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The L. fermentum U-21 strain, known for secreting chaperones into the extracellular milieu, emerges as a promising candidate for the development of novel therapeutics termed disaggregases for Parkinson's disease. Our study focuses on characterizing the secreted protein encoded by the C0965_000195 locus in the genome of this strain. Through sequence analysis and structural predictions, the protein encoded by C0965_000195 is identified as ClpL, homologs of which are known for their chaperone functions. The chaperone activity of ClpL from L. fermentum U-21 is investigated in vivo by assessing the refolding of luciferases with varying thermostabilities from Aliivibrio fischeri and Photorhabdus luminescens within Escherichia coli cells. The results indicate that the clpL gene from L. fermentum U-21 can compensate for the absence of the clpB gene, enhancing the refolding capacity of thermodenatured proteins in clpB-deficient cells. In vitro experiments demonstrate that both spent culture medium containing proteins secreted by L. fermentum U-21 cells, including ClpL, and purified heterologically expressed ClpL partially prevent the thermodenaturation of luciferases. The findings suggest that the ClpL protein from L. fermentum U-21, exhibiting disaggregase properties against aggregating proteins, may represent a key component contributing to the pharmabiotic attributes of this strain.

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Automatic parsing of syntactic information by the human brain is a well-established phenomenon, but its mechanisms remain poorly understood. Its best-known neurophysiological reflection is the so-called early left-anterior negativity (ELAN) component of event-related potentials (ERPs), with two alternative hypotheses for its origin: (1) error detection, or (2) morphosyntactic prediction/priming. To test these alternatives, we conducted two experiments using a non-attend passive design with visual distraction and recorded ERPs to spoken pronoun-verb phrases with/without agreement violations and to the same critical verbs presented in isolation without preceding pronouns. The results revealed an ELAN at ∼130-220 ms for pronoun-verb gender agreement violations, confirming a high degree of automaticity in early morphosyntactic parsing. Critically, the strongest ELAN was elicited by verbs outside phrasal context, which suggests that the typical ELAN pattern is underpinned by a reduction of ERP amplitudes for felicitous combinations, reflecting syntactic priming/predictability between related words/morphemes (potentially mediated by associative links formed during previous linguistic experience) rather than specialised error-detection processes.

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The main aim of this study was to validate 500 true-false general-knowledge questions in Russian. These norms are valuable to researchers in many fields, as is shown by the impact and relevance of similar norms available in other languages. Although the Russian language is widely spoken, there are no norms available in this language for this type of questions. True-false questions are very useful for measuring semantic memory, among other topics, in neurocognitive studies where there is a trade-off between experimental time and the need for many trials. These types of experimental materials are heavily rooted in cultural background knowledge, making the mere translation from one language to another insufficient. The present research aims to fill this gap. One hundred fifty-five participants answered 500 true-false general knowledge questions split over several consecutive days and three topics: Social Sciences, Natural Sciences, and Culture & Sport. The participants' task was to indicate whether the statements were true or not, as well as the confidence they had in the correctness of their answer. Despite obtaining questions on each of the topics covering all difficulty levels, grouped analyses showed that Social Science's accuracy was higher than for Natural Science's or Culture & Sport questions. In relation to confidence, the grouped perceived difficulty was higher for questions about Culture & Sports when compared with the other two topics. Thus, this study reports and makes available a large pool of Russian true-false general knowledge questions covering different levels of difficulty.

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Aim: This study is mainly devoted to determining the ability of ∆FN3.1 protein fragments of Bifidobacterium (B.) longum subsp. longum GT15, namely two FN3 domains (2D FN3) and a C-terminal domain (CD FN3), to bind to tumor necrosis factor-alpha (TNF-α). Methods: Fragments of the fn3 gene encoding the 2D FN3 and CD FN3 were cloned in Escherichia (E.) coli. In order to assess the binding specificity between 2D FN3 and CD FN3 to TNFα, we employed the previously developed sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. The trRosetta software was used to build 3D models of the ∆FN3.1, 2D FN3, and CD FN3 proteins. The detection of polymorphism in the amino acid sequences of the studied proteins and the analysis of human gut-derived bacterial proteins carrying FN3 domains were performed in silico. Results: We experimentally showed that neither 2D FN3 nor CD FN3 alone can bind to TNFα. Prediction of the 3D structures of ΔFN3.1, 2D FN3, and CD FN3 suggested that only ΔFN3.1 can form a pocket allowing binding with TNFα to occur. Polymorphism analysis of amino acid sequences of ΔFN3.1 proteins in B. longum strains uncovered substitutions that can alter the conformation of the spatial structure of the ΔFN3.1 protein. We also analyzed human gut-derived bacterial proteins harboring FN3 domains which allowed us to differentiate between those containing motifs of cytokine receptors (MCRs) in their FN3 domains and those lacking them. Conclusion: Only the complete ∆FN3.1 protein can selectively bind to TNFα. Analysis of 3D models of the 2D FN3, CD FN3, and ΔFN3.1 proteins showed that only the ΔFN3.1 protein is potentially capable of forming a pocket allowing TNFα binding to occur. Only FN3 domains containing MCRs exhibited sequence homology with FN3 domains of human proteins.

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The process of thermocatalytic conversion of pine ethanol lignin in supercritical ethanol was studied over NiCu/SiO2 and NiCuMo/SiO2 catalysts bearing 8.8 and 11.7 wt.% of Mo. The structure and composition of ethanol lignin and the products of its thermocatalytic conversion were characterized via 2D-HSQC NMR spectroscopy, GC-MC. The main aromatic monomers among the liquid products of ethanol lignin conversion were alkyl derivatives of guaiacol (propyl guaiacol, ethyl guaiacol and methyl guaiacol). The total of the monomers yield in this case was 12.1 wt.%. The temperature elevation up to 350 °C led to a slight decrease in the yield (to 11.8 wt.%) and a change in the composition of monomeric compounds. Alkyl derivatives of pyrocatechol, phenol and benzene were observed to form due to deoxygenation processes. The ratio of the yields of these compounds depended on the catalyst, namely, on the content of Mo in the catalyst composition. Thus, the distribution of monomeric compounds used in various industries can be controlled by varying the catalyst composition and the process conditions.

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Investigation of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, we have identified and biochemically characterized three aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B in the strain Streptomyces rimosus ATCC 10970 (producing oxytetracycline), which is resistant to most natural aminoglycoside antibiotics. In the presented work, it was shown that the resistance of this strain to other AGs is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2') subfamily. Induction of the expression of the gene, designated by us as aac(2')-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and increases minimum inhibitory concentrations of these antibiotics.

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BACKGROUND: The early diagnosis of endometriosis in adolescents is not developed. OBJECTIVE: We aim to conduct clinical, imaging, laparoscopic and histological analyses of peritoneal endometriosis (PE) in adolescents in order to improve early diagnosis. METHODS: In total, 134 girls (from menarche to 17 years old) were included in a case-control study: 90 with laparoscopically (LS) confirmed PE, 44 healthy controls underwent full examination and LS was analyzed in the PE group. RESULTS: Patients with PE were characterized with heredity for endometriosis, persistent dysmenorrhea, decreased daily activity, gastrointestinal symptoms, higher LH, estradiol, prolactin and Ca-125 (<0.05 for each). Ultrasound detected PE in 3.3% and MRI in 78.9%. The most essential MRI signs are as follows: hypointense foci, the heterogeneity of the pelvic tissue (paraovarian, parametrial and rectouterine pouch) and sacro-uterine ligaments lesions (<0.05 for each). Adolescents with PE mostly exhibit initial rASRM stages. Red implants correlated with the rASRM score, and sheer implants correlated with pain (VAS score) (<0.05). In 32.2%, foci consisted of fibrous, adipose and muscle tissue; black lesions were more likely to be histologically verified (0.001). CONCLUSION: Adolescents exhibit mostly initial PE stages, which are associated with greater pain. Persistent dysmenorrhea and detected MRI parameters predict the laparoscopic confirmation of initial PE in adolescents in 84.3% (OR 15.4; <0.01), justifying the early surgical diagnostics and shortening the time delay and suffering of the young patients.

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Knowledge of language, its structure and grammar are an essential part of our education and daily activities. Despite the importance of language in our lives, linguistic theories that explain how the language system operates are often disconnected from our knowledge of the brain's neurocognitive mechanisms underpinning the linguistic function. This is reflected, for example, in the inclusion of abstract and often controversial elements into theories of language. Here, we discuss the case of the so-called null constituent and its smallest and the most controversial variant - the zero morpheme, a hypothetical morphosyntactic device that has no overt physical (phonological or orthographic) expression. Focusing on the putative inflectional zero morpheme, we discuss the theoretical origins and pitfalls of this approach and advocate the important role for neurobiological research that could try to elucidate the neurocognitive reality of such constructs in linguistic communication.

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During verbal communication, interlocutors rely on both linguistic (e.g., words, syntax) and extralinguistic (e.g., voice quality) information. The neural mechanisms of extralinguistic information processing are particularly poorly understood. To address this, we used EEG and recorded event-related brain potentials while participants listened to Russian pronoun-verb phrases presented in either male or female voice. Crucially, we manipulated congruency between the grammatical gender signaled by the verbs' ending and the speakers' apparent gender. To focus on putative automatic integration of extralinguistic information into syntactic processing and avoid confounds arising from secondary top-down processes, we used passive non-attend auditory presentation with visual distraction and no stimulus-related task. Most expressed neural responses were found at both early (150 ms, ELAN-like) and late (400 ms, N400-like) phrase processing stages. Crucially, both of these brain responses exhibited sensitivity to extralinguistic information and were significantly enhanced for phrases whose voice and grammatical gender were incongruent, similar to what is known for ERPs effects related to overt grammatical violations. Our data suggest a high degree of automaticity in processing extralinguistic information during spoken language comprehension which indicates existence of a rapid automatic syntactic integration mechanism sensitive to both linguistic and extralinguistic information.

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Bifidobacteria are some of the major agents that shaped the immune system of many members of the animal kingdom during their evolution. Over recent years, the question of concrete mechanisms underlying the immunomodulatory properties of bifidobacteria has been addressed in both animal and human studies. A possible candidate for this role has been discovered recently. The PFNA cluster, consisting of five core genes, pkb2, fn3, aaa-atp, duf58, tgm, has been found in all gut-dwelling autochthonous bifidobacterial species of humans. The sensory region of the species-specific serine-threonine protein kinase (PKB2), the transmembrane region of the microbial transglutaminase (TGM), and the type-III fibronectin domain-containing protein (FN3) encoded by the I gene imply that the PFNA cluster might be implicated in the interaction between bacteria and the host immune system. Moreover, the FN3 protein encoded by one of the genes making up the PFNA cluster, contains domains and motifs of cytokine receptors capable of selectively binding TNF-α. The PFNA cluster could play an important role for sensing signals of the immune system. Among the practical implications of this finding is the creation of anti-inflammatory drugs aimed at alleviating cytokine storms, one of the dire consequences resulting from SARS-CoV-2 infection.

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Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli, the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: IL-1ß, IL-6, IL-10, TNFα. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNFα.

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The gut microbiota (GM), which contains thousands of bacterial species, is a reservoir of antibiotic resistance genes (ARGs) called resistome. Early life exposure to antibiotics alters significantly the composition and function of the gut microbiota of children, which may trigger symptoms of autism spectrum disorder (ASD). This is because the GM plays an important role in the bidirectional communication between the gut and the brain and influences the brain normal functioning through multiple pathways. The goal of this article is to study the distribution of ARGs in the GM of 3- to 5-year-old healthy children and children with ASD living in Moscow, Russia. The metagenomic analysis of samples from both groups revealed differences in the signatures between them. The signatures consisted of the bacterial genera and aminoglycoside, ß-lactam, macrolide, and tetracycline resistance genes that they harbored. Our results show an increase in ARGs in the resistome of the GM of children with ASD. These findings emphasize the negative influence of early-life antibiotic therapy. We found three ARGs, aac(6')-aph(2''), cepA-49, and tet(40), which could serve as markers of ASD. The additional functions carried out by the enzymes, encoded by these genes, are being discussed.

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In this study, we identified a new gene (aph(3″)-Id) coding for a streptomycin phosphotransferase by using phylogenetic comparative analysis of the genome of the oxytetracycline-producing strain Streptomyces rimosus ATCC 10970. Cloning the aph(3″)-Id gene in E.coli and inducing its expression led to an increase in the minimum inhibitory concentration of the recombinant E.coli strain to streptomycin reaching 350 µg/ml. To evaluate the phosphotransferase activity of the recombinant protein APH(3″)-Id we carried out thin-layer chromatography of the putative 32P-labeled streptomycin phosphate. We also performed a spectrophotometric analysis to determine the production of ADP coupled to NADH oxidation. Here are the kinetic parameters of the streptomycin phosphotransferase APH(3″)-Id: Km 80.4 µM, Vmax 6.45 µmol/min/mg and kcat 1.73 s-1. We demonstrated for the first time the ability of the aminoglycoside phototransferase (APH(3″)-Id) to undergo autophosphorylation in vitro. The 3D structures of APH(3″)-Id in its unliganded state and in ternary complex with streptomycin and ADP were obtained. The structure of the ternary complex is the first example of this class of enzymes with bound streptomycin. Comparison of the obtained structures with those of other aminoglycoside phosphotransferases revealed peculiar structure of the substrate-binding pocket reflecting its specificity to a particular antibiotic.

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Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding.

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Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of Mycobacterium tuberculosis in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of M. tuberculosis was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of M. tuberculosis strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of M. tuberculosis. The possibility of using our set of genes for genotyping using PCR is also demonstrated.

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Analysis of the Bifidobacterium longum subsp. infantis ATCC 15697 genome sequence for the presence of toxin-antitoxin genes revealed two relBE-like operons, three relB-mazF-like operons, one relB-vapC-like operon, one solitary gene coding for the MazF toxin and one gene coding for the RelB antitoxin. An attempt to clone the selected relE and mazF toxin genes from B. longum subsp. infantis ATCC 15697 revealed their toxic effects on Escherichia coli, which could be neutralized by coexpression of these toxins with their cognate antitoxins. The only two toxin proteins, RelE and VapC, that were found to be non-toxic to E. coli, were overproduced and purified. Electrophoretic assays showed that both RelE and VapC possessed direct endoribonuclease activity. The expression levels of toxin genes in B. longum subsp. infantis ATCC 15697 increased during the nutrient starvation and entry into the late stationary phase. The two relBE bicistronic operons relE2-relB1 and relE1-relB4 from B. longum subsp. infantis ATCC 15697 were cloned and overexpressed in B. longum subsp. longum NCC2705 strain. The strain B. longum NCC2705 [pCESH80::relE1-relB4] showed a significantly decreased growth rate with later onset of the log phase and decreased cells density in the stationary phase.

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Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.

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