[Safety and efficacy of laparoscopic approach for widespread appendicular peritonitis]. / Vozmozhnosti laparoskopicheskogo metoda v lechenii rasprostranennogo appendikulyarnogo peritonita.

OBJECTIVE: To analyze treatment outcomes in patients with acute appendicitis complicated by widespread peritonitis. MATERIAL AND METHODS: The study included 165 patients acute appendicitis complicated by widespread peritonitis. Inclusion criteria: acute appendicitis complicated by widespread peritonitis MIP grade 1-2 in reactive or toxic phase (grading system by Simonyan K.S.), abdominal cavity index ≤16. Exclusion criteria: MIP grade 3, terminal phase, abdominal cavity index ≥17. RESULTS: Analysis of postoperative data revealed no correlation between surgical approach and incidence of postoperative intra-abdominal abscesses and infiltrates. In the main group, intra-abdominal abscesses occurred in 4.9% of patients (n=5), infiltrates - 12.8% (n=13). In the control group, these parameters were 4.6% (n=2) and 18.2% (n=8), respectively. We have developed and introduced into clinical practice a differentiated approach to surgical treatment of widespread appendicular peritonitis based on laparoscopic data. Abdominal cavity was intraoperatively assessed. The proposed method included 5 criteria with establishment of appropriate points (min 3, max 14). In case of total score 3-8, laparoscopic approach was preferred. Overall score 9-11 required laparoscopic surgery with subsequent elective repeated laparoscopy, ≥12 scores - intraoperative conversion and open surgery. Thus, subject to the rules of surgical intervention, the number of intra-abdominal complications between laparoscopic and open methods is equalized. CONCLUSION: The developed differentiated surgical strategy for patients with appendicular peritonitis is effective and reduces the incidence of wound infection, extra-abdominal complications, and hospital-stay, as well as contributes to early rehabilitation of patients.

Application of alternative de novo motif recognition models for analysis of structural heterogeneity of transcription factor binding sites: a case study of FOXA2 binding sites.

The most popular model for the search of ChIP-seq data for transcription factor binding sites (TFBS) is the positional weight matrix (PWM). However, this model does not take into account dependencies between nucleotide occurrences in different site positions. Currently, two recently proposed models, BaMM and InMoDe, can do as much. However, application of these models was usually limited only to comparing their recognition accuracies with that of PWMs, while none of the analyses of the co-prediction and relative positioning of hits of different models in peaks has yet been performed. To close this gap, we propose the pipeline called MultiDeNA. This pipeline includes stages of model training, assessing their recognition accuracy, scanning ChIP-seq peaks and their classification based on scan results. We applied our pipeline to 22 ChIP-seq datasets of TF FOXA2 and considered PWM, dinucleotide PWM (diPWM), BaMM and InMoDe models. The combination of these four models allowed a significant increase in the fraction of recognized peaks compared to that for the sole PWM model: the increase was 26.3 %. The BaMM model provided the main contribution to the recognition of sites. Although the major fraction of predicted peaks contained TFBS of different models with coincided positions, the medians of the fraction of peaks containing the predictions of sole models were 1.08, 0.49, 4.15 and 1.73 % for PWM, diPWM, BaMM and InMoDe, respectively. Thus, FOXA2 BSs were not fully described by only a sole model, which indicates theirs heterogeneity. We assume that the BaMM model is the most successful in describing the structure of the FOXA2 BS in ChIP-seq datasets under study.

[Laparoscopy in emergency abdominal surgery]. / Videolaparoskopiya v ekstrennoi khirurgii organov bryushnoi polosti.

OBJECTIVE: To evaluate the possibilities of laparoscopy in the diagnosis and treatment of acute abdominal surgical diseases. MATERIAL AND METHODS: A retrospective analysis of laparoscopic procedures in 4655 patients with confirmed or suspected acute abdominal surgical diseases for the period 2008-2017 was performed. Laparoscopy was applied to confirm or to determine unclear diagnosis. RESULTS: Diagnosis was established and confirmed in 4526 (97.2%) patients. Advisability of laparoscopic surgery was confirmed in 3091 (68.3%) patients, laparotomy - in 491 (10.8%) patients. Surgical treatment was not required in 944 (20.9%) patients. Laparoscopic procedures were performed in 3050 (98.7%) patients, 41 (1.3%) patients required conversion to laparotomy. Laparoscopic approach failed to define diagnosis in 129 (2.8%) patients that required conversion to diagnostic laparotomy. CONCLUSION: Laparoscopic was valuable to establish the diagnosis and determine surgical strategy, diagnose concomitant diseases, perform operations including simultaneous ones.

Architecture of Promoters of House-Keeping Genes in Polytene Chromosome Interbands of Drosophila melanogaster.

This is the first study to investigate the molecular-genetic organization of polytene chromosome interbands located on both molecular and cytological maps of Drosophila genome. The majority of the studied interbands contained one gene with a single transcription initiation site; the remaining interbands contained one gene with several alternative promoters, two or more unidirectional genes, and "head-to-head" arranged genes. In addition, intricately arranged interbands containing three or more genes in both unidirectional and bidirectional orientation were found. Insulator proteins, ORC, P-insertions, DNase I hypersensitive sites, and other open chromatin structures were situated in the promoter region of the genes located in the interbands. This area is critical for the formation of the interband, an open chromatin region in which gene transcription and replication are combined.

[Laparoscopy for perforated gastroduodenal ulcers]. / Videolaparoskopiia pri perforativnykh gastroduodenal'nykh iazvakh.

AIM: To assess laparoscopy in diagnosis and treatment of patients with perforated gastroduodenal ulcers. MATERIAL AND METHODS: There were 273 patients with perforated gastroduodenal ulcers who underwent laparoscopy at the Sklifosovsky Research Institute for Emergency Care in 2010-2016. Sample included patients with clinical and instrumental diagnosis of perforated gastroduodenal ulcers, suspected hollow organ perforation including perforated ulcer and other acute abdominal diseases followed by perforated ulcer. RESULTS: Laparoscopy confirmed diagnosis of perforated gastroduodenal ulcer in 82.5% of patients with clear preoperative diagnosis and in 54.2% of patients with suspected perforated ulcer. Video-assisted closure of the ulcer is possible in 81.7% of patients. Any endoscopic procedure should be started from diagnostic measures. Diagnostic laparoscopy followed by curative laparotomy in 14.6% of patients was able to clarify diagnosis, suture technically difficult perforated ulcer and prevent possible complications.

Genetic Organization of Open Chromatin Domains Situated in Polytene Chromosome Interbands in Drosophila.

New data on the organization of genes entirely located in the open domains for chromatin transcription and occupying only one chromosome structure (interband) were obtained. The characteristic features of these genes are the small size (on average, 1-2 kb), depletion of the replicative complex proteins in the regulatory region, and the presence of specific motifs for binding transcription factors, as compared to the genes occupying two structures (interband and gray band). The biological function of these genes is associated primarily with the processes of gene expression and RNA metabolism.

Auxin regulates functional gene groups in a fold-change-specific manner in Arabidopsis thaliana roots.

Auxin plays a pivotal role in virtually every aspect of plant morphogenesis. It simultaneously orchestrates a diverse variety of processes such as cell wall biogenesis, transition through the cell cycle, or metabolism of a wide range of chemical substances. The coordination principles for such a complex orchestration are poorly understood at the systems level. Here, we perform an RNA-seq experiment to study the transcriptional response to auxin treatment  within gene groups of different biological processes, molecular functions, or cell components in a quantitative fold-change-specific manner. We find for Arabidopsis thaliana roots treated with auxin for 6 h that (i) there are functional groups within which genes respond to auxin with a surprisingly similar fold changes and that (ii) these fold changes vary from one group to another. These findings make it tempting to conjecture the existence of some transcriptional logic orchestrating the coordinated expression of genes within functional groups in a fold-change-specific manner. To obtain some initial insight about this coordinated expression, we performed a motif enrichment analysis and found cis-regulatory elements TBX1-3, SBX, REG, and TCP/site2 as the candidates conferring fold-change-specific responses to auxin in Arabidopsis thaliana.

Hidden heterogeneity of transcription factor binding sites: A case study of SF-1.

Steroidogenic factor 1 (SF-1) belongs to a small group of the transcription factors that bind DNA only as a monomer. Three different approaches-Sitecon, SiteGA, and oPWM-constructed using the same training sample of experimentally confirmed SF-1 binding sites have been used to recognize these sites. The appropriate prediction thresholds for recognition models have been selected. Namely, the thresholds concordant by false positive or negative rates for various methods were used to optimize the discrimination of steroidogenic gene promoters from the datasets of non-specific promoters. After experimental verification, the models were used to analyze the ChIP-seq data for SF-1. It has been shown that the sets of sites recognized by different models overlap only partially and that an integration of these models allows for identification of SF-1 sites in up to 80% of the ChIP-seq loci. The structures of the sites detected using the three recognition models in the ChIP-seq peaks falling within the [-5000, +5000] region relative to the transcription start sites (TSS) extracted from the FANTOM5 project have been analyzed. The MATLIGN classified the frequency matrices for the sites predicted by oPWM, Sitecon, and SiteGA into two groups. The first group is described by oPWM/Sitecon and the second, by SiteGA. Gene ontology (GO) analysis has been used to clarify the differences between the sets of genes carrying different variants of SF-1 binding sites. Although this analysis in general revealed a considerable overlap in GO terms for the genes carrying the binding sites predicted by oPWM, Sitecon, or SiteGA, only the last method elicited notable trend to terms related to negative regulation and apoptosis. The results suggest that the SF-1 binding sites are different in both their structure and the functional annotation of the set of target genes correspond to the predictions by oPWM+Sitecon and SiteGA. Further application of Homer software for de novo identification of enriched motifs in ChIP-Seq data for SF-1ChIP-seq dataset gave the data similar to oPWM+Sitecon.

The expansion of heterochromatin blocks in rye reflects the co-amplification of tandem repeats and adjacent transposable elements.

BACKGROUND: A prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained. RESULTS: Using a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences. CONCLUSION: The large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms.

Translation efficiency in yeasts correlates with nucleosome formation in promoters.

Elongation efficiency index (EEI) was suggested earlier to estimate gene expression efficiency by nucleotide context of coding sequence in unicellular organisms. We have analyzed association between EEI and nucleosome formation potential (NFP) in 5' regulatory regions upstream translation initiation site (TIS) from two yeast species. Theoretical estimations of NFP based on DNA sequence were obtained by Recon method. Experimental estimation of nucleosome occupancy was obtained by high-throughput sequencing data of nucleosomal DNA in Saccharomyces cerevisiae . For the sample of all genes correlation coefficient was calculated between two vectors: vector of NFP values for fixed position relative to TIS and vector of EEI values. Profiles of correlation coefficients of NFP and EEI were counted in (-600; +600) regions relative to TIS for gene sequences extracted from GenBank. We found regions of strong negative dependence between NFP and EEI for all genes as well as for 10% highly expressed genes in Schizosaccharomyces pombe (10% of EEI-highest genes). At the same time, we found positive dependence between NFP and EEI for all genes and for low expressed genes in S. cerevisiae (10% of EEI-lowest genes). The association between NFP and EEI could be explained by evolutionary selection of context characteristics of nucleotide sequences for gene expression optimization.

Bioinformatical and experimental approaches to investigation of transcription factor binding sites in vertebrate genes.

The development of computer-assisted methods for transcription factor binding sites (TFBS) recognition is necessary for study the DNA regulatory transcription code. There are a great number of experimental methods that enable TFBS identification in genome sequences. The experimental data can be used to elaborate multiple computer approaches to recognition of TFBS, each of which has its own advantages and limitations. A short review of the characteristics of computer methods of TFBS prediction based on various principles is presented. Methods used for experimental monitoring of predicted sites are analyzed. Data concerning DNA regulatory potential and its realization at the chromatin level, obtained using these methods, are discussed along with approaches to recognition of target genes of certain transcription factors in the genome sequences.

Is the activity of partially agonistic MHC:peptide ligands dependent on the quality of immunological help?

CD8(+) cytotoxic T lymphocytes (CTL) are important for the immunological control of infections and tumours. Engagement of the T-cell receptor (TCR) with major histocompatibility complex (MHC) class I/peptide complexes on antigen-presenting cells (APC) is the key interaction, which initiates the process of T-cell activation. Depending on the affinity of this interaction, different arrays of signalling pathways and functional outcomes can be activated in the specific T cells. Molecular alterations in the peptide bound to the MHC class I can lead to a lower affinity of the MHC:TCR interaction resulting in incomplete or qualitatively different T-cell responses. Altered peptide ligands (APL) exhibiting such activity are referred to as partial agonists and often occur naturally through genetic instability, which affects T-cell epitopes derived from rapidly mutating viruses or tumour-associated cellular antigens. Partial agonists are usually viewed as peptide variants, which escape efficient CTL recognition. Our recent data suggest that APL can not only trigger incomplete activation but also induce and modulate intrinsic T-cell programmes leading to the shut-off of specific CTL responses. This APL-induced suppression appears to be more prominent in the absence of immunological help, suggesting that under conditions of immune deregulation APL may actively inhibit CTL responses against infectious agents or tumours. In this review, we discuss experimental data supporting this model and possible role of APL-induced immunosuppression in different pathological conditions.

Pharmacological disintegration of lipid rafts decreases specific tetramer binding and disrupts the CD3 complex and CD8 heterodimer in human cytotoxic T lymphocytes.

Accumulating evidence strongly supports the role of lipid rafts in the regulation of T-lymphocyte activation, but the organization and molecular composition of these cholesterol- and sphingolipid-rich membrane microdomains in different subsets of T cells remain poorly investigated. Here, we show that pharmacological disruption of lipid rafts in human CD8+ cytotoxic T-lymphocyte (CTL) clones disturbs the integrity of CD3 complex and CD8 heterodimer, without affecting the reactivity with T-cell receptor (TCR)-specific antibodies. This demonstrates that interaction with completely assembled CD3 complex is not required for the stable expression of TCR at the cell surface. The effect of raft disruption on CD3 and CD8 expression correlates with failure to bind specific tetrameric complexes by a proportion of surface TCR molecules. However, the interaction of specific tetramer with the rest of surface TCR pools appears to be unaffected, demonstrating that TCR-signalling complexes may differ in their requirement for cholesterol to stably maintain their composition and to rearrange for efficient tetramer binding. Together with previously published data, our results support the existence of molecular and/or structural heterogeneity of lipid rafts that may play an important role in controlling distinct functional properties of T-cell subsets.

Cytolytic T cell reactivity to Epstein-Barr virus is lost during in vitro T cell expansion.

In the setting of allogeneic hematopoietic stem cell transplantation, ex vivo culturing of donor T lymphocytes is a necessary step for processes such as gene modification. Often the aim is to enable control of undesired alloreactivity after in vivo administration of the cultured cells. However, it is not fully understood how T cell reactivity against donor and third-party targets is affected by the ex vivo cell culturing process. We have assessed how the activity of anti-Epstein Barr virus (EBV)-specific T lymphocytes from healthy EBV-seropositive donors is affected by in vitro cell culturing. Peripheral blood mononuclear cells (PBMCs) were expanded in X-VIVO 15 culture medium supplemented with 5% human serum. The cells were stimulated by either OKT3 (10 ng/ml) and interleukin (IL)-2 (500 U/ml) or by using anti-CD3/CD28-coated immunomagnetic beads and IL-2 (100 U/ml). Induction of polyclonal EBV-specific cytotoxic T lymphocyte cultures was attempted by stimulation of the in vitro-expanded cells at different time points during the cell expansion process, with pre-established autologous EBV-transformed lymphoblastoid cell lines (LCLs). While EBV-specific cytotoxic T lymphocytes (CTL) were generated from untreated PBMCs of 5 healthy donors, EBV-specific cytotoxicity was significantly decreased or absent in CTL cultures established from in vitro-expanded PBMCs. Our results indicate that the ex vivo cell expansion process itself significantly reduces the activity and/or the number of EBV-specific T cells. Additional stimulation with CD28 antibodies could not prevent this effect. Because T cell depleted bone marrow or stem cell grafts are known to contribute to the development of post transplant lymphoproliferative disorders, this should be taken into consideration if one considers expanding and administering PBMCs in conjunction with a T cell-depleted stem cell grafts.

Nucleosome formation potential of eukaryotic DNA: calculation and promoters analysis.

MOTIVATION: A rapid growth in the number of genes with known sequences calls for developing automated tools for their classification and analysis. It became clear that nucleosome packaging of eukaryotic DNA is very important for gene functioning. Automated computer tools for characterization of nucleosome packaging density could be useful for studying of gene regulation and genome annotation. RESULTS: A program for constructing nucleosome formation potential profiles of eukaryotic DNA sequences was developed. Nucleosome packaging density was analyzed for different functional types of human promoters. It was found that in promoters of tissue-specific genes, the nucleosome formation potential was essentially higher than in genes expressed in many tissues, or housekeeping genes. Hence, capability of nucleosome positioning in the promoter region may serve as a factor regulating gene expression. AVAILABILITY: The program for nucleosome sites recognition is included into the GeneExpress system; section 'DNA Nucleosomal Organization', http://wwwmgs.bionet.nsc.ru/mgs/programs/recon/.

Nucleosome formation potential of exons, introns, and Alu repeats.

A program for constructing nucleosome formation potential profile was applied for investigation of exons, introns, and repetitive sequences. The program is available at http://wwwmgs.bionet.nsc.ru/mgs/programs/recon/. We have demonstrated that introns and repetitive sequences exhibit higher nucleosome formation potentials than exons. This fact may be explained by functional saturation of exons with genetic code, hindering the localization of efficient nucleosome positioning sites.