Microbial communities as indicators of marine ecosystem health: Insights from coastal sediments in the eastern Adriatic Sea.

Considering the adaptability and responsiveness of microorganisms to environmental changes, their indicator potential is still not acknowledged in European directives. This comprehensive study examined the changes of microbial communities in sediments and a range of geochemical parameters from pristine and anthropogenically impacted coastal areas in the eastern Adriatic Sea. Various analytical methods found evidence of sediment contamination (high toxicity level, enrichments of metals, tributyltin) in certain areas, leading to the categorization of sediments based on the level of anthropogenic disturbance. Prokaryotes were identified as the most promising group of microbes for further research, with specific bacterial families (Rhodobacteraceae, Ectothiorhodospiraceae, Cyclobacteriaceae) and genera (Boseongicola, B2M28, Subgroup 23, Sva0485, Thiogranum) proposed as potential indicators of environmental status. Finally, predictive models were developed to identify key indicator variables for assessing anthropogenic impact in sediments. This research represents an essential step toward incorporating microbial communities into assessments of benthic environmental health.

Specific detection of cyclobutane pyrimidine dimers in phytoplankton DNA by a non-radioactive assay based on T4-endonuclease V digestion.

The effect of artificial and natural UV irradiation on DNA in marine phytoplankton Isochrysis galbana monoculture was investigated. The presence of cyclobutane pyrimidine dimers (CPDs) in unlabelled I. galbana DNA was detected by a non-radiometric alkaline filter elution assay after T4-endonuclease V digestion. The quantity of CPDs was estimated by alkaline agarose gel electrophoresis. Precise determination of the amount of DNA in the presence of I. galbana pigments was achieved by oxazole yellow homodimer (YOYO) dye. T4-endonuclease V-sensitive sites frequency (ESS/kb), measured after exposure to 2-40 kJ m(-2) of artificial UV light, increased in a dose-dependent manner. Twelve hours after irradiation cell culture growth was disrupted, and 50% of initial DNA damage in the cells was observed. After 1 h of sunlight exposure, the incidence of CPDs increase significantly. Prolonged exposition to sunlight decrease CPDs incidence due to efficiency of I. galbana DNA repair mechanisms. The presence of water-soluble crude oil fraction (WSOF) affected DNA repair efficiency resulting in accumulation of CPDs in I. galbana DNA.

A microplate assay for DNA damage determination (fast micromethod).

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 microg tissue per well), or in environmental samples for genotoxicity assessment.