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Metal contamination coupled with aridity is a major challenge for remediation of abiotic stressed soils throughout the world. Both biochar and beneficial bacteria showed a significant effect in bioremediation; however, their conjugate study needs more exploration. Two rhizobacteria strains Serratia sp. FV34b and Pseudomonas sp. ASe42b isolated from multi-metal and drought stressed sites showed multiple plant-growth-promoting attributes (phosphate solubilization, indole-3-acetic acid, siderophore, and ammonia production). Both strains were able to tolerate a high concentration of Cd along with being resistant to drought (−0.05 to −0.73 MPa). The seldom studied biomass of Amaranthus caudatus L. was used for biochar preparation by pyrolyzing it at 470 °C for 160 min under limited oxygen and then using it for the preparation of biochar-based microbial cell composites (BMC)s. To check the efficiency of BMC under Cd stress (21 mg kg−1 soil) and drought, a pot-scale study was conducted using Brassica napus L. for 47 days. Both the BMC5 (Biochar + Serratia sp. FV43b) and BMC9 (Biochar + Pseudomonas sp. ASe42b) improved the seed germination, plant biometrical (shoot and root biomass, length of organs) and physiological (photosynthetic pigments, proline, malondialdehyde, and relative water content) parameters under drought (exerted until it reaches up to 50% of field capacity) and Cd-spiked soil. However, for most of them, no or few significant differences were observed for BMC9 before and after drought. Moreover, BMC9 maximized the Cd accumulation in root and meager transfer to shoot, making it a best bioformulation for sustainable bioremediation of Cd and drought stressed soils using rapeseed plant.

The mineral deposits’ development is accompanied by significant disturbances of soil and vegetation in large areas. The study of initial stages of disturbed lands natural overgrowing makes it possible to assess patterns of vegetation formation. Forest phytocoenoses formation on serpentine substrates in the Middle Urals, Russia (taiga zone, subzone of southern taiga) was studied in this work. The research was carried out at four dumps of Anatol’sko-Shilovsky asbestos deposit. It was found that forest communities with the dominance of Pinus sylvestris L. are formed on dump sites with the predominance of overburden rocks. The herb-shrub layer of these phytocoenoses was dominated by Calamagrostis arundinacea (L.) Roth. The slopes of dumps and areas with compacted soil are overgrown with sparse undersized forest vegetation dominated by P. sylvestris and Betula pendula Roth. In herb-shrub layer of these areas, the most common were relict species Dendranthema zawadskii (Herbich) Tzvel. and rare for the Urals species, Epipactis atrorubens (Hoffm.) Besser (orсhid) and Thymus talijevii Klok. & Des.-Shost. The geobotanical and soil investigations have shown that all studied parameters (such as humidity, pH value, Mg:Ca ratio) influenced the formation of forest phytocoenoses. In general, the formation of forest vegetation on serpentine dumps is a very long process and it depends on specific climatic and soil conditions.

The results of the study of anatomical and morphological parameters of Pinus sylvestris L., growing on the serpentine dumps of Anatol’sko-Shilovsky Mining, are presented. Adaptive morphological and anatomical changes that contribute to the survival of the species in extreme environmental conditions have been identified. A significant decrease in P. sylvestris morphometic parameters (tree height, the annual growth of the tree, and the branches) under dump conditions were established. Under the influence of unfavorable factors on the dumps (lack of nutrients and water, high rockiness of the substrate), the length and the cross-sectional area of the needles decreased. Of the anatomical features, it is important to note a decrease in the number of resin ducts with a tendency to increase their diameter.

Metal tolerant plant growth-promoting bacteria capable of synthesizing IAA from tryptophan, solubilizing phosphates and converting protein nitrogen into ammonia were isolated from plants growing on contaminated soils.

The study is devoted to adventive macrophyte Lemna gibba L. (duckweed) adaptive reactions to pollution of water by heavy metals. Under the high technogenic impact on aquatic ecosystems, some invasive species quickly spread and crowd out of native flora. In this regard, studies of the physiological and biochemical mechanisms of these species tolerance, contributing to their successful resettlement are of particular importance. The results of two years of research (July 2016–2017) on the study of accumulative abilities in relation to heavy metals (nickel, copper, zinc, manganese and iron) and some morphophysiological parameters of L. gibba – one of the adventive flora representatives of the Middle Urals is presented. Plant material and surface water samples were taken from two water bodies of the Sverdlovsk region, differing in levels of technogenic impact: the Iset River (Aramil town) and its tributary the Sysert River (Dvurechensk town). It was found that Cu, Ni and Mn concentration in the Iset River were on average 1.5 times and Zn – 4.0 times higher than that in the Sysert River. It was shown that the fronds of the studied macrophyte from the habitat with an increased water pollution (the Iset River) were distinguished by a more significant accumulation of metals, larger mesophyll cells, a higher content of photosynthetic pigments and increased activity of catalase and polyphenol oxidase enzymes compared to plants from the water body with a lower level of pollution (the Sysert River). It is concluded that the physiological and biochemical adaptations revealed in L. gibba increase its competitiveness and play an important role in the development of new territories, including technologically disturbed ones.

The objective of this study was to compare eco-physiological and morphological parameters of a regionally endangered orchid species, Epipactis atrorubens (Hoffm. ex Bernh.) Bess., growing in two forest communities (on serpentine and granite outcrops) of the Middle Urals, Russia. Biodiversity, dominance, and phytocoenosis studies showed the colonization of a wide range of plant species on both sites. The physicochemical properties of the soil, chemical composition and morphological features of E. atrorubens, growing under technogenic conditions (asbestos deposits), on serpentine outcrops and in the natural environment of the granite massif were studied for the first time. The serpentine substrate differed from the granite one by its greater stoniness, circumneutral pH and lower contents of available nitrogen and phosphorus. Extremely high concentrations of magnesium were found in the serpentine soil, some 79 times higher than in the granite substrate. High concentrations of nickel (94 times), chromium (59 times), cobalt (17 times), and iron (4 times) were found in the serpentine substrate, higher than in the granite substrate. The differences between the sites for available metal contents and for root and shoot metal contents were significantly less. Concentrations of most of the metals in the roots were higher than in the shoots. Despite higher metal concentrations and lower nitrogen and phosphorus levels in serpentine soils, E. atrorubens had a larger population and greater viability compared to those growing on granite. Plants on serpentine outcrops were characterized by the formation of a larger number of fruits, greater root lengths and thicker leaf blades, compared to plants on granites. The well-developed orchid mycorrhizae contributed to the survival of this species under unfavorable serpentine conditions. Hence, serpentine outcrops formed due to the mining of asbestos could be a suitable substrate for the light-demanding E. atrorubens due to its capacity to adapt to dry, rocky, nutrient-depleted soils and limited competition from other plants. © 2019, The Author(s). The work was supported by the Ministry of Science and Higher Education of the Russian Federation in the framework of the State Task of UrFU No. 6.7696.2017.

In previous decades, some species of the Orchidaceae family have been found growing in man-made habitats. Neottia ovata is one of the most widespread orchids in Europe, however it is quite rare in Russia and is included in several regional Red Data Books. The purpose of this study was to compare the chemical composition and morphophysiological parameters of N. ovata from two forest communities of the Middle Urals, Russia: natural and transformed (fly ash dump of Verkhnetagil’skaya Thermal Power Station) for determining orchid adaptive features. The content of most of the studied metals in the underground parts (rhizome + roots) of N. ovata was considerably higher than in the leaves, which diminished the harmful effect of toxic metals on the aboveground organs. The adaptive changes in the leaf mesostructure of N. ovata such as an increase in epidermis thickness, the number of chloroplasts in the cell, and the internal assimilating surface were found for the first time. The orchids from the fly ash deposits were characterized by a higher content of chlorophyll b and carotenoids than plants from the natural forest community that evidenced the compensatory response on the decrease in chlorophyll a. The ability of N. ovata from the transformed habitat to maintain a relatively favorable water balance and stable assimilation indexes further contribute to its high viability. The study of orchid adaptive responses to unfavorable factors is necessary for their successful naturalization and introduction into a new environment. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. The reported study was partly funded by RFBR and the Government of Sverdlovsk region, project number 20-44-660011 and the Ministry of Science and Higher Education of the Russian Federation as part of state task of the Ural Federal University, FEUZ-2020-0057.

The accumulation of heavy metals (Cu, Ni, Zn, Mn, and Fe), parameters of the photosynthetic apparatus, and flavonoid content in leaves of two widespread species of submerged hydrophytes (Elodea canadensis Michx. and Potamogeton perfoliatus L.) from habitats with different levels of pollutions have been studied. The investigations are carried out in the surroundings of the metallurgical plant in the town of Revda, Sverdlovsk oblast, Russia (Revda River, upstream and downstream the confluence of the sewage). It is shown that hydrophytes from polluted habitats (impact site) differ from the plants of a less polluted zone (background site) by a higher accumulation of heavy metals (HMs), greater leaf thickness, and larger cells and chloroplasts. Plants from the polluted site are characterized by a high content of photosynthetic pigments, while flavonoid content in the leaves is lower. The identified trends indicate a high adaptive potential of these species of plants and their ability to inhabit in an aquatic environment polluted with metals and other pollutants.

The present investigation is the first in situ comparative study for the identification of Ni and Cu accumulation strategies involved in Odontarrhena obovata (syn. Alyssum obovatum (C.A. Mey.) Turcz.) growing in Cu-rich smelter-influenced (CSI) and non-Cu-influenced (NCI) sites. The total and Na2EDTA (disodium ethylenediaminetetraacetic acid)-extractable metal concentration in soils and plant tissues (roots, stem, leaves and flowers) were determined for CSI and NCI sites. High concentrations of total Ni, Cr, Co and Mg in the soil suggest serpentine nature of both the sites. In spite of high total and extractable Cu concentrations in CSI soil, majority of its accumulation was restricted to O. obovata roots showing its excluder response. Since the translocation and bioconcentration factors of Ni > 1 and the foliar Ni concentration > 1000 μg g−1, it can be assumed that O. obovata has Ni hyperaccumulation potential for both the sites. No significant differences in chlorophyll content in O. obovata leaves were observed between studied sites, suggesting higher tolerance of this species under prolonged heavy metal stress. Furthermore, this species from CSI site demonstrated rather high viability under extreme technogenic conditions due to active formation of antioxidants such as ascorbate, free proline and protein thiols. The presence of Cu in higher concentration in serpentine soil does not exert detrimental effect on O. obovata and its Ni hyperaccumulation ability. Thus, O. obovata could act as a putative plant species for the remediation of Cu-rich/influenced serpentine soils without compromising its Ni content and vitality.

This volume contains the reports presented on the Scientific Conference with international participation MATTEX 2018 held at Konstantin Preslavsky University of Shumen in October 2018. The reports contain the results of the research of lecturers, PhD students, specialists and undergraduate students at Konstantin Preslavsky University of Shumen, other Higher Education Institutions, companies and research organizations. The conference was partially supported by the Scientific Research Fund under contract DPMNF01/23-31.08.2018г., project RD-08-146/08.02.2018 of Shumen University and Paşabahçe Bulgaria, Targovishte.

The data on the accumulation of metals and the content of low molecular weight antioxidants in the leaves of Typha latifolia L. naturally growing in the vicinity the Karabash copper smelter (Russia, Chelyabinsk region) are presented. It has been shown that prolonged technogenic exposure caused the development of chronic oxidative stress which was accompanied by the activation of the synthesis of free proline, phenolic compounds and thiol-containing compounds in the leaves of T. latifolia. Altogether, T. latifolia showed high adaptive potential to grow under prolonged extreme technogenic pollution.

Organic fertilizers became a better alternative to chemical fertilizers in modern agricultural practices however, contamination of copper (Cu) from organic fertilizer is still a major concern for the globe. Plant growth promoting (PGP) microorganisms showed their efficiency to combat with this problem and thus Cu tolerant PGP endophytes from roots of Odontarrhena obovata (Alyssum obovatum) growing on Cu smelter contaminated serpentine soil were explored in present study. Out of twenty-four isolates, Pseudomonas lurida strain EOO26 identified by 16s rRNA gene sequencing was selected to check its efficacy for Cu-remediation. The strain EOO26 showed multi-metal tolerance, drought resistance and exhibited PGP attributes such as 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophore and ammonia production. Significant production of indole-3-acetic acid and phosphate-solubilization under different Cu concentration (0–100 mg L−1) at varying pH (5.0–8.0) suggests potentiality of this strain to work effectively under wide range of abiotic stress conditions. Plant growth experiment (pH 6.8 ± 0.3) in copper spiked soil suggested a significant increase in length and dry weight of root and shoot of sunflower (Helianthus annuus) after inoculation with strain EOO26. Plants inoculated with strain EOO26 resulted in increase in Cu uptake by 8.6-fold for roots and 1.9-fold for leaves than uninoculated plants. The total plant uptake in inoculated Cu treatment was 2.6-fold higher than uninoculated one, which is much higher than the previously reported Cu accumulating plants. The excellent adaptation abilities and promising metal removal efficiency strongly indicate superiority of strain EOO26 for phytoremediation of Cu-contamination and may work effectively for Cu removal from contaminated soils. © 2020 Elsevier Ltd Science and Engineering Research Board, SERB; Department of Science and Technology, Government of West Bengal, DST: INT/ RUS / RFBR /363; Ministry of Science and Higher Education of the Russian Federation The authors acknowledge the work support by RFBR , Russia (Project No. 19-516-45006) and DST , India (INT/ RUS / RFBR /363) and the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 02.A03.21.0006). L.B.B thankful to the Science and Engineering Research Board (SERB), India for providing National Post-Doctoral Fellowship (Grant No. PDF/2017/001074 ).

Background. The systematic self-control of students' health state allows optimizing the educational process organization of physical education and contribute improving the functioning of body systems. One of the common methods of such observation is the functional Martine's test, which provides an opportunity to investigate the dynamics of changes in blood pressure and heart rate between the resting state and every minute for five minutes after the load. In most cases, this load characteristic sufficiently fully reflects the student's cardiovascular system state. The determination of the characteristic patterns of such states will allow offering a mechanism for assessing the functional state of the circulatory system. The regular Martine's testing will allow observing the dynamics and evaluating changes in the body during the observation period.Objective. Creation of an automated system for assessing of a student’s circulatory system functional state changes.Methods. The students database of the National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” created during the research by the Department of Physical Education, was used for the study. To determine the functional patterns, separately for boys and girls, cluster analysis was used by the "k-means" method in the parameter space of the blood pressure and heart rate measured during the functional Martine's test. As a result, 7 clusters of states for young men and 8 clusters for girls were obtained. Clusters differ significantly in the nature of the response of blood pressure and heart rate to the load test. The centroids of the resulting clusters were further considered as functional patterns (the most typical representatives) of the body's response to the test physical load. The assessment of the circulatory system functional state is calculated by comparing the student test data with the previously defined functional patterns by the minimum Euclidean distance criterion. Conclusions about the functional state of the system are formed.Results. The clusters of the circulatory system functional states in the extended space of blood pressure and heart rate parameters of functional Martine's test are obtained. The clusters correspond to the normal states and different stages of the regulatory reserves reduction of the body. The algorithm allowing to consider features of an organism functional state at essential deviation of Martine's test indicators from a certain functional pattern is developed. This admits controlling the individual level of physical activity, adapting the training program and identifying conditions requiring additional medical control.Conclusions. As a study result, the automated system for assessing the functional state of technical university students circulatory system using Martine's test was developed. The system relates observation to one of the study sample clusters that were obtained using the "k-means" method. For additional information on the state of the circulatory system, the average radius of the cluster is used, since objects that are far removed from the center may have properties similar to those of a neighboring cluster. The developed system provides an opportunity for screening control the students cardiovascular system state during the educational process., Проблематика. Систематические самостоятельные наблюдения студентов за состоянием своего здоровья позволяют оптимизировать организацию учебного процесса физического воспитания и способствуют улучшению функционального состоянию систем организма. Одной из распространенных методик такого наблюдения является функциональная проба Мартине, которая предоставляет возможность исследовать динамику изменения артериального давления и пульса в период между состоянием покоя и на каждой минуте после нагрузки в течение пяти минут. Указанная нагрузочная характеристика в большинстве случаев достаточно полно отражает состояние сердечно-сосудистой системы студента. Определение характерных паттернов таких состояний позволит предлагать механизм оценки функционального состояния системы кровообращения. Регулярное проведение пробы Мартине позволит наблюдать динамику и оценивать изменения в организме за период наблюдений.Цель. Создание системы автоматизированной оценки изменений функционального состояния системы кровообращения студентов.Методика реализации. Для исследования использовалась база данных студентов Национального технического университета Украины “Киевский политехнический институт имени Игоря Сикорского”, созданная в процессе исследований кафедры физического воспитания. Для определения функциональных паттернов, отдельно для юношей и девушек, применялся кластерный анализ методом “k-средних” в пространстве параметров артериальное давление–пульс, измеренных в процессе проведения пробы Мартине. В результате были получены 7 кластеров состояний для юношей и 8 кластеров для девушек. Кластеры существенно отличаются по характеру реакции показателей артериального давления и пульса на выполнение теста. Центроиды полученных кластеров в дальнейшем рассматривались как функциональные паттерны (типичные представители) реакции организма на тестовую физическую нагрузку. Оценкa функционального состояния системы кровообращения рассчитывается посредством сравнения показателей теста студента с ранее определенными функциональными паттернами по критерию минимального евклидового расстояния. Формируются заключения о состоянии системы.Результаты. Получены кластеры функциональных состояний системы кровообращения в расширенном пространстве параметров артериальное давление–пульс пробы Мартине. Кластеры соответствуют состояниям нормы и различным стадиям снижения регуляторных резервов организма. Разработан алгоритм, позволяющий учитывать особенности функционального состояния организма при существенном отклонении показателей пробы Мартине от определенного функционального паттерна. Это дает возможность контролировать индивидуальный уровень физических нагрузок, адаптировать программу тренировок и выявлять состояния, требующие дополнительного медицинского контроля.Выводы. В результате проведенного исследования была разработана система автоматизированной оценки функционального состояния кровообращения студентов технического университета с применением пробы Мартине. Система относит наблюдения в один из кластеров обучающей выборки, полученных с использованием метода “k-средних”. Для дополнительной информации о состоянии системы кровообращения используется средний радиус кластера, поскольку объекты, сильно удаленные от центра, могут иметь свойства, близкие к свойствам соседнего кластера. Разработанная система позволяет как осуществлять контроль за состоянием системы кровообращения единовременно, так и проводить скрининговый контроль группы студентов., Проблематика. Систематичні самостійні спостереження студентів за станом свого здоров’я дають змогу оптимізувати організацію навчального процесу фізичного виховання і сприяють поліпшенню функціонального стану систем організму. Однією з поширених методик такого спостереження є функціональна проба Мартіне, яка надає можливість досліджувати динаміку зміни артеріального тиску і пульсу в період між станом спокою і на кожній хвилині після навантаження протягом п’яти хвилин. Вказана навантажувальна характеристика в більшості випадків досить повно відображає стан серцево-судинної системи студента. Визначення характерних патернів таких станів дасть змогу пропонувати механізм оцінки функціонального стану системи кровообігу. Регулярне проведення проби Мартіне дасть можливість спостерігати динаміку й оцінювати зміни в організмі за період спостережень.Мета. Створення системи автоматизованої оцінки змін функціонального стану системи кровообігу студентів.Методика реалізації. Для дослідження використовувалася база даних студентів Національного технічного університету України “Київський політехнічний інститут імені Ігоря Сікорського”, створена в процесі досліджень кафедри фізичного виховання. Для визначення функціональних патернів, окремо для юнаків і дівчат, застосовувався кластерний аналіз методом “k-середніх” у просторі параметрів артеріальний тиск–пульс, виміряних у процесі проведення проби Мартіне. В результаті було отримано 7 кластерів станів для юнаків і 8 кластерів для дівчат. Кластери істотно відрізняються за характером реакції показників артеріального тиску і пульсу на виконання тесту. Центроїди отриманих кластерів у подальшому розглядалися як функціональні патерни (типові представники) реакції організму на тестове фізичне навантаження. Оцінкa функціонального стану системи кровообігу розраховується у порівнянні показників тесту студента з раніше визначеними функціональними патернами за критерієм мінімальної евклідової відстані. Формуються висновки про стан системи.Результати. Отримано кластери функціональних станів системи кровообігу в розширеному просторі параметрів артеріальний тиск–пульс проби Мартіне. Кластери відповідають станам норми і різним стадіям зниження регуляторних резервів організму. Розроблено алгоритм, що дає змогу враховувати особливості функціонального стану організму при суттєвому відхиленні показників проби Мартіне від певного функціонального патерну. Це дає можливість контролювати індивідуальний рівень фізичних навантажень, адаптувати програму тренувань і виявляти стани, що вимагають додаткового медичного контролю.Висновки. Розроблено систему автоматизованої оцінки функціонального стану кровообігу студентів технічного університету із застосуванням проби Мартіне. Система відносить спостереження в один із кластерів навчальної вибірки, отриманих із використанням методу “k-середніх”. Для додаткової інформації про стан системи кровообігу використовується середній радіус кластера, оскільки об’єкти, сильно віддалені від центра, можуть мати властивості, близькі до властивостей сусіднього кластера. Розроблена система дає змогу як здійснювати контроль за станом системи кровообігу одноразово, так і проводити скринінговий контроль групи студентів.

Usage of biofertilizers is one of the important components of integrated nutrientmanagement, as they are renewable source of plant nutrients, ecologically safecompared to chemical fertilizers and cost effective. Silicate solubilizing bacteria(SSB) can play an efficient role not only in solubilizing insoluble forms of silicatesbut also potassium and phosphates, hence increasing soil fertility and therebyenhancing plant productivity. The aim of this study was to investigate the influenceof SSB-enriched biofertilizer on the structural and functional parameters ofphotosynthetic apparatus of Brassica juncea (L.) Czern. The pure culture of SSBwas isolated from the clay substrate, cultivated on Zak–Alexandrov medium andidentified as Bacillus sp. To obtain the biofertilizer, SSB culture (0.6*108 cfu mL-1)was mixed with steriled peat (1:1, v/w) and dried at 35–40°C. Plants were grownfrom seeds during two months in the pots with adding SSB-enriched biofertilizer tothe mixture of clay and soil (1:10, w/w). The clay substrate plus peat without SSBwas used as a control. It was found that addition of SSB-enriched biofertilizer toclay substrate significantly increased the content of total nitrogen, phosphorus andpotassium in the leaves of B. juncea. The thickness of mesophyllic layer and thenumber of mesophyll cells were increased on average by 24 %. It was correlatedwith a sharp increase of photosynthetic pigment content and CO2 uptake (1.5–2.0times). We can conclude that SSB-enriched biofertilizer improves thephotosynthetic function of B. juncea.

The impact of sewage water from a pulp and paper mill on the anatomical and biochemical parameters of submerged and semisubmerged plants has been assessed. The pro-oxidant and antioxidant plant responses and changes in their anatomical parameters in relation to industrial pollution are detected. Submerged plants are more subjected to a negative impact of sewages than semisubmerged plants.

In the present study, two fresh water plant species Egeria densa (Planch.) Casp. and Ceratophyllum demersum L. were subjected to separate and combined action of urea (2mМ) and metals (Ni and Cu, 10μM) to investigate the phytoremediation potential of these two submerged macrophytes during short-term experiments (48h). Both submerged macrophytes demonstrated high accumulative potential for Ni and Cu (average bioconcentration factors were 2505 for Ni and 3778 for Cu). The urea (2 mM) was not significantly toxic for studied plant species. Futhermore, urea worked as an additional source of nitrogen and stimulated some metabolic processes such as the synthesis of photosynthetic pigments, soluble proteins, non-enzymatic antioxidants, and activated some enzymes. Adding urea to the metals increased their accumulation in both macrophytes (on average by 35% for Ni and 15% for Cu). Combined action of urea and Ni did not have a significant effect on antioxidant response, but caused a sharp increase of urease activity (4 folds on an average) in both plants. The copper exerted a stronger toxic effect on both studied macrophytes compared to nickel. Adding urea to copper in some cases diminished the toxic action of this metal. Study concludes that the responses of E. densa and C. demersum to urea and metal action (separate and combined) were depended on the type of pollutant and the activity of antioxidant defence system. Therefore, the studied aquatic macrophytes found to be potential phytoremediators of water bodies, the addition of an organic nitrogen source in the form of urea in environmentally relevant concentration will increase the efficiency of phytoextraction of metals.

The antioxidant activity of two common species of aquatic plants (Batrachium trichophyllum (Chaix) Bosch. and Potamogeton alpinus Balb.) with different “accumulation strategies” has been studied. It is shown that the leaves of Batrachium trichophyllum, which accumulate metals in large amounts, have a higher intensity of proand antioxidant processes than Potamogeton alpinus.

Kinetics of the metabolism of the heterocyclic amino acid histidine exposed to the L-histidine ammonia-lyase enzyme has been investigated and the technology of extraction of histidine biotransformation products (urocanic acid and ammonia) from casein hydrolyzates enabling the subsequent use of these hydrolyzates as a milk protein concentrate for the production of specialized dietary products for the nutrition of histidinemia patients has been developed.

This paper analyzes the effect of exogenous urea in increased concentration gradient (0, 100, 500 and 1,000 mg L-1) on photosynthetic pigments (measured spectrophotometrically), uptake of 14CO2 (using radioisotope), and urease activity (by measuring ammonia with Nessler's reagent) in leaves of Elodea densa Planch. We have observed that low concentration of urea (100 mg L-1) stimulates the accumulation of photosynthetic pigments and intensifies photosynthesis in E. densa, whereas high concentration (1,000 mg L-1) suppresses these processes. Urease activity increased by approximately 2.7 and 8 fold when exogenous urea concentrations were 100 and 500 mg L-1, respectively. However, exogenous urea in high concentration (1,000 mg L-1) decreased urease activity by 1.5 fold compared to the control. The necessity of mitigating urea and other nitrogen-containing compounds (NH3 from urea) in water bodies has been discussed with emphasis on the potential for phytoremediation of urea using common water weed viz. E. densa. © 2013 Springer-Verlag Berlin Heidelberg.

Aquatic macrophytes, viz. Sagittaria sagittifolia L., Lemna gibba L., Elodea canadensis Michx., Batrachium trichophyllum (Chaix.) Bosch., Ceratophyllum demersum L. and Potamogeton sp. (P. perfoliatus L., P. alpinus Balb., P. crispus L., P. berchtoldii Fieber, P. friesii Rupr., P. pectinatus L.) were collected from 11 sites for determining their metal accumulation and thiols content. Cu(2+), Ni(2+), Mn(2+), Zn(2+), and Fe(3+) exceeded maximum permissible concentrations in chosen sites. Significant transfer of metals from water to leaves is observed in the order of Ni(2+)Cu(2+)Zn(2+)Fe(3+)Mn(2+). The maximum variation of bioconcentration factor was noticed for manganese. The accumulation of heavy metals in leaves was correlated with non-protein and protein thiols, confirming their important role in metal tolerance. The largest contribution was provided by Cu(2+) (on the average r = 0.88, p0.05), which obviously can be explained as an important role of these ions in thiols synthesis. Increased synthesis of thiols in the leaves allows the usage of SH-containing compounds as biomarkers of metal tolerance. Considering accumulation of metals and tolerance, B. trichophyllum, C. demersum and L. gibba are the most suitable species for phytoremediation of highly multimetal contamination, while E. canadensis and some species of Potamageton are suitable for moderately metal-polluted sites.

Selected cellular responses for urea (5 mM), Ni (100 μM), and Cu (100 μM) treatments and their combined effects on the leaves of a submerged macrophyte Brazilian elodea (Egeriadensa Planch. Casp.) were studied for 4 days. It was observed that the high dose of urea (5 mM) had a significant toxic effect on some physiological and biochemical characteristics of E. densa such as the content of chlorophyll a and the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase. The elevated level of lipid peroxidation was accompanied by the accumulation of proline and ascorbate. A substantial increase in the amount of proline and urease activity was observed in Urea + Ni-treated leaves. The ascorbate content significantly increased in all the treated plants which correlated with ascorbate peroxidase activity. Urea with Cu ions affected the E. densa photosynthetic pigment system and urease activity adversely. The catalase activity was inhibited by the action of all the pollutants studied, especially in Urea + Cu-treated plants, whereas the peroxidases activity (APX and GPX) increased under the Cu and Urea + Cu action. The results presented indicate that the addition of the high dose of urea (5 mM) to the heavy metals studied may enhance the toxic effects in E. densa leaves, especially in the case of copper. © 2016, Botanical Society of Sao Paulo.

Urea being a fertilizer is expected to be less toxic to plants. However, it was found that urea at 100 mg L(-1) caused the oxidative stress in Elodea leaves due to the formation of reactive oxygen species (ROS) and lipid peroxidation that are known to stimulate antioxidant pathway. Urea at a concentration of 500 and 1000 mg L(-1) decreased low-molecular-weight antioxidants. In this case, the antioxidant status of plants was supported by the activity of antioxidant enzymes such as superoxide dismutase and guaiacol peroxidase. A significant increase in the soluble proteins and -SH groups was observed with high concentrations of urea (30-60 % of control). Thus, the increased activity of antioxidant enzymes, low-molecular-weight antioxidants, and induced soluble protein thiols are implicated in plant resistance to oxidative stress imposed by urea. We found that guaiacol peroxidase plays an important role in the removal of the peroxide in Elodea leaves exposed to 1000 mg L(-1)of urea.

The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc–preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium- and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76–85 in the MIR, the incorporation level of HBc–preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.

Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.

We examined the uptake of five heavy metals (Cu, Fe, Ni, Zn, and Mn) in Ceratophyllum demersum L. (hornwort) and Potamogeton alpinus Balb. (pondweed) from Iset' river, Ural region, Russia. This study was conducted in a territory that is highly urbanized where the surface waters are contaminated by a wide spectrum of pollutants. The environmental situation in this territory drastically deteriorated due to anthropogenic activity. The water quality in most of the water bodies in the Ural region is rather poor. In a comparative study of C. demersum and P. alpinus, differential accumulation pattern was noted for heavy metals (HMs). Higher amounts of HMs accumulated in C. demersum compared to P. alpinus. Also it was shown that in leaves of C. demersum there were high amount of total phosphorus, nitrogen, organics acids and ash; high activity of guaiacol peroxidase; high content of nonenzymatic antioxidants viz., flavonoids, ascorbate, glutathione and proline; high amount of thiols (soluble and membrane bound) compared to P. alpinus.

The representatives of the family Orchidaceae Juss. are often used as a source of natural antioxidants, including phenolic compounds, which play an important role in plant resistance under stressful conditions. This study investigates the content of lipid peroxidation products and soluble phenolic compounds in flowering plants of Platanthera bifolia (L.) Rich. growing in natural (forest park) and transformed (fly ash dumps of Thermal Power Stations) ecosystems of the Middle Urals, Russia, as well as the content of flavonoids at different stages of orchid development. Research has shown that in disturbed habitats, P. bifolia is capable of forming abundant populations containing a significant portion of the flowering plants. Additionally, flowering orchids from fly ash dumps contained an average 20 % more lipid peroxidation products, which indicated a shift in the redox balance towards oxidative processes. An increase by 2.4 times on average in the content of phenolic compounds, particularly flavonoids, was observed at all developmental stages of the plants growing in the transformed ecosystems. Regardless of the growing conditions, the non-flowering mature individuals were characterized by a minimum content of flavonoids, probably due to pre-generative metabolic restructuring. Yet, in the period of orchid blooming, the flavonoid content in their leaves increased again in all study sites. At the same time, the flavonoid proportion of the total soluble phenolic compounds was 42 % in the natural habitat, increasing to 66 % on average in the transformed ecosystems. Thus, flavonoids are involved in the protective adaptive responses of P. bifolia, not only ensuring the survival of this orchid but also contributing to the implementation of its ontogenetic program

Aquatic ecosystems are very sensitive to industrial impacts, and, therefore, it is increasingly important to study the mechanisms underlying the tolerance of aquatic organisms to water pollution. Heavy metals (HMs) are among the most common and toxic pollutants of aquatic ecosystems. They have a particularly strong effect on macrophytes, which are in close contact with the aquatic environment and can accumulate metals in considerable quantities. Hydrocharis morsus-ranae L. is a floating macrophyte (pleistophyte) with a high capacity for accumulation of HMs. The aim of the present study was to assess the effect of industrial pollution on the redox reactions in H. morsus-ranae and to identify the role of low molecular weight antioxidants in adaptation of this macrophyte to unfavorable conditions. A comparative analysis of the physiological and biochemical characteristics of H. morsus-ranae from two (reference and impacted) water bodies was carried out. The study revealed an increased level of lipid peroxidation products in the leaves of H. morsus-ranae under industrial impact, which indicates oxidative stress. Nevertheless, this floating plant demonstrated fairly high resistance to adverse conditions, due to the synthesis of non-enzymatic antioxidants such as proline and soluble protein thiols. Revealing the response of macrophytes to pollution of water bodies will help predict the state of aquatic ecosystems with an increase in anthropogenic pressure

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.

Previous studies have shown that the progression of hepatitis B virus-related liver disease in long-term immunosuppressed kidney transplant recipients is associated with the accumulation of virus variants carrying in-frame deletions in the central part of the core gene. A set of naturally occurring core protein variants was expressed in Escherichia coli in order to investigate their stability and assembly competence and to characterize their antigenic and immunogenic properties. In addition, a library of core gene variants generated in vitro with deletions including the major immunodominant region (MIR) of the core protein was investigated. The position and length of deletions determined the behaviour of mutant core proteins in E. coli and their assignment to one of the three groups: (i) assembly-competent, (ii) stable but assembly-incompetent and (iii) unstable proteins. In vivo core variants with MIR deletions between amino acids 77 and 93 belong to the first group. Only proteins with the shortest deletion (amino acids 86-93) showed stability and self-assembly at the same level as wild-type cores, and they showed reduced antigenicity and immunogenicity. Mutants with deletions extending N-terminally beyond residue G73 or C-terminally beyond G94 were found to be assembly-incompetent. We suggest that G73 and G94 are involved in the folding and the native assembly of core molecules, whereas the intervening sequence determines the antibody response. Depending on their ability to form stable proteins or to assemble into particles, core mutants could contribute to liver cell pathogenesis in different ways.

Core particles of hepatitis B virus (HBV) are able to improve the immunogenicity of foreign sequences exposed on the particle surface. The insertion site in the core antigen of HBV (HBcAg) determines the surface presentation and thus the immunogenicity of the foreign sequence. For direct comparison of the value of potential insertion sites in the core antigen, we constructed vectors allowing insertions of a model marker epitope DPAFR. This epitope was inserted at the N-terminus, the c/e1 loop, behind amino acid (aa) 144 and behind aa 183 (DPAF only). In addition, we generated a mosaic construct allowing the co-expression of HBcAg and a HBcAg/DPAFR fusion protein due to a suppressor tRNA-mediated readthrough mechanism. All 6 constructs allowed the formation of chimaeric or mosaic core-like particles. Western blot analyses and a direct ELISA demonstrated the presence of the DPAFR sequence in the chimaeric and mosaic particles. Competitive ELISA and immune electron-microscopic data suggested the c/e1 loop as the insertion site of choice for presenting foreign sequences on the surface of chimaeric HBV core particles. However, the N-terminal fusion also allowed partial surface exposure of the DPAFR motif. In contrast, in particles of constructs carrying the DPAFR insert at aa position 144 or 183, respectively, the epitope seemed not to be surface accessible.

Hantaviruses are rodent-born agents which are pathogenic in humans causing haemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. To induce a protective immunity against a European hantavirus (Puumala) we constructed chimaeric hepatitis B virus (HBV) core particles carrying defined fragments of the Puumala virus nucleocapsid protein. After immunisation of bank voles, the natural host of Puumala virus, with core particles possessing an insertion of the N-terminal part of Puumala virus nucleocapsid protein, four of five animals were protected against subsequent virus challenge. The results show that the major protective region of the nucleocapsid protein is located between amino acids 1 and 45 and that chimaeric HBV core-like particles are useful carries of foreign protective epitopes.

Spatial and immunochemical elucidation of hepatitis B core antigen suggested unique organization of its major immunodominant region (MIR) localized within the central part of molecule around amino acid residues 74-83. This superficial loop was recognized as the most prospective target for the insertion of foreign epitopes ensuring maximal antigenicity and immunogenicity of the latter. MIR allowed a substantial capacity of insertions up to about 40 amino acid residues without loss of the capsid-forming ability of core particles. Vector capacity as well as structural behavior and immunological fate of inserted epitopes were dependent on their primary structure. Special sets of display vectors with retained but cross-sectioned MIR as well as with uni- and bidirectionally shortened MIR have been investigated.

Hepatitis B virus (HBV) core antigen (HBcAg) particles purified from Escherichia coli were probed in a competition enzyme immunoassay (EIA) with a panel of 16 murine monoclonal antibodies (MAbs) directed to different forms of core protein. The linear binding sites of the MAbs were mapped by combination of solid-phase and competition EIA using synthetic peptides covering the complete sequence of HBV core protein. Relative accessibilities of the linear binding sites at the HBcAg surface were investigated by comparing reactivities in solution of the MAbs to (i) two genetic variants of particulate HBcAg, (ii) denatured core protein, and (iii) synthetic peptides mimicking the appropriate linear binding sites. Further, accessibilities of HBV preS1 and preS2 epitopes (introduced into core protein at positions 77 or 144) at the surface of chimeric HBcAg particles were investigated. The previously described surface localization of core protein region 78-83 at the core particle surface was confirmed. In addition, another region, encompassing residues 127-133, was found to occupy a surface position at particulate HBcAg, whereas regions 9-20 and 133-145 were exposed after denaturation of the core protein and at synthetic peptides but not at particulate HBcAg.

Human hepatitis B virus core protein expressed in E. coli assembles into two sizes of particle. We have determined their three-dimensional structures by electron cryomicroscopy and image processing. The large and small particles correspond to triangulation number T = 4 and T = 3 dimer clustered packings, containing 240 and 180 protein subunits, respectively. The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell. The native viral core particle packages RNA and is active in reverse transcription to DNA. The holes we observe may provide access for the necessary small molecules. Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

Hepatitis B virus (HBV) core particles carrying the amino-terminal 120 amino acids (aa) of the nucleocapsid (N) protein of the hantaviruses Dobrava, Hantaan or Puumala have been demonstrated to be highly immunogenic in mice when complexed with adjuvants. Here we demonstrate that even without adjuvant, these chimeric particles induced high-titered, and strongly cross-reactive N-specific antibody responses in BALB/c and C57BL/6 mice. The induced N-specific antibodies represented all IgG subclasses. Pre-existing core-specific antibodies did not abrogate the induction of an N-specific immune response by a hantavirus N insert presented on core particles. Therefore, chimeric core particles should represent promising vaccine candidates even for anti-core positive humans.

In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a C-terminally truncated HBc (HBcΔ) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBcΔ mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain [J Gen Virol 1997;78:2049–2053]. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein. To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of ‘non-mosaic’ HBcΔ fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein. Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBcΔ particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa. The mosaic particles formed exhibited PUUV-N protein antigenicity. Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1–114, aa 1–213 and aa 340–433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1–114 or aa 1–213 of the N protein. Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated. In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and ‘problematic’, e.g. hydrophobic, hantavirus sequences.

Objectives: In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35–98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP). Methods: The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied. Results: The HBs*-E7(35–54) protein expressing E7 residues 35–54 between residues 139 and 142 of the HBs carrier formed HBs-like particles in Saccharomyces cerevisiae. The HBcΔ-E7(35–98), but not the frCP-E7(35–98), ensured VLP formation in Escherichia coli. In Balb/C mice, the HBs*-E7(35–54) VLPs predominantly induced an anti-E7 antibody, but not anti-HBs carrier response, whereas the HBcΔ-E7(35–98) VLPs induced a lower anti-E7 compared to anti-HBc carrier response. The frCP-E7(35–98) protein elicited equally high antibody responses to both E7 and frCP carrier. Analysis of the immunoglobulin G isotype profile of the antibodies induced by the E7-carrying chimeras showed that the HBs and frCP derivatives were capable of eliciting the Th1 and Th2 subsets of T helper cells, whereas the HBc-derived chimeras elicited only the Th2 subset. Conclusions: The HBs and HBc, but not frCP carriers support an efficient outcome for VLPs carrying the HPV16 E7 epitopes. All chimeric proteins may be regarded as potential vaccine candidates.

Negative impacts of agricultural activity on water bodies cause the necessity to regulate anthropogenic loads by means of water protective measures implementation at watersheds. The measures should be aimed at supporting the balance between the natural stability potential and the level of anthropogenic impact. Identification of permissible impacts on watersheds and water protective measures selection should be preceded by watershed state evaluation. The method of evaluation of rural catchment areas state both in terms of anthropogenic environmental impact and geo-ecological stability has been developed. Summarized indicators reflecting the most significant natural conditions and specialities of economic activities at catchment areas are proposed as the principle criteria for the evaluation. The method was testing using the results of field investigations conducted in rural watersheds various in terms of anthropogenic impact and natural characteristics. Correlations between parametres of nutrient delivery into water bodies and summarized indicators were obtained. Usage of this method will help to estimate the degree of rural impact on surface water quality and elaborate ecologically optimal management of economic activities at rural watersheds to protect water bodies.