Poster Abstracts

Ferric iron bioregeneration by communities of acidophiles growing at different temperatures during two-step leaching of sulfidic zinc concentrate

Maxim Muravyov

Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

email address: [email protected]

A promising approach for processing of the low-grade sulfidic zinc concentrates is a biohydrometallurgical method based on the combination of ferric leaching, bio-oxidation and bioregeneration of leaching solutions. High-temperature (80 °C) chemical leaching of the concentrate containing 43.3% of zinc, 2.03% of copper and 13.6% of iron allowed to recover 92% of zinc and 52% of copper. The leach solution contained 24.2 g/L of ferrous iron, 10 g/L of zinc and 0.3 g/L of copper. Ferrous iron is required to be oxidized in this solution for further steps of metal extraction and the chemical leaching. Three consortia of iron- and sulfur-oxidizing microorganisms grown at 35, 40 and 45 °C were tested to regenerate the leach solution in the presence of leach residue containing 30% of elemental sulfur (in order to prevent an increase in pH). The consortium grown at 40 °C showed the highest rate of ferrous iron oxidation (5.4 g/(L day)). According to the results of metagenomic analysis, Leptospirillum ferriphilum, Sulfobacillus thermotolerans and S. thermosulfidooxidans, Acidithiobacillus caldus, Ferroplasma acidiphilum and Acidiplasma sp. predominated in the chemolithotrophic consortium; organoheterotrophic Cuniculiplasma sp. was also present.

Funding: The study was supported by the Russian Science Foundation [project 18-74-00003].

Etiological structure of ARVI in Kazakhstan in the epidemic season 2017–2018

Tatyana Glebova, Nailya Klivleyeva, Nuray Ongarbayeva, Assem Baimukhametova, Nurbol Saktaganov, Galina Lukmanova and Mira Shamenova

The Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan

email address: [email protected]

The goal of this study is to assess the epidemic potential of influenza and ARVI in the Republic of Kazakhstan. In 2017–2018 epidemiological season, 2372 nasopharyngeal swabs were obtained from ill persons in the healthcare facilities located in various regions of Kazakhstan. Molecular genetic studies of swabs in RT-PCR showed the presence of influenza A RNA in 18.27% of cases, type B influenza in 0.36%. Subtyping of RNA of influenza virus A/H1N1 detected in 10.44% of samples, A/H3N2 in 5.10%. A molecular genetic study of the samples for the presence of ARVI caused a predominance of respiratory syncytial infection, which accounted for 22.72%. The percentage of positive samples for rhinovirus infection was 9.06%, and other ARVI had a share of 3.04 to 0.89%. Thus, primary screening of nasopharyngeal swabs in RT-PCR thereby established circulation of influenza A/H1N1, A/H3N2 and B viruses and pathogens causing ARVI of mixed etiology. A virological study of clinical specimens in chicken embryos isolated 38 hemagglutinating agents identified in RT-PCR and HIA as influenza viruses A/H1N1pdm.

Study of the 2019 A/H1N1 influenza virus susceptibility to chemotherapy drugs in ovo

Galina Lukmanova, Nailya Klivleyeva, Tatyana Glebova, Nurbol Saktaganov, Nuray Ongarbayeva and Assem Baimukhametova

The Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan

email address: [email protected]

The susceptibility of 10 Kazakhstan 2019 A/H1N1 influenza viruses to etiotropic chemotherapy drugs was studied in a system of developing chicken embryos. Remantadine (an ion channel blocker) and Tamiflu (a neuraminidase inhibitor) were used as anti-influenza drugs. The susceptibility of viruses to various concentrations of antiviral drugs was evaluated by the degree of reproductive suppression of 100 EID50/0.2 mL of virus per chicken embryo. The dose of the drug which suppressed the virus titer twice in the HA assay as compared with the control was considered an inhibitory concentration at which virus replication was inhibited by 50% (IC50). Although determining the drug resistance of influenza A/H1N1 virus isolates circulating in Kazakhstan in 2019, it was found that IC50 values of Tamiflu did not exceed 2.31 ± 0.5 μg/mL for all 10 isolates. Five viruses were found to be susceptible to remantadine (IC50 was 25–50 μg/mL), the other five viruses under study were completely resistant to this drug. High-level susceptibility of all investigated viruses to oseltamivir (Tamiflu) was therefore revealed. About 50% of the isolates were susceptible at high doses and 50% were resistant to the member of the adamantane class (remantadine).

Synergistic activity of herbal extracts and antibiotics combinations against clinical strains of E. coli isolated from canine urinary infections

Constantin Ioan Mates, Silvana Popescu, Emoke Pall, Jovan Bojkovski, Cosmin Muresan and Mihaela Niculae

Department Clinics, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Department of Animal Hygiene and Welfare, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Life Science Institute, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia

email address: [email protected]

The medicinal plants biological properties assessment represents a worldwide growing trend aimed at identifying whole products or biologically active compounds that represent a viable alternative to antimicrobial agents. The study’s objective was to investigate the in vitro activity of certain herbal extracts and antibiotics combinations tested against clinical strains of E. coli isolated from dogs with urinary infections (n = 10). The study was carried out considering a preliminary evaluation of both essential oils and/or ethanolic extracts derived from herbal species belonging to Lamiaceae family by diffusion method Kirby–Bauer. Selected herbal products were further tested using a broth microdilution technique, based on that minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were established; furthermore, these extracts were checked in combination with gentamicin and choramphenicol. The overall analysis indicated promising valences for the antimicrobial potential towards both susceptible and antibiotic resistant strains. Bacterial strains and herbal products type dependent susceptibility differences were recorded, with the most active product being the essential oils > ethanolic extracts derived from Thymus vulgaris L. > Salvia officinalis L. > Melissa officinalis L. Also, augmented antibacterial activity was pointed out essential oils + antibiotic combinations. These results suggest herbal extracts’ potential for developing herbal-based therapeutic products.

The Mediterranean diet during pregnancy is relevant for the antioxidant properties of breast milk

Elisabetta Albi, Alessandra Mirarchi, Michela Codini, Carmela Conte, Antonio Boccuto, Laura Cerquiglini, Stefania Troiani, Federica Filomena Patria, Stefania Baldoni, Samuela Cataldi, Maria Rachele Ceccarini and Tommaso Beccari

Department of Pharmaceutical Science, University of Perugia, Italy

Struttura Complessa di Neonatologia e Terapia Intensiva Neonatale– Azienda Ospedaliera Santa Maria della Misericordia, Perugia, Italy

email address: [email protected]

Free radicals are unstable and highly reactive molecular species that can either donate or accept an electron to/from other molecules, behaving as oxidants or reductants. They are produced essentially from normal metabolic processes or from external sources as rays, chemicals and others. A balance between free radicals and antioxidants is essential for cell life. If redox homeostasis is altered by a free radical overpowering, biologically relevant damages are induced. Milk naturally contains antioxidant properties. We carried out a study on the antioxidant power of milk samples by Oxygen Radical Absorbance Capacity (ORAC) assay. The results showed that of 30 women, 18 had followed a balanced Mediterranean diet (M), 4 a diet with a prevalence of vegetables (pV), 4 a diet with a prevalence of cereals and meat (pCM) and 4 with a prevalence of meat and fish (pMF). All M milks exhibited a similar antioxidant activity, significantly lower values were present in pV and significantly higher in pCM and pMF. Of 11 formulated milks, only 3 had a value comparable to M whereas 2 were comparable to pV, 3 to pMF and 3 had very high values. In particular, the biological BBmilk (Buona) had a value very close to that of M.

The influence of phytohormones on the morphogenesis in callus tissues of Artemisia glabella

Mukarram Zhussip , Serzhan Amanov, Askhat Kuatbaуev, Gulmira Satybaldyeva, Aizhan Utarbayeva, Kazbek Shupshibayev, Ainagul Kaliyeva, Aliya Bitkeyeva and Yelena Krasnopyorova

S. Toraighyrov Pavlodar State University, Pavlodar, Kazakhstan

The Karaganda State University of the name of academician E.A. Buketov, Karaganda, Kazakhstan

S. Seifullin Kazakh AgroTechnical University, Nur-Sultan, Kazakhstan

Innovative University of Eurasia, Pavlodar, Kazakhstan

email address: [email protected]

It was found the effect of phytohormones on various morphological reactions of callus tissues of Artemisia glabella. With using the different methods the scheme of obtaining regenerant plants was revealed. Adding only auxins to medium content mainly to the formation of roots and there is an increase of callus mass. Rhizogenesis was observed when IAA was used in concentrations up to 2 mg/L. The use of cytokinins led to the induction of hemogenesis in the callus tissue. The maximum number of regenerants was obtained on a medium with a BAP of 1–2 mg/L. Increasing the concentration of cytokinins above 2 mg/L inhibited the process of hemogenesis.When combining different classes of phytohormones, the formation of structures similar to embryos–embryoids was observed. The predominance of kinetin over IAA in the medium contributes to the formation of embryoid-like structures. Optimal use of a combination was obtained at concentration of kinetin of 2 mg/L and IAA of 2 mg/L. It should be noted that subsequent subcultivation of embryoids on a medium with BAP at concentration of 1 mg/L leads to maturation of embryoids and the formation of a bush with multiple shoots.

The effect of the combination of betulin and nobiletin on the formation of Th2 cellular immunity at experimental influenza infection

Aizhan Turmagambetova, Andrey Bogoyavlenskiy, Vladimir Berezin, Pavel Alexyuk, Madina Alexyuk, Nadezhda Sokolova, Elmira Omirtaeva and Irina Zaitceva

Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan

email address: [email protected]

The use of immunostimulating preparations for vaccination is limited by the ability of these preparations to stimulate Th2 cellular immunity. Therefore, the research of stimulating of this link of immunity is an integral part of the study of immunotropic preparations. We studied the effect of the combination of betulin–nobiletin on the expression of the Th2 cellular immunity genes. Cytokines IL-4 and IL-10 were selected as markers. To study the expression of tested genes, a single immunization of white outbreed mice was carried out with the tested combination of betulin–nobiletin in a mixture with purified glycoprotein antigens (HA, NA) (GPAs) of the influenza virus strain A/Almaty/8/98 (H3N2). The dose of influenza virus GPAs was 10 μg/mouse; the dose of the studied immunostimulating combination was 45 μg/mouse. Macrophages were collected from animals 3 days after immunization. Level of selected cytokine genes expression was determined in real time PCR. The expression activity of the IL-4 and IL-10 Th2 cellular immunity genes after administration of betulin–nobiletin combination increased from four to eight times. Thus, immunostimulating combination of betulin–nobiletin enhances the expression of Th2 cellular immunity genes, which makes it possible to use such combinations in the development of new immunotropic preparations.

NGS and metagenomic studies of acute respiratory viral infections

Elmira Anarkulova, Andrey Bogoyavlenskiy, M. Amanbayeva, Madina Alexyuk, Aizhan Turmagambetova and Vladimir Berezin

Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan

Abai Kazakh National Pedagogical University, Almaty, Kazakhstan

email address: [email protected]

Acute respiratory viral infections (ARVIs) are prevalent worldwide. The main reason is the multiple cause of the disease. More than 80 etiological agents have been identified that ARVIs cause belonging to different families (Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Pneumoviridae, Reoviridae etc). The ratio of pathogens among risk groups of children under 12 years old to adults after 50 varies within a few percent but makes up equal shares for ortho-, reo-, and pneumoviruses (∼75%). The remaining viruses of this group account for no more than 25% of the number of cases. We carried out a comparative research of environmental samples using the method of massive parallel sequencing to study the presence of ARVIs pathogens reads. Wastewater samples were collected from the filtration fields near Almaty. Genome libraries were prepared in accordance with the Illumina protocol recommendations. Sequencing was performed using HiSeq 2000. Analysis of the results was carried out using the Kaiju program. Our results are quite unexpected. Frequency of orthomyxoviruses was 33% which did not contradict the standard diagnosis; adenoviruses making up about 26% of ARVIs came in second. Frequency of each remaining families did not exceed 10%. Thus, for better statistics among ARVIs are necessary additional metagenomic studies of environmental samples.

The therapeutic activity of some xanthones in experimental influenza infection

Andrey Bogoyavlenskiy, Aizhan Turmagambetova, Vladimir Berezin, Pavel Alexyuk, Madina Alexyuk, Yergali Moldakhanov, Kuralay Akanova, Elmira Anarkulova, Nadezhda Sokolova, Elmira Omirtaeva and Irina Zaitceva

Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan

email address: [email protected]

The search for new anti-influenza medicines remains one of the main problems of virology. With a rather successful bioinformation screening of chemical compounds, only a few reach the pharmacy for the ability to suppress the reproduction of influenza virus infection (IVI), because most of selected drugs by this way do not have therapeutic properties. We studied the ability of some xanthones to block the reproduction of influenza in the model of experimental IVI in animals. Therapeutic effect of gartanin and mangosteen was studied at a dose of 10 mg/kg against influenza virus strain A/Almaty/8/98 (H3N2). Therapeutic effect of xanthones was evaluated by quantitative determination of influenza virus matrix protein gene in lungs of mice 7 days after the onset of acute IVI against the background of therapeutic use of studied xanthones orally for 3 days after animal’s infection. Matrix protein gene in the samples was quantified in real-time PCR using TaqMan probe. Therapeutic activity of the studied xanthones was largely dependent on compound structure. Gartonin is able to block the reproduction of experimental IVI of animals by 40%. Therapeutic activity of mangosteen was 60%. Thus, xanthones are rather promising group of compounds for the development of new anti-influenza medicines.

EGCG and Polyphenon E protection of retinal pigment epithelial cells under light-induced stress conditions

Madalina Oana Negrean, Dumitrita Rugina, Andrea Bunea and Adela Pintea

University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

email address: [email protected]

Reactive oxygen species can be generated in retina by high level of irradiation in the presence of photosensitizers. Here, we investigated the protective effects of epigallocatechin gallate (EGCG) and Polyphenon E on the oxidative status of RPE cells photosensitized with Rose Bengal (RB) and green light. The human retinal pigmented epithelial D407 cells, previously treated with EGCG (100 μM) or Polyphenon E (100 μg/mL, equivalent of 130 μM EGCG) in completed growth medium for 24 h, were incubated with 500 nM RB for 1 h, after that being exposed to light (580 nm filter) for 30 min. Simultaneous exposure of RPE cells to RB and light determined a significant decrease of cell viability (50%), activity of superoxide dismutase (SOD), catalase (CAT), gluthatione peroxidase (GPx) and concentration of reduced glutathione (GSH), whereas the level of intracellular ROS and lipid peroxides (MDA) were significantly increased. Pre-treatment of RPE cells with EGCG and Polyphenon E had a positive effect on the cell’s viability, activity of SOD and on the concentration of GSH, whereas the activity of CAT and GPx were not significantly changed. Both treatments significantly reduced the level of ROS (fivefolds for EGCG) and MDA compared to positive control.

Surgical treatment of diaphragmatic relaxations

Vlad Cristian Dobrea , Ioan Cordoș, Cristian Paleru, Mimi Dobrea  and Anca Alexandra Popescu

Faculty of Medicine, U.M.F. Carol Davila, Bucharest, Romania

Pneumoftiziology Institute Marius Nasta, Bucharest, Romania

Faculty of Veterinary Medicine, University of Agriculture and Veterinary Medicine, Bucharest, Romania

email address: [email protected]

In some pathological situations, eventration of the diaphragmatic dome occurs and it is named diaphragmatic relaxation. Currently, video-assisted thoracic surgery (VTAS) is a minimally invasive alternative to standard thoracotomy. This study was performed on four patiens with diafragmatic relaxation and three cases of diaphragmatic hernia. VATS was performed with diaphragmatic plication and anchorage at 9 or 10 intercostal space. Diaphragmatic relaxation was left sided in three patients and right sided in only one patient. All patients were over 50 years old, they were smoker, 2/4 of them worked in noxious environments or had a history of diaphragmatic hernia. The mean operation time was about 3 h. The mean hospital stay was 8–9 days. All patiens had symptoms relieved and the mortality was 0. Two from three cases of diaphragmatic hernia were hiatal hernias and one Morgagni Larrey hernia diagnosed at maturity. Only one case was associated with a previous esophagoplasty surgery. These patients were surgically resolved. VATS is minimally invasive alternative to conventional surgery. This technique produced significant functional improvements of operated patients.

Expression of intrinsically disordered proteins in heterologous hosts

Karen S. Sarkisyan

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

email address: [email protected]

Intrinsically disordered proteins do not form a uniform native structure, instead they exist in a number of alternative conformations, depending on the external conditions, such as acidity, salt concentration, presence of the partner protein or post-translational modifications. They represent an important group of proteins whose structure–function connection is not well understood. We aim to develop a high throughput assay for testing the functionality of the intrinsically disordered protein 106094 from tardigrade Hypsibius dujardini, which is critical for the ability of this organism to survive complete drying. As a first step towards this goal, we have tested various conditions and genetic designs for expression of the gene 106094 in yeast Saccharomyces cerevisiae. Here, we report the comparison of assayed genetic designs and desiccation protocols, as well as the effect of expression of gene 106094 on the fitness of yeast.

Funding: This work was supported by the Russian Science Foundation project 19-74-10102.

Dairy cattle fertility related to semen deposition place

Pîtiu Alexandra Mădălina and Tăpăloagă Paul Rodian

Engineering and Management of Vegetal and Animal Resources Doctoral School, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania

Faculty of Animal Science, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania

email address: [email protected]

The experiment was carried out in two dairy facilities, A and B, on 45 adult cows. The deposition place of the semen was in farm A, the bifurcation of the uterine horns was in farm B, the uterine horn corresponding to the evolving Graff follicle. The artificial insemination was performed once, in the optimum moment, approximately 10–12 h after the first signs of heat were detected. The method used was the recto-vaginal one. Following the artificial insemination, it was found that the percentage of fertility for cows where the deposition place of the semen was at the bifurcation of the uterine horns was 55%, and in the other, in the corresponding uterine horn Graff in evolution, was 65%. The lowest average number of artificial inseminations was 1, 42 inoculation/animal on farm B. It was found that the lowest service-period recorded was on farm B of 82.38 days, and the highest service-period recorded was within farm A of 106.18 days. It is recommended that the farm A be closely monitored for the animals to see the first signs of heat and to reduce the period of service to the optimum of 80 days.

Influenza type B virus isolated in Kazakhstan in 2018

Mira Shamenova, Galina Lukmanova, Nailya Klivleyeva, Assem Baimukhametova, Tatyana Glebova, Nuray Ongarbayeva, Nurbol Saktaganov and Baiken Baimakhanova

The Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan

email address: [email protected]

Wide circulation of influenza type B viruses of two genetic lineages (B/Victoria/2/87-like and B/Yamagata/16/88-like) is currently observed around the world. Data from the Kazakhstan monitoring studies confirm global statistics on the circulation of influenza B viruses in recent years. The proportion of laboratory-confirmed type B influenza during the 2014–2015, 2016–2017 and 2017–2018 epidemic seasons exceeded 30% [Smagul et al., 2018; Sagymbay et al., 2019]. In 2018, influenza virus strain B/Almaty/08/2018 was isolated from a nasopharyngeal swab obtained from a healthcare facility located in the Almaty region. A study of the antigenic relationships of this strain showed its kinship with B/Victoria/2/87-like viruses. According to molecular biological characteristics, this strain is also similar to Victoria-lineage influenza B viruses. The nucleotide sequence of the hemagglutinin gene of strain B/Almaty/08/2018 revealed 99% identity with the corresponding genes of viral strains isolated in Africa and North America (B/Kenya/010/2016, B/Florida/02/2017, B/Maryland/04/2017, B/Maine/06/2018) in 2016–2017, which indicates the global distribution of such strains.

Diversity of archaea of the Small Aral Sea

Madina Alexyuk, Andrey Bogoyavlenskiy, Pavel Alexyuk, Yergali Moldakhanov and Vladimir Berezin

Laboratory of antiviral protection, LLC "Research and Production Center for Microbiology and Virology", Almaty, Kazakhstan

email address: [email protected]

Archaea are common in all types of ecosystems. They play an important role in global biogeochemical cycles. Even though archaeal diversity has been studied extensively, there is little data characterizing the community of archaea in the inland and marginal seas. Thus, the experimental results obtained in the research of the Aral Sea microbiome will expand the knowledge of the ecological and phylogenetic diversity of archaea for this type of ecosystem. The aim of the research was to study archaeal diversity in the Small Aral Sea in Butakov Bay. Seawater samples were collected during May of 2019 in volume of 5 L. Samples were filtered through 3-μm pore size filters, then were concentrated by ultracentrifugation. Isolated nucleic acid from concentrated virus containing material was sequenced on Illumina MiSeq. Data were analyzed using the Kaiju program using the non-redundant protein database with default parameters as a reference. Taxonomic classification of metagenomic data showed that among all archaeal sequences identified, up to 53.8% belonged to the Euryarchaeota phylum, 39.7% belonged to the TACK group combining Thaumarchaeota (33.6%) and Crenarchaeota (6.1%) phyla, the DPANN group (6%) was represented by archaeal sequences of Parvarchaeota and Pacearchaeota phyla, unclassified archaea were present in amount of 5%.

Broiler meat water-holding capacity in industrial refrigerated conditions

Solomon Lucian, Ciobanu Marius Mihai, Munteanu Mugurel and Boișteanu Paul Corneliu

Department of Fundamental Sciences in Animal Husbandry, University of Agricultural Sciences and Veterinary Medicine, Iaşi, Romania

email address: [email protected]

Water-holding capacity influences meat tenderness, succulence, firmness and aspect with possible effects for improving meat quality or its economical value. This technological parameter could be externalized as aggregation potential of water, as expressible moisture or effective dropping, each of them having different implications. So, water-binding capacity represents the maximum water quantity, which muscular protein could retain. The greatest part of inner-muscular water (between 88% and 95%) is trapped in intercellular spaces between actin and myosin filaments, only a small part (5%–12%) being into the myofibril ones. The study aimed to evaluate three refrigeration regimes differentially by temperature and air current flow (L1 = 1 °C, 3.5 m/s; L2 = 3 °C, 5.5 m/s; L3 = 5 °C, 7.5 m/s) on three cut anatomical regions (breast, upper thighs and lower thighs), in order to characterize the weight losses through refrigeration. Weight losses by refrigeration at muscular samples gathered from poultry carcasses from experimental batches presented mean values between 0.49% and 1.12%; batch L3 presenting increases of weight losses at refrigeration, followed in a descended order by L2 and L1. At carcass level, the highest weight losses were attributed to upper thigh musculature, followed at small differences by pectoral musculature and the one of lower thigh.

Quantitative evaluation of heavy metals (Pb and Cd) in roe deer meat (Capreolus capreolus L) from N-E area of Romania

Munteanu Mugurel, Ciobanu Marius Mihai, Postolache Alina Narcisa  and Boișteanu Paul Corneliu

Department of Fundamental Sciences in Animal Husbandry, University of Agricultural Sciences and Veterinary Medicine, Iaşi, Romania

Cattle Breeding Research Station from DANCU, Iași, Romania,

email address: [email protected]

Intense concern in recent years for quantitative and toxicological identification and evaluation, especially for heavy metals from animal origin products represent a side that needs to be studied continuously, especially for game meat, nutritional toxicity of those mineral elements (mainly Pb and Cd) being directly influenced by their variations in habitat water, air and soil. In animals, accumulation of heavy metals depends on their concentration in food, exposure period of by the age of animal. The goal of the study is motivated by the inexistence of information regarding appreciation of contamination degree with heavy metals of game meat, having in view the limited checkout of nourishment of game animals exploited in their natural environment, as well as the polluting environmental factors. Analyze of heavy metal content of lyophilized muscular tissue samples was realized by atomic absorption spectrometric method (AAS), on a GBC-AVANTA type atomic absorption spectrometer. The effectuated analysis confirms the presence of xenobiotic heavy metals into cervid meat. The evaluated mean concentrations being under the admissible maximum limit, varying between 0.021 and 0.039 mg/kg DM for Cd and 0.026 and 0.06 mg/kg DM for Pb. This obtained data are a direct consequence of bioaccumulation of heavy metals.

Isolation of pathogens of fungal diseases (oomycetes: Saprolegniales) from sterlet larvae

Anna Aleksandrovna Tolkacheva, Ekaterina Petrovna Anokhina and Olga Sergeevna Korneeva

Voronezh state university of engineering technologies, Voronezh, Russia

email address: [email protected]

In artificial fish breeding facilities, there is often a massive growth of pathogens of fungal diseases, the most common of which are phycomycetes, in particular, Saprolegniales. Considering the importance of preserving the viability of fish embryos, our research group was tasked with finding substances that are effective and safe in the treatment and prevention of saprolegneosis in sturgeon caviar and, in particular, sterlet (Acipenser ruthenus) caviar. The objective of the study was the infected sterlet larvae obtained from the fish farm and water samples from the fishery basins. To obtain a pure culture of the causative agent of saprolegniosis, the pathological material was repeatedly washed in sterile water and by successive subcultures on agar (meat-peptone agar, potato-dextrose agar and wort-agar). In parallel, the studies were carried out by the ‘bait’ method with water samples in order to obtain the genital organs (oogonia and antheridia) of fungi necessary for species identification. Species identification was carried out according to the CEJP (1959). The identified fungi were assigned to two genera: Saprolegnia and Achlya. In the future, we will refine the species of the isolated fungi by sequencing. Then we will select substances that inhibit the growth of pathogens of fungal diseases.

Ecological–genetic monitoring of feijoa forms (Feijoa Sellowiana Berg) in western Georgia

Nino Kedelidze, Davit Baratashvili, Nino Lomtatidze, Neli Khalvashi, Giuli Katamadze and Irina Nakashidze

Institute of Phytopathology and Biodiversity, Batumi Shota Rustaveli State University, Batumi, Georgia

Department of Biology, Faculty of Natural Sciences & Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia

email address: [email protected]

Alteration of the process of mitotic cell division is one of the most sensitive processes. Accordingly, it can be stimulated by environmental agents, starting with a slight change in temperature, ending with the influence of the most influential chemical and physical factors. Mitotic activity (mitotic index) was studied in feijoa, which is widespread in three ecological zones of Georgia (Adjara, Guria, Samegrelo). Commonly accepted methods were used for cytogenetic studies. The mitotic activity of cell division was significantly highest (6.0%–7.6%) in the forms of the Samegrelo region. In the Adjara, mitotic activity was higher in both study forms than in control. Based on comparative analysis, it is suggested that the Samegrelo region has a statistically significant higher rate of mitotic activity than the other areas. There are many reasons, including environmental conditions: soil, climatic conditions (humidity, temperature, etc.), which cause the high activity of cell division. We thought that this may be caused by a high level of radiation that may be connected to the Chernobyl accident.

Enhancer of prokaryotic translation promotes protein expression in tobacco leaves

Anna S. Nizkorodova, Neilya S. Polymbetova and Bulat K. Iskakov

M. Aythozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan

email address: [email protected]

The epsilon element (ε) is a sequence (5′-UUAACUUUA-3′) located in the 5′-UTR of T7 phage gene 10. In case it precedes Shine-Dalgarno (SD) sequence this element (and its variation εII) enhances protein translation in bacteria 2–3 times compared with SD alone. The hypotheses explaining ε-element’s mechanism of action is that it forms complimentary bounds with 16S rRNA’s 45th hairpin and thereby promotes the initiation of translation. The sequence of the 45th hairpin in 16S and 18S rRNAs is very alike. Thus, according to the aforesaid hypothesis, ε-element should also enhance protein expression in eukaryotic cells. To verify this assumption, we’ve obtained genetic constructs based on pCambia2300 encoding β-glucuronidase (GUS) gene interrupted with castor bean catalase 2 intron-2 under the control of 35S-promoter and nos-terminator, and differing in their 5′-UTR (ε + SD, εII + SD, 5′-UTR of potato virus Y as a positive control). As a negative control plasmid was used without any 5′-UTR. These constructs were used for vacuum agroinfiltration in Nicotiana tabacum leaves. The measurement of GUS-activity in total protein samples was performed 3 days after the infiltration. All constructs showed reliable increase in GUS-activity compared with negative control: potato virus Y 5′-UTR – 2.7; ε + SD – 2.2; εII + SD – 1.4 times.

Genetic diversity of HMW-GS in diploid, tetraploid and hexaploid Triticum species

Zdenka Gálová, Milan Chnapek, Zelmira Balážová, Dana Rajnincová, Martin Vivodík, Andrea Špalekováand Magdaléna Lacko Bartošová

Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agricultural, Nitra, Slovakia

Department of Sustainable Agriculture and Herbology, Faculty of Agrobiology and Food Resources, Slovak University of Agricultural, Nitra, Slovakia

email address: [email protected]

The genetic variation of HMW glutenin subunits in twenty-five accessions of three Triticum species were investigated using SDS-PAGE. Out of these, 13 genotypes were hexaploid, ten tetraploid and two diploid, respectively. Three HMW-GS (0, 6 + 8) were observed in diploid wheat. Six alleles were found (0, 6 + 8, 7 + 8, 20) in tetraploid wheat. Five HMW-GS were assessed in hexaploid wheat, while from loci Glu-A1 was detected subunit 1, from loci Glu-B1 subunit pair 7 + 9 and from loci Glu-D1 two pairs of HMW-GS subunits 5 + 10 and 2 + 12. Technological quality of hexaploid varieties was detected by calculation of Glu-score, which varied from 7 to 9. Dendrogram of electrophoretical spectra glutenins divided analysed wheats in two main clusters. Hexaploid genotypes were grouped into cluster I, which was divided into two subclusters (A, B). All tetraploid, both diploid and five hexaploid varieties were grouped into cluster II. Hexaploid varieties from cluster B were further grouped into more subclasses. Two hexaploid genotypes were independently grouped into subcluster A. In subcluster B, three hexaploid genotypes were grouped independently and two diploid varieties were separated from tetraploid genotypes. All alleles of HMW-GS in Triticum species could be further utilized through hybrid in the quality improvement of common wheat.

Funding: This work was co-funded by European Community under project no. 26220220180: Building Research Centre “AgroBioTech", by grant VEGA No. 1/0246/18 and KEGA project No. 025SPU-4/2018.

Posbiotic exopolysaccharides biosynthesized by selected lactic acid bacteria strains for gluten-free sourdough

Bogdan Pacularu Burada, Luminița Anca Georgescu and Gabriela Elena Bahrim

Department Food Science, Engineering, Applied Biotechnology and Aquaculture, Dunarea de Jos University, Galati, Romania

email address: [email protected]

Lactic acid bacteria strains are able to biosynthesize various metabolites that can be exploited as valuable postbiotics in the food products manufacturing processes. Fermentation conducted by selected lactic acid bacteria starters increases the shelf-life of the food products and supports the healthy lifestyle of the consumers. Actual preoccupations regarding healthy, tasty and nutritious foods focus on the identification of microbial sources of exopolysaccharides that can successfully substitute hydrocolloids and thickening agents. This work aimed to identify and select exopolysaccharide producers among 60 new isolated strains of lactic acid bacteria. Exopolysaccharides producing strains were determined by cultivation on solid and broth culture media with sucrose and glucose. Six strains were selected as good producers by cultivation on the broth fermentation medium supplemented with saccharides, followed by quantification of the total sugars after fermentation. The results of phenol-sulfuric acid assay revealed that these lactic acid bacteria strains produced exopolysaccharides amounts ranging between 17.854 and 23.193 mg/mL. In the future, additional studies will be carried out in order to optimize the biotechnological parameters of the polymer biosynthesis that can enhance the quality and functionality of a gluten-free sourdough bread.

Funding: This work was supported by the project "EXPERT", Contract no. 14PFE/17.10.2018.

Study of oxidative processes and changes in the phase state of lipids in damaged nerves under the action of resveratrol

Aleksandra Evgenevna Pyanzina, Sergey Ivanovich Pinyaev, Elena Viktorovna Chudaykina, Alexandra Yurievna Sorokina, Tatyana Pavlovna Kuzmenko, Natalya Sergeevna Krutova and Victor Vasilievich Revin

Department of Biotechnology and Biology, Ogarev Mordovia State University, Saransk, Russia

email address: [email protected]

One of the most important problems of regenerative medicine is the restoration of damaged nerves. A peripheral nerve injury triggers a cascade of processes. First, LPO are intensifying. Second, LPO leads to a change in the phase properties of the membrane. Third, a violation of the phase state of lipids leads to a change in the activity of membrane-bound enzymes, functional characteristics and receptors. It was shown that there is an intensification of oxidative processes in case of damage to somatic nerves. MDA accumulates, the phase properties of the lipid bilayer change. Under the same conditions, multidirectional changes in the activity of enzymes occur, SOD activity decreases, CAT activity increases. Resveratrol reduces the amount of MDA in both areas of the damaged nerve. In addition, under the action of resveratrol, the activity of SOD and CAT tends to return control values. Similar patterns are observed when resveratrol acts on the phase state of lipids with damage to somatic nerves. It can be argued that when a nerve is damaged, profound changes occur both in the lipid component and in the antioxidant system. Resveratrol has a stabilizing effect on the studied parameters, but for their full recovery requires a long time.

The effect of a plant extract or in mixture with a prebiotic on microbiota of cattle rumen

Galimzhan Duskaev, Baer Nurzhanov, Albert Rysaev and Shamil Rakhmatullin

Federal Research Centre of Biological Systems and Agro-technologies of the Russian Academy of Sciences, Orenburg, Russia

email address: [email protected]

The purpose of this research was to study the synergistic effects of plant and prebiotic substances on ruminal microflora. The effect of the prebiotic (Glucomannan + β-Glucans) (Pr), Quercus cortex (QC) extract, including together with Pr against the background of control (C) on the microbiota of cattle rumen was assessed (n = 12; 12 months, diet: 65% hay, 35% grain feed, QC 0.5%, Pr 0.1%). Microbiota of bacteria were analyzed using high throughput sequencing (Illumina). Bioinformatical analysis of the results was made in application PEAR. An analysis of the data showed that Pr contributed to an increase in the bacteria of Bacteroidetes by 24.3%, Firmicutes by 17.6%, Saccharibacteria by 16.1%; QC + Pr increased the number of bacteria of Bacteroidetes and Fibrobacteres by 40.5 (p ˂ 0.05) and 16.3%, the lowest number of Clostridia was noted (of all variants); against QC, the number of bacteria of the phylum Bacteroidetes increased by 48.0% (p ˂ 0.05), the number of bacteria Firmicutes decreased by 16.4%. It is necessary to continue studying the synergistic effects of plant extracts on microbiome in order to find an alternative to antibiotics.

Funding: This research was performed with financial support from the Russian Science Foundation (16-16-10048).

Researches on the level of mycotoxin contamination of some cereals used in the production of compound feed in Romania

Dragos Mihai Lapusneanu, Gabriela Frunza, Cristina Radu Rusu, Daniel Simeanu and Ioan Mircea Pop

Department of Fundamental Sciences in Animal Husbandry, University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania

email address: [email protected]

Mycotoxins are some of the most toxic chemicals, considered as natural contaminants. The aim of the study is to quantitatively determine the mycotoxins from corn and wheat grains used for the production of compound feed. The samples were analyzed during January–May 2019. Quantitative determination of mycotoxins was performed by ELISA technique. The results showed that all 33 samples of corn and 12 of wheat were below the maximum limit imposed by European Union legislation. To determine the quantity of aflatoxin B1 in maize, 26 samples were analyzed, for 8 samples (30.7%) being undetectable; for wheat, 8 samples were analyzed, for 3 samples (37.5%) being undetectable. For six maize samples analyzed, the T-2 toxin had values between 0.005 and 0.076 ppm; for four wheat samples it had values between 0.007 and 0.016 ppm. The content of deoxynivalenol (DON) ranged from 0.035 to 0.355 ppm for seven maize samples, for one sample being undetectable; for wheat, the content in DON ranged from 0.080 to 0.192 ppm for three samples analyzed. The zearalenene content ranged from 0.0015 to 0.051 ppm for eight samples, for one sample being undetectable; for wheat, the values of 0.028 ppm and 0.066 ppm were identified for two samples.

Regeneration and conservation of valuable medicinal plant species in vitro

Olga Molkanova, Darya Egorova, Yuri Gorbunov and Tatyana Konovalova

Tsitsin Main Botanical Garden of Russian Academy of Sciences, Moscow, Russia

email address: [email protected]

Medicinal plants are valuable resources of chemical compounds. Many species have become endangered due to destruction of natural habitats and intensive exploitation. Tissue culture methods are widely applied to conserve biological diversity of medicinally important plant species. The micropropagation technologies for more than 20 plant species were improved at all stages. Micropropagation protocols for cloning of some medicinal plants belonging to the genera Aralia L., Aristolochia L., Codonopsis Wall., Epilobium L., Stevia Cav., Potentilla L. were developed. The plantlets were obtained via organogenesis and somatic embryogenesis at 22 ± 2 °C and 2–3 klux light intensity. Direct regeneration through adventitious shoot formation was common to most studied species. Indirect regeneration through somatic embryogenesis was observed in the relict endemic species Oplopanax elatus (Nakai) Nakai. Leaf and petioles explants taken from O. elatus were used to induce somatic embryos from embryogenic callus on Murashige and Skoog basal medium supplemented with 1.0 mg/L thidiazuron and 1.0 mg/L 2,4-dichlorophenoxyacetic acid. The viable explants were stored under conditions inhibited their growth (at 4–12 °C and 0.2–0.3 klux light intensity). The work was carried out in accordance to Institutional research project № 18-118021490111-5.

Genetic variability of maize cultivars based on gene-targeted and microsatellite markers

Zelmira Balazova, Martin Vivodik, Zdenka Galova and Tomas Vyhnanek

Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

Department of Plant Biology, Faculty of AgriSciences, Mendel University in Brno, Brno, Czech Republic

email address: [email protected]

The aim of our work was to analyze DNA polymorphism in the collection of 20 maize cultivars Zea mays. The genetic diversity of 20 maize cultivars coming from different American countries (Mexico, Peru, Brasil, Argentina) using 14 STMS (Sequence Tagged Microsatellite Site) and 10 SCoT (Start Codon Targeted) markers was evaluated. Average values of PIC for STMS markers and SCoT markers were higher than 0.6, which means high polymorphism detected in the chosen maize genotypes. Hierarchical cluster analysis using UPGMA algorithm based on microsatellite and SCoT marker systems was used to construct dendrogram. Twenty maize genotypes were divided into clusters. Clustering partially reflected geographic origin of the studied maize genotypes in the dendrogram. Microsatellite and SCoT marker systems were altogether used as a powerful tool for assessment of genetic diversity in maize cultivars. Both molecular markers were sufficiently reproducible, polymorphic and able to differentiate analyzed genotypes of maize and indicate the genetic relationships between them.

Funding: This work was supported by grant KEGA (no. 025SPU4/2018).

Genetic diversity of castor based on RAPD, SCoT and SSR markers

Martin Vivodik, Zelmira Balazova  and Zdenka Galova

Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

email address: [email protected]

The present study is focused on estimation of genetic distance between 56 Tunisian castor genotypes, based on 18 RAPD, 37 SCoT and 20 SSR markers. The ricin genotypes were obtained from 12 regions of Tunisia. PCR amplification of DNA using 18 RAPD primers produced 145 DNA fragments that could be scored in all 56 genotypes of Tunisian castor. All 145 amplified bands were polymorphic. PCR amplification using 37 SCoT primers produced 268 DNA fragments that could be scored in all 56 genotypes of Tunisian castor. Of the 268 amplified bands 230 were polymorphic. PCR amplification of DNA using 20 primers for SSR analysis produced 141 DNA fragments that could be scored in all 56 genotypes of castor. Average PIC for each marker technics was higher than 0.75 that means high polymorphism. The dendrogram of 56 Tunisian castor genotypes based on 18 RAPD, 37 SCoT and 20 SSR markers using UGMA algorithm was constructed. Clustering partially reflected geographic origin of studied castor genotypes in the dendrogram. Results showed the utility of RAPD, SCoT and SSR markers for estimation of genetic diversity of castor genotypes leading to genotype identification.

Funding: This work was supported by grant KEGA (no. 025SPU4/2018).

Paper-based сollection of buccal cell human DNA

Kymbat Mukhtarova, Sabina Atavliyeva, Pavel Tarlykov and Yerlan Ramankulov

Nazarbayev University School of Medicine, Nur-Sultan, Kazakhstan

National Center for Biotechnology, Nur-Sultan, Kazakhstan

email address: [email protected]

This study presents a domestic kit suitable for genetic analysis, including a collection of human buccal cells, and long-term storage of nucleic acids on a piece of paper. A filter paper with a density of 85 g/m² was selected. It was treated with chemicals that make up the lysing solution (e.g. EDTA, Tris buffer and N-lauroylsarcosine), which contributes to the destruction of cells and prevents bacterial growth. A total of 25 buccal cell samples were collected on the treated paper. The samples had a median DNA yield of 20 ng/μL (range 0.5–45 ng/μL) according to a Qubit fluorimeter. Real-time PCR was performed on the ABO gene of erythrocyte surface proteins (rs8176746 A > C) and was successful in all samples. Amplification of the first hypervariable segment of human mtDNA with a product size of 572 bp was carried out. Multiplex genotyping of 15 STR regions and the amelogenin locus was done using the AmpFlSTR Identifiler assay. As a result, it was confirmed that the paper card could be used for collecting and isolating DNA from buccal epithelium for genetic and other experiments.

Funding: This study was supported by the Ministry of Education and Science of the Republic of Kazakhstan (grant no. АР05130240).

Ficin complexes with chitosan nanoparticles

Tatyana Belyaeva , Victoria Koroleva, Marina Holyavka, Svetlana Pankova, Svetlana Olshannikova, Farhat Sakibaev, Alena Belenova, Dmitry Vandyshev, Alexander Pashkov  and Valeriy Artyukhov

Department of Biophysics and Biotechnology, Voronezh State University, Voronezh, Russia

Department of Biology, Voronezh State Medical University named after N.N. Burdenko, Voronezh, Russia

Department of Pharmaceutical Chemistry and Pharmaceutical Technology, Voronezh State University, Voronezh, Russia

Department of Organic Chemistry, Voronezh State University, Voronezh, Russia

email address: [email protected]

Ficin (EC 3.4.22.3) is a proteolytic enzyme destroying bacterial films. Chitosan has properties such as biocompatibility, antimicrobial, antitumour and antiviral activities. The aim of the work was to obtain ficin complexes with chitosan nanoparticles and chitosan nanoparticles with the addition of ascorbic acid for further use in biomedicine and veterinary medicine. Nanoparticles of pure and ascorbic-acid-doped chitosan were formed with an average size of 136 and 165 nm, respectively. The next step was the preparation of ficin complexes with chitosan nanoparticles and chitosan nanoparticles with the addition of ascorbic acid 334 and 439 nm in size, respectively. The activity of ficin complex with chitosan nanoparticles and chitosan nanoparticles with ascorbic acid was 28% and 38% of the catalytic ability of the native enzyme, but ficin in complex with nanoparticles was more stable than soluble enzyme. On the fifth day, the activity of the enzyme was more 40% of the initial for chitosan nanoparticle complexes of both types, whereas the free ficin retained its catalytic ability at 25% level.

Funding: This work was financially supported in the form of a grant from the President of the Russian Federation for state support to young Russian scientists–doctors of sciences MD-1982.2020.4.

Increase of antibiotic activity by the method of induced mutagenesis

Khasenova Almagul Khamitovna, Zere Turlybayeva, Amankeldy Sadanov, Almagul Khasenova, Aliya Kalbayeva and Ramilya Galimbayeva

Limited Liability Company “Research and Production Center for Microbiology and Virology", Almaty, Kazakhstan

email address: [email protected]

The widespread of resistant microorganisms makes screening of new natural antibiotics relevant. Actinomycetes with antagonistic properties against laboratory collection Gram-positive (Bacillus subtilis ATCC 6633) and Gram-negative (Comamonas terrigena ATCC 8461) test-microorganisms from the samples of North Kazakhstan’s medicinal plants’ rhizosphere were isolated. Actinomycetes with high antagonistic activity underwent induced mutagenesis with the aim of increasing antibiotic yield and antibiotic activity. Cultures were treated with mutagenic solution of N-nitroso-N-ethyl urea in 0.1 M of phosphate buffer (pH = 8.0) with 300 microgram/mL for 60–180 min at 30°С by the standard technique of Miller, 1976. Samples were taken from reaction blend every hour, then immediately diluted by sterile phosphate buffer and 100 μL of spore suspension were distributed to source nutrient medium and cultivated at 28°С for 5–6 days. It was established that the most active clones appear after two and more hours of mutagen influence to the spore suspension. All the mutant strains are noted to have statistically significant (p < 0.05) increase of antagonistic activity compared to initial strains. Genetic stability of obtained clones was studied by sequence inoculations on agar nutrient medium with the control inoculation of each of them. The perspective mutant strains were identified by molecular-genetic analysis.

Radial basis function network: an efficient tool for modelling the hydrogen production from sugarcane bagasse using supercritical water gasification process

Carmen Riverol and Antonio Delgado

Chemical Engineering Department, University of West Indies, St. Augustine, Trinidad and Tobago

JC Engineering, Av. Principal Los Palos Grandes, Caracas, Venezuela

email address: [email protected]

The emerging technology Supercritical Water Gasification has a great potential for recycling biomass for the production of synthesis gas with a higher percentage of hydrogen. A good advantage is that the Supercritical Water Gasification does not require drying; thus, biomass with high percentage of moisture (>50%) suggests that greater energy efficiency can be achieved. In this article, experiments were performed using sugarcane bagasse or/and wood as feedstock. The effects of the temperature, pressure and residence time are investigated in the process of biomass gasification with carbon as catalyst, reforming (27.6 MPa) and producing hydrogen along with CO, CH₄ and CO₂. A Radial Basis Function Network (RBFN) was designed to predict the results. The proposed method herein is based on clustering of input space vectors and computing weights of Euclidian distances and histogram equalization within each cluster will determine the centre and width of each receptive field for the estimation of the risk parameters. The results depicted an 92.4% accuracy where 40%–60% of the biomass were converted under subcritical conditions, fuel gas with >50 vol% H₂, 40% CH₄ and up to 10% of higher hydrocarbon species with complete fuel utilization at temperatures higher than 923 K.

Regularities of dimeric complexes formation by exo- and endoinulinases

Anastasia Dubovitskaya , Farhat Sakibaev, Marina Holyavka, Victoria Koroleva, Tatyana Belyaeva, Svetlana Pankova, Svetlana Olshannikova, Alena Belenova, Pavel Karlov, Dmitry Vandyshev , Alexander Pashkov, Alexey Slivkin  and Valeriy Artyukhov

Department of Biophysics and Biotechnology, Voronezh State University, Voronezh, Russia

Resource Center “Molecular Structure of Matter”, Sevastopol State University, Sevastopol, Russia

Department of Biology, Voronezh State Medical University named after N.N. Burdenko, Voronezh, Russia

Department of Pharmaceutical Chemistry and Pharmaceutical Technology, Voronezh State University, Voronezh, Russia

Department of Organic Chemistry, Voronezh State University, Voronezh, Russia

email address: [email protected]

The amino acid composition of conserved sequences of inulinases, the localization of amino acid residues included in their composition and the regularities of its change as a result of in silico dimerization were studied. For the majority of inulinases presented in this work, the greatest contribution to the change in the composition of the regions of amino acid residues local accumulation during dimerization is made by transitions of hydrophobic residues in the surface/inner part of the molecule. The uneven distribution of charged and hydrophobic amino acid residues on the surface of molecules with the formation of local accumulation areas was revealed. It was shown that for exoinulinases from Aspergillus ficuum, A. niger and A. awamori and endoinulinases from A. niger and A. fumigatus negatively charged carriers are optimal. They are likely to bind to sites located in the region of the non-catalytic C-terminal domain. Hydrophobic polymers are the least promising for the adsorption of all inulinases discussed in this work.

Funding: This work was financially supported in the form of an internal grant from Sevastopol State University (no. 42-01-09/90/2020-2).

Transient expression and immunogenicity of M2e peptide of avian influenza virus bearing from hepatitis B virus-like particles produced in Nicotiana benthamiana

Gergana Geneva Zahmanova, , Katerina Hristova Takova, Rumyana Panova Valkova, Valentina Todorova Toneva and Ivan Nikiforov Minkov,

Department of Plant Physiology and Molecular Biology, University of Plovdiv, Plovdiv, Bulgaria

Center of Plant System Biology and Biotechnology, Plovdiv, Bulgaria

Institute of Molecular Biology and Biotechnologies, Plovdiv, Bulgaria

email address: [email protected]

The immunogenicity of small proteins such as M2e peptide of influenza virus can be significantly increased by their presentation on particulate carriers such as the virus-like particles (VLPs). The core antigen of hepatitis B virus (HBcAg) is highly immunogenic and spontaneously assembles into VLPs during its expression in several heterologous systems. The aim of the study is the development of a plant expression system for the production of chimeric HBcAg/M2e VLPs and the characterization of their immunogenicity. In this study, we inserted the 23 aa of M2e peptide into the immunodominant loop of HBcAg. The recombinant protein was expressed in Nicotiana benthamiana plants using the Cowpea Mosaic Virus-based vector pEAQ-HT. The yield of rHBcAg/M2e protein after purification was 50–80 µg per 1 g of green leaf biomass. Electron microscopy showed that plant produced chimeric protein spontaneously assembled into VLPs with ∼26 nm diameter. The immunization of mice with purified HBcAg/M2e VLPs induced high levels of M2e-specific serum antibodies.This study demonstrates efficiency of plant produced VLPs and we believe that plant-produced vaccines could find application for human and veterinary vaccine design.

Funding: This research was funded by the Horizon 2020, PlantaSYST project/739582 and the Bulgarian Science Fund, DNTC-Russia 02/6.

Gamma lactone and vanillic acid change the microbiome of broiler blind appendage

Elvina Shajahmetova, Dmitry Deryabin and Galimzhan Duskaev

Federal Research Centre of Biological Systems and Agro-technologies of the Russian Academy of Sciences, Orenburg, Russia

email address: [email protected]

The research objective was to study the effect of new plant substances on poultry microflora. The effect of gamma lactone (Eucalyptus viminalis) (GL), Vanillic acid (Quercus cortex) (VA) and GL + VA against the background of control (C), on the microbiota of cecum of broilers (Arbor Acres, n = 120; control – basic diet (BD); GL (0.07 mL/bird/day); VA (0.07 mL/bird/day); GL + VA. Microbiota of bacteria were analyzed using high throughput sequencing (Illumina). Bioinformatical analysis of the results was made in application PEAR. The inclusion of GL in the diet increases the phylum Bacteroidetes (32.3%) in the intestine, decreases Firmicutes (8.3%) and Verrucomicrobia (21.9%); VA – increases Bacteroidetes (17.4%) and Firmicutes (11.8%), decreases Verrucomicrobia (27.0%); GL + VA – increases Bacteroidetes (18.9%) and Firmicutes (9.1%). An increase in Rikenellaceae family and a decrease in Lactobacillaceae and Verrucomicrobiaceae are found in GL; VA increases Ruminococcaceae; GL + VA increases Rikenellaceae. Some separate substances from extracts of medicinal plants can significantly change the intestinal microbiome, a search for all alternatives to antibiotics.

Funding: This research was performed with financial support from the Russian Science Foundation (16-16-10048).

Development of a new controller using head myoelectric potential

Yasuhiro Hayakawa  and Soki Marumoto

Department of Control Engineering, Nara College, National Institute of Technology, Nara, Japan

email address: [email protected]

Tablet-type terminals such as smart phones are widely used. Thus, many industries have been developing many kinds of products that use the terminal as an input device. However, the terminals are operated by finger’s motion. As the results, the people who cannot handle a hand finger freely cannot operate the terminal device. Therefore, it is necessary that we will develop a new controller to operate devices without the use of a hand finger. In order to develop the controller, we use myoelectric potential signals as input signals to the controller. In this study, we use the myoelectric potential signals of a head part as input signals to replace fingers. First, we observe myoelectric potential signals in multiple head muscles and consider diversification of signals by combining them. As the results, in order to clarify the dependence of myoelectric signals, a measuring device that can simultaneously measure myoelectric signals at multiple sites is manufactured, and simultaneous measurement experiments of myoelectric signals are performed. Furthermore, we reduce the size of the controller circuit by introducing a digital filter with a microcontroller. Through some experimental results, we cleared the effectiveness of the proposed controller to drive some devices.

Induction of shoot regeneration in vitro cultures of endangered species ‘Malacocarpus crithmifolius’

Valentina Kadamatovna Mursaliyeva and Akjunus Altaevna Imanbayeva

Mursaliyeva V.K. Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan

Imanbayeva A.A. Mangyshlak Experimental Botanical Garden, Aktau, Kazakhstan

email address: [email protected]

Relict plant ‘Malacocarpus crithmifolius’ is included in IUCN Red List as an endangered Iranian-South Turonian species with low population size and a limited range, so its preservation is very important. The research aim was to first establish ‘in vitro’ and micropropagation of ‘M. crithmifolius’. The vegetative and generative shoot explants and Murashige & Skoog medium (MS) with 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA), indole-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D) were used. The type of organogenesis in regard to explants and the effects of hormonal treatments on shoot formation frequency, the average number of shoots and callusogenesis were investigated. Direct organogenesis was observed only at generative explants on the MS with BAP and IBA or IAA. The buds conglomerate was initiated in leaf axil under inflorescences after a week of cultivation. Multiple shoot formation was reached by multiple sub-cultivation of revealed buds and adventitious shoots. The multiplication rates at MS with 4 mg/L BAP + 0.25 mg/L IBA and MS at 1.5 mg/L BAP + 0.1 mg/L IAA insignificantly differed from each other and composed in mean 5.85 for three passages after 12-week culture. Furthermore, applying of developed ‘in vitro’ regeneration techniques will allow preventing the disappearance of the genetic resources ‘M. crithmifolius’.

Molecular and genetic analysis of some cereals and pseudocereals proteomes

Dana Rajnincová, Milan Chňapek, Andrea Špaleková, Želmíra Balážová, Martin Vivodík, Katarína Klubicová, Martin Morovič,and Zdenka Gálová

Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

Centers of plant biology and biodiversity Slovak Academy of Sciences, Nitra, Slovakia

Department of Zoology, Constantine the Philosopher University, Nitra, Slovakia

Department of Anthropology, Constantine the Philosopher University, Nitra, Slovakia

email address: [email protected]

The aim of the study was to analyze the proteome of the winter wheat (Triticum aestivum L.), common oat (Avena sativa) and common buckwheat (Fagopyrum esculentum Moench.) genotypes by immunochemical methods and to detect the presence of potentially allergenic proteins. Western blot analysis revealed the strongest reaction of polyclonal antibody with wheat proteins with a molecular weight from 25 to 50 kDa. The strongest signal was detected in oat proteins with a molecular weight of 20 kDa and 40–55 kDa. No signal was detected in buckwheat, which was confirmed by ELISA results. The oat variety Zvolen and all three analyzed buckwheat varieties fulfil the criteria for labelling as gluten-free food. In 2DE gel of wheat, 221 protein spots were quantified by PDQuest, of which 10 proteins were identified. In the sample of oat, 168 protein spots were quantified, of which five were identified. Buckwheat proteins were separated into 208 protein spots in the 2DE gel, of which six were identified. By comparing the data with the database Allergome, we detected wheat allergens Tri a 26, Tri a 33, Tri a 36, Tri a alpha Gliadin, Tri a 20, oat allergens Ave s 11S and buckwheat allergen Fag e 1.

Funding: This work was co-funded by European Community under project no 26220220180: Building Research Centre “AgroBioTech", by grant VEGA No.1/0246/18 and KEGA project No. 025SPU-4/2018.

Enhanced ointments with bioactive compounds and lactic acid bacteria

Elena Enachi , Monica Boev, Vasilica Barbu and Gabriela Bahrim

Department Food Science, Engineering, Applied Biotechnology and Aquaculture, Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Galati, Romania

Department of Pharmaceutical Sciences, Faculty of Medicine and Pharmacy, Dunarea de Jos University of Galati, Galati, Romania

email address: [email protected]

Today, biologically active compounds are amongst the most important natural compounds that possess several beneficial activities. The aim of this study was to determine and to optimize the ability of some Lactobacillus probiotic strains to develop on a minimal fermentative medium, enriched with Aloe vera cryodessicated powder, as the active ingredient, a mixture that was further incorporated into a pure bioactive pharmaceutical ointment base. The ointment mixture was studied with modern qualitative and quantitative techniques of analysis like FTIR and HPLC. The experimental ointment samples were tested to assess their adhesive capacity to an epithelial cells line and to evaluate their antimicrobial activity against clinical pathogens that usually colonize the skin’s surface. The proposed functional pharmaceutical product could be used for several skin disorders such as atopic dermatitis, minor injuries, uncomplicated first or second degree burns, psoriasis or eczema. The formulation of an innovative ointment using probiotic lactic bacteria and biologically active plant compounds opens up new research perspectives and offers the possibility of diversifying the topic use pharmaceuticals.

Funding: This research was supported by the project "EXPERT", Contract no. 14PFE/17.10.2018 and by the project "ANTREPRENORDOC", Contract 431 no. 36355/23.05.2019, financed by The Human Capital Operational Programme 2014–2020 (POCU), Romania.

Microencapsulated powders from black rice with high nutritional functionality

Carmen Alina Bolea, Elena Enachi, Gabriela Bahrim and Nicoleta Stănciuc

Department Food Science, Engineering, Applied Biotechnology and Aquaculture, Dunarea de Jos University of Galati, Galati, Romania

email address: [email protected]

The undertaken study aimed to obtain a functional powder composed of the biologically active compounds from black rice extract and probiotic microorganisms. Whey and inulin proteins were used to encapsulate the black rice flour’s compounds, the micro-encapsulation being achieved by cryodesiccation. Prior to the microencapsulation step, the black rice flour’s extract was characterized in terms of the total anthocyanins content, the total flavonoids content, the total polyphenols content and the antioxidant activity. In addition, the aforementioned determinations and the encapsulation efficiency were also assessed for the obtained powder and additionally the influence on the growth and viability of the probiotic Lactobacillus acidophilus strain was also highlighted. In order to evaluate the stability of the anthocyanins from black rice flour, the microencapsulated powder was analyzed to evaluate whether the present bioactives were still stable for a period of one month. Moreover, the in vitro release profile of anthocyanins in the simulated gastric and intestinal juices was also studied. All the results demonstrated that the microencapsulated powder has the potential to be used as a functional food ingredient, not only because of its health benefits but also for its prebiotic effect.

Funding: This work was supported by the project "EXPERT", Contract no. 14PFE/17.10.2018 and by the project "ANTREPRENORDOC", Contract 431 no. 36355/23.05.2019, financed by Programme 2014-2020 (POCU), Romania.

Extraction of lycopene from tomato peels with supercritical carbon dioxide and the antioxidant effect

Ionica Dima (gheonea), Iuliana Aprodu, Liliana Mihalcea, Gabriela Râpeanu, and Nicoleta Stănciuc

Department of Food Science, Food Engineering, Biotechnology and Aquaculture, Faculty of Food Science and Engineering, “Dunarea de Jos” University of Galati, Galati, Romania

email address: [email protected]

In the food industry, lycopene has several important applications as a natural pigment responsible for the deep-red characteristic colour of tomato fruits. Lycopene possesses significant beneficial effects by protecting the cells against oxidative damage and thereby decreasing the risk of chronic diseases. The study’s objectives were to optimize the supercritical carbon dioxide extraction parameters and to determine the antioxidant activity. The experiments were carried out at pressures and temperatures ranging from 20 to 50 MPa and 343 to 353 K, respectively, without any modification. The CO₂ flow rate was maintained between 2.5 mL/min and 4.5 mL/min. The extracts were analyzed by UV–visible spectroscopy. The best results were obtained under the following extraction parameters: temperature of 343 K, pressure of 40 MPa, CO₂ flow rate of 2.5 mL and 155 min. Only the ABTS radical scavenging activity was found to be significant and highly correlated to the carotenoids content of the tomato peels’ extracts. The ABTS activity was expressed in terms of µg Trolox/mL. The carotenoids bright red colour highlights their use as natural food colourants that are regarded not only as high antioxidant compounds but also as valuable replacers of artificial food dyes hence increasing the products’ functionality.

New applications of styrylpyridinium derivatives as potent antifungal drugs or fluorescence probes

Marie Kodedová , Simona Vaitkienė, Rimantas Daugelavičius  and Hana Sychrová

Institute of Physiology CAS, BIOCEV, Praha, Czech Republic

Department of Biochemistry, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania

email address: [email protected]

The incidence of Candida glabrata infections increases every year due to its higher resistance to commonly used antifungal drugs. Therefore, novel treatments for fungal infections are urgently needed. We characterized the mechanism of action of eight styrylpyridinium derivatives on C. glabrata cells. These drugs caused dose-dependent damage of the plasma membrane and were transported by Cdr pumps out of the cells. Some derivatives exhibited strong synergistic effects with calcineurin inhibitor FK506 or terbinafine, which blocks synthesis of ergosterol. Not only antifungal, but also spectroscopic properties of styrylpyridinium compounds were dependent on their structure. We measured absorption and fluorescence spectra of styrylpyridinium derivatives and determined their localization in yeast cells. The tested styrylpyridines, depending on their molecular structure, can be used as new fluorescence vital dyes or vacuolar membrane probes, because they are able to accumulate in dead cells or tonoplasts. Moreover, their fluorescence characteristics differ from the properties of current commercial vacuolar markers and allow multi-colour staining. In summary, styrylpyridinium compounds may find their applications as new antifungal drugs or fluorescence dyes in yeast research.

Acknowledgment: We thank Prof. Duburs for kindly providing styrylpyridinium compounds and Prof. Sanglard for C. glabrata strains.

Funding: This work was supported by LQ1604 NPUII provided by MEYS, CZ.1.05/1.1.00/02.0109 BIOCEV provided by ERDF and MEYS, and also by the Research Council of Lithuania (No. 09.3.-LMT-K-712-02-0200).

Investigations on the use of microencapsulated anthocyanins from eggplant peel as functional ingredient for pastry products

Georgiana Horincar, Gabriela Râpeanu and Iuliana Aprodu

Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Galati, Romania

email address: [email protected]

This study aimed the microencapsulation of anthocyanin-rich extract from eggplant peel, followed by incorporation of the purple coloured powder in the pastry cream as functional ingredient. The matrix used for anthocyanins encapsulation consisted of whey proteins and pectin, and the encapsulation efficiency was 94%. The microencapsulated anthocyanin extract (MAE) and the pastry cream enriched with MAE were further characterized in terms of phytochemicals content and antioxidant properties. Total monomeric anthocyanins (TMA), total phenolic content (TPC), total flavonoid content (TFC) and radical scavenging activity were determined. The addition of different levels of MAE to the pastry cream resulted in the linear increase of the phytochemicals content: TMA increased from 0 to 0.136 mg delphinidin-3-glucoside/g DW, TFC from 0.113 to 0.345 mg catechin equivalents/g DW and TPC from 0.949 to 7.473 mg gallic acid equivalents/g DW. Moreover, when compared to the control sample, the 32 times increase of the antioxidant activity was registered for the pastry cream supplemented with 10% MAE (2.010 mM Trolox equivalents/g DW). These results suggest that a promising use of microencapsulated eggplant extract as functional ingredient for pastry cream.

Valorization of fat pork and okara through SSF fermentation with Yarrowia lipolytica and Lactobacillus paracesei by using the Plackett–Burman design

Elena Enachi, Mihaela Cotârleţ and Gabriela Bahrim

Department Food Science, Engineering, Applied Biotechnology and Aquaculture, Faculty of Food Science and Engineering, Dunarea de Jos University, Galati, Romania

Cross-Border Faculty, Dunarea de Jos University, Galati, Romania

email address: [email protected]

This study reports the identification of the biotechnological parameters that influence the semi-solid fermentation of food by-products (okara and pork fat) with a mixed culture of Yarrowia lipolytica MIUG D5 and Lactobacillus paracasei MIUG BL21 in order to obtain a tribiotic product with applications in food industry. The studied independent variables such as the concentrations of the okara, pork fat, glycerol, yeast and lactic acid bacteria inoculums and the fermentation time were analyzed. The Response Surface Methodology and Plackett–Burman experimental design (PB) were employed to design the experimental matrix by using the Design Expert software (Version 7.0, Stat-Ease Inc., MN, USA). The antioxidant activity (DPPH method) and the antimicrobial activity (as the growth inhibition percentage) were undertaken as the analyzed responses. The okara and pork fat fermented product showed an antioxidant potential between 6.77 and 17.78 mM TE/g, and also an antimicrobial activity against Aspergillus niger (24–64 mm).

Funding: This research was supported by the project "EXPERT", Contract no. 14PFE/17.10.2018 and by a grant of the Romanian Ministry of Research and Innovation, CCCDI-UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0569-PRO-SPER (10PCCI), within PNCDI programme.

Some biotechnological aspects of Prunus persica hybrid forms obtaining

Irina Mitrofanova, Nina Lesnikova Sedoshenko, Olga Mitrofanova, Irina Zhdanova and Anatoliy Smykov

Department of Plant Developmental Biology, Biotechnology and Biosafety, FSFIS The Nikita Botanical Gardens - National Scientific Center of the RAS, Yalta, Russia

email address: [email protected]

Peach is one of the most valuable stone fruit crops in the world. New peach forms and cultivars tolerant to viruses have the possibility to be created by integrated usage of breeding, biotechnological and virological methods. Crosses were performed based on the results of virological analysis. By using embryoculture method the hybrid embryos ‘Progress’ × ‘Persey’ and ‘Babygold 7’ × ‘Persey’ were isolated and introduced to in vitro conditions. Vigorous seedlings from mature embryos were obtained on hormone-free Monnier and MS culture media after preliminary stratification at a temperature of 4 ± 1 °C without light. The ‘Progress’ × ‘Persey’ embryos demonstrated a high regeneration frequency, up to 78.6%–90.4%. The germination rate of immature embryos at the heart and torpedo stages was 8.4%–19.9% on Monnier and MS culture media with 0.4 mg/L kinetin and 0.1 mg/L GA₃. In the treatments without stratification, an abnormal seedlings was formatted, which characterized by the death of the apical part and the underdevelopment of the roots. The use of microcutting made it possible to divide such seedlings and place them on B5 medium for the induction of direct organogenesis and further micropropagation.

Funding: This study was funded by a research grant no. 19-16-00091 of the Russian Science Foundation.

In vitro regeneration of the rare plant Aconitum lasiostomum Rchnb. ex Besser

Olga Mitrofanova, Irina Mitrofanova, Nina Lesnikova Sedoshenko, Svetlana Chelombit and Marina Rudenko

Department of Plant Developmental Biology, Biotechnology and Biosafety, FSFIS The Nikita Botanical Gardens - National Scientific Center of the RAS, Yalta, Russia

email address: [email protected]

Aconitum lasiostomum Rchnb. ex Besser (Ranunculaceae) is a rare species, which is included in the Red Book of the Russian Federation and regional Red Books of some Eastern European countries. Aconitine, extracted from roots of various Aconitum species, is used to treat a wide range of diseases. Our previous studies showed that A. lasiostomum inflorescences, leaves and fruits contain high concentrations of phenolic substances with great variety of the components. In vitro morphogenetic capacity of aconite was realized via direct regeneration on MS culture medium with 0.1–0.25 mg/L BAP, 0.1 mg/L IBA and 0.1 mg/l GA₃. Further subcultures of explants on MS medium with 1.3 mg/L TDZ, the increase of the rates of adventitious buds formation and microshoot regeneration was promoted. The number of microshoots formed was up to 10–14 per explant. On MS medium with 1.1–2.5 mg/L 2,4-D the morphogenic callus was formed. Thus, the use of biotechnological methods let us to study the regeneration capacity in A. lasiostomum in order to further plant propagation and preservation in vitro, in situ and ex situ.

Funding: This study was funded by ST No. 0829-2019-0038 FSFIS “NBG-NSC” and done on the base of the Unique Scientific Installation "PHYTOBIOGEN".

Antibacterial activity of aqueous solutions of essential oils as an indicator for their content

Nelly Georgieva , Nevena Lazarova Zdravkova, Yoana Todorova, Denitsa Tsanova and Dimitar Peshev

Department of Biotechnology, University of Chemical Technology and Metallurgy, Sofia, Bulgaria

Department of Chemical Engineering, University of Chemical Technology and Metallurgy, Sofia, Bulgaria

email address: [email protected]

According to market forecasts, annual consumption of essential oils on the world market is growing steadily, reaching 403 thousand tonnes in 2025. The steam distillation of the plant materials to produce essential oils generates vast amounts of aqueous fractions as by-products. Certain amounts of essential oils remain dissolved in these fractions, predetermining their biological activity. If discarded into the environment they have a negative impact on the ecological balance. At the same time, these aqueous fractions are an attractive source of high value-added substances if subjected to valorization. A preliminary appraisal of their quality is required prior to both disposal and valorization. In the present work, antibacterial activity is suggested as an indicator of the content of essential oils in the analyzed products, as far as they are known for their bactericidal potential. To prove the concept, antibacterial activity of pure essential oils dissolved in water in different concentrations was determined using Gram-negative Escherichia coli K12 and Gram-positive Bacillus subtilis 356 bacterial strains. The minimum inhibitory concentrations of essential oils and antibacterial activity using the agar-diffusion method were determined.

Funding: This work was supported by the Bulgarian National Science Fund (contract KP-06-H37/14).

Characterization of Romanian honeys according to their physicochemical parameters and FTIR spectroscopy

Giorgiana Valentina Blaga(costea) , Elena Enachi, Andreea Veronica Dediu, Rodica Mihaela Dinică, Vasilica Barbu and Camelia Vizireanu

Faculty of Food Science and Engineering, “Dunărea de Jos” University, Galati, Romania

Faculty of Science and Environment, “Dunărea de Jos” University, Galati, Romania

email address: [email protected]

The aim of this study was to characterize monofloral and multifloral honey samples through various physicochemical parameters. For this purpose, eighteen honey samples were collected directly from beekeepers in 2015 from different Romanian regions and with different botanical origins. Microscopic analysis of the pollen in the honey was used to determine its botanical origin. Physicochemical analysis included the determination of pH, electrical conductivity, moisture, ash and colour parameters: L*, a*, b*. The FTIR-ATR analysis of the honey samples allowed the identification of several bands characteristic to different organic compounds functional groups present in these samples. The obtained results showed considerable differences in the physicochemical parameters, between honeydew and blossom honeys. Concerning the physicochemical parameters, all the samples met the EU Council Directive 2001/110 for all the parameters.

Funding: The authors are grateful for the technical support offered by Center MoRAS developed through Grant POSCCE ID 1815, cod SMIS 48745 (www.moras.ugal.ro).

Genetic polymorphism of glutenin subunits with high-molecular weight in grain Triticum aestivum L., Triticum spelta L. and Triticum durum DESF

Milan Chňapek, Želmira Balážová, Andrea Špaleková, Dana Rajnincová, Janka Drábekováand Zdenka Gálová

Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

Department of Mathematics, Faculty of Economics and Management, Slovak University of Agriculture, Nitra, Slovakia

email address: [email protected]

The collection of 45 genotypes of bread wheat (Triticum aestivum L.), 33 genotypes of spelt wheat (Triticum spelta L.) and 18 genotypes of durum wheat (Triticum durum DESF.) were analyzed by SDS-PAGE. Variability of electrophoretic spectra of storage proteins was monitored and based on this a dendrogram of relationships was prepared. Bread wheat genotypes were found to be homogenous and nine electrophoretical profiles were detected. Bread wheat genotype with component composition of HMW-GS 0, 7 + 9, 5 + 10 was the most frequent (78%) of all genotypes. The genotypes Magvas, SK 76, Favorit and Astela revealed the highest Glu-score value (9). Secalin block – Gld 1B3, a marker for resistance against wheat rust and also a marker for worse technological quality was observed in gliadin spectra of 12 classified bread wheat genotypes. Genotypes of spelt wheat were characterized as a multiline and 43 different individual HMW-GS were identified. The highest Glu-score value (8) achieved 10 genotypes. However, evaluated durum wheat genotypes had lower Glu-score value (2) than other wheat cultivars, which confirmed their applicability for pasta production. We have constructed dendrogram of relationships on the basis of the storage proteins, which divided collection of bread wheat into two clasters and four subclasters.

Funding: This work was co-funded by European Community under project no. 26220220180: Building Research Centre “AgroBioTech", by grant VEGA No.1/0246/18 and KEGA project No. 025SPU-4/2018.

Laboratory-based high-resolution X-ray tomography for three-dimensional visualizing the natural diatoms

Qiong Li, Jürgen Gluch and Ehrenfried Zschech

Fraunhofer Institute for Ceramic Technologies and Systems IKTS, Dresden, Germany

email address: [email protected]

Biomaterials, with natural features of hierarchical organization and adaptability, inspire the design of more reliable, efficient, biocompatible and environmentally friendly materials. Therefore, it is very important to understand the hierarchical structure of these natural biomaterials. Here, an advanced technique of laboratory-based high-resolution transmission X-ray microscopy (TXM) is used to investigate the whole diatoms without prior staining. The major advantage of this technique for biological specimens is providing three-dimensional (3D) information without extensive sample preparation procedures. Diatom frustules and natural diatoms were nondestructively imaged by TXM. The detailed 3D structural information of diatoms imaged non-invasively demonstrates the excellent capabilities to study complicated hierarchical biological structures by TXM. Furthermore, a sample holder set-up is described for the TXM system to visualize the diatoms three dimensionally in quasi-living state allowing further promising studies of living species.

Evaluating the diversity and cellulolytic potential of Black Sea shipworm (Teredo navalis) gill symbionts

Irina Lascu, Maria Preda, Ioana Mereuță, Ileana Stoica, Iulia Chiciudean and Ana Maria Tănase

Department of Genetics, Faculty of Biology, University of Bucharest, Bucharest, Romania

email address: [email protected]

In order for biocatalytic cellulose depolymerization to gain footing in the biofuel and biosynthesis industries, the cellulolytic enzymes used must be efficient enough to make these processes economically viable. Such enzymes are likely to be produced by microorganisms from specialized environments, in this case, tissues of the xylotrophic marine bivalve Teredo navalis. Symbiotic bacteria located in the shipworms’ gills secrete various cellulases, enabling the digestion of ingested wood particles. In this study, gill tissue from T. navalis specimens collected from the Romanian shore of the Black Sea was used for a cellulose-enriched culture, total DNA extraction and pure strain isolation. The shipworm specimens’ phylogeny/phylogeography was investigated via cytochrome-oxidase subunit I (cox1) gene sequence. DNA metabarcoding of the cellulose-enriched culture gave a detailed inventory of symbiont diversity. Pure isolates were tested for their ability to produce endoglucanase (EC 3.2.1.4) through Congo Red staining and DNS assays, with enzyme activity tested at various temperatures. Cellulase-producing strains were taxonomically classified by 16S rRNA gene sequencing. Our results have great potential to be employed in biorefinery applications.

Funding: This work was supported by the Romanian National Authority for Scientific Research and Innovation, CCCDI – UEFISCDI, project number 1/2018, ERANET-Marine Biotechnology METAMINE, within PNCDI III.

Variation of total phenolic content, organic acids, antiradical and antimicrobial activity in callus cultures of highbush blueberry (Vaccinium corymbosum)

Dora Livkiša , Anna Ramata Stunda, Valda Valkovska, Mārtiņš Borodušķis, Elza Kaktiņa, Baiba Silamiķele, Anete Borodušķe and Nils Rostoks

Faculty of Biology, University of Latvia, Riga, Latvia

Faculty of Chemistry, University of Latvia, Riga, Latvia

email address: [email protected]

Known for its antioxidant activity, blueberry (Vaccinium corymbosum) is becoming more popular for consumption and various applications in medicine and nutrition. Tissue cultures, genetic engineering and other biotechnological solutions allow to overcome the availability limitations set by plant seasonality. In this study, we looked at the differences in total phenolic content (TPC), antiradical activity (ARA), antimicrobial activity and quantity of selected organic acids in leaves of field-grown plants and in vitro callus cultures grown on Murashige-Skoog or Woody plant (WP) media supplemented with varying concentrations and combinations of growth regulators. TPC varied from 83 mg/g dry weight (DW) to 142 mg/g DW in leaves of ‘Bluecrop’ and ‘Duke’, respectively, and correlated with ARA which was higher in ‘Duke’. In callus cultures, the highest ARA and the highest TPC of 94 mg/g DW was observed in ‘Bluecrop’ grown on WP medium with 2,4-dichlorophenoxyacetic acid. High levels of chlorogenic and quinic acids were found in the leaves of all tested varieties, while callus cultures exhibited relative increase of malic acid and contained oxalic acid. Both leaves and callus cultures showed antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis. MIC and MBC values correlated with organic acid contents and TPC.

Aspects regarding the influence of some hormonal parameters in different physiological stages in ewes

Simona Călin (Nicolae), Iuliana Codreanu, Ramiro Simion and Mario Darius Codreanu

Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine Bucharest, Bucharest, Romania

email address: [email protected]

The aim of this research is to highlight the influence of the physiological status on some hormonal parameters (T3, T4 and prolactin) in ewes and the importance of correcting these parameters in order to reach the maximum productive potential. The hormones were dosed 5 days antepartum, at the parturition day, 5 and 10 days post-parturition. The T4 blood concentration (32.7 ± 2.01 ng/mL) decreased statistically significant at parturition (p < 0.05) compared to 5 days antepartum (63.9 ± 2.81 ng/mL). The T3 mean values were higher during lactation compared to late gestation, but without statistically significant differences (p > 0.05). The important variations observed in these two hormones indicate an energetic imbalance in early lactation, an increase of their levels being considered, an indicator for tissue protein catabolism as they stimulate oxygen consumption, protein metabolism and milk production. At parturition, the prolactin blood concentration increased very significantly (p < 0.01) reaching a peak (439 ± 72.3 ng/mL) and gradually decreasing 5 and 10 days post-parturition. The increase of the prolactin concentration mean value is associated with the imminent approach of parturition, when the mammary gland is in full lactogenesis process and with the development of the maternal behaviour through its action on the central nervous system.

Synthesis and investigations on new conjugates of modified BIM-23052

Dancho Danalev , Ivan Iliev, Tatyana Dzimbova, Tamara Pajpanova, Inna Sulikovska, Zdravka Zaharieva , Ivo Grabchev  and Emilia Naydenova

University of Chemical Technology and Metallurgy, Sofia, Bulgaria

Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad., Sofia, Bulgaria

South-West University “Neofit Rilski”, Blagoevgrad, Bulgaria

Institute of Molecular Biology “Acad. Roumen Tsanev”, Bulgarian Academy of Sciences, Bulgaria

Testing center Global Test Ltd., Sofia, Bulgaria

 Department of Chemistry and Biochemistry, Physiology, and Pathophysiology, Sofia University "St. Kliment Ohridski", Sofia, Bulgaria

email address: [email protected]

Recently, a new approach for drug design is used for combining two or more pharmacological effects of different subunits in a single ligand. Bivalent ligand approach would maintain the strength of the multidrug therapy (synergistic effect, lower doses and weaker side effects) and overcome the weakness of a co-administration. Herein, we report design, synthesis and some investigations on the modified with halogenated amino acids BIM-23052 analogues [1], which are combined with second pharmacophore. The latter is molecule with proven anti-tumour properties. The target molecules are synthesized by standard SPPS, Fmoc-strategy. In addition, in vitro 3T3 Neutral Red Uptake (NRU) Cytotoxicity assay are done. Mean IC50 values for newly synthesized hybrid structure are obtained. The same peptide conjugates were tested on a panel of human and mouse tumour cell lines (MCF-7, MDA-MB-231 and EAC) for antitumour activity. The selectivity index is calculated. The anticancer activity, some modelling investigations and hydrolytic stability will be also discussed.

Funding: The work was supported by National Science Fund, Ministry of Education and Science, Bulgaria; Project: DN19/17, 20.12.2017.

Purification, applications and cross-reactivity of polyclonal antibodies to fungal luciferase

Andrey Yurievich Gorokhovatsky, Tatyana Vladimirovna Chepurnykh, Ekaterina Sergeevna Shakhova, Vladislav Denisovich Soloviev, Konstantin Victorovich Purtov  and Ilia Victorovich Yampolsky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, Russia

National Research Nuclear University MEPhI, Moscow, Russia

email address: [email protected]

The recently discovered luciferase from the fungus Neonathopanus nambi, nnLuz belongs to the new family of light-emitting enzymes utilizing 3-hydroxyhispidin as a substrate of bioluminescent reaction. Besides nnLuz, eight homologous luciferases from various species of fungi are known to date. nnLuz was successfully expressed in bacteria, yeast, plant and mammalian cells making it a promising reporter in a growing field of biotechnological applications. Here, we report the purification, characterization and some applications of rabbit polyclonal antibodies to nnLuz. Recombinant nnLuz purified after expression in Escherichia coli cells was used for the immunization of animals. Specific antibodies were purified from immune serum by affinity chromatography using nnLuz covalently linked to agarose beads. For the immunosorbent preparation, nnLuz was purified after expression in Pichia pastoris cells. We demonstrated successful use of antibodies to nnLuz in immunocytochemistry (ICC) and western-blot (WB) applications. It was shown that the antibodies display a broad cross-reactivity towards other fungal luciferases allowing their specific detection.

Funding: This work was supported by Russian Science Foundation grant 16-14-00052P.

Russian-manufactured eptacog alfa activated immunogenicity assessment during inhibitor-associated hemophilia treatment

Dmitry Kudlay , Alexander Shuster, Anton Borozinets, Mariya Zhilyaeva  and Alexander Kazarov

JSC GENERIUM, Moscow, Russia

LLC IBC Generium, Volginsky, Russia

email address: [email protected]

Twenty hemophilia A patients with inhibitors were enrolled in two-stage study to evaluate Russian-manufactured eptacog alfa-activated immunogenicity. Binding ADA were revealed through bridge-ELISA approach, with immobilized eptacog alfa activated as capturing agent and biotin-linked eptacog alfa activated as detecting reagent. To assess ADA neutralizing effect against eptacog alfa activity, clotting and chromogenic assays were consecutively applied. Two patients were specified as ADA-positive at the 1st study stage, both titered as 1:40. Being resampled at the 2nd stage, only one of them remained ADA-positive, with titer reduced to 1:10. Meanwhile, another patient, previously being ADA-negative, produced ADA titered as 1:10 after occasional plasma-derived FIX treatment. Mild neutralizing effect of ADA was affirmed through chromogenic assay for both ADA-positive patients at the 2nd study stage. The use of chromogenic, instead of clotting, assay, along with sampling of blood serum instead of plasma, supported more sensitive and precise ADA assessment. Neither FVII level decrease, nor loss of treatment efficacy associated with ADA onset was indicated during whole study, suggesting no apparent clinical effect of revealed low-titer ADA to eptacog alfa activated.

Special features of in vitro culture of valuable Lavandula angustifolia Mill. cultivars

Irina Zdanova, Valentina Brailko and Irina Mitrofanova

Department of Plant Developmental Biology, Biotechnology and Biosafety, FSFIS The Nikita Botanical Gardens - National Scientific Center of the RAS, Yalta, Russia

email address: [email protected]

The aim of this investigation is the optimization of in vitro micropropagation in selected promising Lavandula angustifolia Mill. cultivars ‘Belyanka’, ‘Prima’, ‘Record’ and ‘Sineva’. These cultivars from the Nikita Botanical Gardens’ aromatic plant collection are linalool and linalyl acetate-linalool type from predominance of linalool, and the average yield of essential oil is 0.8%–2.2%. For biotechnology experiments, the primary explants were taken from in vitro lavender collection. The apical segments of the cultivars were placed on MS medium with 0.7 mg/L BAP or 0.7 mg/L kinetin, 0.25 mg/L NAA at 25 ± 1 °C, 16-h photoperiod with light intensity 37.5 μmol/m²/s. For subcultures, MS medium with 0.5 mg/L BAP or 0.5 mg/L kinetin, 0.25 mg/L NAA was used. Microshoots were subcultured every 21 days. Buds development was induced after 6–8 days. All studied cultivars demonstrated high morphogenetic capacity. After each subculture, 1–5 adventitious microshoots per explant were formed. The microshoot height varied 1.0–3.2 cm and 8–25 leaves per microshoot were formed. Chlorophyll fluorescence induction parameters and the content of chlorophyll indicated the normal functional state of the vegetative organs of the microshoots and plantlets.

Funding: This study was funded by a research grant No. 19-76-00023 of the Russian Science Foundation.

Estimation of the efficiency of DNA isolation methods from Rosa damascena Mill. leaves

Valentina Brailko, Irina Mitrofanova, Ilya Bulavin, Oksana Grebennikova, Nina Lesnikova Sedoshenko, Natalia Ivanova and Olga Mitrofanova

Department of Plant Developmental Biology, Biotechnology and Biosafety, FSFIS The Nikita Botanical Gardens - National Scientific Center of the RAS, Yalta, Russia

email address: [email protected]

Genomic investigations of essential oil roses are especially relevant, as there is no information on Rosa damascеna Mill. in the international databases NCBI and GDR. The object of this study was to obtain high-molecular weight DNA from leaves of in vitro propagated essential oil rose plants (‘Raduga’, ‘Tavrida’, ‘Lany’, ‘Kooperatorka’, ‘Festivalnaya’, ‘Dzhalita’, ‘Krymskaya Krasnaya’, ‘Ukraina’, ‘Aura’, ‘Kazanlykskaya’) by using phenol–chloroform extraction and commercial kits (PureLink® Plant Total DNA Purification Kit, GeneJET Plant Genomic DNA Purification Kit) with subsequent DNA analysis on spectrophotometer (NanoDrop-Lite, Thermo) and by capillary electrophoresis (Agilent 4200 TapeStation, Germany). High-molecular weight DNAs (46,512–60,000 bp) were obtained with phenol/chloroform method for the cultivar ‘Festivalnaya’ and the GeneJET Plant Genomic DNA Purification Kit for the cultivars ‘Festivalnaya’, ‘Raduga’, ‘Dzhalita’ and ‘Aura’. The DNA concentration ranged from 12.0 to 148.2 ng/μL, the ratio A260/280 was 1.37–1.54. These isolation methods were more effective compared to the PureLink® Plant Total DNA Purification Kit, the use of which made it possible to detect DNA from 7,800 to 12,251 bp with a concentration of no more than 29.2 ng/μL and the ratio A260/280 from 1.65 to 2.04.

Funding: The study was supported by the ST No. 0829-2019-0038 FSFIS “NBG-NSC”.

Whole-genome sequencing of the rare endemic Heracleum ligusticifolium M.Bieb.: new looking insight to the largest Apiaceae genus

Olga Krivenko, Irina Mitrofanova, Olga Kuleshova, Valentina Brailko, Ilya Bulavin and Olga Mitrofanova

Kurchatov Genomic Center - NBG-NSC, FSFIS The Nikita Botanical Gardens - National Scientific Center of the RAS, Yalta, Russia

email address: [email protected]

Next-generation sequencing technologies allowing to assemble high-quality genomes at a reasonable cost open new prospects and opportunities for different plant investigations. Up-today genome-wide information is available for a rather limited range of model objects and economically valuable plants but the most part of the natural plant molecular diversity remains a mystery. Heracleum is the largest genus of the Apiaceae family with a broad spectrum of phytochemistry and biological activities but without any whole-genome sequencing data which could allow a targeted search for new substances, decipher and analyze the biochemical pathways of their synthesis on a genomic basis. The entire genome of the Crimean rare endemic species H. ligusticifolium cultured in vitro has been sequenced using an Oxford Nanopore MinION and Illumina NextSeq550 instruments with more than ×30 and ×60 covering correspondingly. Hybrid genome assembling is in the process. It is going to be used as a tool to target search and annotation of genes responsible for unique biochemical decisions allowing the species to successfully survive far beyond the optimum ranges of the environmental conditions.

Funding: This study was supported by the grant No. 075-15-2019-1670 of the Ministry of Science and Higher Education of the Russian Federation.

Influence of mediators secreted by mesenchymal stem cells on the sensitivity of bladder cancer cells to ciprofloxacin

Karolina Balik, Małgorzata Maj, Paulina Modrakowska, Karolina Matulewicz and Anna Bajek

Department of Tissue Engineering, The Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Torun, Poland

email address: [email protected]

Despite intensive investigation, influence of adipose-derived stem cells (ASCs) on cancer cells remains unclear. Molecules synthesized by ASCs may be involved in the development of resistance to anticancer agents such as ciprofloxacin. The immortalized human adipose-derived stem cells (ASC52telo) were used to explore the impact of secreted factors on the bladder cell line (HB-CLS-1). For this purpose, we used conditioned media. MTT test was used to measure inhibitory concentration and cell viability. Cell proliferation was assessed using synthetic thymidine analogue that incorporated into DNA of dividing cells. Inhibitory concentrations IC10, IC50 and IC90 of ciprofloxacin against HB-CLS-1 were 145 µM, 347 µM and 1000 µM. ASCS-CM cultures increased HB-CLS-1 proliferation after 48 and 72 h, respectively, by 65% and 46% (<0.05) and viability respectively by 5% and 8% (<0.05) compared to control. Incubation HB-CLS-1 with ASC-CM reduced sensitivity to ciprofloxacin at concentration corresponding to IC50 by 21% (<0.05) and increased at IC10 by 33% (<0.05). Obtained results indicate that the factors synthesized by ASCs interact with cancer cells by increasing their viability and proliferation. Furthermore, it has been shown that ASCS-CM affects cancer cell’s sensitivity to anticancer agents. Our research indicates the need to further deepen the knowledge about the interactions between cancer and stem cells.

Properties of fungal luciferase purified after expression in Pichia pastoris cells

Konstantin Viktorovich Purtov , Andrey Yurievich Gorokhovatsky, Tatyana Vladimirovna Chepurnykh, Juliana Anatolyevna Mokrushina, Margarita Nikolaevna Baranova, Sergey Aleksandrovich Goncharuk  and Ilya Viktorovich Yampolsky

Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, Russia

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, IBCh, Moscow, Russia

email address: [email protected]

Luciferase nnLuz was recently cloned from fungus Neonothopanus nambi. The enzyme utilizes substrate 3-hydroxyhispidin to generate light as a result of bioluminescence reaction. Here, we describe the expression under control of the pGAP promoter of nnLuz fused with C-terminal 6His-tag in Pichia pastoris cells. It was shown that recombinant nnLuz accumulated in the membrane fraction of the cells. Active luciferase was solubilized with mild detergents and further purified using metal-affinity and gel-filtration chromatography resulting in 90% purity, as judged by SDS-PAGE. In the presence of 3-hydroxyhispidin, recombinant nnLuz emits light in a bioluminescence reaction with a glow-type kinetic and a pH optimum around pH 8.0. Bioluminescence spectrum of the purified enzyme has the maximum at 535 nm, identical to that observed upon bioluminescence of N. nambi mycelium. In gel-filtration chromatography, elution volume of detergent-solubilized nnLuz corresponds to 60 kDa protein, indicating possible dimerization of the enzyme. Far-UV circular dichroism spectrum of the nnLuz allowed estimating extent of the secondary structure of the detergent-solubilized recombinant protein.

Funding: This work was supported by Russian Science Foundation grant 16-14-00052P.

The efficiency of the aeration process as the main energy consumer in a wastewater treatment plant

Ramona Ionela Zgavarogea, Andreea Maria Iordache, Mihaela Iordache and Roxana Elena Ionete

National Research and Development Institute for Cryogenics and Isotopes Technologies - ICSI Ramnicu Valcea, Valcea, Romania

email address: [email protected]

Aeration is the most energy-intensive operation in wastewater treatment, amounting to 40%–80% of plant energy costs required for an efficient treatment of wastewater. This work refers to the experimental testing of a porous elastic membrane aeration device to be used in wastewater treatment plants. Therefore, a testing stand was done to analyze the elastic membrane diffusers fixed on the base of the aeration basin. For a more accurate approximation of the airflow introduced into the basin, diaphragms with different sizes were individually attached to the blower discharge pipe, namely for the first experiments a standardized diaphragm with a diameter of 39.31 mm was used, while a diaphragm with lower diameter, of 17.46 mm, was set for the second round of experiments. The most representative parameter that characterize a pneumatic aeration device is the specific oxygenation capacity, for which the following values emerged: between 14.5 and 24.1 g O₂/m³ air, using the exponential method, and between 13.6 and 24.7 g O₂/m³ air, using the natural logarithm method from oxygen deficiency. A minimum efficiency of 15% was obtained. Thus, our study tested and evaluated the efficiency of the aeration solution with elastic membranes, showing its feasibility for application.

Development of a new form of the medicine Roseofungin-AC® for the treatment of vaginal candidiasis

Baiken Baimakhanova, Zere Turlybayeva, Asya Balgimbaeva, Gaukhar Tugelbai, Lyudmila Trenozhnikova, Liliya Ibragimova and Amankeldy Sadanov

Limited Liability Company “Research and Production Center for Microbiology and Virology", Almaty, Kazakhstan

email address: [email protected]

The pharmaceutical development of the compositions for a new form of the medicine Roseofungin-AC® in accordance with the Kazakhstan State Pharmacopoeia was carried out. Based on the matrix method, 70 compositions with active substance were calculated. For the development of the Roseofungin-AC® suppository, the 2% concentration of the active pharmaceutical substance, providing a stably high growth suppression of 15 clinical Candida strains (C. albicans R-11, R-25, R-28, R-29, R-33, R-41, R-44, R-46, R-50; C. krusei R-19, R-47; C. tropicalis R-5. R-39; C. glabrata R-17; C. parapsilosis R-14) was selected. The compositions nos. 2, 11, 45 that included the drug substance Roseofungin, polyethylene glycols 400 and 1500, vitepsol, solid confectionery fat and dimethyl sulfoxide as a solvent showed the highest activity against all clinical Candida strains, providing 19–25 mm zones of Candida growth inhibition. The composition no. 2 showed the highest activity with inhibition zones of 24–25 mm equal to the control Roseofungin 2% solution. This composition was selected for the new drug form of Roseofungin-AC® as a suppository.

Funding: The work was carried out within the framework of the MES RK grant project AR05131866 "Development of a new form of the Roseofungin-AS preparation for the treatment of vaginal candidiasis".

Obtaining a new medicinal preparation for the treatment of vaginal candidiasis

Amankeldy Sadanov, Zere Turlybayeva, Asya Balgimbaeva, Gaukhar Tugelbai, Baiken Baimakhanova, Lyudmila Trenozhnikova, Liliya Ibragimova and Aliya Kalbayeva

Limited Liability Company “Research and Production Center for Microbiology and Virology", Almaty, Kazakhstan

email address: [email protected]

The object of research is antibiotic roseofungin produced by strain Streptomyces roseoflavus var. roseofungini AS-20.14. As a result of research, a collection of test-microorganisms of clinical Candida fungal strains was created on the basis of 15 isolates from patients with vaginal candidiasis from Almaty region. Fungi clinical strains of Candida genus after identification were assigned to five species: Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, Candida parapsilosis. Accumulation of medicinal substance ‘Roseofungin’ in an amount of 500 g was carried out. All parameters of obtained medicinal substance correspond to Analytical Normative Document. ‘Roseofungin’ is stored in a dry, dark place at a temperature of 2–8 °C. The high antifungal activity of ‘Roseofungin’ to clinical strains of vaginal candidiasis pathogens has been established, the minimum inhibitory concentration is in the range of 1.66–2.5 μg/mL. The essence of the novelty of the results of work lies in the development of a new medicinal form of medical preparation for the treatment of vaginal candidiasis based on the original polyene antibiotic roseofungin. The results will create the necessary basis for the development of new antifungal medicinal preparations that can improve the condition of patients in Kazakhstan and beyond.

Novel photoactivatable fluorescent proteins from the salp Thalia democtratica

Tatyana Vladimirovna Chepurnykh , Nikolai Nikolaevich Sluchanko, Andrey Yurievich Gorokhovatsky, Milena Aleksandrovna Dobronos and Ilia Viktorovich Yampolsky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

email address: [email protected]

Thalia democratica is a planktonic tunicate of Salpidae family forming long, stringy colonies in warm seawater. T. democratica possesses both bioluminescence (a glow in the dark) and fluorescence (under sunlight). Here we report cloning, expression, purification and characterization of two novel fluorescent proteins from T. democratica. Analysis of transcriptome data revealed two ORFs (ThFP-1 and ThFP-2) with high homology to GFP-like protein family. Each of the proteins was cloned and purified after being expressed in Escherichia coli cells. Absorption spectra of purified proteins showed maxima at 400 and 488 nm for ThFP-1, and at 337 and 510 mm for ThFP-2. Fluorescence emission maxima were 512 nm for ThFP-1 and 527 nm for ThFP-2. Illumination with violet-blue light resulted in a reversible significant increase of long-wavelength bands of the absorption spectra of the proteins. The effect of photoactivation could be repeated multiple times. Gel-filtration chromatography of both proteins showed concentration-dependent oligomeric state transitions.

Funding: This work was supported by the President grant for leading scientific schools LS-2605.2020.4.

Mutagenesis of plant type III polyketide synthases and 4-coumarate-CoA ligases

Kseniia Palkina , Nadezhda Markina, Karen Sarkisyan and Ilya Yampolsky

Planta LLC, Moscow, Russia

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russian Federation

email address: [email protected]

Plant type III polyketide synthases (PKS) with additional enzyme 4-coumaroyl:CoA ligase (4CL) produce structurally diverse bioactive polyketides. One of them is hispidin – anti-inflammatory, antioxidant substance and also the precursor of fungal luciferin. We have chosen mutagenesis as a common way to increase enzyme activity. Hispidin biosynthesis is the important step of the fungal bioluminescence and its optimization can broaden the range of its applications as a very useful reporting biotechnological tool. We have created several mutant variants of PKS from Hydrangea macrophylla and two isoforms of 4CL from Arabidopsis thaliana for expression in yeast and mammalian cells. Moreover, we have demonstrated their functionality in heterologous hosts as a stage of fungal bioluminescence pathway. We are planning to identify the best mutant pair of PKS and 4CL and choose it for our future work in yeast and mammalian cells.

Funding: Work is supported by RFBR grant 18-29-08049.

Enhancement of phenolic acids content of grape pomace as a consequence of treatment with Ganoderma resinaceum

Gordana Šelo, Mirela Planinić, Marina Tišma, Josipa Grgić, Srećko Tomas and Ana Bucić Kojić

Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia

email address: [email protected]

The genus Ganoderma belongs to the Basydiomycota division, family Ganodermataceae. Fungi of Ganoderma genus are known for their healing properties and prevention of various diseases in humans. They have been used in traditional Asian medicine since ancient times. Ganoderma resinaceum is white rot fungus, which can degrade plant cell wall by performing selective delignification or simultaneous rot. The aim of this work was to enhance the extractability of phenolic acids from grape pomace (GP) after solid-state treatment with G. resinaceum for 15 days. Extraction of phenolic acids from GP was performed with 50% ethanol. Ten phenolic acids were analyzed by UHPLC. Biological treatment had a positive effect on production of seven phenolic acids. Initial content of phenolic acids such as gallic acid (GA), protocatechuic acid (PHA), syringic acid (SA), o-coumaric acid (o-CuA), p-hydroxybenzoic acid (p-HBA), p-coumaric acid (p-CuA) and vanillic acid (VA) in the untreated GP were 0.200 mg/gdb, 0.134 mg/gdb, 0.079 mg/gdb, 0.007 mg/gdb, 0.005 mg/gdb, 0.005 mg/gdb, 0.034 mg/gdb, respectively. After biological treatment, the content was higher up to 1.59-, 2.03-, 1.25-, 1.15-, 1.25-, 2.09- and 1.21-fold for GA, PHA, SA, o-CuA, p-HBA, p-CuA and VA, respectively.

Gene expression and RNAi-mediated knockdown of the chitin synthase from leaf-cutting ant

Dulce Helena Ferreira Souza, Ariele Cristina Moreira, Renato Lajarim Carneiro, Mariana Francisco Fracola, Kelli Cristina Micocci  and Odair Correa Bueno

Chemistry Departament, UFSCar, São Carlos, Brazil

Center for Social Insect Studies, UNESP, Rio Claro, Brazil

email address: [email protected]

Chitin synthase (CHS) is the only enzyme specifically associated with chitin synthesis and it has been described as the key enzyme for this mechanism. Here two CHS transcripts from the leaf-cutting ant Atta sexdens were identified. RT-qPCR revealed that expression of CHS transcripts (AsCHS-A) was highest in pupae, followed by larvae and workers. Two splice variants of AsCHS-A (AsCHS-A1 and AsCHS-A2), probably under regulation of different promoters, were identified in all developmental stages. Both transcripts are clustered within class A insect CHSs, generally related to chitin synthesis in epidermis and in ectodermal cells. AsCHS-A1 shares the same expression pattern as total transcripts (AsCHS-A) and represents most of the CHS transcripts in larvae and pupae, whereas AsCHS-A2 is the main CHS transcript in workers. AsCHS-A1 has topology expected for insect CHSs, while the predicted AsCHS-A2 topology, with a missing A domain, is similar to some fungal CHSs. RNAi-mediated gene silencing in pupae revealed that low reduction in CHS transcript levels (18%) was enough to cause morphological changes in the pupa exoskeleton and impair the process of cuticle sclerotization. To our knowledge, this work was the first to show the feasibility of using RNA interference techniques on leaf-cutting ants.

Cloning, expression and characterization of a chitinase gene from leaf-cutting ant Atta sexdens

Kelli Cristina Micocci, Amanda Dutra Sanchez, Jéssica Lincoln Pettinatti, Melissa Tomaz Soares, Bruna Soares Dionizio, Ariele Cristina Moreira, Dulce Helena Souza  and Odair Correa Bueno

Center for the Study of Social Insects, UNESP, Rio Claro, Brazil

Department of Chemistry, UFSCar, São Carlos, Brazil

Department of Physics, Chemistry and Mathematics, UFSCar, Sorocaba, Brazil

email address: [email protected]

Chitin, a polymer formed by monomers of β-1,4-N-acetylglucosamine, is degraded by enzymes called chitinases which are expressed in a wide variety of organisms including bacteria, fungi, insects, plants and animals. Chitinases have different functions, such as nutrition, morphogenesis and defense against pathogens; in addition, they are widely used to biocontrol in bioinsecticides. Eight groups of insect chitinases are described and classified according to the presence/absence of catalytic site and auxiliary domains on soluble substrates, the CBM (carbohydrate-binding module). Two chitinase DNA fragments from leaf-cutting ant Atta sexdens (Chit3.1 and Chit3.2) were amplified by polymerase chain reaction (PCR). Chit3.1 and Chit3.2 cDNA were cloned in the pPICZαA vector for expression in Pichia pastoris. Chit3.1 (a peptide containing one catalytic and one CBM domain) was secreted to the extracellular medium, in its soluble and functional form, being purified by nickel column affinity chromatography. Characterization tests showed that the Chit3.1 enzyme has maximum activity at pH 5.0 and 55 °C, when tested against chitin substrate. Insecticide capacity of the chitinases has been initialized using the Spodoptera frugiperda insect (in vivo) as experimental model. The anti-fungal capacity of chitinases will be performed against Aspergillus fumigatus, Cryptococcus neoformans, Cryptococcus gattii, Candida albicans and Candida glablata.

Lignocellulosic biomass delignification using fungal laccases to improve biological hydrogen production

Bruna Soares Dionizio, Camila Abreu Borges da Silva Rabelo, Maria Bernadete Amancio Varesche, Edenir Rodrigues Pereira-Filho and Dulce Helena Ferreira de Souza

Chemistry Department, UFSCar, São Carlos-SP, Brazil

Department of Hydraulics and Sanitation, EESC-USP, São Carlos-SP, Brazil

email address: [email protected]

Laccases are multicopper oxidases (MCOs) capable of oxidizing a variety of substrates, such as lignin. Lignin is a polymer formed from phenylpropanoids with complex structure and high-molecular weight, which makes it difficult to use biomass for the production of biofuels. In this study, laccases expressed by the fungus Chaetomium cupreum URM 5066 were used to delignify sugarcane bagasse (SCB) and then to facilitate saccharification and fermentation for H₂ production by Clostridium butyricum CCT7470. The variables of the enzymatic process were evaluated by statistical methods. Using a univariate mode, the optimal pH (4) and temperature (50 °C) were determined. Employing a 2³ factorial design with central and axial points, three variables were optimized: the incubation time, SCB and enzyme concentrations, with results of 2 h 44 min, 23.4 g/L and 0.234 U/L, respectively. The SCB delignified by the laccases increased the H₂ production in approximately 98%, when compared to SCB control, without treatment by laccases, both fermented by C. butyricum. In addition, scanning electron microscopy assays demonstrated that there were structural changes in the SCB delignified by enzymes. Thus, according to the results obtained, laccases help to delignify, consequently, maximize utilization of SCB for biofuels productions, such as H₂.

The importance of the correct selection of range variables in the pretreatment of lignocellulosic material

Juan Camilo Acosta Pavas, Nataly Bonilla Ospina and Angela Adriana Ruiz Colorado

Departamento de Procesos y Energía, Universidad Nacional de Colombia, Medellín, Colombia

email address: [email protected]

The use of agroindustrial waste allows the generation of value-added products such as glucose syrups, and other metabolites of great importance in the food, energy and pharmaceutical industries. These residues, especially, lignocellulosic materials can be processed in several stages; therefore, it’s possible to obtain glucose. One of the most determined is the pretreatment stage, the residue is subjected to different conditions in order to break the lignin and releasing the cellulose and hemicellulose. In this work, an analysis of the effect that has the correct selection of the variables that can influence the pretreatment of sugarcane and maralfalfa grass by the response surface method is carried out. The variables time and solids percentage were selected, varying between 5:15 min and 5:15% wt/wt, respectively, with a fixed temperature of 150 °C. For agro-industrial waste a R² 82.74% and $R_{\text{adjusted}}^2$ = 70.41% for sugarcane and R² = 89.61% and $R_{\text{adjusted}}^2$ = 82.19% for maralfalfa Grass were obtained. For sugarcane, an optimum point were obtained in 10.93 min and 9.79% wt/wt, for maralfalfa grass in 17.07 min and 16.64% wt/wt, which indicates that maralfalfa grass need an adjustment of the ranges of the selected variables so that an optimum point is obtained within the ranges indicated for this industrial waste.

Biocompounds evaluation for Cornus mas fruits by ultrasound-assisted extraction

Iuliana Maria Enache, Gigi Coman, Oscar Benito Román, Sagrario Beltrán Calvo  and Liliana Mihalcea

Faculty of Food Science and Engineering, Dunarea de Jos University, Galati, Romania

Faculty of Science, University of Burgos, Burgos, Spain

email address: [email protected]

Cornelian cherry Cornus mas L. is a valuable forest fruit rich in bioactive compounds and can be consumed by humans for various ways: fresh fruits, decoction, fermented, galenical or infusion from all botanical parts of the plant (root, bark, leaves, flower and fruits). In Romania, the Cornelian cherry is widely located from the plains to the mountains due to his resistance to diseases and pests. In our study, the freeze-dried cornelian cherry fruits from the spontaneous culture were investigated by ultrasound-assisted extraction method for 15 samples using a BOX-Behnken model with three independent variables (amplitude, pulses and time). The results were modelated using the software Design-Expert 6.0 Trial. In our study, the highest value for phenolic content were obtained at 60% amplitude, 0.75 pulse and 12.5 min (27.645 mg GAE/g), the flavonoid content at 40% amplitude, 0.75 pulse and 5 min (2.792 mg QE/g) and antioxidant activity (31.630 mg Trolox/g) at 80% amplitude, 0.75 pulse and 15 min. The studies suggested that the cornelian cherry fruits from spontaneus culture could be a great source of phytonutrients and open the posibility to be used in new food products formula (compotes, jams, jellies, juices, liqueurs, syrups, pickles, wines).

Funding: This work was supported by two mobility grants of the Romanian Ministery of Research and Innovation, CNCS - UEFISCDI, project number PN-III-P1-1.1-MC-2019-1905 and PN-III-P1-1.1-MC-2019-1908, within PNCDI III.

New fungal luciferin analogs: Optimized synthetic approach

Maria S. Lyakhovich, Andrey I. Bubyrev, Andrey Y. Gorokhovatsky, Anastasia A. Andrianova, Renata I. Zagitova, Aleksandra S. Tsarkova, Ilya V. Yampolsky  and Zinaida M. Kaskova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

Planta LLC, Moscow, Russia

email address: [email protected]

Bioluminescence is light emission by living organisms due to oxidation of a substrate called luciferin assisted by an enzyme called luciferase. Bioluminescence becomes more and more popular for the development of analytical methods in vitro and in vivo, including real-time bioimaging. Bioimaging in vivo is most efficient when luciferin–luciferase systems have their emission maxima close to the near-infrared transparency window of biological tissues. The bioluminescent properties of these systems can be modulated both by mutations in luciferase and chemical modifications of luciferin. The fungal luciferin, or 3-hydroxyhispidin, belongs to the styrylpyrone subfamily of polyketide natural products family. It consists of an α-pyrone cycle and an aromatic moiety, linked through a double bond. It was shown previously that fungal luciferin is functionally modular: while the pyrone fragment undergoes enzymatic oxidation, the aromatic part and the linker, unaffected by bioluminescent reaction, influence spectral characteristics of emitted light. Using the optimized synthetic method, we have obtained a series of fungal luciferin analogs with modulated chemiluminescence activities and spectral characteristics. A strong shift of chemiluminescence emission maxima into the red region was observed for one compound.

Funding: This study was supported by Russian Science Foundation, grant no. 18-73-00347.

Apoptosis screening in leukemia as a new insight in therapeutics development

Maida Hadzic, Sanin Haveric and Lejla Pojskic

Institute for Genetic Engineering and Biotechnology University of Sarajevo, Sarajevo, Bosnia and Herzegovina

email address: [email protected]

Apoptosis is a complex, genetically controlled, regulatory mechanism for maintenance of cellular homeostasis. Deregulation of apoptosis causes an imbalance between cell proliferation, survival and death, and plays an important role in the development of solid and hematological tumours. One of the leading approaches in antitumour treatment of leukemia is based on the apoptosis induction via extrinsic or intrinsic pathways in order to target and eliminate tumour cells. Anti-leukemic strategies include cellular and molecular monitoring of apoptosis triggered by some novel agents and potential therapeutics. Techniques based on supravital staining and microscopy analysis present the first step in apoptosis screening giving the clear distinction between different stages of cell death. Various molecular techniques have been developed for the detection of caspases, regulators of apoptosis, which include many forms and types of enzyme activity assays, western blot analyzes, immunoprecipitation and immunohistochemistry. In addition, PCR microarray-based methods for apoptotic markers can detect differences in the expression of genes encoding key ligands, receptors, intracellular modulators and transcription factors involved in the regulation of apoptosis. Nowadays, a new generation of small molecular inhibitors is being developed such as BH3 mimetics that are considered as promising weapon of leukemia cell destruction and targeted therapy improvement.

Studies on the development of a new functional product for children

Dan Cosmin Misu, Daniela Istrati, Oana Constantin, Camelia Vizireanu, Eugenia Pricop and Geanina Ghenea

Faculty of Food Science and Engineering, “Dunarea de Jos” University, Galati, Romania

email address: [email protected]

The main objective of this study was to create a functional syrup-type product designed to increase children’s immunity, made from honey, sea buckthorn fruits (Hippophaë rhamnoides), rosehip fruits (Rosa canina) and Nigella sativa seeds oil. The raw materials used to obtain the syrup were physico-chemical analyzed to highlight the main natural constituents. According to the experimental data obtained, three types of syrup were developed and stored for 28 days at ambient temperature. The syrups were analyzed at certain time intervals in terms of physico-chemical, microbiological and sensory characteristics. Particular attention was paid to the evaluation of total polyphenols, flavonoids, vitamin C content, antioxidant capacity, TBA index and sensorial characteristics. The analyzed samples recorded a significant amount of vitamin C (on average 830 mg/100 g) with a decrease of the vitamin C content by approximately 5% until the end of the storage period. Microbiological tests revealed the stability of the product during the entire storage period. Experimental data revealed that the syrups analyzed can be a functional food, a natural alternative to processed syrups, with balanced nutritional and sensory characteristics, a superior antioxidant activity, which recommends it in the diet of all consumers concerned with maintaining health and increasing immunity.

Funding: This work was supported by the project "EXPERT", financed by the Romanian Ministry of Research and Innovation, Contract no. 14PFE/17.10.2018.

Healthy eating choices and habits of teenagers: identification of influence factors

Camelia Vizireanu, Daniela Ionela Istrati, Aura Daraba and Aurica Baroian

Department Food Science, Food Engineering, Biotechnology and Aquaculture, Dunarea de Jos University, Galati, Romania

email address: [email protected]

Undergraduates from a Buzau County college were used as a cohort for evaluating eating habits and dietary choices, potential knowledge of nutritional significance of their food ratios in relationship to early onset of obesity and associated diseases among youngsters. Questionnaires with specific indicators were used: gender (females: 54%; males: 46%), age (between 15 and 19 years old), average height based on gender (163 cm: females; 175 cm: males), the place of residence (22%: urban area, 78%: rural area). Average weight indicator (females: 55.9 kg and males: 68.3 kg) showed that 61% of respondents had a normal weight and 27% of the respondents were overweighed. The type of undertaken courses was used as respondents’ knowledge indicator of the healty eating habits (32% of students enrolled in environmental protection courses, and 68% enrolled in public alimentation courses). Undergraduates (52%) were oscillating when evaluating the quality of their eating choices and habits, 31% of respondents were very sure on their adopted healthy eating choices and habits, only 17% were displaying unhealthy eating choices and habits. The analysis of responses indicated that balanced food ratios can be optimally formulated to correspond to healthy alimentary habits and choices of young individuals during their transition to adulthood.

Romanian vegetal-origin extracts utilized for therapeutic purposes: characterization and antimicrobial activity

Aura Daraba, Camelia Vizireanu, Daniela Istrati, Oana Constantin and Mihaela Jantea

Department Food Science, Food Engineering, Biotechnology and Aquaculture, Faculty of Food Science and Engineering, University “Dunarea de Jos”, Galati, Romania

email address: [email protected]

Plants and fruits hydroalcoholic extracts, essential oils, aqueous extracts and tincture were selected from Romanian producers and evaluated from phytochemical composition and antimicrobial activity standpoints. Sage and garlic were evaluated as hydroalcoholic extracts and essential oils. Grapefruit was evaluated as aqueous extract (seeds) and as essential oil (peel). Oregano and cloves were evaluated as essential oils. Basil was evaluated as tincture and as essential oil. The GS/MS analysis indicated each essential oil has an unique combination in biologically active compounds and contains one major active compound depending on the used raw materials. The antimicrobial activity was tested against Bacillus cereus ATCC 10876, Listeria innocua, Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 using agar well diffusion and by determining the minimally inhibitory concentration (MIC). Sterile water and 40% vol ethanol were used as control samples. The inhibition zone indicated that the essential oils displayed a higher antibacterial activity than hydroalcoholic/aqueous extracts, and tincture against B. cereus, L. innocua and E. faecalis. Grapefruit hydroalcoholic extract, essential oils of grapefruit, basil, oregano, clove, sage, garlic and sage tincture displayed selectively, depending on the target pathogen, a high antibacterial activity (MIC < 10 µL/mL and MIC < 50 10 µL/mL).

Undergraduate students’ alimentary preferences: optimizing the food ratios

Daniela Istrati, Camelia Vizireanu, Aura Daraba and Florentina Trifan

Faculty of Food Science and Engineering, Department Food Science, Food Engineering, Biotechnology and Aquaculture, University “Dunarea de Jos”, Galati, Romania

email address: [email protected]

Alimentary behaviour of teenagers from Galati County was studied within in household and school environments. Optimization of food ratios for identifying the factors that influence the teenagers’ have been performed based on specific questionnaires. Studied cohort was respresented by 120 ungraduates with ages between 15 and 18. Within the cohort 92% were males and 8% females who reside in urban (35%) and rural (65%) environments. The corporal weight revealed that 80% of undergraduates were within normal range, 5% were under-weighted, 10% were over-weighted and 5% were obese. During teenage stages, alimentary habits and attitudes suffer negative variations and modifications in that the daily meals become irregular (22% do not serve breakfast, 15% eat dinner after 11 pm o’clock); snack-type products (chips, bagles etc.) are preffered by 31%; 24% eat on a daily basis fast-food meals, and 33% drink daily soft drinks. The consumption of legumes (dried-40%; fresh-30%), meat and meat products (18%), milk and dairy products (28%), eggs (14%) and cereals (12%) is below the recommended daily quantities. Our study indicates that teenagers must be educated and informed with regard to a healthy diet and the education must start from the households and continued throughout primary and secondary education levels.

Virtual screening for hTAS2Rs ligands: a predictive model using AdaBoost and ECFP

Gerardo Sebastián De León, Eleonore Fröhlich  and Sharareh Salar Behzadi

Research Center Pharmaceutical Engineering GmbH, Graz, Austria

Center for Medical Research, Medical University of Graz, Graz, Austria

University of Graz, Department of Pharmaceutical Technology, Graz, Austria

email address: [email protected]

The aim of the study is to create a ligand-based predictive model for screening hTAS2Rs (bitterness receptors) using 2D fingerprints in an AdaBoost ensemble with Decision Trees as weak learners and validate the performance of the model on a set external to the training set. The ligands and negative examples were obtained from BitterDB, SupersweetDB and Fenaroli’s handbook of flavor ingredients. Data curation was performed to remove salts, disconnected structures and repeated structures. The ECFP6-1024bit were created using the RDKit package. The model was trained using the library scikit-learn. A fivefold cross validation was used for the selection of the hyperparameters of the model. The model’s performance was evaluated using f1 score on the external set UNIMI. The model was trained with 700 estimators and a max depth = 1, showing a cross-validation accuracy of 0.81 and a training accuracy of 0.87. The accuracy and the f1 score of the trained model, based on the UNIMI set validation, were 0.70 and 0.71. The confusion matrix obtained was TN, FP, FN, TP = 18, 14, 2, 20. The increase of non-bitter molecules database would most likely improve the performance of the model due to the relative high number of false positives.

Funding: Authors thank HERMES-Johannes-Burges-Stiftung for funding this project.

Viral metagenomic studies of wild birds

Kobey Karamendin, Aidyn Kydyrmanov, Klara Daulbayeva, Kasymbekov Ermukhammet, Seidalina Aigerim, Khan Elizaveta and Sayatov Marat

Department of Virology, Scientific and Production Center of Microbiology and Virology, Almaty, Kazakhstan

email address: [email protected]

Wild aquatic birds are considered the main reservoir of all subtypes of avian influenza viruses. In this study, samples from wild birds were collected in 2014–2015 in Kazakhstan. Avian influenza viruses belonging to seven different subtypes were identified. Samples from wild birds were obtained during seasonal hunting, as well as during banding by ornithologists. Hemagglutinating agents were isolated in 9- to 10-day-old chicken embryos. To get rid of host’s nucleic acids, allantoic fluid was treated with Turbo DNAse and RNAse A before RNA extraction. Viral RNAs were isolated using the QIAamp Viral RNA Mini kit in accordance with the manufacturer’s recommendations. Complementary DNA from RNA was synthesized using random hexamers. The libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina, USA). Sequencing for viral metagenome (virome) was carried out using MiSeq sequencer. Bioinformatic analysis was carried out using Geneiuos Prime software. As a result, the complete genomes of influenza A viruses of the following subtypes H1, H3, H5, H9, H10, H13 and H16 circulating among wild birds were obtained. Thus, metagenomic study using massive parallel sequencing technology is an effective tool for detecting influenza viruses of various subtypes in samples with the simultaneous obtaining their complete genomes.

Vegetable pea paste Pisum sativum fortified with hemp protein powder Cannabis sativa physical, chemical and sensorial characterization

Eugenia Mihaela Pricop, Octavian Baston, Daniela Istrati, Camelia Vizireanu and Oana Constantin

Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Galati, Romania

email address: [email protected]

The effect of hemp protein powder addition on the physical, chemical and sensorial attributes of vegetable pea-based paste composition was investigated. The ingredients used to produce the vegetable paste are: peas, vegetables (carrot, onion and bell pepper), hemp protein powder, lemon juice, vegetable fats and spices. Hemp protein powder was added in different proportions (5%–15%) and is combined very well with green peas to achieve a balanced protein profile in terms of amino acid content. According to the experimental data, the protein content of the vegetable pasta fortified with different quantities of hemp protein powder increased from 23% (dry weight) to 27% (dry weight). In the lipid content ω-3 and ω-6 essential fatty acids were identified which gives the product high nutritional quality. The vegetable pasta fortified with 15% hemp protein powder had the highest protein, fat, ash, fiber content. Our product was sensory examined by a team of 15 trained panelists. The trained panelists appreciated the product fortified with 10% hemp protein powder for the natural appearance, the distribution and combination of vegetables used, the pleasant aroma of the product and the unique texture of the product.

Funding: This work was supported by the project "EXPERT", financed by the Romanian Ministry of Research and Innovation, Contract no. 14 PFE/17.10.2018.

Modern membrane mimetic systems for studying GPCRs

Polina Khorn, Anastasia Gusach, Aleksandra Luginina, Olga Sukhacheva, Lev Vasilenko, Valentin Borshchevskiy, Aleksey Mishin and Vadim Cherezov

Research Center for Molecular Mechanisms of Aging and Age Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia

Bridge Institute, USC Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, California, USA

email address: [email protected]

G-protein coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in human genome, targeted by over 30% of therapeutic drugs on the market. Structural and biophysical studies of GPCRs are essential for understanding the mechanisms of ligand recognition and signalling. The most common membrane mimetic systems for GPCRs are detergent micelles and membrane scaffold protein (MSP) based nanodiscs that have been widely used for structure determination and for studying protein conformational dynamics. Nevertheless, selecting the most optimal membrane mimetic system for diverse applications is still a challenge. Ideally, this environment should stabilize the protein, be compatible with its purification procedure and enable the study of its structural and functional properties while the protein shows native behaviour. New generations of membrane mimicking systems that include native nanodiscs built by copolymers like diisobutylene maleic acid (DIBMA), covalently circularized nanodiscs and Salipro may satisfy these requirements. Here, we present protocols for incorporation of the human adenosine A2A receptor and cysteinyl leukotriene receptors (1 and 2 types) into different membrane mimetic systems and provide a comparison of different properties for receptors incorporated in classical and novel mimetic systems.

Funding: This work was supported by the Russian Science Foundation project no. 197400088.

Synthesis of plant receptor-like kinases involved in interaction with pathogenic and beneficial fungi in heterologous system and their functional analysis

Olga A. Pavlova, Irina V. Leppyanen, Igor V. Senderskiy, Viacheslav V. Dolgikh, Igor A. Tikhonovich and Elena A. Dolgikh

All-Russia Research Institute for Agricultural Microbiology, St.-Petersburg, Russia

All-Russia Research Institute for Plant Protection, St.-Petersburg, Russia

email address: [email protected]

Plants are able to efficiently distinguish the pathogenic and beneficial fungi. Released from fungal cell walls under the influence of plant chitinases, long-chain chitooligosaccharides (COs) (n ≥ 6) stimulate strong defense reactions, whereas short-chain CO4-5 and Myc factors secreted by arbuscular mycorrhiza (AM) fungi induce symbiosis development. The LysM-receptor-like kinase PsLYK9 was shown to be important for interaction with pathogenic and AM fungi and, the most likely, involved in CO perception in pea plants. The probable explanation that the PsLYK9 may interact with various co-receptors to recognize COs differing in the polymerization degree. Among possible candidates for the role of co-receptors, a few LysM-RLKs were identified. The synthesis of these proteins with functional activity was performed using the Bac-to-Bac baculovirus expression. Extracellular domains of PsLYK9, PsLYR4 and PsLYR3 with N-terminal 6xHis tags were synthesized using the pFastBac1 vector inserted in bacmid in DH10Bac E. coli cells and the bacmids were used to transform the Sf9 insect cells. All tested recombinant proteins showed accumulation in the soluble form. The functional activity of affinity purified synthesized receptors was analyzed using the microscale thermophoresis and their capacity to form complexes was checked in pull-down assays.

Funding: The work was supported by RSF (16-16-10043).

Purification and characterization of the blue protein from Rhizostoma pulmo

E. S. Korunova, A. Yu. Gorokhovatsky, T. V. Chepurnykh, Yu. A. Mokrushina, R. Kh. Ziganshin, A. S. Tsarkova and I. V. Yampolsky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

email address: [email protected]

Marine invertebrates display a wide range of colourations. It was previously shown that the blue colour of jellyfish Rhizostoma pulmo is caused by a chromoprotein. This protein, rpulFKz1, contains a Frizzled cysteine-rich domain and a Kringle domain. However, the nature of the chromophore remains unknown. The aim of this work was to obtain rpulFKz1 for crystal structure analysis. We developed a method for purification of rpulFKz1 from R. pulmo jellyfish tissues which included differential precipitation with ammonium sulfate, ion-exchange and gel-filtration chromatography. The chromoprotein was characterized by an absorption spectrum with the maxima at 420, 588 and 624 nm. The electrophoretic mobility of the purified protein in denaturing conditions corresponded to 32 kDa. In-gel treatment of the protein band with periodate and Schiff’s reagent resulted in a characteristic staining and indicated protein glycosylation. In gel-filtration chromatography, elution volume of rpulFKz1 corresponded to the dynamic equilibrium between monomeric and dimeric forms. Using thermal shift assay in the presence of the Sypro Orange fluorescent dye, the optimal buffer conditions ensuring highest protein stability were found. These conditions were used for crystallization of rpulFKz1 protein.

Funding: This work was supported by the President grant for leading scientific schools LS-2605.2020.4.

The development of functional products from carrots and lactic acid bacteria

Mihaela Turturica, Carmen Alina Bolea, Gabriela Elena Bahrim, Nicoleta Stanciuc, Elena Enachi and Vasilica Barbu

Department Food Science, Engineering, Applied Biotechnology and Aquaculture, Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Galati, Romania

email address: [email protected]

Carrots (Daucus carota L.) are root vegetables grown worldwide and represent important sources of bioactive compounds such as vitamins, polyphenols, carotenoids and dietary fibers. Carrots are among the most consumed vegetables in a fresh-cut form due to their versatility and nutritional composition. The carrot matrix was used in a fresh or dried form or as a freeze-dried powder. In order to obtain high-functional products, a 10¹² CFU/g concentration of lactic acid bacteria was sprayed onto the vegetal tissue. The viability of the lactic acid bacteria was evaluated for all the products over a period of 21 days through microbiological culture methods. The antioxidant capacity revealed a high-antioxidant activity of the products. Moreover, the total polyphenols content showed a high content of bioactive compounds. The obtained food products enriched with lactic acid bacteria can be considered as new and innovative functional food products that imprint several beneficial properties to the human body.

Funding: This research was supported by the project "EXPERT", Contract no. 14PFE/17.10.2018 and by the project "ANTREPRENORDOC", in the framework of Human Resources Development Operational Programme 2014-2020, financed from the European Social Fund under the contract number 36355/23.05.2019 HRD OP/380/6/13 – SMIS Code: 123847.

Production of xylanase during cultivation of Trametes versicolor on sugar beet pulp

Marina Tišma, Gordana Šelo, Ana Marija Dukarić, Anja Bošnjaković, Ana Bucić Kojić and Mirela Planinić

Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia

email address: [email protected]

Sugar beet pulp (SBP) is a by-product of the sugar beet (Beta vulgaris L.) processing in sucrose production and is primarily used as animal feed. Around 20 million tons of SBP is produced in Europe each year. The dry basis of SBP is mainly composed of polysaccharides (cellulose, hemicellulose and pectin), along with protein, and small amounts of lignin, fat and ash. Hemicellulose is consisted of xylan, a heteropolysaccharide substituted with monosaccharides (l-arabinose, d-galactose, d-mannoses) and organic acids (acetic, ferulic and glucuronic acid), interwoven together with glycosidic and ester bonds. Xylanase (endo-1,4-β-d-xylanase, xylanohydrolase, EC 3.2.1.8) is an important xylan degrading enzyme involved in the random cleavage of xylan to produce xylose, xylobiose and xylo-oligosaccharides. In this work, SBP was used as a substrate for Trametes versicolor cultivation in solid-state conditions during 8 days for the purpose of xylanase production. Xylanase activity of 318 U/g DM was reached after 8 days of cultivation under following initial process conditions: T = 27 °C, pH 4.5, m (SBP) = 25 g, m (added water) = 60 cm³, five mycelial plugs (Ø 1 cm).

Funding: This work was supported by the European Regional Development Fund (ERDF) [grant number KK.01.1.1.04.0107].

Regioselective synthesis of linoleic acid-based hydroperoxides with in situ oxygen generation

Valentin Gala Marti, Anna Coenen and Ulrich Schoerken

Faculty of Applied Natural Sciences, Cologne University of Applied Sciences, Leverkusen, Germany

email address: [email protected]

Lipoxygenases catalyze the hydroperoxidation of linoleic acid with molecular oxygen leading either to the 9- or 13-hydroperoxides, which are interesting precursors for further processing. Splitting of the 13-hydroperoxide by, e.g. hydroperoxide lyase leads to volatile C6-aldehydes, alcohols and C12-oxoacids. The volatiles are marketed by the flavour and fragrance industry as green notes and the bifunctional oxoacids are interesting bio-based intermediates for the chemical industry. Hydroperoxides were obtained with soybean lipoxygenase in moderate to good yields with oxygen bubbling at substrate concentrations below 30 mM. The 13-regiospecificity dropped with increasing linoleic acid concentration and decreasing yield. Therefore, soybean LOX1 was cloned and the expression in E. coli was optimized to obtain pure enzyme with high 13-specificity. After Ni-NTA affinity chromatography active fractions of the purified His-tagged enzyme were obtained. The development of an enzymatic cascade for a green synthesis route to hydroperoxides is envisaged starting from safflower oil in combination with lipase hydrolysis and catalase mediated in situ oxygen supply to suppress foaming caused by gasification. Lipase screening showed that the enzymes from Candida rugosa are suitable catalysts under the LOX reaction conditions. First results applying catalase reveal that the peroxidation with LOX is comparable to that of gas bubbling experiments.

Heterologous synthesis of plant LysM-receptor-like kinase K1 in insect cells and binding assay with ligand

Olga A. Pavlova, Anna N. Kirienko, Igor V. Sendersky, Viacheslav V. Dolgikh  and Elena A. Dolgikh

All-Russia Research Institute for Agricultural Microbiology, Saint Petersburg, Russia

All-Russia Research Institute for Plant Protection, Saint Petersburg, Russia

email address: [email protected]

Studying the symbiosis between legume plants and nitrogen-fixing bacteria of the order Rhizobiales is impossible without understanding of the signal perception mechanisms and functional response to them. The rhizobial lipo-chitooligosaccharide signals called Nod factors were shown to be perceived by lysin motif receptor-like kinases (LysM-RLK) in plants. Previously, we revealed the LysM-RLK K1, which is important for symbiosis development in pea Pisum sativum L. The formation of the complex between K1 and co-receptor SYM10 was shown to be essential for pea-rhizobial symbiosis initiation, but binding with Nod factors was not verified. In current research, the binding between the LysM-RLK K1 and the Nod factors was analyzed by means of the microscale thermophoresis. With this aim the Bac-to-Bac baculovirus expression of recombinant protein with N-terminal 6xHis tag was performed in Sf9 insect cells. The sequence encoding the extracellular domain of K1 was cloned in pFastBac1 vector, inserted in bacmid in DH10Bac E. coli cells and used for transformation of insect cells. Recombinant protein was detected in cell debris and solubilized with 1% Triton X-100. The functionality of the solubilized protein was tested using the Tycho system and verified in the binding assay with ligand.

Funding: The work was supported by RSF (16-16-10043).

Origin of fibrillar sphere and its potential impact on genome activation

Michal Benc, Frantisek Strejcek, Alexandra Bartkova, Amelie Bonnet-Garnier  and Jozef Laurincik

Constantine the Philosopher University in Nitra, Nitra, Slovak Republic

UMR BDR, INRAE, ENVA, Université Paris Saclay, Jouy-en-Josas, France

email address: [email protected]

It is well accepted that fibrillar sphere (NLB) plays key role in processes occured immediately after fertilization. Our observations indicate that the origin and specific composition of NLB has potential impact on regulation of genome activation. Our recent results show significant differences in relative protein content of NLB between mouse and porcine oocytes. These findings were supported by interspecies nucleolotransfer. Low level of protein content in porcine NLB was increased by two porcine NLBs injected into a previously enucleolated mouse oocyte. This step supported the development of a mouse parthenogeneticaly produced embryo derived from an enucleolated oocyte. However, this was followed by delayed localization of UBF and fibrillarin to embryonic nucleoli in interspecies nucleolotransfer embryos. In addition, our new data show that minor zygotic genome activation is delayed too. Therefore, the origin and specific composition of NLB has possible impact on timing of embryonic genome activation.

Funding: This work was supported by the Slovak Research and Development Agency under the Contract no. SK-FR-19-0010 and also by the Scientific Grant Agency VEGA [VEGA 1/0167/20].

Acknowledgement: The authors thank to Institute of Animal Science (Prague – Uhrineves, Czech Republic) for providing the biological material.

The evolution of suspected poisonings in honey bee families in Romania during 2016–2020 beekeeping active seasons

Savu Vasilica, Şapcaliu Agripina, Tache Bogdan, Zaharia Roxana, Tăpăloagă Dana, Lagunovschi Luchian Viorica, Bădic Luiza, Bodescu Dan  and Rădoi Ion It should be written as other authors on other abstracts

Beekeeping Research and Development Institute, Bucharest, Romania

Research and Development Institute for Plant Protection, Bucharest, Romania

University of Agronomic Sciences and Veterinary Medicine Bucharest, Bucharest, Romania

Spiru Haret University, Bucharest, Romania

University of Agronomical Sciences and Veterinary Medicine Iasi, Romania

email address: [email protected]

The aim of this study is to present the evolution of the suspected poisoning in bees during the 2016–2020 active seasons. The bees’ health was monitored and there were sampled dead or alive bees and honeycomb fragments from 911 apiaries across the country. Diagnosis for attempted poisoning with drugs (anti-parasitic products, hydro alcoholic extracts, antibiotics), chemical substances (pesticides, disinfection, antiviral, antifungal substances) and toxic food (honey, pollen, corn syrup) was done corroborating the history, morphological and clinical examinations, but excluding other diseases (viral, bacterial, parasitical). During the study, 48 of the 911 apiaries were identified with suspected poisoning including 3524 bee families. The dynamics of suspected poisonings in bee families ranged between 3.69% (2017) and 44.9% (2019), but the chemical poisonings reached a maximum of 59.6% (2019), the drug ones of 59.5% (2016) and the toxic food of 59.21% (2018). The evolution of the main types of suspected poisonings in bees during 2016 and 2020 was influenced by extreme weather phenomena which affected the bees’ survival and harvesting and also by the existence of some prophylactic measures of fighting against the viral infection Covid 19 (spraying and quarantine).

Funding:This work was supported by project ADER 2.2.1.

β-Casein gene polymorphisms in dairy cows reared in central Italy

Giulia Cinti, Carla Sebastiani, Chiara Arcangeli, Marcella Ciullo, Martina Torricelli, Stefano Fisichella  and Massimo Biagetti It should be written as other authors on other abstracts

R&D Cooperlat Società Cooperativa Agricola, Jesi (Ancona), Italy

Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche-Togo Rosati, Perugia, Italy

email address: [email protected]

The majority of proteins in cow’s milk are caseins, which occur in four groups (α-s1, α-s2, β and k). In this study, we focused on the β-casein allele variants A1 and A2 due to their influence on human health. A1 variant is probably involved in intolerance to milk and some human diseases due to the production of the bioactive peptide β-casomorphin 7 with opioid activity. The β-casein variant A2 is instead desirable in milk because it seems to increase milk digestibility. The difference between A1 and A2 variants is a mutation at position 67 of the β-casein gene, which causes an aminoacid change. Allele frequencies of β-casein gene variants were evaluated by sequencing analysis in 1723 Italian Holstein Friesian dairy cows reared in central Italy. The A1 and A2 alleles were found with frequencies of 30% and 61%, respectively. Regarding the genotype, the homozygous genotype A2/A2 was the most common, with a frequency of 37%, followed by the A1/A2 and A1/A1 genotypes with frequencies of 36% and 10%, respectively. The identification of A2 carrier cows will allow to manage animal couplings with the aim of increasing A2 allele frequency and producing a milk containing only β-casein A2 variant.

A simple HPLC method for screening and quantitation of anabolic-androgenic steroids in model matrices

Zdravka Zaharieva, Dancho Danalev and Dimitar Tanev

Biotechnology Department, University of Chemical Technology and Metallurgy, Sofia, Bulgaria

Testing center Global Test Ltd., Sofia, Bulgaria

email address: [email protected]

The use of dietary supplements is widespread in sport and most athletes competing at the highest level of competition use some form of dietary supplementation. Dietary supplements seem to modify the body appearance and to promote more consistent and intensive training for athletes by allowing a speedy recovery between training sessions and by enhancing competitive performance. Several studies have highlighted that some of the legitimate dietary supplements on the market contain undeclared ingredients prohibited by the International Olympic Committee and of the World Anti-Doping Agency. Contaminants that have been identified include a variety of anabolic androgenic steroids. The most of the present methods for testing of anabolic androgenic steroids rely on GC/MS and LC-MS/MS are developed for monitoring of biological fluids like plasma and urine as well as tissues but they have not been applied to dietary supplements. A simple HPLC method for simultaneously determination of 13 AAS-metyltestosterone, testosterone and its esters (propionate, enanthate), nandrolone and its esters (propionate, decanoate), methanedienone, androstendione, trenbolone and its esters (acetate, enanthate) and boldenone undecylenate was developed. Main analytical parameters include C18 column, DAD at 254 nm and gradient flow with water and methanol.

Impact of adipose tissue storage conditions on the adipose-derived mesenchymal stromal cell isolation yield

Natalia Siedlecka, Daria Balcerczyk, Kamil Szeliski, Tomasz Drewa and Marta Pokrywczyńska

Department of Regenerative Medicine, Cell and Tissue Bank, Chair of Urology, Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland

email address: [email protected]

The aim of the study was evaluation of impact of human adipose tissue preservation conditions on the number of isolated and cultured human adipose-derived mesenchymal stromal cells (hAD-MSCs). Subcutaneous adipose tissue was harvested during liposuction (n = 10). Samples were stored in Biolasol Solution (B), culture medium MEM Alpha (M) and physiological saline (S), at 4 °C for 3, 6,12 and 24 h. Fresh tissue was used as a control sample. The highest number of cells were isolated from the tissue directly after harvesting (624,424.84 ± 195,052.02). The results for tissue stored in M (490,899.87 ± 185,790.07) and in S (449.367.54 ± 164,163.19) were comparable. Significantly lower number of cells was obtained for tissue stored in B (376,767.72 ± 176,349.86; p < 0.05; p < 0.01). The number of viable cells from primary culture was significantly lower for tissue samples after 24 h of storage (30,844.64 ± 15,076.57) compared to other tissue incubation times. Tissue subjected to cell isolation should be processed within the time not exceeding 3 h after harvesting. The Biolasol solution should not be used for tissue storing before isolation of hAD-MSCs.

Funding: The present work was supported by the National Center for Research and Development (NCBR) in Poland under Agreement no. STRATEGMED1/235368/8/NCBR/2014 (Smart AUCI Project) within the Strategic Programme STRATEGMED “Prevention practices and treatment of civilization diseases.”

Additional point mutations in ACAN and GATA4 genes in an atypical achondroplasia patient with platyspondyly and congenital heart defect

Volkan Sonmez, Taner Karakaya, Burcu Albuz, Fatma Silan and Ozturk Ozdemir

Department of Medical Genetics, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey

email address: [email protected]

Achondroplasia is the most common cause of short-limb dwarfism and affected individuals have macrocephaly and characteristic facial features with frontal bossing and midface retrusion. Herein, we aim to present an achondroplastic patient with platyspondyly and congenital heart defect (CHD) who also harboured mutations in ACAN and GATA4 genes. A 2-month-old male infant with achondrplasia had CHD including patent foramen ovale (PFO), muscular ventricular septal defect (VSD), atrial septal defect (ASD) was referred to our outpatient clinic for investigation. Patient’s skeletal survey demonstrated diffuse platyspondyly and ultrasonography of hip joint revealed early stage developmental dysplasia of the hip (DDH). The patient with acondroplasia was referred to us for further genetic evaluation because of atypical symptoms. After DNA isolation from peripheral blood sample, CEA was performed using the Clinical Exome Solution (CES) kit and analyzed with Sophia DDM 5.5.1 and IGV Software. Besides the c.1138G > A(p.Gly380Arg) mutation, additive two mutations in ACAN and GATA4 [c.1702G > A(p.Asp568Asn), c.487C > T(p.Pro163Ser) respectively] were detected as causative likely pathogenic mutations associated with the patient’s presentation. It is substantial for clinicians to assess vigilantly the patients with achondroplasia presenting atypical findings. Just identifying only the mutation in FGFR3 can hinder to delineate the entire molecular characteristics underlying the phenotype. Combined mutations in patients with achondroplasia can serve an important function in counseling about risk and prognosis.

A rare lethal multiple pterygium syndrome caused by a homozygous point mutation In CHRNG gene: a case report

Mehmet Berkay Akcan, Burcu Albuz, Ahmet Kablan, Ozturk Ozdemir and Fatma Silan

Department of Medical Genetics, Çanakkale Onsekiz Mart University Research Hospital, Çanakkale, Turkey

email address: [email protected]

Lethal Multiple Pterygium Syndrome (LMPS) is a rare genetic condition which is characterized by growth retardation, dysmorphic facial appearance, webbing of the neck, elbows, knees and fingers. Here, we present a LMPS patient with a homozygous pathogenic mutation in CHRNG gene. A newborn at 34 weeks’ gestational age was refferred to our medical genetics department with webbing of the skin (pterygium) at the joints, severe joint contractures, hypoplasia of lower extremity and lung hypoplasia. He died shortly after birth. His parents were non-consanguineous and both healthy. In the family story, they had an another son who had similar symptoms and died after birth as our patient. After DNA isolation from peripheral blood sample, the whole exome sequencing (WES) analysis was performed using the xGen Exome Research Panel v2 kit. We detected a homozygous pathogenic frameshift (c.459dupA,p.V154fs*24) mutation on exon5 of CHRNG gene.The parents were heterozygous for the same mutation. Multiple Pterygium Syndrome (MPS) is traditionally divided into lethal which is fatal before birth or shortly after birth and nonlethal (Escobar syndrome [ES]) types. CHRNG associated LMPS is an extremely rare autosomal recessive disorder affecting the skin, muscles and skeleton. There have been approximately 50 cases of this disorder reported in the medical literature. In this report, we aimed to emphasize the importance of advanced genetic techniques in genetic counseling by drawing attention to a rare case.

A novel variant for SQSTM1 gene in a patient with frontotemporal dementia

Ozgul Ocak, Taner Karakaya, Burcu Albuz, Nihan Ecmel Akbas, Volkan Sonmez, Mehmet Berkay Akcan, Fatma Silan  and Ozturk Ozdemir

Department of Neurology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey

Department of Medical Genetics, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey

email address: [email protected]

Frontotemporal dementia (FTD) is a clinical entity emerging with significant changes in personality, social relationships, language; and cognitive impairments of executive functions are more common in the frontal lobe. Herein, we report a patient with FTD shown to carry novel pathogenic variants in SQSTM1 gene. A 56-year-old female patient with recently showing up personality changes and demential findings was consulted to assess whether there was an underlying genetic etiology. Cranial MRI scan confirmed subtle atrophy fitting the frontal region. The patient had no symptoms of any other disease. Clinical exome sequencing (CES) was carried out on the İllumina® instrument according to the manufacturer’s (Sophia-Genetics®) protocols.The data were examined using IGV, Sophia DDM platforms for the interpretation corresponding to the patient’s consent. The novel mutations c.97G > T(p.A33S) and c.73T > C(p.F25L) of SQSTM1 were detected by CES and then confirmed with Sanger sequencing. The mutations were predicted to be likely pathogenic according to the ACMG criteria. We could not be able to conduct family segregation analysis because of unavailable relatives. According to the current knowledge, although only a single pathogenic mutation harbouring SQSTM1 was enough for resulting in FTD, It is possible to say that combined effects of the two mutations result in significant implications for the proband.

A homozygous nonsense mutation in MTHFR gene causes a severe phenotype: hypotonia and hydrocephalus

Burcu Albuz, Umit Ali Malcok, Fatma Silan and Ozturk Ozdemir It should be written as other authors on other abstracts

Department of Medical Genetics, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey

Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey

email address: [email protected]

Methylene tetrahydrofolate reductase deficiency (MTHFRD) is a common inborn error of folate metabolism among enzyme defiencies and caused by mutations in the MTHFR gene. The phenotype is highly variable, from severe neurologic findings in infant to asymptomatic adult. Here, we report a homozygous nonsense mutation in MTHFR gene in a hypotonic infant. A 1-month-old girl with hypotonia and nutrition problems was referred to our medical genetics department. She was born at 38 weeks of gestation. Her parents were nonconsanguineous but originated from same village. She was admitted to the neonatal intensive care unit at the age of 14 days. Seizures began and brain operations were performed because of hydrocephalus and supratentorial giant arachnoid cyst in this process. Echocardiography showed patent ductus arteriosus and aberrant band in the left ventricle. A homozygous nonsense, c.1072 C > T,(p.Arg358*) mutation in MTHFR gene was detected in whole exom sequencing.Her parents were heterozygous for the same mutation. MTHFR is a key enzyme of the folate cycle. MTHFR deficiency is a autosomal recessive disorder potentially treatable when properly diagnosed at early stages.The mutation of our patient was first reported in a male child with similar symptoms as our case in a Turkish family. Our case is important to raise awareness among clinicians about MHFRD patients.

The monitoring result of heavy metals in river water in the coastal area of Adjara

Nino Lomtatidze, Nargiz Alasania, Nino Kedelidze, Marine Gvianidze and Giuli Katamadze

Department of Biology, Faculty of Natural Sciences & Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia

email address: [email protected]

The population uses the rivers for domestic and agricultural purposes; therefore, the periodic monitoring of heavy metals at these sites is essential. We investigated the content of heavy metals in the rivers waters (Chorokhi, Mejinistskali, Cuba water, Bartskhana, Kintrishi) in the coastal area of Adjara. Accordingly, we analyzed the samples of river water at different seasons of the year. The research was carried out by atomic absorption spectrometer. The study showed an increased amount of lead (0.02 mg/L, p = 0.0001) in the Mejinistskali River based on the heavy metals content in rivers water during all periods. The high concentrations of cadmium (0.006 mg/L, p = 0.006) revealed in Kintrishi. Notably, increased lead content (0.02 mg/L; p = 0.00067) revealed in Kintrishi; we have thought that the permissible amount of heavy metals in the studied rivers during the fall-spring period should be linked to the increased amount of precipitation.

An NMR-based profile for fruits origin discrimination

Oana Romina Botoran , Marius Miricioiu, Diana Costinel, Roxana Elena Ionete  and Gabriel Lucian Radu

Politehnica University of Bucharest, Bucharest, Romania

National Research and Development Institute for Cryogenics and Isotopic Technologies - ICSI Rm. Valcea, Râmnicu Vâlcea, Romania

email address: [email protected]

Fruits have a unique fingerprint owing to a specific chemical profile characterized by different metabolites depending on the genotype and natural growth environment. This study attempted to discriminate fruits into groups considering their chemical composition obtained by ¹H/¹³C-NMR profiling and deuterium content of sugars measured by SNIF-NMR. For that, 17 types of fruits originating from Romania, during 2013–2017 harvest years, were considered. The ¹H/¹³C-NMR compositional profile of fruit juices correlated with the chemometric analysis of data allowed a correct classification in proportion of 100%, based on their origin and harvest year, highlighting the NMR signals important for differentiation. Due to their distinct composition, berry fruits separated very well from the stone fruits. When observing the deuterium content of sugars, some fruits (e.g. blueberries, raspberries, peaches, sour cherries and grapes) showed the influence of the environmental conditions (geographical and year of harvest), while others maintained their isotopic fingerprint regardless of the environment (e.g. strawberries, blackberries, apricots, cherries, pear). Emphasis is placed on the use of an integrated approach based on the compositional and isotopic fingerprint constructed from minor components of the fruit and a statistical model for data evaluation and interpretation that provides efficient means of combating fraud in this sector.

Plant function progress and challenges: using tree rings C-O isotope fingerprint in the study of climate change

Oana Romina Botoran, Constantin Nechita, Marius Miricioiu, Diana Costinel and Roxana Elena Ionete

National Research and Development Institute for Cryogenics and Isotopic Technologies - ICSI Rm. Valcea, Râmnicu Vâlcea, Romania

National Research and Development Institute for Silviculture "Marin Dracea", Voluntari, Romania

email address: [email protected]

Woodland environments and their component species have been involved in many evolutionary processes due to climate change, wherefore deepening knowledge with respect to the vegetation history and the response of species and ecosystems to these changes is basic in adopting dynamic preservation strategies. In this work, the stable isotope technique was used as a powerful approach for assessing the impact on biogeochemical processes at different scales based on the information stored by water and carbon in the plant. We aimed the development of a simple model to accurately estimate climate proxy information from six trees belonging to the Pinus spp. originating from northeastern Romania. Therefore, δ¹³C and δ¹⁸O values were determined in tree ring cellulose, in order to obtain the strongest climate signal. Negative relationships were noted between δ¹³C values and the mean annual temperature, as well as for δ¹⁸O and precipitation. Also, was envisanged the relationship between climate and the stable isotopic composition of pine populations growing in the same climatic microregion. The observed trends in stable isotope records emphasize the importance of studying the biogeochemical processes to achieve an appropriate prediction model for the reaction of plant metabolism to climate change.

An approach for targeted analysis of cell-free DNA fragmentation in liquid biopsies

Anastasia Koval, Daria Rodionova, Mikhail Chanyshev, Konstantin Blagodatskikh and Dmitry Shcherbo

Molecular Oncology Laboratory, Institute of Translational Medicine, Pirogov Russian National Research Medical University, Moscow, Russia

email address: [email protected]

Circulating nucleic acids are intensively studied as a component of liquid biopsies in cancer. A vast selection of methods has been developed in the past decade to derive sequence information from cell-free DNA (cfDNA) with high precision. In contrast, the layers of epigenetic information still require the development of sophisticated and reliable analysis approaches. The non-random fashion of cfDNA cleavage during shedding reflects epigenetic features of the dying cells. Specifically, several recent studies demonstrated the utility of cfDNA fragmentation patterns analysis at the whole-genome level for cancer diagnostics. Here we report a method for targeted quantification of DNA fragment end distributions in multiple genomic targets of choice. Single target-specific primer for each locus and incorporation of adapters containing unique molecular identifiers allow decreasing PCR biases while preserving the coordinates of the free ends of the fragments. The proposed approach could be applicable for cfDNA fragmentation evaluation in a target-specific manner, allowing for deep fragment end profiling in genomic regions of interest.

Funding: The study was supported by a grant from the Russian Science Foundation (project no. 20-75-10008).

Juniperus communis cell culture derived ingredients inhibit growth of acne causing bacteria and alleviate acne related inflammation in skin keratinocytes

Elza Kaktina, Baiba Silamikele, Madara Balode, Anna Ramata Stunda, Martins Boroduskis, Lita Grine, Dora Livkisa, Zaiga Petrina  and Reinis Rutkis

Alternative Plants Ltd., Riga, Latvia

Department of Microbiology and Biotechnology, Faculty of Biology, University of Latvia, Riga, Latvia

email address: [email protected]

Juniper Juniperus communis is a rich source of bioactive compounds. The composition of desired compounds varies during the season and is affected by growth conditions. This often makes industrial production of juniper derived compounds challenging. Plant cell cultivation is a valuable tool for efficient and consistent production of J. communis secondary metabolites. In this study, J. communis callus culture was established from surface sterilized in vitro grown internodal stem segments. Callus cultures were developed in photoperiod (16-h light/8-h dark) at 25 ± 1 °C. Effects of different media compositions on callus quality and its extract activity were evaluated. Hydroethanolic extracts of calli were analyzed by HPLC-MS. Total phenolic content (TPC) and DPPH scavenging activity was determined spectrophotometrically. Anti-microbial activity against acne-causing bacteria was evaluated and effects on HaCaT keratinocytes tested. The dominating compounds in callus extracts were dehydroabietic acid and procyanidins in various degrees of polymerization. Extracts inhibited growth of Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acne, Propionibacterium granulosum. In P. acne, lysate stimulated HaCaT keratinocytes extracts dose dependently stimulated expression of SOD1 and downregulated expression of TNFa and IL1B. Results show that J. communis callus culture extracts have a high potential for anti-acne applications.

Funding: Research was supported by Competence Center project (no. 1.2.1.1./18/A/007).

Fibrinolytic proteases of micromycetes promising in biomedicine and biodegradation

Svetlana Timorshina, Yelyzaveta Boiko, Elena Kornienko, Anastasia Orekhova and Alexander Osmolovskiy

Lomonosov Moscow State University, Moscow, Russia

email address: [email protected]

Enzymatic cleavage of fibrin, which is a fibrillar protein and the main component of blood clots, is a significant process in the recycling of animal waste and the treatment of thromboembolism. However, these two tasks require different properties of proteolytic enzymes. Micromycetes are known as producers of exoproteases with different substrate specificity. In this regard, a screening for the ability to produce fibrinolytic enzymes was conducted on filamentous fungi of various taxonomic groups. Activity against fibrin and activator activity against plasminogen of proteases was measured using fibrin plates, caseinolytic activity was measured spectrophotometrically. Extracellular enzymes of Sarocladium strictum have the highest fibrinolytic (843.0 U/mL) and plasminogen activator activity (574.3 U/mL) among the studied cultures. Moreover, the proteases of this micromycete show low activity against casein (29.2 μmol Tyr/mL × min), which may indicate their high-substrate specificity. Proteolytic enzymes with such properties are promising in medicine for the treatment of diseases associated with hemostasis system dysfunction. Proteases of Aspergillus chevalieri have lower activity against fibrin (129.9 U/mL) and plasminogen (124.9 U/mL), however this culture has a high caseinolytic activity (398.3 μmol Tyr/mL × min), due to this A. chevalieri can be considered as a producer of enzymes useful for biodegradation.

ELISAs for determination of ochratoxin A by monoclonal anti-ochratoxin A antibody or F(ab')2 fragment

Zlatina Becheva, Yavor Ivanov  and Tzonka Godjevargova

Department of Biotechnology, Prof. d-r A. Zlatarov University, Burgas, Bulgaria

email address: [email protected]

Ochratoxins are possible human carcinogens and development of sensitive immunoassay for its determination in foods is very important task. Ochratoxins can be found in various agricultural commodities. The aim of this study was to develop sensitive ELISAs on the base of mice monoclonal antibody (mAb) against ochratoxin A (OTA) or its F(ab')₂ fragment. The mice mAb was produced in vitro using tissue-culture techniques. The activity of the obtained anti-OTA mAb was measured. F(ab′)₂ fragment of anti-OTA mAb was obtained by pepsin hydrolysis of the produced mAb. A indirect ELISAs were performed by using obtained F(ab')₂ fragment and anti-OTA mAb. Peroxidase-labelled secondary antibody was used. The analytical characteristics of the indirect ELISA with F(ab′)₂ fragment were compared with those with mAb. The linear range of OTA in phosphate buffer at immunoassay with F(ab')₂ fragment was from 0.1 to 100 ng/mL. The linear range of immunoassay with the whole anti-OTA mAb was from 0.1 to 10 ng/mL. The obtained results were compared by certified mice mAb against ochratoxin A. The linear range with the certified mAb was from 1 to 500 ng/mL. The developed ELISAs with obtained F(ab′)₂ fragment or produced anti-OTA mAb have possibility to measure low OTA concentration.

Quercetin affects the expression of microsomal proteins in obese rats

Vladimir Alexandrovich Shipelin, Ilya Evgenevich Sokolov, Natalia Victorovna Kirbaeva and Ivan Vsevolodovich Gmoshinski

Federal Research Centre of Nutrition and Biotechnology, Moscow, Russia

email address: [email protected]

The development of functional foods requires studying the molecular aspects of the metabolic processes regulation in obesity and evaluating the efficiency of their correction with biologically active substances. It is known that the quercetin may alleviate symptoms of a metabolic syndrome caused by consuming an excess of simple carbohydrates and fat. In the present 65-day study, Wistar rats with obesity induced by a high-fat-carbohydrate diet received quercetin at a dose of 50 mg/kg body weight daily. At the end of the experiment, 46 differentially expressed proteins isolated by 2D-electrophoresis from the liver microsomal fraction (MFL) were analyzed by LTQ-Orbitrap mass-spectrometer. The development of obesity contributed to expression in MFL proteins such as heat shock cognate 71 kDa, endoplasmic reticulum chaperone, protein disulfide-isomerase, hemoglobin subunit beta-1, and hemoglobin subunit alpha-1/2, while quercetin supplement cancelled these effects. Excepting the ectopically expressed hemoglobin proteins, other identified proteins play an important role in cell protection from stress, regulating the folding and refolding processes, proteolysis and posttranslational transport. Thus, quercetin in the studied dose has a positive effect on the compensatory homeostasis processes in damaged liver tissue in rats with diet-induced obesity.

Funding: The research was supported by a grant from the Russian Science Foundation (no.17-16-01043).

Antimicrobial activity of selected plant extracts and its possibility to effect human health

Marie Vysoka, Vittorio Lorenzo Mazzarello, Luigi Sciola, Giuseppe Delogu  and Ivana Marova

Faculty of Chemistry, Institute of Food Science and Biotechnology, Brno, Czechia

University of Sassari, Sassari SS, Italy

email address: [email protected]

This study was designed to evaluate the antimicrobial activity and cytotoxicity of selected plant extracts and its possibility to effect human health. Herbal medicines are used widely because of their potent therapeutic efficacy, antioxidant activities and no side effects. In this study, tested samples of herbs were prepared as water extracts and essential oils. All extracts were prepared from dry samples of Curcumin, Euterpe oleracea, Origanum vulgare, Panax Ginseng, Syzygium aromaticum and Zingiber officinale. Antimicrobial effect was tested on Staphylococcus aureus, Candida glabrata and Escherichia coli. Cytotoxicity was tested by colourimetric MTT test on keratinocytes HaCaT and B16F1 and also on other cell lines, e.g. Caco-2, Raji. In all extracts was detected concentration-dependent antimicrobial effect. Apoptosis, the cell’s natural mechanism for death, is a promising target for anticancer therapy. The hallmarks of cancer are present in all cancer cells regardless of the cause or type; these include uncontrolled growth, angiogenesis and apoptosis evasion. The prevention of cancer is one of the main functions of apoptosis. In this study was tested the apoptotic effects of selected plant extracts of Raji cell lines.

Biocompatibility evaluation of novelty bone cement

Bogdan Sevastre, Alexandra I. Dreanca , Baldea Ioana, Marioara Moldovan, Cosmin Pestean, Flaviu Tabaran, Sanda Andrei, Liviu Oana and Ioan Marcus

University of Agricultural Sciences and Veterinary Medicine, Cluj Napoca, Romania

“Iuliu Hațieganu” University of Farmacy and Medicine, Cluj Napoca, Romania

Babes Bolyai University, Cluj Napoca, Romania

email address: [email protected]

Bone cement is a material used to fix implants and fill cavities following traumatic surgical procedures. The purpose of this study was to evaluate the biocompatibility response of bone cement, based on polylactic acid, by evaluating tissular specific bone markers. The effectiveness of the treatment has been investigated using an experimental non-critical femoral defect protocol. Thirty-five Wistar rats were divided into negative and positive controls and experimentally treated groups. Bone tissue samples were collected at 30, 60 and 90 days, postoperatively. Oxidative stress markers from bone proteic extract (spectofotometry), bone markers (TNFα and osteocalcin) (Western blott) and local biomaterial osteointegration were evaluated. Bone cement treatment had a protective role by maintaining higher glutathione levels, thus decreasing cellular damage and osteoresorbtion. The moderate increase in tissue necrosis factor does not prevent the expression of osteocalcin. According to the analysis of local irritability, the tested cement was non-irritative at 3 months postoperatively, also demonstrating in histopathological findings, osteointegrity and osteotransductivity properties. The treatment with polylactic acid based bone cement is osteoinductive, being integrated into a partially mineralized osteoid mass, focally delimited by fibrous connective tissue.

Funding: This project is funded by the Ministry of Research and Innovation of Romania, Contract no. 37PFE/06.11.2018.

Pro- and antioxidant activity of a horse-derived biomaterial after intense physical exertion

Oksana Kolenchukova , Valentina Kratasyuk, Ludmila Stepanova, Alecsandr Kolomeytsev, Arina Fedotova  and Irina litvinova

Siberian Federal University, Krasnoyarsk, Russia

Krasnoyarsk State Agrarian University, Krasnoyarsk, Russia

Scientific Research Institute of Medical Problems of the North FRC KSC SB RAS, Krasnoyarsk, Russia

email address: [email protected]

The aim is to study oxidants in blood, serum and saliva in horses in the training process. Object was saliva of sports horses (n = 12) of the Trakenens breed, specializing in dressage. Testing of horses was carried out before and after training with light, medium and heavy standard physical activity. Heart rate (HR) and respiratory rate (RR) were measured. Saliva, blood serum and phagocytes testing were carried out using the chemiluminescence.: The obtained results characterize the increased activity of the oxidative system of phagocytes (p = 0.048; p = 0.037), serum (p = 0.031; p = 0.043) and saliva (p = 0.015; p = 0.013) in horses at two stages of training activity, as well as the activation of only the antioxidant system in the third stage (p = 0.025; p = 0.047) with maximum physical activity. Showed the presence of a positive dependence between physical activity of medium intensity and increase in RR (r = 0.65, p = 0.05). With a gradual increase in training activity, the horse’s organism adapts to the tested physical stresses, as a result, there is no change in the production of oxidants.

Funding: The reported study was funded by RFBR and Government of the Krasnoyarsk territory, project number 19-416-240001.

Enzymatic synthesis of gold and silver nanoparticles

Lela Martinaga, Katarina Komljenović, Petra Pohanić, Ana Vrsalović Presečki, Roland Ludwig  and Iva Rezić

Department of Applied Chemistry, University of Zagreb Faculty of Textile Technology, Zagreb, Croatia

Department of Reaction Engineering and Catalysis, University of Zagreb Faculty of Chemical Engineering and Technology, Zagreb, Croatia

Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria

email address: [email protected]

The usage of metal nanoparticles with exactly defined characteristics and their synthesis is among the most investigated research topics in the last decades. Gold and silver nanoparticles (NP) are considered as one of the most interesting nanoparticles due to their widespread application in healthcare, electronics, renewable energy and others. By their implementation in the products of the food and textile industry, many improvements in antibacterial, UV protection, flame retardant and/or hydrophobicity properties can be achieved. To overcome disadvantages during the conventional NPs synthesis, i.e. use of harsh chemicals and/or reaction conditions, enzymatic synthesis of Au- and Ag-NPs as a more convenient method was carried out in this research. For that purpose, two oxidoreductases: cellobiose dehydrogenase and glucose dehydrogenase were employed. The impact of the reaction conditions (temperature, enzyme and reactants concentration, reaction medium, pH, light and oxygen) was investigated and optimized. Successfully synthesized NPs were characterized by UV–vis spectroscopy and transmission electron microscope (TEM). The determination of reaction kinetics and model development enabled insight in the reaction mechanism of the enzymatically catalyzed NPs.

Funding: This work was financially supported by the Croatian Science Foundation, Project IP-2019-04-1381 under title “Antibacterial coating for biodegradable medicine materials ABBAMEDICA.”

Genetic markers in the study of threatened with extinction cattle breeds

Madalina Alexandra Davidescu , Andrei Cristian Gradinaru, Mitica Ciorpac, Madalina Henea  and Steofil Creanga

Faculty of Animal Husbandry, University of Agricultural Sciences and Veterinary Medicine of Iasi, Iasi, Romania

Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine of Iasi, Iasi, Romania

Research and Development Station for Cattle Breeding Dancu, Iasi, Romania

email address: [email protected]

Extinction of cattle breeds threatens genetic diversity of livestock species and also, the cattle breed is a significant animal crop for human existence. According to the Food and Agriculture Organization of the United Nations (FAO), about one-third of the recorded livestock species are listed as having a significant chance of extinction and about 1000 have vanished in the last 100 years. The desire to protect genetic diversity is generally recognized for environmental, economic and cultural purposes. Domestic Animal Diversity Information System (DAD-IS) provides with information to track national breeding populations and to make educated decisions on the management of animal genetic capital. Assessing the demographic structure and genetic variability of breeds by knowledge given by molecular markers facilitates the assessment of their likelihood of extinction and the creation of strategies for their management and protection. Indigenous species should be periodically tracked using molecular markers, and management plans should be revised on the basis of community parameters extracted from these analyses. The purpose of this study is to analyze the genetic status of extinct cattle breeds through molecular data analysis.

Prospects for use of the tea catechins enzymatic oxidation products as α-glucosidase inhibitors: in silico study

Varuzhan A. Sarkisyan, Yuliya V. Frolova, Roman V. Sobolev and Alla A. Kochetkova

Laboratory of Food Biotechnology and Foods for Special Dietary Uses, Federal Research Center for Nutrition and Biotechnology, Moscow, Russia

email address: [email protected]

The search for new effective inhibitors of α-glucosidase is one of the actual directions in the treatment of type 2 diabetes mellitus. Tea catechins are well known as potential α-glucosidase inhibitors. Fermentation results in the formation of theasinensins, theaflavins, theanaftoquinones and theacitrines. These compounds are poorly investigated as potential inhibitors. This study aims to identify potential α-glucosidase inhibitors among the products of enzymatic degradation of tea catechins. For this, we applied methods of molecular docking using HADDOCK 2.4 and molecular dynamics using GROMACS 2018. MAL12 enzyme was used as a target, prepared by homology modelling using Modeller. Docking and molecular dynamics of catechins were performed with three MAL12 models obtained from free-energy landscape analysis. Thirty-nine ligands, including acarbose (a known competitive inhibitor), were analyzed. The complexes with the highest docking scores were analyzed by 30 ns molecular dynamics, followed by the free-energy analysis with MMPBSA. Our results show that complexes of theasinensins and theaflavins, such as the epigallocatechin gallate trimer and theaflavate C, are more stable than acarbose complex, which makes them potential inhibitors of α-glucosidase and determines the viability of their targeted enzymatic synthesis.

Funding: This work was supported financially by the Russian Science Foundation (Project no. 19-76-30014).

The growth of Euglena gracilis in the presence of bacteria and fungi without the addition of vitamins

Alexandra Lukáčová, Terézia Beck, Juraj Krajčovičand Matej Vesteg

Department of Biology and Ecology, Faculty of Natural Sciences, Matej Bel University, Banská Bystrica, Slovakia

Department of Biology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Trnava, Slovakia

email address: [email protected]

Euglena gracilis can be used for biotechnological production of feed supplements, biofuels, industrial chemicals and pharmaceuticals. E. gracilis is not able to synthesize thiamine and cobalamin and these vitamins have to be added to media for the growth of axenic cultures. Since these vitamins are expensive, the cultivation of E. gracilis in containers for biotechnological applications is limited by their price. Thiamine can be, however, synthesized by most bacteria, plants and fungi, and cobalamin by some bacterial and archaeal species. This study has revealed that no vitamins need to be added to media, in which E. gracilis is co-cultivated with bacteria Bacillus badius and Lysinibacillus boronitolerans, and the fungus Cladosporium sp. E. gracilis cells are viable and most of them are motile in the co-culture suggesting that these microorganisms can produce these vitamins essential for E. gracilis. Some non-motile viable E. gracilis cells appear to be specifically associated with the fungus in the co-culture suggesting a nutrition exchange between these microorganisms. We propose that the usage of such mixed cultures for biotechnological applications can reduce the price of cultivation media for euglenoid flagellates grown in containers.

Investigation of autoinflammatory genes in patients with conventional treatment-resistant dermatological diseases

Selda Isik Mermutlu, Burcu Albuz, Nihan Ecmel Akbas, Fatma Silan and Ozturk Ozdemir

Department of Medical Genetics, Canakkale Onsekiz Mart University, Canakkale, Turkey

email address: [email protected]

Autoinflammatory syndroms includes a group of diseases that are characterized by recurrent fever and serositis attacks, lymphadenopathy, skin lesions, joint and muscle complaints in adults. In this study, it was aimed to identify the mutational profiles of pleiotropic autoinflammatory genes in some treatment-resistant dermatological diseases. In total, 10 individual DNA samples were genotyped for autoinflammatory panel including target 11 systemic pleiotropic genes (MEFV, TNFRSF1A, ADA2, MVK, NOD2, IL1RN, ELANE, IL10, PSTPIP1, LPIN2, PL CG2) by IonTorrent S5 NGS platform and analyzed with IonReporter and The IGV. Autoinflammatory NGS panel containing 11 genes was studied in 10 patients who were followed up for treatment-resistant hidradenitis supurativa, urticaria, Behçet’s disease and psoriasis. A total of 29 variants were detected in patients and 45% of these variants were pathogenic, likely pathogenic, VUS mutations, which would explain the patients’ symptoms. Mutations that explain the symptoms of patients 38% MEFV, 23%PLCG2, 15%NOD2, 8%IL10, 8%LPIN2, 8% IL1RN gene were detected. %50 LPIN2 mutations detected in Behçet diseases, %40 MEFV, %40 NOD2, %40 PLCG2 mutations detected in hydradenitis supurativa, 100% MEFV, 33% IL10, 33% PLCG2, 33% IL1RN mutations detected in urticaria patients.There are no adequate studies on the effect of autoinflammatory genes in diseases with treatment-resistant skin symptoms, so the diagnosis of patients is delayed. Studies are needed in larger patient groups related to these diseases and we think that our study will contribute to the literature.

The chemical composition and antioxidant activity of Inonotus obliquus and Fomitopsis pinicola extracts

Irina Sergeevna Kiseleva, Alexander Anatolyevitch Ermoshin and Ma Chaomei

Department of Experimental Biology and Biotechnology, Ural Federal University, Ekaterinburg, Russia

College of Life Sciences of Inner Mongolia University, Huhhot, China

email address: [email protected]

The content of 20 phenols and terpenes in ethanol extracts (5 mg/mL) and their antioxidant activity in vitro were studied in Fomitopsis pinicola and Inonotus obliquus – chaga. The total phenols were 3.1 mg/g in Fomitopsis and 4.9 mg/g in chaga. The UHPLC-MRM-MS method revealed caffeic, p-coumaric, chlorogenic and ferulic acids from 0.22 to 17.8 μg/g, catechin (0.5–0.8 μg/g). Resveratrol (8.4 μg/g) was indicated only in chaga. In both species, triterpenic acids and alcohols 20-OH Lucidenic acid N, Ganoderic acid A, Ganoderiol A, D, F, Licidumol A were found. They were previously discovered in ganoderma as anticancer substances. Of particular interest was the detection of ergosterol peroxide that has antimalaria, anti-tuberculosis and anti-cancer activity. Chaga contains 6.6 μg/g and Fomitopsis – 151.7 μg/g, which is comparable to the content in known medicinal fungi. Fomotopsis extract showed a higher NO-quenching activity than gallic acid (28.5% vs. 21.1%), while in chaga – 15.6%. In turn, chaga extract effectively inhibited the formation of superoxide anion (46.1%) compared to rutin (2.9%) and gallate (27.4%), while Fomitopsis did not. The presence of triterpenoids, a high content of ergosterol peroxide and pronounced antiradical and reducing activity makes Fomotopsis promising for biotechnology application.

Degradation of food dyes by white rot fungus Phanerochaete chrysosporium

Martina Vršanská, Andrea Blahutová, Nikola Schlosserová and Stanislava Voběrková

Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic

CEITEC – Central European Institute of Technology, Mendel University in Brno, Brno, Czech Republic

email address: [email protected]

One of the most important and largest group of commercially produced synthetic colours are azo dyes. Their degradation by Phanerochaete chrysosporium is induced by a lignin-degrading enzyme system, which is known for degrading many xenobiotic compounds. This study focuses on the degradation of the five commonly used food dyes by P. chrysosporium. The process has been monitored not only on individual dyes (E102 Tartrazine, E110 Yellow, E122 Azorubin, E124 Ponceau 4R and E123 Amaranth), but also on two commercial food products (Vitacit – lemon and Vitacit – strawberry) containing E102 Tartrazine or E122 Azorubin azo dye. Azorubin dye showed the highest decolourization around 90%, Tartrazine was degraded around 70% and the Ponceau 4R dye was decolourized the least 20% after 20 days of cultivation. The level of decolourization of the powdered beverage solutions was compared with the decolourization of the individual dyes. Results suggest that fungus can be employed as a vital biological tool for developing wastewater treatment systems for decolourization of dye effluents through biosorption or biodegradation.

Crude laccase-based biosensor for the detection of catechin

Polina Nadtochaeva, Ester Badinova, Lukas Richtera  and Stanislava Voberkova

Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic

Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic

Central European Institute of Technology, Mendel University in Brno, Brno, Czech Republic

email address: [email protected]

In this work, a simple laccase biosensor for detection and determination of (+)-catechin concentration was prepared. The native enzyme was isolated from Trametes versicolor followed by immobilization with glutaraldehyde using crosslinked enzyme aggregates (CLEA). Crosslinking of the enzyme helps to maintain its activity and improves stability. The electrocatalytic activity of biosensor was investigated by differential pulse voltammetry using a carbon paste electrode modified with lyophilized laccase. Experimental parameters such as applied potential, pH and amount of the enzyme were optimized. Carbon paste with laccase was characterized by several parameters like storage, reaction and thermal stabilities and temperature optimum. The results show that enzyme paste was stable, with 85% retention of initial activity after 63 days of storage and 61% after exposure temperature 55 °C during 60 min. The prepared biosensor was tested on real samples of green teas and concentrations of catechin were approximately the same in all four kinds of teas and ranged from 19.57 to 24.91 mg/L. The biosensor prepared from native laccase was shown to be a simple, economic and accessible analytical tool with high stability and sensitivity.

Comparison of CD34+ and CD45+ cells enumeration by three different fluorescent methods

Dimitrina Krasteva, Yavor Ivanov, Zlatina Becheva and Tzonka Ivanova Godjevargova

Department of Biotechnology, Prof. d-r A. Zlatarov University, Burgas, Bulgaria

email address: [email protected]

Enumeration of the stem cells and leukocytes in peripheral blood and apheresis samples was performed by using biomarker expression of CD34+ and CD45+ proteins on their cell surface. The aim of this study was simultaneous counting of live stem cells and leukocytes in apheresis samples by using two different fluorescent conjugates. The conjugate between anti-CD34 monoclonal antibody and fluorescent dye DR110 stained stem cells in green colour. The conjugate between anti-CD45 monoclonal antibody and fluorescent dye ATTO620 stained leukocytes in red colour. The conjugates anti-CD34 antibody – DR110 and anti-CD45 antibody – ATTO620 were prepared by carbodiimide method and purified by gel filtration. The counting was performed by using a new automatic fluorescence microscope EasyCounter BC. The dead cells were counted by monomethyne cyanine dye – Sofia Green. The analyses of six apheresis samples were performed. The obtained results by EasyCounter BC were compared with results received by other two standard methods – flow cytometry (Guava easyCyte 8HT) and fluorescent microscopic method (Olympus BX51). The coefficients of variation of the obtained results with EasyCounter BC (3%–6%) were lower than those with the flow cytometer (5%–8%) and with the Olympus (14%–18%).

Spinach leaves extract: technology development and biochemical assessment in vivo

Yuliia Sidorova, Nikita Petrov, Sergey Zorin and Vladimir Mazo

Federal Research Center for Nutrition, Biotechnology and Food Safety, Moscow, Russia

email address: [email protected]

Phytoecdysteroids are biologically active substances used to relieve chronic fatigue syndrome, to improve memory and attention processes. Flavonoids are plant polyphenols with strong antioxidant properties. Complexes of flavonoids and phytoecdysteroids can be isolated not only from medicinal, but also from food plants, e.g. spinach. We have developed the dry extract production method from spinach leaves, which includes the following stages: extraction, ultrafiltration and adsorption on the hydrophobic carrier and enables us to concentrate phytoecdysteroids and flavonoids more than 30 times and remove oxalic acid. We have conducted in vivo study to assess the effect of spinach extract on cognitive functions and stress resistance of rats subjected to immobilization or exercise until exhaustion. The extract prevented the increase of dopamine, noradrenaline and adrenaline in the urine, significantly reduced triglycerides and levelled the growth of AST and ALT, which is possible with increased physical exercise. The result obtained indicates the relative activation of stress-limiting systems of stressed animals when they consume the phytoecdysteroid-containing extract. The observed adaptogenic effect is associated with a balanced adequate change in the level of the most important stress mediators – stress activators and inhibitors.

Funding: The study was supported by Russian Science Foundation, grant no. 19-16-00107.

Biocompatibility study of soil microorganisms

Svetlana Ibragimova, Mariya Milina and Victor Revin

National Research Mordovia State University, Moscow, Russia

email address: [email protected]

High-efficiency characteristic for preparations includes several types of microorganisms and various biologically active substances. In the biological preparation, the titer of active cells depends on the biocompatibility of the studied bacteria. We used bacteria that are characterized by xenobiotic biodegrade, growth-promoting and fungicide properties. With a different combination of the studied bacteria, antagonism with respect to each other is possible. This work is aimed at determining the biocompatibility of the studied bacteria. The biomass level is determined by the combination of the studied bacterial species. The maximum amount of biomass in the variant with P. putida and R. erythropolis was 10.6 g/L, in the variant with P. aureofaciens, B. subtilis and A. vinelandii – 14.7 g/L. In the consortium with all cultures, the maximum biomass value increased 16 times relative to the initial one, the titer was 10¹⁰ CFU/mL, which corresponds to the conditions required for biological preparation. In a consortium, species diversity of bacteria is observed, which indicates about their compatibility. The method of perpendicular strokes showed that P. aureofaciens and B. subtilis were predominant over other cultures.

Funding: This work was supported by the RFBR grant no. 18-416-130003\19.

Inspection of PHB accumulation in cyanobacteria via spectral flow cytometry and fluorescence microscopy

Zuzana Sedrlova , Eva Slaninova, Jakub Drinka, Filip Mravec, Ines Fritz  and Stanislav Obruca

Faculty of Chemistry, BUT, Brno, Czech Republic

IFA-Tulln, University for Natural Resources and Life Sciences, Tulln, Austria

email address: [email protected]

Cyanobacteria are ecologically extremely important phototrophic Gram-negative bacteria capable of oxygenic photosynthesis. They synthesize many interesting metabolites such as glycogen, carotenoids, but the most interesting metabolites in are polyhydroxyalkanoates (PHA). PHA are biodegradable biopolymers with similar properties as petrochemical synthetic plastics. Numerous prokaryotes including cyanobacteria use PHA as a storage of carbon and energy in the form of intracellular granules. PHA can be produced by heterotrophic bacteria as well, such as Cupriavidus necator H16 or Methylobacterium rhodesianum. The main advantage of cyanobacteria is the fact they do not require costly organic substrate and, oppositely, cyanobacteria can fix CO₂. In this work, we focused on involvement of PHB in robustness among various environmentally relevant stress factors. Nowadays, the flow cytometry is a popular technique which can be used to detect biological and chemical characteristics of a microbial population. In this work, we developed methodology to determine PHB content in cyanobacterial strains Synechocystis PCC 6803 and Synechocystis CCALA 192.

Funding: This study was partly funded by the project GA19-19-29651L of the Czech Science Foundation (GACR) and partly funded by the Austrian Science Fund (FWF), project I 4082-B25. Zuzana Sedrlova is Brno Ph.D. Talent Scholarship Holder – Funded by the Brno City Municipality.

FLISAs for determination of staphylococcal enterotoxin A by immobilized monoclonal anti-enterotoxin A antibody or F(ab′)2 fragment on magnetic nanoparticles

Zlatina Rumenova Becheva, Yavor Lukanov Ivanov and Tzonka Ivanova Godjevargova

Department of Biotechnology, "Prof. Dr Assen Zlatarov" University, Burgas, Bulgaria

email address: [email protected]

Foods with high-protein content, such as milk, meat and salads, have been frequently incriminated in staphylococcal enterotoxin (SЕ) outbreaks. Staphylococcal enterotoxin A (SEA) is the most toxic SE. The threat of SEA requires food analyses that measure the lowest possible concentrations. Fluorescent-linked immunosorbent assays (FLISAs), sandwich format, for SEA detection in milk were developed, using immobilized anti-SEA monoclonal antibody or F(ab′)₂ fragment onto magnetic nanoparticles (MNPs). F(ab′)₂ fragment of monoclonal anti-SEA antibody (mAb) was obtained by pepsin hydrolysis. Fluorescent conjugates of the fragment and mAb with fluorescein isothiocyanate (FITC) were prepared and purified. The sandwich FLISAs were performed using the obtained FITC-conjugates. Analytical characteristics of the analyses with F(ab')₂ conjugate were compared with those with mAb conjugate. F(ab')₂ fragment – FITC conjugate detection was with linear range of SEA from 0.5 to 300 pg/mL in buffer, and from 1.0 to 300 pg/mL in milk. While, the analyses with mAb-FITC had linear ranges from 50 in buffer and from 70 in milk to 300 pg/mL SEA in both cases. The detection limit with F(ab')₂ fragment (0.9 pg/mL SEA) was 10 times lower than that with whole mAb (9 pg/mL SEA) in milk. Therefore, the fragment provided a more sensitive method.

DNA analysis of human skeletal remains from the medieval necropoles in Bosnia and Herzegovina

Mirela Dzehverovic, Jasmina Cakar, Edin Bujak, Abdurahim Kalajdzic, Amela Pilav, Lejla Pojskic and Naris Pojskic

University of Sarajevo-Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

Department of Archaeology, University of Sarajevo-Faculty of Philosophy, Sarajevo, Bosnia and Herzegovina

email address: [email protected]

Archaeological sites represent a rich historical and cultural heritage of medieval Bosnia. Among the archaeological sites, the most of them are with stećak tombstone. Stećak is medieval tombstone that appears in the period between the 12th and 16th century AD primarily on the territory of today's Bosnia and Herzegovina. Every stećak is a gravestone, under which one or more people are buried. Because of its unique numbers, wealth of decorative motives and the timeless epitaphs, they are listed on the UNESCO World Heritage List. In cases where there is no historical and archaeological context, skeletal remains found at archaeological sites represent a valuable source of information. Studying the archaeological remains using molecular methods can give more detailed insights into historical times. Analysis of nuclear STRs, Y STRs and mDNA аncient DNA (aDNA) has the potential to identify sex and precise kinship patterns between groups of skeletons. The aim of this study was to obtain usable DNA profiles, then mutually compare them and potentially find kinship relations, and determine haplogroup based on obtained Y-STR profiles and mDNA sequence.

The influence of selected encapsulation techniques on the viability of probiotics

Julie Hoova, Petra Matouskova and Ivana Marova

Faculty of Chemistry, Institute of Food Science and Biotechnology, Brno University of Technology, Brno, Czech Republic

email address: [email protected]

Probiotics as part of human microbiome show health benefits. Probiotics are commonly used in supplements to increase their amount in human gut. Due to low pH and other factors, probiotic viability might be decreased on its way to large intestine. Encapsulation methods and prebiotics might help protecting probiotic growth. Selected probiotic strains, bifidobacteria and lactobacili, were cultivated. The influence of enzymes and various pH on probiotic viability was observed by adding model digest juices. Moreover, probiotic bacteria were encapsulated into alginate particles and/or lyophilized and model digestion was also performed. The influence of different approaches of encapsulation methods on the probiotic viability were determined by using cultivation assay, spectrophotometry or flow cytometer. Prebiotics were added into samples to analyze the effect on viability during model digestion. Probiotic cells were more sensitive to gastrointestinal environment without encapsulation. The probiotic viability was higher after encapsulation treatment and by adding prebiotics. However, lyophilized alginate particles need more time for rehydration to be released to environment after the digestion. Longer storage of probiotics in supplements and probiotic protection can be achieved by combining encapsulation into alginate particles and lyophilization.

Funding: This work was supported by the project Nr. FCH-S-20-6316 of the Faculty of Chemistry, BUT.

Metabolic engineering of the methanol utilization pathway of Pichia pastoris for increased tolerance to formaldehyde toxicity

Julio Berrios, Belén Ponce, Edgar Velastegui, Karlo Guerrero, Claudia Altamirano, Chrispian W. Theron, Sébastien Steels  and Patrick Fickers

School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile

Gembloux Agro-Bio Tech, TERRA Teaching and Research Centre, University of Liege, Gembloux, Belgium

email address: [email protected]

The methylotrophic yeast Pichia pastoris has become a popular host for recombinant protein production. Methanol both induces the synthesis of the recombinant protein and serves as carbon and energy source by the cell. First, it is oxidized to formaldehyde and then it can be either further oxidized to CO₂ (dissimilative pathway) or assimilated to biomass (and also the recombinant protein) through the enzyme dihydroxyacetone synthase (DAS). In bioreactors, methanol feeding during induction phase can lead to formaldehyde accumulation. In order to prevent this accumulation and, at the same time, to favour the carbon to be directed through the assimilation pathway, we have overexpressed the gene DAS1 of Pichia pastoris. We tested the tolerance of engineered strains to 2, 5 and 8 mM of formaldehyde. A growth inhibition model was used to quantify the formaldehyde effect on the maximum specific growth rate (µmax), obtaining an inhibition constant K_i = 13.0 mM in the modified strain (K_i control = 4.6 mM), evidencing an important increase in the tolerance to formaldehyde. Interestingly, the engineered strain evidenced a 30%-increase in µmax, compared to the control. The impact of this modification on the production of recombinant protein is also discussed.

Production of P(3HB-co-4HB) copolymer by new thermophilic isolate Aneurinibacillus sp. H1 in batch, repated-batch and fed-batch cultivation modes of cultivation

Iva Pernicová, Xenie Kouřilová, Ivana Nováčková, Petr Sedláček and Stanislav Obruča

Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic

email address: [email protected]

Extremophilic organisms are promising producers of biotechnological productions, because thanks to living in extreme conditions the biotechnological process is not so demanding on sterility and the biotechnological price of production is cheaper. Extremophiles can live in high temperature, low or high pH, high salinity, etc., or a combination of these extreme conditions. Thermophiles are organisms that live and thrive at higher temperatures. Among them, producers of polyhydroxyalkanoates (PHA) can be found. PHA are microbial polyesters that are fully degradable and biocompatible. They are also a good alternative to petroleum polymers. Using our original protocol to isolate thermophilic PHA producers from natural source, we isolated the bacteria, which, was designated as Aneurinibacillus sp. H1. This bacterium is capable of producing poly(3-hydroxybutyrate) (P3HB) on glycerol (even on waste glycerol), and, furthermore, when supplemented with proper precursors, it is able to produce copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or even terpolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) with very high 4-hydroxybutyrate and 3-hydroxyvalerate fractions. These copolymers reveal superior properties as compared to P3HB copolymer. The purpose of this work was to compare various cultivation scenarios such as batch, repeated-batch and fed-batch to maximize polymer yields maintaining astonishing properties.

Identification, isolation and characterization of substances with antimicrobial activity in Lonomia obliqua hemolinph

Ronaldo Zucatelli Mendonça, Walter José Silveira, Patricia Yumi Hayashida  and Pedro Ismael Silva Junior

Parasitology Laboratory, Institut Butantan, São Paulo, Brazil

Special Laboratory of Applied Toxinology, Institut Butantan, São Paulo, Brazil

email address: [email protected]

Resistant bacterial strains are a serious public health problem killing thousands of patients annually, making the search for new antibiotics an urgent necessity. Bioprospection from insect exudates has become a frequent source for new antibiotics. The objective of this work was the identification, isolation and chemical characterization of molecules with antimicrobial action in the hemolymph of Saturniidae caterpillars. Hemolymph obtained was centrifuged, inactivated, sterilized and applied on Sep-Pack®C18 column. Antimicrobial assays were performed with Cladosporium herbarum, Escherichia coli, Micrococcus luteus, Salmonella arizonae and Staphylococcus aureus strains. Antimicrobial activity was observed in all bacteria strains. The fractions showing activity were concentrated and subjected to HPLC chromatography. Elution was performed with 0%–80% acetonitrile in 0.05%TFA at a flow rate of 1.5mL/min. Cytotoxic and hemolytic activity was performed. None of the fractions with antimicrobial activity showed cytotoxic or hemolytic activity. The minimum inhibitory concentration was 1–3 µg/mL. Concentrations as low as 0.50 µg/mL were sufficient to neutralize S. aureus. The hemolymph fractions with antimicrobial activity were applied to the mass spectrometer. The final compound showed activity against the resistant hospitalar strains, S. aureus, S. epidermidis, M. luteus, E. coli, Serratia marcescens, showing that hemolymph can be a source of a new antibiotics.

In silico analysis of calpains among pro- and eukaryotic microorganisms

Dominika Vešelényiová, Andrea Patlevičová, Mária Schneiderová and Juraj Krajčovič

Department of Biology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius in Trnava, Trnava, Slovakia

email address: [email protected]

Calpains are enzymes that belong to a superfamily of calcium-dependent cysteine proteases. They have been found to cleave various substrates, which makes them important for many biological processes. Although calpains are well conserved among eukaryotes, most information is coming from studies of mammals and plants, and little attention is paid to calpains in unicellular eukaryotes and prokaryotes. For this reason, we conducted in silico identification of calpain homologs among eukaryotes, especially unicellular flagellates from taxon Euglenozoa. We also searched for calpains in selected cyanobacteria and species from taxon Azgard Archaea, since previous studies reported absence of calpains in Archaea. Our results show that there is a surprising expansion of calpain proteins in euglenozoan microorganisms. We also identified bacterial calpain homologs in several cyanobacterial species, but not in Archaea. We conducted phylogenetic analysis of all identified calpains to better understand their evolutionary relationships and events that gave rise to different calpain sub-families. Taken together, we show that calpains in unicellular eukaryotes are present in high numbers and these species also possess specific calpains that are not present in higher eukaryotes. Insights from Euglenozoa may bring new information about calpain biology, evolution and their possible application in biomedicine and biotechnology.

Application of Salix purpurea bark raffinate in ethanol fermentation

Marcin Walter, Natalia Kordala and Marek Adamczak

Department of Food Biotechnology, University of Warmia and Mazury, Olsztyn, Poland

email address: [email protected]

The aim of the experiments was to use the bark of Salix purpurea after supercritical extraction with CO₂ to obtain a medium for ethanol fermentation. The biomass was subjected to the following processes: alkaline pre-treatment, enzymatic hydrolysis with cellulase, xylanase and β-glucosidase, detoxification and fermentation with Saccharomyces cerevisiae 7. Raw material and raffinate were characterized by fiber composition analysis using Fibertec™ 1020 (FOSS). The sugar and ethanol concentrations were determined using HPLC-RI and GC-FID, respectively. The content of reducing sugars after enzymatic hydrolysis of the raw material and raffinate was 30.1 and 28.1 g/dm³, respectively. Alcohol content was 1.45% (vol:vol) and 1.46%, respectively and the fermentation efficiency was 78.0% and 83.6%. The application of the detoxification step resulted in a 32% higher ethanol content compared to samples without detoxification. The results indicated the possibility of the use of Salix purpurea bark raffinate for bioproduct synthesis.

Funding: This work was co-financed by the National Centre for Research and Development, BIOSTRATEG3/344253/2/NCBR/2017. Marcin Walter is a recipient of a scholarship from the Interdisciplinary Doctoral Studies Programme in Bioeconomy (POWR.03.02.00-00-I034/16-00), which is funded by the European Social Fund.

Imagistic CT analysis of experimental bone defects treated with PLA-derived bone cement

Andra Popescu, Mărioara Moldovan, Codruţa Sarosi, Amalia Neagu, Bogdan Sevastre, Cosmin Pestean, Liviu Oana, Alexandra Dreanca, Robert Purdoiu and Ioan Marcus

University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Babes Bolyai University, Cluj-Napoca, Romania

email address:[email protected]

Bone cement is a biomaterial that confers consistency and strength in bone defects, providing mechanical resistance to the area. The aim of the study was to monitor the influence of a newly synthetized bone cement derived from polylactic acid (PLA) on the evolution of the reparatory bone process, by evaluating the imagistic analysis of defects. In the study, rats with non-critical femoral defects were divided in two groups, PLA treated group and control group. After surgery, all animals underwent CT scans at 0, 14, 30, 60 and 90 days postoperatively. At these times, bone density and measurements of the defects were made with Syngo Somaris CT program. During the entire length of the study, the PLA rats displayed a persistent zone of hyperattenuation, while, in the first 14 days, an increased bone density of the PLA group was observed. At 90 days, although the defect size was larger in the PLA group, we noticed a tendency towards cortical bone regeneration presenting reduced hypoattenution, affrontation of the defect edges and increased density. In conclusion, CT scans allowed non-invasive in vivo measurements of healing stages of bone defects. The tested bone cement offered stability and durability, which may consolidate the bone tissue.

Bioprospecting for polar region polyhydroxyalkanoic acid producers: evaluation of popular screening methods

Malgorzata Marta Rogala, Jakub Grzesiak and Jan Gawor

Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

email address: [email protected]

Polyhydroxyacids (PHAs) are bacterial storage materials of enormous biotechnological potential. Prospecting for PHA producers is therefore of key importance. Two hundred bacterial isolates originating from different polar region habitats have been screened using gene-based and physiology-dependent methods (epifluorescent microscopy and agar plate), including PHA synthesis induction by nitrogen starvation with sugars, sugar alcohols, short- and long-chain fatty acids as substrates. Jaccards similarity coefficient showed moderate overlap between those methods (0.18–0.55). Arbitrarily, the combination of nitrogen starvation, Nile red staining and microscopy was chosen as the most reliable technique for screening for PHA producers. Based on this, contribution of PHA producing isolates was as high as 66%. Taxonomically, PHA producers belonged to the class Betaproteobacteria (95% of PHA positive isolates within the group) and the phylum Actinobacteria (78% PHA positives). Most promising for bioprospecting were low-nutrient habitats like glaciers (92% positive isolates) and soils of postglacial origin (80%).

Changes in self-reported depressive symptoms and objectively measured kynurenine pathway in mild depression: evidences for human studies using therapeutic horticulture intervention

Timea Buru, Éva Kállay, Loredana Elena Olar, Răzvan Ştefan, Erzsébet Buta, Maria Cantor and Ionel Papuc

Faculty of Horticulture, University of Agriculture Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Department of Psychology, Babes Bolyai University, Cluj-Napoca, Romania

Faculty of Veterinary Medicine, University of Agriculture Sciences and Veterinary Medicine, Cluj-Napoca, Romania

email address: [email protected]

In accordance that today 70% of people in Europe are living in urban areas, one of the reasons for which 38% of the population are more likely to develop a mental illness may be the side effect of urbanization. Moreover, available data indicate that 20% of those who suffer from major depression are treatment-resistant. In several research studies, tryptophan metabolites were associated with mental health, especially symptoms of anxiety and/or depression, evidenced by the imbalance in the kynurenine pathway. Therefore, the present study evaluates the results obtained after attending several therapeutic horticulture sessions by people with mild levels of depression. Using a non-invasive method, spectrofluorimetric analyses evaluate the urinary biomarker contributions before and after attending therapeutic horticulture session. Analyzing the effect of an antidepressant therapy as TH at participants with depression, the obtained results reveal changes in the urinary biomarker contributions in the experimental compared to control group, but the results did not indicate statistical changes. The present research study emphasizes that people who are attending several therapeutic horticulture sessions can improve their mental health, evidenced by higher level of subjective well-being measured with the 5-item WHO subjective well-being questionnaire and a lower depression level measured with Beck Depression Inventory.

The new PII protein PotN from Lactobacillus hilgardii interacts with transcriptional factor, the ATPase subunit of polyamine ABC transporter and ketoacid dehydrogenase subunit

Zalina I. Iskhakova, Darya E. Zhuravleva, Karl Forchhammer  and Airat R. Kaymov

Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia

Interfaculty Institute of Microbiology and Infection Medicine, Eberhard-Karls University Tübingen, Tübingen, Germany

email address: [email protected]

PII proteins coordinate carbon/nitrogen metabolism of bacteria and archaea by regulating the activity of transcription factors, key metabolic enzymes and transporters in response to intracellular level of ATP, ADP and 2-ketoglutaric acid. Among more than 200 species of Lactobacilli, only 4 of them carry the PII protein gene located in the potABCD operon, which encodes spermidine/putrescine ABC transporter. Based on this unique genetic background, low identity with other PII proteins and nucleotides binding ability, PII protein from Lactobacillus hilgardii was designed as PotN, representing the new subfamily of PII proteins. Immunoprecipitation and co-elution from crude extract have shown that PotN interacts with transcriptional factor GlnR and ketoacid dehydrogenase subunit, AcoB. As well PotN is capable to interact with PotA, the ATPase subunit of polyamine ABC transporter. The in vitro pull-down assay and Microscale Thermophoresis (MST) have shown that ADP positively affects the PotN-GlnR interaction, while negatively regulates PotN-PotA. Neither ATP nor ADP has significant effect on PotN-AcoB interaction. These data suggest that PotN apparently regulates the activity of GlnR and PotA in response to energy availability for the cell.

Funding: The work was supported by the grant of the President of the Russian Federation for young scientists (MD-572.2020.4).

Influence of the medium on the amino acid composition of feed yeast Candida utilis

Nelli Atykyan  and Victor Revin

National Research Mordovia State University, Moscow, Russia

email address: [email protected]

Yeast protein is rich in certain essential amino acids, such as lysine and methionine, which are limited in most feeds. For obtaining cheap yeasts protein commonly producers are cultivate on waste products of processing industry – molasses, whey, stillage, etc. The aim of the work was to study the influence of the composition of nutrient media on the growth and amino acid composition of feed yeast proteins of Candida utilis. As a result of a comparative analysis of nutrient media based on molasses, stillage, defective grain in various variations used for cultivation of C. utilis fodder yeast, the maximum biomass yield (near 36 g/L) was obtained on a medium with corn flour, pre-treated with amylolytic enzymes. Under these conditions, C. utilis yeast accumulated a high content of such essential amino acids as tryptophan, methionine and phenylalanine. The content of the remaining essential amino acids was negligible. Of the amino acids that are replaced, a high content of glutamic acid and asparagine is still noted. The amount of amino acids in C. utilis yeast grown on cornmeal is 20% higher compared to the variant grown on molasses and stillage.

Funding: This work was supported by the grant of RFBR (18-44-130002\19).

Multiplex qPCR-HRM with intercalating dye: a possible way to verify red fruit presence in food

Lenka Fialova, Denisa Romanovska, Anna Plaskova, Frantisek Papala, Andrea Nemcova and Ivana Marova

Faculty of Chemistry, Brno University of Technology, Brno, Czechia

email address: [email protected]

Certain species of red fruit, e. g. strawberries, raspberries and blueberries, are expensive components of fruit juices and purees. As a consequence, these products are subject to food fraud. The content of red fruit in them can be lower than declared, or these fruit species can be substituted for cheaper ones. This work explored and expanded a multiplex-PCR based approach to food authentication which has previously been applied to the authentication of buffalo meat products by Sakaridis et al. [1]. We successfully used triplex qPCR-HRM with amplicone detection by intercalating dye, and with primers specific for strawberry (Fragaria annanassa), red raspberry (Rubus idaeus) and berries of the Vaccinium genus to prove or disprove the presence of strawberry, red raspberry and bilberry in both model and commercial fruit purees.

[1] Sakaridis I, Ganopoulos I, Argiriou A, et al. A fast and accurate method for controlling the correct labeling of products containing buffalo meat using high resolution melting (HRM) analysis. Meat Sci. 2013; 94(1):84–88.

Proposal of some measures to improve the fattening management at swine

Gelu Huzau, Diana Marin, Alexandru Tiberiu Dekany, Cornelia Petroman, Elisabeta Bianca Panduru and Ioan Petroman

Faculty of Management and Rural Tourism, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania”, Timisoara, Romania

email address: [email protected]

In order to reduce the stress produced by heat at fatty swine exploited in the semi-intensive system, we propose some measures to improve the management in the fattening sector: floor cooling by splashing water, intensifying the natural ventilation in the shelters, shelter microclimate control, cooling of the shed of dripping humidifiers, reducing thermal stress by establishing optimum density on the speakers. In order to improve the maintenance conditions in the fattening shelters, the optimal parameters of the density of the animals from the stalls will be established, correlated with the production indices, and we propose the following measures: establishing the useful surface per youth head and the pig fattening recommended for the type of speaker and the optimal number of fatty pigs recommended for accommodation on the speaker (maximum of young pigs must not exceed 8 heads, returning on each pig a usable area of 0.310 ± 0.001 square meters). In order to improve the speaker management, the following conditions must be respected: feed with granulated feed, feed at discretion because it ensures the achievement of large increases and behaviour of beatitude, rest and lack of agitation.

Genome of starter culture bacteria Kocuria salsicia reveals its technological properties and safety for usage in meat industry

Evgenii A. Konorov, Mikhail Y. Minaev, Konstantin A. Kurbakov and Oksana A. Kuznetsova

V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russian Federation

email address: [email protected]

Kocuria salsicia is used as a starter culture for dry sausage production in Russian Federation. Qualified Presumption of Safety (QPS) list does not include this species; however, some of its strains are approved for meat industry in Russia. We sequenced and assembled a starter culture genome for an investigation of potential risk for usage in food industry. Assembly has 2472 predicted protein-coding genes, 51 tRNA genes and 3 rRNA clusters. Phylogenetic analysis of rRNA clusters confirmed species identification of starter culture as K. salsicia. Protein-coding genes were annotated by Blast2GO with GO and KEGG metabolic pathway analysis. In addition, we performed search of virulence factors and antibiotic resistance genes using VFDB and CARD databases. There were no strong hits for neither pathogen virulence factor nor resistance determinants. We also performed search for biogenic amine production proteins and found arginine decarboxylase gene. Analysis of starter culture genome can provide information about its safety for usage in food industry and its biochemical properties important for technological process. Although this information in many cases should be confirmed by other methods, genomics largely narrows a set of necessary tests and gives clues about culture properties that are difficult to find by other methods.

Combinatory effects of sorafenib and berberine treatment on hepatocellular carcinoma cell line, Hep3B

Bihter Ozdemir, Urun Ukan, Firdevs Cansu Atilgan, Murat Ozpolat and Huseyin Cimen

Yeditepe Proteomics and Mass Spectrometry Laboratory (YediPROT), Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkiye

email address: [email protected]

Sorafenib is a tyrosine kinase inhibitor and approved by Food and Drug Administration (FDA). Recent studies have shown that anti-angiogenic therapies increase the amount of hypoxia-inducible factor 1 alpha (HIF1α) in cancer cells, which leads to chemoresistance in the later stages. Hypoxic niche formation is one of the most basic features of cancer cells and can be defined as essentially oxygen deficient. The formation of a hypoxic niche elevates HIF1α in cancer cells. HIF1α is directly related to the Warburg effect, which is defined as metabolic shift from oxidative phosphorylation to glycolysis, mainly in cancer cells. Berberine, herbal extract, is widely used in traditional Chinese medicine to treat liver diseases. Our studies have shown that berberine has significant effects on p53-null liver cancer cells (Hep3B), particularly in the mitochondrial context. In this study, we aimed to increase the long-term effect of sorafenib and to achieve a better treatment method by employing the suppressor effect of berberine on HIF1a. We examined the effects of the combination of sorafenib and berberine on the mitochondrial energy pathway and total cell proteome. We demonstrated that angiogenesis is suppressed and the rate of mitochondrial damage is drastically elevated in combined treatment.

Biobased carrageenan films enriched with orange essential oil and trehalose – physical and chemical characteristics

Simona Jancikova, Dani Dordevic, Dani Dordevic, Petr Sedlacek  and Bohuslava Tremlova

Department of Vegetable Foodstuffs Hygiene & Technology, Faculty of Veterinary Hygiene & Ecology, University of Veterinary & Pharmaceutical Sciences Brno, Brno, Czech Republic

Department of Technology & Organization of Public Catering, South Ural State University, Chelyabinsk, Russia

Faculty of Chemistry, Brno University Technology, Brno, Czech Republic

email address: [email protected]

The main aim of this study was to evaluate basic physical and chemical characteristics of biobased carrageenan films as a biodegradable replacement of plastic films for food packaging. The films were enriched by orange essential oil (1%) and trehalose (0.5; 1 and 3%). The basic material characteristics were analyzed by measuring changes of thickness (mm), strength (MPa), breaking strain (%). Chemical and physico-chemical structure of the films was investigated by methods of molecular spectroscopy (ATR FTIR, FT-Raman) and crystallography (XRD). It was found that with addition of orange essential oil the thickness was increased and this was also supported by addition of trehalose. The strength of films decreased with addition of essential oil, on the other hand the breaking strain increased. It can be said, that the addition of orange essential oil has positive impact to elasticity of films so they can be better use as a replacement of plastic non-biodegradable packaging.

Funding: This research was supported by project FVHE/Tremlová/ITA2019 from University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic.

Multiple sclerosis treatment with glatiramer acetate induces dynamic changes of DNA methylation in peripheral blood mononuclear cells

Ivan Kiselev, Ludmila Danilova, Marsel Kabilov, Alexey Boyko, Olga Kulakova and Olga Favorova

Pirogov Russian National Research Medical University, Moscow, Russia

email address: [email protected]

To investigate DNA methylation changes in peripheral blood mononuclear cells of four female multiple sclerosis (MS) patients during glatiramer acetate (GA) treatment, we analyzed their genome-wide DNA methylation profiles immediately before the first use of GA, 4.2 ± 0.5 and 7.9 ± 0.5 months after starting GA treatment. DNA samples of four sex- and age-matched healthy women were analyzed for comparison. Fourteen differentially methylated sites (DMS) showed significant methylation changes accompanying MS therapy with GA. All of them demonstrated gradual increase in methylation level during therapy. Ten out of 14 identified DMS are located in/near genes DST, CGREF1, TMEM134, MIR9-3HG, COL16A1, ATRN, ARHGAP12, TESK1, LDB1 and SORBS1. Statistical overrepresentation test identified nine Reactome pathways over-represented in this set of genes on a nominal significance level. All of the identified pathways are linked with the cytoskeleton reorganization and function. Four of them (‘Collagen formation’, ‘Cell junction organization’, ‘Cell-Cell communication’ and ‘Regulation of cytoskeletal remodelling and cell spreading’) withstood FDR correction for multiple comparisons.

Funding: The study was funded by Russian Foundation for Basic Research according to the research project no. 17-00-00295 (17-00-00206, 17-00-00208 and 17-00-00210).

Study regarding the place of poultry products in consumers’ shopping basket from Bihor County, Romania

Marinela Lidia Bold, Diana Marin, Cornelia Petroman, Loredana Vaduva and Ioan Petroman

Faculty of Management and Rural Tourism, Banat’s University of Agricultural Sciences and Veterinary Medicine“King Michael I of Romania”, Timisoara, Romania

email address: [email protected]

The consumption and its components in the studied area, Bihor County, reflects the population level in terms of education, culture and civilization, but also the quality of social life. Regarding the place in monthly shopping basket of the poultry meat, there is an increase regarding the consumption among men in all age segments, the largest increase can be observed at the 41–50 age segment, where 61 respondents have stated that the quantity of poultry purchased monthly have increased compared to the previous period. If we speeak about women in all age groups, the quantity of meat and poultry products have known increases, the largest quantity of meat in the shopping basket is found at the age segment of 31–40 years and 41–50 years, but the most small decrease in quantity is recorded at the age segment over 60 years. In men cases, in the age group of 21–30 years, the amount of meat consumed increased and in women cases in the same age segment it was maintained at the same level because this age category has a different eating behaviour, usually serving the meal at fast food.

Microencapsulation and chromatographic analysis of plant extracts

Mateusz Ostolski and Marek Adamczak

Department of Food Biotechnology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

email address: [email protected]

Plant extracts are a rich source of polyphenolic compounds. Phytoextracts can be used as a food additive, but the effectiveness of the chemical compounds present in the extracts depends on their stability and bioavailability. The aim of the experiment was to determine the chemical composition and properties of supercritical CO₂ extracts obtained from the bark of Salix purpurea, Salix viminalis and Populus nigra × P.Maximowiczii. The extracts were used for microencapsulation by extrusion or spray-drying. The total concentration of polyphenols, flavonoids and DPPH radical-scavenging activity was determined. Chromatographic analysis was carried out by using the HPLC system coupled with a mass spectrometer consisting of a triple quadrupole, an ion trap and an ion source of electrospray ionization. Microencapsulation was carried out by extrusion and spray-drying. Thirteen chemical compounds from the phenolic acid and flavonoid classes were identified. Improvement of the extract microencapsulation efficiency was obtained by using a higher concentration of sodium alginate and by modification of the hardening solution composition (the microencapsulation yield was ∼77%–90%). The highest yield of microencapsulation (∼98%) was obtained by the spray drying of a maltodextrin and extract solution.

Funding: This work was co-financed by the National Centre for Research and Development, BIOSTRATEG3/344253/2/NCBR/2017.

Automation in 3D cellular system in live-imaging with microfluidic technology CELLviewer®

Gabriele Brecchia, Arnauld Capuzzo, Simone Pasqua, Daniele Gazzola, Daniele Vigo, Matteo Bertelli  and Giulio Curone

Department of Veterinary Medicine, University of Milano, Milano, Italy

CELLdinamycs, Bologna, Italy

MAGI Euregio, Bolzano, Italy

email address: [email protected]

Cells grown in a monolayer tend to flatten on the bottom of the plate by adhering and spreading on the horizontal plane without expanding into the vertical dimension. Microfluidic technology helps 3D crops in suspension, while microfluidics also optimize by avoiding n 3D cell cultures by avoiding adhesion. The aim of the work was to test some of the potential offered by CELLviewer® technology on some 3D models such as, complexes of a single cell population or aggregated systems in co-culture, modulating the environment extracellular with concomitant and repeated observation of these patterns spontaneous or induced phenomena by Nanotechnology. CELLviewer® ability to culture samples is the assessment using fluorescence markers that are selectively permeable to live and/or specific probes to stain dead cell. Live-imaging of 3D models in microfluidic represents alternative and innovative solution to cover gaps in traditional 2D models. A better understanding of 3D model will help further in vitro systems for studyng cells and tissue functions, as well as better translation to new therapeutic approaches.

How to overcome butanol stress in its native producer ‘Clostridium beijerinckii’ NRRL B-598

Petra Patakova , Barbora Branska, Karel Sedlar, Jan Kolek, Maryna Vasylkivska, Katerina Jureckova, Jana Musilova  and Ivo Provaznik

Department of Biotechnology, University of Chemistry and Technology, Prague, Czechia

Department of Biomedical Engineering, Brno University of Technology, Brno, Czechia

email address: [email protected]

The main bottleneck of 1-butanol production by acetone-butanol-ethanol (ABE) fermentation by different species of clostridia such as ‘Clostridium beijerinckii’ is low attainable butanol titer. This is mainly caused by low butanol tolerance, which results in severe product inhibition. For increase of butanol tolerance, deep understanding of butanol stress mechanisms is essential. Transcriptomic profiling during standard and butanol shocked ABE fermentations was used to find key mechanisms of butanol induced stress response. Butanol shock resulted in upregulation of heat shock protein genes as well as the genes for cyclopropanation of membrane fatty acids and formation of plasmalogens. In addition, there was observed upregulation of several putative butanol efflux pumps genes. Although, it is questionable whether butanol is actively pumped out of producing cells, profile of efflux activity during solventogenic phase under standard conditions in comparison to the conditions under which the solventogenesis was impaired seems to be an indirect evidence for this activity. Furthermore, a new flow cytometric assay based on direct efflux of ethidium bromide was used for measurement of efflux capacity which also confirmed that efflux pumps might be involved in another not well understood mechanism of butanol tolerance.

Funding: This work was supported by grant GACR (17-00551S).

Phytofiltration of uranium-contaminated waters in natural field conditions

Paulo J. C. Favas and João Pratas

University of Trás-os-Montes e Alto Douro, UTAD, Quinta de Prados, Vila Real, Portugal

MARE – Marine and Environmental Sciences Centre, University of Coimbra, Coimbra, Portugal

Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal

email address: [email protected]

This study has been performed to evaluate the phytofiltration potential of native aquatic flora grown in waters enriched with uranium (U) in several regions in the Northern, Central and Southern of Portugal. In the studied areas, U was detected in the surface waters at concentrations ranging from 0.23 to 1.217 μg/L. At nine of the studied sites, the U concentration exceeded the provisional guideline value (30 µg/L) indicated by the WHO. High U bioaccumulation levels were observed in several aquatic plant species at a magnitude much higher than the concentration in the surrounding water. The highest concentrations were found in the submerged species and the lowest in the free floating species. Among the submerged species, highest U concentrations were observed in Fontinalis antipyretica, Callitriche stagnalis, Callitriche hamulata, Ranunculus peltatus, Callitriche lusitanica and Ranunculus trichophyllus. Among the rooted emergent species, high U concentrations were observed in rhizomes/roots of Typha latifolia and Juncus effusus, and in the aerial parts of Myosotis secunda, Juncus effusus, Apium nodiflorum, Galium palustre, Oenanthe crocata and Rorippa sylvestris. The free-floating species Lemna minor also showed good ability to accumulate U. In general, these species showed very high values of bioconcentration factor, revealing a remarkable phytofiltration ability.

Effect of different nitrogen sources on the growth of culture A. vinelandii D-08 and biosynthesis of polysaccharide levan

Nadezhda Rakova, Nikolay Novokuptsev and Victor Revin

Department of Biotechnology and Biology, Ogarev Mordovian State University, Saransk, Russia

email address: [email protected]

Work to enhance the biosynthesis of the unique polysaccharide levan is carried out by scientists around the world. Levan has a wide range of properties. Microbial polysaccharide solutions are compatible with salts and surfactants, and also have good heat dissipation, acid and alkaline stability and the ability to form films. It is known that the studied bacterium can actively increase the biomass and produce the levan on various carbon sources. One of the important components for the growth of bacteria is nitrogen. Therefore, the aim of this work was to study the effect of various nitrogen sources on the growth of Azotobacter vinelandii D-08 culture and the biosynthesis of polysaccharide levan. In the course of research, A. vinelandii D-08 was cultivated on nutrient media with the addition of organic nitrogen sources (yeast extract, peptone, urea) and non-organic ((NH₄)₂SO₄, KNO₃). Of organic nitrogen sources, the yeast extract had the greatest stimulating effect on bacterial growth and biosynthesis of polysaccharide levan, and among non-organic ones – ammonium sulfate. From an economic point of view, it is better to use a yeast extract as this observed increase in the maximum biomass and yield of exopolysaccharide levan.

Hydrogel sorbents of heavy metals based on Azotobacter vinelandii alginate and zeolite

Evgeniy Vasilevich Balakin, Victor Vasilevich Revin and Vitalina Victorovna Shutova

Department of Biotechnology and Biology, National Research Mordovia State University, Saransk, Russia

email address: [email protected]

Polysaccharides as sorbents of xenobiotic have recently attracted increased attention of researchers. We used alginate synthesized by Azotobacter vinelandii on molasses media. Based on a polysaccharide and polyvinyl alcohol by the type of interpenetrating network, the hydrogel sorbent was obtained. The ability of the hydrogel to efficiently sorb zinc and cadmium ions has been revealed. At the same time, with an increase of polysaccharide purification degree, the metal ions adsorption efficiency also increased zinc by 1.7 times, cadmium by 2 times. This is probably due to the presence of culture medium impurities in the crude alginate that shield the polysaccharide and polyvinyl alcohol functional groups and prevent the formation of bonds between pollutants and hydrogel. It was also revealed that the maximum concentration of adsorbed zinc in the hydrogel occurs only on the 3rd day, and for cadmium after 60 min. At the second stage, hydrogels with alginate, polyvinyl alcohol and zeolite were obtained. The sorption ability of the obtained hydrogels with respect to copper ions increased sharply compared to the control. It is assumed that alginate-based hydrogel materials are promising sorbents for xenobiotic treatment of industrial effluents.

Funding: This work was supported by the RFBR (Russia), project 18-29-05054.

Comparative analysis of NK1.1 positive CIK cells expansion after stimulation by IL-2 and IL-15

Madina Zhunussova, Aliya Sekenova , Assel Issabekova, Marzhan Zhumabekova and Vyacheslav Ogay

Stem cells laboratory, National Center for Biotechnology, Nur-Sultan, Kazakhstan

Department of General Biology and Genomics, L.N.Gumilyov Eurasian National University, Nur-Sultan, Kazakhstan

email address: [email protected]

CIK cells are type of ex vivo expanded effector lymphocytes. To induce CIK cells’ proliferation PBMCs (peripheral blood mononuclear cells) from spleen and lymph nodes were stimulated by IFN-γ, anti-CD3 and IL-2 or IL-15 addition. In the group of cells obtained from murine spleen, the proliferation rate was 4.7 times higher among cells cultivated with IL-2 in comparison with cells cultivated with IL-15. However, the cells obtained from lymph nodes didn’t show significant changes in proliferation. Also, one of the CIK cells marker NK1.1 was studied. We found that 16% of cells obtained from lymph nodes and cultivated with IL-2, and 48% of cells treated with IL-15 were NK 1.1 positive. The proportion of cells expressing the NK1.1 marker isolated from the spleen and treated with IL-2 and IL-15 were 8% and 17%, respectively. Obtained results demonstrate the utilization of PBMCs obtained from lymph nodes and cultivated with IL-15 is the best way to induce CIK cells proliferation.

Funding: Supported by a grant AP05135467 "Development of production technology of dendritic vaccines and cytokine-induced killer cells for combination therapy in colorectal cancer" from the Ministry of Education and Science of the Republic of Kazakhstan.

Isolation of copolymers of polyhydroxyalkanoates (PHAs) from Aneurinibacillus sp. H1

Eva Slaninova, Kristyna Krupickova, Petr Sedlacek  and Stanislav Obruca

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, BUT, Brno, Czechia

Material Research Centre, Faculty of Chemistry, BUT, Brno, Czechia

email address: [email protected]

Polyhydroxyalkanoates (PHAs) are native polyesters accumulated as intracellular granules by numerous bacteria with the content up to 90% wt of cell dry weight. These compounds are biodegradable and biocompatible which make them more suitable in comparison with conventional plastics. Especially, copolymers have great potential due to better mechanical properties in comparison with homopolymers such as poly-3-hydroxybutyrate (P3HB). We used thermophilic isolate which was taxonomically classified as Aneurinibacillus sp. H1, PHA producing bacterial strain capable of synthesis of copolymer P(3HB-co-4HB). For isolation of homopolymers of PHAs, chloroform is mostly used. Using this solvent for extraction results in product which is still pure and without any physical changes. On the other hand, chloroform does not among environmental-friendly solvents.. Therefore, we used several alternative solvents such as acetone, isopropanol, tetrahydrofuran, ethylacetate to recover PHA from bacterial biomass. We used various techniques for characterization of efficiency of isolation, structural differences and molar mass of copolymer P(3HB-co-4HB) such as GC, ATR-FTIR, eSEM and SEC-MALS. Structural changes of films of isolated material were visible at a glance where number and size of microstructural pores were dependent on used solvent. This material with specific properties could be used in various branches of industry (food chemistry, medicine, etc.).

Impact of plant extracts on the activity of β-galactosidase and α-glucosidase

Magdalena Sniezawska  and Marek Adamczak

Department of Food Biotechnology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

email address: [email protected]

Plant extracts are a rich source of polyphenolic compounds with high-biological activity. The components of plant extracts affect the activity of enzymes that determine human health in different ways. Their potential use in the treatment of type II diabetes and associated diseases such as obesity, hyperlipidemia, hypertension and cardiovascular diseases has increasingly been analyzed. The purpose of the experiments was to determine the influence of plant extracts on the activity of β-galactosidase and α-glucosidase. The supercritical CO₂ extracts from bark, wood and a mixture of wood and bark from Salix viminalis, Salix purpurea, Populus nigra × P. maximowiczii, Helianthus salicifolius and Miscanthus giganteus were used. The activity of the β-d-galactosidase and α-glucosidase was determined using 2-nitro-phenyl-β-d-galactopyranoside and 4-nitrophenyl-β-d-glucopyranoside, respectively. Generally, the addition of extracts has a strong inhibitory effect on α-glucosidase enzyme. Extracts from the bark of S. viminalis, S. purpurea, P. nigra × P. maximowiczii caused about 100% inhibition of the enzyme and the plant extracts generally stimulated the activity of the β-galactosidase. The highest enzyme activity 175–8500 JAG/µg GAE was obtained in a medium with an extract from wood of P. nigra.

Funding: This work was co-financed by the National Centre for Research and Development (NCBiR), BIOSTRATEG3/344253/2/NCBR/2017.

Trends in the consumer behaviour of animal products from Mehedinti County

Eugen Zoican, Diana Marin, Marinela Lidia Bold, Ioan Petroman, Loredana Văduva and Cornelia Petroman

Faculty of Management and Rural Tourism, Banat’s University of Agricultural Sciences and Veterinary Medicine“King Michael I of Romania”, Timisoara, Romania

email address: [email protected]

The food problem has become more acute and has acquired quantitatively and qualitatively new features and dimensions, from a local or national problem, it has become a global problem, encompassing all levels of contemporary society, the population from the area under analysis, Mehedinți County has become increasingly aware of the importance of consuming quality food for health insurance. The issue of consumption has become extremely complex, which is why we highlight the original and novel aspects related to the consumption of animal products, how consumers try to meet their consumption needs. We find that consumers of animal products are oriented towards diversity, variety, change and permanent renewal in terms of consumed products. The tastes and demands of consumers vary profoundly from one individual to another, this diversity must be manifested through the offer of products offered on the market. A significant change in consumer behaviour refers to the preference of consumers with middle-income, who are experiencing a decline in purchasing power, purchasing products not necessarily from commercial spaces. The case study realized is actually a statistical survey, which was based on the questionnaire, applied to consumers from Mehedinti County, the questions referring to preferences regarding of animal products.

Extraction of bioactive compounds from seeds of Echinocystis lobata

Olga Shelepova and Yulia Vinogradova

Laboratory of Plant Physiology and Biochemistry, Tsitsin Main Botanical Garden of Russian Academy of Sciences, Moscow, Russia

Laboratory of Natural Flora, Tsitsin Main Botanical Garden of Russian Academy of Sciences, Moscow, Russia

email address: [email protected]

The most important in terms of use in traditional folk medicine is Echinocystis lobata (Michx.) Torr. et A. Gray. The main pharmacological effect of E. lobata is analgesic effect (for stomach pain, kidney diseases, rheumatism, for chills and fever). We have found that seeds of E. lobata may be a valuable source of lipids, such as polyunsaturated fatty acids (nearly 75% of the weight). The HPLC chromatographic profile of oil obtained from seeds of E. lobata revealed the presence of eight major compounds, mainly conjugated linolenic acid, with a concentration of 59%–67% of the total fatty acids content. The antioxidant activity of the seeds extract was assessed by the DPPH method. The extract showed a high antioxidant activity. The pericarp, the stem and the leaves of the plant are also a good source of phenolic compounds, which can be useful to protect from oxidative damage by acting on reactive oxygen. The results of the study highlight the importance of products obtained from the non-traditional plants, these products that can be harnessed as rich and important sources of biologically active compounds.

Funding: The work was supported by the Ministry of education and science of Russian Federation (project 19-119080590035-9).

Oxidative stress in type 2 diabetes

Arailym Yessenbekova , Botagoz Ussipbek, Nurzhanyat Ablaikhanova, Iryna Rusanova, Aizada Kozhakhmetova, Zura Yessimsiitova, Dinara Batyrbayeva  and Lyazzat Umbetyarova

Department of Biophysics and Biomedicine, Al-Farabi Kazakh National University, Almaty, Kazakhstan

University of Granada, Granada, Spain

Asfendiyarov Kazakh National Medical University, Almaty, Kazakhstan

email address: [email protected]

This study is based on data collected from 70 patients with type 2 diabetes mellitus. The average age is 50 years and disease duration is 5 years. Glycated hemoglobin (HbA1c) – 8.9%. The control group of 20 patients matched by sex and age. The diagnosis for diabetes mellitus 2 and the degree of compensation for carbohydrate metabolism were established using recommendations from WHO and the National Standards for patients with diabetes mellitus. HbA1c was detected using a Bio-Rad liquid chromatography and standard kits (France). Total antioxidant activity (TAA) were measured using induced chemiluminescence. The collected data were processed using the STATISTICA 6.0 (StatSoft, Inc., USA). It was found that activation of free radical oxidation and statistically significant increase in intensity of free radical oxidation 1.772 ± 0.064 (p < 0.001) and in the control group by 1.572 ± 0.142. Reduced TAA 0.033 ± 0.005 (p = 0.003) were in this group of patients compared to control group 0.038 ± 0.005. This suggests that the development of free radical oxidation in a quiescent state is staged, where oxidized modified proteins are inactive. However, further generation of radicals activates oxidative modification of proteins, which stimulates lipid peroxidation, serves as an additional source of free radicals, inactivates antioxidant enzymes.

Changes in the morphology of red blood cells and the state of cyto-skeleton proteins under experimental hypoxia

Natalya Yuryevna Ratnikova, Victor Vasilevich Revin, Natalia Vasilevna Gromova, Elvira Sergeevna Revina, Nadezhda Victorovna Revina, Igor Pavlovich Grunyushkin, Vera Ivanovna Inchina, Vasilisa Mikhailovna Kudryashova and Julia Nikolaevna Fedkina

Department of Biotechnology and Biology, Ogarev Mordovia State University, Saransk, Russia

email address: [email protected]

The method of AFM spectroscopy during hypoxia revealed a change in the shape of cells and the transition of erythrocytes to echinocytes and stomatocytes. Incubation under conditions of erythrocytes hypoxia with quercetin led to an increase in the proportion of transitional and echinocytic forms in the samples, and the diameter and thickness of the cells increased. In the presence of resveratrol, discocytes prevailed in the samples, and the diameter and thickness of the cells did not differ from the control. The main erythrocyte membrane proteins were isolated by electrophoresis. During hypoxia, the amount of spectrin, ankirin, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) decreased, and proteins of bands 4.1 and 4.2 were absent. When flavonoids were added under hypoxic conditions, the amount of spectrin and GAPDH increased, proteins of band 4.1 and 4.2 were absent, and the content of band 3 protein and ankyrin decreased. The actin content does not change in all samples. Thus, hypoxia causes changes at all levels of the organization of erythrocytes, and this may be the cause of functional disorders of the oxygen-transporting properties of hemoglobin and, subsequently, the complete degradation of erythrocytes. The use of flavonoids in this case has a protective effect on erythrocytes.

Funding: This work was supported by the Russian Science Foundation, Grant No. 15-15-10025.

The effect of natural polyphenols on lipid peroxidation in red blood cells during hypoxia

Tatyana Olegovna Oshkina, Nadezhda Viсtorovna Revina, Natalia Vasilevna Gromova, Elvira Sergeevna Revina, Igor Pavlovich Grunyushkin, Vera Ivanovna Inchina, Victor Vasilevich Revin, Vera Vasilevna Vdovina, Natalya Alexandrovna Ermusheva and Tatyana Nikolaevna Shcherbakova

Department of Biotechnology and Biology, Ogarev Mordovia State University, Saransk, Russia

email address: [email protected]

In erythrocytes under experimental hypoxia, the lipid peroxidation products were determined – the content of diene conjugates (DC) and the concentration of TBA-active products (TBARS). The effect of natural polyphenolic compounds (quercetin and resveratrol) on the state of oxidative processes in red cells was also studied. It was shown that under hypoxia there was a significant increase in the number of lipid peroxidation products. When incubated under similar conditions, but with the additional presence of quercetin and resveratrol, there was a decrease in the amount of DC, and in the case of resveratrol, almost to the level of normoxia. The amount of TBARS under hypoxic conditions varied in a similar way. The presence of antioxidants in the incubation medium led to a decrease in the number of TBARS, but the level of control was never achieved. The intensification of oxidative processes in the lipid bilayer leads to both a change in the composition of fatty acids of individual phospholipids and a phase shift in the state of the bilayer of erythrocyte membranes. The changes taking place contribute to a decrease in the oxygen-binding properties of hemoglobin. Compounds of a flavonoid nature (in particular, resveratrol) had a protective effect under hypoxia.

Funding: This work was supported by the Russian Science Foundation, Grant No. 15-15-10025.

Effect of sorafenib and sodium dichloroacetate combination on human neuroblastoma cell line, SH-SY5Y

Urun Ukan, Bihter Ozdemir, Firdevs Cansu Atilgan, Murat Ozpolat and Huseyin Cimen

Yeditepe Proteomics and Mass Spectrometry Laboratory (YediPROT), Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkiye

email address: [email protected]

Sorafenib (SOR) is a kinase inhibitor known to suppress angiogenesis and proliferation in a vast range of tumour types. SOR approved by the Food and Drug Administration (FDA) for the treatment of advanced renal cell carcinoma (RCC), and recent studies showed promising effects for neuroblastoma too. Besides, sodium dichloroacetate (DCA) is a pyruvate dehydrogenase kinase inhibitor long employed in the therapy of diseases related to mitochondrial metabolism. DCA is known to reverse the Warburg effect and thus subsequently induces apoptosis in neoplastic cells including neuroblastoma. The main objective of this study is to assess the effect of sorafenib and sodium dichloroacetate combination on human neuroblastoma cell line, SH-SY5Y, through re-directing energy metabolism to favour oxidative phosphorylation over aerobic glycolysis and blocking angiogenesis. After SOR and DCA co-treatment on SH-SY5Y cells, annexin V/PI staining showed the late-apoptotic cell number to be elevated around 70%. Moreover, tube formation assay has diminished angiogenesis compared to control cells. In conclusion, SOR and DCA co-treatment has been proven to be synergistic, more effective in inducing apoptosis for human neuroblastoma cell line.

High-level expression of fungus Neonothopanus nambi hispidin-3- hydroxylase in E. coli

Andrei Gerasimov, Rogozhkin Sergei  and Shakhova Ekaterina

Department of biomolecular chemistry, Shemyakin-Ovchinnikov institute of bioorganic chemistry of the RAS, Moscow, Russian Federation

Department of biotechnology, Vyatka state university, Kirov, Russian Federation

email address: [email protected]

The fungus Neonothopanus nambi is the first eukaryotic bioluminescent organism with fully discovered enzymes, involved in luciferin biosynthesis. One of the crucial reactions in this pathway is luciferin formation by hydroxylation of hispidin catalyzed by hispidin-3-hydroxylase (H3H). Therefore, the production of functional enzyme suitable for structural and kinetic investigations is an important task. For this purpose, we have engineered six constructions containing H3H gene with hexahistidine extension sequence at 5′-end and several auxiliary sequences, such as SUMO, thioredoxin, HELIX for better protein solubility and PelB and NSP4 leader sequences for periplasmic expression. E. coli BL21(DE3) and BL21(DE3) pLysS strains were used for recombinant protein production. We have found that H3H expression level dramatically increases in case of SUMO fusions, but the products form inclusion bodies. Coexpression target protein with GroEL/S chaperon and decreasing of cultivation temperature to 15 °C had led to the appearance H3H solubility. Sumo-H3H has been purified by IMAC chromatography and it showed specificity activity to produce luciferin from hispidin. The yield of soluble purity target protein is 200 mg per liter.

Funding: This work was supported by Russian Foundation for Basic Research grant 18-34-20134.

Newly designed primers targeting limonene synthase

Denisa Romanovská, Lenka Fialová and Ivana Márová

Faculty of Chemistry, Institute of Food Science and Biotechnology, Brno University of Technology, Brno, Czech Republic

email address: [email protected]

Cosmetic products with ingredients of plant origin promise beneficial effects on the customer health. Those may contain allergens that belong to the group of 24 fragrance allergens that can negatively affect the health of sensitive individuals. One of these allergens is d-limonene present for example in citrus fruits. The presence of plant DNA in the cosmetic product can be detected using techniques of molecular biotechnology, even if it is present in very small quantity. New primers Lim2 F/R, targeting limonene synthase, were designed to confirm the presence of the citrus DNA in a leave-on cosmetic product. The DNA of Citrus limon was isolated from the leave-on cosmetic product and the specific sequence was amplified using real-time PCR. The presence of amplicons of specific size was confirmed using agarose electrophoresis.

Air quality microbiological studies in straw bale and reed houses

Jane Raamets, Aime Ruus, Mari Ivask and Lembit Nei

Tartu College, Tallinn University of Technology, Tartu, Estonia

email address: [email protected]

‘Green’ building materials such as reed and straw are of increasing popularity. They both are CO₂ neutral materials with excellent properties, but they also involve potential hazard, being food sources for microorganisms, as they contain cellulose, hemicellulose and lignin. Active microbial community can pose danger for the indoor environment and human health. Fungi play an important role in the degradation of organic matter. They are able to grow on both straw and reed. Samples were collected from indoor and outdoor air according to ISO standard 16000-18 using Microbio MB2 air samplers. Two different media were exploited – Malt Extract Agar and Dichloran (18%) Glycerol Agar. In the bedrooms of straw bale and reed houses culturable fungi revealed seasonal variations. Mean CFU (colony forming unit) values were always higher in reed houses. Seasonally, CFU values were higher in summer and lower in winter. Microbial genera were the same in both type houses. Members of the genus Cladosporium predominated at both types of houses in spring, summer and autumn. In winter, members of the genus Penicillum predominated in all samples. Most of detected species may present health risk for the occupants of these houses.

LAMP2 mutations causing of dilated cardiomyopathy in women

Larysa Sivitskaya, Tatiyana Vaikhanskaya, Nina Danilenko, Oleg Davydenko and Nikolai Zhelev,

Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, Belarus

Republican Scientific and Practical Centre of Cardiology, Minsk, Belarus

School of Medicine, University of Dundee, Scotland, UK

Medical University Plovdiv, Plovdiv, Bulgaria

email address: [email protected]

LAMP2 encodes a lysosomal-associated membrane protein 2 participating in lysosome maintenance and adhesion. Mutations in LAMP2 cause X-linked genetic disease with a poor prognosis – Danon disease. However, in women they commonly manifest as phenocopy of dilated cardiomyopathy (DCM) and thus mask the real mechanism of pathology. We investigated a cohort of women with DCM using NGS and identified 15% of carriers of truncating and splicing mutations in LAMP2. Their initial symptoms of DCM varied over a wide range of ages (16–51 years old). Nevertheless, heart failure progressed rapidly in all cases. Heart transplantation was required for 6 months on average after DCM diagnostics. We estimated the X-chromosome inactivation (XCI) pattern in the heart of women and found a relationship between XCI and DCM onset: a higher level of mutated X-chromosome inactivation in tissue slows down the disease presentation. We suggest that LAMP2 mutations are underestimated as causes of DCM in women. Carriers of truncating mutations in LAMP2 have a poor prognosis and require a simple and rapid method for their identification.

The importance of quality control in medical analyses, according the model implemented in Intermedica laboratory

Ina Toska  and Anila Mitre

Intermedica Center, Sofia, Bulgaria

email address: [email protected]

Accreditation means: do the right things right, describe how you do it, do it as you describe, evaluate everything. It does not guarantee success, but is only one step along quality journey. Quality management systems in labs are internal and external quality control. IQC is the action of detecting errors that would invalidate the medical usefulness of laboratory results. It is based on Westgard rules, it enables the detection of problems and assures the precision of clinical laboratory tests. An IQC requires results from two levels of quality control measured everyday. The mean and 2 SD limits are established for each analyte at both levels. SD values are plotted on a Levey–Jennings chart and SD is used to observe the deviations from the mean. External QC allows laboratories to monitor their accuracy and to compare performance with other laboratories. Nowadays, there exist special agencies responsible for external QC that send samples with unknown concentration. Their values are sent to the responsible agency that compiles a detailed report to evaluate the quality of run. How is implemented internal and external quality control in Cobas 6000 analyzer? Qualityis never an accident: result of high intention, sincere effort, intelligent direction, skillful execution.

Stable isotopes signatures in fruits as tool to inferred climate variations

Oana Romina Botoran, Diana Costinel, Nicoleta Stanciuc and Roxana Elena Ionete

National Research and Development Institute for Cryogenics and Isotopic Technologies - ICSI Rm. Valcea, Romania

Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Galati, Romania

email address: [email protected]

The isotopic signature of some bio-elements integrated into the plant tissue (e.g. oxygen of water, carbon of sugars) was used in this work as sensitive climatic proxies to reveal plant–environment interactions, since it stores information related to seasonal and annual variations of precipitation/humidity and temperature. We explored the variation of δ¹³CC₂H₆O, δ¹⁸OH₂O and (D/H)methyl/methylene in 132 fruit samples from 6 genus, sampled between 2013 and 2017, in correlation with the spatial distribution of the aridity index calculated according to De Martonne, Pinna combinative index and UNEP. The aridity index shows that susceptibility to desertification is present and pronounced in the warm part of the year. A strong relationship was found between the values (D/H) methylene and seasonal De Martonne aridity index, as well as between the De Martonne and Pinna indices, confirming their similar spatial distribution. Moreover, during the studied period was observed a specific pattern linked to a gradual decline in rainfall and an ascend in temperature, which further widened the gap between precipitation and agricultural water demand. Thus, our results reveal the high potential for estimating the growing season temperatures based on fruits isotopic signature, approach that could contribute as support for improved strategies in agriculture relative to climate change.

Identification of a novel family of bacterial lipolytic enzymes

Alisa Gricajeva, Justas Šidiškis and Lilija Kalėdienė

Department of Microbiology and Biotechnology, Institute of Biosciences, Life Sciences Center, Vilnius, Lithuania

email address: [email protected]

Lipolytic enzymes (LE) belong to a large class of enzymes named serine hydrolases that are distinguished by a presence of a nucleophilic serine (nSer) in their active site. Most of the bacterial LE have the nSer located in a conservative GXSXG pentapeptide (X – any amino acid (aa) residue), GDSL motive or less frequently in a motive specific to the LE structurally resembling β-lactamases/DD-peptidases. Conservative aa motives and biochemical properties of bacterial LE are used for their classification. In this work, a putative carboxylesterase (EstAG1) from Staphylococcus saprophyticus AG1 with previously uncharacterized pentapeptide and nSer location motive was identified. Enzyme having no close homologues had unconventional GDGTG pentapeptide and nSer located in a SPXYD (Y – hydrophobic residue) sequence previously unspecified for the LE. The putatively catalytic nSer and other aa related to catalytic function were investigated and validated by site-directed mutagenesis and activity analysis of the mutant recombinant enzymes. Some of the EstAG1 (wt) biochemical characteristics are also presented. Phylogenetic analysis revealed that EstAG1 homologues are characteristic to other Staphylococcus spp as well. Based on the low aa sequence identities, unique conservative aa motives and phylogenetic analysis results, EstAG1 belongs to a new family of bacterial LE.

The effect of clobetasol on the protein composition in damaged nerves during regeneration

Mariya Yurievna Gladysheva, Tatyana Pavlovna Kuzmenko, Elvira Sergeevna Revina, Ilya Vladimirovich Syusin and Victor Vasilievich Revin

Department of Biotechnology and Biology, Ogarev Mordovia State University, Saransk, Russia

email address: [email protected]

In the field of regenerative medicine, the problem of repairing damaged nerve tissue remains unresolved. In order to find optimal conditions for the regeneration process, physiologically active compounds are also used, which increase and optimize the metabolism in the nerve cell. Directly in this regard, glucocorticoids, in particular clobetasol, can be used. After an injury, processes are activated in the cell that trigger the synthesis of neurotrophic factors and surface proteins, which, interacting with various classes of receptors, nerve growth factors activate signaling pathways, in particular, such as the phosphoinositide pathway, which promotes the differentiation and survival of neurons. It has been shown that under the influence of clobetasol on damaged somatic nerves, the synthesis of structural proteins and proteins associated with axonal growth in the nerve cell proceeds more intensively. During the initiation of regeneration processes, the number of cytoskeleton proteins in the proximal part of damaged nerves increases markedly, in particular, the synthesis of H-neurofilaments increases by two times. Thus, we can conclude that clobetasol is one of the factors that enhance the regenerative processes in the somatic nerves of rats.

Development of digital polymerase chain reaction

Martina Gaňová and Pavel Neužil

Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic

email address: [email protected]

During the last 20 years, microfluidics has proven to be a powerful nucleic acid (NA) analysis tool in applications such as point-of-care diagnostic methods, which critically depended on miniaturization. From simple systems they evolved into complex devices integrating sample processing, NA amplification and product detection using samples with an ultra-small volume, resulting in inexpensive screening. There are even techniques which cannot be conducted without microfluidics, such as digital polymerase chain reaction (dPCR) developed in 1999. We demonstrate a concept of droplet real-time PCR (qPCR) which will be developed into dPCR. For droplet real-time detection, the amplification is conducted in 0.3-µL master mixture droplet containing target gene encapsulated in 2 µL of mineral oil. For digital detection, a chip-based system with microwell sample dispersion will be developed. The 3 µL of master mix solution with DNA will be pipetted on a silicone chip and covered with a hydrophobically treated glass. We improved the features of both devices where the experimental setup for qPCR and dPCR is the same, except the detectors. Our detection system is more economic thanks to the use of a recyclable microchip, lower consumption of components and shortened detection time, which makes it attractive for point-of-care applications.

Optimization of solid-state fermentation of red mold rice for antiviral activity

Jiwon Yang  and Youn-Je Park

Department of Food Science and Technology, Kongju National University, Yesan, South Korea

email address: [email protected]

Red mold rice is an oriental traditional food fermented by Monascus sp. with rice. Monascus sp. is called as red mold or red yeast, and it has been known to produce monacolin K reducing cholesterol levels. In this study, a fermentation process of red mold rice was investigated to optimize to produce stronger antiviral activity against Ebstein–Barr virus(EBV). Steamed rice was cultured with Monascus ruber by solid-state fermentation in a flask for 40 days, and was extracted with 70% ethanol. When the concentration of inoculum was 8% (vol/wt), the antiviral activity of red mold rice extract was highest. Red mold rice extract fermented at 28 °C had a stronger antiviral activity than those at 25 °C and 30 °C. When the initial pH was adjusted to 6.5, antiviral activity of red mold rice extract was stronger than pH 6.0 or 7.0. Although antiviral activity of red mold rice extract gradually increased until 25 days of fermentation, it increased a little after 25 days. Antiviral activity of red mold rice extract was not identical with the pattern of monacolin K amounts. In conclusion, antiviral activity of red mold rice extract was highest at the 25th day after fermentation at 28 °C, pH 6.5.

Functional composites for biomedical applications

Oksana Olegovna Khramova , Natalia Borisovna Nazarova, Alexandra Iurevna Liyaskina, Vladimir Viktorovich Revin, Elena Vladimirovna Liyaskina, Guang Yang, Sabu Thomas  and Victor Vasilevich Revin

National Research Mordovia State University, Saransk, Russia

College of Life Science and Technology of Huazhong University of Science and Technology (HUST), Wuhan, China

Mahatma Gandhi University, Kottayam, Kerala, India

email address: [email protected]

Development of novel functional materials for biomedicine has attracted more and more attentions in recent years. Bacterial cellulose (BC) is an ideal biomaterial due to its unique properties such as ultrafine nanofibre network structure, large surface area, great water holding capacity and biocompatibility. Chitosan, a natural amino polysaccharide, possesses many outstanding properties, such as the biocompatibility, healing promotion and especially antibacterial properties. Fusidic acid has been widely used in the systemic and topical treatment of staphylococcal infections, including strains resistant to penicillin and other antimicrobials, making it an alternative for the treatment of diseases caused by MRSA strains. The aim of this study was to obtain a biocomposite of BC, chitosan and fusidic acid in the form of hydrogels and aerogels. The subject of the experiment was BC, produced by bacteria Komagataeibacter sucrofermentans B-11267. The structure and physico-chemical properties of biocomposites were analyzed by AFM, FTIR spectroscopy and X-ray diffraction. The antibacterial activity of biocomposites was investigated by diffusion method. The resulting composites have high antibiotic activity against Staphylococcus aureus and can be used for biomedical applications.

Funding: The work is supported by the Russian Foundation for Basic Research (no. 19-515-80002\19).

Silver nanoparticles for wound healing application

Federica Filomena Patria , Maria Rachele Ceccarini, Elisabetta Albi, Beccari Tommaso, Saboktakin Mohammad Reza, Michela Codini and Carmela Conte

Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy

NanoBMat Company, GmbH, Department Nanomedicine and Nanotechnology, Hamburg, Germany

email address: [email protected]

After tissue damage, a wound healing process occurs to ensure a rapid tissue repair and to prevent secondary infections by enhancing the quality of the patient’s life. Wound healing is an essential and complex process that requires specific molecular mechanisms and sequential events in time. The process includes three overlapping phases: hemostasis/inflammation, proliferation and remodelling. An aberration of process can seriously impair the normal physical function. Nowadays, new drugs and technologies are emerging in order to promote wound healing. The application of silver nanoparticles (AgNPs) is one of the potential therapeutic strategies recently investigated. The aim of the work was to study the molecular mechanisms involved in the use of AgNPs in skin regeneration. Thus, we performed an in vitro study in keratinocytes and fibroblasts treated with AgNPs by Wound Healing Assay. The results showed a positive effect of AgNPs on keratinocytes proliferation and fibroblasts differentiation. Moreover, it was evident that AgNPs requires neutral sphingomyelinase specifically for its action in fibroblasts differentiation.

Application of liposomes and PHB-liposome particles with encapsulated plant and microalgae extracts in cosmetics and food industry

Renata Pavelková, Sara Ramírez Acosta, Petra Matouškováand Ivana Márová

Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic

Department of Chemistry, Faculty of Experimental Sciences, Campus El Carmen, University of Huelva, Huelva, Spain

email address: [email protected]

Plants are sources of a broad spectrum of compounds potentially useable as antioxidants and UV protectants in cosmetics as well as in the food industry. This work aims at the extraction and preservation of chosen plant and microalgae extracts into liposomes itself and into liposome particles enriched by PHB to prolong their activity and stability. Extracts were characterized in terms of polyphenols, flavonoids, antioxidant activity, UV protective properties, and packed into particles. Prepared samples were tested by MTT assay on HaCaT and Caco-2 cell lines if they alter cell viability. These particles were characterized based on their encapsulation efficiency. Particle size and size distribution were observed by dynamic light scattering and stability in solution by zeta-potential. All the prepared samples showed moderate or high encapsulation efficiency. Extracted active compounds had high-antioxidant activity as well as UV protective effects. A major part of prepared particles did not possess a cytotoxic effect on human cell lines. Encapsulation of active compounds from plants and microalgae into liposomes and PHB/liposomes can be an interesting way how to prolong their activity. Besides UV protecting effect for natural cosmetics they can find use as a preservative in cosmetics as well as in the food industry.

Decolouration of selected diazo dyes by unicellular algae

Pavel Chaloupsky, Martina Kolackova, Martin Zahalka, Anna Janova, Veronika Hynstova, Marketa Vaculovicova and Dalibor Huska

Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic

Central European Institute of Technology, Mendel University in Brno, Brno, Czech Republic

Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic

email address: [email protected]

Presented work focuses on prospective application of selected algae for azo dyes biodegradation. Azo dyes are widely utilized chromophores and make up more than 70% of organic dyes employed. Currently used conventional treatment of industrial effluents is either costly or severely inefficient and innovation in degradation technology has to be addressed. We examined multiple unicellular alga for capability to degrade and absorb diazo dyes (Congo Red and Evans Blue) and we found that some species are capable of decolouration within short period. Highest decolouration rate was observed in Desmodesmus armatus and Chlorella sorokiniana where decolouration reached 90% in 48 h. Other species were capable of decolouration only to some extent. Within this work, we also developed methodology for rapid analysis of the dyes and respective degradation products by HPTLC and capillary electrophoresis.

Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 826312. The financial support of the Mendel University Grant Agency, the Czech Republic (AF-IGA2020-IP034) is acknowledged.

Testing of candidate protective compounds for skin regeneration

Radu Albulescu, Roxana Madalina Stoica, Adrian Albulescu, Iulian Terchescu, Georgeta Neagu and Andrei Zbuchea

National Institute for Chemical-Pharmaceutical Research and Development – ICCF, Bucharest, Romania

email address: [email protected]

Skin regeneration is a very important process, mainly after severe burns. We have tested in vitro and in vivo a natural extract and the pharmaceutical formulation based on it, in the form of a topical application ointment. The in vitro testing included testing of cytotoxic effects on 3T3 cells, testing of proliferative effects on keratinocytes, as well as on human fibroblasts. In vivo testing included testing of toxicity (LD50), of potential deleterious effects on genetic material (micronuclei assay on mouse bone marrow), and testing of specific effects on thermal burns on rats. The in vitro stage revealed that the extracts presented no risk upon exposure of the cells. There were significant accelerations of cell growth upon exposure of human keratinocytes to the extracts, as well as to the pharmaceutical formulation, and similar effects were achieved upon exposure of fibroblasts to either extracts or formulated product. In vivo studies revealed that the extract could be classified in the class of ‘practically nontoxic’ and demonstrated no deleterious effects on DNA. Functional testing on burned skin produces a complete skin regeneration in 12–14 days, compared to controls (more than 18 days). We conclude that the product is safe and efficient for skin regeneration.

Funding: This research work was carried out with the support of National Institute for Chemical-Pharmaceutical Research &Development, ICCF-Bucharest.

On the degradation of some widely used pharmaceuticals during sewage sludge composting

Lembit Nei, Egge Haiba and Sandra Victoria Nei

Tartu College, Tallinn University of Technology, Tartu, Estonia

Tartu University Hospital, Tartu, Estonia

email address: [email protected]

Sewage sludge contains organic pollutants. Composting is the most cost-effective way of treating sewage sludge. Composting technology has an impact on the degradation rate of some pharmaceuticals and personal care products, whereas some of them are quite persistent. Metformin and diclofenac almost fully degrade during a one month composting period. However, the degradation of triclosan, which is a widely used antibacterial agent, does not take place with sufficient speed: ca 25% of its molecules remained ‘unaffected’ under comparable composting conditions. Due to this, the composting period should be longer (at least one year) or more intensive. A remarkable increase in microbial activity was attained when adding ethanol to the compost mixture, enabling us to shorten the composting period. Still, there are pollutants that do not degrade during sewage sludge composting. Carbamazepine is persistent during this process. As composting leads to the decrease of dry mass, the concentration of carbamazepine rises. It has been claimed that adding organic carbon into sewage sludge compost may partially initiate the degradation of carbamazepine. Our LC-MS/MS determinations did not confirm this. In conclusion, as a rule, the more soluble the organic pollutant is in water, the better it degrades during sewage sludge composting.

Preliminar characterization of peptides in toxines from venom of Euscorpius italicus

Concetta Caglioti, Federico Palazzetti and Bernard Fioretti

Department of Chemistry, Biology and Biotechnologies, University of Perugia, Perugia, Italy

email address: [email protected]

Specimens of Euscorpius italicus of both genders have been bred. A first sample of toxin from a female subject has been tested by SDS-PAGE electrophoresison acrylamide gel to separate the bands. A further test will be carried out on the toxin from the male subject to verify possible differences in weight and structure between the genders. A non-invasive method to take samples of toxin consists in inducing the scorpion sting a parafilm fixed on an Eppendorf. The venom is eventually collected on the bottom of the Eppendorf. From the electrophoresis, the following bands, estimated by molecular weight marker, result: at 66,200 Da could be assigned to an enzyme, probably a protease or a lipase; at 31,000 Da a band must still be assigned; bands corresponding to molecular weights lower than 14,000 Da are probably related to toxins that activate ionic channels. After electrophoresis, a size-exclusion chromatography on column packed with dextran polymers (Sephadex) will be performed. The choice of Sephadex will depend on the weight of the bands to be separated. Finally, a sequencing of the isolated fractions to determine their structures and an activity test on the ionic channels will be channels will be carried out.

The preparation of biologically active lactoferrin conjugated with iron oxide nanoparticles and its influence on cytotoxicity of healthy and tumour cell lines

Nikola Mannova and Katerina Polakova

Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic

Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc, Olomouc, Czech Republic

email address: [email protected]

Effective therapy of tumour diseases is an actual topic worldwide thanks to the fact that cancer remains one of the leading causes of death. Quickly progressing field of nanotechnology brings us new ways of development of effective anti-cancer therapy. This research paper introduces a new concept of therapeutic and diagnostic agent, which combines the known anti-carcinogenic and anti-inflammatory properties of dairy protein lactoferrin and benefits of superparamagnetic iron oxide nanoparticles. The goal of this research is to find out, whether this complex shows anti-carcinogenic properties in vitro or have any other influence on healthy or tumour cell lines. Results obtained by two methods monitoring the cytotoxicity (MTT assay, flow-cytometry) confirmed the biocompatibility of the complex. The viability of healthy and tumour cells didn’t drop below 80% for concentration of SPIO-Lf to 500 µg/mL. Preliminary results of experiments monitoring the oxidative stress denoted no effect and no increase of ROS compared to control cells. We managed to prepare stable and biocompatible complex SPION-Lf, which can be used as contrast agent with high affinity to inflammatory and cancer cells. This agent can be used in targeted diagnostics (e.g. magnetic resonance imaging) with possible therapeutic effects.

Comparative tissue distribution between principal routes of administration of citrate-silver nanoparticles

Antonella De Luca, Ivana Ferri, Francesco Ragonese, Lorenzo Monarca, Federica Patria, Chiara Pennetta, Costantino Ferdinando, Angelo Sidoni  and Bernard Fioretti

Department of Chemistry, Biology and Biotechnologies, University of Perugia, Perugia, Italy

Department of Experimental Medicine, Section of Anatomic Pathology and Histology, University of Perugia, Perugia, Italy

Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy

email address: [email protected]

Citrate-silver nanoparticles (AgNPs) have been widely applied in different fields in our daily life, such as biomedical, as they have antimicrobial and anticancer properties. The purpose of our present study is to investigate the different tissue distribution after several pathways of delivery of AgNPs (size 10 nm) in mice by inductively coupled plasma-mass spectrometry (ICP-MS) and by autometallography 6 and 24 h following last intravenous (i.v.) and intraperitoneal (i.p.) administration. The spleen was the organ with the highest levels of Ag+. Most of the AgNPs were detected in the red pulp region of the spleen, followed by the liver, were presumably they were captured by Kupffer cells. Also, the Ag + levels were within the detectable range in the lung and in the kidney. In addition, the Ag + levels were within the detectable range in brain of ICP-MS, but they were not detected by Autometallography. After intragastric administration it was not possible to detect any trace of nanoparticles or Ag+. After intranasal instillation, Ag + and AgNPs were present only in the lung. Our histological results demonstrate that AgNPs are well tolerated by tissues and that their visualization in light microscopy is reliable and predictive of wider application in clinical studies

Case report: A very rare genetic disease -Pycnodysostosis

Kübra Baysal , Mikail Demir, Damla Badur Mermer, Dilek Çiçek, Gül Direk  and Munis Dündar

Department of Medical Genetics, Erciyes University, Kayseri, Turkey

Department of Pediatric Endocrinology and Metabolism, Erciyes University, Kayseri, Turkey

Department of Pediatric Endocrinology and Metabolism, Mardin Public Hospital, Mardin, Turkey

email address: [email protected]

Pycnodysostosis (OMIM 265800) is a rare autosomal recessive disorder characterized by short stature, cranial dysplasia, clavicular dysplasia, acro-osteolysis of the terminal phalanges, osteosclerosis with increased bone fragility. The estimated prevalence of pycnodysostosis is 1 per 1.7 million. Pycnodysostosis is caused by mutations in the cathepsin K (CTSK) gene located at chromosome 1q21. It encodes a lysosomal cysteine protease which is critical for bone resorption and remodelling. Here, we described one Turkish male patient who has short stature and repeated fractures. He was the twelfth child of thirteen siblings, of parents who were third-degree cousins. At the clinical evaluation, his weight was 38 kg (<3rd centile), height 142 cm (<3rd centile) and head circumference 52 cm (3rd centile). The physical examination showed frontal bossing, prominent eyes with blue sclerae, beaked nose, micrognathia, short and bolded fingers. X-rays of the hands showed increased bone density, brachydactyly and acroosteolysis of distal phalanges. Radiography of the skull showed open fontanelles, abnormal widening of suturae cranii and obtuse mandibular angle. The patient’s clinical and radiographic features were compatible with pycnodysostosis, which was confirmed by gene studies identifying the homozygote, missense, c.953G > A (p.Cys318Tyr) variant in CTSK gene. This variant has been observed in individuals with pycnodysostosis but not previously reported from Turkey.

A rare microcephaly and malignancy syndrome, Nijmegen breakage syndrome: a case report

Kubra Kiranatlioglu, Selcan Ozturk, Serife Ebru Ozudogru, Neslihan Basgoz, Mehmet Canpolat, Tamer Gunes, Ekrem Unal  and Munis Dundar

Department of Medical Genetics, Erciyes University, Kayseri, Turkey

Division of Pediatric Neurology, Department of Pediatrics, Erciyes University, Kayseri, Turkey

Division of Neonatology, Department of Pediatrics, Erciyes University, Kayseri, Turkey

Division of Pediatric Hematology and Oncology, Department of Pediatrics, Erciyes University, Kayseri, Turkey

email address: [email protected]

Nijmegen breakage syndrome is a rare autosomal recessive disorder caused by the pathogenic alterations in the NBN gene. Chromosomal instability and sensitivity to ionizing radiation play a role in the pathophysiology. The main findings are microcephaly, pre- and postnatal growth retardation, dysmorphic facial features, recurrent infections, minor skeletal anomalies and increased risk of developing malignant diseases. When patients turn twenty years old, more than forty percent of them develop malignancies. This syndrome is estimated to affect one in one hundred thousand newborns worldwide. It is thought to be more frequent in Slavic populations in Eastern Europe. In this report, we investigated a case of a one-year-old girl with microcephaly, prenatal growth retardation, micrognathia, large dysplastic ears and partial syndactyly of the fourth and fifth toes. The patient was referred to our centre with a pre-diagnosis of Seckel syndrome. After a physical examination, we performed clinical exome sequencing in the patient. As a result of clinical exome sequencing analysis, we detected a frameshift homozygous pathogenic mutation in the NBN gene and associated this mutation with Nijmegen breakage syndrome. Early diagnosis of these patients is crucial for following up on these patients for immune deficiencies and malignancies.

A novel variant in SALL4 in a patient with Duane-radial ray syndrome

Firat Ozcelik, Hande Kulak, Nuriye Coskun, Selcan Ozturk, Sefer Kumandas  and Munis Dundar

Department of Medical Genetics, Erciyes University, Kayseri, Turkey

Division of Pediatric Neurology, Department of Pediatrics, Erciyes University, Kayseri, Turkey

email address: [email protected]

Duane-radial ray syndrome (DRRS, Okihiro syndrome) is a rare genetic condition that is caused by the pathogenic alterations in the SALL4 gene and is inherited in an autosomal dominant fashion. The syndrome is characterized by ocular anomalies (Duane anomaly), upper limb anomalies, and renal anomalies. SALL4 gene appears to be important for the normal development of the eyes, heart and limbs. It plays a key role in stem-cell maintenance and self-renewal. We report a 15-year-old Turkish male patient with a novel variant in the SALL4 gene. He was referred to our clinic with bilateral aplasia of the thumbs, bilateral aplasia of the radii, bilateral thenar hypoplasia, webbed and short neck, limited eye movements, limited arm extension and horseshoe kidney. Using Next-Generation Sequencing, we performed a gene panel that includes 4493 protein-coding genes’ exons and exon-intron borders. The results revealed a heterozygous nonsense variant c.670C > T (p.Gln224Ter) in the SALL4 gene (NM_001318031). The variant was classified as ‘pathogenic’ according to the ACMG 2015 guideline for interpretation of sequence variants.To our knowledge, there was no previous study reporting this variant. A case with isolated thenar hypoplasia was reported in Turkey. However, this is the first reported Duane-Radial Ray Syndrome patient in Turkey.

The combination of resveratrol and N-acetylcysteine modulate T-cell differentiation during PHA stimulation

Francesco Ragonese, Antonella De Luca, Lorenzo Monarca, Filomena De Falco, Daniele Sorcini, Francesco Sordini, Beatrice Del Papa, Rossana Iannitti, Paolo Sportoletti  and Bernard Fioretti

Department of Chemistry, Biology and Biotechnologies, University of Perugia, Perugia, Italy

Department of Experimental Medicine, University of Perugia, Perugia, Italy

Hematology Division, Department of Medicine, University of Perugia, Perugia, Italy

S&R Farmaceutici S.p.A Bastia, Umbra, Italy

email address: [email protected]

Modulation of the immune response is one of the major issues in alleviating immune-mediated damage associated to infection. Lymphocytes cells differentiating into subsets is an important element to take into account when studying a molecules with immunomodulatory and anti-inflammatory properties. Here, we evaluated the effects of resveratrol (RSV, 3 µM) and N-acetylcysteine (NAC, 30 µM) alone or in combination for 48 h on the expression of cytokines and process of lymphocyte differentiation in naive human peripheral blood mononuclear cells (PBMC) and after exposure to PHA (10 μL/mL). The novel observations of this study was that the combination of RSV and NAC can influence the Th1/Th17 balance and contribute to the induction of CD4 + CD25 + FOXP3 T (Treg) cells and inhibit the Th17 axis after phytohemagglutinin (PHA) stimulation (24 h). Gene expression analysis also showed that the treatment with RSV + NAC modulated the expression of IL-10 and downregulated IL-17. These findings suggest that RSV together with NAC differentially alter the immune cell activation status and cytokine response, providing support for the promotion of immune surveillance in resting conditions and immune tolerance after 24 h stimulation.

Evolutionary engineering approach for adaptation of PHA producing strain Halomonas halophila to levulinic and acetic acid

Ivana Novackova, Vendula Chatrna, Eva Slaninova, Jaromir Porizka, Petr Sedlacek  and Stanislav Obruca

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, BUT, Brno, Czech Republic

Department of Physical and Applied Chemistry, Faculty of Chemistry, BUT, Brno, Czech Republic

email address: [email protected]

Evolutionary engineering provides approaches useful for obtaining of microorganisms with desired characteristics on phenotype level (e.g. more effective growth, ability of utilization of different carbon sources, etc.). Characteristics of polyhydroxyalkanoates (PHAs), microbial biodegradable and biocompatible polymers, strongly depend on monomer composition. These materials could be advantageously used as an alternative of traditional petrochemical plastics. Evolved microbial strains obtained during evolutionary engineering experiments could be used for production of PHA with required properties for selected purposes. Promising halophilic PHA producer Halomonas halophila (CCM 3662) was exposed to microbial inhibitors commonly presented in hydrolysates of lignocellulosic biomass, namely acetic and levulinic acid. Multiple serial transfers of cell cultures in Erlenmeyer flasks after 48 h were provided within adaptation experiments for more than 80 passages. Every passage has been basically characterized and selected preserved ones were also compared with wild-type strain considering potential of PHAs accumulation, effectivity of utilization of organic acids, testing of robustness and others features.

Funding: This work was supported by project GA 19-20697S of Czech Science Foundation (GAČR) and also by Brno Ph.D. Talent – Funded by the Brno City Municipality.

Polyhydroxyalkanoates’ production by thermophilic genera Caldimonas and Tepidimonas

Xenie Kourilova, Michaela Vidlakova, Iva Pernicova, Ivana Novackova and Stanislav Obruca

Faculty of Chemistry, Institue of Food Science and Biotechnology, Brno University of Technology, Brno, Czech Republic

email address: [email protected]

Polyhydroxyalkanoates (PHAs) are bacterial polyesters and many prokaryotic microorganisms accumulate them in the form of intracellular granules. From an industrial point of view, PHAs are particularly interesting because of their properties, which are reminiscent of some synthetic polymers. However, PHAs are completely biodegradable and biocompatible. For this reason, PHAs are generally considered to be an ecological alternative to synthetic polymeric materials. Still, there is a major drawback in PHA’s commercial use. It’s the production cost. There are two main ways how to reduce the price and increase competitiveness. One possibility is the use of waste substrates and the other is the use of extremophilic microorganisms, in our case specifically thermophilic. The thermophilic strains of the genera Caldimonas and Tepidimonas, which have not been explored so far, present great potential in PHA production. They are generally Gram-negative, rod-shaped cells and their optimum growth is around 50 °C. The aim of this work was to create a basic comprehensive overview of the biotechnological potential of these genera. Production conditions were tested at various temperatures, carbon substrates and precursors suitable for the synthesis of polymers with excellent properties. Based on our results, these genera can be considered as promising PHA producers.