Mechanisms of Nanosecond Pulsed Electric Field (NsPEF)-Induced Cell Death in Cells and Tumors

The evolution of pulse power technology from high power physics to biology and medicine places nanosecond pulsed electric fields (nsPEFs) in positions for in vitro and in vivo applications as non-ligand agonists that not only bypass plasma membrane receptors for induction of intracellular signaling pathways, but also bypass intracellular oncogenic impasses to induce cell death by regulated mechanisms. Based on work reviewed here, a likely scenario for cell and tumor demise includes nsPEF-induced permeabilization of the plasma membrane, Ca2+ influx, dissipation of the mitochondrial membrane potential, which is likely due to events beyond permeabilization of the inner mitochondrial membrane, cytochrome c release and activation of caspase-dependent and -independent cell death mechanisms. In vivo, nsPEF-treated orthotopic rat N1-S1 hepatocellular carcinoma tumors exhibit caspase-9 and caspase-3 positive and –negative tumor cells, indicating intrinsic apoptotic and non-apoptotic cell death. Interestingly, after N1-S1 tumor ablation and clearance, rats are resistant to challenge injections of the same N1-S1 tumor cells, indicating a protective, vaccine-like effect that appears to be due to innate and/ or adaptive immune responses that are under further investigation. This provides additional impetus to further develop nsPEF ablation as a cancer therapy

Biofortification of common bean for higher iron concentration

Common bean (Phaseolus vulgaris L.) is a staple food of smallholder farmers and poor urban consumers in Latin America and eastern-southern Africa among whom iron deficiency is frequent. Bean was domesticated in Mexico and the southern Andes, creating two distinct gene pools. Evaluation of a core collection of 1,441 entries revealed average concentrations of 55 mg kg−1 iron. A breeding target was set at 44 mg kg−1 iron above the level in a local check variety, while 50% of goal or a 22 mg kg−1 advantage was accepted as “biofortified.” In a bioefficacy trial among college-age women in Rwanda, high iron beans improved iron status and enhanced cognitive ability, brain function, and work efficiency. However, breeding progress has been slow, likely due in part to homeostatic mechanisms whereby organisms moderate iron and zinc uptake. This phenomenon may represent resistance to increasing concentration of these elements. Crosses between gene pools may “jumble” genes for homeostasis and permit high levels. A second breeding strategy is the use of sister species that evolved in iron-poor environments and that could be more receptive to iron uptake. Future breeding may also increase attention on improving bioavailability through mechanisms such as non-or-slow darkening grain or low phytate mutants. Changing dietary patterns in developed countries could increase iron deficiency and create demand for iron biofortified beans

Cell Responses Without Receptors and Ligands, Using Nanosecond Pulsed Electric Fields (nsPEFs)

Stephen J Beebe Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk VA, USAThe plasma membrane is a lipid bilayer that surrounds and shelters the living structural and metabolic systems within cells. That membrane is replete with transmembrane proteins with and without ligand binding sites, oligosaccharides, and glycolipids on the cell exterior. Information transfer across this structure is closely controlled to maintain homeostasis and regulate cell responses to external stimuli. The plasma membrane is contiguous with the endoplasmic reticulum (ER) and nuclear membranes. A number of proteins form ER–mitochondria junctions, allowing interorganelle communications, especially for calcium transport. Transport mechanisms across these membranes include nongated channels or pores; single-gated channels for ion transport; carrier molecules for facilitated diffusion; and pumps for active transport of ions and macromolecules. During the activation of these transport systems, "pores" are formed through protein structures that transiently connect the intracellular and extracellular milieu. These pores are nanoscale structures with diameters of 0.2−4.0 nm. However, there can also be maligned movements of molecules across the plasma membranes. Staphylococcus aureus protein α-toxin and Streptococcus pyogenes protein streptolysin O both create pores that allow unsolicited molecular transfer across membranes that disrupts vital functions. Cytotoxic T-cells permeabilize the invading cell membranes with perforin, creating pores through which granzymes can induce apoptosis. These pores have a lumen of 5–30 nm with the majority at 13–20 nm.

Bioelectrics in Basic Science and Medicine: Impact of Electric Fields on Cellular Structures and Functions

Bioelectrics is a new interdisciplinary field that investigates electric field effects on cell membranes and other cellular components. It incorporates four main technologies, including electroporation, nanosecond pulsed electric fields, picosecond pulsed electric fields and cold plasmas. The parent technology in Bioelectrics is electroporation, which uses milli- and/or micro-second electric pulses to permeabilize cells and tissues, for delivery of membrane impermeable molecules. It is now being used for electro-gene delivery, with vascular endothelial growth factor, for revascularization in wound healing and cardiovascular and peripheral vascular disease. Plasmids expressing IL-12 are being delivered for immune system activation in melanoma treatment, now in phase II clinical trials. DNA vaccine delivery by electroporation is also being investigated. More recently, electroporation has been extended to include nanosecond pulsed electric fields (nsPEFs), a pulse power technology that was originally designed for military applications. It stores intense levels of electric energy, and then unleashes nanosecond bursts of instantaneous power into cells and tissues, creating unique intracellular conditions of high power and low, non-thermal energy. It is presently being used for cancer ablation of skin and internal tumors, and for platelet activation for wound healing in injury and diabetes. An extension of nsPEFs is to make the pulses even shorter, using picosecond pulsed electric fields. This is being developed as an imaging system to detect cancer and other aberrant tissues, using an antenna. The fourth technology is cold plasmas or ionized gasses, a fourth state of matter. Applications of these ionized gases are being developed for decontaminating wounds, water, food and surfaces. Other possible applications that are of specific interest, but not yet fully investigated, and/or developed, are pain control, fat ablation and decontamination of indwelling catheters. This review will outline some applications of Bioelectrics, with greatest focus on nsPEF effects on cells in vitro and tumors in vivo

Nanosecond Pulsed Electric Fields: A New Stimulus to Activate Intracellular Signaling

When new technologies are introduced into the sci-entific community, controversy is expected and both ex-citement and disappointment enrich the lives of those who initiate the new ideas. It becomes the mission of the “inventors ” to embrace the burden of proof to estab-lish their ideas and convince the skeptics and disbeliev-ers who will undoubtedly temper their enthusiasm and test their patience. While open mindedness is generally a scientific motto, those who review patents, manuscripts, and grants do not always readily practice it, even when the evidence is convincingly presented; old ideas and concepts often die hard. So it has been and still is in many instances as engineers, physicists, biologists, and physicians pursue innovative ideas and novel technolo-gies. So what is “Bioelectrics”? It is the application of ultra-short pulsed electric fields to biological cells, tissues, and organs. More specifically, it is the analysis of how these bi-ological systems respond to high electric fields (10–100 s of kV/cm) when applied with nanosecond (1–300) dura-tions. Compressing electrical energy by means of pulsed power techniques allows the generation of ultrashort (bil-lionth of a second) electrical pulses [1]. Because the pulses are so short the energy density is quite low and there-fore nonthermal. However, the power is extremely high generating billions of watts. This can be compared to a coal power plant, which generates less than billion watts, but does it continuously. For example, for a 10 ns, 40 kV, 10Ω pulse generator, the power provided by the pulse is 160MW, however, the energy is only 1.6 J. Depositing thi

Nanosecond Pulsed Electric Field (nsPEF) Ablation as an Alternative or Adjunct to Surgery for Treatment of Cancer

Surgery as resection or transplantation remains a fundamental means for cancer treatment and often offers an opportunity for a cure. However, surgery is not always possible because of tumor proximity to blood vessels or ducts or when a patient is not healthy enough to undergo surgery. Application of nanosecond pulsed electric fields (nsPEFs) is a new approach to treat cancer using pulse power technology that was originally designed for military purposes. This novel approach deposits extremely short pulses of high power, low energy electric fields into malignant tissues using electrodes to encompass tumors. Pre-clinical studies show that treatments are effective and without local or systemic side effects, including absences of scarring. Pre-clinical trials for basal cell carcinoma are completed, but results have not been published. For treating internal tumors, electric fields can be delivered by catheter electrodes and laparoscopy procedures. Here we present a review of the literature using nsPEFs for cancer ablation and present some recent work from the author’s laboratory. We demonstrate efficacy for treatment of an ectopic mouse (Hepa-1- 6) and an orthotopic rat (N1-S1) Hepatocellular Carcinoma (HCC). NsPEFs eliminate tumors by mechanisms in the presence of active caspases (apoptosis) as well as in absences of active caspases (necrosis/necroptosis). Treatment also breaches small vessels, but spares larger vessels and ducts. NsPEF treatments also reduce angiogenesis as determined by decreases in Vascular Endothelia Growth Factor (VEGF). Microvascular density markers (CD-31, CD-34 and CD-105) are significantly decreased after treatment, limiting new blood vessel formation and reinforcing tumor cell demise. Furthermore, initial challenge studies show that mice are resistant to re-introduction of the same tumor cells after treatment, suggesting that nsPEFs induces immunogenic cell death and possible host cell immune responses after treatment. NsPEF ablation of cancer targets at least three hallmarks of cancer (evasion of apoptosis, angiogenesis maintenance and immune surveillance) and provides an effective alternative or adjunct therapy for cancers in skin and internal organs

Editorial Board

This nursery was organized in response to the need for improving adaptation and obtaining genetic information on the resistance or reaction to the different stress environments (biotic and abiotic) to which beans are subjected in production areas. Results are given for the best black- and red-seeded lines of the 1987 VIDAC, covering characteristics such as 100 seeds, days to flowering and physiological maturity, wt. of and response to diseases such as rust (Uromyces phaseoli), bacterioses (Xanthomonas phaseoli), BGMV, and web blight (Thanatephorus cucumeris). The yield potential and genetic adaptation of the best materials over different sites are also analyzed. Information on these national and regional trials should be collected in an efficient regional information and documentation center. (CIAT)Este vivero se organizo con base en la necesidad de conseguir una mejor adaptacion e informacion genetica sobre la resistencia o la reaccion a los diferentes ambientes de estres (bioticos o abioticos) a los cuales se somete el frijol en las areas de produccion. Se presentan los resultados de las mejores lineas de semilla negra y semilla roja de los VIDAC de 1987, para las caracteristicas de peso de 100 semillas, dias a floracion y a madurez fisiologica, y respuesta a enfermedades como roya (Uromyces phaseoli), bacteriosis (Xanthomonas phaseoli), BGMV y mustia hilachosa (Thanathephorus cucumeris). Tambien, se examinan el potencial de rendimiento y la adaptacion genetica de los mejores materiales en diferentes sitios. Es necesario reunir gran cantidad de informacion sobre estos ensayos nacionales y regionales en un centro de informacion y documentacion regional eficiente. (CIAT