[Algorithm for Physiological Interpretation of Transcriptome Profiling Data for Non-Model Organisms].

Modern techniques of next-generation sequencing (NGS) allow obtaining expression profile of all genes and provide an essential basis for characterizing metabolism in the organism of interest on a broad scale. An important condition for obtaining a demonstrative physiological picture using high throughput sequencing data is the availability of the genome sequence and its sufficient annotation for the target organism. However, a list of species with properly annotated genomes is limited. Transcriptome profiling is often performed in the so-called non-model organisms, which are those with unknown or poorly assembled and/or annotated genome sequences. The transcriptomes of non-model organisms are possible to investigate using algorithms of de novo assembly of the transcripts from sequences obtained as the result of RNA sequencing. A physiological interpretation of the data is difficult in this case because of the absence of annotation of the assembled transcripts and their classification by metabolic pathway and functional category. An algorithm for transcriptome profiling in non-model organisms was developed, and a transcriptome analysis was performed for the basidiomycete Lentinus edodes. The algorithm includes open access software and custom scripts and encompasses a complete analysis pipeline from the selection of cDNA reads to the functional classification of differentially expressed genes and the visualization of the results. Based on this algorithm, a comparative transcriptome analysis of the nonpigmented mycelium and brown mycelial mat was performed in L. edodes. The comparison revealed physiological differences between the two morphogenetic stages, including an induction of cell wall biogenesis, intercellular communication, ion transport, and melanization in the brown mycelial mat.

Pathogen-induced conditioning of the primary xylem vessels - a prerequisite for the formation of bacterial emboli by Pectobacterium atrosepticum.

Representatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant-Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific 'multicellular' structures - bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen-induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum-caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host-pathogen integration, since the formation of these structures requires the action of both partners.

Depolymerization of lignosulfonates by submerged cultures of the basidiomycete Irpex consors and cloning of a putative versatile peroxidase.

Lignosulfonates are abundantly available byproducts of the paper and pulping industry, and they therefore represent a promising feedstock for new sustainable processes. For industrial applications of lignosulfonates, their molecular weight distribution is a critical factor. In order to decrease the average molecular weight of lignosulfonates, Seventeen basidiomycetes were screened for their capability to depolymerize lignosulfonates from spent sulfite liquor (SSL) in surface and liquid cultures. Five basidiomycetes polymerized the lignosulfonates under the selected conditions. Only Irpex consors was found to efficiently degrade calcium lignosulfonates when SSL (0.5%, w/w) was used as the sole carbon and nitrogen source. The average molecular weight of the lignosulfonates was reduced from ∼26 to ∼4 kDa as determined by size exclusion chromatography (SEC) within two weeks. Various extracellular enzyme activities of I. consors were determined over the culture period. High peroxidase activities were correlating with a high degradation rate and the culture was harvested at the day of highest peroxidase activity. A putative versatile peroxidase was isolated by fast protein liquid chromatography (FPLC) and its encoding cDNA was cloned.

[Activity and expression of laccase, tyrosinase, glucanase, and chitinase genes during morphogenesis of Lentinus edodes].

Activation of expression of the lcc4 and tir genes encoding laccase and tyrosinase was observed during transition of a xylotrophic basidiomycete Lentinus edodes from the vegetative to the generative growth stages. This was especially pronounced in the brown mycelial mat (the stage preceding formation of the fruiting bodies). Development of this structure was shown to be associated with a sharp increase of laccase and tyrosinase activities, as well as with rearrangements in the phenol oxidase complex. Formation of the tissues with thickened cell walls was associated with enhanced expression of the chi and exg1 genes encoding chitinase and glucanase, respectively. Exogenous treatment of the vegetative mycelium with laccase preparation from the brown mycelial mat promoted formation of this morphological structure. Activation of the lcc4, tir, chi, and exg1 genes may be used as a marker of readiness to fruition in xylotrophic fungi.

[The effect of topology of quorum sensing-related genes in Pectobacterium atrosepticumon their expression].

In prokaryotic genomes, the neighboring genes are often located on the complementary DNA strands and adjoin each other by their 5'- or 3'-ends or even overlap by their open reading frames. It was suggested that such gene topology hasfunctional purpose providing the regulation of their expression. For those genes that overlap by their coding 3'-termini this assumption has not been confirmed experimentally. In a broad group of bacteria that belong to proteobacteria such a convergent gene arrangement is typical for functionally connected quorum sensing-related genes "P" and "R" that encode synthases of N-acyl homoserine lactones and their sensors, respectively. In the present study on the example of overlapping quorum sensing-related genes of plant pathogenic bacterium Pectobacterium atrosepticum SCRI1043--expI and expR it was shown that the topology of these genes determines the regula- tion of their expression.

Composition and antimicrobial activity of the skin peptidome of Russian brown frog Rana temporaria.

A nano-HPLC-ESI-OrbiTrap study involving HCD and ETD spectra has been carried out to clarify the composition of the skin peptidome of brown Russian frogs Rana temporaria. This approach allowed determinantion of 76 individual peptides, increasing 3-fold the identified portion of the peptidome in comparison to that obtained earlier with FTICR MS. A search for the new bradykinin related peptides (BRPs) was carried out by reconstructing mass chromatograms based on the ion current of characteristic b- and y-ions. Several peptides were reported in the secretion of R. temporaria for the first time. The overall antibacterial activity of the skin secretion in general and of one individual peptide (Brevinin 1Tb) was determined using PMEU Spectrion (Portable Microbe Enrichment Unit) technology. The inhibitory effects of these peptides on Staphylococcus aureus and Salmonella enterica Serovar typhimutium were equal in scale to that reported for some antibiotics.

Structure of 10He low-lying states uncovered by correlations.

The 0+ ground state of the 10He nucleus produced in the 3H(8He,p)10He reaction was found at about 2.1±0.2 MeV (Γ∼2 MeV) above the three-body ^{8}He+n+n breakup threshold. Angular correlations observed for ^{10}He decay products show prominent interference patterns allowing us to draw conclusions about the structure of low-energy excited states. We interpret the observed correlations as a coherent superposition of a broad 1- state having a maximum at energy 4-6 MeV and a 2+ state above 6 MeV, setting both on top of the 0+ state "tail." This anomalous level ordering indicates that the breakdown of the N=8 shell known in 12Be thus extends also to the ^{10}He system.

Quantitative analysis of SLC34A2 expression in different types of ovarian tumors.

AIM: The main purpose of this study was to estimate the SLC34A2 gene expression in normal ovary and different types of ovarian tumors. METHODS: We have investigated SLC34A2 gene expression level in papillary serous, endometrioid, unspecified adenocarcinomas, benign tumors, and normal ovarian tissues using real-time PCR analysis. Differences in gene expression were calculated as fold changes in gene expression in ovarian carcinomas and benign tumors compared to normal ovary. RESULTS: We have found that SLC34A2 gene was highly expressed in well-differentiated endometrioid and papillary serous ovarian carcinomas compared to low-differentiated endometrioid carcinomas, benign serous cystoadenomas and normal ovary. Analysis of SLC34A2 gene expression according to tumor differentiation level (poor- and well-differentiated) showed that SLC34A2 is up-regulated in well differentiated tumors. CONCLUSION: Upregulation of SLC34A2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis.

Mass spectrometric study of bradykinin-related peptides (BRPs) from the skin secretion of Russian ranid frogs.

Amphibian skin secretion is known to contain biologically active peptides. Bradykinins and related peptides (BRPs) can be found in these animals, while frogs from the genus Rana are considered to be leaders in the levels and variety of these peptides. A reasonable rationalization of this fact is that bradykinins are efficient defense compounds against predators. Forty-four various BRPs have been identified in the skin secretions of five ranid frog species (R. ridibunda, R. lessonae, R. esculenta, R. temporaria, R. arvalis) from the Zvenigorod region (Moscow district, Russia). Some of these peptides are already known, but the novel ones constitute a significant portion. An interesting group of novel peptides was isolated from R. lessonae. These are bradykinin analogues bearing a tyrosine residue in the 5th or 8th position. [Arg(0), Trp(5), Leu(8)]bradykinin and [Thr(6), Leu(8)]bradykinin that had been isolated from fish and avian species, respectively, were also detected in the frog secretion, supporting the predator defense hypothesis. Furthermore, a novel group of BRPs named 'lessonakinins' was discovered in R. lessonae and R. esculenta. All of them include the [Arg(0), Trp(5), Leu(8)]bradykinin sequence and have some structural resemblance to the precursor of this peptide cloned by Chen and coworkers recently. However, the C-terminal part of the lessonakinins does not match the sequence predicted by Chen, demonstrating possible incompleteness of information obtained by cDNA cloning.

Novel natural peptides from Hyla arborea schelkownikowi skin secretion.

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano-electrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron-capture dissociation (ECD) and collision-induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b-ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins.

[Identification and characterization of non-cultivated forms of enterobacteria Erwinia carotovora in continuously incubated cultures].

AIM: To determine overall number as well as number of viable cells in continuously incubated cultures of E. carotovora by methods of confocal microscopy and quantitative PCR-analysis. MATERIALS AND METHODS: Strain E. carotovora atroseptica SCRI1043 was grown on LB medium to density 2x10(9) CFU/ml. Cells were aggregated by centrifugation and transferred on fresh LB medium, containing alkyloxybenzol, or on the AB medium, which was deficient on phosphorus and carbon. BacLight LIVE/ DEAD kit in combination with confocal laser microscopy as well as quantitative PCR were used for the determination of the number of viable cells. RESULTS: Total number and number of viable cells in cultures on AB medium was high (10() - 10(9) and 10(7) - 10(8) cells/ml respectively) up to 3 - 5 months of cultivation. Though, number of cultivated cells significantly decreased in all variants of the experiment. Number of viable cells in such cultures was several orders greater than genomic copies detected by PCR. Efficacy of DNA amplification increased after dialysis and deproteinization of samples. CONCLUSION: Loss of cultivation ability when number of viable bacteria is high points to possible switch of E. carotovora cells in non-cultivated state under unfavourable conditions. We assume that it is accompanied by formation of low-molecular components and DNA-bound proteins in cells, which inhibit PCR.

Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region.

A high-performance liquid chromatography nano-electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI-FTMS) approach involving recording of collision-activated dissociation (CAD) and electron-capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid-throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring ('rana box'). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin-related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda.

[A new type of breast image upon dual-energy x-ray exposure].

The new possibilities of using scattered x-ray radiation in mammology are presented by the example of obtaining the new type of a highly informative breast image, by identifying the distribution of an effective atomic number by dual-energy exposure.

Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges.

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.

Chemical characterization of element 112.

The heaviest elements to have been chemically characterized are seaborgium (element 106), bohrium (element 107) and hassium (element 108). All three behave according to their respective positions in groups 6, 7 and 8 of the periodic table, which arranges elements according to their outermost electrons and hence their chemical properties. However, the chemical characterization results are not trivial: relativistic effects on the electronic structure of the heaviest elements can strongly influence chemical properties. The next heavy element targeted for chemical characterization is element 112; its closed-shell electronic structure with a filled outer s orbital suggests that it may be particularly susceptible to strong deviations from the chemical property trends expected within group 12. Indeed, first experiments concluded that element 112 does not behave like its lighter homologue mercury. However, the production and identification methods used cast doubt on the validity of this result. Here we report a more reliable chemical characterization of element 112, involving the production of two atoms of (283)112 through the alpha decay of the short-lived (287)114 (which itself forms in the nuclear fusion reaction of 48Ca with 242Pu) and the adsorption of the two atoms on a gold surface. By directly comparing the adsorption characteristics of (283)112 to that of mercury and the noble gas radon, we find that element 112 is very volatile and, unlike radon, reveals a metallic interaction with the gold surface. These adsorption characteristics establish element 112 as a typical element of group 12, and its successful production unambiguously establishes the approach to the island of stability of superheavy elements through 48Ca-induced nuclear fusion reactions with actinides.

Beam wandering in the atmosphere: the effect of partial coherence.

The effect of a random phase screen on laser beam wander in a turbulent atmosphere is studied theoretically. The photon distribution function method is used to describe the photon kinetics of both weak and strong turbulence. By bringing together analytical and numerical calculations, we have obtained the variance of beam centroid deflections caused by scattering on turbulent eddies. It is shown that an artificial distortion of the initial coherence of the radiation can be used to decrease the wandering effect. The physical mechanism responsible for this reduction and the applicability of our approach are discussed.

[Scattered X-ray radiation in diagnosis of mammary gland diseases].

The possibility of estimating the effective atomic number distribution from the ratio of the scattering and total absorption coefficients is considered. To illustrate the use of this technique in mammology, the results of measurement of microcalcinate and cholesterol distribution are discussed. It is suggested that tomographic examination of mammary gland should include detection of scattered radiation.

Doubly magic nucleus (108)(270)Hs162.

Theoretical calculations predict 270Hs (Z=108, N=162) to be a doubly magic deformed nucleus, decaying mainly by alpha-particle emission. In this work, based on a rapid chemical isolation of Hs isotopes produced in the 26Mg+248Cm reaction, we observed 15 genetically linked nuclear decay chains. Four chains were attributed to the new nuclide 270Hs, which decays by alpha-particle emission with Qalpha=9.02+/-0.03 MeV to 266Sg which undergoes spontaneous fission with a half-life of 444(-148)(+444) ms. A production cross section of about 3 pb was measured for 270Hs. Thus, 270Hs is the first nucleus for which experimental nuclear decay properties have become available for comparison with theoretical predictions of the N=162 shell stability.