An improved microtiter plate assay to monitor the oxidative burst in monocot and dicot plant cell suspension cultures

BACKGROUND: A screening method for elicitor and priming agents does not only allow detecting new bioactive substances, it can also be used to understand structure–function relationships of known agents by testing different derivatives of them. This can not only provide new lead compounds for the development of novel, more environment-benign, bio-based agro-chemicals, it may eventually also lead to a better understanding of defense mechanisms in plants. Reactive oxygen species (ROS) are sensitive indicators of these mechanisms but current assay formats are not suitable for multiplex screening, in particularly not in the case of monocot systems. RESULTS: Here we describe continuous monitoring of ROS in 96-well microtiter plates using the chemiluminescent probe L012, a luminol derivative producing chemiluminescence when oxidised by ROS like hydrogen peroxide, superoxide, or hydroxyl radical that can thus be used as an indicator for these ROS. We were able to measure ROS in both monocot (Oryza sativa) and dicot (Medicago truncatula) cell suspension cultures and record dose dependencies for the carbohydrate elicitors and priming agents ulvan and chitosan at low substrate concentrations (0.3–2.5 µg/ml). The method was optimized in terms of cell density, L012 concentration, and pre-incubation time. In contrast to the single peak observed using a cuvette luminometer, the improved method revealed a double burst in both cell systems during the 90-min measuring period, probably due to the detection of multiple ROS rather than only H2O2. CONCLUSION: We provide a medium throughput screening method for monocot and dicot suspension-cultured cells that enables direct comparison of monocot and dicot plant systems regarding their reaction to different signaling molecules.<br

The genetic status of Sorocea bonplandii in the highly fragmented forest in southern Brazil.

Sorocea bonplandii is a dioecious sub canopy plant species with an abundant and widespread distribution in the Atlantic Forest, Southern Brazil. It is traditionally used for art crafts and popular medicine. We have investigated the genetic status of S. bonplandii in one large and six small populations of the fragmented Atlantic Forest in southern Brazil. In spite of population fragmentation, the allozyme electrophoretic analysis involving 23 loci of 12 enzymes from 420 plants revealed unusually high frequencies of heterozygous genotypes and a balanced distribution, in agreement with Hardy-Weinberg proportions. Very little genetic differentiation among populations was detected. The observed excess of heterozygotes suggests that selection favors heterozygous plants. Although S. bonplandii is a dominant sub-canopy species which strongly depends on ombrophilous environmental conditions, its genetic structure and diversity do not appear to suffer with forest fragmentation. All of these characteristics, in addition to its medicinal uses, suggest that this species is a potential resource for sustainable management of tropical forests in southern Brazil

Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture

Выбор оптимальных геолого-технических мероприятий для интенсификации притока нефти на Х нефтяном месторождении

Рассматриваемое месторождение находится на завершающем этапе разработки. На основании анализа его геологических условий и технологических показателей разработки и анализа выработки запасов нефти были определены факторы, негативно влияющие на процесс разработки месторождения: высокая обводненность, неравномерный профиль вытеснения, наличие высокопроницаемых высокобводненных каналов и слабопроницаемых участков пласта с невыработанными целиками нефти. В соответствии с условиями месторождения предложен комплекс геолого-технических мероприятий, направленный на борьбу с выявленными негативными факторами разработки: зарезка боковых стволов с многостадийным гидроразрывом пласта на добывающих скважинах, полимерное заводнение – на нагнетательных скважинах.The considered field is at the final stage of development. From the analysis of its geological conditions and technological indicators of development and analysis of the production of oil reserves, factors were identified that negatively affect the process of field development: high water cut, uneven displacement profile, the presence of highly permeable high-water-cut channels and poorly permeable reservoir sections with unexploited oil reserves. In accordance with the conditions of the field, a set of geological and technical measures was proposed aimed at combating the identified negative development factors: sidetracking with multi-stage hydraulic fracturing at production wells, polymer flooding at injection wells

Исследование микроструктуры безобжиговых периклазоуглеродистых огнеупоров при использовании в качестве заполнителя различного вида периклаза

У статті представлено результати досліджень мікроструктури периклазовуглецевих зразків, у яких в якості наповнювача використовували різні види периклазу. Петрографічні дослідження показали, що зразки щільні та міцні, як на плавленому, так і на спеченому периклазі.In clause the results of researches of microstructure magnesia-carbon refractors are submitted, at which in quality filler used different kind magnesia. Microstructures of samples strong and dense, both on melted, and on sintered periclase have shown, that

Physicochemical characterization of FRET-labelled chitosan nanocapsules and model degradation studies

Sub-micron o/w emulsions coated with chitosan have been used for drug delivery, quorum sensing inhibition, and vaccine development. To study interactions with biological systems, nanocapsules have been fluorescently labelled in previous works, but it is often difficult to distinguish the released label from intact nanocapsules. In this study, we present advanced-labelling strategies based on Förster Resonance Energy Transfer (FRET) measurements for chitosan-coated nanocapsules and investigate their dissolution and degradation. We used FRET measurements of nanocapsules loaded with equimolar concentrations of two fluorescent dyes in their oily core and correlated them with dynamic light scattering (DLS) count rate measurement and absorbance measurements during their disintegration by dissolution. Using count rate measurements, we also investigated the enzymatic degradation of nanocapsules using pancreatin and how protein corona formation influences their degradation. Of note, nanocapsules dissolved in ethanol, while FRET decreased simultaneously with count rate, and absorbance was caused by nanocapsule turbidity, indicating increased distance between dye molecules after their release. Nanocapsules were degradable by pancreatin in a dose-dependent manner, and showed a delayed enzymatic degradation after protein corona formation. We present here novel labelling strategies for nanocapsules that allow us to judge their status and an in vitro method to study nanocapsule degradation and the influence of surface characteristics

Исследование состава и свойств нефтей месторождений Западной Сибири

В данной работе выполнены исследования в области состава и свойств высокопарафинистых нефтей месторождений Западной Сибири. Цель работы: исследование физико-химических свойств нефти со временем эксплуатации скважин месторождений Западной Сибири, для выбора наиболее выгодного метода транспортировки. Для достижения поставленной цели изучили химический состав, промысловую подготовку нефти, рассмотрели проблемы, возникающие при перекачке и способы транспортировки парафинистой нефти, исследовали изменение физико – химических свойств парафинистых нефтей Верхне-Салатского месторождения эксплуатационной скважины № 123.The purpose of the work: study of changes in physical and chemical properties of oil over the time of operation of wells of fields of Western Siberia, in order to choose the most profitable method of transportation. In order to achieve this goal, the chemical composition, field preparation of oil were studied, problems arising during pumping and methods of transportation of paraffinic oil were considered, changes in physical and chemical properties of paraffinic oils of the Verkhne-Salatskoye field of production well No. 123 were investigated

Разработка системы электропитания малого космического аппарата

Объектом исследования является системы электропитания малого космического аппарата. Целью ВКР является проектирование системы электропитания малого космического аппарата (МКА) для дистанционного зондирования Земли. В результате исследования определены: характеристики малых космических аппаратов; структура системы электроснабжения; проведен расчет элементов схем; рассмотрены производственная и экологическая безопасность проекта; рассчитаны затраты на проектирование.The object of study is the power supply system of a small spacecraft. The aim of the WRC is to design a power system for a small spacecraft (MCA) for remote sensing of the Earth. The study identified: characteristics of small spacecraft; power system structure; Calculation of circuit elements; considered industrial and environmental safety of the project; Design costs are calculated

Transkingdom mechanism of MAMP generation by chitotriosidase feeds oligomeric chitin from fungal pathogens and allergens into TLR2-mediated innate immune sensing.

Chitin is a highly abundant polysaccharide in nature and is linked to immune recognition of fungal infections and asthma in humans. Ubiquitous in fungi and insects, chitin is absent inmammals and plants and, thus, represents a microbeassociatedmolecular pattern (MAMP). However, highly polymeric chitin is insoluble, which potentially hampers recognition by host immune sensors. In plants, secreted chitinases degrade polymeric chitin into diffusible oligomers, which are "fed to" innate immune receptors and co-receptors. In human and murine immune cells, a similar enzymatic activity was shown for human chitotriosidase (CHIT1), and oligomeric chitin is sensed via an innate immune receptor, Toll-like receptor (TLR) 2. However, a complete system of generating MAMPs from chitin and feeding them into a specific receptor/co-receptor-aided sensing mechanism has remained unknown in mammals. The effect of the secreted chitinolytic host enzyme, CHIT1, on the TLR2 activity of polymeric chitin preparations from shrimps, house dust mites and the fungal pathogen Candida albicans was assessed in vitro using cell lines and primary immune cells. Moreover, the regulation of CHIT1 was analyzed. Here, we show that CHIT1 converts inert polymeric chitin into diffusible oligomers that can be sensed by TLR1/TLR2 co-receptor/receptor heterodimers, a process promoted by the lipopolysaccharide binding protein (LBP) and CD14. Furthermore, we observed that Chit1 is induced via the b-glucan receptor Dectin-1 upon direct contact of immortalized human macrophages to the fungal pathogen Candida albicans, whereas the defined fungal secreted aspartyl proteases, Sap2 and Sap6, from C. albicans were able to degrade CHIT1 in vitro. Our study shows the existence of an inducible system of MAMP generation in the human host that enables contact-independent immune activation by diffusible MAMP ligands with a striking similarity to the plant kingdom. Moreover, this study highlights CHIT1 as a potential therapeutic target for TLR2-mediated inflammatory processes that are fueled by oligomeric chitin

Transcriptomics reveal a mechanism of niche defense: two beneficial root endophytes deploy an antimicrobial GH18‐CBM5 chitinase to protect their hosts

Effector secretion is crucial for root endophytes to establish and protect their ecological niche. We used time-resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root-associated bacteria. We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization. Some effectors responded to both plants and microbes, suggesting dual roles in intermicrobial competition and plant-microbe interactions. A subset of putative antimicrobial effectors, including a GH18-CBM5 chitinase, was induced exclusively by microbes. Functional analyses of this chitinase revealed its antimicrobial and plant-protective properties. We conclude that dynamic effector gene expression underpins the ability of Sebacinales to thrive in diverse ecological niches with a single fungal chitinase contributing substantially to niche defense