Subgenomic replicons of the flavivirus Kunjin: construction and applications

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA Delta ME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon Delta ME/76 with a deletion of 76 nucleotides in the 3'UTR of Delta ME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon Delta ME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of Delta ME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (Delta ME/76CAT[+])- and minus (Delta ME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental Delta ME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of Delta ME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the Delta ME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system

Приложение для мониторинга затопления территории при обрушении плотины в Лаосе в 2018 г. на основе данных SAR Sentinel-1A и объектно-ориентированного метода

Flood disasters seriously threaten the survival and development of human beings. Monitoring the changes of water bodies during floods and estimating the affected area is essential for comprehensive and accurate analysis of disaster information. Recently, radar satellite data has been increasingly used for flood monitoring, since in this case, cloudiness is not an obstacle to estimating the flood area. In this paper Sentinel-1 ground range detected (GRD) data was selected to estimate the inundated area after the Xe-Pian Xe-Namnoy Dam breach in Laos at the end of July 2018. The flooded Hinlat area and the Xe-Pian Xe-Namnoy reservoir were selected as the study area for flood inundation extent monitoring, because this area is characterized by bare land, agricultural land, and residential land with complex topography and geomorphology. The study area is located in the Bolaven Plateau, is a highland region in southern Laos. One of the reasons for the flooding of the study area is an elevation difference between upper reaches and downstream of the river. Several reaches with a convex profile and knickpoints because of the geologic control when draining the plateau represent the undeveloped longitudinal profile of the Vang Ngao River. The main channel of the Vang Ngao River is dug into Mesozoic fluvial sandstones, which resist scouring by the flood. The eCognition software is used to organize the process of extracting information about the flood zone. The object-oriented approach and the threshold method are combined to extract information about the reservoir. First, SNAP software is used to pre-process Sentinel-1A SAR images. Then, the eCognition multi-scale segmentation method is used to determine the best segmentation scale, for iterative testing and comparative analysis of experimental results, taking into account the characteristics of the object and a priori knowledge. After sensitivity analysis of the flooded area image features and other features, the VH-polarized backscattered mean features were selected to construct a knowledge base for flooded area extraction to differentiate water and non-water bodies. At the same time, the modified bare soil index (MBI) and the terrain relief were combined to remove the influence of bare land and mountain shadow on the extraction results to achieve the 2018 dam collapse flood monitoring in Laos. Finally, the extent and area of the affected region were analyzed and the changes of water bodies before and after the disaster were mapped. The study shows that the monitoring results of Sentinel-1A SAR data are more consistent with the actual situation and have significant advantages in flood hazard monitoring and assessment, which can effectively carry out large-scale flood inundation extent monitoring

Cellular and humoral immunity in children with asthma

Asthma – is a chronic inflammatory disease of the airways, which most researchers attribute to allergic diseases which are based on immunological mechanisms, essentially even at present the subject of numerous studies. The aim of our study was to determine the cellular and humoral immunity in children with various forms of asthma. We have comprehensively examined 200 children with asthma aged 6 to 17 years. Found that state humoral and cellular immunity in patients with asthma differs depending on the form of the disease, such as serum IgA was at 24.68% higher at nonatopic asthma compared with atopic its form. Phagocytic index in children with asthma in nonatopic 2.92 times higher than in atopic asthma, indicating the presence of an infectious factor. Marked changes in cellular and humoral immunity depending on the content of 25 (OH) D3 and cathelitcidin LL-37. Thus, the value of IgA was 1.75 times higher in patients with the optimal content of 25 (OH) D3 in the serum, unlike the parameters in children with failure and deficiency of this vitamin. Found that in children with asthma containing cathelitcidin LL-37 inthe serum of more than 51.9 pg / ml phagocytic index at 44.74% higher compared to patients in whom serum content cathelitcidin LL-37 was less than 17 55 pg / ml

trans-complementation analysis of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal half in RNA replication

Recently we described rescue of defective Kunjin virus (KUN) RNAs with small deletions in the methyltransferase and RNA polymerase motifs of the ns5 gene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as helper (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). We have now extended our previous observations and report successful trans-complementation of defective KUN RNAs with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. Replication of full-length KUN RNAs with 3'-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) nucleotides in the ns5 gene was complemented efficiently in transfected repBHK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complemented less efficiently, and RNA with an even larger deletion of 2,279 nucleotides (84%) was not complemented at all. Chimeric KUN genomic RNA containing 87% of the KUN ns5 gene replaced by the corresponding sequence of the dengue virus type 2 ns5 gene was unable to replicate in normal BHK cells but was complemented in repBHK cells. These results demonstrate for the first time complementation of flavivirus RNAs with large deletions (as much as 75%) in the RNA polymerase gene and establish that translation of most of the N-terminal half of NS5 is essential for complementation in trans. A model of formation of the flavivirus replication complex implicating a possible role in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KUN and on reported data with other flaviviruses

Subgenomic flaviviral RNAs: what do we know after the first decade of research

The common feature of flaviviral infection is the accumulation of abundant virus-derived noncoding RNA, named flaviviral subgenomic RNA (sfRNA) in infected cells. This RNA represents a product of incomplete degradation of viral genomic RNA by the cellular 5′-3′ exoribonuclease XRN1 that stalls at the conserved highly structured elements in the 3′ untranslated region (UTR). This mechanism of sfRNA generation was discovered a decade ago and since then sfRNA has been a focus of intense research. The ability of flaviviruses to produce sfRNA was shown to be evolutionary conserved in all members of Flavivirus genus. Mutations in the 3′UTR that affect production of sfRNAs and their interactions with host factors showed that sfRNAs are responsible for viral pathogenicity, host adaptation, and emergence of new pathogenic strains. RNA structural elements required for XRN1 stalling have been elucidated and the role of sfRNAs in inhibiting host antiviral responses in arthropod and vertebrate hosts has been demonstrated. Some molecular mechanisms determining these properties of sfRNA have been recently characterized, while other aspects of sfRNA functions remain an open avenue for future research. In this review we summarise the current state of knowledge on the mechanisms of generation and functional roles of sfRNAs in the life cycle of flaviviruses and highlight the gaps in our knowledge to be addressed in the future

Modification of the epicuticular waxes of plant leaves due to increased sunlight intensity

Climatic changes observed around the world in recent years are associated with an increase in the solar radiation intensity and temperature and reduction in the humidity. Fluctuations of environmental factors significantly change the conditions for the existence of plants, which dictates the need for adaptive reactions of plant organisms at the different levels of their organization. Such dangerous processes as excessive heating of the surface of plant leaves and water loss can be prevented by the formation of a cuticle, which is a complex composition consisting of cutin and the soluble intracuticular and epicuticular waxes. We suggested that the structure, component composition and properties of the cuticle of trees undergo adaptive changes due to microclimatic conditions in different parts of the tree crown. The study was aimed at the identification and evaluation of light-induced differences in the accumulation and composition of leaf epicuticular waxes of Ulmus trees (native U. minor Mill. and alien U. pumila L.), and was conducted in 2018–2019 in Dnipro city located in the steppe zone of Ukraine. Analysis of the waxes’ chloroform extracts was carried out using GC Shimadzu 2010 PLUS equipped with a flame ionization detector and capillary column SP-2560. The highest amount of epicuticular waxes (12.23 ± 0.39 µg/cm2) was on the sunlit leaves of U. pumila, and wax deposits on the sunned leaves exceeded twice those on the shaded leaves in both U. minor and U. pumila. Long-chain hydrocarbons detected in the epicuticular waxes of both elm species were represented by free fatty acids, aldehydes, alcohols, and n-alkanes in various ratios. In the epicuticular waxes of U. minor, fatty acids dominated both on shaded and sunned leaves, while alkanes together with alcohols were the main components in U. pumila waxes, especially on the sunlit leaves. According to our results, local high illumination of leaves in the crown of both elm species caused increase in share of long-chain alkanes (1.2–1.9 times), but simultaneous reduction of the content of free fatty acids (1.5–16.8 times) in the epicuticular waxes’ composition. General patterns of the leaf epicuticular waxes’ modification due to increased solar radiation and air temperature can indicate the adaptive metabolic responses of woody plants to changing climatic conditions

Morpho-ecological structure of oribatid mite (Acariformes, Oribatida) communities in the forest litter of recultivated areas

The study of morpho-ecological organization of oribatid mite communities (Acariformes, Oribatida) inhabiting forest litter of recultivated areas in steppe zone conditions of Ukraine was performed. The role of the forest and forest floor litter in optimization of the ecological situation on degraded lands was demonstrated. The function of environment creation by oribatids, as primary destructors of dead plant matter, supporting such ecosystem services as soil fertility improvement and nutrients turnover was highlighted. The research was performed within different stratigraphic types of bulk edaphotops in the recultivated plot of “Pavlogradskaya” colliery (Pavlograd, Dnipropetrovsk region, Ukraine) planted with red juniper (Juniperus virginiana L.). Withdrawal and collection of mites was performed with thermoeclector. For determination of the domination structure in the mite communities, the Engelmann scale was used. Adaptive (morpho-ecological) groups of oribatid mites were diagnosed by Krivolutsky. It was established that the number of species of oribatid mites in the forest litter of the studied red juniper plantation varied from 16 to 25. Average density of oribatid mites varied from 4,720 to 25,327 ind./m2. Among such morpho-ecological groups as soil surface inhabitants, small soil pore inhabitants, deep soil forms, floor litter inhabitants and unspecified forms, identified in the coniferous litter, the share of unspecified forms increased from loess-like loam type (21% of total amount) to Calcic Chernozem types with different stratigraphy (41.0%, 70.0% and 70.4% accordingly). Deep soil forms in the forest floor litter of the studied red juniper plots were not identified for any of recultivation types. The obtained results expand our understanding of the role of oribatid mites in the processes of ecological rehabilitation of disturbed ecosystems in the conditions of modern nature management

Glutathion-S-Transferase activity in zea mais seedlings under high temperature and heavy metals combine action

У модельному експерименті досліджено зміни активності глутатіон-S-трансферази (GST) [КФ 2.5.18] у зерні й органах проростків кукурудзи за комбінованої дії високої температури, солей свинцю та кадмію. Виявлено тканиноспецифічність динаміки активності ферменту за спільного впливу двох чинників. Показано залежність рівня ферментативної активності в органах проростків залежно від впливу іонів кадмію та свинцю на фоні короткочасної дії гіпертермії. Зроблено висновок про посилення процесу детоксикації важких металів у проростках за умов тривалого впливу високої температури. В модельном эксперименте исследованы изменения активности глутатион-S-трансферазы (GST) [КФ 2.5.18] в зерне и органах проростков кукурузы при комбинированном воздействии высокой температуры, солей свинца и кадмия. Выявлена тканеспецифичность динамики активности фермента при совместном воздействии двух факторов. Показана зависимость уровня ферментативной активности в органах проростков от воздействия ионов кадмия и свинца на фоне крат­ковременного влияния гипертермии. Сделан вывод об усилении процесса детоксикации тяжелых металлов в проростках в условиях долговременного воздействия высокой температуры.In model experiment the changes of Glutathione-S-Transferase (GST) activity in Zea maise seeds and seedlings under high temperature, and lead and cadmium ions combine action were investigated. Tissue-specific dynamics of enzymatic activity under two factors’ combine action was revealed. The dependence of enzyme’s activity level in seedlings’ organs from influence of cadmium and lead ions under short-term action of high temperature was shown. The enhancing of heavy metals detoxification process in seedlings under long-term action of high temperature was concluded.

Дослідження впливу солей 2-((4-R-3-(морфолінометилен)-4H-1,2,4-триазол-5-іл) тіо)ацетатних кислот на ріст і розвиток паростків кукурудзи

Topicality. Agricultural industry urgently requires new and effective growth stimulating remedies. 1,2,4-triazole derivatives have had exhibited themselves as active fungicides and growth stimulators and the salts of 2-(4-R-3-R1-1,2,4-triazole-5-yltio)acetic acid are quite promising in this aspect.Aim. To determine the impact on quality indicators of Galatea hybrid corn sprouts germination after the processing with solutions of 2-((4-R-3 (morfolinomethylen)-4H-1,2,4-triazole-5-yl)thio) acetic acid salts.Materials and methods. The objects of our research were 10 new substances derivative 1,2,4-triazole derivatives. Further it was studied the impact of these compounds on the performance and germination of the “Galatea” hybrid corn seed in 2016 harvest. We have used auxin as the study comparison and distilled water was used as a control.Results and discussion. The study had found that these compounds could differently influence on the growth and development of corn sprouts. It was mentioned that on the growth stimulating activity intensiveness can influence the replacement of amino group at the N4 nitrogen atom of 1,2,4-triazole cycle and the type of cation which is combined with acids.Conclusions. In the result of our experiment it was determined the prospects of further investigations of synthesized compounds as a growth stimulators and noted appropriateness of “structure action” dependences could be integrated into future research.Актуальность. Сельскохозяйственная промышленность остро нуждается в создании новых и эффективных ростстимулирующих средств. Производные 1,2,4-триазола уже успели проявить себя как активные стимуляторы роста и фунгициды, а соли 2-(4-R-3-R1-1,2,4-триазол-5-илтио)ацетатных кислот являются весьма перспективными в этом плане.Целью данных исследований является определение влияния на качественные показатели прорастания ростков кукурузы гибрида Галатея после их обработки растворами солей 2-((4-R-3-(морфолинометилен)-4H-1,2,4-триазол-5-ил)тио)ацетатных кислот.Материалы и методы. Объектами наших исследований были 10 новых веществ, производных 1,2,4-триазола. В дальнейшем проведено исследование влияния указанных соединений на показатели энергии прорас-тания и всхожести семян кукурузы гибрида «Галатея» урожая 2016 г. Как эталон исследования использовался ауксин, а контролем служила дистиллированная вода.Результаты и их обсуждение. В результате исследования установлено, что указанные соединения поразному способны влиять на рост и развитие ростков кукурузы. Отмечено, что на интенсивность ростстимулирующей активности влияют как заместители при N4 атоме азота 1,2,3,4-триазолового цикла, так и характер катиона, который связан с кислотами.Выводы. В результате эксперимента установлено перспективность дальнейших исследований синтезированных веществ в качестве ростстимуляторов, а отмеченные закономерности зависимости «структура-действие» могут быть интегрированы в дальнейшие исследования.Актуальність. Сільськогосподарська промисловість гостро потребує створення нових та ефективних ріст-стимулюючих засобів. Похідні 1,2,4-триазолу вже встигли проявити себе як активні стимулятори росту та фунгіциди, а солі 2-(4-R-3-R1-1,2,4-триазол-5-ілтіо)ацетатних кислот є досить перспективними в цьому плані.Метою даних досліджень є визначення впливу на якісні показники проростання паростків кукурудзи гібриду Галатея після їх обробки розчинами солей 2-((4-R-3-(морфолінометилен)-4H-1,2,4-триазол-5-іл)тіо)ацетатних кислот.Матеріали та методи. Об’єктами наших досліджень були 10 нових речовин, похідних 1,2,4-триазолу. В подальшому проведено дослідження впливу вказаних сполук на показники енергії проростання та схожості насіння кукурудзи гібриду «Галатея» врожаю 2016 р. Як еталон дослідження використовувався ауксин, а контролем слугувала дистильована вода.Результати та їх обговорення. В результаті проведеного дослідження встановлено, що вказані сполуки по-різному здатні впливати на ріст та розвиток паростків кукурудзи. Відмічено, що на інтенсивність рістстиму-люючої активності впливають як замісники за N4 атомом азоту 1,2,4-триазолового циклу, так і характер катіону зв’язаного з кислотами.Висновки. В результаті експерименту встановлено перспективність подальших досліджень синтезованих речовин в якості рістстимуляторів, а відмічені закономірності залежності «структура-дія» можуть бути інтегровані в подальші дослідження

Cuticular wax composition of mature leaves of species and hybrids of the genus Prunus differing in resistance to clasterosporium disease

The interaction of a host plant with pathogen implies an extremely complex process involving the outer waxy layer of the cuticle, cutin, cell membrane, and intracellular structures. However, the initial contact between plants and pathogens takes place in cuticular waxes covering the surface of leaves, stems and fruits. Despite many findings on the role of plant epicuticular waxes, there is a gap in the understanding of the relationship between individual compounds and their functions. The pathogenic fungus Clasterosporium carpohilum (Lev.) Aderh. parasitizes the tissues of many stone fruit trees, damaging leaf and fruit surface. The aim of this work was to find out if the quantity and composition of leaf epicuticular wax could be responsible for the resistance to clasterosporium disease. The study of differences of plants in fungal resistance was carried out on species and hybrids of the genus Prunus from the collection of the Botanical Garden of Oles Honchar Dnipro National University (Dnipro city, Ukraine). The chloroform extracts of epicuticular waxes from the surface of mature leaves were analyzed by gas chromatography connected to mass-spectrometry. GC/MS assay was performed using Shimadzu GCMS-QP 2020 El equipped with capillary column (5% diphenyl/95% dimethyl polysiloxane), and helium as a carrier gas. Mass Spectrum Library 2014 for GSMS was used to identify the separated compounds of the wax extracts. The maximum total wax amount on the adaxial and abaxial leaf surface of hybrid 2 was twice the minimum wax accumulation for hybrid 4. Overall, 20 individual compounds belonging to six hydrocarbon classes were identified. Leaf epicuticular wax composition both in Prunus persica (L.) Batsch and P. dulcis (Mill.) D. A. Webb, and hybrids was dominated by long-chain n-alkanes with even carbon number (77.6–90.9% of total sum). The alkenes’ class was represented only by 17-pentatriacontene detected in the wax of both Prunus species. Octadecanaldehyde was found in epicuticular wax of P. dulcis while absent in wax of the more resistant species P. persica. Prime alcohols 1-tetradecanol and 1-hexacosanol were detected in leaf waxes of hybrid 4 and P. dulcis respectively. The ester class contained seven compounds found in leaf epicuticular waxes of both plant species and all hybrid forms. The identification of phthalic acid esters in leaf wax extracts was unexpected, and the phthalates’ origin is discussed. Strong positive correlation between leaf damage and tetrapentacontane content in epicuticular waxes could presumably be the result of infection-induced metabolism reprogramming in epidermal cells of infected leaves