A MULTIPLEX PCR ASSAY FOR THE DIFFERENTIAL DETECTION OF OPISTHORCHIS FELINEUS AND METORCHIS BILIS.

Opisthorchis felineus and Metorchis bilis are two common small worms that parasitize in the gallbladder and bile ducts of the liver of humans and carnivores. These parasites have a severe impact on health and are considered pathogens of serious diseases worldwide, such as cholangiocarcinoma. However, there are still no commercially available molecular diagnostic kits capable of simultaneously detecting these parasites in humans. Therefore, the study aimed to develop a multiplex PCR analysis that will differentially determine these two opisthorchiasis infections in one reaction. Two specific primer pairs for a multiplex polymerase chain reaction (PCR) were designed based on corresponding mitochondrial genome sequences. The multiplex assay detection limit was assessed by serial dilutions of the genomic DNAs of trematode worms examined. Naturally, infected samples of human bile and feces were tested using the developed assay. A multiplex PCR assay was developed based on mitochondrial DNA that accurately and simultaneously identifies two trematode species in one reaction using specific fragment sizes of 307 and 252 bp for O. felineus and M. bilis, respectively. The optimal reaction conditions, specificity, and sensitivity of the multiplex PCR assay were investigated. The lowest DNA concentration detected was 100 pg for M. bilis and O. felineus in a 25µl reaction system. This study provides an efficient tool for the simultaneous detection of O. felineus and M. bilis. The proposed multiplex PCR assay will be potentially useful in epidemiological studies, diagnosis, and treatment of this mixed opisthorchiasis infection.

Morphological re-description and molecular characterization of Chloromyxum thymalli (Myxosporea: Chloromyxidae) infecting the gall bladder of Hovsgol grayling Thymallus nigrescens.

Chloromyxum thymalli infects the gall bladder of fishes belonging to the genus Thymallus. Here, we provide morphological, histological, and molecular data to complete the original description. Rounded plasmodia measured from 25 to 30 µm in diameter and contain 8 developing spores at most. Spores are typical of the genus Chloromyxum. Mature spores are subspherical, (mean ± SD) 9.05 ± 0.08 µm (range: 7.75-10.03 µm) long, and 8.81 ± 0.09 µm (range: 7.19-10.01 µm) wide. Four pyriform polar capsules are equal in size, 3.4 µm long and 2.7 µm wide on average. Histopathological analysis showed that presporogonic stages were found in the lamina propria under the epithelial cells of the host's gall bladder and destroyed the integrity of the epithelium. The obtained SSU rDNA sequences of C. thymalli did not match any available sequences in GenBank. Phylogenetic analysis showed that C. thymalli forms a sister cluster with C. truttae, with a subclade of Chloromyxum spp. that infect the gall bladder of freshwater teleosts.

[Role of opisthorchis felineus on induction of bile duct cancer].

Opisthorchis felineus (Trematoda) is widespread in the Russian Federation, especially in Siberia, and other countries of Europe. Infestation of endemic area population with O. felineus reaches 80%. On animal models of the infection of closely related Opisthorchis viverrini combined with the nitrosamines' intake it has been shown that the parasite induces cholangiocarcinoma. However carcinogenic potential of O. felineus is still poorly studied. The present study is aimed to investigate the role of O. felineus in cholangiocarcinoma carcinogenesis in hamster treated additionally by dimethylnitrosamine (DMN). Golden hamsters were divided into 4 groups (15 specimens in the control group and 20 for other groups): (I) untreated control, (II) 12.5 ppm DMN solution intake, (III) infected with 50 metacercariae of O. felineus and (IV) infected with 50 metacercariae of O. felineus and 12.5 ppm DMN solution intake. According to the histological data, in the. O. felineus-infested group significant hyperplastic and dysplastic biliary changes were found considered as a precancerogenic state. Such pathological changes of bile ducts were more severe in group treated with both factors, with cholangiocarcinoma being found out at 18th week in all the animals of this group. These results demonstrate that O. felineus could play promoting role in two-step model in cholangiocarcinoma carcinogenesis and may be used to define the O.felineus group in the International Agency for Research on Cancer classification of agents, mixtures and exposures (IARC categories).

[Secretome of the adult liver fluke Opisthorchis felineus].

The opisthorchiasis caused by Opisthorchis felineus, the Siberian liver fluke remains a serious public health problem in Russia and Eastern Europe. Proteomic identification of the proteins in the excretory-secretory products (ESPs) released by O. felineus is an important key for the investigation of host-parasite interactions and understanding the mechanisms involved in parasite survival within the host. In the ESP of O. felineus we have identified 37 proteins using high-resolution proteomics approach (LTQ-FT-ICR mass spectrometer). The O. felineus secretes either excretes a complex mixture of proteins including: glycolytic enzymes (enolase, aldolase, fructose-1 ,6-bisphosphatase and other); detoxification proteins (4 isoform of glutathione S-transferases, Cu/Zn superoxide dismutase, thioredoxin peroxidase, thioredoxin); cytoskeletal proteins (beta tubulin and paramyosin); a number of proteases (cathepsin F, B1, leucin aminopeptidase 2); protease inhibitors (putative cys1 protein, leukocyte elastase inhibitor), binding proteins (ferritin, myoglobin, FABP) and other. In the O. felineus ESP we also identified Of-HDM protein belonging to a novel family "helminth defence molecules" (HDMs). The O. felineus proteins identified in this study provide necessary information for the further investigation of molecular mechanisms of opisthorchiasis pathogenesis and some of them would be of interest as potential antigens for vaccine and immunodiagnostics development and as potential new anthelmintic drug targets.

[The reagents kit to detect Metyorchis biis, Opisthorchis viverrini and Clonorchis sinensis, Opisthorchis felineus--agents of opisthorchiasis using technique of polymerase chain reaction in real-time].

The helminths Opisthorchis felineus, Opisthorchis viverrini, Clonorchis sinensis, Metorchis bilis are the agents of opisthorchiasis. The actual diagnostic of parasitic diseases based on microscope analysis of samples of human feces to detect presence of ova of parasites suffers of many shortcomings, in particular low sensitivity especially at earlier stages. The purpose of this study was to compare results of detection of parasites using both classical technique and technique of specific differentiation based on extraction of nucleic acids from samples of human feces and implementation of reaction of amplification of the chosen fragment of DNA with detection of products of polymerase chain reaction in the real time. The study detected 150 out of 165 positive samples and also 6 out of 37 negative samples both validated by coproovoscopy.

[Molecular genetic identification and phylogeny of Daphnia species (Crustacea, Cladocera) from water bodies of Lake Chany basin].

The data on the molecular genetic identification of Daphnia species from the water bodies of Lake Chany basin are presented. Phylogenetic relationships between these species have been established. The fragments of the mitochondrial DNA 16S and 12S genes were used as genetic markers. According to the data obtained, the water bodies examined were inhabited by five Daphnia species, including Daphnia (Daphnia) galeata Sars, D. (D) longispina O. E Müller, D. (D) curvirostris Eylmann, D. (D) pulex Leydig, and D. (Ctenodaphnia) magna Straus. In addition, a group of longispina-like individuals that form a separate phylogenetic cluster was identified.

[DNA diagnosis of mixed invasions of Opisthorchis felineus and Metorchis bilis by polymerase chain reaction].

Opisthorchiasis is one of the significant naturofocal diseases in Russia. The diagnosis of opisthorchiasis may mask some diseases, such as opisthorchiasis, metorchiasis, and clonorchiasis - biohelminthoses that are induced by various representatives of the family Opisthorchiidae-Opisthorchis felineus/O.viverrini, Metorchis bilis, and Clonorchis sinensis, respectively. Coproovoscopy and serologic methods fail to accurately define the species-specific affiliation; in this connection the identification of opisthorchids, by using DNA diagnostic techniques, becomes urgent. The present paper gives the results of development of DNA diagnosticum, which differentiates parasitic diseases induced by O. felineus and M. bills. The ribosomal RNA gene cluster fragment incorporating internal transcribed spacer 2 (ITS2) was used as a diagnostic marker. A system for diagnosing opisthorchiasis was developed as a multiplex polymerase chain analysis and tested on 37 patients infected with various species of opisthorchids.

[The analysis of structure of insulin-dependent regulatory contours of mature adipocyte].

Insulin is the important regulator of adipose cell metabolism and activates the branched out network of the signaling pathways supervising glucose transport, glycogen synthesis, lipogenesis stimulation, lipolysis inhibition and adipokine secretion. The purpose of our work is the analysis of structure of regulatory contours providing the response of mammalian adipocytes to insulin. With use of computer technology GeneNet adipocyte regulatory-effector network has been reconstructed. The network generalizes experimental data concerning the main insulin-dependent signaling pathways and their targets in a context insulin-sensitive metabolic processes and transcription events. Analysis of the network revealed positive and negative regulatory contours including MAP kinase-, Cbl/TC10- and P13K-dependent signaling pathways. Regulatory contours functioning with participation of transcription factors SREBP-1c, PPARgamma/RXRalpha, C/EBPalpha, FOXO1 are defined also. The major effectors of regulatory contours are glucose carrier GLUT4, and kinase mTOR.

[Real-time polymerase chain reaction and its potential clinical application].

The review introduces real-time polymerase technique, a modern high-tech method, to the medical community. Physico-chemical principles of the method, its main stages, and variants of techniques applying fluorescent reporter platforms are considered. Equations used for quantitative estimation, methods of data processing are given; absolute and relative quantification of specific targets is considered. The special part of the review is dedicated to clinical application of the method by the example of infectious and oncological diseases.

[Molecular-cytogenetic characteristics of B-chromosomes in chironomid (Diptera, Chironomidae)]. / Molekuliarno-tsitogeneticheskaia kharakteristika B-khromosom khironomid (Diptera, Chironomidae).

Morphological and molecular study of B-chromosomes of three Chironomus species (siblings Ch. borokensis and Ch. phumosus from plumosus group, and Ch. heterodentatus from obtusidens group) was carried out. Morphological similarity of B-chromosome banding pattern and telomer-centromeric region banding pattern of chromosome IV in Ch. borokensis was shown. Polytene B-chromosomes of Ch. borokensis and Ch. heterodentatus were microdissected, and their DNA was amplified using degenerate oligonucleotide primer polymerase chain reaction. Comparative analysis of the localization of homologous B-chromosome DNA sequences of A- and B-polytene chromosomes was made using in situ fluorescence hybridization. It has been shown that B-chromosomes in the studied species are composed mainly of repetitive DNA sequences homologous to sequences of centromeric and telomeric DNA of A-chromosomes, and also these of the mobile element NLRCthl. The B-chromosome DNA, homologous to sequences of DNA mobile element, was scattered on A-chromosomes (more than 100 sites). No ribosomal DNA repeats were identified in B-chromosome. Heterologous FISH of B-chromosome DNA to polytene A-chromosomes of Ch. thummi, a species lacking B-chromosomes, enabled us to reveal the presence of numerous sites homologous to DNA of B-chromosomes. These are mainly mobile element sites. An origin of B-chromosomes and peculiarities of their organization in chironomids are discussed.

[Molecular genetic analysis of Thrithorax-like gene encoded transcriptional factor GAGA in Drosophila melanogaster]. / Molekuliarno-geneticheskii analiz gena Trithorax-like, kodiruiushchego transkriptsionnyi faktor GAGA Drosophila melanogaster.

The Drosophila melanogaster Trithorax-like (Trl) gene is classed with the trx-G genes and codes for several isoforms of the GAGA transcription factor (GAF) which regulates expression of homeotic and numerous other genes. GAF acts as a transcriptional antirepressor, i.e., its interaction with nucleosomal DNA results in the open chromatin conformation in promoter gene regions. The regions thereby become accessible to other transcription factors. As mutations of the Trl gene enhance position effect variegation and disturb chromosome segregation in mitosis and meiosis, GAF is thought to play another, more significant role in determining the chromatin structure. To study the molecular basis of its pleiotropic effect, the Trl gene was subjected to a structural analysis. The genomic Trl gene was sequenced, the sizes of its exons and introns was established, and a complex structure of the 5' and 3' gene regions was demonstrated. The Trl13C, Trl62, DfTrlR67, and DfTrlR85 mutations were exactly mapped. In addition, four insertions of the P element were identified as Trl alleles (Trll(3)s2325, TrlEP(3)3184, TrlEP(3)3191, and TrlEP(3)3609). The viability at various developmental stages was studied in homozygotes for the Trl mutations and in interallelic compounds. The following lethality stages were established: hatching, (Trl13C, DfTrlR85, TrlEP(3)3609), larval molts (Trll(3)s2325), pupation, metamorphosis (DfTrlR67, Trl62), and eclosion (several compounds).

[Computer analysis and recognition of Drosophila melanogaster gene promoters]. / Komp'iuternyi analiz i raspoznavanie promotorov genov Drosophila melanogaster.

A new method for recognizing eukaryotic gene promoters was based on their partition and on analysis of correlations of dinucleotide frequencies for each individual fragment. The method was used to recognize the TATA-containing and TATA-less promoters of Drosophila melanogaster genes. Dinucleotide context was correlated with conformational and physicochemical DNA properties in promoter fragments. Mean values of several parameters proved to dramatically change on transition from the TATA box to its GC-rich flanks. In TATA-less promoters, specific properties were revealed in the DPE region. The method was employed in a promoter recognition program, which is available through Internet.

[The effect of some mutations in the Trl gene on mitosis in embryonal and larval tissues and egg chamber morphology in Drosophila melanogaster]. / Vliianie nekotorykh mutatsii po genu Trl na mitoz v émbrional'noi i lichinochnykh tkaniakh i morfologiiu iaitsevykh kamer u Drosophila melanogaster.

A study was made of three insertional mutations (Trl13C, Trls2325, and TrlEP(3)3184) located in the second intron of the Trithorax-like (Trl) gene for the GAGA transcription factor (GAF). Their cytological effects were analyzed in oogenesis, early embryonic development, and in larval development (96-108 h) in cells of nervous ganglia and imaginal disks. Notwithstanding an interallelic difference in expression, all three P-element insertions proved to be dominant as far as the examined parameters were concerned. The most substantial defects were the formation of "granular" chromatin during the interphase and mitosis and high proportions of cells with hypercondensed chromatin (which were arrested at the G2/M boundary) and cells with abnormal chromosome segregation. A higher frequency of egg chambers with trophocytes defective in number and in chromatin condensation was observed in females carrying the mutant Trl gene. The defects were assumed to result from poor coordination of the chromosome and cell cycles and, including, the nuclear and centrosomal cycles in embryonic development and the cycles of chromosome condensation and spindle formation in cells of larval imaginal disks and nervous ganglia.

Expression of Drosophila melanogaster gene encoding transcription factor GAGA is tissue-specific and temperature-dependent.

The GAGA factor (GAF) of Drosophila melanogaster encoded by the Trithorax-like gene is known to maintain expression of many Drosophila genes including homeotic ones, through configuration remodeling of local chromatin. The complicated transcript pattern of the GAF gene has been revealed at all stages of development. The study of GAF gene expression in whole flies and in salivary glands and in the brains with adjacent imaginal disks of the third instar larvae showed tissue-specific variations in transcript patterns and dependence of these patterns on the temperature of development (14-37 degrees C).

[Cloning and cytogenetic localization of evolutionarily conserved genomic sequences which are expressed in the head of the Drosophila melanogaster imago]. / Klonirovanie i tsitogeneticheskaia lokalizatsiia évoliutsionno konservativnykh, ékspressiruiushchikhsia v golovakh imago genomnykh posledovatel'nostei Drosophila melanogaster.

Screening of Drosophila melanogaster genomic library was carried out using mouse brain polysomal poly(A)+RNA. As a result, 100 clones were selected, among which 14 clones were picked up after hybridization with fly head poly(A)+RNA. It follows therefore, that these clones contain evolutionary conserved sequences which are expressed in Drosophila fly heads. Analysis of these 14 clones revealed RNA-coding fragments. Comparison of their expression in heads and bodies of Drosophila was carried out. Using in situ hybridization we determined the localization of selected 14 sequences on polytene chromosomes. The possibility of further analysis of some clones to study developmental and functional processes in neural system of Drosophila is discussed.

[Cytogenetic localization of Acph-1 and Lap-1 genes in virilis group Drosophila]. / Tsitogeneticheskaia lokalizatsiia genov Acph-1 i Lap-1 u Drosophila gruppy virilis.

Gene Acph-1 coding for acid phosphatase was localized on Drosophila virilis chromosome 2 in the region 20A-20E and in D. texana and D. imeretensis homologous regions of the chromosomes 2. Gene Lap-1 coding for leucine aminopeptidase was linked to Acph-1 gene and localized in the 20E-20G region for these Drosophila species. The cytogenetical localization of two genes was determined after analysing recombinant chromosomes by isozymic and cytological methods in the progeny of interspecific hybrids.