Vol 12, No 2 (2020)


Fecal Metabolites As Non-Invasive Biomarkers of Gut Diseases

Zhgun E.S., Ilyina E.N.


Recent studies have shown the importance of the human intestinal microbiome in maintaining a healthy gastrointestinal tract, as well as in the development of pathological processes. The intestinal microbiome manifests itself primarily as fecal metabolites. In the past decade, there has been growing interest in studying its composition, which for the most part had to do with the possibility of using the metabolomic analysis in clinical diagnosis. In contrast to the comprehensive description of blood serum, urine, saliva, and cerebrospinal fluid metabolites, data on fecal metabolites is sparse. Despite the instrumental and methodological achievements in the metabolomic analysis in general, the analysis of fecal metabolome remains less well developed, mainly because of the inhomogeneity of its composition and the lack of standardized methods for collecting, processing, and analyzing fecal samples. This review summarizes data on methods for studying and describing various groups of fecal metabolites. It also assesses their potential as tools in the diagnosis of gastrointestinal diseases.

Acta Naturae. 2020;12(2):4-14
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Palette of Luciferases: Natural Biotools for New Applications in Biomedicine

Kotlobay A.A., Kaskova Z.M., Yampolsky I.V.


Optoanalytical methods based on using genetically encoded bioluminescent enzymes, luciferases, allow one to obtain highly sensitive signals, are non-invasive, and require no external irradiation. Bioluminescence is based on the chemical reaction of oxidation of a low-molecular-weight substrate (luciferin) by atmospheric oxygen, which is catalyzed by an enzyme (luciferase). Relaxation of the luciferin oxidation product from its excited state is accompanied by a release of a quantum of light, which can be detected as an analytical signal. The ability to express luciferase genes in various heterological systems and high quantum yields of luminescence reactions have made these tools rather popular in biology and medicine. Among several naturally available luciferases, a few have been found to be useful for practical application. Luciferase size, the wavelength of its luminescence maximum, enzyme thermostability, optimal pH of the reaction, and the need for cofactors are parameters that may differ for luciferases from different groups of organisms, and this fact directly affects the choice of the application area for each enzyme. It is quite important to overview the whole range of currently available luciferases based on their biochemical properties before choosing one bioluminescent probe suitable for a specific application.

Acta Naturae. 2020;12(2):15-27
pages 15-27 views

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts

Markina N.M., Kotlobay A.A., Tsarkova A.S.


Heterologous pathways are linked series of biochemical reactions occurring in a host organism after the introduction of foreign genes. Incorporation of metabolic pathways into host organisms is a major strategy used to increase the production of valuable secondary metabolites. Unfortunately, simple introduction of the pathway genes into the heterologous host in most cases does not result in successful heterologous expression. Extensive modification of heterologous genes and the corresponding enzymes on many different levels is required to achieve high target metabolite production rates. This review summarizes the essential techniques used to create heterologous biochemical pathways, with a focus on the key challenges arising in the process and the major strategies for overcoming them.

Acta Naturae. 2020;12(2):28-39
pages 28-39 views

The Molecular and Cellular Mechanisms Associated with a Microvascular Inflammation in the Pathogenesis of Heart Failure with Preserved Ejection Fraction

Ovchinnikov A.G., Arefieva T.I., Potekhina A.V., Filatova A.Y., Ageev F.T., Boytsov S.А.


Heart failure with preserved ejection fraction (HFpEF) is a severe disease with an often unfavorable outcome. The prevalence of HFpEF continues to increase, while effective treatment options remain elusive. All the medical strategies used to improve the outcome in a heart failure with reduced ejection fraction proved ineffective in HFpEF, which was probably due to the different mechanisms of development of these two types of heart failure and the diversity of the HFpEF phenotypes. According to the current paradigm of HFpEF development, a chronic mild pro-inflammatory state causes a coronary microvascular endothelial inflammation, with further myocardial fibrosis and diastolic dysfunction progression. This inflammatory paradigm of HFpEF has been confirmed with some evidence, and suppressing the inflammation may become a novel strategy for treating and managing HFpEF. This review summarizes current concepts about a microvascular inflammation in hypertrophied myocardium and provides a translational perspective of the anti-inflammatory and immunomodulatory approaches in HFpEF.

Acta Naturae. 2020;12(2):40-51
pages 40-51 views

Molecular Mechanisms and Clinical Manifestations of Catecholamine Dysfunction in the Eye in Parkinson’s Disease As a Basis for Developing Early Diagnosis

Pavlenko T.А., Chesnokova N.B., Nodel M.R., Kim A.R., Ugrumov M.V.


This review provides information on the non-motor peripheral manifestations of Parkinson’s disease (PD) associated with a pathology of the visual analyzer and the auxiliary apparatus of the eye. The relationship between neurodegenerative processes that take place in the brain and in the eye opens new prospects to use preventive ophthalmologic examination to diagnose PD long before the characteristic motor symptoms appear. This will encourage the use of neuroprotective therapy, which stops, or at least slows down, neuronal death, instead of the current replacement therapy with dopamine agonists. An important result of an eye examination of patients with PD may be a non-invasive identification of new peripheral biomarkers manifesting themselves as changes in the composition of the lacrimal fluid.

Acta Naturae. 2020;12(2):52-62
pages 52-62 views

Research Articles

A New Solid-Phase Immunosorbent for Selective Binding of Desmoglein 3 Autoantibodies in Patients with Pemphigus Vulgaris

Abramova T.V., Spilevaya M.V., Kubanov A.A.


Autoantibodies, immunoglobulins G (IgG) against the desmosomal proteins desmogleins 1 and 3, play a significant role in the pathogenesis of pemphigus vulgaris. The basic therapy for pemfigus includes systemic corticosteroids, but their use should be as brief as possible because of the severe side effects. In cases of corticosteroid-resistant pemfigus, adjuvant therapy, in particular extracorporeal methods, is used. The most effective and safest extracorporeal therapy is immunosorbtion. Immunosorbtion is based on the removal of pemphigus antibodies from the blood using an affinity sorbent during a therapeutic apheresis procedure. Existing immunosorbents are nonselective and increase the risk of infection. We designed an immunosorbent based on an agarose matrix, Affi-Gel 15, and human recombinant desmoglein 3, as a ligand, for a selective removal of autoantibodies from pemphigus patients’ sera. It was shown on a pemphigus experimental model in vivo (neonatal Balb/c mouse model) and in vitro that the immunosorbent can effectively remove desmoglein 3-associated autoantibodies. The experimental results demonstrate that the solid-phase matrix immunosorbent Affi-Gel 15–Dsg3 is a promising product for the development of pemphigus therapy.

Acta Naturae. 2020;12(2):63-69
pages 63-69 views

Rare Cases of IDH1 Mutations in Spinal Cord Astrocytomas

Konovalov N.A., Asyutin D.S., Shayhaev E.G., Kaprovoy S.V., Timonin S.Y.


A low occurrence rate of spinal cord gliomas (4.3% of primary and glial CNS tumors) and the associated difficulties in building statistically significant cohorts of patients considerably slow down the development of effective approaches to the treatment of spinal cord tumors compared to brain tumors. Despite our extensive knowledge regarding IDH mutations in intracranial tumors, mutations of this gene in spinal cord astrocytomas remain poorly understood. In this study, we report on five cases of identified mutations in the IDH1 gene in spinal cord astrocytoma cells, two of which are unique, as they have never been previously described in CNS gliomas.

Acta Naturae. 2020;12(2):70-73
pages 70-73 views

Effect of the Substrate Structure and Metal Ions on the Hydrolysis of Undamaged RNA by Human AP Endonuclease APE1

Kuznetsova A.A., Novopashina D.S., Fedorova O.S., Kuznetsov N.A.


Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyze the phosphodiester bond on the 5′-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidine–purine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.

Acta Naturae. 2020;12(2):74-85
pages 74-85 views

Comparative Analysis of the Effects of Upconversion Nanoparticles on Normal and Tumor Brain Cells

Mishchenko T.A., Mitroshina Е.V., Smyshlyaeva А.S., Guryev Е.L., Vedunova М.V.


Glioma is the most aggressive type of brain tumors encountered in medical practice. The high frequency of diagnosed cases and risk of metastasis, the low efficiency of traditional therapy, and the usually unfavorable prognosis for patients dictate the need to develop alternative or combined approaches for an early diagnosis and treatment of this pathology. High expectations are placed on the use of upconversion nanoparticles (UCNPs). In this study, we have produced and characterized UCNPs doped with the rare-earth elements ytterbium and thulium. Our UCNPs had photoluminescence emission maxima in the visible and infrared spectral regions, which allow for deep optical imaging of tumor cells in the brain. Moreover, we evaluated the toxicity effects of our UCNPs on a normal brain and glioma cells. It was revealed that our UCNPs are non-toxic to glioma cells but have a moderate cytotoxic effect on primary neuronal cultures at high concentrations, a condition that is characterized by a decreased cellular viability and changes in the functional metabolic activity of neuron-glial networks. Despite the great potential associated with the use of these UCNPs as fluorescent markers, there is a need for further studies on the rate of the UCNPs accumulation and excretion in normal and tumor brain cells, and the use of their surface modifications in order to reduce their cytotoxic effects.

Acta Naturae. 2020;12(2):86-94
pages 86-94 views

Recombinant Bispecific Antibodies to the Human ErbB2 Receptor and Interferon-Beta

Panina A.A., Rybchenko V.S., Solopova O.N., Balabashin D.S., Yakimov S.A., Aliev T.K., Dolgikh D.A., Sveshnikov P.G., Kirpichnikov M.P.


The development of and research into new therapies that can selectively and effectively destroy tumor cells that overexpress the ErbB2 receptor is a pressing task. Recently, research into the use of type I interferons in the treatment of cancer has intensified. Cytokine therapy is aimed at activating the cells of the immune system to fight tumors, but it has drawbacks that limit its use because of a number of side effects the severity of which varies depending on the dosage and type of used cytokine. At the moment, a number of studies are being conducted regarding the use of IFNβ in oncology. The studies are aimed at mitigating the systemic action of this cytokine. The immunocytokine complex made of a bispecific antibody against the ErbB2 receptor and recombinant IFNβ developed in this study underlies the mechanism meant to avoid the systemic action of this cytokine. Part of this study focuses on the development of full-length antibodies that bind to the ErbB2 receptor on the one hand, and bind and neutralize IFNβ, on the other hand, which allows us to consider the antibodies as a means of cytokine delivery to tumor cells.

Acta Naturae. 2020;12(2):95-104
pages 95-104 views

The SNCA-Rep1 Polymorphic Locus: Association with the Risk of Parkinson’s Disease and SNCA Gene Methylation

Iakovenko E.V., Abramycheva N.Y., Fedotova E.Y., Illarioshkin S.N.


Neurodegeneration in Parkinson’s disease is characterized by the accumulation of alpha-synuclein, a protein encoded by the SNCA gene, in neurons. In addition to mutations, many polymorphisms have been identified in this gene, and one of these is a dinucleotide microsatellite: SNCA-Rep1. The mechanisms by which specific configurations of SNCA-Rep1 may contribute to the development of this disease have yet to be clarified. In our study, a relationship between long SNCA-Rep1 alleles and Parkinson’s was confirmed in the Russian population. Long allelic variants of SNCA-Rep1 were shown to be associated with the hypomethylation of the CpG-sites in intron 1 of the SNCA gene. Long variants of SNCA-Rep1 are supposed to exert their effect through the hypomethylation of a transcriptionally significant region of this gene. Hypomethylation is usually associated with increased expression, which, in turn, contributes to alpha-synuclein accumulation in neuronal cytoplasm, with the latter being the main molecular marker of Parkinson’s disease. Further studies are needed to establish a relationship between our finding and SNCA gene expression.

Acta Naturae. 2020;12(2):105-110
pages 105-110 views

Short communications

ASIC1a Inhibitor mambalgin-2 Suppresses the Growth of Leukemia Cells by Cell Cycle Arrest

Bychkov M.L., Shulepko M.A., Vasileva V.Y., Sudarikova A.V., Kirpichnikov M.P., Lyukmanova E.N.


Although tyrosine kinase inhibitors have brought significant success in the treatment of chronic myelogenous leukemia, the search for novel molecular targets for the treatment of this disease remains relevant. Earlier, expression of acid-sensing ion channels, ASIC1a, was demonstrated in the chronic myelogenous leukemia K562 cells. Three-finger toxins from the black mamba (Dendroaspis polylepis) venom, mambalgins, have been shown to efficiently inhibit homo- and heteromeric channels containing the ASIC1a subunit; however, their use as possible antitumor agents had not been examined. In this work, using the patch-clamp technique, we detected, for the first time, an activation of ASIC1a channels in the leukemia K562 cells in response to an extracellular pH decrease. Recombinant mambalgin-2 was shown to inhibit ASIC1a activity and suppress the proliferation of the K562 cells with a half-maximal effective concentration (EC50) ~ 0.2 μM. Maximum mambalgin-2 inhibitory effect is achieved after 72 h of incubation with cells and when the pH of the cell medium reaches ~ 6.6. In the K562 cells, mambalgin-2 caused arrest of the cell cycle in the G1 phase and reduced the phosphorylation of G1 cell cycle phase regulators: cyclin D1 and cyclin-dependent kinase CDK4, without affecting the activity of CDK6 kinase. Thus, recombinant mambalgin-2 can be considered a prototype of a new type of drugs for the treatment of chronic myelogenous leukemia.

Acta Naturae. 2020;12(2):111-116
pages 111-116 views

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