Journal articles on the topic 'Degree Discipline: Biological Active Molecules'

Several techniques to assisting in the drug design and discovery stages have been developed during the last several decades. The bulk of these techniques aimed to find novel chemical entities that had the greatest significant interaction with the targeted receptors or enzymes while providing the least degree of risk of unwanted interactions. This approach, on the other hand, is time-consuming and expensive, as it requires the screening of thousands of molecules for biological activity, with only one making it to market. The prodrug strategy, in which the active drug molecule is disguised by a promoiety to change its undesirable characteristics, is one of the most appealing and promising methods. The folate receptor (FR)-targeted systems may also open the path for more advanced drug conjugates, especially because this receptor is now being targeted by a variety of technological innovations, including nanoparticles, small molecules, and protein-based technologies, resulting in a wealth of experience in the discipline.

A reduction in the planet’s biodiversity requires an active response by politicians, environmental activists, and scientists. Modern biological education should provide an opportunity to acquire the knowledge and skills necessary to solve complex tasks targeted at preserving and restoring vulnerable species habitats and ecosystems. Students study conservation biology at many universities around the world with this as their goal. For the first time in Russia, a Master’s Conservation Biology course for biology students was developed and tested at Novosibirsk State University. This primer course (108 hours) includes lectures, discussions, excursions, elements of gamification, combines auditorium and online classes, uses social networks for additional communication with students, and experienced practitioners. The course has been highly rated by students and can be expanded to include a larger audience.

Natural products have historically contributed to drug discovery as a source of bioactive molecules, due to their great diversity and structural complexity. They have provided “lead” molecules for the development of drugs in different therapeutic areas, with a very prominent representation in the treatment of pain and inflammation, coagulation disorders, metabolic disorders, as well as in the treatment of cancer and infectious diseases. In recent decades there has been a paradigm shift in drug discovery strategies that has allowed the identification of new active natural products in therapeutic targets. Combinatorial Chemistry and biological tests (High Throughput Screening), together with the development of computational techniques, have contributed decisively to the design and optimization of libraries of natural product derivatives based on their biological activity. In parallel, technological advances in the field of Omics sciences and in data processing lead to a multidimensional approach in the drug discovery process. These powerful tools will allow the analysis of the pharmacological potential of natural products and their derivatives for the conversion of these molecules to active products with low toxicity. In the Precision Medicine era, natural products continue to be molecules with great potential in pharmaceutical development, since, unlike other therapeutic strategies, they have a favorable cost-benefit ratio, which will allow their future use in this discipline.

The synthesis of a new xanthine nitric oxide donor (TSP-81) has been discussed. The designed compound includes two structural moieties - theophylline (1,3-dimethylxanthine) and acetaminophen (4-hydroxyacetanilide) linked by the nitric oxide donor alkyl chain as a spacer. The compound has been characterized by microanalysis (CHN), 1H-NMR, 13C-NMR, FT-IR, UV-vis, TG and DTG. The thermal behaviour showed that TSP-81 melts with decomposition, in four steps, the most important ones being the 2nd one (the registered weight loss being 17.6 %) and the 3rd one (with a registered weight loss of 30.4 %). The toxicity degree, the anti-inflammatory effect and the ability of releasing nitric oxide of the TSP-81 have also been evaluated. The biological assays established that TSP-81 exhibits enhanced biological properties such as lower toxicity and higher anti-inflammatory effect in reference with theophylline and acetaminophen, the drugs used as parents molecules. The TSP-81 is approximately 2 times more active than theophylline and 4 times more active than acetaminophen in reducing cotton pellet-granuloma formation. Furthermore, the release of nitric oxide (NO) appears to have an important contribution to enhancing the anti-inflammatory effect.

Many of the similarity-based virtual screening approaches assume that molecular fragments that are not related to the biological activity carry the same weight as the important ones. This was the reason that led to the use of Bayesian networks as an alternative to existing tools for similarity-based virtual screening. In our recent work, the retrieval performance of the Bayesian inference network (BIN) was observed to improve significantly when molecular fragments were reweighted using the relevance feedback information. In this paper, a set of active reference structures were used to reweight the fragments in the reference structure. In this approach, higher weights were assigned to those fragments that occur more frequently in the set of active reference structures while others were penalized. Simulated virtual screening experiments with MDL Drug Data Report datasets showed that the proposed approach significantly improved the retrieval effectiveness of ligand-based virtual screening, especially when the active molecules being sought had a high degree of structural heterogeneity.

Although significant differences in activity between optical isomers have been recognized in many types of pesticides, the role of stereoselectivity has not been fully characterized for one of the most important classes of commercial herbicides, those that inhibit photosynthetic electron transport. This report describes experiments in which optically active α-methylbenzylamine or sec-butylamine was used as starting material for the synthesis of optically active triazine and urea herbicides. The biological activities of the compounds were determined in two in vitro chloroplast assays - competition for specifically bound [14C]atrazine and inhibition of photosystem II-medi- ated dye reduction - as well as in whole plant phytotoxicity. In both in vitro assays the (-)-isomer of the N-a-methylbenzyl triazine was about 15-fold more active than the (+)-isomer, and the racemate fell in between and was of about the same potency as atrazine. The same relative activities were also seen for in vivo phytotoxicity. The a-methylbenzyl urea derivatives were much less herbicidally active, but the in vitro assays were able to discriminate between the optical isomers. In both assays, the (-)-isomer of the urea was much more active than the (+)-isomer, with the racemate intermediate. Steric factors play a critical role in the degree of this chiral discrimination, since in both the corresponding triazines and ureas, the optically active molecules synthesized from the enantiomers of 2-butylamine showed only slight differences in activity. Saturation of the phenyl ring of the a-methylbenzyl triazines resulted in molecules which still showed substantial differences in activity related to chirality, further supporting the importance of steric factors, rather than electronic, in this chiral discrimination.

Chitosan displays a dual function, acting as both an active ingredient and/or carrier for pharmaceutical bioactive molecules and metal ions. Its hydroxyl- and amino-reactive groups and acetylation degree can be used to adjust this biopolymer’s physicochemical and pharmacological properties in different forms, including scaffolds, nanoparticles, fibers, sponges, films, and hydrogels, among others. In terms of pharmacological purposes, chitosan association with different polymers and the immobilization or entrapment of bioactive agents are effective strategies to achieve desired biological responses. Chitosan biocompatibility, water entrapment within nanofibrils, antioxidant character, and antimicrobial and anti-inflammatory properties, whether enhanced by other active components or not, ensure skin moisturization, as well as protection against bacteria colonization and oxidative imbalance. Chitosan-based nanomaterials can maintain or reconstruct skin architecture through topical or systemic delivery of hydrophilic or hydrophobic pharmaceuticals at controlled rates to treat skin affections, such as acne, inflammatory manifestations, wounds, or even tumorigenesis, by coating chemotherapy drugs. Herein, chitosan obtention, physicochemical characteristics, chemical modifications, and interactions with bioactive agents are presented and discussed. Molecular mechanisms involved in chitosan skin protection and recovery are highlighted by overlapping the events orchestrated by the signaling molecules secreted by different cell types to reconstitute healthy skin tissue structures and components.

Holistic medicine is an interdisciplinary field of study that integrates all types of biological information (protein, small molecules, tissues, organs, external environmental signals, etc.) to lead to predictive and actionable models for health care and disease treatment. Despite the global and integrative character of this discipline, a comprehensive picture of holistic medicine for the treatment of complex diseases is still lacking. In this study, we develop a novel systems pharmacology approach to dissect holistic medicine in treating cardiocerebrovascular diseases (CCDs) by TCM (traditional Chinese medicine). Firstly, by applying the TCM active ingredients screened out by a systems-ADME process, we explored and experimentalized the signed drug-target interactions for revealing the pharmacological actions of drugs at a molecule level. Then, at a/an tissue/organ level, the drug therapeutic mechanisms were further investigated by a target-organ location method. Finally, a translational integrating pathway approach was applied to extract the diseases-therapeutic modules for understanding the complex disease and its therapy at systems level. For the first time, the feature of the drug-target-pathway-organ-cooperations for treatment of multiple organ diseases in holistic medicine was revealed, facilitating the development of novel treatment paradigm for complex diseases in the future.

Environmental engineering education has been an active option for engineers from all disciplines for nearly 50 years at the graduate level. Some graduate programs expanded to integrate students with undergraduate science degrees with the engineering programs, since the cross discipline interaction is required outside the academic programs. In the mid-1980s interest increased to such a level that undergraduate programs began to form. Several of these programs have been accredited in their various forms recognizing the diversity of the field and those presenting the programs. The progression from graduate-degree-based specializations to broad-based undergraduate programs reflects both the increased knowledge in the field and the increased demand for professional engineers capable of responding to public health and environmental protection issues. Graduate programs greatly expand fundamental knowledge of physical, chemical, and biological processes and their application to protection problems. Of course, the doctorate is dedicated to the development of significant new knowledge. This paper defines several of the basic components of the environmental engineering profession and the educational process needed to produce qualified environmental engineers.Key words: environmental engineering, education, courses, undergraduate environmental engineering, graduate environmental engineering.

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

The majority of the effects observed upon envenomation by scorpaenoid fish species can be reproduced by the cytolysins present in their venoms. Fish cytolysins are multifunctional proteins that elicit lethal, cytolytic, cardiovascular, inflammatory, nociceptive, and neuromuscular activities, representing a novel class of protein toxins. These large proteins (MW 150–320 kDa) are composed by two different subunits, termed α and β, with about 700 amino acid residues each, being usually active in oligomeric form. There is a high degree of similarity between the primary sequences of cytolysins from different fish species. This suggests these molecules share similar mechanisms of action, which, at least regarding the cytolytic activity, has been proved to involve pore formation. Although the remaining components of fish venoms have interesting biological activities, fish cytolysins stand out because of their multifunctional nature and their ability to reproduce the main events of envenomation on their own. Considerable knowledge about fish cytolysins has been accumulated over the years, although there remains much to be unveiled. In this review, we compiled and compared the current information on the biochemical aspects and pharmacological activities of fish cytolysins, going over their structures, activities, mechanisms of action, and perspectives for the future.

Abstract Objective The aim of the study is to explore the molecular mechanism of Yadanzi (Brucea javanica) in the treatment of glioblastoma (GBM) by using the methods of bioinformatics and network pharmacology. Methods The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature retrieval method were applied to obtain the active ingredients of Yadanzi (Brucea javanica), and to predict the relevant targets of the active ingredients. The GBM-related targets were retrieved and screened through the Gene Expression Profiling Interactive Analysis (GEPIA) database, and mapped to each other with the targets of the components of Yadanzi (Brucea javanica) to obtain the intersection targets. The GBM differentially expressed gene targets were imported into the String database to obtain the protein interaction relationship, the Cytoscape software was used to draw the protein interaction network, the Cytobba and MCODE plug-ins were used to screen the core genes and important protein interaction modules, and the GEPIA database was applied to make survival analysis of the core genes. The network map of “active ingredients-targets” was constructed through the Cytoscape 3.6.1 software. Gene Ontology (GO) biological function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis for GBM differentially expressed genes were performed through the DAVID database. Results Through TCMSP and literature retrieval, 23 potential active ingredients and 129 related targets were obtained from Yadanzi (Brucea javanica). In the GEPIA database, 247 GBM differentially expressed genes were screened, including 113 up-regulated genes and 134 downregulated genes. After mapping with the targets related to the active ingredients of Yadanzi (Brucea javanica), six intersection targets were obtained, that is, the potential action targets of Yadanzi (Brucea javanica) in treating GBM, including MMP2, HMOX1, BIRC5, EGFR, CCNB2, and TOP2A. Cytoscape software was applied to build an “active ingredient-action target” network. Two active ingredients and five action targets of β-sitosterol (BS) and luteolin were found, and the targets were mainly concentrated in BS. It was found by KEGG pathway enrichment analysis that GBM differentially expressed genes were mainly involved in signaling pathways related to Staphylococcus aureus infection, phagosome formation, tuberculosis and systemic lupus erythematosus and other infectious and autoimmune diseases. It was found by GO enrichment analysis that the GBM differentially expressed genes mainly involved such biological processes (BP) as the processing and presentation of exogenous antigenic peptides and polysaccharide antigens through MHC II molecules, γ-interferon-mediated signaling pathways, extracellular matrix composition, and chemical synapses transmission; it involved cellular components such as cell junctions, axon terminal buttons, extracellular space, vesicle membranes for endocytosis, and MHC II protein complexes; molecular functions such as calcium-mediated ionic protein binding, MHC II molecular receptor activity, immunoglobulin binding, and phospholipase inhibitor activity were also involved. Survival analysis was conducted by GEPIA on the top 37 core targets in degree value, and a total of five genes related to GBM prognosis were obtained. Among them, FN1 and MMP2 were highly expressed while GABRD (γ-aminobutyric acid A receptor delta subunit), RBFOX1, and SLC6A7 were expressed at a low level in cancer patients. Conclusion The pathogenesis of GBM is closely related to the human immune system, and BS and luteolin may be the main material basis of Yadanzi (Brucea javanica) for the treatment of GBM and the improvement of prognosis. The molecular mechanism may be related to the physical barrier formed by destroying the tumor cell stromal molecules and its involvement in tumor immune response.

In modern conditions, nanomaterials, especially nanoparticles of metals and nonmetals, are increasingly used in various industries. Due to their unique properties, in particular, the ability of nanoparticles to exhibit an enzyme-like effect they are widely used in biology, medicine, biotechnology, the food industry and agriculture. Important advantages of nanoparticles are their size, which enables specific properties to be present: their large surface area, the ability to transfer molecules and the ability to protect them from degradation and release over a long time, the location of action and the specificity of interaction with biological structures. Nanoparticles play a special role in the processes of neutralizing the active forms of oxygen. It has been established that a number of nanoparticles, in particular, Fe, Mn, Zn, Ce, Si and Se oxides, have an enzyme-like activity mimicking that of some enzymes. By changing the degree of oxidation, these particles can regenerate and continuously catalyze the reaction of neutralizing superoxide anion radicals, thus fulfilling the function of SOD and being the first link in protecting tissues and cells from oxidative stress in physiological and pathological conditions. It is proved that nanoparticles Mn3O4, Fe3O4, Co3O4, CeO2, LaCoO3 and other elements can effectively dispose of hydrogen peroxide and other peroxides, showing catalase-like and peroxidase-like activity. Nanozymes are characterized that exhibit the activity of oxidases, peroxidases and phosphatase. The prospect of using mimetics for complex in vitro analyzes of high-sensitivity biomarker disease detection is shown. The possibility of effective multi-use of nanoparticles as antioxidants is indicated. There are good prospects for further research on properties and the use of polyfunctional particles that are easily synthesized, reliable and inexpensive. More work is needed to determine the interaction of enzymomimetics with biological molecules such as proteins, carbohydrates and lipids, and also to take into account the peculiarities of their metabolism, clearance, degradation, biocompatibility and side effects, since individual nanoparticles have the potential to be deposited in separate organs.

Background: Spread of COVID-19 attains a crucial transition in reveling its pandemic across the boundaries. In combating the infection caused by SARS-CoV-2, there is a spectrum of ideal strategies that have been adopted globally, of which repurposing of approved drugs considerably having high clinical relevance. 3-chymotrypsin-like protease (3CL pro) is considered to be the potential target for the researchers as it is highly essential for cleavage of polyprotein to get 16 nonstructural proteins (called nsp1-nsp16). These proteins are highly essential for viral replication and hence become a primary target for enzyme inhibitors. 3CL pro, having a structural projectile helical chain with biologically active site involved in processing viral polyproteins that are evolved from RNA genome translation. Objective: The major objective of the present investigation is to evaluate the enzyme inhibition potential of FDA approved therapeutic leads in targeting 3CLpro that medicates the viral replication. Methods: Docking calculations were carried out for an array of FDA approved molecules which leads to a notable few molecules such as Emtricitabine, Oseltamivir, Ganciclovir, Chloroquine, Baricitinib, Favipiravir, Lopinavir, Ritonavir, Remdesivir, Ribavirin, Tenofovir, Umifenovir, Carbapenam, Ertapenem and Imipenam which have both specificity and selectivity in terms of binding efficiency against 3CL proenzyme. Results: A combinatorial evaluation employing in-silico screening shows a major lead for remdesivir which possesses a substantial affinity to 3CL pro binding on core amino acid residues, such as Leu 27, His 41, Gly 143, Cys 145, His 164, Met 165, Glu 166, Pro 168 and His 172 which share the biological significance in mediating enzymatic action. Results of docking simulation by Autodock over a host of FDA approved molecules show high degree of selectivity and specificity in the increasing order of binding capacity; Remdesivir> Ertapenem> Imipenam> Tenofovir> Umifenovir> Chloroquine> Lopinavir> Ritonavir> Emtricitabine> Ganciclovir> Baricitinib> Ribavirin>Oseltamivir>Favipiravir> Carbapenam. Conclusion: Till date, there is no known cure attained for treating COVID-19 infection. In conclusion, lead molecules from already approved sources provoke promising potential which grabs the attention of the clinicians in availing potential therapeutic candidate as a drug of choice in the clinical management of COVID-19 time-dependently.

Isoindoline-1,3-dione derivatives constitute an important group of medicinal substances. In this study, nine new 1H-isoindole-1,3(2H)-dione derivatives and five potential pharmacophores were obtained in good yield (47.24–92.91%). The structure of the new imides was confirmed by the methods of elemental and spectral analysis: FT–IR, H NMR, and MS. Based on the obtained results of ESI–MS the probable path of the molecules decay and the hypothetical structure of the resulting pseudo-molecular ions have been proposed. The physicochemical properties of the new phthalimides were determined on the basis of Lipiński’s rule. The biological properties were determined in terms of their cyclooxygenase (COX) inhibitory activity. Three compounds showed greater inhibition of COX-2, three compounds inhibited COX-1 more strongly than the reference compound meloxicam. From the obtained results, the affinity ratio COX-2/COX-1 was calculated. Two compounds had a value greater than that of meloxicam. All tested compounds showed oxidative or nitrosan stress (ROS and RNS) scavenging activity. The degree of chromatin relaxation outside the cell nucleus was lower than the control after incubation with all test compounds. The newly synthesized phthalimide derivatives showed no cytotoxic activity in the concentration range studied (10–90 µM). A molecular docking study was used to determined interactions inside the active site of cyclooxygenases.

The concurrent use of muscle relaxants and nonsteroidal anti-inflammatory drugs (NSAIDs) is a promising treatment for painful muscle hypertonia and convulsive states.Objective: to identify the most effective and safe synergist combinations of tolperisone and NSAIDs.Material and methods. A differential chemoreactome analysis was employed to evaluate the effects of the muscle relaxant tolperisone and five NSAIDs (dexketoprofen, etoricoxib, meloxicam, naproxen, and diclofenac). The biological activities of the molecules under study were assessed in five sections: 1) inhibition of the proteins of prostaglandin and leukotriene metabolism; 2) inhibition of the effects of the transcription factor nuclear factor kappa, tumor necrosis factor-, and other anti-inflammatory mechanisms; 3) inhibition of excessive blood coagulation and platelet aggregation; 4) vasodynamic effects; 5) antitumor properties on cell lines in culture.Results and discussion. Based on the differences in the pharmacological activity profiles of tolperisone and NSAIDs under study, the investigators identified the most promising synergistic combinations, in which both active ingredients complemented each other as effectively and safely as possible. The obtained estimates of the degree of synergism of various combinations of tolperisone and NSAIDs hold that the most promising antithrombotic, and antitumor effects.Conclusion. The results of this study will help adequately choose combinations of muscle relaxants and NSAIDs in patients with muscle hypertonia, which will be able to improve the efficiency and safety of treatment.

Abstract Background: CD3-engaging DART molecules direct T cells to tumor-expressed antigens. Flotetuzumab, a CD123 x CD3 bispecific DART molecule that targets the differential expression of the IL-3 receptor alpha chain (CD123) on acute myeloid leukemia (AML) blasts and leukemic stem cells, is currently being investigated in a phase 1 study in relapsed/refractory AML and myelodysplastic syndrome (MDS), with initial evidence of clinical activity1. A common adverse event in redirected T-cell killing interventions is cytokine release syndrome (CRS), a Cmax-associated event that may limit their therapeutic window. Owing to its short circulating half-life, flotetuzumab is administered as continuous infusion, which affords prolonged exposure with limited Cmax excursions. In contrast, long-acting Fc-bearing, CD3-engaging molecules would eliminate the need for continuous dosing, but would be especially impacted by CRS, given the high Cmax levels required to maintain adequate trough concentrations over several days or weeks. We previously reported the development of a CD3-engaging DART molecule with reduced affinity for CD3 that maintained maximal target cell killing and T-cell proliferation, albeit at higher concentrations than its wild-type (WT) counterpart, but with reduced cytokine release2. We report here further optimization and validation of the approach. Methods: Anti-CD3 variants spanning a range of affinities were formatted as Fc-bearing DART molecules with CD123- or CD19-targeting arms and evaluated for cell binding as well as for antigen-dependent induction of T-cell proliferation, cytolytic activity and cytokine release. A therapeutic index (TI) was determined from the ratio of in vitro cell killing activity to that of target cell-induced cytokine release. In vivo anti-tumor activity was assessed in human immune cell-reconstituted mouse tumor models or in mice expressing the epitope recognized by the CD3 arm of the DART proteins (human CD3 k/i mice). Safety, pharmacokinetic (PK) and biological activity of selected molecules was evaluated in cynomolgus monkeys. Results: Six Fc-bearing DART molecules (from a panel of 23 CD123 x CD3 DART variants) were selected for characterization. These molecules spanned a relatively small interval of low CD3 affinities, but varied greatly in activity, from inactive at all concentrations tested to being capable of lysing tumor cells, including primary leukemic and MDS blasts, to the same maximal level of killing observed with the parental WT DART molecule. Higher concentrations of the active variants were needed, owing to a deliberate design incorporating decreased CD3 binding. At levels capable of complete tumor cell lysis, active DART variants showed greatly reduced pro-inflammatory cytokine release (e.g., IL-2, IFN-γ and TNF-α) in vitro and in human PBMC-reconstituted mice compared to the WT version. Molecules were ranked based on their TI and a version with a highly favorable index was engineered as a B-cell targeting CD19 x CD3 DART molecule to conveniently ascertain its biological activity in cynomolgus monkeys. The CD19 x CD3 DART variant demonstrated good tolerability at single doses up to 30 mg/kg. B-cell depletion in the circulation and lymphoid tissues occurred at 1 mg/kg and achieved maximal depletion at ≥10 mg/kg, equivalent to the degree of reduction observed with 0.1 mg/kg of the WT molecule. However, only minimal circulating pro-inflammatory cytokines (including IL-6) were observed at all doses of the DART variant, while a substantial increase in IL-2, IL-6 and IFN-γ (among others) was observed with 0.1 mg/kg of the WT counterpart. The PK profile of the DART variant in cynomolgus monkeys appeared linear in the 1-30 mg/kg interval, with a half-life of 5 to 10 days, consistent with possible clinical dosing at weekly or longer intervals. Conclusions: A next-generation CD3-engaging Fc-bearing DART platform was generated through CD3 arm affinity modulation. These DART variants feature extended PK and an expanded TI suitable for potential clinical development in hematological malignancies. 1. Uy et al, Blood 2017, vol. 130, Suppl 1 637 2. Huang et al, Keystone Symposia: Antibodies as Drugs, 2/25-3/1/2018, Whistler, BC, Canada Disclosures Bonvini: MacroGenics: Employment, Equity Ownership. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Huang:MacroGenics, Inc.: Employment, Equity Ownership. Lam:MacroGenics, Inc.: Employment, Equity Ownership. Kaufman:MacroGenics, Inc.: Employment, Equity Ownership. Liu:MacroGenics, Inc.: Employment, Equity Ownership. Alderson:MacroGenics, Inc.: Employment, Equity Ownership. Stahl:MacroGenics, Inc.: Employment, Equity Ownership. Brown:MacroGenics, Inc.: Employment, Equity Ownership. Li:MacroGenics, Inc.: Employment, Equity Ownership. Sharma:MacroGenics, Inc.: Employment, Equity Ownership. Tamura:MacroGenics, Inc.: Employment, Equity Ownership. Johnson:MacroGenics, Inc.: Consultancy, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership.

Abstract Abstract 3353 Fucoidans are sulfated polysaccharides extracted from brown algae. Fucoidans have a wide variety of biological activities including pro- and anticoagulant activities, which occur at different concentration ranges. Therefore, fucoidans have also been described as non-anticoagulant sulfated polysaccharides (NASPs, Liu et al. Thromb Haemost 2006; 95:68). Fucoidans have complex structures due to their large molecular weight (Mw), wide Mw distribution, variable degree and pattern of sulfation, diverse monosaccharide composition, branching of the sugar chain and different monomer linkages. This structural complexity challenges identification of the components responsible for fucoidan activities. The aim of the presented study was to fractionate Fucus vesiculosus (F.v.) fucoidan by size and to de- and oversulfate it to obtain compounds of varying Mw or degree of sulfation (DS), respectively. The fucoidans were then to be analyzed for their pro-and anticoagulant activities and structure, and the effect of modified fucoidan on the target protein Tissue Factor Pathway Inhibitor (TFPI) investigated. Two approaches were applied to generate F.v. fucoidan with different structural properties to the original. Firstly, fucoidan was fractionated by size using ultrafiltration. The Mw of the resulting fractions ranged from 8–180 kD. NMR, elemental analysis and HPAEC showed that other structural features, such as sulfate content and monosaccharide composition, were similar to those of the original fucoidan. Thrombin generation (CAT) assays showed an EC50 for procoagulant activity of 0.3–3 μg/mL; aPTT increased by 50% at 4–25 μg/mL. Generally, higher Mw increased procoagulant activity. Below 15 kD, this activity was markedly reduced. The minimum active length of F.v. fucoidan was 70 carbohydrate units. De- and oversulfated F.v. fucoidans were used to investigate the impact of charge on pro- and anticoagulant activities. In the CAT assay, oversulfated fucoidans showed improved procoagulant activity with an EC50 of 0.09–0.12 μg/mL, while desulfated fucoidans demonstrated reduced procoagulant activity compared to the original. A DS of 0.5 was estimated to be the limit for procoagulant activity. Inhibition of TFPI by fucoidan was assessed with a dilute prothrombin time assay (dPT) with added full-length TFPI. TFPI-blocking activity was mainly dependent on the DS and less on the fucoidans' Mw. Interestingly, oversulfation also stimulated an undesired activation of the contact pathway. The presented study determined the minimal structural requirements for procoagulant activity of fucoidan molecules and identified features causing undesired biological effects. Disclosures: No relevant conflicts of interest to declare.

Abstract. Organic aerosols generated from the smoldering combustion of wood critically impact air quality and health for billions of people worldwide; yet, the links between the chemical components and the optical or biological effects of woodsmoke aerosol (WSA) are still poorly understood. In this work, an untargeted analysis of the molecular composition of smoldering WSA, generated in a controlled environment from nine types of heartwood fuels (African mahogany, birch, cherry, maple, pine, poplar, red oak, redwood, and walnut), identified several hundred compounds using gas chromatography mass spectrometry (GC-MS) and nano-electrospray high-resolution mass spectrometry (HRMS) with tandem multistage mass spectrometry (MSn). The effects of WSA on cell toxicity as well as gene expression dependent on the aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) were characterized with cellular assays, and the visible mass absorption coefficients (MACvis) of WSA were measured with ultraviolet–visible spectroscopy. The WSAs studied in this work have significant levels of biological and toxicological activity, with exposure levels in both an outdoor and indoor environment similar to or greater than those of other toxicants. A correlation between the HRMS molecular composition and aerosol properties found that phenolic compounds from the oxidative decomposition of lignin are the main drivers of aerosol effects, while the cellulose decomposition products play a secondary role; e.g., levoglucosan is anticorrelated with multiple effects. Polycyclic aromatic hydrocarbons (PAHs) are not expected to form at the combustion temperature in this work, nor were they observed above the detection limit; thus, biological and optical properties of the smoldering WSA are not attributed to PAHs. Syringyl compounds tend to correlate with cell toxicity, while the more conjugated molecules (including several compounds assigned to dimers) have higher AhR activity and MACvis. The negative correlation between cell toxicity and AhR activity suggests that the toxicity of smoldering WSA to cells is not mediated by the AhR. Both mass-normalized biological outcomes have a statistically significant dependence on the degree of combustion of the wood. In addition, our observations support the fact that the visible light absorption of WSA is at least partially due to charge transfer effects in aerosols, as previously suggested. Finally, MACvis has no correlation with toxicity or receptor signaling, suggesting that key chromophores in this work are not biologically active on the endpoints tested.

The safety of antibiotics is still debatable. The properties of large molecules to give insoluble compounds with metal ions leads to a decrease of absorption of active components and most often loss of biological activity. Depending on the presence of functional groups in the side chain, the formation of complexes of different strength is possible. The properties of a number of antibiotics to interact with calcium ions allow us to expect the interaction of these antibiotics with the surface of the tooth enamel along the calcium centers. The alkaline environment of the oral cavity and the excess of calcium ions in the saliva, in comparison with phosphate residues, make it possible to make available the calcium centers of the hydroxyapatite crystal. The strength of this complex will be determined by the structure and functional substituents of the antibiotic molecule and will not depend much on its charge. In this article the effect of broad-spectrum antibiotics on the remineralizing ability of saliva in relation to tooth enamel based on the experiments performed on teeth is discussed. The analysis of the degree of remineralization function of saliva was carried out by means of spectrophotometric determination of the stability of calcium complexes with individual broad-spectrum antibiotics at different multiplicity of individual components. As a result of setting up an experiment to restore tooth enamel after treatment with these solutions of antibiotics, the characteristic assessment of which is the presence of sites of remineralization and their size on the surface of the tooth enamel for certain periods of time and in comparison with the standard, it was concluded that antibiotic solutions reduce the remineralization ability of saliva to restore the outer surface of the enamel of teeth.

Whey proteins has the highest biological value among all proteins, but the main disadvantage of their use in food technologies is the presence of antigenic epitopes in the molecules that can cause allergic reactions in the human body. The most efficient way to reduce the allergenicity of whey proteins is their enzymatic hydrolysis, which leads to the destruction of antigenic sites and the release of biologically active peptides, including those with antioxidant effects. The purpose of the research is the determination of the whey proteins hydrolysis efficiency in an ultrafiltration concentrate (UF-concentrate) of cheese whey to reduce its allergenicity, as well as to establish the antioxidant activity of the obtained hydrolysate. The experimental studies were carried out at the Core Facilities Centre "Structural and Functional Research of Proteins and RNA" at the FSBSI "Institute of Protein of the Russian Academy of Sciences", as well as "Control and Management of Energy-efficient Projects" at FSBEI HE VSUET. Evaluation of the effectiveness of exposure to whey proteins in the UF-concentrate of cheese whey was carried out by the molecular weight distribution, length and charge of hydrolysis products. ?-lactoglobulin’s derivatives containing from 5 to 17 amino acid residues with a molecular weight of 561 to 1943 Da were found in the finishing hydrolysate. At the same time, hydrolysis made it possible to increase the mass fraction of short-chain peptides, including those with antioxidant properties. As a result of the whey proteins proteolysis in the UF-concentrate of cheese whey, its antioxidant activity increased by 2 times. The degree of hydrolysis of the main allergenic protein, ?-lactoglobulin, was 90-91%. The obtained hydrolysate of whey proteins can be recommended for use in the technology of various assortment groups of dairy products for dietary food with reduced allergenicity and antioxidant effect.

The achievements of modern domestic pharmacy clearly prove the prospects of searching for biologically active molecules among 1,2,4-triazole derivatives. The aim of our work was to predict the safest compound, select it and unambiguously prove the structure of a new promising molecule, to investigate some parameters of its toxicity. Materials and methods. X-ray diffraction analysis was performed in the laboratory of the Institute of Single Crystals of the National Academy of Sciences of Ukraine (Kharkiv). Computer methods were used to build “structure – toxicity” models and predict LD50 using already created models GUSAR, TEST. The degree of toxicity (DL50) and the approximate doses for the subacute experiment were determined by studying the acute toxicity of the test chemical and the injectable solution based on it. Determination of acute toxicity parameters by intragastric administration was performed on white rats, aged 3-4 months, body weight 200-220 g. Results. Due to the study of the toxicity of 1,2,4-triazole derivatives by non-experimental methods using GUSAR and TEST models, it was found that the test compounds could be classified as low-toxic substances. The compound is an organic salt that exists in the crystal as a solvate with one molecule of methanol and two molecules of water. When studying the structure of the crystal, it was found that the crystal is in the pinacoidal triclinic syngony. According to the results of the studies, it was found that after a single intragastric administration of the compound in doses of 1000, 3000 and 5000 mg / kg, all animals remained alive for 14 days. The basis of the biological action of chemical compounds is the violation of several biochemical processes. We found that the studied blood constants, on the background of the use of newly synthesized substance, underwent some changes. Conclusions. According to the assessment of the toxicity of the drug “VPK-434” when administered intragastrically to laboratory rats, it was found that in accordance with SOU 85.2-37-736: 2011 the test substance belongs to the IV class of toxicity (low toxicity). It was found that the average lethal dose of DL50 of the test substance by intramuscular administration to rats is 1666.66 mg / kg body weight. It was studied that some abnormalities in the hematopoietic system (increase in the number of leukocytes, including eosinophils), liver and kidney function (increased activity of transaminases, decreased serum concentrations of total protein, urea and creatinine) and changes in mineral metabolism of experimental animals groups, on the background of receiving 10 multiple doses of the study drug, was short-term, and the restoration of the functional state of the body of rats could be said as early as 4-5 days after discontinuation of the drug into their body

Abstract Mesenchymal stromal cells (MSCs) are multipotent progenitors, capable to differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, and chondrocytes. Thanks to their capacity to modulate immune responses and promote tissue repair, MSCs are considered a potential novel treatment for autoimmune and inflammatory diseases, including Crohn’s disease (CD). So far, the biological and functional properties of MSCs derived from bone marrow (BM) of CD patients have not been characterized. Aim of this study was to evaluate the feasibility of isolating and expanding ex vivo MSCs from BM of 7 patients with active CD (CD-MSCs; 5 males, median age 32 years, range 18–59), as well as to characterize these cells for their clonogenic efficiency, proliferative capacity, morphology, immunophenotype by flow cytometry, differentiation potential into osteoblasts and adipocytes, biosafety and ability to suppress in vitro the proliferation of autologous and allogeneic peripheral blood mononuclear cells (PBMCs). The properties of CD-MSCs were compared with those of BM-MSCs isolated from 4 healthy donors (HD-MSCs; 2 males, median age 33 years, range 16–47). Platelet lysate (PL, 5%) was employed as culture supplement to stimulate MSC growth. MSCs were successfully isolated and expanded ex vivo from BM of all 7 CD patients. The colony-forming unit-fibroblast (CFU-F) frequency after 10-day culture and the proliferative capacity were comparable in CD- and HD-MSCs. CD-MSCs showed the typical spindle-shaped morphology and ability to differentiate into osteoblasts and adipocytes. As regards the immunophenotype, CD-MSCs displayed the characteristic panel of surface markers (positivity for CD90, CD73, CD105, HLA A-B-C and negativity for CD34, CD45, CD14, CD80, CD31 molecules), with the exception of the expression of variable levels of HLA-DR at early passages (P1–P3) in culture. MSCs of 5 CD patients were propagated in long-term in vitro culture. CD-MSCs ceased their growth at variable passages (from P7 to P24) and entered a senescence phase. Senescent CD-MSCs were monitored for up to 8 weeks, without showing any change in morphology and/or proliferation rate. Results of array-Comparative Genomic Hybridization (array-CGH) demonstrated that CD-MSCs expanded in vitro do not show imbalanced chromosomal rearrangements. CD-MSCs were able to reduce in vitro PHA- and OKT3-stimulated proliferation of autologous PBMCs by up to 40% and 35%, respectively. The same degree of inhibition was observed when HD-MSCs were tested against both HD-PBMCs and CD-PBMCs. MSCs can be easily isolated and expanded ex vivo from BM of CD patients in the presence of a PL-auditioned medium; these cells exhibit similar morphological, phenotypic, and functional properties to those of HD-MSCs. These cells maintain a normal genetic asset after extensive ex vivo culture, as demonstrated by array-CGH experiments. The variable expression of HLA-DR in CD-MSCs at early passages might be related to the state of systemic inflammation of CD patients with active disease. In view of these results, autologous CD-MSCs can be considered as innovative, potentially anti-inflammatory, and reparative strategy for cell-therapy approaches in CD patients’ refractory to conventional treatments.

Perfectly projected and impeccably created, the endocrine system precisely regulates the most delicate immune processes. The immune and neuroendocrine systems are two essential physiological components of mammalian organisms important for protection from the infection and disease on one hand, and on the other, for regulation of metabolism and other physiological activities; namely, the evidence has been found indicating that there is active and dynamic collaboration of these systems in the execution of their designated functions [1, 2,4]. These interactions occur at many stages of embryonic and neonatal development, and they are a continual part of normal homeostatic balance necessary to preserve health. There is communication between neuroendocrine and immune system via cytokines, neurotransmitters and peptide hormones which act, in both systems, through the same receptor molecules (Scheme 1). Many investigators have reported the increased thymic weight in experimental animals due to both castration and adrenalectomy [4]. The discovery from 1898 revealing that thymus was enlarged in castrated rabbits has been considered the embryo of hybrid medical discipline, i.e. the immunoendocrinology [1]. In the actual literature, at least in that available to us, it has not been noted that the appearance of the eunuchs, i.e. the castrates, stimulated the analytical approach to this phenomenon. Endocrine influences appear to be a part of bidirectional circuitry, namely, thymic hormones also regulate the release of hormones from the pituitary gland. Physiologically, thymus is under neuroendocrine control. It is apparent that the circulating levels of distinct peptide hormones are necessary to maintain a series of biological functions related both to micro environmental and lymphoid cells of the organ. The neuroendocrine control of the thymus appears to be extremely complex, with apparent presence of complete intrathymic biological circuitry involving the production of pituitary hormones, as well as the expression of their respective receptors by thymic cell [7-9]. The influence of gonadectomy on the humoral immunity has been controversial. All investigations agree that women have higher titres of all classes of circulating antibodies than men [1, 3]. The application of estrogens stimulated the formation of antibodies in the circulation [17]. Then, if there were no sex glands, the immune response of the individual would be enhanced. Both the cellular and the humoral immune response is more powerful in the adult normal women than in men of the same age. The immune response is different in different sexes meaning that there is a sexual dimorphism. This difference has not been noted before the puberty [4]. It has been noticed that the substitution therapy has alleviated the late skin hypersensitivity [9], The estrogens have also curtailed the rejection time of the transplant and all reactions in which T-effector lymphocytes have been involved. NK-cells and T-lymphocytes activities have been decreased by the action of estrogens, as well as the release of thymus hormones [27]. Cortical RE cells express a surface antigen, gp200-MR6, which plays a significant role in thymocyte differentiation [7, 9]. irrespectively of which pathway may be triggered by neuroendocrine factors, the effects are pleiotropic and result in modulation of the expression of several genes in different cell types. Thymic neuroendocrine polypep-tides are the source of self-antigens presented by MHC molecules enabling the differentiation of haematopoietic stem cells [10]. Thymic nurse cells also produce thymosins beta 3 and beta 4 and display a neuroendocrine cell specific immunophenotype (IP): Thy-1+, A2B5+, TT+TE4+, UJ13/A+, UJ127.11+, UJ167.11+, S181.4+ and presence of common leukocyte antigen (CLA+) [7,16]. GH enhances thymocyte release from TNCs, as well as the reconstitution of these lymphoepithelial complexes [11]. Similar to its role as a regulator of bone metabolism through regulating osteoprotegerin (OPG) production, the estrogen is involved in the processes of thymocyte development although aromatase mRNA has not been detectable in the thymus. While the increase of TNC number during lactation may be linked to the process of reconstruction of the thymic lymphoid population, the increased activity of lymphoepithelial interactions on GD14 may be associated with thymic engagement in pregnancy-induced immune processes [27,29] The major antigens in the experimental autoimmune hypophysitis in rats are growth hormone, thyrotropin, and luteinizing hormone [12]. The intrathymic T-lymphocyte selection is a complex, multistep process, influenced by several functionally specialised RE cells and under immuno-neuroendocrine regulation control reflecting the dynamic changes of the mammalian organism. In HIV-1-infected adults treated with growth hormone [25 ], thymic mass and circulating naive CD4 T cells are increased. The treatment would be easier for the diseased, as well as to us, the physicians, if we were aware of two millennia old wisdom - that the disease is a visit of God.

Abstract Introduction Mantle cell lymphoma (MCL) patients often presents at later stages and progress through its disease course by frequent involvement of multiple dissemination sites including spleen, liver, bone marrow (BM), peripheral blood (PB), and gastrointestinal tract (GI). This devious behavior translates into high degree of clinicopathologic heterogeneity, which may compromise therapies and promote relapse. Therefore, dissecting the cellular and molecular profiling and trafficking is critical in understanding the role of tissue tropism and evolution patterns contributing to its biological behavior. Since it is almost unfeasible to perform spatiotemporal collection in patients, in this study we took advantage of PDX models with serial samples and single cell transcriptomic profiling to address this important biology issue for the first time on MCL. Method Orthotopic PDX models (n = 6) were established via intravenous (IV) inoculation of primary MCL patient samples collected from PB (n = 5) or from LN (n = 1). These mouse models displayed similar dissemination patterns as the parental tumors. Cells from the predominant site of generation 1 (G1) were used to pass onto next generations (up to G9). For heterotopic PDX models, subcutaneous (SC) models were generated in parallel from two independent lines (up to G6) and exhibited predominant tumor growth at primary injection site with tumor spread to secondary sites only at very late stage. PDX samples from IV models (spleen, liver, BM, PB) and SC models across generations (n = 36) were collected and subjected to scRNA-seq profiling together with parental patient samples (n = 6) and healthy donor PBMC samples (n = 2). Results All six PDX models at G1 faithfully mirrored parental samples by displaying similar cancer hallmarks. Interestingly, MYC and OXPHOS signaling were predominantly and progressively augmented with each IV passage, and to a lesser extent across SC passages, suggesting a higher degree of selection and evolution processes during orthotopic passage. With spatial collection at distinct dissemination sites (spleen, liver, BM and PB) within same generations, we revealed that heterogenous transcriptomic profiles were more evident across tissues than generations. Specifically, cancer hallmarks such as MYC (NES = 8.4, FDR < 0.01), OXPHOS (NES = 8.9, FDR < 0.01) and mTORC1 (NES = 6.6, FDR < 0.01) signaling were highly enriched in cells from PB, and to a lesser extent in spleen and liver when compared to the cells in BM. More intriguingly, 55-60% of tumor cells in PB clustered together and showed enhanced cancer hallmarks for tumor migration and invasion (NES = 7.9, FDR < 0.01), higher de-differentiation scores (cytoTRACE) and G0/G1 cell cycle stage. This suggests that these cells are quiescent, de-differentiated and disseminative. Importantly, a small fraction of cells from spleen (5-18%) and liver (12-18%), but not in BM, showed similar characteristics and clustered together with those from PB. Histopathologic analysis showed that tumor cells could be detected in blood only after cells settled and expanded in the spleen, liver or BM, whereas dissemination to LN, GI tract, lung and kidney were even later events. Therefore, it is likely that these disseminative MCL cells originate from tissues and represent the tumor seed cells for disease dissemination. More interestingly, the top differential expressed genes (DEGs) in these seed cells were also significantly upregulated in ibrutinib-resistant patients (p < 0.01), compared to that in ibrutinib-sensitive patients based on bulk RNA sequencing (n = 69). This indicates that these seed cells are more resistant to ibrutinib and may drive therapeutic relapse. Targetable molecules are under active investigation to eradicate this ibrutinib-resistant seed cells. Conclusion MCL tissue tropism results in distinct transcriptomic profiles. A special cell population of tumor seed cells was identified to be quiescent, de-differentiated and disseminative, and may drive tumor spread, disease progression and therapeutic resistance (Figure 1). These observations provide biological insights into MCL disease progression in multiple MCL sites. Figure 1 Figure 1. Disclosures Wang: InnoCare: Consultancy, Research Funding; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Pharmacyclics: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; OMI: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; Oncternal: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Chinese Medical Association: Honoraria; Celgene: Research Funding; Imedex: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria; BioInvent: Research Funding; Physicians Education Resources (PER): Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Moffit Cancer Center: Honoraria; Newbridge Pharmaceuticals: Honoraria; Lilly: Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Genentech: Consultancy; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; Mumbai Hematology Group: Honoraria; CStone: Consultancy; Bayer Healthcare: Consultancy; Anticancer Association: Honoraria; Scripps: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Clinical Care Options: Honoraria; BGICS: Honoraria; Molecular Templates: Research Funding.

Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.

The article highlights the main achievements, problems and directions of the further development of the landing stock of Ukraine, the prospects of scientific research of Institute of Animal breeding and Genetics nd. a. M.V.Zubets of the NAAS in the areas of breeding, genetics, biotechnology of reproduction and preservation of the gene pool of farm animals. Institute is the initiator of four dairy herds (Ukrainian Red-and-White, Black-and-White, Red and Brown dairy bread) and four meat (Ukrainian, Volyn, Polissya and Southern meat) breeds of cattle. Its employees carry out scientific support of regional livestock development programs, development of systems for the creation and management of commercial herds of dairy and beef cattle, which contributes to solving the global food problem, and to ensure the nutrition security of Ukrainian population. The newly created Ukrainian Black-and-White, Red-and-White and Red dairy breeds for the predominantly intra-species breeding improvement and limited access to the gene pool of the Holstein breeding breed should remain the main areas of the breeding improvement of domestic dairy cattle breeding. The existing breeding system in cattle in Ukraine does not meet international standards and practically does not work in a complex way, and it threatens the final destruction of domestic breeding livestock, a significant dependence of the country on the import of breeding resources. To solve the problem, a new structure of the breeding service with a clear definition of the organizational basis for the management of tribal affairs and functional responsibilities of the subjects of its implementation was proposed, the formation of a centralized national information base for the identification, registration, origin and performance of animals, the keeping of state books of breeding animals as the basis estimation of their genetic value, and its realization is entrusted to the state enterprise created at the institute on Main scientific-production informational-elective center in livestock. Promising areas for farm animal breeding research are grouped into gene identification and the degree of development of quantitative attributes (QTL), early prediction and evaluation of breeding value of animals using markers (MAS). Research on molecular genetics is aimed at improving genetic analysis methods at individual and population levels, monitoring herds of cattle according to different types of genetic markers. Genetic systems for testing animals in 9 loci quantitative attributes, which are involved in the formation of qualitative indicators of dairy and meat productivity. A work is under way to test animals for the polymorphism of the BoLA-DRB3 gene of the major histocompatibility complex in animal populations for resistance to or susceptibility to mastitis. Biotechnology research focuses on reproductive biology methods, first of all, manipulations with gametes of farm animals, in vitro fertilization of pre-matured oocytes of cows and pigs, and others. The technology of obtaining oocyte cumulus complexes from ovaries of animals, the conditions of their storage, cultivation and fertilization out of the organism, which allows receiving a much larger number of embryos for both scientific and practical purposes, is developed. A separate direction is the work to improve the biotechnological methods of reproduction of farm animals using nanomaterials. It is based on the application in cryopreservation and sperm production of sperm and ovules of various variants of biologically active substances that are applied to highly dispersed silica molecules (albumin of blood serum of cattle, N-acetylneuramic acid – UFS / BSA / NANA). In order to monitor and preserve the diversity of genetic resources of agricultural animals in Ukraine, a complex of works under NAAS scientific program "System of work in populations and preservation of biological diversity of genetic resources of farm animals" ("Preservation of gene pool of breeds") with a coordination center on the basis of Institute of Animal breeding and Genetics nd. a. M.V.Zubets of NAAS. The research resulted in the development of the Program for the preservation of the gene pool of local and endangered breeds of farm animals in Ukraine for 2017–2025, in which the methodological bases for preservation of the gene pool were generalized, animal breeds were classified according to the criteria of risk, the minimum sizes of herds (real and virtual) of faulting species were substantiated, the minimum the size of subsidies for the proper functioning of small-numbered breeds, general methodological approaches to assessing the specificity of genetic resources are specified.

Abstract Introduction FDA approval for CD19 chimeric antigen receptor (CAR) T therapy with Brexucabtagene Autoleucel (BA) for relapsed mantle cell lymphoma (MCL) is a milestone advance. However, most patients relapsed after Bruton's tyrosine kinase (BTK) inhibitor ibrutinib therapy and CAR T therapy, making it urgent and essential to uncover the underlying mechanisms that govern resistance to ibrutinib and BA, as well as potential therapeutic targets. Method Two cohorts of primary patient samples were longitudinally collected at pre- and post-ibrutinib therapy (cohort 1, n = 12, samples = 28) or at pre- and post-BA therapy (cohort 2, n = 15, samples = 39) and subject to single-cell RNA sequencing (Figure 1A). Two healthy PBMC samples were included as normal controls. All 67 patient samples were divided into four major groups based on clinical outcomes: samples with fast response to ibrutinib (IBN-S), samples with slow response (IBN-Slow), samples with resistance (IBN-R), and samples with dual resistance to ibrutinib and BA (Dual). Data was integrated across cohorts and experimental batches. In-silico isolation of B cells was followed by differential gene expression analysis using a mixed model with random effect accounting for inter-patient variation. Gene Set Enrichment Analysis (GSEA) was performed to link clinical outcomes to dysregulated cancer hallmarks. Trajectory analysis further modeled the sequential changes resulting in different clinical outcomes. Copy number variation (CNV) analysis was performed to infer chromosomal aberrations. Results A high degree of transcriptomic heterogeneity was observed in tumor cells among patients within the same or across different clinical outcomes. Differential gene expression analysis revealed a set of outcome-specific genes across all patients. A list of 37 genes was consistently altered between Dual and IBN-R samples across both cohorts (FDR<0.1). The co-enrichment of OXPHOS and MYC pathways was linked to both ibrutinib resistance and BA resistance in an additive manner. Progressive co-enrichment was detected in different contrasts, including IBN-Slow vs IBN-S, IBN-R vs IBN-Slow plus IBN-S, and Dual vs IBN-R (Figure 1B, FDR<0.05). To capture the transitions of biological processes between different clinical outcomes, we performed trajectory analysis. The major trajectory stemmed from normal to IBN-S samples, then branched into IBN-Slow and IBN-R/Dual, which further branched into IBN-R and Dual (Figure 1C). A list of differentially expressed genes near the bifurcation point of trajectories (IBN-R/Dual) was identified (adjusted p-value < 2e-16) . To further understand the evolution associated with therapeutic resistance at the genomic level, we conducted the CNV analysis. We then quantified the genome instability score using chromosomal aberrations and observed a significant positive association with tumor aggressiveness (ANOVA test, p-value < 2e-16). Dual samples had the highest score, followed by IBN-R, IBN-Slow, and IBN-S (Figure 1D). Of note, chr22p gain was exclusive to the CAR T-resistant samples (4 out of 6), with specific aberrations in immunoglobulin lambda variable (IGLV) and constant (IGLC) genes, suggesting its association with BA resistance. Together with previously identified DEGs, targetable molecules are under active investigation for therapeutic development to overcome BA resistance. Conclusion In this study, we found co-enrichment of the OXPHOS and MYC pathways in each resistant cohort, suggesting their predominant role in ibrutinib resistance and BA resistance. Trajectory analysis detected genes differentially expressed at the branch point, which may represent novel early drivers of therapeutic resistance and disease progression. Acquired genome instability is positively correlated with tumor aggressiveness. The exclusive chr22p gain and immunoglobulin lambda alterations in Dual samples may suggest novel therapeutic targets to overcome the resistance for relapsed MCL patients. Collectively, our findings gained novel insight into the underlying mechanisms of resistance to ibrutinib and BA, as well as therapeutic vulnerabilities that can be targeted to overcome resistance. Figure 1 Figure 1. Disclosures Jain: Kite: Consultancy; Lilly: Membership on an entity's Board of Directors or advisory committees. Wang: Hebei Cancer Prevention Federation: Honoraria; Dava Oncology: Honoraria; Genentech: Consultancy; Bayer Healthcare: Consultancy; Clinical Care Options: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Imedex: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; InnoCare: Consultancy, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Mumbai Hematology Group: Honoraria; DTRM Biopharma (Cayman) Limited: Consultancy; Epizyme: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; VelosBio: Consultancy, Research Funding; CStone: Consultancy; BGICS: Honoraria; OMI: Honoraria; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; CAHON: Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Chinese Medical Association: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Moffit Cancer Center: Honoraria; Newbridge Pharmaceuticals: Honoraria; Scripps: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria.

The book is intended for students studying medical and biological specialties. CHAPTER I. EPIGENETICS INTRODUCTION The science of epigenetics looks at the mechanisms of molecular modifications of histones and DNA that can regulate gene activity without affecting the nucleotide sequences in the DNA molecule. Recognized epigenetic regulators are DNA methylation, post-translational modifications of histones, and non-coding RNAs (nkRNAs). One of the most important differences between eukaryotic cells and prokaryotes is the presence of a complex nucleo-protein chromatin complex in eukaryotes. It is in this form that the DNA molecule is stored in our cells. On the one hand, the complex structural organization of chromatin provides a compact arrangement of DNA in the cell nucleus. On the other hand, chromatin is directly involved in the process of regulating gene expression. At the same time, the nucleosome depicted in Fig. 1 (a structural and functional unit of chromatin) is considered as a key component in the processes of regulating gene expression. The nucleus of the nucleosome is 8 histone proteins (octamers). The nucleus of the nucleosome consists of two copies of each of the histone proteins H2A, H2B, H3 and H4. The DNA chain, which includes 147 nucleotides, folds 1.65 times around the octamer of histones. The nucleosomes are arranged as a linear array along the DNA molecule in the form of "beads on a string". The linker section of DNA connecting adjacent nucleosomes (transcriptionally inactive) is sealed with H1-histone protein. The length of the linker section is 30 nm. Moreover, the site of the beginning of transcription is usually located inside the nucleosome. Consequently, the nucleosome serves as a gene repressor, preventing the initiation of transcription. That is, chromatin provides a total repression of genes. In contrast, transcription becomes possible as a result of chromatin remodeling factors that enable the "dismantling" of nucleosomes or otherwise alter their structure and organization. Thus, the repression (inactivation) of genes begins with wrapping the DNA molecule around the histones in the nucleosome, and the liberation of genes from repression (activation) involves freeing DNA from binding to histone proteins and unfolding DNA by chromatin remodeling factors (Lorch Y., Kornberg R. D., 2017). Thanks to this mechanism, selective expression of only those genes that are needed at a given time by the cell or tissue is possible. It should be emphasized that nucleosome repression extends not only to transcription, but also to most other biological processes associated with the DNA molecule, such as replication, mitotic division, repair of double-strand breaks, and maintenance of telomeres. Thus, epigenetic mechanisms control various physiological and pathological processes by regulating the expression of the corresponding genes by changing the availability of epigenetic control systems to chromatin. The scope of application of epigenetic research methods is rapidly expanding. Currently, we are witnessing the active introduction of epigenetic approaches in the field of practical medicine aimed at diagnosing and treating dangerous human diseases. CHAPTER II. TRANSCRIPTION FACTORS INTRODUCTION For the first time, the existence of transcription factors was revealed on the basis of a discovery that made it possible to establish in vitro purified RNA polymerase-II can initiate transcription on the DNA template in the presence of a cell extract (Weil P. A. et al., 1979). Further research aimed at the fractionation and identification of the general transcription factors (GTF) required to initiate transcription in vitro has identified similar factors in rats, Drosophila, and yeast and substantiated the assumption that GTFs are indeed "common" factors necessary for the expression of genes transcribed by RNA polymerase II. is highly conserved in a number of eukaryotic organisms (Matsui T. et al., 1980). We only mention RNA polymerase II because only this type of enzyme has the ability to synthesize mRNA. Whereas RNA polymerase I is responsible for the synthesis of pro-rRNA, and RNA polymerase III is responsible for the synthesis of tRNA and other non-coding cell RNAs. Meanwhile, the regulation of transcription in eukaryotes is quite complex, since it depends on chromatin remodeling complexes (Burns L. G., Peterson C. L., 1997) and covalent modification of histone proteins (Natsume-Kitatani Y., Mamitsuka H., 2016). In transcription initiation, the immediate target of GTF is a well-defined promo zone of a structural gene. In the structure of the promotra of eukaryotes, the main elements and regulatory elements can be distinguished. The main elements of the promotra (bark promoter, see Fig. 2.1) can be attributed to the site for assembling the transcription initiation complex (PIC), including the TATA sequence located above from the transcription start site (TSS ), and an initiating sequence (Inr) covering the start site. Promoters may include a TATA unit, an initiator sequence (Inr), or both (Hampsey M., 1998). A third major element, the downstream promoter element (DPE), was originally described in Drosophila and is located about 30 p.p. below TSS. The DPE promoter element appears to function in conjunction with the Inr element as a binding site for the transcription factor TFIID on non-TATA promoters. According to current research, the cellular (main) promoters of multicellular organisms that control the initiation of transcription by RNA polymerase II may contain short sequences of nucleotides called cow promoter elements (motifs) (e.g., the TATA block, the initiator (Inr), and the lower element of the cow promoter (DPE)) that recruit RNA polymerase II through a common transcription initiation mechanism (Dreos R. et al., 2021). The authors report that the classes of Promoters of Inr+DPE are not only present in the genome of Drosophila and humans and are structurally similar to each other, but may also be common to different species of multicellular organisms. The most studied element of the cow promoter is the TATA box, but the TATA box is found only in about 10-20% of multicellular cortical promoters. Therefore, along with the TATA sequence, it is necessary to name other possible key DNA sequences known as cortical promoter elements, which include: BRE, MTE, TST and DPE sequences. The two BRE (TFIIB recognition element) motifs are located either above (BREu) or below (BREd) elements of the TATA box. It should be emphasized that TBP, TATA box, and BRE demonstrate high levels of conservatism in the range from archaebacteria to humans (Kadonaga J. T., 2012). In doing so, BREu as well as BREd have both positive and negative effects on transcription activity. The downstream core promoter element (DPE) was detected in the analysis of non-TATA gene promoters in Drosophila. The MTE element (motif ten element), which is located directly in front of the DPE, was identified as an overrepresented sequence of a cow promoter called "motif 10" and then discovered, that it is a functional element of a cow promoter. The MTE and DPE motifs exhibit high conservatism in the range from Drosophila to humans, and both motifs appear to be directly recognized by the subunits of the main transcription factor TFIID, TAF proteins that resemble histone proteins in structure. In turn, the TCT sequence regulates the transcription of ribosomal protein genes in Drosophila and humans. Although there are no universal cortical promoter elements that are present in all promoters, the concept of a cow promoter of nuclear RNA polymerase II can be defined as a minimum stretch of DNA that is sufficient to accurately initiate transcription by RNA polymerase II (Kadonaga J. T., 2012; Haberle V., Stark A., 2018). It should be noted that the results of modern research will constantly supplement the list of all new components of the cow promoter, for example, DNA-replicatedrelated element (DRE), Ohler 1,6 and 7 motifs (Danino Y. M. et al., 2015; Haberle V., Stark A., 2018). According to the authors, the bark promoter may be transformed in the course of evolution. Due to this, gene expression levels can be modulated by the composition of cow promoter elements. Such modulation is directly achieved through the emergence of combinations of new elements of the cow promoter, as a result of which an additional level of transcription regulation is realized. To summarize the above facts, transcription is usually initiated at a specific position, the Transcription Initiation Site (TSS), at the 5' end of the gene. The TSS site is embedded in a bark promoter, which is a short sequence spanning 50 base pairs above and 50 below TSS. The cortical promoter serves as a binding platform for the components required to initiate transcription, including RNA polymerase II and related common transcription factors (GTFs). Regulatory elements. The cortical promoter is sufficient to initiate transcription, but such transcription has low basal activity, which can be further activated, generally by more distally arranged regulatory elements called enhancers (discussed below). Enhancers bind regulatory proteins known as transcription factors, recruit transcription cofactors, and can further enhance transcription. CHAPTER III. CELL SIGNALING PATHWAYS INTRODUCTION In a multicellular organism, the work of each cell is regulated by a large number of signals. These signals can be formed both in the organism itself, reflecting the specific needs of a living organism (metabolic state, stages of development, differentiation, reproduction), and in the form of a reaction to the effects of the external environment. The implementation of each of these signals encompasses all the biological and biochemical processes that lead from the cell's perception of the signal to the cell's response. A signal to a cell is something that is recognized by a specific receptor, which in turn can initiate a response to that signal. A receptor is a structure that recognizes a signal, interprets the specificity of a signal, and translates it into the cell in the form of intracellular signaling molecules, a cascade of protein phosphorylation, and other pathways. Thus, signaling to the cell begins as soon as the signaling molecule (ligand) binds to its receptor – a protein with a complementary structure on the transmembrane protein or inside the cell. Growth factors, hormones, cytokines, neurotransmitters, components of the extracellular matrix, etc. The chemical nature of the ligands is diverse, including small molecules such as lipids (prostaglandins, steroid hormones), proteins (for example, peptide hormones, cytokines and chemokines, growth factors)., complex polymers of sugars (for example, β-glucan and zymosan) and their combinations (for example, proteoglycans), nucleic acids, etc. Binding of the ligand induces conformational changes in the receptor and is then translated into the cell by activating cascades of secondary messengers (kinases, phosphatases, GTPases, ions and small molecules such as cAMP, cGMP, diacylglycerol, etc.). Thus, the message is transmitted from the membrane to the nucleus, where the processes of gene expression, subsequent translation and targeting of the protein to the cell membrane and other organelles are triggered. There are two main types of receptors – membrane (transmembrane) cell receptors and intracellular receptors. Membrane receptors are located on the plasma membrane and have a separate extracellular domain binding ligand, a transmembrane domain that is hydrophobic in nature, and a cytoplasmic domain. Cell surface receptors can be divided into G-protein-bound receptors, tyrosine kinase-bound receptors, and ionotropic receptors. When the ligand binds, plasma receptors undergo conformational changes in their extracellular domain and activate enzymatic mechanisms associated with the cytoplasmic domain, usually kinases, phosphatases and adapter proteins. These proteins can be covalently bound to the receptor and are capable of producing secondary messengers for subsequent signal transmission. Intracellular receptors can be nuclear receptors (estrogen receptor, glucocorticoid receptor, progesterone receptor, retinoic acid receptor, thyroid hormone receptor, etc.), cytoplasmic receptors or receptors located on the membranes of organelles (mitochondria, endoplasmic reticulum and Golgi apparatus). Thus, information (ligand) received on the cell surface (e.g., through a membrane receptor) is transformed by specific enzyme systems associated with the plasma membrane receptor and transmitted in the form of secondary messengers to intracellular targets. All of these components form the path of signal transmission to the cell. However, a certain set of effector proteins, enzymes and substrates that implement cellular signals form this signaling pathway (signaling cascade). Recently, however, there has been growing evidence that not only the signaling proteins themselves play an extremely important role in the regulation of cellular signaling, but also theso-called scaffold proteins ("platform proteins", adaptor proteins), which coordinate the assembly of multicomponent protein complexes. Scaffold proteins can bind several elements of one signaling pathway into a single complex, thereby modulating the efficiency of transmission of the corresponding signal. Binding and by bringing two or more signaling proteins closer together, these platform proteins direct the flow of information in the cell, activating, coordinating and regulating signaling events in regulatory networks (Skovorodnikova P.A. et al., 2017). According to the literature, several types of scaffold proteins have been described, which cover a wide range of functions. This group of proteins is usually divided into three main categories (Fig. 1): simple proteins that bind two functionally dependent proteins (adaptors), larger multi-domain proteins designed to bind a large number of proteins and regulate their activity by complex mechanisms (scaffold⁄anchoring proteins) and proteins specialized for initiating signaling cascades by localizing certain proteins-components of signaling pathways on the cell membrane (docking proteins) ( Buday L., Tompa P, 2010) The presence of such protein platforms increases the efficiency and selectivity of the signaling pathway, and also allows the formation of regulatory feedback. e ultimate target of cell signaling pathways are transcription factors that regulate gene expression and ultimately allow the resulting signal to be converted into a change in cellular activity (Brivanlou A. H., Darnell J. E., 2002). Most signaling pathways initiate a cascade of several intracellular signaling molecules that eventually form activation proteins or transcription repressors designed to bind to a specific DNA sequence. Eukaryotic transcription factors, like other proteins, are transcribed in the nucleus, but then their translation takes place in the cytoplasm. Signal transmission to the cell is a multifactorial system, which is based on nodular complexes of special proteins of signaling cascades. However, none of the signaling pathways in the cells work in isolation. The interaction of signaling mechanisms is inevitable in complex complexes, when the system perceives a combination of stimuli (hormones, cytokines, growth factors and pathogenic ligands), but at the same time preserves the accuracy of signal transmission (Saini N., Sarin A., 2021). It is well known that a relatively small number of signaling pathways control the development of all cell types in mammals (Brivanlou A. H., Darnell J. E., 2002). Combinations and time of action of the main signaling pathways determine decisions about the fate of the cell, including events such as cell differentiation in the process of ontogenesis (Li R., Elowitz M.V., 2019; de Roo J. J. D., Staal F. J. T., 2020) and cell malignancy (Dreesen O., Brivanlou A.N., 2007; Skovorodnikova P.A. et al., 2017). Consider some of the cell signaling pathways that are most important medically important. CHAPTER IV. MOLECULAR BIOLOGY OF THE TUMOR: MECHANISMS OF INITIATION, PROMOTION AND PROGRESSION INTRODUCTION Tumor diseases occupy a leading place, both in terms of morbidity and mortality. However, despite the advances in the study of molecular genetic patterns, many unresolved questions remain. On the one hand, the spectrum of molecular markers makes it possible to diagnose, predict the course, degree of malignancy, rate of tumor progression and predict a possible response to the therapy. On the other hand, those processes that occur at the molecular level are not characterized by stability, they are dynamic and are associated with a change in the genetic profile - the appearance of many clones of tumor cells with a different set of properties. The heterogeneity of tumor diseases simultaneously complicates the strategy of managing such patients, creating the prerequisites for further study of the molecular genetic characteristics of tumor cells.

Plants for medicinal purposes are considered as the main source of health care for the majority of the world population. In order to promote medicinal plants in Algeria, the present work aimed to assess the physico-chemical characterization, hemolytic, and antioxidant activities of Ammodaucus leucotrichus essential oil of an endemic plant from south-west of Algeria. The plant was harvested from the region of Bechar (south-west of Algeria). The oil was obtained by hydrodistillation method using Clevenger apparatus. The antioxidant activity of the oil was carried out using DPPH scavenging assay, and its effect on erythrocyte cells was evaluated by measuring the hemolytic degree. The obtained results showed that this oil presented a low antioxidant activity compared to positive control (ascorbic acid). The hemolytic activity was very low since the oil diluted to 1/10, while it was low in the pure state. 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Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. 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IntroductionIf human beings have become a geophysical force, capable of impacting the very crust and atmosphere of the planet, and if geophysical forces become objects of study, presences able to be charted over millions of years—one of our many problems is a 'naming' problem. - Bethany NowviskieThe anthropocene "denotes the present time interval, in which many geologically significant conditions and processes are profoundly altered by human activities" (S.Q.S.). Although the narrative and terminology of the anthropocene has not been officially legitimized by the scientific community as a whole, it has been adopted worldwide by a plethora of social and cultural studies. The challenges of the anthropocene demand interdisciplinary efforts and actions. New contexts, situations and environments call for original naming propositions: new terminologies are always illegitimate at the moment of their first appearance in the world.Against the background of the naming challenges of the anthropocene, we will map the emergence and tell the story of a tiny world within the world of media studies: the world of the term 'nanomedia' and its hyphenated sister 'nano-media'. While we tell the story of the uses of this term, its various meanings and applications, we will provide yet another possible interpretation and application to the term, one that we believe might be helpful to interdisciplinary media studies in the context of the anthropocene. Contemporary media terminologies are usually born out of fortuitous exchanges between communication technologies and their various social appropriations: hypodermic media, interactive media, social media, and so on and so forth. These terminologies are either recognised as the offspring of legitimate scientific endeavours by the media theory community, or are widely discredited and therefore rendered illegitimate. Scientific legitimacy comes from the broad recognition and embrace of a certain term and its inclusion in the canon of an epistemology. Illegitimate processes of theoretical enquiry and the study of the kinds of deviations that might deem a theory unacceptable have been scarcely addressed (Delborne). Rejected terminologies and theories are marginalised and gain the status of bastard epistemologies of media, considered irrelevant and unworthy of mention and recognition. Within these margins, however, different streams of media theories which involve conceptual hybridizations can be found: creole encounters between high culture and low culture (James), McLuhan's hybrid that comes from the 'meeting of two media' (McLuhan 55), or even 'bastard spaces' of cultural production (Bourdieu). Once in a while a new media epistemology arises that is categorised as a bastard not because of plain rejection or criticism, but because of its alien origins, formations and shape. New theories are currently emerging out of interdisciplinary and transdisciplinary thinking which are, in many ways, bearers of strange features and characteristics that might render its meaning elusive and obscure to a monodisciplinary perspective. Radical transdisciplinary thinking is often alien and alienated. It results from unconventional excursions into uncharted territories of enquiry: bastard epistemologies arise from such exchanges. Being itself a product of a mestizo process of thinking, this article takes a look into the term nanomedia (or nano-media): a marginal terminology within media theory. This term is not to be confounded with the term biomedia, coined by Eugene Thacker (2004). (The theory of biomedia has acquired a great level of scientific legitimacy, however it refers to the moist realities of the human body, and is more concerned with cyborg and post-human epistemologies. The term nanomedia, on the contrary, is currently being used according to multiple interpretations which are mostly marginal, and we argue, in this paper, that such uses might be considered illegitimate). ’Nanomedia’ was coined outside the communications area. It was first used by scientific researchers in the field of optics and physics (Rand et al), in relation to flows of media via nanoparticles and optical properties of nanomaterials. This term would only be used in media studies a couple of years later, with a completely different meaning, without any acknowledgment of its scientific origins and context. The structure of this narrative is thus illegitimate, and as such does not fit into traditional modalities of written expression: there are bits and pieces of information and epistemologies glued together as a collage of nano fragments which combine philology, scientific literature, digital ethnography and technology reviews. Transgressions Illegitimate theories might be understood in terms of hybrid epistemologies that intertwine disciplines and perspectives, rendering its outcomes inter or transdisciplinary, and therefore prone to being considered marginal by disciplinary communities. Such theories might also be considered illegitimate due to social and political power struggles which aim to maintain territory by reproducing specific epistemologies within a certain field. Scientific legitimacy is a social and political process, which has been widely addressed. Pierre Bourdieu, in particular, has dedicated most of his work to deciphering the intricacies of academic wars around the legitimacy or illegitimacy of theories and terminologies. Legitimacy also plays a role in determining the degree to which a certain theory will be regarded as relevant or irrelevant:Researchers’ tendency to concentrate on those problems regarded as the most important ones (e.g. because they have been constituted as such by producers endowed with a high degree of legitimacy) is explained by the fact that a contribution or discovery relating to those questions will tend to yield greater symbolic profit (Bourdieu 22).Exploring areas of enquiry which are outside the boundaries of mainstream scientific discourses is a dangerous affair. Mixing different epistemologies in the search for transversal grounds of knowledge might result in unrecognisable theories, which are born out of a combination of various processes of hybridisation: social, technological, cultural and material.Material mutations are happening that call for new epistemologies, due to the implications of current technological possibilities which might redefine our understanding of mediation, and expand it to include molecular forms of communication. A new terminology that takes into account the scientific and epistemological implications of nanotechnology applied to communication [and that also go beyond cyborg metaphors of a marriage between biology and cibernetics] is necessary. Nanomedia and nanomediations are the terminologies proposed in this article as conceptual tools to allow these further explorations. Nanomedia is here understood as the combination of different nanotechnological mediums of communication that are able to create and disseminate meaning via molecular exchange and/ or assembly. Nanomediation is here defined as the process of active transmission and reception of signs and meaning using nanotechnologies. These terminologies might help us in conducting interdisciplinary research and observations that go deeper into matter itself and take into account its molecular spaces of mediation - moving from metaphor into pragmatics. Nanomedia(s)Within the humanities, the term 'nano-media' was first proposed by Mojca Pajnik and John Downing, referring to small media interventions that communicate social meaning in independent ways. Their use of term 'nano-media' proposes to be a revised alternative to the plethora of terms that categorise such media actions, such as alternative media, community media, tactical media, participatory media, etc. The metaphor of smallness implied in the term nano-media is used to categorise the many fragments and complexities of political appropriations of independent media. Historical examples of the kind of 'nano' social interferences listed by Downing (2),include the flyers (Flugblätter) of the Protestant Reformation in Germany; the jokes, songs and ribaldry of François Rabelais’ marketplace ... the internet links of the global social justice (otromundialista) movement; the worldwide community radio movement; the political documentary movement in country after country.John Downing applies the meaning of the prefix nano (coming from the Greek word nanos - dwarf), to independent media interventions. His concept is rooted in an analysis of the social actions performed by local movements scattered around the world, politically engaged and tactically positioned. A similar, but still unique, proposition to the use of the term 'nano-media' appeared 2 years later in the work of Graham St John (442):If ‘mass media’ consists of regional and national print and television news, ‘niche media’ includes scene specific publications, and ‘micro media’ includes event flyers and album cover art (that which Eshun [1998] called ‘conceptechnics’), and ‘social media’ refers to virtual social networks, then the sampling of popular culture (e.g. cinema and documentary sources) using the medium of the programmed music itself might be considered nano-media.Nano-media, according to Graham St John, "involves the remediation of samples from popular sources (principally film) as part of the repertoire of electronic musicians in their efforts to create a distinct liminalized socio-aesthetic" (St John 445). While Downing proposes to use the term nano-media as a way to "shake people free of their obsession with the power of macro-media, once they consider the enormous impact of nano-technologies on our contemporary world" (Downing 1), Graham St John uses the term to categorise media practices specific to a subculture (psytrance). Since the use of the term 'nano-media' in relation to culture seems to be characterised by the study of marginalised social movements, portraying a hybrid remix of conceptual references that, if not completely illegitimate, would be located in the border of legitimacy within media theories, I am hereby proposing yet another bastard version of the concept of nanomedia (without a hyphen). Given that neither of the previous uses of the term 'nano-media' within the discipline of media studies take into account the technological use of the prefix nano, it is time to redefine the term in direct relation to nanotechnologies and communication devices. Let us start by taking a look at nanoradios. Nanoradios are carbon nanotubes connected in such a way that when electrodes flow through the nanotubes, various electrical signals recover the audio signals encoded by the radio wave being received (Service). Nanoradios are examples of the many ways in which nanotechnologies are converging with and transforming our present information and communication technologies. From molecular manufacturing (Drexler) to quantum computing (Deutsch), we now have a wide spectrum of emerging and converging technologies that can act as nanomedia - molecular structures built specifically to act as communication devices.NanomediationsBeyond literal attempts to replicate traditional media artifacts using nanotechnologies, we find deep processes of mediation which are being called nanocommunication (Hara et al.) - mediation that takes place through the exchange of signals between molecules: Nanocommunication networks (nanonetworks) can be used to coordinate tasks and realize them in a distributed manner, covering a greater area and reaching unprecedented locations. Molecular communication is a novel and promising way to achieve communication between nanodevices by encoding messages inside molecules. (Abadal & Akyildiz) Nature is nanotechnological. Living systems are precise mechanisms of physical engineering: our molecules obey our DNA and fall into place according to biological codes that are mysteriously written in our every cell. Bodies are perfectly mediated - biological systems of molecular communication and exchange. Humans have always tried to emulate or to replace natural processes by artificial ones. Nanotechnology is not an exception. Many nanotechnological applications try to replicate natural systems, for example: replicas of nanostructures found in lotus flowers are now being used in waterproof fabrics, nanocrystals, responsible for resistance of cobwebs, are being artificially replicated for use in resistant materials, and various proteins are being artificially replicated as well (NNI 05). In recent decades, the methods of manipulation and engineering of nano particles have been perfected by scientists, and hundreds of nanotechnological products are now being marketed. Such nano material levels are now accessible because our digital technologies were advanced enough to allow scientific visualization and manipulation at the atomic level. The Scanning Tunneling Microscopes (STMs), by Gerd Binnig and Heinrich Rohrer (1986), might be considered as the first kind of nanomedia devices ever built. STMs use quantum-mechanical principles to capture information about the surface of atoms and molecules, allowed digital imaging and visualization of atomic surfaces. Digital visualization of atomic surfaces led to the discovery of buckyballs and nanotubes (buckytubes), structures that are celebrated today and received their names in honor of Buckminster Fuller. Nanotechnologies were developed as a direct consequence of the advancement of digital technologies in the fields of scientific visualisation and imaging. Nonetheless, a direct causal relationship between nano and digital technologies is not the only correlation between these two fields. Much in the same manner in which digital technologies allow infinite manipulation and replication of data, nanotechnologies would allow infinite manipulation and replication of molecules. Nanocommunication could be as revolutionary as digital communication in regards to its possible outcomes concerning new media. Full implementation of the new possibilities of nanomedia would be equivalent or even more revolutionary than digital networks are today. Nanotechnology operates at an intermediate scale at which the laws of classical physics are mixed to the laws of quantum physics (Holister). The relationship between digital technologies and nanotechnologies is not just instrumental, it is also conceptual. We might compare the possibilities of nanotechnology to hypertext: in the same way that a word processor allows the expression of any type of textual structure, so nanotechnology could allow, in principle, for a sort of "3-D printing" of any material structure.Nanotechnologies are essentially media technologies. Nanomedia is now a reality because digital technologies made possible the visualization and computational simulation of the behavior of atomic particles at the nano level. Nanomachines that can build any type of molecular structure by atomic manufacturing could also build perfect replicas of themselves. Obviously, such a powerful technology offers medical and ecological dangers inherent to atomic manipulation. Although this type of concern has been present in the global debate about the social implications of nanotechnology, its full implications are yet not entirely understood. A general scientific consensus seems to exist, however, around the idea that molecules could become a new type of material alphabet, which, theoretically, would make possible the reconfiguration of the physical structures of any type of matter using molecular manufacturing. Matter becomes digital through molecular communication.Although the uses given to the term nano-media in the context of cultural and social studies are merely metaphorical - the prefix nano is used by humanists as an allegorical reference of a combination between 'small' and 'contemporary' - once the technological and scientifical realities of nanomedia present themselves as a new realm of mediation, populated with its own kind of molecular devices, it will not be possible to ignore its full range of implications anymore. A complexifying media ecosystem calls for a more nuanced and interdisciplinary approach to media studies.ConclusionThis article narrates the different uses of the term nanomedia as an illustration of the way in which disciplinarity determines the level of legitimacy or illegitimacy of an emerging term. We then presented another possible use of the term in the field of media studies, one that is more closely aligned with its scientific origins. The importance and relevance of this narrative is connected to the present challenges we face in the anthropocene. The reality of the anthropocene makes painfully evident the full extent of the impact our technologies have had in the present condition of our planet's ecosystems. For as long as we refuse to engage directly with the technologies themselves, trying to speak the language of science and technology in order to fully understand its wider consequences and implications, our theories will be reduced to fancy metaphors and aesthetic explorations which circulate around the critical issues of our times without penetrating them. The level of interdisciplinarity required by the challenges of the anthropocene has to go beyond anthropocentrism. Traditional theories of media are anthropocentric: we seem to be willing to engage only with that which we are able to recognise and relate to. Going beyond anthropocentrism requires that we become familiar with interdisciplinary discussions and perspectives around common terminologies so we might reach a consensus about the use of a shared term. For scientists, nanomedia is an information and communication technology which is simultaneously a tool for material engineering. For media artists and theorists, nano-media is a cultural practice of active social interference and artistic exploration. However, none of the two approaches is able to fully grasp the magnitude of such an inter and transdisciplinary encounter: when communication becomes molecular engineering, what are the legitimate boundaries of media theory? If matter becomes not only a medium, but also a language, what would be the conceptual tools needed to rethink our very understanding of mediation? Would this new media epistemology be considered legitimate or illegitimate? Be it legitimate or illegitimate, a new media theory must arise that challenges and overcomes the walls which separate science and culture, physics and semiotics, on the grounds that it is a transdisciplinary change on the inner workings of media itself which now becomes our vector of epistemological and empirical transformation. A new media theory which not only speaks the language of molecular technologies but that might be translated into material programming, is the only media theory equipped to handle the challenges of the anthropocene. ReferencesAbadal, Sergi, and Ian F. Akyildiz. "Bio-Inspired Synchronization for Nanocommunication Networks." Global Telecommunications Conference (GLOBECOM), 2011.Borisenko, V. E., and S. Ossicini. What Is What in the Nanoworld: A Handbook on Nanoscience and Nanotechnology. Weinheim: Wiley-VCH, 2005.Bourdieu, Pierre. "The Specificity of the Scientific Field and the Social Conditions of the Progress of Reason." Social Science Information 14 (Dec. 1975): 19-47.---. La Distinction: Critique Sociale du Jugement. Paris: Editions de Minuit, 1979. Delborne, Jason A. "Transgenes and Transgressions: Scientific Dissent as Heterogeneous Practice". Social Studies of Science 38 (2008): 509.Deutsch, David. The Beginning of Infinity. London: Penguin, 2011.Downing, John. 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Ljubljana, Slovenia: Peace Institute, 2008. 7-16.Qarehbaghi, Reza, Hao Jiang, and Bozena Kaminska. "Nano-Media: Multi-Channel Full Color Image with Embedded Covert Information Display." In ACM SIGGRAPH 2014 Posters. New York: ACM, 2014. Rand, Stephen C., Costa Soukolis, and Diederik Wiersma. "Localization, Multiple Scattering, and Lasing in Random Nanomedia." JOSA B 21.1 (2004): 98-98.Service, Robert F. "TF10: Nanoradio." MIT Technology Review April 2008. Shanken, Edward A. "Artists in Industry and the Academy: Collaborative Research, Interdisciplinary Scholarship and the Creation and Interpretation of Hybrid Forms." Leonardo 38.5 (Oct. 2005): 415-418.St John, Graham. "Freak Media: Vibe Tribes, Sampledelic Outlaws and Israeli Psytrance." Continuum: Journal of Media and Cultural Studies 26. 3 (2012): 437–447.Subcomission on Quartenary Stratigraphy (S.Q.S.). "What Is the Anthropocene?" Quaternary.stratigraphy.org.Thacker, Eugene. Biomedia. 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Most of us are aware that ozone is basically a treatment of pure oxygen and its effect on the human body is even better. As our human body is made up of 70% of water, we used ozone in many different way, one of them is what we know as ozonated water. As ozone when mix in water it diluted thoroughly and highly purifies the water, as it is soluble in water. It maintain its tri-oxygen identity. Biological Properties of Ozonated Water: • It kills viruses, bacteria, fungi and algae on contact. • It breakdown harmful synthetic chemicals into less dangerous molecules. • It purifies the blood by rupturing the cell wall of the microorganisms. • It kills some cancer cells, slows tumor growth, and may stop the spread of cancer. • It provides more oxygen to the brain. • It boosts the immune system. Preparation of Ozonated Water: In order to prepare this, we first use the glass cylinder filled with approximately ¾ of pure water (mineral water) through which O2-O3 gas mixture has to be bubbled continuously for at least 5-10 minutes by using diffuser to achieve saturation. The unused ozone flows out via silicon tubing in to the destructor and is converted to oxygen. As ozone physically dissolve in water therefor it's concentration is equally to approximately ¼ of total O3 concentration in gas. If put at 20 degree, half-life of it is only 9 hours and if put at room temperature half-life is approximately 4-5 hour, only if maintained properly. The stability of ozonated water depends largely on the temperature and also ionic contents and Ph. of water. Colder the water more ozone is absorbed and retained for a longer rate. Drinking Ozonated water Local Treatment Dental use Allergies Ankle sprain Aphthous stomatitis Cancers Athlete's Foot Candida Gastritis, indigestion Fresh/recent wounds Gum disease Candidiasis Burns - arms & legs Mouth ulcers Headaches Herpes zoster and simplex Wound treatment Viral infections Pains due to bad peripheral Disinfection after tooth blood circulation extraction & dental workIndication of Ozonated Water: Ozonated water is helpful in following diseases : Drinking ozonated water is highly energetic water. It has to be drunk immediately on empty stomach. On regular use of ozonated water will establish high level of oxygenation in the body.Topical application: Ozonated water is basically applied on account of its pain relieving, disinfectant and anti-inflammatory effects as well as tissue activating property in acute & chronic injuries with & without infection. Ozonated water also being used intra operatively for rinsing. The healing time for primary scar is shortened and irritation free.Dental use: Ozonated water strongly inhibited accumulation of experimenter dental plaque in vitro. Ozone destroys this niche in which acid loving bacteria grow. Ozone also destroys the protein coat over the lesion, which effectively protect the niche. Ozonized water can offer an efficient non antibiotic agent for the control of microorganism prior to replantation of contaminated avulsed teeth. Ozonated Oil and Products : Ozone therapy has been in India since over 2 decades. Every year, the awareness among people increases rapidly due to the factor that ozone has help a lot of people in curing the disease and medical problem. As ozone has been in India for over 2 decades now, there is a body that takes care and helps in not only promoting ozone but also manufacturing and distributing ozone product. Till the date Ozone Forum Of India have come out with eight different product and still researching and working in introducing more products, and we are still working on research and development of further new products. Ozonated oíl is made by using pure vegetable sesame oil and it prepare by bubbling the O2-O3 gas mixer for about 100 to 200hrs. After this it becomes more viscous. Due to prolong ozonation, it results in the formation of ozonides and peroxide which remain stable for 1 year at room temperature and for 2 year in refrigerator. In India, after completion of full procedure in order to check the degree of ozonation we use viscosity measurement and peroxide value, finally the viscosity increase up to 60% indicating that double bonds in oil has reacted with ozone to form more bulky molecules. For peroxide value which is maintain between 300 to 2000 by checking peroxide, represents the quantity of peroxide expressing in mili equivalent of active oxygen contained in 1kg of the oil. As ozonides and peroxide are the main active ingredients therefore triozonide become stable and comes into contact with the warm exudate of the wood. This then, slowly discomposes and generate reactive oxygen species (ROS) and lipid oxidation products (LOPs). This explains the prolonged disinfectentant and stimulatory activity. Ozonized oil helps in: Controlling bleeding, Disinfecting the lesion, Inhibiting the proliferation of potentially infective organisms, Improving metabolism, Stimulatory tissue regeneration, . . Thus, FAST HEALINGIn India, we have eight (8) different types of ozonized products: RAPID HEAL Oil is pure vegetable sesame oil with peroxide value is about more than 1500. It has a powerful germicidal effect and hence helps in all kind of skin related disease, non-healing ulcer, burns and dental pathology. PAIN RELIEF is healthy and safe massaging oil with main active ingredient is ozonized oil. It helps in relieving all kind of musculoskeletal pain. PAIN BALM is an instant pain relieving products. It is an extremely effective balm for body ache and joint pain. As ozonized oil is a major ingredient and due to which it helps in inflammation and reduce swelling and pain instantly. HAIR REVIVE is an effective hair oil for nourishing scalp and hair and makes hair healthy and smooth and shiny. Due to ozonated oil is an active ingredient it is very effective in dandruff and dry scalp. ANTIACIDITY OIL is excellent oil for the treatment of all kind of acidity related problem of the body. Using a unique formula with ozone, it helps in maintaining a good health by removing anaerobic germs from intestinal tract. At the same time it makes the immunity of the body stronger. SKIN BLOSSOM is a unique formula of cream with Ozonated oil is an active ingredient. Due to the pollution people's skin are effected in major way, therefore we suggest you the skin blossom, which detoxified and hydrated the skin. It also repair the skin Tissue and helps in sun burn and protect the skin. MIRAKLE CREAM makes your skin young, bright and cleaner and smoother. With the unique formula with ozone it superoxide's the skin, which in true Increase the metabolic rate and energy of the skin and promotes cell healing. As miracle also act as antioxidant, on using regularly, it helps in reducing Wrinkle, aging spots and damage of dull skin, which in turn skin looks younger.

<p align="center"><strong>DETERMINATION OF ANTIOXIDANT ACTIVITY IN COLOR PLANT EXTRACTS <em>IN VITRO</em></strong></p><p align="center"><strong>G.R. Lamazian, I.M. Sytnik, P.A. Chernovol, I.S. Chekman, M.V. Khaitovych</strong></p><p align="center">Bogomolets National Medical University, Kyiv</p><p><strong>Introduction.</strong> To date, the relationship between free radical oxidation and the development of most pathological processes - endocrinology and cardiovascular diseases is proved. Modern living conditions, the negative impact of environmental factors, emotional stresses, genetic factors - all these are a prerequisite for initiating redox balance disturbances in the body. A key role in this regard belongs to reactive oxygen species (ROS), which cause development of oxidative stress (OS). Recently, the antioxidant properties of medicinal plants (MP) in the prevention and correction of the OS are actively studied. It is commonly known that MP is a source of polyphenolic compounds - powerful natural antioxidants that inhibit free radical oxidation chain reaction through inhibition of oxidative enzymes, the formation of metal chelates or neutralization of radicals.</p><p>The aim of this work was to study the antioxidant activity (AOA) of <em>Hypericum perforatum (L.)</em> and <em>Citrullus colocynthis (L.) Shrad.</em> extracts on the model of superoxide radical inhibition <em>in vitro.</em></p><p><strong>Investigation methods.</strong> Determination of AOA was carried out spectrophotometrically according to the change in absorbance of studied extracts in a model test system of adrenaline autoxidation in an alkaline medium at a wavelength of 340 nm. Assessment of AOA was conducted by the degree of inhibition of superoxide radical, taking into account the impact of extracts’ color that absorbs certain wavelength in the visible spectrum.</p><p><strong>Results and discussion.</strong> Investigated extracts show high AOA in a range of all concentrations, preventing the formation of superoxide radical in vitro. It was somewhat higher in <em>H. perforatum</em> versus <em>C. colocynthis.</em> It should be noted that the AOA of extracts depends on their concentration. The highest value of AOA for <em>H. perforatum</em> (95,77%) is detected at a concentration of 0.5 mg/ml. <em>C. colocynthis</em> showed maximum AOA (88.62%) at a concentration of 0,5˟10-3 mg/ml.</p><p>Values of absorbance after ten minutes exposure indicate that with time the AOA of extracts decreases due to their multi-component composition and interinfluence of biologically active substances, which can be both antagonists and synergists.</p><p>It is known that the antioxidant effect of MP is achieved due to nonenzymatic molecules represented mainly by polyphenolic compounds – flavonoids, phenolcarbonic acids, catechins, stilbenes etc. Activation of cell protective mechanisms by plants polyphenols significantly reduces the manifestations of the OS. Thus, a number of epidemiological and experimental studies indicate that regular action of natural polyphenols improves the quality of patients’ lives with diabetes, Alzheimer's disease, hypertension, metabolic syndrome.</p><p><strong>Conclusions. </strong></p><p>1. In was established that the plant extracts of <em>H. perforatum (L.)</em> and <em>C. colocynthis (L.) Shrad.</em> possess AOA in a range of all concentrations.</p><p>2. Extracts activity depends on the concentration. The highest value of AOA for <em>H. perforatum</em> (95,77%) is detected at a concentration of 0.5 mg/ml and <em>for C. colocynthis </em>(88.62%) - at a concentration of 0,5˟10-3 mg/ml.</p><p>3. The decrease in AOA with time (10 minutes exposure) was revealed, which may be due to the interinfluence of biologically active substances in extracts.</p><p><strong>References</strong></p><p>1. Significance of antioxidant potential of plants and its relevance to therapeutic applications / D. M. Kasote, S. S. 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