Micropropagation of berry crops for creation of germplasm cryobanks.

One of the main stages of cryopreservation of meristematic tissues in vegetative plants is a clonal micropropagation, which includes isolating the explants of the raw material in vitro and optimizing the culture medium for micropropagation. As the result of our studies, the optimal periods for in vitro micropropagation are: first - isolation of explants from initiated shoots of dormant buds (blackcurrants and raspberries) in January-March; the second - from actively growing shoots (blackcurrants and raspberries) in May-June, from the formed mustache (strawberry) in July-August. The optimal drugs for sterilization of raspberry explants are: a) 0.1% HgCl2 (6 min), then 3% H2O2 (15 min); b) chlorine-containing bleach «Domestos¼ in the dilution of H2O 1:9 (10 min). For blackcurrant: a) 0.1% HgCl2 (5 min) in combination with 0.1% fungicide "Topaz" (30 min); b) 0.1% HgCl2 (5 min) in combination with the treatment with KMnO4 (30 min); c) "Domestos" in the dilution of H2O 1:5 (20 min). For strawberry: a) 0.1% HgCl2 (6 min) followed by treatment with 3% H2O2 10 (min); b) 1% deochlor (7 min), 3% H2O2 (10 min); c) "Domestos" in the dilution of H2O 1:5 (8 min) with subsequent treatment 0,1% HgCl2 -7 min, then 0,20 mg/l КМnO4 - 30 min. Optimal compositions of culture media for micropropagation of blackcurrant - Murashige and Skoog (MS) medium with 0.5 mg L-1 BAP, 0.5 mg L-1 GA3, 0.1 mg L-1 IBA and 20 g L-1 glucose. For raspberry -MS medium with 0.5 mg L-1 BAP, 0.1 mg L-1 IBA, 10 mg L-1 iron chelate and 30 g L-1 sucrose. For strawberry - MS medium with 0.3 mg L-1 BAP, 0.01 mg L-1 IBA, 0.2 mg L-1 GA3, 10 mg L-1 iron chelate and 30 g L-1 sucrose. Based on these studies, the cryobank was created, which include the germplasm of in vitro meristematic tissues in 66 cultivars, hybrids and wild-growing forms of blackcurrant, raspberry and strawberry. Therefore, the aim of the research was to obtain aseptic plants, clonal micropropagation and the creation of a cryogenic collection of germplasm based on the developed technology.

Prediction of massive coronary thrombosis of the infarct-related artery in ST-elevation myocardial infarction.

Aim    To identify clinical, laboratory and angiographic predictors for development of massive coronary thrombosis in patients with ST-segment elevation myocardial infarction (STEMI).Material and methods    This prospective, single-site study included 137 patients with STEMI (mean age, 66.5±13.2 years). Among these patients, 59 were in the group of massive coronary thrombosis and 78 patients were in the group of minor thrombosis. To identify predictors for the development of massive coronary thrombosis, medical history data, blood count and biochemistry, coagulogram, and angiography data were analyzed. A predictive model was constructed using the method of binary logistic regression followed by a search for the optimum value of the prognostic function with a ROC analysis. Results    The study showed statistically significant roles of total bilirubin, platelets, prothrombin ratio (PTR), activated partial thromboplastin time (APTT), and presence of inferior myocardial infarction in prediction of massive coronary thrombosis in STEMI. The model sensitivity was 71.2 %, specificity 75.6 %, and overall diagnostic efficacy 73.7 %.Conclusion    The predictive model for the development of massive coronary thrombosis in STEMI based on laboratory and instrumental data potentially allows assessing the thrombus load in the infarction-involved coronary artery and determining the optimum tactics of percutaneous coronary intervention in patients with STEMI. This reduces the probability of distal embolization with fragments of the disintegrated thrombus and improves the prognosis of STEMI patients both during the stay in the hospital and in the long-term. According to results of this study, the prognostic model for massive coronary thrombosis in STEMI based on such indexes as the platelet count, PTR, APTT, total bilirubin, and presence of inferior myocardial infarction provides accurate predictions in 73.7 % of cases. Independent predictors of massive coronary thrombosis were inferior myocardial infarction and total bilirubin.

The Genomics of Cannabis and Its Close Relatives.

Cannabis sativa L. is an important yet controversial plant with a long history of recreational, medicinal, industrial, and agricultural use, and together with its sister genus Humulus, it represents a group of plants with a myriad of academic, agricultural, pharmaceutical, industrial, and social interests. We have performed a meta-analysis of pooled published genomics data, andwe present a comprehensive literature review on the evolutionary history of Cannabis and Humulus, including medicinal and industrial applications. We demonstrate that current Cannabis genome assemblies are incomplete, with ∼10% missing, 10-25% unmapped, and 45S and 5S ribosomal DNA clusters as well as centromeres/satellite sequences not represented. These assemblies are also ordered at a low resolution, and their consensus quality clouds the accurate annotation of complete, partial, and pseudogenized gene copies. Considering the importance of genomics in the development of any crop, this analysis underlines the need for a coordinated effort to quantify the genetic and biochemical diversity of this species.

Simultaneous Percutaneous Coronary Intervention and Endovascular Closure of Atrial Septal Defect in Adults.

AIM: to assess clinical efficacy and expediency (appropriateness) of simultaneous single stage combined coronary stenting and closure of atrial septal defect. MATERIALS AND METHODS: Of total number of patients who underwent endovascular correction of atrial septal defect (ASD) (n=91), in 6 (6.6 %) the procedure of endovascular repair of secondary ASD was combined with performed at same session oronary stenting. Mean age of these patients was 63±6.4 years. Mean diameter of ASD according to transesophageal echocardiography was 13.7±3.1 мм (from 10 to 17 mm). Two patients had dysplasia of atrial septum with pronounced aneurysmal protrusion in the right atrial cavity. Estimate of coronary arteries (CA) involvement SYNTAX score was 14.5±4.9. RESULTS: At initial stage we performed coronary stenting, then ASD closure with occluder. Technical success of combined endovascular procedures was 100 %. Six ASD occluders were implanted in 6 patients. Mean occluder diameter was 21±7,3 mm. Immediately after occluder implantation complete defect closure was achieved in 5 cases, in one case small residual shunt was observed. CA stenting procedure, in one patient after successful recanalization of chronic CA occlusion, in all cases was fulfilled without complications. At control examination after 13.5±1.5 months complete closure of defects was preserved. In all cases significant reduction of right heart chambers occurred. According to echocardiography right atrial volume decreased from 48.6±5.6 to 32.6±3.3 cm3, right ventricular volume - from 45.2±5.1 to 33.4±3.8 cm3, systolic pulmonary pressure fell from 49.7±8.6 to 32.6±6.9 mm Hg. According to control coronary angiography good effect of endovascular procedures was preserved. Tolerance to exercise rose from 68.5±11.8 до 85.3±12.4 W. CONCLUSION: Same time CA stenting and endovascular ASD closure appears to be safe and effective procedure. The strategy used was not associated with additive risk for a patient and shortened duration of hospital stay.

[Brucellosis in the Stavropol Territory: Results of 15-year follow-up of epidemiological and clinical features].

AIM: To analyze the epidemiological situation of brucellosis in the Stavropol Territory and to determine the specific features of its acute form. MATERIALS AND METHODS: The epidemiological situation of brucellosis in the Stavropol Region was retrospectively analyzed using the 2000-2014 official statistics of the Russian Federation and Stavropol Territory for brucellosis cases, individual documents of the Russian Federal Service for Supervision of Consumer Rights Protection and Human Welfare Directorate in the Stavropol Territory and Russian Federation, and medical records of the Brucellosis Department, Stavropol Hospital Two. RESULTS: Brucellosis is a social problem in a group of focal zoonotic infections in the Russian Federation. In the North Caucasian Federal District, the largest number of new human brucellosis cases was notified in the Republic of Dagestan (59.3%) and Stavropol Territory (27.4%). The incidence of brucellosis in the Stavropol Territory was 5-10-fold higher than that in the Russian Federation as a whole. The true prevalence of brucellosis was 25% greater than the statistical data in terms of its clinical forms that were not included in official registration. Infection occurred in humans by contact with large and small cattle in 49.7% of cases. The disease mainly affected men (78.3%) and able-bodied people (67.7%); 78.5% of the patients seeking medical advice and as high as 79.9% of those being covered by serological examination of groups at risk for brucellosis were detected. There was a declining trend for the number of occupational brucellosis cases. At the same time, brucellosis accounted for 75% of the territory's total number of occupational diseases. In the past decade, brucellosis has exhibited a relatively benign course (91.3% for its moderate form), by predominantly involving the osteoarticular system (63%). Non-compliance with outpatient antibacterial therapy is one of the factors for brucellosis chronization. 28.2% of the patients who had undergone a complete therapy cycle developed chronic brucellosis less frequently (p < 0.005) than those who had stopped treatment for different reasons. CONCLUSION: Registration of brucellosis among humans is a result of the unfavorable epizootic situation of animal brucellosis. The highest incidence of brucellosis is observed in the northeastern areas of the Stavropol Territory, which border the Republic of Dagestan. The true prevalence of brucellosis greatly exceeds the official statistical data. Active detection of infection and timely diagnosis will be able to determine the real epidemic situation of brucellosis. Continuity of management and follow-up will provide a reduction in medical and social losses associated with this infection.

Growth medium alterations improve in vitro cold storage of pear germplasm.

BACKGROUND: Development of new fruit cultivars is dependent on genetic resource collections such as those at the Pomological Garden of the Institute of Horticulture and Viticulture near Almaty, Kazakhstan. The pear germplasm collection of the Pomological Garden contains 615 cultivars and three species. In vitro cold storage of the collection would provide additional security to the field collection. OBJECTIVE: This study was designed to improve medium-term in vitro storage of pear germplasm. METHODS: Shoots of seven pear cultivars (Pyrus communis L.) were stored in plastic five-section bags at 4 degree C and a 10-h photoperiod (7 µmol/m2/s). Treatments included medium with four carbohydrate sources (3% sucrose, 2% or 3% mannitol, or 2% sucrose + 2% mannitol) with 0.5 mg/l BAP and 0.1 mg/l IBA or without plant growth regulators (PGRs) and at three Murashige and Skoog (MS) nitrogen concentrations (100%, 50% or 25%). RESULTS: Pear shoots remained viable for 9 to 15 months without repropagation on the control MS medium with 3% sucrose without PGRs. There were significant impacts of cultivar and treatment on the duration of cold storage. Shoots of 'Mramornaya' remained viable (rating of ≥ 2) for 27 months with PGRs and 2% sucrose + 2% mannitol compared to 12 months for the PGR + 3% sucrose treatment. Talgarskaya Krasaviza stored for 18 months on 2% sucrose + 2% mannitol while all other treatments lasted only 6 to 9 months. Treatments with 0.5 or 1 mg/l abscisic acid (ABA) with 3% sucrose increased storage duration as did reducing the concentration of nitrogen in the medium to 25% without PGRs and with 3% sucrose.

Biomonitoring the genotoxicity of environmental factors with transgenic plants.

All organisms must react to constantly changing surroundings. Environmental factors are thus powerful forces continuously shaping the genomes of all species. Induced genetic changes can be followed using a biomonitor - a living organism that reacts to a given compound in the environment. A vital but challenging task is identifying organisms with which to study the influence of changing environmental conditions. Plants are especially valuable biomonitors. Here, we describe the use of transgenic plant systems to evaluate the genotoxicity of chemical and radiological compounds. We evaluate the potential of further transgene-based systems for studying somatic and germ-line mutations.

A sensitive transgenic plant system to detect toxic inorganic compounds in the environment.

We describe a transgenic plant-based assay to study the genetic effects of heavy metals. Arabidopsis thaliana plants carrying a beta-glucuronidase (GUS) marker gene either with a point mutation or as a recombination substrate were used to analyze the frequency of somatic point mutations and homologous recombination in whole plants. Transgenic test plants sown on media contaminated by the salts of the heavy metals Cd2+, Pb2+, Ni2+, Zn2+, Cu2+, and As2O3 exhibited a pronounced uptake-dependent increase in the frequencies of both somatic intrachromosomal recombination and point mutation. The test was applied to monitor the genotoxicity of soils sampled in sites contaminated with several heavy metals. Our results indicate that this is a highly sensitive system for monitoring metal contamination in soils and water.

A new hyperrecombinogenic mutant of Nicotiana tabacum.

We have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six- to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non-homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma-irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up-regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.

Genome-wide variation of the somatic mutation frequency in transgenic plants.

In order to analyse the frequency of point mutations in whole plants, several constructs containing single nonsense mutations in the beta-glucuronidase (uidA) gene were used to generate transgenic Arabidopsis thaliana plants. Upon histochemical staining of transgenic plants, sectors indicative of transgene reactivation appeared. Reversion frequencies were in the range of 10(-7)-10(-8) events per base pair, exceeding the previous estimates for other eukaryotes at least 100-fold. The frequency was dependent on the position of the mutation substrate within the transgene and the position of the transgene within the Arabidopsis genome. An inverse relationship between the level of transgene transcription and mutation frequency was observed in single-copy lines. DNA-damaging factors induced the mutation frequency by a factor of up to 56 for UV-C, a factor of 3 for X-rays and a factor of 2 for methyl methanesulfonate. This novel plant mutation-monitoring system allowed us to measure the frequencies of point mutation in whole plants and may be used as an alternative or complement to study the mutagenicity of different environmental factors on the higher eukaryote's genome.

[Information analysis of spinal ganglia]. / Informatsionnyi analiz spinnomozgovykh gangliev.

Information parameters (entropia and redundancy) of cervical and thoracic spinal ganglia of albino rat foetuses, mature animals (cat and dog) and human subjects were analysed. Information characteristics of spinal ganglia were shown to be level-specified and to depend on their functional peculiarities. Information parameters of thoracic spinal ganglia of man and different animals are specie specified and may be used in assessment of morphological structures as information systems.

Plants experiencing chronic internal exposure to ionizing radiation exhibit higher frequency of homologous recombination than acutely irradiated plants.

Ionizing radiation (IR) is a known mutagen responsible for causing DNA strand breaks in all living organisms. Strand breaks thus created can be repaired by different mechanisms, including homologous recombination (HR), one of the key mechanisms maintaining genome stability [A. Britt, DNA damage and repair in plants, Annu. Rev. Plant. Phys. Plant Mol. Biol., 45 (1996) 75-100; H. Puchta, B. Hohn, From centiMorgans to basepairs: homologous recombination in plants, Trends Plant Sci., 1 (1996) 340-348.]. Acute or chronic exposure to IR may have different influences on the genome integrity. Although in a radioactively contaminated environment plants are mostly exposed to chronic pollution, evaluation of both kinds of influences is important. Estimation of the frequency of HR in the exposed plants may serve as an indication of genome stability. We used previously generated Arabidopsis thaliana and Nicotiana tabacum plants, transgenic for non-active versions of the beta-glucoronidase gene (uidA) [P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recombination in whole plants, EMBO J., 13 (1994) 484-489; H. Puchta, P. Swoboda, B. Hohn, Induction of homologous DNA recombination in whole plants, Plant, 7 (1995) 203-210.] serving as a recombination substrate, to study the influence of acute and chronic exposure to IR on the level of HR as example of genome stability in plants. Exposure of seeds and seedlings to 0.1 to 10.0 Gy 60Co resulted in increased HR frequency, although the effect was more pronounced in seedlings. For the study of the influence of chronic exposure to IR, plants were grown on two chemically different types of soils, each artificially contaminated with equal amounts of 137Cs. We observed a strong and significant correlation between the frequency of HR in plants, the radioactivity of the soil samples and the doses of radiation absorbed by plants (in all cases r0.9, n=6, P<0.05). In addition, we noted that plants grown in soils with different chemical composition, but equal radioactivity, exhibited different levels of HR, dependent upon the absorbed dose of radiation. Remarkably, we observed a much higher frequency of HR in plants exposed to chronic irradiation when compared to acutely irradiated plants. Although acute application of 0.1-0.5 Gy did not lead to an increase of frequency of HR, the chronic exposure of the plants to several orders of magnitude lower dose of 200 muGy led to a 5-6-fold induction of the frequency of HR as compared to the control.

Radiation hazard caused by the Chernobyl accident in inhabited areas of Ukraine can be monitored by transgenic plants.

The genetic impact of the 1986 accident at the Ukraine Chernobyl Nuclear Power Plant (NPP) on populations of living organisms has yet to be fully assessed. Monitoring of the genotoxicity of polluted soils is a key element in the disaster management program. We used Arabidopsis thaliana and Nicotiana tabacum plants transgenic for a reporter gene revealing homologous recombination to study the genetic effects of chronic low-dose radiation stemming from the soil in inhabited areas of Ukraine where contamination by the accident ranges from 1 to 40 Ci/km2. We noted a significant dose-dependent increase of homologous recombination in plants cultivated in the affected inhabited areas, proving the persistently high genotoxicity of the radioactively contaminated soils.

Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident.

To evaluate the genetic consequences of radioactive contamination originating from the nuclear reactor accident of Chernobyl on indigenous populations of plants and animals, it is essential to determine the rates of accumulating genetic changes in chronically irradiated populations. An increase in germline mutation rates in humans living close to the Chernobyl Nuclear Power Plant site, and a two- to tenfold increase in germline mutations in barn swallows breeding in Chernobyl have been reported. Little is known, however, about the effects of chronic irradiation on plant genomes. Ionizing radiation causes double-strand breaks in DNA, which are repaired via illegitimate or homologous recombination. We make use of Arabidopsis thaliana plants carrying a beta-glucuronidase marker gene as a recombination substrate to monitor genetic alterations in plant populations, which are caused by nuclear pollution of the environment around Chernobyl. A significant (p<0.05) increase in somatic intrachromosomal recombination frequencies was observed at nuclear pollution levels from 0.1-900 Ci/km2, consistent with an increase in chromosomal aberrations. This bioindicator may serve as a convenient and ethically acceptable alternative to animal systems.

The Allium cepa chromosome aberration test reliably measures genotoxicity of soils of inhabited areas in the Ukraine contaminated by the Chernobyl accident.

The accident on the Chernobyl Nuclear Power Plant reactor IV in April 1986 led to the release of an enormous amount of radioactive material into the biosphere and to the formation of a complex pattern of nuclear contamination over a large area. As a consequence more than 5 million km2 of the soil in the Ukraine became contaminated with more than 1 Ci/km2 [1,2]. An assessment of the genetic consequences of the nuclear pollution is one of the most important problems. We applied the Allium cepa test to estimate the impact on plant chromosomes of nuclear pollution in the inhabited zones of the Ukraine. We tested soil from the obligatory resettlement zone (zone 2), where the mean density of pollution is 15-40 Ci/km2; zones of enhanced radiological control-zone 3, 5-15 Ci/km2 and zone 4, 1-5 Ci/km2. We found a dose-dependent increase in the fraction of aberrant mitoses from control values of 1.6 +/- 0.9% up to 23.8 +/- 5.0%, and a corresponding monotonous decrease of the mitotic index from 49.4 +/- 4.8% to a limiting value of 22.5 +/- 4.0% at pollution levels exceeding 35 Ci/km2 (activity of the soil samples exceeding 6000 Bq/kg, respectively). We observed a strong, significant correlation of 137Cs activity of soil samples with the percentage of chromosomal abnormalities, r = 0.97 (P < 0.05), and with the mitotic index, r = -0.93 (P < 0.05), in the roots of A. cepa, respectively. The results showed high toxicity and genotoxicity of radioactively polluted soils and confirmed the efficiency of the A. cepa test as a quick and inexpensive biological test for ecological and genetic risk assessment in the 'Chernobyl' zones.