the identification of distinct subgroups among ASD patients requires research on large samples reflecting the clinical and biological diversity of patients with ASD, and the use of unified approaches for such studies. An integrated approach, including clinical observation, clinical-psychological assessment of patient behavior, study of anamnesis and description of individual molecular profiles should become a new strategy for stratifying and subgrouping patients with ASD for clinical pharmacotherapeutic trials, as well as for evaluating its efficiency.
The proteasome interactome and its role in the mechanisms of brain plasticity
Authors: Buneeva O. A , Kopylov A. T , Medvedev A. E
Published: April 2024

Proteasomes are highly conserved multienzyme complexes responsible for the proteolytic degradation of short-lived, regulatory, misfolded, and damaged proteins. They play an important role in the processes of brain plasticity, and the decrease in their function is accompanied by the development of neurodegenerative pathology. Studies performed in different laboratories both on cultured mammalian and human cells and on preparations of the rat and rabbit brain cortex revealed a large number of proteasome-associated proteins. Since the identified proteins belong to certain metabolic pathways, multiple enrichment of the proteasome fraction with these proteins indicates their important role the proteasome functioning. Extrapolation of the experimental data, obtained on various biological objects, to the human brain, suggests that proteasome-associated proteins account for at least 28% of the human brain proteome. The proteasome interactome of the brain contains a large number of proteins involved in the assembly of these supramolecular complexes, regulation of their functioning, and intracellular localization, which can be changed at different conditions (for example, during oxidative stress) or in different phases of the cell cycle. In the context of the molecular functions of the Gene Ontology (GO) Pathways, the proteins of the proteasome interactome mediate cross-talk between components of more than 30 metabolic pathways annotated in terms of GO. The main result of these interactions is the binding of adenine and guanine nucleotides, crucial for realization of the nucleotide-dependent functions of the 26S and 20S proteasomes. Since the development of neurodegenerative pathology is often associated with a regioselective decrease in the functional activity of proteasomes, a positive therapeutic effect will obviously be provided by factors increasing the proteasomal activity. In any case, the pharmacological regulation of brain proteasomes seems to be realized through changes in the composition and/or activity of proteins associated with proteasomes (deubiquitinase, PKA, CaMKIIα, etc.).
The role of lipid in the regulation of neuroglial interactions
Authors: Galkina O. V , Vetrovoy O. V , Krasovskaya I. E , Eschenko N. D
Published: April 2024

Lipids are an extremely heterogeneous group of compounds, resulting in a wide variety of biological functions they perform. The traditional view of lipids as important structural components of the cell and compounds playing a trophic role is currently being supplemented by information on the possible participation of lipids in signaling, not only intracellular, but also intercellular. The review article discusses current data on the role of lipids and their metabolites formed in glial cells (astrocytes, oligodendrocytes, microglia) in the communication of these cells with neurons. In addition to the metabolic transformations of lipids in each type of glial cells, special attention is paid to the lipid signal molecules (phosphatidic acid, arachidonic acid and its metabolites, cholesterol, etc.) and the possibility of their participation in the implementation of the synaptic plasticity, as well as in other possible mechanisms associated with the realization of the neuroplasticity. The generalization of these new data can significantly expand knowledge about the regulatory functions of lipids in neuroglial relationships.
Sympathetic innervation and endogenous catecholamines in neuromuscular preparations of muscles of different functional profiles
Authors: Dmitrieva S. A , Vologin S. G , Tsentsevitsky A. N , Arkhipov A. Y , Khuzakhmetova V. F , Sibgatullina G. V , Bukharaeva E. A
Published: April 2024

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and β adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in the modulation of the synaptic function.
Genetic architecture of Parkinson’s disease
Authors: Shadrina M. I , Slominsky P. A
Published: April 2024

Year 2022 marks the 25th anniversary of the first mutation in familial autosomal dominant Parkinson’s disease. Over the years, our understanding of the role of genetics in the pathogenesis of familial and idiopathic forms of Parkinson’s disease has expanded significantly - a number of genes for the familial form of the disease have been identified, and DNA markers of an increased risk of developing a sporadic form of the disease have been identified. But, despite all the successes achieved, we are far from an accurate assessment of the contribution to the development of the disease as a whole, both genetic factors and (and even more so) epigenetic factors. The review summarizes the information accumulated to date on the genetic architecture of Parkinson’s disease and formulates issues that need to be addressed and are primarily related to the assessment of epigenetic factors in the pathogenesis of the disease.
Neuronal exosomes as a new signaling system
Authors: Yakovlev A. A
Published: April 2024

Every year more and more works are devoted to the study of neuronal exosomes. The potential of exosomes as diagnostic markers for neurodegenerative diseases has been significantly explored., and similar marker search patterns have been adopted for the study of psychiatric pathologies. The fundamentals of exosome biogenesis in different cell types have been elucidated., the physiological significance of exosomes is being actively studied., and many aspects of signaling with their participation are being elucidated. At the same time., data have been accumulated pointing to the role of exosomal signaling as an important element of interneuronal communication. Do we have enough evidence to call exosomes a new non-canonical neurotransmitter in the brain? This discussion work is devoted to answering this question., in which the author presents to the scientific community the concept of the possible role of brain exosomes as a signaling system.
Brain CoA and acetyl CoA system in mechanisms of neurodegeneration
Authors: Moiseenok A. G , Kanunnikova N. P
Published: April 2024

The processes of biotransformation of pantothenic acid (Pan) in the biosynthesis and hydrolysis of CoA, the key role of pantothenate kinase (PANK) and CoA synthetase (CoASY) in the formation of the priority mitochondrial pool of CoA, with a high metabolic turnover of the coenzyme and limited transport of Pan across the blood-brain barrier are considered. The system of acetyl-CoA, a secondary messenger, the main substrate of acetylation processes, including the formation of N-acetylaspartate and acetylcholine, post-translational modification of histones, determines the protection of neurons from degenerative signals and cholinergic neurotransmission. The biochemical mechanisms of neurodegenerative syndromes in PANK and CoASY defects and the possibility of correcting the development of CoA biosynthesis in knockout models for these enzymes are described. The data of a post-mortem study of the brain of patients with Huntington’s and Alzheimer’s diseases are presented, proving Pan deficiency in the CNS, which is especially pronounced in pathognomonic neurostructures. In the frontal cortex of patients with Parkinson’s disease, combined immunofluorescence of anti-CoA- and anti-tau protein was detected, reflecting CoAlation during dimerization of the tau protein and its redox sensitivity. The redox activity and antioxidant properties of the precursors of CoA biosynthesis were confirmed in vitro on synaptosomal membranes and mitochondria in the modeling of aluminum neurotoxicosis, accompanied by a decrease in the level of CoA in the CNS. The ability of CoA biosynthesis precursors to stabilize the glutathione pool in neurostructures, in particular, in the hippocampus, is considered as a pathogenetic protection mechanism when exposed to neurotoxins, the development of neuroinflammation and neurodegeneration, and justifies the combined use of Pan derivatives (for example, D-panthenol) and glutathione precursors (N-acetylcysteine). Taking into account the discovery of new functions of CoA - redox-dependent processes of CoAlation of proteins, the possible association of oxidative stress and deficiency of Pan (CoA) in neurodegenerative pathology, the study of the bioavailability and biotransformation of Pan derivatives, in particular, D-panthenol, 4′-phospho-pantetheine, its acylated derivatives and compositions with redox pharmacological compounds are promising as potential etiopathogenetic agents.
Pathogenetic mechanisms of mental disorders: endogenous intoxication
Authors: Uzbekov M. G
Published: April 2024

There is described the existence of the syndrome of endogenous intoxication in patients with mental disorders. Oxidative stress, middle-mass endotoxic molecules, disorders in functional properties of the blood albumin and the thiols of the albumin, disturbances in the state of the neurotrophic factors and in the activity of several enzymes, including monoamine oxidase and semicarbazide-sensitive amine oxidase, contribute to the development of the endogenous intoxication. We introduce the scheme of the possible pathogenetic mechanisms of the development of the endogenous intoxication and the ways of its overcoming.
Activation of embryonic gene transcription in neural precursor cells derived from induced pluripotent stem cells of patients with Parkinson’s disease
Authors: Fedoseyeva V. B , Novosadova E. V , Nenasheva V. V , Novosadova L. V , Grivennikov I. A , Tarantul V. Z
Published: April 2024

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases in the world. Despite numerous studies, the causes of this pathology remain completely unknown. This is, among other things, due to the difficulty of obtaining biological material for analysis. Neural cell cultures derived from induced pluripotent stem cells (iPSCs) serve as a great potential for studying the molecular events underlying the pathogenesis of PD. This paper presents the results of bioinformatic analysis of data obtained using RNA-seq technology in the study of neural precursors (NP) obtained from iPSCs of healthy donors and patients with PD carrying various mutations that are a common cause of familial PD. This analysis showed that in NP cells obtained from PD patients, unlike healthy donors, the level of transcription of genes actively expressed in the nervous system at the embryonic stage of development was significantly increased. Bioinformatic data are generally confirmed by real-time PCR. The data obtained suggest that one of the causes of PD may be an abnormal process of enhancing the expression of embryonic genes during differentiation of neural cells (dematuration).
Recombinant human cyclophilin A in combination with adoptive T cell therapy improves the efficacy of cancer immunotherapy in experimental models in vivot;
Authors: Kalinina A. A , Kazansky D. B , Khromykh L. M
Published: April 2024

Adoptive T-cell therapy (ACT) is successfully applied in cancer treatment, however, its efficiency can be limited by a low viability, short persistence time, and loss of functional activity of T cells after adoptive transfer. The search for novel immunomodulators that can improve the viability, expansion, and functions of T cells after their infusion with the minimal side effects could contribute to the development of more efficient and safe ACT strategies. Recombinant human cyclophilin A (rhCypA) is of particular interest in this respect, as it exhibits pleiotropic immunomodulatory activity and stimulates both innate and adaptive anti-tumor immunity. Here, we evaluated the effect of rhCypA on the efficacy of ACT in the mouse EL4 lymphoma model. Lymphocytes from transgenic 1D1a mice with an inborn pool of EL4-specific T cells were used as a source of tumor-specific T cells for ACT. In models of immunocompetent and immunodeficient transgenic mice, the course (3 days) rhCypA administration was shown to significantly stimulate EL4 rejection and prolong the overall survival of tumor-bearing mice after adoptive transfer of lowered doses of transgenic 1D1a cells. Our studies showed that rhCypA significantly improved the efficacy of ACT by enhancing the effector functions of tumor-specific cytotoxic T cells. These findings open up the prospects for the development of innovative strategies of adoptive T-cell immunotherapy for cancer using rhCypA as an alternative to existing cytokine therapies.
Prediction of adverse effects of drug-drug interactions on the cardiovascular system based on the analysis of structure-activity relationships
Authors: Sukhachev V. S , Ivanov S. M , Dmitriev A. V
Published: April 2024

The co-administration of drugs often leads to drug-drug interactions, which may be accompanied by various adverse drug reactions that pose a threat to the life and health of patients. The effect caused by adverse drug reactions from the cardiovascular system is one of the most significant manifestations of drug-drug interaction. Clinical assessment of adverse drug reactions resulting from drug-drug interaction between all drug pairs used in therapeutic practice is not possible. The purpose of this work was to build models using structure-activity analysis to predict the effects of adverse drug reactions on the cardiovascular system, mediated by interactions between drug pairs when they are taken together. Data on adverse effects resulting from drug-drug interaction were obtained from the DrugBank database. The data on drug pairs that do not cause the corresponding effects, necessary for building accurate structure-activity models, were obtained from the TwoSides database, which contains the results of the analysis of spontaneous reports. Two types of descriptors were used to describe a pair of drug structures: PoSMNA descriptors and probabilistic estimates of the prediction of biological activities obtained using the PASS program. Structure-activity relationships were established using the Random Forest method. Prediction accuracy was calculated by means of a five-fold cross-validation. The highest accuracy values were obtained using PASS probabilistic estimates as descriptors. The area under the ROC curve was 0.94 for bradycardia, 0.96 for tachycardia, 0.90 for arrhythmia, 0.90 for ECG QT prolongation, 0.91 for hypertension, 0.89 for hypotension.
Biochemical features of X or Y chromosome bearing spermatozoa for sperm sexing
Authors: Pozdyshev D. V , Kombarova N. A , Muronetz V. I
Published: April 2024

This review presents information on the biochemical features of spermatozoa bearing an X or Y chromosome, enabling to obtain a sperm fraction with a pre-defined sex chromosome. Almost the only technology currently used for such separation (called sexing) is based on sperm fluorescence-activated cell sorting in regard to DNA content. Besides its applications, this technology made it possible to analyze the properties of isolated populations of spermatozoa bearing an X or Y chromosome. In recent years, a number of works have appeared proving the existence of differences between these populations at the transcriptome and proteome level. It is noteworthy that these differences are primarily related to energy metabolism and structural proteins of the flagellum. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between spermatozoa with different sex chromosomes. Sperm sexing is a widespread part of the protocol of artificial insemination of cows with cryopreserved semen, it allows to increase the proportion of offspring of the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.
Detection of specific RNA targets by multimerization
Authors: Sakhabutdinova A. R , Chemeris A. V , Garafutdinov R. R
Published: April 2024

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to an essential role of RNA in realization of genetic information and diseases development. Here, we report on an approach for detection of RNA targets based on a particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was approved using genetic material of SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate SARS-CoV-2 RNA-positive samples from SARS-CoV-2 negative samples with high reliability. The proposed technique determines detection of RNA even in samples, which undergone multiple freezing.
Mechanism of D-cycloserine inhibition of D-amino acid transaminase from Haliscomenobacter hydrossis
Authors: Bakunova A. K , Matyuta I. O , Nikolaeva A. Yu , Boyko K. M , Popov V. O , Bezsudnova E. Yu
Published: April 2024

D-cycloserine inhibits pyridoxal-5′-phosphate (PLP)-dependent enzymes. The inhibition efficiency depends on the organization of their active center and the mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of enzyme similarly to substrate amino donor, and the interaction is predominantly reversible. Inhibition products include hydroxyisoxazole-pyridoxamine-5′-phosphate, oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate, etc. For some enzymes the formation of a stable aromatic product - hydroxyisoxazole can lead to irreversible D-cycloserine inhibition at certain pH value. The aim of this work was to study the mechanism of D-cycloserine inhibition of PLP-dependent D-amino acid transaminase from the bacterium Haliscomenobacter hydrossis. Spectral methods revealed several products of the interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5′-phosphate and cyclic or open forms of D-cycloserine, pyridoxamine-5′-phosphate. The formation of hydroxyisoxazole-pyridoxamine-5′-phosphate was not observed. The 3D structure of the complex of transaminase with D-cycloserine was obtained by X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5′-phosphate and D-cycloserine in the cyclic form was found; the ketimine occupied two positions and was coordinated via hydrogen bonds with different active site residues. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and the activity of transaminase from H. hydrossis can be restored by adding an excess of keto substrate as well as by adding an excess of cofactor. The results obtained confirm the reversibility of D-cycloserine inhibition and the conversion of various adducts of D-cycloserine and PLP into each other.
Femtosecond exciton relaxation in chlorosomes of the photosynthetic green bacterium Chloroflexus aurantiacus
Authors: Yakovlev A. G , Taisova A. S , Fetisova Z. G
Published: April 2024

In the green bacteria Chloroflexus (Cfx.) aurantiacus, the process of photosynthesis begins with the absorption of light by chlorosomes, peripheral antennas consisting of thousands of bacteriochlorophyll c (BChl c) molecules combined into oligomeric structures. In this case, excited states are formed in BChl c, the energy of which migrates along the chlorosome towards the baseplate and further to the reaction center, where the primary charge separation occurs. Energy migration is accompanied by nonradiative electronic transitions between numerous exciton states, that is exciton relaxation. In this work, we studied the dynamics of exciton relaxation in Cfx. aurantiacus chlorosomes using difference femtosecond spectroscopy at cryogenic temperature (80 K). Chlorosomes were excited by 20 fs light pulses at wavelengths from 660 to 750 nm, and the difference (light-dark) kinetics were measured at a wavelength of 755 nm. Mathematical analysis of the data obtained revealed the kinetic components with characteristic time constants of 140, 220, and 320 fs responsible for the exciton relaxation. As the excitation wavelength decreased, the number of components and their relative contribution increased. Theoretical modeling of the results obtained was carried out on the base of the cylindrical model of BChl c aggregates. Nonradiative transitions between groups of exciton bands were ascribed by the system of kinetic equations. The model in which the energetic and structural disorder was taken into account turned out to be the most adequate.
Display of oligo-α-1,6-glycosidase from Exiguobacterium sibiricum at the surface of Escherichia coli cells
Authors: Shingarova L. N , Petrovskaya L. E , Kryukova E. A , Gapizov S. S , Dolgikh D. A , Kirpichnikov M. P
Published: April 2024

Cell-surface display based on the use of anchor motifs of outer membrane proteins allows the exposure of target peptides and proteins on the surface of microbial cells. Previously, we obtained and characterized recombinant oligo-α-1,6-glycosidase of the psychrotrophic bacterium Exiguobacterium sibiricum (EsOgl) which demonstrated high catalytic activity. It has also been shown that the autotransporter AT877 from Psychrobacter cryohalolentis and its deletion variants effectively exhibit the 10th domain of type III fibronectin (10Fn3) on the surface of Escherichia coli cells. The aim of the work was to obtain an EsOgl display system on the surface of bacterial cells based on AT877. The genes of the hybrid autotransporter EsOgl877 and its deletion mutants EsOgl877Δ239 and EsOgl877Δ310 were constructed. The enzymatic activity of EsOgl877 was investigated and it was found that the cells expressing this protein retained about 90% of the maximum activity in the range of 15-35°C. It was shown that activity of the cells containing EsOgl877Δ239 and EsOgl877Δ310 was 2.7 and 2.4 times higher, respectively, than of the cells expressing full-sized AT. Analysis of cells expressing shortened variants of EsOgl877 after treatment with proteinase K showed that the passenger domain is also localized on the cell surface. The obtained results can be used for optimization of the display systems of oligo-α-1,6-glycosidase and other heterologous proteins on the surface of E. coli cells.
Recombinant endopeptidases IdeS and IdeZ and their potential applications
Authors: Boksha I. S , Lunin V. G , Danilova T. A , Poponova M. S , Polyakov N. B , Lyashchuk A. M , Konstantinova S. V , Galushkina Z. M , Ustenko E. V
Published: April 2024

IdeS and IdeZ endopeptidases are streptococcal virulence factors that specifically cleave IgG heavy chains and represent an interest from biotechnological, medical, and veterinary points of view. The genes encoding IdeS and IdeZ endopeptidases (the ideS gene was cloned from a Streptococcus pyogenes collection strain, and S. zooepidemicusideZ gene was synthesized) were cloned and expressed in Escherichia coli heterologous expression system, and 6His-tag affinity domain was introduced into the amino acid sequence of each endopeptidase. IdeS and IdeZ enzymes were isolated and purified by metal affinity chromatography. The resulting IdeS and IdeZ are apparently homogeneous on polyacrylamide gel electrophoresis and are active against human and animal IgG. The specificity of human IgG cleavage by endopeptidases IdeS and IdeZ was confirmed by polyacrylamide gel electrophoresis. Application of recombinant IdeZ was demonstrated for immunological analysis of equine “strangles” (diagnostics and specific antibodies’ titer determination in the blood). Thus, IdeZ can be used in veterinary medicine and sanitary microbiology for the diagnosis of infections caused by S. equi and S. zooepidemicus in addition to their applications in medicine and biotechnology.
The role of microRNA in the regulation of cellular responses to hypoxia
Authors: Silina M. V , Dzhalilova D. Sh , Makarova O. V
Published: April 2024

The impact of hypoxia causes changes in the transcription of genes that contribute to the adaptation of cells to a lack of oxygen. The main mechanism regulating the cellular response to hypoxia is the activation of a group of transcription factors of the HIF (Hypoxia-Inducible Factor) family, which include several isoforms and control the expression of more than a thousand genes. HIF activity is regulated at various levels, including by small non-coding RNA molecules called microRNAs (miRNAs). miRNAs regulate the cellular response to hypoxia by influencing the activation of HIF, its degradation, and the translation of proteins dependent on it. At the same time, HIF also affects miRNA biogenesis. Data on the relationship of a particular HIF isoform with miRNA are contradictory, since studies are performed using different cell lines, different types of experimental animals and clinical material, as well as at different oxygen concentrations and different durations of hypoxic exposure. In addition, HIF expression may be affected by the initial resistance of organisms to lack of oxygen, which is not taken into account in studies. This review analyzes data on the effect of hypoxia on the biogenesis and functioning of miRNAs, as well as the effect of microRNAs on mRNAs of genes involved in adaptation to oxygen deficiency. Understanding the mechanisms of the relationship between HIF, hypoxia, and miRNA is necessary to develop new approaches to personalized therapy for diseases accompanied by oxygen deficiency.
Effects of chronic combined treatment with ketanserin and fluoxetine in B6.CBA-D13Mit76C recombinant mice with abnormal 5-НТ1a receptor functional activity
Authors: Tsybko A. S , Kondaurova E. M , Zalivina E. V , Blaginya V. O , Naumenko V. S
Published: April 2024

The recombinant B6.CBA-D13Mit76C mouse strain is characterized by altered sensitivity of 5-HT1A receptors and greater Htr1a gene transcription. Recently, we found that in B6.CBA-D13Mit76C mice, chronic fluoxetine treatment produced pro-depressive effect in a forced swim test. Since 5-HT2A receptor blockade may be beneficial in treatment-resistant depression, we investigated the influence of chronic treatment (14 days, intraperitoneally) with selective 5-HT2A antagonist ketanserin (0.5 mg/kg), fluoxetine (20 mg/kg), or fluoxetine + ketanserin on the behavior, functional activity of 5-HT1A and 5-HT2A receptors, serotonin turnover, and transcription of principal genes of the serotonin system in the brain of B6.CBA-D13Mit76C mice. Ketanserin did not reverse the pro-depressive effect of fluoxetine. Fluoxetine, ketanserin, and fluoxetine + ketanserin decreased functional activity of 5-HT1A receptors and Htr1a gene transcription in the midbrain and hippocampus. Additionally, all the tested drug regimens decreased mRNA levels of Slc6a4 and Maoa in the midbrain. These changes were not accompanied by a significant shift in the levels of serotonin and its metabolite 5-HIAA. Notably, ketanserin upregulated enzymatic activity of tryptophan hydroxylase 2 (TPH2). Thus, despite some benefits (reduced Htr1a, Slc6a4, and Maoa transcription and increased TPH2 activity), prolonged blockade of 5-HT2A receptors failed to ameliorate the adverse effect of fluoxetine during abnormal functioning of 5-HT1A receptors.
Molecular bases of signaling processes regulated by cryptochrome sensory photoreceptors in plants
Authors: Fraikin G. Ya , Belenikina N. S , Rubin A. B
Published: April 2024

The blue-light sensors cryptochromes compose the widespread class of flavoprotein photoreceptors, regulating in plants signaling processes underlying their development, growth, and metabolism. In several algae cryptochromes may act not only as sensory photoreceptors but also as photolyases, catalyzing the repair of UV-induced DNA lesions. Cryptochromes bind FAD as the chromophore in the photolyase homologous region (PHR) domain and contain the cryptochrome C-terminal extension (CCE), which lacks in photolyases. Photosensory process in cryptochrome is initiated by photochemical chromophore conversions, including the formation of FAD redox forms. In a state with the chromophore reduced to neutral radical (FADH•), photoreceptory protein undergoes phosphorylation, conformational changes, and disengagement of the PHR domain and CCE with the subsequent formation of oligomers of cryptochrome molecules. Photooligomerization is a structural basis of functional activity of cryptochromes, which determines the formation of their complexes with variety of signaling proteins, including transcriptional factors and transcription regulators. Interactions in such complexes change the protein signaling activity, leading to gene expression regulation and plant photomorphogenesis. In recent years, multiple papers, reporting novel, more detailed information about molecular mechanisms of above-mentioned processes were published. The present review largely focuses on the analysis of data contained in these publications, particularly regarding structural aspects of cryptochrome transitions into photoactivated states and regulatory signaling processes mediated by cryptochrome photoreceptors in plants.
Multiple non-canonical base-stacking interactions as one of the major determinants of RNA tertiary structure organization
Authors: Metelev V. G , Baulin E. F , Bogdanov A. A
Published: April 2024

Interplane (stacking) interactions of heterocyclic bases of nucleotide residues (n.t.) of RNA are one of the most important factors in the organization of its secondary and tertiary structure. Most of these (canonical) interactions are carried out between neighbors in the polynucleotide chains of RNA. However, with the accumulation of data on the atomic tertiary structures of a wide variety of RNAs and their complexes with proteins, it became clear that RNA nucleotide residues that are not neighbors in their polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of n.t. can interact with the help of base stacking (non-canonical). This paper presents an exhaustive database of such elements and their environment in the macromolecules of natural and synthetic RNAs. They were called nonadjacent base-stacking elements (NA-BSE). The analysis of these data showed that the NA-BSE forming nucleotides, on average, account for about a quarter of all nucleotides of a particular RNA, therefore, they should be considered as real motifs in their tertiary structure. The classification of NA-BSE by types of localization in RNA macromolecules is carried out. It is shown that the structure-forming role of NA-BSE consists in the compact folding of single-stranded RNA loops, in the transformation of double-stranded bulges into imperfect helices, as well as in the binding of RNA regions removed in their primary and secondary structure.
Lipoxygenase in a giant sulfur bacterium: an evolutionary solution for size and complexity?
Authors: Kurakin G. F
Published: April 2024

Discovery of Thiomargarita magnifica - an exceptionally large giant sulfur bacterium - urges us to pay additional attention to the giant sulfur bacteria and to revisit our recent bioinformatic finding of lipoxygenases in the representatives of the genus Beggiatoa. These close relatives of Thiomargarita magnifica meet the similar size requirements by forming multicellular structures. We hypothesize that their lipoxygenases are a part of the oxylipin signaling system that provides high level of cell-to-cell signaling complexity which, in turn, enables them to reach large sizes.
Expression, intracellular localization and maturation of cysteine cathepsins in renal embryonic and cancer cell lines
Authors: Frolova A. S , Tikhomirova N. K , Kireev I. I , Zernii E. Yu , Parodi A. , Ivanov K. I , Zamyatnin A. A
Published: April 2024

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.
Superoxide anion radical generation in photosynthetic electron transport chain
Authors: Kozuleva M. A , Ivanov B. N
Published: April 2024

The review analyzes the data available in the literature on the rates, characteristics and mechanisms of oxygen reduction to a superoxide anion radical at the sites of the photosynthetic electron transport chain where this reduction can occur. The existing assumptions about the role of the components of these sites in this process are critically examined, with use of thermodynamic approaches and the results of recent studies. The process of O2 reduction at the acceptor side of PSI, which is considered the main place of this process in the photosynthetic chain, is described in detail. The evolution of the photosynthetic apparatus in the context of controlling the leakage of electrons to O2 is considered. The reasons limiting the application of the results obtained with isolated segments of the photosynthetic chain to estimate the rates of O2 reduction at the corresponding sites in the intact thylakoid membrane are discussed.
Analysis of transferrin in urine in patients with bladder cancer using nanobodies
Authors: Sachko A. M , Goryainova O. S , Ivanova T. I , Nikolaeva I. Yu , Tarnopolskaia M. E , Bychkov A. Yu , Gaas M. Ya , Vorob'ev N. V , Kaprin A. D
Published: April 2024

It is known that the saturation ratio of transferrin (Tf) with iron in human blood is an important clinical parameter. Specific antibodies can be used to analyze subtle changes in the relative abundance of different forms of transferrin potentially associated with a pathological process. Recently, the authors of this study were able to obtain and characterize highly specific single-domain antibodies (nanobodies) that predominantly recognize the iron-saturated (holo-Tf) or iron-unsaturated (apo-Tf) form of transferrin. In this work, under conditions closer to physiological than in previous experiments, we further demonstrated that these unique nanobodies have extremely high differential binding specificity for different forms of Tf in different human biological fluids. Using these nanobodies, we were able to analyze for the first time the relative abundance of transferrin forms in urine samples from patients with bladder cancer (BC). We have shown that an increase in the concentration of total Tf in urine samples normalized for creatinine is associated with the degree of progress and growth of malignancy of BC. In the samples of healthy donors and in the early stages of BC (G1), Tf is detected in much smaller amounts (compared to later stages) and only with additional concentration of the studied samples. For most of the studied urine samples from BC patients, it is expected (as previously shown in the case of Tf in the blood of terminal ovarian cancer patients) that the concentration of apo-Tf is clearly higher than holo-Tf, especially in the case of the most advanced muscle-invasive BC. It was a surprise for us that approximately equal amounts of apo-Tf and “holo-Tf” were found in the urine samples of some patients with BC. We hypothesized that the “holo-Tf” fraction in this case can be largely represented by “secondary complexes” formed by apo-Tf in combination with ions other than Fe3+, which accumulate in the urine of some cancer patients and are able to bind to apo-Tf, changing its conformation towards holo-Tf. Using inductively coupled plasma ionization mass spectroscopy (ICP-MS), we obtained the first results confirming our hypothesis. The “holo-Tf” preparation in these urine samples was found to be highly enriched in zinc and nickel. Also in this preparation, a relative enrichment in cadmium is observed, but it is present in much lower concentrations. The data obtained indicate that the nanobody used, recognizing predominantly the iron-saturated form of transferrin (holo-Tf), is also capable of binding transferrin in association with other metal ions that are different from iron. This ability could potentially open up new possibilities in studies of the relative abundance of various metal ions in association with transferrin in human biological fluids in normal and pathological conditions.
Long-term experimental hyperglycemia does not impair macrovascular endothelial barrier integrity and function in vitro
Authors: Khapchaev A. Y , Antonova O. A , Kazakova O. A , Samsonov M. V , Vorotnikov A. V , Shirinsky V. P
Published: April 2024

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.
Recent developments in bioprocessing of recombinant antibody fragments
Authors: Zelenovic N. , Filipovic L. , Popovic M.
Published: April 2024

Biotechnological and biomedical applications of antibodies have been on a steady rise since the 1980s. As unique and highly specific bioreagents, monoclonal antibodies (mAbs) have been widely exploited and approved as therapeutic agents. However, the use of mAbs has limitations for therapeutic applications. Antibody fragments (AbFs) with preserved antigen-binding sites have a significant potential to overcome the disadvantages of conventional mAbs, such as heterogeneous tissue distribution after systemic administration, especially in solid tumors, and Fc-mediated bystander activation of the immune system. AbFs possess better biodistribution coefficient due to lower molecular weight. They preserve the functional features of mAbs, such as antigen specificity and binding, while at the same time, ensuring much better tissue penetration. An additional benefit of AbFs is the possibility of their production in bacterial and yeast cells due to the small size, more robust structure, and lack of posttranslational modifications. In this review, we described current approaches to the AbF production with recent examples of AbF synthesis in bacterial and yeast expression systems and methods for the production optimization.
Reconstitution of calcium channel protein Orai3 into liposomes for functional studies
Authors: Liang Ch. , Wu F.
Published: April 2024

Store-operated calcium entry (SOCE) is the main mechanism for the Ca2+ influx in non-excitable cells. The two major components of SOCE are stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. SOCE requires interaction between STIM1 and Orai. Mammals have three Orai homologs: Orai1, Orai2, and Orai3. Although Orai1 has been widely studied and proven to be essential for numerous cellular processes, Orai3 has also attracted a significant attention recently. The gating and activation mechanisms of Orai3 have yet to be fully elucidated. Here, we expressed, purified, and reconstituted Orai3 protein into liposomes and investigated its orientation and oligomeric state in the resulting proteoliposomes. STIM1 interacted with the Orai3-containing proteoliposomes and mediated calcium release from them, suggesting that the Orai3 channel was functional and that recombinant STIM1 could directly open the Orai3 channel in vitro. The developed in vitro calcium release system could be used to study the structure, function, and pharmacology of Orai3 channel.
Upregulation of YciM expression reduces endotoxin contamination of recombinant proteins produced in Escherichia coli cells
Authors: Bobrovsky P. A , Kharlampieva D. D , Kirillin S. A , Brovina K. A , Grafskaia E. N , Lazarev V. N , Manuvera V. A
Published: April 2024

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.