Soil Alveolata diversity in the undisturbed steppe and wheat agrocenoses under different tillage.

Microeukaryotes are vital for maintaining soil quality and ecosystem functioning, however, their communities are less studied than bacterial and fungal ones, especially by high throughput sequencing techniques. Alveolates are important members of soil microbial communities, being consumers and/or prey for other microorganisms. We studied alveolate diversity in soil under the undisturbed steppe (US) and cropped for wheat using two tillage practices (conventional, CT, and no-till, NT) by amplifying the ITS2 marker with ITS3_KYO2/ITS4 primers and sequencing amplicons using Illumina MiSeq. A total of 198 Alveolata OTUs were identified, with 158 OTUs attributed to the Ciliophora phylum, containing five classes: Litostomatea, Spirotrichea and Oligohymenophorea, Nassophorea and Phyllopharyngea. Litostomatea and Phyllopharyngea were more abundant in US as compared with CT and NT. The observed OTU richness was higher in US than in CT and NT. The ß-biodiversity of soil ciliates also very distinctly differentiated the US field from CT and NT. In the US, Nassophorea and Spirotrichea correlated positively with sand and negatively with clay, silt and SOM contents. This is the first report about soil ciliates diversity in Siberia as assessed by metabarcoding technique. The revealed clear effect of land use on the relative abundance of some taxa and a lack of tillage effect suggest the importance of the quantity and quality of plant material input for shaping the prey for ciliates. The ITS-metabarcoding technique was used for the first time in the research of ciliates diversity; further studies, embracing diverse aspects of soil ciliates by combining -omics methodology with the traditional one, are needed to get a better insight on the ecological roles of the main ciliate taxa in the complex soil system.

[DNA Methylation Profile of CD14+ Monocytes Changes in Primary Progressive Multiple Sclerosis].

Multiple sclerosis (MS) is a chronic autoimmune inflammatory and neurodegenerative disease of the central nervous system, which is characterized by significant clinical heterogeneity. Primary progressive MS (PPMS) develops in 10-15% of patients. Unlike the most common relapsing-remitting form of MS, PPMS involves steady progress of neurodegeneration and, as a consequence, a persistent gradual increase in neurological symptoms. The peculiarities of epigenetic regulation of gene expression may be one of the reasons for the differences in the pathogenesis of the two MS forms. DNA methylation is one of the key epigenetic mechanisms, which remains almost unexplored in different cell populations of PPMS patients. The goal of this work was to identify differential methylation profiles of the CpG sites in the CD14+ monocyte DNA, which characterize PPMS. A genome-wide analysis of DNA methylation in PPMS patients and healthy individuals has identified 169 differentially methylated positions (DMPs), 90.5% of which were hypermethylated in PPMS patients. More than half of all DMPs are located in/near known genes and within CpG islands and their neighboring regions, which indicates their high functional significance. We have found six differentially methylated regions (DMRs) in the OR2L13, CAT, LCLAT1, HOXA5, RNF39, and CRTAC1 genes involved in inflammation and neurodegeneration, which indicates active epigenetic regulation of their expression.

[A comparison of DNA methylation profiles of blood mononuclear cells in patients with multiple sclerosis in remission and relapse]. / Sravnenie profilei metilirovaniya DNK mononuklearnykh kletok krovi bol'nykh rasseyannym sklerozom v stadiyakh remissii i obostreniya.

OBJECTIVE: To study the whole-genome DNA methylation profiles of peripheral blood mononuclear blood cells (PBMCs) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and relapse in order to assess the contribution of this epigenetic mechanism of gene expression regulation to the activity of the pathological process. MATERIAL AND METHODS: Eight patients with RRMS in remission and 6 patients in relapse were included in the study. Methylation levels of DNA CpG sites in PBMCs were analyzed using Infinium HumanMethylation450 BeadChip DNA microarrays. RESULTS: Seven differentially methylated positions (DMPs) were identified, of which 3 were hypermethylated (cg02981003, cg18486102, cg19533582) and 4 were hypomethylated (cg16814680, cg1964802, cg18584440, cg08291996) during RRMS relapse. Five DMPs are located in protein-coding genes (GPR123, FAIM2, BTNL2, ZNF8, ASAP2), one in microRNA gene (MIR548N), and one in an intergenic region. For all identified DMPs, we observed a change in DNA methylation levels of more than 20% (range 20.2-57.5%). Hierarchical clustering of DNA samples on the heatmap shows their clear aggregation into separate clusters corresponding to RRMS patients in the stages of relapse and remission. CONCLUSION: For the first time it was shown that during relapse and remission of RRMS there are differences in the DNA methylation profile that allow discrimination between these clinical stages. These data indicate the involvement of the epigenetic mechanism of DNA methylation in the activation of the pathological process in RRMS.

[Genome-Wide Analysis of DNA Methylation in Cd4+ T Lymphocytes of Patients with Primary Progressive Multiple Sclerosis Indicates Involvement of This Epigenetic Process in the Disease Immunopathogenesis].

The pathogenesis of multiple sclerosis (MS), a chronic disease of the CNS, includes autoimmune and neurodegenerative components. In most cases, patients develop relapsing-remitting MS (RRMS), while 10-15% of patients develop primary progressive MS (PPMS), which differs from RRMS in the mechanisms of the pathological process, some demographic, and some clinical characteristics. These differences may be explained by the epigenetic regulation of gene expression in PPMS including DNA methylation as one of the key epigenetic processes. The features of DNA methylation in various cell populations in PPMS patients remain understudied. The goal of this study is to identify differentially methylated CpG sites (DMSs) of the genome of CD4+ T lymphocytes, which characterize PPMS. The study included eight treatment-naive PPMS patients and eight healthy controls. Genome-wide analysis of DNA methylation of CD4+ T lymphocytes was performed using high-density DNA microarrays. We have identified 108 DMSs, which distinguish PPMS patients from healthy controls. In PPMS patients 81% of the DMSs are hypermethylated. More than a half of the identified DMSs are located in known genes in CpG islands and adjacent regions, which indicates a high functional significance of these DMSs in PPMS development. Analysis of the overrepresentation of DMS-containing genes in the main biological processes demonstrates their involvement in the regulation of cell adhesion to the extracellular matrix and the development of the immune response, i.e., antigen processing and presentation, and development of the immune system. Genome-wide analysis of DNA methylation in CD4+ T lymphocytes of PPMS patients indicates the involvement of this epigenetic process in the immunopathogenesis of the disease. These results may help better understand the pathogenesis of this severe form of MS.

Soil microbiota and microarthropod communities in oil contaminated sites in the European Subarctic.

The present comprehensive study aimed to estimate the aftermath of oil contamination and the efficacy of removing the upper level of polluted soil under the conditions of the extreme northern taiga of northeastern European Russia. Soil samples from three sites were studied. Two sites were contaminated with the contents of a nearby sludge collector five years prior to sampling. The highly contaminated upper soil level was removed from one of them. The other was left for self-restoration. A chemical analysis of the soils was conducted, and changes in the composition of the soil zoocoenosis and bacterial and fungal microbiota were investigated. At both contaminated sites, a decrease in the abundance and taxonomic diversity of indicator groups of soil fauna, oribatid mites and collembolans compared to the background site were found. The pioneer eurytopic species Oppiella nova, Proisotoma minima and Xenyllodes armatus formed the basis of the microarthropod populations in the contaminated soil. A complete change in the composition of dominant taxonomic units was observed in the microbiota, both the bacterial and fungal communities. There was an increase in the proportion of representatives of Proteobacteria and Actinobacteria in polluted soils compared to the background community. Hydrocarbon-degrading bacteria-Alcanivorax, Rhodanobacter ginsengisoli, Acidobacterium capsulatum, and Acidocella-and fungi-Amorphotheca resinae abundances greatly increased in oil-contaminated soil. Moreover, among both bacteria and fungi, a sharp increase in the abundance of uncultivated organisms that deserve additional attention as potential oil degraders or organisms with a high resistance to oil contamination were observed. The removal of the upper soil level was partly effective in terms of decreasing the oil product concentration (from approximately 21 to 2.6 g/kg of soil) and preventing a decrease in taxonomic richness but did not prevent alterations in the composition of the microbiota or zoocoenosis.

CYP2C19*17 May Increase the Risk of Death Among Patients with an Acute Coronary Syndrome and Non-Valvular Atrial Fibrillation Who Receive Clopidogrel and Rivaroxaban.

INTRODUCTION: The aim of this study is to assess the influence of gene CYP2C19, CYP3A4, CYP3A5 and ABCB1 polymorphisms on clopidogrel antiplatelet activity, rivaroxaban concentration equilibrium, and clinical outcomes among patients with acute coronary syndrome and non-valvular atrial fibrillation. METHODS: In the multicenter prospective registry study of the efficacy and safety of a combined antithrombotic therapy 103 patients with non-valvular atrial fibrillation both undergoing or not a percutaneous coronary intervention were enrolled. The trial assessed the primary outcomes (major bleeding, in-hospital death, cardiovascular death, stroke\transient ischaemic attack, death/renal insufficiency) and secondary outcomes (platelet reactivity units (PRU), rivaroxaban concentration). RESULTS: For none of the clinical outcomes when combined with other covariates, the carriership of polymorphisms CYP3A5*3 rs776746, CYP2C19*2 rs4244285;*17 rs12248560, ABCB1 3435 C>T, ABCB1 rs4148738 was significant. None of the markers under study (CYP3A5*3 rs776746, CYP2C19*2 rs4244285, *17 rs12248560, ABCB1 3435 C>T, ABCB1 rs4148738) has proven to affect rivaroxaban equilibrium concentration in blood plasma among patients with atrial fibrillation and acute coronary syndrome. CONCLUSION: In situations of double or triple antithrombotic rivaroxaban and clopidogrel therapy among patients with atrial fibrillation and acute coronary syndrome, the genetic factors associated with bleeding complications risk (CYP2C19*17) may prove to be clinically relevant.

Assessment of the Phenylketonuria (PKU)-Associated Mutation p.R155H Biochemical Manifestations by Mass Spectrometry-Based Blood Metabolite Profiling.

Homozygous siblings with different treatment histories represent an excellent model to study both the phenotypic manifestation of mutations and the efficacy of therapy. We compared phenylketonuria (PKU) manifestations in two different gender siblings who were homozygous carriers of a rare phenylalanine hydroxylase (PAH) mutation, p.R155H, subjected to different treatments. PKU caused by mild mutations may be easily underdiagnosed if the diagnosis is based solely on the phenylalanine (Phe) blood concentration. One of the described patients is an example of this diagnostic error. For reducing diagnostic errors, we suggest the use of more elaborate methods in screening practice, in particular mass spectrometric analysis of blood metabolites, the efficiency of which is demonstrated in the present study.

Prevalence of Atrial Fibrillation and use of Oral Antithrombotic Therapy in Patients with Acute Coronary Syndrome.

PURPOSE: To assess the prevalence of atrial fibrillation (AF) and use of antithrombotic agents in adult patients with acute coronary syndrome (ACS). MATERIALS AND METHODS: We consecutively enrolled all ACS patients (n=1155) who were hospitalized in two Moscowbased percutaneous coronary intervention centers (each center performs over 500 PCIs a year) between October 2017 and February 2018. AF was diagnosed in 204 patients (17.7%). The risk of thromboembolic complications was assessed using the CHA2DS2-VASc Score. The risk of hemorrhagic complications was assessed using the HAS-BLED Score. The data were processed using StatSoft Statistica 10.0 and IBM SPSS Statistics v.23 software. RESULTS: The prevalence of diagnosed AF was 13.6%, while the prevalence of undiagnosed AF was 4.1%. Of the 179 discharged patients with AF, only 2 had a low risk of ischemic stroke (IS). One hundred and fifty patients (83.8%) eligible for oral anticoagulant therapy received oral anticoagulants. Patients with diagnosed AF were administered oral anticoagulants (OACs) significantly more often than patients with undiagnosed AF [125 (91.9%) vs. 25 (58.1%), р<0.001]. Novel oral anticoagulants (NOACs) were administered four times more often than vitamin K antagonists [120 (80.0%) vs. 29 (19.3%), р<0.001]. Rivaroxaban was used in 51.3% of cases. Of the 29 patients treated with warfarin, only 3 (10.3%) achieved the target international normalized ratio (INR) at discharge. Of the 107 patients who underwent percutaneous coronary intervention (PCI), 77 patients (80%) received an OAC and two antiplatelet agents (with 74% receiving this three-agent therapy for one month), 11 patients (10.3%) received an OAC and an antiplatelet agent, and 18 patients (16.8%) received two antiplatelet agents. The only antiplatelet agent used as part of the three-agent therapy was clopidogrel. The three-agent therapy without PCI was administered in 43.1% of cases. CONCLUSION: We found that the prevalence of AF in patients with ACS was high. The fact that doctors administered NOACs suggests that they are aware of the need to use these agents to prevent thromboembolic complications in AF patients.

A comparison of the adaptations of strains of Lymantria dispar multiple nucleopolyhedrovirus to hosts from spatially isolated populations.

The adaptation of pathogens to either their hosts or to environmental conditions is the focus of many current ecological studies. In this work we compared the ability of six spatially-distant Lymantria dispar (gypsy moth) multiple nucleopolyhedrovirus (LdMNPV) strains (three from eastern North America and three from central Asia) to induce acute infection in gypsy moth larvae. We also sequenced the complete genome of one Asian (LdMNPV-27/0) and one North American (LdMNPV-45/0) strain which were used for bioassay. We found that all of the North American virus strains, with the exception of one, demonstrated higher potency than the Asian virus strains, either in North American (Lymantria dispar) larvae or, in Asian (Lymantria dispar asiatica) larvae. Complete genome sequencing revealed two gene deletions in the LdMNPV-27/0 strain: the virus enhancin factor gene (vef-1) and the baculovirus repeated orf gene (bro-p). These deletions were not seen in the LdMNPV-45/0 strain nor in other American strains available in archiving systems. We also found deletions of the bro-e and bro-o genes in LdMNPV-45/0 strain but not in the LdMNPV-27/0 strain. The phylogenetic inference with an alignment of the 37 core gene nucleotide sequences revealed the close relationship of the LdMNPV-45/0 strain with other American strains accessed in GenBank (Ab-a624 and 5-6) while the LdMNPV-27/0 strain was clustered together with the LdMNPV-3054 strain (isolated in Spain) instead of predicted clustering with LdMNPV- 3029 (isolated in Asia). Our study demonstrated that first, different LdMNPV isolates from the same metapopulations of L. dispar exhibit little or no difference in the degree of virulence towards host larvae and second, that locality of host population is not an important driver of LdMNPV virulence. Virulence of LdMNPV is determined only by viral genetics. The genetic differences between North American and Central Asian virus strains are discussed.

Whole-Genome DNA Methylation Analysis of Peripheral Blood Mononuclear Cells in Multiple Sclerosis Patients with Different Disease Courses.

Multiple sclerosis (MS) is a severe neurodegenerative disease of polygenic etiology affecting the central nervous system. In addition to genetic factors, epigenetic mechanisms, primarily DNA methylation, which regulate gene expression, play an important role in MS development and progression. In this study, we have performed the first whole-genome DNA methylation profiling of peripheral blood mononuclear cells in relapsing-remitting MS (RRMS) and primary-progressive MS (PPMS) patients and compared them to those of healthy individuals in order to identify the differentially methylated CpG-sites (DMSs) associated with these common clinical disease courses. In addition, we have performed a pairwise comparison of DNA methylation profiles in RRMS and PPMS patients. All three pairwise comparisons showed significant differences in methylation profiles. Hierarchical clustering of the identified DMS methylation levels and principal component analysis for data visualization demonstrated a clearly defined aggregation of DNA samples of the compared groups into separate clusters. Compared with the control, more DMSs were identified in PPMS patients than in RRMS patients (67 and 30, respectively). More than half of DMSs are located in genes, exceeding the expected number for random distribution of DMSs between probes. RRMS patients mostly have hypomethylated DMSs, while in PPMS patients DMSs are mostly hypermethylated. CpG-islands and CpG-shores contain 60% of DMSs, identified by pairwise comparison of RRMS and control groups, and 79% of those identified by pairwise comparison of PPMS and control groups. Pairwise comparison of patients with two clinical MS courses revealed 51 DMSs, 82% of which are hypermethylated in PPMS. Overall, it was demonstrated that there are more changes in the DNA methylation profiles in PPMS than in RRMS. The data confirm the role of DNA methylation in MS development. We have shown, for the first time, that DNA methylation as an epigenetic mechanism is involved in the formation of two distinct clinical courses of MS: namely, RRMS and PPMS.

A comparative study on microbiota from the intestine of Prussian carp (Carassius gibelio) and their aquatic environmental compartments, using different molecular methods.

AIMS: The aim of this study was to evaluate, via various molecular methods, the possible correlations between microbial community structure of Prussian carp and the environmental compartments of their habitat. METHODS AND RESULTS: Microbial communities in the intestine and environmental compartments were studied using PCR-screening, cloning and next-generation high-throughput sequencing of the 16S ribosomal RNA genes. The 16S rDNA metagenomic sequencing showed higher bacterial diversity in comparison with clone libraries, while group-specific PCR showed positive detection of nine bacteria phyla. Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria and Actinobacteria were most abundant both in the intestine and habitat environments. The comparative analyses reveal that the bacterial community in the Prussian carp intestine is most similar to that identified from the chironomid. CONCLUSIONS: This study demonstrated some differences between molecular methods and showed advantages and limitations associated with them. These differences have the potential to reduce bias in results obtained from analysis of the community structure. The advantages of each molecular technique can be used for a better understanding of microbial diversity. The microbiota of Prussian carp intestine is most similar to those from the chironomids. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the diversity of the intestinal microbiota in an economically important aquaculture species, the Prussian carp (Carassius gibelio). The results provide significant information to discuss possible functions of these bacteria for further understanding of Prussian carp health.

The enhancin gene: One of the genetic determinants of population variation in baculoviral virulence.

It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion.

[Mutation analysis of the phenylalanine hydroxylase gene of phenylketonuria patients of Kemerovskaya Oblast' and Saha Republic].

Phenylketonuria (PKU) associated mutations in phenylalanine hydroxylase (PAH) gene were identified by direct DNA sequencing in 46 PKU patients and members of their families from Kemerovskaya Region and Saha Republic. Mutations found included both widespread known mutations (R158Q, R252W, R261Q, P281L, IVS10-11G>A, R408W, IVS12+1G>A) and several rare mutations (IVS2+5G>A, R155H, Y168H, W187R, E221_D222>Efs, A342T, Y386C, IVS11+1G>C). We observed the increase in diversity of PKU-associated alleles in the populations studied, probably due to their complex mixed ethnic structure.

[Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips].

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3') probes GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of A-C and G-T mutations in the binding site (imperfect duplexes) or a C-G pair (perfect duplex) affects the change in fluorescence level to a considerably lesser degree.

[An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes].

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.