ArdA genes from pKM101 and from B. bifidum chromosome have a different range of regulated genes.

The ardA genes are present in a wide variety of conjugative plasmids and play an important role in overcoming the restriction barrier. To date, there is no information on the chromosomal ardA genes. It is still unclear whether they keep their antirestriction activity and why bacterial chromosomes contain these genes. In the present study, we confirmed the antirestriction function of the ardA gene from the Bifidobacterium bifidum chromosome. Transcriptome analysis in Escherichia coli showed that the range of regulated genes varies significantly for ardA from conjugative plasmid pKM101 and from the B. bifidum chromosome. Moreover, if the targets for both ardA genes match, they often show an opposite effect on regulated gene expression. The results obtained indicate two seemingly mutually exclusive conclusions. On the one hand, the pleiotropic effect of ardA genes was shown not only on restriction-modification system, but also on expression of a number of other genes. On the other hand, the range of affected genes varies significally for ardA genes from different sources, which indicates the specificity of ardA to inhibited targets. Author Summary. Conjugative plasmids, bacteriophages, as well as transposons, are capable to transfer various genes, including antibiotic resistance genes, among bacterial cells. However, many of those genes pose a threat to the bacterial cells, therefore bacterial cells have special restriction systems that limit such transfer. Antirestriction genes have previously been described as a part of conjugative plasmids, and bacteriophages and transposons. Those plasmids are able to overcome bacterial cell protection in the presence of antirestriction genes, which inhibit bacterial restriction systems. This work unveils the antirestriction mechanisms, which play an important role in the bacterial life cycle. Here, we clearly show that antirestriction genes, which are able to inhibit cell protection, exist not only in plasmids but also in the bacterial chromosomes themselves. Moreover, antirestrictases have not only an inhibitory function but also participate in the regulation of other bacterial genes. The regulatory function of plasmid antirestriction genes also helps them to overcome the bacterial cell protection against gene transfer, whereas the regulatory function of genomic antirestrictases has no such effect.

Gene Expression in the Three-Spined Stickleback (Gasterosteus aculeatus) of Marine and Freshwater Ecotypes.

Three-spine stickleback (Gasterosteus aculeatus) is a well-known model organism that is routinely used to explore microevolution processes and speciation, and the number of studies related to this fish has been growing recently. The main reason for the increased interest is the processes of freshwater adaptation taking place in natural populations of this species. Freshwater three-spined stickleback populations form when marine water three-spined sticklebacks fish start spending their entire lifecycle in freshwater lakes and streams. To boot, these freshwater populations acquire novel biological traits during their adaptation to a freshwater environment. The processes taking place in these populations are of great interest to evolutionary biologists. Here, we present differential gene expression profiling in G. aculeatus gills, which was performed in marine and freshwater populations of sticklebacks. In total, 2,982 differentially expressed genes between marine and freshwater populations were discovered. We assumed that differentially expressed genes were distributed not randomly along stickleback chromosomes and that they are regularly observed in the "divergence islands" that are responsible for stickleback freshwater adaptation.

Differential miRNA expression in the three-spined stickleback, response to environmental changes.

miRNAs play important role in the various physiological and evolutionary processes, however, there is no data allowing comparison of evolutionary differences between various ecotypes adapted to different environmental conditions and specimen demonstrating immediate physiological response to the environmental changes. We compared miRNA expression profiles between marine and freshwater stickleback populations of the three-spined stickleback to identify the evolutionary differences. To study the immediate physiological response to foreign environment, we explored the changes induced by transfer of marine sticklebacks into freshwater environment and vice versa. Comparative analysis of changes in miRNA expression suggested that they are driven by three independent factors: (1) non-specific changes in miRNA expression under different environmental conditions; (2) specific response to freshwater conditions in the marine stickleback ecotype; (3) specific response to extreme osmotic conditions for both marine and freshwater ecotypes during the contact with non-native environment. Gene Ontology enrichment analysis of differential expressed miRNA targets supports our current hypothesis.

[Gene mutations in patients with hereditary cavernous malformations]. / Analiz mutatsii genov u bol'nykh s nasledstvennymi kavernoznymi mal'formatsiiami TsNS.

AIM: To identify mutations in cerebral cavernous malformation (CCM) genes in patients with hereditary and sporadic CCMs in the Russian population. MATERIAL AND METHODS: Blood samples from 73 randomly selected patients, including 29 MRI-confirmed familial cases, 8 clinically confirmed familial cases and 38 so-called sporadic cases, were examined. A search for large deletions/duplications was performed using multiplex ligation-dependent probe amplification (MPLA). For MLPA-negative samples, the whole genome sequencing was performed to search for single nucleotide polymorphisms (SNP). RESULTS: Deletions in three genes (ССМ1, ССМ2, ССМ3) were identified in 14 patients, including 5 without definitely established familial type, in whom the familial character of disease was not confirmed by clinical and neuroimaging results. SNP mutations were found in 13 patients, CCM gene mutations in 27. Mutations were detected in 91.7% of familial cases. In two patients, new CCM3 deletions were identified. Gene distribution was as follows: 60.7 for CCM1, 32.2 for CCM2 and 7.1% for CCM3. In two members of a family with hereditary CCMs, no high effect mutations in the known CCM genes were found. Patients with mutations had greater severity of disease. Two patients with CCM3 mutations demonstrated the most aggressive clinical course. De novo formation and growth of CCM were observed only in patients with mutations. CONCLUSION: The distribution of pathogenic mutations in known CCM genes is consistent with other large-scale studies. Familial CCMs are associated with more severe disease course and may be caused by mutations beyond the known CCM genes.

Metagenomic analysis of microbial community of a parasitoid wasp Megaphragma amalphitanum.

The vast majority of multicellular organisms coexist with bacterial symbionts that may play various roles during their life cycle. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) belongs to the smallest known insects whose size is comparable with some bacteria. Using 16S rRNA gene sequencing and Whole Genome Sequencing (WGS), we described microbiota diversity for this arthropod and its potential impact on their lifecycle. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX2363723 and SRX2363724. We found that small body size and limited lifespan do not lead to a significant reduction of bacterial symbionts diversity. At the same time, we show here a specific feature of microbiota composition in M. amalphitanum - the absence of the Rickettsiaceae family representatives that are known to cause sex-ratio distortion in arthropods and well represented in other populations of parasitoid wasps.

Individual genome sequencing identified a novel enhancer element in exon 7 of the CSFR1 gene by shift of expressed allele ratios.

The sequencing of individual genetic information may provide a powerful tool for elucidating the mechanism by which individual SNPs affect promoter function. Here, we assessed the genome of a Russian male that was previously sequenced. The RNA-Seq data from blood cells revealed 234 candidate transcripts with shifts of greater than 1.5-fold from equal biallelic transcription. Of these genes, the CSF1R gene had variations in genic regions that affected the association of RORalpha with its target binding site in vivo. The results of a reporter assay confirmed that a single nucleotide substitution, rs2228422, within the RORalpha recognition motif altered the ability of the enhancer to regulate CSF1R gene transcription. Notably, 31% of Europeans and only 3% of Asians are homozygous for a RORalpha responsive "A" allele, but no association with diseases of rs2228422 has been found thus far.

Analysis of the Mitochondrial Genome of a Novosvobodnaya Culture Representative using Next-Generation Sequencing and Its Relation to the Funnel Beaker Culture.

The Novosvobodnaya culture is known as a Bronze Age archaeological culture in the North Caucasus region of Southern Russia. It dates back to the middle of the 4th millennium B.C. and seems to have occurred during the time of the Maikop culture. There are now two hypotheses about the emergence of the Novosvobodnaya culture. One hypothesis suggests that the Novosvobodnaya culture was a phase of the Maikop culture, whereas the other one classifies it as an independent event based on the material culture items found in graves. Comparison between Novosvobodnaya pottery and Funnelbeaker (TRB) pottery from Germany has allowed researchers to suggest that the Novosvobodnaya culture developed under the influence of Indo-European culture. Nevertheless, the origin of the Novosvobodnaya culture remains a matter of debate. We applied next-generation sequencing to study ~5000-year-old human remains from the Klady kurgan grave in Novosvobodnaya stanitsa (now the Republic of Adygea, Russia). A total of 58,771,105 reads were generated using Illumina GAIIx with a coverage depth of 13.4x over the mitochondrial (mt) DNA genome. The mtDNA haplogroup affiliation was determined as V7, suggesting a role of the TRB culture in the development of the Novosvobodnaya culture and supporting the model of sharing between Novosvobodnaya and early Indo-European cultures.

[Oligoglycine surface structures: molecular dynamics simulation].

The full-atomic molecular dynamics (MD) simulation of adsorption mode for diantennary oligoglycines [H-Gly4-NH(CH2)5]2 onto graphite and mica surface is described. The resulting structure of adsorption layers is analyzed. The peptide second structure motives have been studied by both STRIDE (structural identification) and DSSP (dictionary of secondary structure of proteins) methods. The obtained results confirm the possibility of polyglycine II (PGII) structure formation in diantennary oligoglycine (DAOG) monolayers deposited onto graphite surface, which was earlier estimated based on atomic-force microscopy measurements.

Combining two technologies for full genome sequencing of human.

At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

[Use of DIR-PCR for elaboration of molecular markers of intraspecies bacterial groups as exemplified by Bacillus thuringiensis].

A recently developed PCR-fingerprinting method, the so-called DIR (diverged inverted repeats)-PCR, was used for quick search for molecular markers of Bacillus thuringiensis subspecies carrying the cry1 genes. The analysis of the fingerprints obtained by this method made it possible to reveal PCR fragments characteristic of the subspecies that produce proteins toxic for insects of the order Lepidoptera. Cloning and sequencing of these fragments allowed systems of SCAR (sequence characterized amplified region) primers to be designed, which are specific to the above group of B. thuringiensis strains. Comparison of the specific fragments with sequences available in the GenBank database revealed their homology with the rpoC gene family and the adjacent spacer region, suggesting chromosomal localization of these markers. This increases the reliability of the designed system of SCAR primers, because plasmids may be lost or transferred by transformation between closely related strains. It was demonstrated that the DIR-PCR method allows markers to be developed that are linked to diagnostic genotypic and phenotypic characteristics of bacteria.

[Spontaneous and promoted association of linear oligoglycines].

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)m-OH(m = 3-5). At the same time, oligoglycylamides H-(Gly)m-NH2 (m = 3-5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.

[Obtaining of intrapopulational dissociants of some bacilli and the use of DIR-PCR for their identification]. / Poluchenie vnutripopuliatsionnykh dissotsiantov nekotorykh batsill i primenenie metoda DIR-PTSP dlia ikh identifikatsii.

The paper is the first to suggest methods for rapid obtaining and genotypic identification of phenotypic (colonial-morphological) dissociants of bacterial cultures. For revelation of the potential dissociation ability and obtaining of dissociants, the use of bacterial cyst-like refractile cells (CRC) is recommended. These cells are characterized by enhanced variability; upon their first passage, an abrupt increase in the dissociation index is observed as a result of the emergence of cells that form morphologically different types of colonies. The approaches elaborated were tested with Bacillus cereus, B. subtilis, and B. licheniformis, for which colonial-morphological dissociants of various types were obtained after the first passage of CRC (both of those formed in the developmental cycle of bacteria and of those arising as a result of artificial increase of the concentration of anabiosis autoinducers in the cultivation medium). The genomic distinctions between dissociants of B. cereus and B. subtilis were estimated using polymerase chain reaction with a primer system designed based on the analysis of nucleotide sequences of complete prokaryotic genomes available in the GenBank database (DIR-PCR). The application of the suggested method allowed distinctions between the genomes of dissociants of Bacillus cereus and B. subtilis to be revealed, which is in agreement with the hypothesis that suggests reversible intragenomic rearrangements to be the basis of bacterial dissociation into subpopulations.

[The surgical risk factors in patients with nonmalignant space-occupying lesions of the liver and the importance of immunological studies in assessing their extent]. / Faktory khirurgicheskogo riska u bol'nykh s nezlokachestvennymi ob''emnymi obrazovaniiami pecheni i znachenie immunologicheskikh issledovanii v otsenke ego stepeni.

Results of surgical treatment of benign new growths of the liver (BVML) depend not only on timely diagnosis of local growth but on precise analysis of surgical risk factors (SR) as well as accurate definition of its significance. The proposed classification helps to evaluate factors of SR in these patients. Results of the investigation showed that in majority of patients (94.7%) with BVML without complications immune system was were not suppressed. The fact may be considered as a favourable sign, demonstrating potential immunologic safety of patients in definite period of BVML development. Patients with lowered parameters of immunologic status made up a group of a high risk to develop inflammatory and septic complications in postoperative period. Definite correlation between the extent of immunodeficiency and frequency of postoperative complications has established the necessity of action to prevent the provocation of immunological status disturbances.

[Immunological indices of "drainage" blood used for compensation of blood loss]. / Immunologicheskie pokazateli "drenazhnoi" krovi, ispolzuemoi dlia vospolneniia krovopoteri.

The authors discuss experience in compensation for blood loss in patients with ischemic heart disease after aortocoronary shunting using drainage blood without or with donor blood (80 persons) and donor blood alone (60 persons). Mild changes of immunity cell factors were revealed and no differences in the concentration of class A, M, and G immunoglobulins and phagocytosis as compared to the indices recorded before the operation and 24 hours after it. Analysis of postoperative infectious-inflammatory complications showed their incidence to be less in patients who received drainage blood without or donor blood (23%) than in those given transfusion of only donor blood (55%). The results of the study allow drainage blood to be recommended for reinfusion.

[Microsurgical allotransplantation of fetal bone on a vascular peduncle: immunological aspects]. / Mikrokhirurgicheskaia allotransplantatsiia plodnoi kosti na sosudistoi nozhke: immunologicheskie aspekty.

The authors studied the immunological profile of three patients after transplantation of bone allobrephotransplants with their microsurgical revascularization in the recipient zone. A reaction of rejection of the transplanted brephobone did not occur in any of the three cases. In transplantation of a complex brephobone the soft tissues overlying it, rejection of the soft tissues was encountered but there were no signs of bone rejection. The data of immunological monitoring correlate with the results of the clinicolaboratory and histological studies.

[The immunological indices of patients with echinococcosis undergoing preoperative hemoexfusions]. / Immunologicheskie pokazateli u bol'nykh s ékhinokokkozom pri provedenii predoperatsionnykh gemoéksfuzii.

The immunologic parameters were studied in 38 patients with hydatid disease of the liver and abdomen before and after a single hemoexfusion (HE) in a volume of 400 ml. Among the patients who were examined there were those with uncomplicated hydatid disease of the liver (group I, second clinical stage, 25 persons), patients with suppuration of hydatid cysts (group 11, 10 persons), and two patients with multiple hydatid cysts complicated by their suppuration and calcification with involvement of large areas of the liver. Comparative analysis of immunological parameters in patients before and in the first 24 hours after HE showed a difference between the groups of patients and the controls. The parameters were restored to their initial values in patients of both groups 3-5 days after HE. The results of the study allow the conclusion that in screening patients with hydatid disease of the liver and abdominal cavity for preoperative HE a strict individual approach is necessary with the patient's general condition, the clinical form of the disease, and complications on the part of the cysts being taken into account.

[A system for the clinico-immunological prognosis of infectious-inflammatory complications in planned surgery]. / Sistema kliniko-immunologicheskogo prognozirovaniia infektsionno-vospalitel'nykh oslozhnenii v planovoi khirurgii.

Postoperative infectious complications in planned surgery are a pressing problem. The preoperative condition of the patient and his immune system play an important role in their development. Fifty patients of the "risk" group were selected from the standpoint of classification of the etiological factors of secondary immunodeficiency states. Fifteen patients who had no symptoms classified as SIDS risk factors formed the control group. The immune status was examined before and on the third day after the operation in all patients of the main and control groups. Various infectious complications developed after the operation in 23 patients of the risk group. No such complications occurred in the control group. The preoperative immune status of the risk group patients differed significantly from that of the controls in 13 of 19 parameters. The patients of the main and control groups formed the instructing selection. An expert system of preoperative clinico-immunological prognostication of postoperative infectious-inflammatory complications was created on oasis of the methods of the theory of image recognition and instructing selection. The system was tested in a direct clinical experiment on 66 patients. The prognoses were correct in 89% of cases. It was thus proved that symptoms classified as SIDS risk factors influence the initial immunity status and the course of the postoperative period in planned surgery. An accurate, sensitive, and specific method for prognosticating postoperative infectious complications is suggested.

[Early postoperative rejection crises after transplantation of the kidney from the living related donors]. / Rannie posleoperatsionnye krizy ottorzheniia pri peresadke pochki ot zhivogo rodstvennogo donora.

The frequency of the development of rejection crises in the early postoperative period in 32 recipients of allogeneic kidneys from alive related donors was analysed depending on the histocompatibility according to the antigens of the HLA-AB system and the nature of the immunosuppressant therapy. The results of the analysis showed that the frequency of rejection crises was 100% when the donor and recipient were compatible in 1-2 antigens, and 40% in compatibility according to 3-4 antigens. The incidence of rejection crises was 71.4% among patients who received the standard immunosuppressant therapy (corticosteroids + azathioprine) and 36% among those given also sandimmune. Analysis of the frequency of rejection crises according to the ABO blood group system to which the donor-recipient pair occurred, showed that rejection crises occurred most frequently among patients with A (II) blood group.

[Characteristics of immunologic homeostasis in the late period after reconstructive operations on the heart valves]. / Osobennosti immunnogo gomeostaza v otdalennom periode posle rekonstruktivnykh operatsii na klapanakh serdtsa.

The immune status, i.e. T and B lymphocytes and their populations, was assessed after heart valve replacement or reconstruction in 70 patients with acquired heart defects. In 30%, the immune status was found to be normal which corresponded to inactive rheumatic process in these patients; 40% showed activated immunity manifested by increased T-active, T-total, T-helper, and T-suppressor numbers compared to the first group, which corresponded to activation of a rheumatic process in these patients. The rest 30% patients showed suppressed immunity, i.e. diminished subpopulations of T lymphocytes and B lymphocytes. These changes were detected in patients with circulatory insufficiency and progressive septic endocarditis. This allows for a conclusion that determination of the status of immunity and its correction are essential for cardiac surgery patients.