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Background Nanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 μs pulses and to investigate the effects of electric pulses on cisplatin structure. Materials and methods Chinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Results Nanosecond pulses equivalent to 8 × 100 μs pulses were established in vitro based on membrane permeabilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and accumulating cisplatin intracellularly as 8 × 100 μs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin. Conclusions Equivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 μs pulses.

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In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacterium Synechocystis sp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA from Anabaena combined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising the nucA gene fused to a variant of the copM promoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring of Synechocystis TA pairs ssr1114/slr0664 and slr6101/slr6100 for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including a nrsB variant, were characterized by beta-galactosidase reporter assay.

Povečano prepustnost celične membrane zaradi izpostavitve celic oz. tkiv električnemu polju imenujemo elektroporacija. Povzroči vrsto sprememb v celici, od strukturnih in kemijskih sprememb v celični membrani, strukturnih sprememb proteinov oz. proteinskih kompleksov, prenosa snovi v celice in iz njih do aktiviranja signalnih poti in popravljalnih mehanizmov; ob določenih pogojih sproži tudi celično smrt. S pojmom elektropermeom označujemo tako permeabilizirano celico med ali tik po dovajanju električnih pulzov kot tudi vse poznejše procese, ki ostanejo aktivni še nekaj časa potem, ko ni več mogoče opaziti povečanega transmembranskega transporta snovi, za katere je celična ovojnica običajno neprepustna, torej tudi po času, ko že ugotavljamo, da je zaceljena. Elektroporacija se uporablja na številnih področjih, vključno z ablacijo tkiv, gensko elektrotransfekcijo za vnos plazmidov v celice ter elektrokemoterapijo. Aplikacije elektroporacije v medicini so učinkovite in varne, vendar so zaradi delovanja električnih pulzov lahko prisotni tudi določeni neželeni stranski učinki, predvsem mišično krčenje in akutna bolečina. Za optimiziranje parametrov elektroporacije in s tem rezultatov na elektroporaciji temelječih terapij je ključnega pomena nadaljnja razjasnitev osnovnih mehanizmov elektroporacije in vplivov posameznih parametrov električnega polja na elektropermeom. Namen prispevka je predstaviti celovit pregled mehanizmov elektroporacije ter elektropermeoma, tj. celičnega odgovora na elektroporacijo.