Electroporation-based therapies are increasingly used in medicine, yet the molecular mechanisms underlying electroporation-induced cell death remain incompletely understood. Here, we investigated gene expression changes associated with apoptosis, pyroptosis, necroptosis, and autophagy 4 and 24 h after cells were treated by pulsed fields using monopolar and high frequency bipolar waveforms of increasing amplitudes in vitro. Our results confirmed that electroporation-induced cell death is a dynamic process and demonstrated involvement of both accidental and regulated cell death pathways. Apoptosis-related markers were predominantly associated with early post-treatment responses, whereas pyroptosis- and necroptosis-related markers were more pronounced at later timepoints. Notably, necroptosis exhibited the strongest transcriptional response among all investigated pathways. In contrast, autophagy-related changes were more consistent with cellular stress responses than with activation of autophagic cell death. These findings provide new insight into the temporal dynamics and molecular mechanisms underlying electroporation-induced cell death.
Lipid bilayers are not limited to the plasma membrane but also constitute the membranes of numerous intracellular organelles, where they possess similar physicochemical properties while performing distinct biological functions. One such example is the outer mitochondrial membrane, which is exposed to the cytosol rather than the extracellular environment and therefore experiences a markedly different ionic composition. In this study, we investigated the effect of a simplified cytosol-mimicking ionic buffer on the physicochemical properties of a model outer mitochondrial membrane. Particular attention was paid to magnesium ions (Mg2+), the most abundant free divalent cations in the cytosol. We demonstrate that the presence of Mg2+ significantly alters the properties of the model membrane despite its composition being limited to zwitterionic lipids. In particular, Mg2+ shifts the interfacial electrostatic response of the membrane, highlighting its substantial influence on the electrochemical properties of the model outer mitochondrial membrane.
Direct electron transfer (DET) in oxidoreductase-based bioelectrocatalysis offers energy efficiency and selectivity over mediated systems. However, realizing its full catalytic potential is limited by an insulating protein matrix surrounding redox cofactors, which necessitates precise electronic coupling and a minimal electron tunneling distance to the electrode surface. This review evaluates strategies for the oriented immobilization of redox metalloenzymes, with the scope limited to studies that meet strict DET criteria, excluding systems in which the observed bioelectrocatalytic signal may originate from a dissociated cofactor acting as a built-in diffusive mediator. Analysis of leading case studies, curated by the chemistry of the enzyme-support interface, reveals that enzyme loading, orientation, and electronic coupling are distinct variables that must be deconvoluted rather than conflated, and that orientation control does not by itself guarantee enhanced performance. These distinctions expose a central bottleneck of the field, namely, the capacity to verify interfacial architecture lags behind the capacity to design it. Empirically, enzyme orientation on surfaces is still assessed predominantly through indirect electrochemical readouts that cannot separate improved alignment from a simple increase in the number of electroactive enzymes. Closing this gap will require operando, single-molecule-resolved characterization benchmarked against dynamic simulation.
