Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltagesensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is ~1 nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca2+ mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1–60), and amplitudes (15–60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-μs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.

We explored how the effect of plasma membrane permeabilization by nanosecond-duration electric pulses (nsEP) depends on the physical characteristics of exposure. The resting membrane resistance (R m ) and membrane potential (MP) were measured in cultured GH3 and CHO cells by conventional whole-cell patch-clamp technique. Intact cells were exposed to a single nsEP (60 or 600 ns duration, 0-22 kV/cm), followed by patch-clamp measurements after a 2-3 min delay. Consistent with earlier findings, nsEP caused long-lasting R m decrease, accompanied by the loss of MP. The threshold for these effects was about 6 kV/cm for 60 ns pulses, and about 1 kV/cm for 600 ns pulses. Further analysis established that it was neither pulse duration nor the E-field amplitude per se, but the absorbed dose that determined the magnitude of the biological effect. In other words, exposure to nsEP at either pulse duration caused equal effects if the absorbed doses were equal. The threshold absorbed dose to produce plasma membrane effects in either GH3 or CHO cells at either pulse duration was found to be at or below 10 mJ/g. Despite being determined by the dose, the nsEP effect clearly is not thermal, as the maximum heating at the threshold dose is less than 0.01 °C. The use of the absorbed dose as a universal exposure metric may help to compare and quantify nsEP sensitivity of different cell types and of cells in different physiological conditions. The absorbed dose may also prove to be a more useful metric than the incident E-field in determining safety limits for high peak, lowaverage power EMF emissions.

Background-Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8-and 9-μs EP.Methods-Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.Results-10-ns EP caused apoptotic or necrotic death within 2-20 hrs. Survival (S, %) followed the absorbed dose (D, J/g) as: S=αD (−K) , where coefficients K and α determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas α was cell type-and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.Conclusions-1.8-and 9-μs EP cause cell death efficiently and indiscriminately (LD 50 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD 50 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms.General significance-Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

Human dermal fibroblasts exhibit comparable cellular and molecular effects when exposed to THz radiation and hyperthermic stress. These findings suggest that radiation at 2.52 THz generates primarily thermal effects in mammalian cells. Therefore, we conclude that THz-induced bioeffects may be accurately predicted with conventional thermal damage models.

In this study, we determined the LD50 (50% lethal dose) for cell death, and the ED50 (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD50 varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD50 lower than the ED50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

MicroRNAs (miRNAs) are a class of small RNAs that play a critical role in the coordination of fundamental cellular processes. Recent studies suggest that miRNAs participate in the cellular stress response (CSR), but their specific involvement remains unclear. In this study, we identify a group of thermally regulated miRNAs (TRMs) that are associated with the CSR. Using miRNA microarrays, we show that dermal fibroblasts differentially express 123 miRNAs when exposed to hyperthermia. Interestingly, only 27 of these miRNAs are annotated in the current Sanger registry. We validated the expression of the annotated miRNAs using qPCR techniques, and we found that the qPCR and microarray data was in well agreement. Computational target-prediction studies revealed that putative targets for the TRMs are heat shock proteins and Argonaute-2-the core functional unit of RNA silencing. These results indicate that cells express a specific group of miRNAs when exposed to hyperthermia, and these miRNAs may function in the regulation of the CSR. Future studies will be conducted to determine if other cells lines differentially express these miRNAs when exposed to hyperthermia.

Terahertz spectrometers and imaging systems are currently being evaluated as biomedical tools for skin burn assessment. These systems show promise, but due to their size and weight, they have restricted portability, and are impractical for military and battlefield settings where space is limited. In this study, we developed and tested the performance of a compact, light, and portable THz time-domain spectroscopy (THz-TDS) device. Optical properties were collected with this system from 0.1 to 1.6 THz for water, ethanol, and several ex vivo porcine tissues (muscle, adipose, skin). For all samples tested, we found that the index of refraction (n) decreases with frequency, while the absorption coefficient (μ(a)) increases with frequency. Muscle, adipose, and frozen/thawed skin samples exhibited comparable n values ranging between 2.5 and 2.0, whereas the n values for freshly harvested skin were roughly 40% lower. Additionally, we found that the freshly harvested samples exhibited higher μ(a) values than the frozen/thawed skin samples. Overall, for all liquids and tissues tested, we found that our system measured optical property values that were consistent with those reported in the literature. These results suggest that our compact THz spectrometer performed comparable to its larger counterparts, and therefore may be a useful and practical tool for skin health assessment.