Determination of volume averaging correction factors using an elliptical absorbed dose model for Gamma Knife Perfexion.

PURPOSE: The purpose of this study is to calculate volume averaging correction factors for detectors used in the dosimetry of Gamma Knife's narrow photon beams, and to determine the impact of volume averaging on the field output correction factor. METHODS: Simulations of different Gamma Knife fields were done using elliptical dose model formalism with newly introduced fit functions. To determine volume averaging correction factors a calculation of the absorbed dose over the volume of the detector was performed. The elliptical dose model was tested with respect to absorbed dose distribution for different volumes and compared with the calculations of Leksell GammaPlan v.11.3.1. RESULTS: The largest differences in absorbed dose calculated by the elliptical model and Leksell GammaPlan are 2.25%, 1.5%, and 0.6% for 16, 8, and 4 mm field sizes, respectively. Volume averaging correction factors were determined for six ionization chambers, five semiconductor detectors, a diamond, and two plastic scintillator detectors. In general, for all examined detectors the impact of volume averaging is more pronounced for smaller field sizes. All studied ionization chambers had a larger volume than other detectors, therefore the volume averaging correction factors for ionization chambers are larger for all investigated field sizes. Besides the fact that plastic scintillator detectors can be considered tissue-equivalent, volume averaging correction factor should be applied. CONCLUSION: Volume averaging correction factors for different detectors are determined and suitable detectors for dosimetry of Gamma Knife's narrow photon beams are recommended. It is shown that volume averaging has a dominant contribution to a field output correction factor.

Bioprospecting of Less-Polar Fractions of Ericaria crinita and Ericaria amentacea: Developmental Toxicity and Antioxidant Activity.

Ericaria crinita and Ericaria amentacea from the Adriatic Sea (Croatia) were investigated with respect to the presence of less-polar compounds for the first time after fractionation by solid-phase extraction (SPE). The composition of less-polar fractions of freeze-dried E. crinita (FdEc) and E. amentacea (FdEa) were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). The major identified compounds were: amides of higher aliphatic acids (palmitoleamide, linoleamide, palmitamide, oleamide and erucamide) and related compounds, carotenoid (fucoxanthin), chlorophyll derivatives (pheophytin a and b and their derivatives) and higher terpenes (loliolide, isoamijiol with its oxidation product), ß-stigmasterol and (3ß,6α)-14-methylergosta-8,24(28)-diene-3,6-diol). The toxic effects observed on the less-polar fractions obtained from Ericaria species on zebrafish Danio rerio embryos could be associated with the high abundance of all five detected amides. The antioxidant activity of the fractions was evaluated by means of five independent assays, including the reduction of the radical cation (ABTS), the oxygen radical absorbance capacity (ORAC), ferric-reducing antioxidant power (FRAP), the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay and the Folin-Ciocalteu method. A higher antioxidant activity of E. amentacea in comparison to that of the E. crinita fractions was found with IC50 concentrations of 0.072 and 1.177 mg/mL, respectively. The correlation between the activity and the chemical composition revealed that the synergistic effect of different compounds impacted their antioxidant response.

Bioprospecting of Less-Polar Constituents from Endemic Brown Macroalga Fucus virsoides J. Agardh from the Adriatic Sea and Targeted Antioxidant Effects In Vitro and In Vivo (Zebrafish Model).

The endemic brown macroalga Fucus virsoides J. Agardh from the Adriatic Sea was in the focus of the present research. The volatiles of fresh (FrFv) and air-dried (DrFv) samples of F. virsoides obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were analyzed by gas chromatography equipped with flame ionization detector and mass spectrometry (GC-FID/MS). The major HS-FrFv compound was pentadecane (61.90-71.55%) followed by pentadec-1-ene (11.00-7.98%). In HS-DrFv, pentadec-1-ene was not present, and few lower aliphatic compounds appeared, as well as benzaldehyde and benzyl alcohol. In HD-FrFv, particularly abundant were alkenes (such as pentadec-1-ene (19.32%), or (E)-pentadec-7-ene (8.35%)). In HD-DrFv, more oxidation products were present (e.g., carbonyl compounds such as tridecanal (18.51%)). The fatty acids profile of freeze-dried sample (FdFv) after conversion to methyl esters was determined by GC-FID, and oleic acid was dominant (42.28%), followed by arachidonic acid (15.00%). High-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (HPLC-ESI-HRMS) was used for the screening of less polar fractions (F3 and F4) of F. virsoides. Mono- and diglycerides of stearic, palmitic, oleic, and arachidonic acids were found. Terpenoids and steroids comprised the compounds C20H30(32)O2 and C29H48O(2). Among carotenoids, fucoxanthin was identified. Chlorophyll derivatives were also found (C55H74(72)N4O(5-7)), dominated by pheophytin a. The antioxidant activity of the fractions was investigated by in vitro assays (oxygen radical absorbance capacity (ORAC), reduction of radical cation (ABTS•+), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay, and ferric reducing antioxidant power (FRAP)) and by in vivo zebrafish model (along with fish embryotoxicity). In vitro experiments proved good radical scavenging abilities of F3 and F4 fractions, which were additionally supported by the protective effect against hydrogen peroxide-induced oxidative stress in zebrafish embryos.

Bioprospecting of Coralline Red Alga Amphiroa rigida J.V. Lamouroux: Volatiles, Fatty Acids and Pigments.

Due to the lack of phytochemical composition data, the major goals of the present study on Amphiroa rigida J.V. Lamouroux were to: (a) investigate and compare volatilome profiles of fresh and air-dried samples obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) followed by gas chromatography and mass spectrometry (GC/MS) analysis; (b) determine fatty acids profile by gas chromatography with flame ionization detector (GC-FID); (c) obtain the pigment profiles of semipurified extracts by high performance liquid chromatography (HPLC) and (d) evaluate the antioxidant and antimicrobial activities of its less polar fractions. The comparison of headspace of fresh (FrAr) and air-dried (DrAr) samples revealed many similarities regarding the presence and abundance of the major (heptadecane and pentadecane) and minor compounds. The hydrodistillate (HD) of DrAr profile was quite different in comparison to HD-FrAr. The predominant compound in HD-FrAr was (E)-phytol. In HD-DrAr, its percentage was approximately one-half reduced, but the abundance of its degradation product phytone and of unsaturated and oxygenated compounds increased indicating more intense fatty acid decomposition and oxidation during drying. The fatty acid determination revealed that the most dominant was palmitic acid (42.86%) followed by eicosapentaenoic acid (19.14%) and stearic acid (11.65%). Among the pigments, A. rigida contained fucoxanthin (0.63 mg g- 1 of dry fraction), lutein (5.83 mg g- 1), ß-carotene (6.18 mg g-1) and chlorophyll a (13.65 mg g-1). The analyzed less polar fractions of A. rigida exhibited antioxidant scavenging activity with diammonium salt of 2,2'-azino-bis (3-ethylbenzthiazolin-6-yl) sulfonic acid (ABTS) assay up to 3.87 mg g-1 trolox equivalents (TE), and with the oxygen radical absorbance capacity (ORAC) assay up to 825.63 µmol g-1 TE (with carotenoids as the major contributors).

Possibility of Human Gender Recognition Using Raman Spectra of Teeth.

Gender determination of the human remains can be very challenging, especially in the case of incomplete ones. Herein, we report a proof-of-concept experiment where the possibility of gender recognition using Raman spectroscopy of teeth is investigated. Raman spectra were recorded from male and female molars and premolars on two distinct sites, tooth apex and anatomical neck. Recorded spectra were sorted into suitable datasets and initially analyzed with principal component analysis, which showed a distinction between spectra of male and female teeth. Then, reduced datasets with scores of the first 20 principal components were formed and two classification algorithms, support vector machine and artificial neural networks, were applied to form classification models for gender recognition. The obtained results showed that gender recognition with Raman spectra of teeth is possible but strongly depends both on the tooth type and spectrum recording site. The difference in classification accuracy between different tooth types and recording sites are discussed in terms of the molecular structure difference caused by the influence of masticatory loading or gender-dependent life events.

Fish photobacteriosis-The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida.

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent.

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study.

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO2 stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92-94%, DNA may undergo a structural transition into a more compact form.

Synthesis and Biological Evaluations of Granulatamide B and its Structural Analogues.

BACKGROUND: While granulatamides A and B have been previously isolated, their biological activities have been only partially examined. The aim of this study was to synthesize granulatamide B (4b), a tryptamine-derivative naturally occurring in Eunicella coral species, using the well-known procedure of Sun and Fürstner and its 12 structural analogues by modifying the side chain, which differs in length, degree of saturation as well as number and conjugation of double bonds. METHOD: The prepared library of compounds underwent comprehensive assessment for their biological activities, encompassing antioxidative, antiproliferative, and antibacterial properties, in addition to in vivo toxicity evaluation using a Zebrafish model. Compound 4i, which consists of a retinoic acid moiety, exhibited the strongest scavenging activity against ABTS radicals (IC50 = 36 ± 2 µM). In addition, 4b and some of the analogues (4a, 4c and 4i), mostly containing an unsaturated chain and conjugated double bonds, showed moderate but non-selective activity with certain IC50 values in the range of 20-40 µM. RESULT: In contrast, the analogue 4l, a derivative of alpha-linolenic acid, was the least toxic towards normal cell lines. Moreover, 4b was also highly active against Gram-positive Bacillus subtilis with an MIC of 125 µM. Nevertheless, both 4b and 4i, known for the best-observed effects, caused remarkable developmental abnormalities in the zebrafish model Danio rerio. CONCLUSION: Since modification of the side chain did not significantly alter the change in biological activities compared to the parent compound, granulatamide B (4b), the substitution of the indole ring needs to be considered. Our group is currently carrying out new syntheses focusing on the functionalization of the indole core.

The influence of copper-doped mesoporous bioactive nanospheres on the temperature rise during polymerization, polymer cross-linking density, monomer release and embryotoxicity of dental composites.

OBJECTIVES: Composites with copper-doped mesoporous bioactive nanospheres (Cu-MBGN) were developed to prevent secondary caries by imparting antimicrobial and ion-releasing/remineralizing properties. METHODS: Seven experimental composites containing 1, 5 or 10 wt% Cu-MBGN, the corresponding inert controls (silica) and bioactive controls (bioactive glass 45S5) were prepared. The temperature rise during light curing, cross-linking density by ethanol softening test, monomer elution and their potential adverse effects on the early development of zebrafish Danio rerio was investigated. RESULTS: Materials combining Cu-MBGN and silica showed the highest resistance to ethanol softening, as did the bioactive controls. Cu-MBGN composites showed significant temperature rise and reached maximum temperature in the shortest time. Bisphenol A was not detected, while bis-GMA was found only in the control materials and TEGDMA in the eluates of all materials. There was no increase in zebrafish mortality and abnormality rates during exposure to the eluates of any of the materials. CONCLUSIONS: The composite with 5 wt% Cu-MBGN combined with nanosilica fillers showed the lowest ethanol softening, indicating the polymer's highest durability and cross-linking density. Despite the TEGDMA released from all tested materials, no embryotoxic effect was observed.

Estimating risk of cardiovascular pharmaceuticals in freshwaters using zebrafish embryotoxicity test - statins threat revealed.

Cardiovascular pharmaceuticals (CVPs) are globally present in inland waters and have also been found in the sediment and plasma of fish from the Sava River, Croatia. Based on the previous research, CVPs amiodarone (AMI), ramipril (RAM), simvastatin (SIM), and verapamil (VER) have been selected for this study. Their effect has been investigated, individually and in a mixture, on the development of the zebrafish embryo Danio rerio (Hamilton, 1822) within the first 96 h of development. Upon exposure to environmentally relevant concentrations of tested CVPs (0.1, 1, and 10 µg/L) zebrafish survival and development as apparent from observed morphological abnormalities, heartbeat rates and changes in behavior, hatching success, larval length and oxidative stress level were monitored. The CVP causing the highest mortality and pathological changes was SIM (1 and 10 µg/L), which corresponds well with the observed effects during zebrafish exposure to CVPs' mixtures (4 and 40 µg/L). All pharmaceuticals affected cardiac function and decreased heart rate. SIM (1 µg/L), VER and RAM (10 µg/L) decreased larval length, while induced oxidative stress was recorded in the SIM- and VER-exposed specimens. Behavioral alterations of zebrafish were observed only in AMI-treated group (10 µg/L). Our amino acid sequence comparison and structural and docking analysis showed a highly conserved binding site between human and zebrafish HMG-CoA reductase for SIM and its main metabolite simvastatin acid. Using these ecotoxicological bioassays on a zebrafish model with particular emphasis on sublethal endpoints, the risk of CVPs, especially statins, for fish in inland waters has been identified.

Fish liver damage related to the wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) continuously release a complex mixture of municipal, hospital, industrial, and runoff chemicals into the aquatic environment. These contaminants are both legacy contaminants and emerging-concern contaminants, affecting all tissues in a fish body, particularly the liver. The fish liver is the principal detoxifying organ and effects of consistent pollutant exposure can be evident on its cellular and tissue level. The objective of this paper is thus to provide an in-depth analysis of the WWTP contaminants' impact on the fish liver structure, physiology, and metabolism. The paper also gives an overview of the fish liver biotransformation enzymes, antioxidant enzymes, and non-enzymatic antioxidants, their role in metabolizing xenobiotic compounds and coping with oxidative damage. Emphasis has been placed on highlighting the vulnerability of fish to xenobiotic compounds, and on biomonitoring of exposed fish, generally involving observation of biomarkers in caged or native fish. Furthermore, the paper systematically assesses the most common contaminants with the potential to affect fish liver tissue.

Intra-Species Variations of Bioactive Compounds of Two Dictyota Species from the Adriatic Sea: Antioxidant, Antimicrobial, Dermatological, Dietary, and Neuroprotective Potential.

The marine environment has a significant impact on life on Earth. Organisms residing in it are vital for the ecosystem but also serve as an inexhaustible source of biologically active compounds. Herein, the biodiversity of two brown seaweeds, Dictyota dichotoma and Dictyota fasciola from the Adriatic Sea, was evaluated. The aim of the study was the determination of differences in compound composition while comparing their activities, including antioxidant, antimicrobial, and enzyme inhibition, in connection to human digestion, dermatology, and neurological disorders. Chemical analysis revealed several terpenoids and steroids as dominant molecules, while fucoxanthin was the main identified pigment in both algae. D. dichotoma had higher protein, carbohydrate, and pigment content. Omega-6 and omega-3 fatty acids were identified, with the highest amount of dihomo-γ-linolenic acid and α-linolenic acid in D. dichotoma. Antimicrobial testing revealed a dose-dependent inhibitory activity of methanolic fraction against Escherichia coli and Staphylococcus aureus. Moderate antioxidant activity was observed for both algae fractions, while the dietary potential was high, especially for the D. fasciola dichloromethane fraction, with inhibition percentages of around 92% for α-amylase and 57% for pancreatic lipase at 0.25 mg/mL. These results suggest that Dictyota species might be a potent source of naturally derived agents for obesity and diabetes.

Effect of seasonal temperature shifts on body homeostasis, biochemical parameters and chemical composition of green ormer (Haliotis tuberculata) in the Northern Adriatic.

Seasonal responses of green ormer in terms of antioxidant capacity and lipid peroxidation, proximate and fatty acid tissue composition, trace and macro elements concentrations over the seasons were studied in relation to temperature shifts in the Northern Adriatic Sea. Overall antioxidative defenses (SOD, TBARS, TAS, LDH) varied significantly (p < 0.001) according to seasons (primarily spring and summer). The proportions of overall SFA were highest in summer. The proportions of MUFA increased in autumn, with significant differences between genders in spring and summer, and spring, summer and autumn for C18:1n7 and C20:4n6. The only fatty acid lacking significant variation between seasons was C22:5n3. Significant overall differences were observed in summer vs. winter samples for As, Ba, Co, Ni, Mn, Pb, Sb, and Se content in soft tissues, however, gender variations were not significant. The data obtained in the study are of utmost importance for the management of this under-investigated species.

Magnesium ions reversibly bind to DNA double stranded helix in thin films.

Effects of magnesium (Mg2+) ions on the stability and structural properties of double-stranded DNA are vitally important for DNA folding and functional behavior. Complementing our previous study on highly hydrated thin films of DNA with sodium counterions, with no buffer (pH ≈ 6) and surrounded with Mg2+ cations, here we use Fourier transform infrared spectroscopy and band shape analysis to explore in detail the vibrational signatures of DNA-magnesium interaction in the case when DNA charges are neutralized solely by Mg2+ cations, hereafter called MgDNA. Ion atmosphere has been controlled by the magnesium to phosphate molar concentration ratio r which varied between 0.0067 and 10. For r = 0 we find that spectral features in the base region remain similar as in DNA, whereas changes in the backbone region indicate that the B conformation becomes fully stabilized. With increasing r a pronounced structural reshaping occurs in the phosphate backbone region indicating a blue shift of the asymmetric band, while the symmetric band does not show any displacement in frequency. The band shape analysis of overlapping peaks in the respective phosphate regions demonstrates that the number of constituent modes as well as their positions in frequency do not change, whereas their intensities and bandwidths display disparate changes. The results reflect a variety of local environments at the DNA backbone due to a heterogeneous ion atmosphere with randomly distributed magnesium ions and local patterns of hydrogen bonds which change with increasing r. Remarkably, after crowded r = 10 ion atmosphere is depleted, Mg induced spectral changes vanish and structural features of MgDNA (r ≈ 0) are fully restored. Overall results strongly suggest that in MgDNA on highly hydrated thin films the hydrogen-base pairing remains preserved and that Mg2+ ions, similar to sodium ions, retain their mobility and interact with double helix via water-mediated electrostatic forces.

In Vivo and In Vitro Antioxidant Activity of Less Polar Fractions of Dasycladus vermicularis (Scopoli) Krasser 1898 and the Chemical Composition of Fractions and Macroalga Volatilome.

The present research is a comprehensive investigation of Dasycladus vermicularis (Scopoli) Krasser 1898 from the Adriatic Sea (Croatia) regarding volatilome-volatile organic compounds (VOCs, mostly nonpolar compounds) and less polar nonvolatile compounds for the first time. Headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were used showing the great volatilome variability among fresh (HS-FrDV and HD-FrDV) and dried (HS-DrDV and HD-DrDV) samples after GC-MS analysis. Aromatic aldehydes were dominant in both fresh and air-dried HS samples with benzaldehyde as the most abundant in fresh samples and decreasing 2.7-3.7 times after drying together with 2-phenylbut-2-enal that was not present after drying. Aliphatic compounds (unsaturated hydrocarbons in HS-FrDV; saturated hydrocarbons in HS-DrDV) were also present. C11-hydrocarbons (dictyopterpene C' and dictyopterpene D') were detected in HS-FrDV. (E)-Phytol was the most dominant compound in HD-FrDV and HD-DrDV. Diterpene alcohols (cembra-4,7,11,15-tetraen-3-ol and (Z)-falcarinol) and sesquiterpene alcohol, cubenol, were dominant in HD-FrDV, and their abundance decreased after drying. C13-norisoprenoides (α-ionone and ß-ionone) increased after drying. Aliphatic compounds were present in both HD-FrDV and HD-DrDV samples. The less polar nonvolatile compounds in the obtained fractions F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C55H74N4O5-7), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represented the majority of identified compounds. By implementing both in vitro and in vivo assays for antioxidant activity determination, F3 showed a higher activity than F4. Inhibitory concentrations (IC50) for F3 and F4 were 498.00 ± 0.01 µg/mL and 798.00 ± 0.81 µg/mL, respectively, while a 1.5-fold reduction in the ROS level was observed after pre-treatment of zebrafish larvae with 45 µg/mL of F3.

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00 mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00 mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00 mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Shewanella spp. from wastewater treatment plant-affected environment: isolation and characterization.

Bacteria from the genus Shewanella are inhabitants of marine and freshwater ecosystems, recognized fish spoilage bacteria, but less known as fish disease agents. Shewanella spp. isolated from fish living in waters close to effluents of a wastewater treatment plant (WWTP) were not previously characterized. We have tested Shewanella isolates from WWTP-affected waters and related fish. Genotypic characterization identified most strains as S. baltica and S. oneidensis. In order to investigate the sensibility and accuracy of their MALDI-TOF MS identification, they were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and duration. We analyzed their antimicrobial susceptibility on a panel of antimicrobial drugs and capacity for biofilm production. With a view to demonstrate their capacity to produce fatty acids, we assessed the impact of different culture media on their lipid profile. We performed zebrafish embryotoxicity tests to simulate the environmental infection of the earliest life stages in S. baltica-contaminated waters. The best MALDI-TOF MS identification scores were for strains cultivated on TSA for 24 h at 22 °C and with supplementation of 1.5% NaCl. Less than 17% of isolates demonstrated antimicrobial resistance. Most isolates were weak biofilm producers. Strain-to-strain variation of MIC and MBC was low. The major fatty acids were C15:0, C16:0, C16:1, C17:1, and iC15:0. Exposure of Danio rerio to different S. baltica concentrations induced severe effects on zebrafish development: decreased heartbeat rate, locomotor activity, and melanin pigmentation. S. baltica passed through chorionic pores of zebrafish.

Combining short-term bioassays using fish and crustacean model organisms with ToxCast in vitro data and broad-spectrum chemical analysis for environmental risk assessment of the river water (Sava, Croatia).

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

Less Polar Compounds and Targeted Antioxidant Potential (In Vitro and In Vivo) of Codium adhaerens C. Agardh 1822.

Codium adhaerens from the Adriatic Sea (Croatia) was comprehensively investigated regarding less polar compounds for the first time. Although there are several phytochemical studies on C. adhaerens from other regions, this is the first report on volatile organic compounds (VOCs) from fresh (FrCa) and air-dried (DrCa) samples. The novelty is also related to its targeted antioxidant potential in vitro and in vivo. The main aims were to: (a) identify and compare VOCs of FrCa and DrCa obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD); (b) determine fatty acid (FA) composition of freeze-dried sample (FdCa); (c) determine the composition of less polar fractions of FdCa by high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI-HRMS); and (d) comprehensively evaluate the antioxidant activity of the fractions by four in vitro assays and in vivo zebrafish model (including embryotoxicity). Significant changes of VOCs were found after air drying. ω6 FAs were present in higher content than ω3 FAs indicating C. adhaerens as a good source of dietary polyunsaturated FAs. The results obtained in vivo correlate well with in vitro methods and both fractions exerted similar antioxidative responses which is in agreement with the high abundance of present biomolecules with known antioxidant properties (e.g., fucoxanthin, pheophytin a, and pheophorbide a). These results suggest that C. adhaerens might be a potent source of natural antioxidants that could be further used in the research of oxidative stress-related diseases.

Utilization of the zebrafish model to unravel the harmful effects of biomass burning during Amazonian wildfires.

Amazonian wildfires in 2019 have raised awareness about rainforest burning due to increased emissions of particulate matter and carbon. In the context of these emissions, by-products of lignin thermal degradation (i.e. methoxyphenols) are often neglected. Methoxyphenols entering the atmosphere may form intermediates with currently unknown reaction mechanisms and toxicity. This study for the first time provides a comprehensive insight into the impact of lignin degradation products [guaiacol, catechol], and their nitrated intermediates [4-nitrocatechol, 4,6-dinitroguaiacol, 5-nitroguaiacol] on zebrafish Danio rerio. Results revealed 4-nitrocatechol and catechol as the most toxic, followed by 4,6DNG > 5NG > GUA. The whole-organism bioassay integrated with molecular modeling emphasized the potential of methoxyphenols to inhibit tyrosinase, lipoxygenase, and carbonic anhydrase, consequently altering embryonic development (i.e. affected sensorial, skeletal, and physiological parameters, pigmentation formation failure, and non-hatching of larvae). The whole-organism bioassay integrated with in silico approach confirmed the harmful effects of lignin degradation products and their intermediates on aquatic organisms, emphasizing the need for their evaluation within ecotoxicity studies focused on aquatic compartments.