669 Application of In-cell Western Blot to Study Burn-induced Cardiac Dysfunction

Introduction Traditional Western blotting (WB) may not reflect true target protein amount due to interference from enzymic activity during sample preparation. In-Cell Western (ICW) blotting has proved to have advantages over the traditional WB. However, ICW has not been used to study burn-induced cardiac dysfunction. This study will explore the efficacy of ICW in the study of burn-induced cardiac dysfunction. Methods Human cardiomyocytes (Ac16) were cultured in serum from rats with/without 60% of total body surface area (TBSA) burn injury. Cultured cells were permeabilized by Tritons-100 X, 1st anti-targeted protein antibodies were added and incubated, and, finally, two fluorescence-conjugated 2nd antibodies (one red and one blue) were added and incubated. LI-COR Odyssey CLx scanner was employed to obtain images and LI-COR Image Studio 4.0 software was used to analyze image and GraphPad Prism 8.0 software was used to determine image density. Results ICW proved to be a good tool to study burn injury. Our ICW data showed that burn serum resulted in 1) increased cardiomyocyte PARP1 (leading to increased protein acetylation); 2) increased damage of cardiomyocyte mitochondrial DNA replication (via increase in mitochondrial Fis1and BCBN proteins and decrease in parkin and POLG proteins); and 3) a decrease of cardiomyocyte mitochondrial biogenesis (with increase in inflammation-related proteins, apoptosis-related proteins, and decrease Brf2-ARE-related proteins). Conclusions This study provides preliminary evidence that ICW may be a novel tool to study burn injury and confirms that burn-injury induced cardiomyocyte mitochondrial dysfunction occurs via damage of mitDNA replication, involving inflammation, apoptosis, and mitochondrial biogenesis pathways.

Recombinant expression of lytic polysaccharide monooxygenase and its functional characterization

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that can act on crystalline polysaccharides directly, which plays a critical role in cellulose degradation. In addition to reports on its structure and mechanism of action, it is important to study the auxiliary activity 9 (AA9) characteristics from different resources to support the mechanism research. The gene encoded ToLPMO9A was cloned from Trichoderma orientalis EU7-22 and first heterologously expressed in Pichia pastoris GS115. Both metal ions and reducing agent concentrations showed an important effect on ToLPMO9A. The ToLPMO9A exhibited maximal activity at 60 °C and a 6.0 pH. In addition, ToLPMO9A showed substrate specificity. The matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis showed that ToLPMO9A cleaved the glycosidic bonds at C1 and C4/C6 position via oxidation. Concerning the synergistic effects on enzymic activity, ToLPMO9A exhibited promotion with endo-glucanase or exo-glucanase, but inhibition with β-glucosidases. In conclusion, ToLPMO9A could be a good choice for enzyme cocktails and provide theoretical support for subsequent action mechanisms and broader applications.

The characteristics of glucose homoeostasis in grass carp and Chinese longsnout catfish after oral starch administration: a comparative study between herbivorous and carnivorous species of fish

An oral starch administration trial was used to evaluate glucose homoeostasis in grass carp (Ctenopharyngodon idella) and Chinese longsnout catfish (Leiocassis longirostris Günther). Fish were administered with 3 g of a water and starch mixture (with 3:2 ratio) per 100 g body weight after fasting for 48 h. Fish were sampled at 0, 1, 3, 6, 12, 24 and 48 h after oral starch administration. In grass carp, plasma levels of glucose peaked at 3 h but returned to baseline at 6 h. However, in Chinese longsnout catfish, plasma glucose levels peaked at 6 h and returned to baseline at 48 h. The activity of intestinal amylase was increased in grass carp at 1 and 3 h, but no significant change in Chinese longsnout catfish was observed. The activity of hepatic glucose-6-phosphatase fell significantly in grass carp but change was not evident in Chinese longsnout catfish. The expression levels and enzymic activity of hepatic pyruvate kinase increased in grass carp, but no significant changes were observed in the Chinese longsnout catfish. Glycogen synthase (gys) and glycogen phosphorylase (gp) were induced in grass carp. However, there was no significant change in gys and a clear down-regulation of gp in Chinese longsnout catfish. In brief, compared with Chinese longsnout catfish, grass carp exhibited a rapid increase and faster clearance rate of plasma glucose. This effect was closely related to significantly enhanced levels of digestion, glycolysis, glycogen metabolism and glucose-induced lipogenesis in grass carp, as well as the inhibition of gluconeogenesis.

EFFECT OF NITROGEN AND HUMIC FERTILIZERS ON THE BIOCHEMICAL STATE OF OIL‐CONTAMINATED CHERNOZEM

Aim. In this paper, we aim to assess the effect of nitrogen and humic fertilizers on the biochemical state of oil‐contaminated chernozem.Methods. In order to simulate the oil pollu‐ tion, chernozem was exposed to oil doses constituting 1, 5 and 10% of the soil mass for 30, 60 and 90 days. For simulating bioremediation of oil‐contaminated chernozem, the following fertilizers were used: potassium and sodium humates, urea and nitroammophos. Nitrogen fertilizers – urea and nitroammophos having a nitrogen content of 46% and 15%, respectively – were applied to the soil for the purposes of restoring the equilibrium between carbon and nitrogen. Humic fertilizers (potassium and sodium humates) were applied to the soil for stimulating the indigenous oil destructive microbiota. In order to assess the biological activity of the soil, we determined catalase activity, invertase activity, as well as CO2 emission intensity.Results. The effect of urea, nitroammophos, potassium and sodium humates on the enzymatic activity and CO2 emissions of ordinary chernozem, which had been exposed to various doses of oil (1, 5 and 10% of the soil mass) for 90 days, was studied in a model experiment. Following the introduction of nitroammophos into soil with low levels of oil pollution, catalase activity decreased, whereas respiration and invertase activity increased. Urea introduced into the soil contaminat‐ ed with a 10% dose of oil stimulated catalase activity. At oil concentrations of 1 and 5%, the introduction of potassium and sodium humates had a stimulating effect on enzymic activity and carbon dioxide evolution.Conclusions. It is advisable to use the intensity of CO2 emissions released from the soil, as well as the invertase activity for diagnosing the state of chernozem con‐ taminated with oil (5‐10%) following the introduction of nitrogen and humic ameliorants. At lower doses of oil, it is advisable to assess the state of the soil following the introduction of nitrogen fertilizers by catalase activity.

Enzymatic Activity of Chernozem Typical Depending on The Main Cultivation of Soil and Fertilizer

All agrotechnical and agrochemical measures aimed at increasing soil fertility have an effect on enzyme activity. Measures such as soil cultivation, the introduction of organic and mineral fertilizers into the soil,activate or suppress the enzymatic processes. The research relies on the intensity and direction of biochemical processes in the soil, containing a series of methods, the use of which has enabled the establishment of a favorable enzymatic active layer of arable chernozem typical. Such methods include: field, laboratory, analysis. The level of differentiation of protease, urease, phosphatase, amylase, catalase, depending on the cultivation of soil and fertilizer, was revealed in the process of comparative analysis of enzymic activity of typical black soil in sunflower agrocenosis. Field experiments on the study of fermentative activity of typical black currant were conducted during 2012-2016. The object of research was soil cover. It was established that the activation of biochemical processes in the soil is ensured by the organo-mineral fertilizer system - compost 4.5 t + N40P48K54 + 3.5 t by-products and seed weight per hectare of crop rotational area. Application of the mineral fertilizer system reduces the enzymatic activity of typical black soil. The highest activity of the protease and catalase passes in the arable layer for powered-unpowered cultivation. Higher phosphatase activity was observed for differentiated soil cultivation. Research on this problem should be continued in order to establish a relationship between the parameters of enzymatic activity of soil with the structure of microbial cenosis of typical black soil of different systems of basic cultivation and fertilization

INFLUENCE OF SINGLE AND MIXED CROPS OF PERMANENT GRASS ON SOIL ENZYMIC ACTIVITY IN THE CONDITIONS OF FOREST-STEPPE OF THE MIDDLE VOLGA REGION

The research is aimed at the development of effective methods to restore soil fertility under intensive agrogenic loads. The studies were conducted in the fields of the Department of Plant Cultivation and Agriculture from 2016 to 2018. The following variants of single-species and mixed crops of perennial grasses were studied: 1. awnless brome; 2. crested wheat grass; 3. awnless brome + smooth brome; 4. crested wheat grass + Agropyron; 5 awnless brome + smooth brome + Hungarian sainfoin; 6. crested wheat grass + Agropyron + Hungarian sainfoin. Medium soil samples were taken from the experimental field from all variants of single-species and mixed crops of perennial grasses in triplicate in three periods: beginning, middle, and end of the growing season. Samples were taken from depth of 0-20 cm and 20-40 cm. Bacteria number was assessed by sowing special soil mixture on the solid sterile MPA media according to the method of J. Sega. Enzyme activity was determined by the method of A. Sh. Galstyan. When studying the activity of PPO, a high activity of this enzyme was observed in the soil layer of 20-40 cm. The analysis of the activity of the enzyme peroxidase has various indicators both between the studied variants and in the soil section. The high activity of the enzymes was noted in the variants «awnless brome + smooth brome» and «crested wheat grass + Agropyron + Hungarian sainfoin». In the process of research, a correlation was found between peroxidase activity and the number of bacteria. The highest coefficient of humification was noted in the «awnless brome», the lowest – «crested wheat grass + Agropyron + Hungarian sainfoin». From 2016 to 2018, in the first variant, a decrease in the coefficient of humification is observed. Thus, of the above options, with the optimal species composition, the following options turned out to be: « awnless brome», «awnless brome +smooth brome», «awnless brome +smooth brome + Hungarian sainfoin». Research data were processed by the dispersion method.

Combined effect of glyphosate, saccharin and sodium benzoate on the gut microbiota of rats

Glyphosate is the main component of many broadly used herbicides due to its safety for humans and animals. It is known that the remains of glyphosate are present in allowable doses in fodders and food products, and, consumrd in low doses, it is found in insignificant amounts in milk, eggs and even in the internal organs (liver, kidneys) of animals. For determining combined impact of glyphosate and the commonest food additives on the composition of microbiota of animals, four groups of laboratory male rats were formed, which during 42 days consumed pure water without any restrictions; 1% aqueous solution of glyphosate; 1% solution of glyphosate in combination with 1% solution of sodium benzoate; 1% solution of glyphosate with 1% solution of saccharin. After killing the animals, 1 g of feces were collected and by serial dilutions with 10–1 to 10–9 sterile physiologic solution, a microbiological analysis was undertaken. Out of each dilution an inoculation of the studied material to the elective growth media was performed, by 0.1 cm3, then the material was incubated in a thermostat (24–72 hours, temperature 37 °С), the results were recorded after 24–72 h. The microorganisms were identified by studying morphological parameters, tinctorial, cultural and enzymic properties. Results are provided in CFU/g (colony-forming unit per gramm) of feces. The impact of glyphosate and glyphosate with food additives led to no changes in the number of Escherichia coli and emergence of this species of microorganism with changed enzymic activity. Also no changes occurred in the number of microorganisms of Bifidobactrium and Lactobacillus spp. Addition of glyphosate, and also glyphosate in combination with saccharin to the diet contributes to broader reproduction of microorganisms of Klebsiella, Citrobacter, Enterobacter and Pseudomonas genera. Mixtures of glyphosate and food additives allow conditionally pathogenic yeast-like Candida fungi (Candida glabrata and C. albicans) to spread more widely in the intestine. Significant fluctuations in the number of Enterococcus spp. bacteria genus were observed: by 80 times within range of each of the three experimental groups of rats with addition of herbicide with benzoate and saccharin to the diet.

Anti-genotoxic and anti-mutagenic activity of Escherichia coli Nissle 1917 as assessed by in vitro tests

Anti-genotoxic or anti-mutagenic activity has been described for a number of Gram-positive probiotic bacterial species. Here we present evidence that Gram-negative Escherichia coli Nissle 1917 (EcN) also displays anti-genotoxic/anti-mutagenic activity, as assessed in vitro by the Comet Assay and the Ames Test, respectively. This activity was demonstrated by use of the mutagens 4-nitroquinoline-1-oxide (NQO), hydrogen peroxide (H2O2) and benzo(a) pyrene (B[a]P). For both assays and all three test agents the anti-genotoxic/anti-mutagenic activity of EcN was shown to be concentration dependent. By the use of extracts of bacteria that were inactivated by various procedures (heat treatment, ultrasound sonication or ultraviolet light irradiation), mechanistic explanations could be put forward. The proposed mechanisms were enforced by treating the bacterial material with proteinase K prior to testing. The mutagen H2O2 is most likely inactivated by enzymic activity, with catalase a likely candidate, while several explanations can be put forward for inactivation of B[a]P. NQO is most likely inactivated by metabolising enzymes, since the formation of the metabolite 4-aminoquinoline could be demonstrated. In conclusion, the in vitro results presented here make a strong case for antimutagenic properties of EcN.

A proposition for a cancer treatment study using radioactive metal co-factor enzymes

Cancer is a serious pathological condition of abnormal cells are gathered in tumors in the body's tissues or organs. Due to their accelerated metabolism, cancer cells require a great demand for energy, protein (cell structure substrates), and metabolic enzyme activity. If the body does not respond adequately to this demand, the metabolic processes of cancer cells will be hampered, and their growth will be limited or even stopped. It is possible to control the metabolic processes of the cancerous tumors by performing one or more of the following approaches: stopping the energy and cell structure substrate supply, inhibiting enzymic activity, and/or destroying cancer cells with external agents (such as radiation and/or chemicals). These approaches have been investigated either in single or combination modes, but so far the results obtained have not been on par with expectations. In this paper, we propose a method of cancer treatment which entails the use of a radioisotope instead of stable metal to break down the structure of metal co-factor enzyme and to deactivate its catalytic function. With a judicious choice of the metal radioisotope, this method is even able to perform all the above-mentioned approaches, and at the same time, giving a much better efficacy in cancer treatment.