[The problem of iodine deficiency in the Chechen republic: assessment of the current state and ways of solution].

Iodine deficiency disorders (IDD) are prevalent and highly morbidity, have hidden progression, severe disabling somatic complications, including cognitive disorders, reproductive losses, and oncopathology. This presents a serious challenge to the healthcare system of the Russian Federation, as it affects over 3 million people. The lack of relevant data on the severity of IDD and the current prevention programs at the regional level necessitates the need for appropriate research and measures in individual subjects of the Russian Federation. AIM: To conduct a comprehensive study to assess the current iodine security of the population of the Chechen Republic, to analyze the prevalence of thyroid pathology and compare it with official statistics, to formulate conclusions about the necessary preventive measures. MATERIALS AND METHODS: In the Chechen Republic, a total of 1239 people were examined, of which 921 were schoolchildren of pre-pubertal age (8-10 years) and 318 were adults. The survey of the adult population was carried out at medical organizations in four districts of the republic (Nadterechny, Shalinsky, Vedensky, Grozny) and included a questionnaire survey, a clinical examination by an endocrinologist with palpation of the thyroid gland, thyroid ultrasound, and a study by a qualitative method of samples of table salt used in households for the presence of iodine.Children's examinations were carried out by the cluster method on the basis of secondary schools in 9 out of 15 districts of the republic and included an examination by an endocrinologist and measurement of anthropometric parameters (height, weight), thyroid ultrasound to evaluate volume, determination of iodine concentration in single portions of urine and qualitative analysis of samples of table salt used in children's nutrition in families for the presence of iodine.The incidence and prevalence of thyroid disease among the population of the Chechen Republic were analyzed using data from official state statistics - form No. 12 «Information on the number of diseases registered in patients living in the area served by the medical institution¼ (ROSSTAT data as of 01.01.2021). RESULTS: According to the results of a survey of 921 pre-pubertal children, the median urinary iodine concentration was 71.3 µg/L (frequency of values below 50µg/L - 17,7%) and varies from 48.9 to 179.2 µg/L in the surveyed areas. According to thyroid ultrasound data, diffuse goiter was detected in 16.4% of the examined children, with goiter frequency ranging from 11.3% to 23.5%. The proportion of iodized salt consumed in schoolchildren's families was 4.2% in all study areas (range of values from 1.3% to 8%), which indicates an extremely low level of using iodized salt by household.According to the results of the examination of the adult population (n=318), structural changes in thyroid tissue were detected in 79.9% (n=254), while the proportion of nodular thyroid pathology being 83% (n=205), with a range of values across different districts of 52.5-80%. CONCLUSION: Based on the obtained data, according to WHO criteria, it can be stated that, overall, the degree of severity of iodine deficiency disorders in the Chechen Republic corresponds to mild severity with a tendency towards moderate severity in several districts of the foothills. The results of the examination of the adult population indicate a high prevalence of thyroid pathology, predominantly nodular, in the Chechen Republic. The data obtained in the course of large-scale research made it possible to initiate the development of necessary medical and organizational measures in the region - a program for the prevention of IDD.

[The development of Graves' disease after long-term hypothyroidism due to Hashimoto's disease].

The main autoimmune thyroid diseases are Hashimoto's thyroiditis (HT) and Graves' disease (GD). Despite the significant differences in a pathogenesis and a clinical picture between HT and GD, the literature describes the cases of the conversion of one autoimmune disease to another, which, according to one version, is associated with a change in the balance between the levels of a stimulating and blocking antibodies to the thyroid-stimulating hormone receptor. At the same time, there are more frequent observations of the transition of GD to HT, and much less often describe, on the contrary, the development of GD against the background of HT. The article presents a clinical case of the conversion of HT to GD. A detailed algorithm of the conservative management according to the «block-replace¼ scheme is described, indicating the results of laboratory and instrumental examination. At the time of describing the clinical case, the result of the treatment can be considered successful. The predictors such as a low level of the thyroid-stimulating hormone receptor and thyroid volume before discontinuation of the thyrostatic therapy suggest a low risk of the recrudescence of GD.According to the authors, the phenomenon of the conversion of one autoimmune thyroid disease to another, in addition to the scientific interest, is important for the practitioners, since a timely change in the diagnostic paradigm can significantly change the treatment strategy and the favorably affect the prognosis of disease, preventing the development of complications.

New Targets of Kunitz-Type Peptide from Sea Anemone Heteractis magnifica.

The interaction of Kunitz-type peptide, HMIQ3c1, from the sea anemone Heteractis magnifica with several serine proteases, including inflammatory proteases, was investigated using the surface plasmon resonance approach. We showed that the recombinant analog of HMIQ3c1 forms sufficiently strong complexes with trypsin (KD = 1.07 × 10-9 М) and chymotrypsin (KD = 4.70 × 10-8 М). Analysis of thermodynamic parameters of HMIQ3c1/chymotrypsin revealed significant contribution of the entropic factor to the complex formation. The formation of specific complexes of HMIQ3c1 with the kallikrein (KD = 2.81 × 10-8 М) and neutrophil elastase (KD = 1.11 × 10-7 М) indicates its anti-inflammatory activity and makes prospects to use the peptide as a potential therapeutic agent.

Inclusion Bodies of Recombinant OmpF Porin from Yersinia pseudotuberculosis: Properties and Structural Characterization.

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.

[10-year risk of fractures (FRAX) in people with diabetes type 2].

AIM: To study indicators of bone mineral densit (BMD) and trabecular bone score (TBS) and to reveal the 10-year fracture risk (FRAX®) taking into account the data obtained in persons with type 2 diabetes (DM2). MATERIALS AND METHODS: A clinical study of the type of case - control. The study included 122 people with and without DM2. All persons were: questionnaires, anthropometry, densitometry, determination of TBS and fracture risk on the FRAX®. RESULTS AND DISCUSSION: Persons with DM2 who underwent a fracture had lower T-score values in all areas except the spine, unlike those with DM2, but without fracture. However, persons with DM2 had a fracture at high values of T-score in vertebrae and hips in comparison with persons without DM. Using the TBS, we did not get a significant difference in any of the examined groups. We also found no differences in the risk of recurrent fractures among women with and without DM2 using FRAX® without densitometry and FRAX® adjusted for TBS. The values of FRAX® by T-score in the group of persons with DM with fractures were significantly lower (p=0.029 for major fractures, p=0.024 for hip fractures) than in persons without DM with fractures. CONCLUSION: Persons with DM2 and fractures have higher BMD values, lower than the FRAX fracture risk values adjusted for the T-score, do not differ significantly in TBS, which determines the difficulties in diagnosis, the need to find additional methods for early diagnosis of increased fracture risk in patients with DM2.

Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization.

The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

[Regulation of Yersinia pseudotuberculosis major porin expression in response to antibiotic stress].

The OmpF porin gene expression in Yersinia pseudotuberculosis in response to antibiotics of two different classes (kanamycin and nalidixic acid) was analyzed using quantitative PCR and a fluorescence reporter system. Both antibiotics downregulated the expression of the ompF gene. The nalidixic acid significantly reduced ompF expression, while kanamycin, for which porins are considered to be an alternative transport route, only slightly reduced the ompF level.

Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin.

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.

Hct-a is a new actinoporin family from the heteractis crispa sea anemone.

Several new actinoporin isoforms with molecular weights of 18995.5 to 19398.7 Da exhibiting a high hemolytic activity were isolated from the tropical sea anemone Heteractis crispa using a combination of liquid chromatography techniques. The actinoporins were demonstrated to occur as mono-, di-, and trimers in aqueous solutions. The sequences of the genes encoding actinoporins were identified, and the amino acid sequences of the new polypeptides belonging to the Hct-A actinoporin family were obtained. The new acinoporins differ in their isoelectric points, the number and localization of charged amino acid residues at the functionally important N-terminal fragment of the molecule, as well as in the charge of a tetrapeptide (amino acid residues 74-77) involved in an electrostatic interaction with the cytoplasmic membrane. A recombinant actinoporin, rHct-A2, with a molecular weight of 19141 Da, pI of 9.64, and hemolytic activity of 4.0 × 104 HU/mg, was obtained. The conductivity of the ion channels formed by rHct-A2 in the BLM was demonstrated to be similar to that of the native actinoporin from H. crispa. The obtained data expand knowledge on the structural and functional relationships of actinoporins and contribute to our understanding of the functioning mechanism of these molecules, which is the basis for the development of compounds with a high biomedical potential. Currently, they are considered as models for obtaining antitumor, antibacterial, and cardiac-stimulating agents.

OmpC-like porin from outer membrane of Yersinia enterocolitica: molecular structure and functional activity.

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the "warm" variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the "cold" variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 ß-strands connected by short "periplasmic" and longer "extracellular" loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the "extracellular" loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.

Detection and genotyping of Helicobacter pylori gene vacA in children with gastroduodenal diseases and in adults with gastric cancer in Vladivostok.

Geographical distribution of individual genotypes of Helicobacter pylori, predominance of virulent types in various regions of Russia, particularly in the Prymorye Territory, remains unclear. We examined 115 children with various gastroduodenal pathology and 33 patients with gastric cancer, of which 57.39 and 60% respectively were infected with H. pylori. All samples positive for H. pylori were further analyzed for gene vacA mosaicism. In all clinical subgroups, variants s1 and m1 predominated; the frequency of genotype s1 was significantly increased (1.3-fold) in the group of cancer patients in comparison with the group of children with gastroduodenal pathology. Three variants of allele combination of signaling and middle regions of the vacA gene (s1m1, s1m2, and s2m2) were revealed; s1m1 was the most frequent in both groups. We suggest that this genotype is a marker of complicated course of gastroduodenitis and a factor of gastric cancer development in local population.

Molecular cloning, isolation, and properties of chaperone Skp from Yersinia pseudotuberculosis.

The skp gene of Yersinia pseudotuberculosis was expressed without its signal sequence in Escherichia coli BL21(DE3) cells. The recombinant protein Skp accumulated in soluble form in the cytoplasm of the producer strain. The protein was isolated and characterized: the molecular weight, isoelectric point, N-terminal amino acid sequence (20 amino acid residues), and the content of the secondary structure elements were determined. Using cross-linking stabilization and high-mass MALDI-TOF mass spectrometric analysis, it was found that rSkp forms a stable homotrimer in solution and interacts with human IgG. Three-dimensional models of the Skp trimer and its complexes with Fc- and Fab-fragments of human IgG1 were constructed by computer modeling.

[Polymorphism of GSTT1 and GSNM1 glutathione transferase genes in smokers and patients at the early stages of chronic obstructive pulmonary disease].

We studied the distribution of deletion polymorphisms of GSTT1 and GSTM1 glutathione transferase genes by PCR in patients with stage I and II chronic obstructive pulmonary disease (COPD) and in smokers without COPD. The significance of the differences in allele and genotype distribution between the groups was estimated by the X2 test and BIOSTAT software package. The study revealed a significant rise in the frequency of deletion polymorphisms of GSTT1 transferase genes in patients with stage I COPD compared with controls. The odd ratio between the groups was 0.6 which suggests a two-fold decrease in the risk of COPD. Simultaneous increase in the frequency of "zero" GSTT1 genotype in patients with stage 2 COPD is indicative of rapid progress of the disease in the presence of homozygous deletion of GSTT1. The difference between the frequency of homozygous deletion of GSTM1 was insignificant. The "zero" GSTT1 genotype in heavy smokers was associated with a decreased risk of COPD and may be regarded as a marker of the diminished impact of smoking on the pulmonary function. The homozygous deletion of GSTT1 in patients at the early stages of COPD suggests the risk of its rapid progress. Deletion polymorphism of GSTT1 glutathione transferase gene is recommended to use as a marker for predictive diagnostics of development and progress of COPD.

[Yersinia pseudotuberculosis mutant OmpF porins with deletions of the external loops: genetic constructions design, expression, isolation and refolding].

Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Mutant proteins in oligomeric form were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was demonstrated to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. Lacking of the loops L1, L6 and L8 didn't affect the conductivity level of Y pseudotuberculosis porin channel as shown using bilayer lipid membrane (BLM) technique. Lacking of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated with use of immunoblotting technique and ELISA.

[Interaction of sea amemone Heteractis crispa Kunitz type polypeptides with pain vanilloid receptor TRPV1: in silico investigation].

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz proteinase inhibitor family. Identified amino acid sequences have point residue substitutions, high degree of homology with sequences of known H. crispa Kunitz family members, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by homologous modeling methods. Analysis of their molecular electrostatic potential enabled us to divide given polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2 and APHC3, which were earlier shown to possess a unique property of inhibiting of the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo in addition to their trypsin inhibitory activity. Molecular docking made possible establishing the spatial structure of the complexes, the nature of the polypeptides binding with TRPV1, as well as functionally important structural elements involved in the complex formation. Structural models have enabled us to propose a hypothesis contributing to understanding the APHC1-3 impact mechanism for the pain signals transduction by TRPV1: apparently, there is an increase of the receptor relaxation time resulted in binding of its two chains with the polypeptide molecule, which disrupt the functioning of the TRPV1 and leads to partial inhibition of signal transduction in electrophysiological experiments.

A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability.

A novel OmpY porin was predicted based on the Yersinia pseudotuberculosis genome analysis. Whereas it has the different genomic annotation such as "outer membrane protein N" (ABS46310.1) in str. IP 31758 or "outer membrane protein C2, porin" (YP_070481.1) in str. IP32953, it might be warranted to rename the OmpN/OmpC2 to OmpY, "outer membrane protein Y", where letter "Y" pertained to Yersinia. Both phylogenetic analysis and genomic localization clearly support that the OmpY porin belongs to a new group of general bacterial porins. The recombinant OmpY protein with its signal sequence was overexpressed in porin-deficient Escherichia coli strain. The mature rOmpY was shown to insert into outer membrane as a trimer. The OmpY porin, isolated from the outer membrane, was studied employing spectroscopic, electrophoretic and bilayer lipid membranes techniques. The far UV CD spectrum of rOmpY was essentially identical to that of Y. pseudotuberculosis OmpF. The near UV CD spectrum of rOmpY was weaker and smoother than that of OmpF. The rOmpY single-channel conductance was 180 ± 20 pS in 0.1 M NaCl and was lower than that of the OmpF porin. As was shown by electrophoretic and bilayer lipid membrane experiments, the rOmpY trimers were less thermostable than the OmpF trimers. The porins differed in the trimer-monomer transition temperature by about 20°C. The three-dimensional structural models of the Y. pseudotuberculosis OmpY and OmpF trimers were generated and the intra- and intermonomeric interactions stabilizing the porins were investigated. The difference in the thermal stability of OmpY and OmpF trimers was established to correlate with the difference in intermonomeric polar contacts.

[Development of a multiplex PCR for detection of the Yersinia genus with identification of pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica)].

To identify the Yersinia genus and pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) in a single reaction a multiplex PCR technique was developed. It was optimized by five compounds of PCR buffer and temperature of primers annealing. The multiplex PCR provides an improved and rapid method for detection of the Yersinia genus and identification of pathogenic species.

[Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane].

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Molecular characteristics of OmpF-like porins from pathogenic Yersinia.

Nonspecific pore-forming proteins (porins) are the major proteins of the outer membrane of Gram-negative bacteria responsible for diffusion of low-molecular-weight compounds. Nucleotide sequences of the OmpF-like porins from the pathogenic bacteria Yersinia pseudotuberculosis (YPS) and Yersinia enterocolitica (YE) were cloned and determined. Values of molecular weights (MW) and isoelectric points (IEP) calculated for these proteins (for OmpF-YPS: MW 37.7 kD, IEP 4.45; for OmpF-YE: MW 39.5 kD, IEP 4.34) are in good agreement with experimental data. The OmpF-like Yersinia porins are highly homologous to each other (83-92%) and also to the OmpF protein from Serratia marcescens (70%); the homology to the OmpF porin from E. coli is significantly lower (52-58%). Multiple alignment of the amino acid sequences of mature OmpF proteins provided the distribution of conservative amino acid residues typical for porins. Moreover, the OmpF-like porins from Yersinia are characterized by the presence of extended regions with high and low homologies, which coincide with the transmembrane domains and "external" loops, respectively, of the topological model of the OmpF porin from E. coli. By predictive methods, the secondary structure of the OmpF-like porins from Yersinia was obtained. This structure is represented by 16 beta-strands connected by short "periplasmic" and longer "external" loops with unordered structure.

[Primary structures of actinoporins from sea anemone Oulactis orientalis].

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with specific primers to the N-terminal regions of the 0. orientalis actinoporin sequences and to the C-terminal region, which is known to be highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were established by sequencing the cDNA clones obtained by the fast amplification of 3'-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain lower hemolytic activities of Or-A and Or-G than that of actinoporins from the tropical species.