P. P. Laktionov

Circulating DNA in the blood and its application in medical diagnosis

Circulating nucleic acids were discovered more than 30 years ago, but did not attract much attention until the past decade. This review summarizes the data on the sources of extracellular DNA circulating in the blood, features of its circulation, and pathways of its removal. The possibility of using circulating DNA in medical diagnosis is discussed.

Transcription factor comr acts as a direct activator in the genetic program controlling spermatogenesis in D. melanogaster

AbstarctIn Drosophila melanogaster differentiation of the male germ cells is accompanied by chromatin rearrangement and activation of the specific genes. These processes are regulated by few transcription factors that belong to two classes, can and aly that form distinct functional complexes. Mechanisms of action of aly and can class transcription factors on gene expression and chromatin state remain unclear. To investigate this question we have built the whole genome binding profile of transcription factor Comr belonging to aly class using the tissue-specific DamID method. Resulting data were correlated with gene expression in comr (aly class) and can (can class) mutant testes. It was shown that Comr is a direct activator for about 300 testis-specific genes. Furthermore a set of genes revealed decreased expression in comr mutants but did not bind Comr protein, suggesting the existence of secondary regulation. Indeed, among the Comr gene targets we found a gene coding an uncharacterized transcription factor that could be a secondary participant in the genetic pathway in spermatocytes. These date allowed us to advance a model of gene activation needed for male gametes differentiation in D. melanogaster.

Generation of blood circulating DNAs: Sources, features of struction and circulation

Extracellular nucleic acids (exNAs) were originally described in blood of healthy and sick people in 1948; however, little attention was paid to them until 1960th. exNAs are intensively studied now, especially during the last five years. The major attention is paid to exNAs as the source of a diagnostic material, whereas mechanisms of their generation, as well as mechanisms responsible for their long-term circulation in blood-stream still need better understanding. Some authors believe that the processes of apoptosis and necrosis represent the main source of blood circulating deoxyribonucleic acids (cirDNA), while others suggest nucleic acid secretion by healthy and tumor cells. Circulating DNA were found to be stable in blood for a long time, escaping from the action of DNA hydrolyzing enzymes, possibly, due to formation of various supramolecular complexes. This review summarizes literature data which support all the theories describing appearance of cirDNA; certain attention is paid to features of circulation and structure of the cirDNA and factors affecting time of DNA circulation in blood.

Data analysis algorithm for DamID-seq profiling of chromatin proteins in Drosophila melanogaster

Analysis of gene expression regulation typically requires identification of genomic sites bound by regulatory proteins. For this purpose, chromatin immunoprecipitation (ChIP) and Dam identification (DamID) methods can be applied to cell lines, whole organisms, or enriched cell populations. In this work, we present modifications to the experimental DamID protocol, as well as a custom data processing algorithm, that allow to confidently identify genomic sites enriched with the proteins of interest. This algorithm is implemented in Perl and is also available as executable files, thereby making DamID analysis relatively straightforward. Finally, we demonstrate how this pipeline performs when fed with real experimental data.

Circulating microRNAs in lung cancer: Prospects for diagnosis, prognosis, and prediction of antitumor treatment efficacy

The review considers the main techniques to extract microRNA (miRNA) from various biological fluids (in particular, the serum and plasma), approaches to the analysis of miRNA concentration and composition, and methods to normalize the results in data analyses. Advantages and drawbacks of the methods are described. Special attention is given to circulating miRNAs, which can be used as markers for minimally invasive diagnosis, prediction of antitumor treatment efficacy, and disease prognosis in lung cancer. The review discusses the prospects and limitations that arise as the clinical significance is evaluated for miRNAs as potential tumor markers and a better understanding is gained for the roles various miRNAs play in the pathogenesis of lung cancer.

MIRA analysis of RARβ2 gene methylation in DNA circulating in the blood in lung cancer.

Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment effi ciency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfi te conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARβ2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.

Genome-wide analysis of SU(VAR)3-9 distribution in chromosomes of Drosophila melanogaster

Histone modifications represent one of the key factors contributing to proper genome regulation. One of histone modifications involved in gene silencing is methylation of H3K9 residue. Present in the chromosomes across different eukaryotes, this epigenetic mark is controlled by SU(VAR)3-9 and its orthologs. Despite SU(VAR)3-9 was discovered over two decades ago, little is known about the details of its chromosomal distribution pattern. To fill in this gap, we used DamID-seq approach and obtained high-resolution genome-wide profiles for SU(VAR)3-9 in two somatic (salivary glands and brain ganglia) and two germline (ovarian nurse cells and testes) tissues of Drosophila melanogaster. Analysis of tissue and developmental expression of SU(VAR)3-9-bound genes indicates that in the somatic tissues tested, as well as in the ovarian nurse cells, SU(VAR)3-9 tends to associate with transcriptionally silent genes. In contrast, in the testes, SU(VAR)3-9 shows preferential association with testis-specific genes, and its binding appears dynamic during spermatogenesis. In somatic cells, the mere presence/absence of SU(VAR)3-9 binding correlates with lower/higher expression. No such correlation is found in the male germline. Interestingly, transcription units in piRNA clusters (particularly flanks thereof) are frequently targeted by SU(VAR)3-9, and Su(var)3-9 mutation affects the expression of select piRNA species. Our analyses suggest a context-dependent role of SU(VAR)3-9. In euchromatin, SU(VAR)3-9 may serve to fine-tune the expression of individual genes, whereas in heterochromatin, chromosome 4, and piRNA clusters, it may act more broadly over large chromatin domains.

Preparation of dendritic cells for cancer immunotherapy

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.

Molecular genetic markers in diagnosis of lung cancer

The review considers the main approaches to the identification of lung cancer (LC) markers, including genetic, epigenetic, protein, transcriptomic, proteomic, metabolic, and miRNA markers. Emphasis is placed on epigenetic markers, which are the most promising because epigenetic changes are among the earliest events in malignant transformation. Special attention is given to circulating tumor markers, which can be detected in easily accessible biological fluids with minimally invasive methods and may be useful for screening the risk groups for LC, diagnosing cancer before its clinical manifestation, monitoring the tumor in remission after therapy, and verifying the diagnosis based on standard clinical and instrumental methods of diagnostics. Extracellular nucleic acids (circulating in blood, circNA) are highlighted as a potential source of material for early diagnosis of LC, prediction of the efficiency of antitumor treatment, posttreatment monitoring, and disease prognosis.

Cell Surface Oligonucleotide-Binding Proteins of Human Squamous Carcinoma A431 Cells

Abstract Affinity modified with Flu-DAP-p(N)16degU oligonucleotide-binding proteins were isolated by affinity chromatography using Ultrogel A2 - anti fluorescein antibodies. After separation by SDS-PAGE the proteins with molecular masses about 68 kDa were MS/MS sequenced and identified as keratin K1, keratin K10, keratin K2e and albumin.