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Individuals of symbiotic green hydra (Hydra viridissima Pallas, 1766) and free-living brown hydra (Hydra oligactis Pallas, 1766) were treated with aqueous solutions of herbicide norflurazon (2x10-7, 2x10-6 mol/L) for 72 h and hydras were also exposed to simultaneous treatment to norflurazon and UVB radiation (λ = 254 nm, 2 min), along with control groups. The experiment was performed in triplicate. Migration and change of body shape (relaxed, contracted), as the evasion signs of deleterious effects, were observed by stereo microscope, and responses to mechanical stimuli were also noted. Comet assay was applied to measure the level of primary DNA damage of exposed green and brown hydras. Beside single and double strand breaks, it detects DNA base alkylation, oxidatively changed bases, DNA-adducts, covalently bound proteins to DNA, cross-linking and base dimers. Each hydra was put into a conical tube containing chilled homogenization buffer (75 mM NaCl, 24 mM Na2EDTA, pH 7.5, +4 °C) and its cells were separated mechanically. The obtained homogenate was mixed with the low melting point agarose, spread across the microscopic slide and left for the gel to polymerize. Preparations were treated with a series of buffers to remove the cell structures and isolate nucleoids. By immersing the slides into alkaline buffer, pH 13.0, DNA denatures rapidly. At the spots of primary damage, DNA strands broke, which led to the formation of relaxed loops and free DNA fragments. In electrophoresis, these loops and fragments are pulled from the nucleoid core toward the anode, and following fluorescent staining, under the microscope, they appear as comets. Comet preparations were analysed under an epifluorescent microscope equipped with CCD camera, and the level of damage was measured by image analysis software for the comet assay. Based on the intensity of fluorescence of the tail compared to the nucleoid, the percentage of DNA affected by primary damage was calculated. The treatment did induce a concentration related increase in primary DNA damage, but no significant difference between green and brown hydra was observed (4.40 % vs. 5.23 %, respectively ; negative controls 1.13 % and 1.17 %, respectively). On the other hand, in the presence of radiation, brown hydras showed significantly higher level of primary DNA damage already when exposed to the lowest concentration compared to the green ones (15.10 % and 3.77 %, respectively), proving the advantage of the symbiotic organism. The increase in damage was concentration dependent in either case.

Rotifers and planktonic crustaceans (cladocerans and copepods) occupy a central role in the food webs of shallow water bodies, connecting autotrophic (algae) with heterotrophic mode in the food webs. Differences between temperate-continental and Mediterranean climates affect variously interactions of abiotic and biotic factors. The aim of this study was to determine: (i) the diversity, abundance and trophic structure of zooplankton ; (ii) zooplankton indices in comparison to the trophy state indices ; (iii) a proposal for measures in the management and conservation of shallow water bodies in two different climates. In nine ponds, 48 species of zooplankton were determined, and rotifers dominated with 36 species. Habitat complexity, macrophyte coverage, and nutrient concentrations were indicated as significant factors in the structuring of zooplankton in shallow water bodies of both climate regions. Zooplankton in the temperate-continental climate was characterized by populations of higher diversity, while in the Mediterranean climate zooplankton was characterized by populations of lower diversity and higher abundance, particularly species of genera Lecane and Brachionus. Results of this study support the importance of zooplankton as a sentinel indicator in assessing the trophic and ecological status of shallow water bodies and provide a basis for determining guidelines in the conservation and management of these sensitive and endangered ecosystems.

Iako je Bosna i Hercegovina postigla značajan napredak na svim područjima u poslijeratnom razdoblju, ona se još bliži statusu kontinuirane opće krize i izložena je velikom riziku pogoršanja situacije pod utjecajem globalne financijske, ekonomske i socijalne krize. Nekoliko je uzroka opće krize: ekonomska situacija, socijalni položaj stanovništva, politička ne- stabilnost i onemugućavanje vladavine prava, koji će ugrozit integraciju Bosne i Hercegovine u Europsku uniju. Pitanja su o kojima ćemo govoriti u ovom radu: Imamo li vladavinu zakona u Bosni i Hercegovini Da li je moguće procesuirati i otpuštati politički neodgovorne ljude Da li je moguće spriječiti različite oblike viktimizacije građana Bosne i Hercegovine zbog različitih oblika zlouporabe vlasti u Bosni i Hercegovini Imamo li "demokracija" u krizi u Bosni i Hercegovini Da li smo žrtve zlouporabe položaja

After 14 years, the Multinational Congress on Microscopy again took place in Croatia, this time in Rovinj, on September 24–29, 2017. MCM 2017 was jointly organized by the Croatian Microscopy Society, with the Ruđer Bošković Institute, Zagreb. MCM 2017 brought together leading experts and young researchers who develop microscopy methods and apply them in the fields of life and material sciences. Altogether, more than 450 delegates from 30 countries participated with a keynote and seven plenary lectures, 42 invited talks and 84 short talks in 3 parallel sections and 175 posters.

Free-living unicellular algal species Chlorella vulgaris Beij. [K&H, 1992] strain SAG 211 - 11b is often found in freshwater habitats and overall shows high adaptability. Turbellaria are one of the traditional sub-divisions of the phylum Platyhelminthes (flatworms). As predators they inhabit freshwater or moist terrestrial environments. They move by cilia on the ventral dermis, allowing them to glide along on a film of mucus. In this experiment were used free-living unicellular photoautotrophic alga C. vulgarisand turbellarians Dugesia gonocephala (Duges, 1830)and Polycelis felina (Dalyell, 1814). The influence of algae on the behavior of turbellarians was assessed. Algae were cultured on a sterile deep stock agar and were growing in test tubes of 16 cm in length and 15 mm in diameter in air chamber under sterile conditions at 24 °C and constant light. By standardizing the method of maintaining algal culture, a constant amount of clonal cultures suitable for performing the experiment was obtained. Turbellarians were isolated from their natural habitat, cultivated at 13.5 °C and fed with aquatic crustaceans Artemia salina. The experiment was conducted under different temperature and light conditions : at 25 °C and exposed to daylight (photoperiod 8 hs light, 16 hs dark) and at 13.5 °C in the dar k. Different number of turbellarians was added in experimental dishes (60 ml): 5 dishes with one specimen in each (first setup with P. felina and second with D. gonocephala ) and 5 dishes with 5 specimens in each (first setup with P. felina and second with D. gonocephala) at both temperatures, including both fed and starved animals separately, and in both sets with constant concentrations of C. vulgaris added to each experimental dish. The results were recorded immediately after the experiment setup and every 1, 8 and 24 hs after the beginning of the experiment. There were 5 replicas for each experiment, including controls. Altogether 170 experimental dishes, 240 P. felina and 240 D. gonocephala individuals were used. For analysis of algae, light and cTEM microscopy were used. After one hour noticed was the sediment of algae formed at the bottom of each experimental dish as well as the inhibition of turbellarians D. gonocephala by Chlorella, mitigating their movement and coordination. In the suspension of algae, D. gonocephala exhibited extremely numb behaviour. Individuals that were sporadically moving had extremely slow and uncoordinated behaviour and this was specifially expressed only on the walls of the glass dish where no algal sediment was present. D. gonocephala that rested, mostly rested in the algal sediment at the bottom of the dish. This phenomenon of inhibition of motion and coordination was also present in P. felina , but it was extremely expressed in D. gonocephala.

Green Hydra (Hydra viridissima Pallas, 1776) is a typical example of an endosymbiotic organism. In gastrodermal myoepithelial cells it contains unicellular photoautotrophic algae. Algae and especially microalgae take an important place as the primary producers and food resources in aquatic ecosystem sand are the basis of many food chains.Class Turbellaria is the most primitive group within the phylum Platyhelminthes. Turbellarians generally locomote by coordinated waves of cilia on a secreted mucus trail, though some species can swim by rhythmic muscle contractions. In this experiment, the interaction between endosimbiotic algae Desmodesmus subspicatus (Chlorophyceae) (Chodat) Hegewald and Schmidt isolated from green hydra and predatory turbellarian species Polycelis felina (Dalyell, 1814) and Dugesia gonocephala (Duges, 1830) was observed at different light and temperature conditions: at 25 °C and exposed to daylight (photperiod 8 hs light, 16 hs dark) and at 13.5 °C in the dark, both separately with starv ed and fed animals. The results were recorded immediately after the experiment setup, and every 1, 8 and 24 hs after the beginning of the experiment, including the controls. Cladoceran Daphnia magna (Straus, 1820) was added to each glass dish as a prey (10 individuals). There were 5 replicas for each experiment, and the controls: altogether 220 dishes and 1540 animals. Hunting nets were visualized by adding the constant amount of algal suspension to e ach glass dish.For analysis of algae, light and cTEM microscopy were used. In this micro-ecosystem, turbellarians were characterized by phenomenon of mucus network emerging by the interaction with algae. The appearance of mucus and the formation of complex network occurred between 1 and 8 hs after the experiment setup.It was observed that the appearance of the nets was intensive due to the presence of algae this way changing the interactions within fresh water micro-ecosystem. The development of the nets was noticed even when only algae were present. But, in interaction with turbellarians especially with starved P.felina at the 13.5 °C this effect was much more intensive. Mostly after 8 hs Daphnia specimens were trapped within the nets but some remained alive and some of them managed to escape. After 24 hs within dishes where the nets were formed the highest density of dead D.magna was observed. There is a clear correlation between D. Magna density, algae and formation of the nets. The interaction of endosimbiotic algae and turbellarians created a mechanism that potentially gives more efficient hunting and stronger predatory role for turbellarians in the fresh water ecosystems. Further microscopic analyses could provide the insight into the complex formations of the hunting nets and food web interactions.

Concentrated CaCl2 and CaBr2 salt solutions of densities up to 2.3 kg L-1 are regularly used to control hydrostatic pressure in oil wells during special operations in the exploration and production of natural gas and crude oil. Various concentrations of high density salts are frequently left in mud pits near the drilling site as waste, polluting fresh and ground waters by spillage and drainage. The toxic effects of these salts have already been observed. This study investigated the effects of CaCl2 and CaBr2 on water flea Daphnia magna Straus in a 21-day reproduction test. The three tested concentrations of CaCl2 (240, 481, and 1925 mg L-1) caused a significant dose-response decrease of reproduction (p

For the first time effects of norflurazon on green (Hydra viridissima Pallas, 1766) and brown hydra (Hydra oligactis Pallas, 1766) in high- and low-light conditions were investigated in order to establish the extent of damage that this substance inflicts, with special emphasis on the "bleaching effect" and the effect on hydra-algae symbiosis. Green hydra is a typical example of an endosymbiotic organism. The gastrodermal myoepithelial cells of green hydra contain endosymbiotic algae. Norflurazon is a selective translocational herbicide that induces a "bleaching effect" on newly developed chloroplasts, resulting in a decrease of photosynthetic activity and viability of the organism. In the experiment, morphological (binocular), cytological and histological (Bouin fixative, dehydration, paraplast embedding, Hämalaun-eosine staining, light microscope) and conventional transmission electron microscopy (cTEM) (glutaraldehyde, dehydration, raisin, uranyl-acetate, Pb-citrate) were used. Depending on the concentration and light conditions, norflurazon caused mortality, deformations, changes in behavior, locomotion and asexual reproduction, changes in the structure of all 3 layers in hydras, changes in the position and shape of endosymbiotic algae inside the hydra body as well as ultrastructural changes of treated hydras and endosymbiotic algae. Under low concentrations of norflurazon the effects on hydra were similar to the controls, while in the highest concentrations especially manifested were antichloroplastal and antimitochondrial effects. Norflurazon caused a great extent of damage and induced deleterious effects also upon other cellular components such as cellular membranes, ER, Golgi apparatus, ribosomes. Newly developed buds in symbiotic green hydras were not bleached. After a recovery period, the green hydra individuals that had survived re-established regular endosymbiosis with algae and recovered completely, whereas brown hydras recovered only partially.

Hydras are simple aquatic organisms, members of the phylum Cnidaria, usually found in unpolluted freshwater. Green hydra (Hydra viridissima Pallas, 1766) is an endosymbiotic and brown hydra (Hydra oligactis Pallas, 1766) is a non-symbiotic species [1]. Aluminium is one of the most abundant elements in the Earth's crust, but its effects upon living organisms have not been much studied. The aim of this research was to explore the effects of aluminium in two closely related hydra species, and to trace the extent of damage caused. For the first time, a modified alkaline comet assay [2] was used to study primary DNA damage in green and brown hydra cells. Five animals of each species were treated under laboratory conditions (21.5°C) with aluminium-sulphate (Al2(SO4)3.18H2O ; 25, 250, 475 mg/L) in aerated aquarium water in glass dishes, 6 cm in diameter, 3.5 cm in height (photoperiod 10 hrs light/15 μ mol/m2s, 14 hrs dark) and compared with the control groups. After 72 hrs, animals from each of the concentrations of Al were transferred into a micro-test-tube containing 10 μ l of chilled distilled water. Hydras were homogenized with a 100 μ l micropipette by sucking the animals in and out several times. Fully frosted slides were coated with 1% and 0.6% normal melting point (NMP) agarose. Homogenized hydras were mixed with 0.5% low melting point (LMP) agarose and placed on the slides. The slides were immersed for 1.5 hrs in freshly prepared ice-cold detergent lysis solution pH 10. Alkaline denaturation was carried out in freshly prepared buffer pH 13.0 for 15 min and electrophoresis for another 10 min at 0.8 V/cm. Slides were neutralized with Tris-HCl buffer pH 7.5, stained with ethidium bromide and examined using epifluorescence microscope Orthoplane (Leitz Wetzlar, Germany), equipped with immersion objective 170/0.17 63/1.30, an excitation filter of 515– 560 nm and a barrier filter of 590 nm (Leitz Wetzlar, Germany). A total of 100 comets were scored per slide. Using a black and white high performance CCD camera 4900 Series (COHU), the microscope image was taken and analysed by Comet Assay II software (Perceptive Instruments Ltd.). Tail length (μ m) and tail intensity were used as a measure of DNA damage. Entire DNA damage evaluation was performed in duplicate. Means of repeated experiments were used to test the difference of the tail length and intensity values between treated and control animals applying the Student’ s t-test (Statistica 5.0, StatSoft, USA). The dependence of the comet assay endpoints on the Al concentration was evaluated by calculating the Spearman correlation coefficient. Multiple regression was performed to test the predictivity of the Al-dose-response. Significance level was set to 0.05. Aluminium toxicity triggered DNA changes. DNA tail length and intensity changes were greater in brown than in green hydras. Since the comet assay detects DNA lesions at a single cell level [3], it enables to observe intercellular differences in the genomic susceptibility. Control cells appear as compact orange spheres, whereas cells bearing the genome damage exhibit more or less dense tail of orange spots resembling the comets in the sky (Fig. 1). Brown hydra was more susceptible to aluminium than green hydra, confirming the evolutionary advantage provided by symbiosis. Due to its higher genetic susceptibility, brown hydra appears to be a better biomarker for aquatic toxicology studies compared with green hydra. Due to its higher correlation and predictivity, the tail intensity was shown to be a more relevant comet assay parameter for DNA damage than tail length. Biomonitoring protocols using hydra and the comet assay could be developed to provide a valuable and rapid method for determining the quality of freshwater. 1. Douglas, A.E., Symbiotic Interactions. Oxford University Press Inc., Oxford & New York (1994) 2. Singh, N.P. et al., Exp. Cell Res. 175 (1988) p184. 3. Collins, A.R., Mol. Biotechnol. 26 (2004) p249. 4. The presented results are a product of a scientific projects “ Molecular phylogeny, evolution and symbiosis of freshwater invertebrates” and “ Genotoxicity of chemical and physical agents of natural and anthropogenic origin” carried out with the support of the Ministry of Science, Education and Sport of the Republic of Croatia. We thank Professor D.H.S. Richardson and the two reviewers for helpful comments on this manuscript. AB Figure 1. Micrographs of green hydra cell (a) and brown hydra cell (b) with highly damaged DNA. Hydras were exposed to the 250 mg/L Al2(SO4)3.18H2O for 72 hours. Micrographs were taken using Comet Assay II software and COHU CCD camera 4900 Series. Magnification 400x.

Background and Purpose: Chlorella test was performed for the first time. The interesting application of a newly proposed test in ecotoxicological research was explained. It was tested using the herbicide norflurazon as bleaching toxicant. The experimental object was green alga Chlorella kessleri Fott et Novak. [K&H, 1992]. Materials and Methods: Algae were treated on the surface of deep stock agar in tubes. Instead of dH2O t the equivalentmeasure of aqueous solution of toxicant was used in the medium. This is the essence of the Chlorella test method. Results: Already after a short period of two days, morphological changes were distinguished macroscopically. Bleaching of algae was present in particular tubes, as the result of the effect of the toxicant upon the algal growth and viability. Conclusions: We propose Chlorella test as a toxicity test that provides easy detection of sublethal, lethal and NOEC. The use of this quick, simple, indicative, cheap and applicative test is proposed as amethod for research on the preliminary effects of environmental pollutants upon green algae in culture.

Although aluminum is one of the most abundant chemical elements in the Earth's crust, its effect on living beings was little known until recently. Since the effect of aluminum ions upon green and brown hydras has not been studied yet, with this technical note we showed how aluminum depositions in symbiotic and asymbiotic hydras could be detected, observed and analyzed. Differential eriochrome cyanine staining on hydras has been performed for the first time, using an appropriately modified technique for detecting the aluminum depositions in hydras. This hydra bioassay was shown to be a useful and exact tool for detection of expected results e.g. aluminum depositions in hydra cells.

Broj 𝜋 s razlogom se može nazvati čudesnim brojem. Već stotinama i tisućama godina fascinira matematičare, a i znanstvenike općenito, svojom zagonetnom beskonačnošću. Što točnije ga odrediti bio je primarni cilj. Kako doći do što točnije formule za izračun, kako ga primijeniti na poljima matematike i arhitekture, problemi su koji su golicali umove znanstvenika kroz povijest. Isto je i danas, kada postoji svojevrsna računalno-algoritamska utrka koja je istovremeno i sprint i maraton – u što kraćem roku doći do što većeg broja točnih decimala koje se sada već broje u stotinama milijarda. There is a reason why number 𝜋 is called the magical number. For more than a couple of hundreds and thousands of years it fascinates mathematicians, and scientists alike with its enigmatic infinity. The prime goal is the more exact value of it. How to get to the more correct formula and how to apply it in a field of math and architecture are problems which intrigued the minds of scientists through the history. Same is today, when there is a kind of computer-algorithm race which at the same time is sprint and marathon – in the least time possible to get to a greater number of decimals which are now already counted in hundreds of billions.

Graphene is a material with extraordinary chemical and physical properties and due to them is emerging as a promising material for a wide range of applications. Among them is the application for electrode material or surface modification of electrochemical sensors [1]. Using voltage cycling in electrolyte solution, it is possible to induce surface changes in electrodes modified with graphene paste. Induced surface changes cause enhancement of electrochemical sensing characteristics. In this study, screen-printed electrode with a graphene paste modified surface is prepared and electrochemical sensing characteristics are investigated. The electrode surface is investigated by Raman spectroscopy, energy- dispersive X-ray spectroscopy, and scanning electron microscopy before and after voltage cycling treatment. After voltage cycling electrode shows enhanced sensing characteristics, higher heterogeneous electron transfer rate, lower electron transfer resistance, lower capacitance, and higher sensitivity. Raman spectroscopy indicates a higher amount of available basal and edge plane graphene, while energy dispersive X-ray spectroscopy indicates the presence of intercalated oxygen species.

With its endocrine and metabolic activity, visceral adipose tissue (VAT) is more than a storage organ. Research shows that VAT has a role in the development of cardiovascular disorders. The influence of epicardial adipose tissue (EAT, a subset of VAT) morphology on cardiovascular function and health is still limited (1). Furthermore, the data on the content of lipids and their metabolites in the EAT depending on the amount of VAT are scarce or non-existent. The aim of this pilot study was to characterize lipids and their metabolites in EAT in relation to visceral obesity. EAT from 6 obese and 6 non-obese subjects (based on waist circumference) was collected post-mortem and homogenized in 20mM ammonium acetate buffer using Dounce homogenizer. Lipid extraction from the homogenate was performed with Blight and Dyer’s two-phase method (2). Both polar and nonpolar phases were imaged in positive and negative imaging mode on Bruker UltrafleXtreme MALDI-TOF device in the 200-1500 m/z range. The matrices used were dihydrobenzoic acid and 9-aminoacridine, respectively. Collected data were analyzed using R statistical software with FELLA and KEGGREST packages. Out of 106 determined putative metabolites, C08320 – Lignoceric acid was 2.37 times more abundant in the obese group (p=0.0396). Out of the 106 molecules, 13.5% were upregulated, 11.9% were downregulated in the obese group and the rest were unchanged compared to the non-obese group. FELLA enrichment revealed alteration in fatty acid biosynthesis and degradation accompanied with upregulation in glycosphingolipid biosynthesis. Preliminary data indicate a metabolic difference in EAT of obese patients. None of the subjects suffered from Gaucher’s disease, so it may be assumed that lignoceric acid is exogenous in nature (from food). This also explains upregulation in glycosphingolipid biosynthesis, lignoceric acid being one of its precursors. The preliminary data showed some changes in EAT of obese patients which are potentially food-related. Larger groups are necessary to draw more definite conclusions.

Cell migration is one of the basic properties of almost all eukaryotic cells and is crucial in a large number of biological processes. The shape of migrating cells is primarily determined by the spatial and temporal dynamics of the intracellular actin cytoskeleton and associated proteins. However, the shape and migratory properties of cells in two-dimensional culture depend also on the specific interactions of cells with the underlying surface. It is known that amoebae Dictyostelium discoideum dynamically change their shape when migrating. In order to quantitatively classify cell shapes, one possible approach is to use descriptors based on invariant moments of mass distribution. For example, elongation (E) is equal to the ratio of the long and short radii of an ellipse that best fits the contour of a cell, and dispersion (D) describes how much mass is dispersed away from the centroid of the cell shape [1]. In order to quantify the dependence of the shape of D. discoideum cells on spatial confinement, we engineered patterned surfaces coated with anti-adhesive co-polymer PLL- g-PEG (polylysine grafted to polyethylene glycol), except for small patches of uncoated glass several square micrometers in size [2]. Contours of fluorescently labelled cells, which were deposited either onto the confined adhesive patches or onto the plain glass coverslips, were obtained by analysing the images obtained by confocal fluorescence microscopy. Comparison of the shape descriptors of confined vs. nonconfined cells showed that ranges of, and ratios between, cell elongation and dispersion values depended on the state of confinement. The methodology developed here will be useful in characterizing the role of proteins involved in the regulation of the cell- substratum adhesion and the cell shape dynamics by analysing the phenotypes of mutant cells devoid of these proteins.