Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations.     

Olfactory system of crustaceans: structural, functional organization and perspectives of ecologo-toxicological studies

Based on our own studies and literature data, we considered peculiarities of the structural-functional organization of the crustacean olfactory system and effect of pollutants on it. There are described changes of behavioral reactions based on chemoreception under conditions of pollution of the aquatic medium. Expedience of study of the crustacean olfactory system as a perspective object for ecologo-toxicological studies is substantiated.     

Adipose tissue inflammation and liver fat in patients with highly active antiretroviral therapy-associated lipodystrophy

In this cross-sectional study, we sought to determine whether gene expression of macrophage markers and inflammatory chemokines in lipoatrophic subcutaneous abdominal adipose tissue and liver fat content are increased and interrelated in human immunodeficiency virus (HIV)-1-positive, highly active antiretroviral therapy (HAART)-treated patients with lipodystrophy (HAART+LD+; n = 27) compared with those without (HAART+LD-; n = 13). The study groups were comparable with respect to age, gender, and body mass index. The HAART+LD+ group had twofold more intra-abdominal (P = 0.01) and 1.5-fold less subcutaneous (P = 0.091) fat than the HAART+LD- group. As we have reported previously, liver fat was 10-fold higher in the HAART+LD+ compared with the HAART+LD- group (P = 0.00003). Inflammatory gene expression was increased in HAART-lipodystrophy: CD68 4.5-fold (P = 0.000013), tumor necrosis factor (TNF)-alpha 2-fold (P = 0.0094), chemokine (C-C motif) ligand (CCL) 2 2.5-fold (P = 0.0024), CCL3 7-fold (P = 0.0000017), integrin alphaM (ITGAM) 3-fold (P = 0.00067), epidermal growth factor-like module containing, mucin-like, hormone receptor-like (EMR)1 2.5-fold (P = 0.0038), and a disintegrin and metalloproteinase domain (ADAM)8 3.5-fold (P = 0.00057) higher in the HAART+LD+ compared with the HAART+LD- group. mRNA concentration of CD68 (r = 0.37, P = 0.019), ITGAM (r = 0.35, P = 0.025), CCL2 (r = 0.39, P = 0.012), and CCL3 (r = 0.54, P = 0.0003) correlated with liver fat content. In conclusion, gene expression of markers of macrophage infiltration and adipose tissue inflammation is increased in lipoatrophic subcutaneous abdominal adipose tissue of patients with HAART-associated lipodystrophy compared with those without. CD68, ITGAM, CCL2, and CCL3 expression is significantly associated with accumulation of liver fat.     

MIC and MBC of quinupristin/dalfopristin of erythromycin-resistant MRSA strains isolated from clinical specimens

The MICs and MBCs of quinupristin/dalfopristin were determined for 22 clinical strains MRSA with inducible type of resistance to MLS-B and for 15 of their derivatives with constitutive resistance to MLS-B. For MRSA strains with inducible resistance to MLS-B the obtained results for quinupristin/ dalfopristin were: MIC50 = 0.25, MIC90 = 0.5, MBC50 = 1.0 and MBC90 = 1.0. Mutants of the same strains characterized with the following values for quinopristin/dalfopristin: MIC50 = 0.5, MIC90 = 1.5, MBC50 = 4.0 and MTC90 = 8.0.     

The frequency of the occurrence of genes ermA, ermB, ermC and msrA/B among methicillin-resistant Staphylococcus aureus strains resistant to erythromycin

The group of 50 clinical MRSA strains resistant to MLS-B was examined for the presence of ermA, ermB, ermC, msrA/B genes by using PCR. Gene ermA was found in 43 strains (86%). 20 of ermA strains demonstrated inducible whereas 23 constitutive type of expression. The gene ermC was present in 15 of examined MRSA strains (30%). The expression of the gene was inducible in the case of 9 and constitutive in the case of 6 of the strains. The msrA/B gene was present in the case of 5 strains (10%). The ermB gene was not detected among the investigated strains.     

Allocation and identification of fibrocytes from human peripheral blood

Fibrocytes are the cells circulating in peripheral blood that synthesize a big number of various factors and take part in the start of reclaiming processes. The wound healing is a result of activity of fibrocytes. It is known that they participate in formation of hypertrophic and kelloid scars. The purpose of the present work was to research specific properties of fibrocytes in vitro. The data obtained testify that these cells really have hematopoietic origin and are undifferentiated. In this connection, while cultivating fibrocytes it is necessary to keep to some specific conditions: the use of the medium specific for stem cells and very high density of cultivation. In 10 days of culturing, the fibrocytes differentiate into fibroblasts. From the general pool of peripheral blood mononuclear cells, only fibrocytes are capable of DNA synthesis but in spite of it proliferative potential of these cells is very low.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

Investigation of the surface heteroorganic antigens of rat hepatoma cells involved in process of cell proliferation

The investigation of antigenic diversion of hepatoma cells resulting from the expression of heteroorganic kidney antigens has been continued. Tumor-associated heteroorganic antigens 110-115 and 125-130 kDa were detected by immunoserum of narrow specificity in fractions of plasmatic membranes of cells of rat ascitic hepatoma Zajdela and cultured hepatoma HTC; the antigen 75-80 kDa was revealed only for hepatoma Zajdela cells. It has been shown by methods of radioisotope analysis and flow DNA-cytometry that heteroorganic antigens 110-130 kDa can be involved in process of cell proliferation.     

The MEK/ERK pathway is essential for maintenance of cytoprotective autophagy in <Emphasis Type="Italic">E1A</Emphasis>+<Emphasis Type="Italic">cHA</Emphasis>-<Emphasis Type="Italic">RAS</Emphasis> transformants after exposure to radiation

Autophagy is a conserved process of protein and organelle degradation that serves to maintain cell viability. Autophagy is frequently induced in response to stress or to exposure to DNA-damaging agents or retinoids, as well as to starvation and deficiency of growth factors. In this work, autophagy induced in E1A+cHA-RAS transformed cells in response to X-ray radiation was studied, with a focus on the role of the MEK/ERK signaling pathway in the regulation of radiation-induced autophagy. It was found that inhibition of the MEK/ERK pathway diminished cell viability and altered the sequence of events in radiation-induced autophagy. In particular, it caused aberrations in its final stages, leading to cytoplasmic accumulation of the p62/SQSTM1 adaptor protein in autophagic cavities of unclear origin. Thus, the MEK/ERK pathway activity is essential for the induction and maintenance of autophagy, increasing the viability of exposed cells in response to radiation.