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The strong electric field required for nanosecond pulsed electric fields (nsPEF) stimulation can evoke cellular effects qualitatively different from the conventional micro- and millisecond pulses. nsPEF may compromise the barrier function of the plasma membrane, endoplasmic reticulum, and mitochondria. Most studies agree that it is caused by the formation of transient aqueous pores, with the effective pore diameter not exceeding 1-1.5 nm ("nanoelectroporation"). This pore size was established by the selective uptake of dye molecules and ions, by blockage of cell swelling using solutes too large to pass through the pores, and by modeling. Nanopores are remarkably stable, with the lifetime on the order of minutes. They are distinguished from "regular" larger pores by complex conductive properties similar to endogenous ion channels, such as voltage sensitivity, current rectification, and cation selectivity.

Stimulation by nsPEF can bypass membrane receptors and ion channels to elicit second messenger Ca2+ and PIP2 signaling and evoke neuromediator release and other downstream effects. Intense nsPEF treatments cause cytoskeleton rearrangements, osmotic stress, cell swelling and blebbing, and apoptotic or necrotic cell death.

We explore the phenomenon of nanoelectroporation in living cells, focusing on the underlying physico-chemical and physiological mechanisms. We also explore nanopore properties and lifetime, as well as many downstream effects of membrane permeabilization.

Nanosecond pulsed electric fields (nsPEF) are a new neuromodulation modality with unique capabilities beyond those of conventional milli- and microsecond stimuli.

Primary effects of nsPEF encompass brief high-amplitude membrane polarization, alteration of membrane proteins, and nanoelectroporation. Depending on the stimulation protocol, nsPEF can elicit or suppress action potentials and activate or inhibit voltage-gated ion channels.

Opening of transient nanopores is a unique method of Ca2+ mobilization while bypassing plasma membrane receptors and channels. Cells interpret Ca2+ transients due to nanopore "leaks" as authentic Ca2+ signals and amplify them by the Ca2+-induced Ca2+-release. Ca2+ mobilization by nsPEF can evoke heart and muscle contraction, stimulate neurosecretion, and activate genes responsible for neuroprotection.

nsPEF expand the toolbox of electrostimulation with novel and fundamentally different capabilities. A combination of classic excitation mechanisms with nanoelectroporation, modulation of ion channels, and effects on organelles offers a choice from stimulatory and inhibitory effects to tissue ablation.

The use of nsPEF may enable a radical advancement of electrostimulation therapies, such as chronic stimulation without electrochemical side effects; transient or permanent inhibition of neural networks; safer and more efficient defibrillation; and targeted neuromodulation at a distance, including non-invasive deep brain stimulation.

Electrical pulses causing diverse biological effects are generated by pulsed power generators, which have discrete components that allow for high hold-off voltages and high output currents. Although generators have different operating principles, they are generally configured in the same structure that includes a charger, an energy storage, a switch, and a load. To generate high power pulses, a pulse generator works in way of "slow charging and fast discharging". At the beginning, the charger pumps a DC current or a pulsed one to the energy storage, which could be a capacitor, an inductor, or the combination of both. Upon completion of charging, the stored energy is released to the load after turning on the switch. The time for discharging could easily be three orders of magnitude shorter than charging. Reducing the discharging time results in a gain of current or voltage, so the pulse's instantaneous power is amplified compared to the average charging power, albeit the total energy remains approximately the same. Current research and development by investigators at the Center for Bioelectrics focus on high voltage, high peak power, tunable, multiphasic, and flexible waveform generators. Picosecond pulse generators that can deliver pulses at 10 MHz or higher are also studied for using these pulses as an effective wireless stimulus.

To understand��bio...��electrics... we must understand how��bio-matter��interacts with��electric��fields. That means, at a fundamental level, how atoms and molecules in biomolecular assemblies — lipid membrane domains, ion channels, nucleosomes, ribosomes,... — interact with electric fields. Because real-time, atomic-resolution observations of living systems are not yet feasible, we use simulations — computer models based on physics and chemistry — to help us form hypotheses, design experiments, and interpret the data we generate in the laboratory.

Molecular dynamics is a versatile, widely used class of modeling tool that enables atomic detail without the computational cost of quantum mechanics. Of course, there is a price to pay in accuracy, and we constantly calibrate our models against real-world controls. We use the methods of molecular dynamics to construct systems of phospholipid bilayer membranes in water to which we apply electric fields of varying durations, amplitudes, and polarities, and in this way we have learned much about the nanoscale physics of electropermeabilization. Building on simple systems consisting of a single phospholipid and water, we are investigating now the effects of including in these systems ions (Na+, K+, Ca2+, Cl⁻), cholesterol, and more complex mixtures of phospholipids and other components of biological membranes, including transmembrane peptides.

At the Center for Bioelectrics we are applying to biomedical targets very high electric fields — megavolts per meter — for very short times — nanoseconds. These electric fields are stronger and shorter than had ever been used in the biology lab or the clinic... or in nature. Since cells and organisms have never in the history of life on earth seen this kind of electrical excitation, they have evolved no specific defense, regulatory, or signaling mechanisms that might be induced by this new kind of stimulation.

After initial nanosecond pulsed electric fields (nsPEF) experiments demonstrated success at decontaminating bacteria, researchers at the Center turned their attention to cancer. At that time, apoptosis was a central topic in cell and cancer biology research. Apoptosis is a programmed cell death, in contrast with unprogrammed necrosis, now called accidental death. The first studies to show that nanosecond pulses induce apoptosis, reduce mouse tumor size, and kill human tumor cells were in 2002 and 2003. Since then, studies at 91��Ƭ�� have shown that nanosecond pulses can kill many types of tumors and furthermore can induce an immune response in treated animals. Ablation with nsPEF of melanoma, breast, liver, and colorectal cancers initiates an antitumor immune response that both assists tumor eradication and prevents the formation of new tumors. Researchers at the Center are currently characterizing this immune response and exploring the efficacy of combination therapies.

Cold atmospheric plasma (CAP) produces reactive oxygen and nitrogen species, ions, and transient electric field, each exhibiting anticancer activity and together amplifying their individual activities to devastate malignant cells. Its viability as an anticancer therapy is illustrated by recent clinical trials of CAP treatment of patients with head and neck cancers. At the Center for Bioelectrics, we focus on inhibitory effects of CAP on cancer metabolism, proliferative signaling, and inflammation and how they may be exploited to address therapy resistance. For example, we recently discover that CAP at an unexpected low-dose regime can simultaneously suppress multiple supply lines to cancer survival (e.g. metabolism, proliferative signaling, angiogenesis), often known as cancer hallmarks, in therapy-resistant malignant cells, with negligible impact on their healthy counterparts, leading to high-rate apoptotic death of malignant cells in vitro and in clinically relevant in vivo models. Findings such as these are being explored to develop novel strategies that can improve the current options for mitigating the risk of tumorigenesis, overcoming drug resistance, and enhancing prognosis of patients who receive current anticancer therapies.

Cold plasma produces diverse biologically active agents, including reactive species, ions, photons, and will affect transient electric fields that are also produced endogenously by eukaryotic cells. This similarity has fueled extensive exploitation of its benefits to human health, leading to cold plasma-based procedures in clinical utility for coagulation and ablation and in clinical trials for treatment of wounds, head and neck cancers, and autoimmune skin disorders. Here at the Center for Bioelectrics, investigators engineer cold plasma chemistry to replicate and leverage beneficial biological effects of endogenous reactive species and ions for novel solutions that can improve the prognosis of patients with cancer (e.g. pancreatic, leukemia, breast, skin cancers), infection, and injured organs. Through multidisciplinary collaboration, we focus on (1) dose-controlled delivery of cold plasma and plasma-activated solution; (2) their effects on the mammalian host's immune response and energy requirement; (3) their molecular and cellular targets in pathological or regenerative tissues; (4) their benefits as a monotherapy for cancer, infection and injury or as a drug-delivery method for gene immunotherapy; and (5) their synergy with other bioengineering platforms such as pulsed electric field. Furthermore, investigators at the Center are interested in cold plasma-based infection control in agriculture and environment settings.

Microorganisms are known to be vulnerable to oxidative stress and membrane poration induced by cold plasma and pulsed electric field (PEF), respectively. With cold plasma and PEF being developed into two complimentary bioengineering technologies in the Center for Bioelectrics, they are being advanced toward patient benefits. Against antibiotic resistance, investigators at the Center have developed a plasma activated solution (PAS) to achieve a 7-log10 reduction of pandrug-resistant bacteria and fungi without harming mammalian cells. This is significant given that these pathogens are resistant to all current antibiotics. Further, PAS is engineered to dismantle bacterial biofilm formed inside lumens of gastrointestinal endoscope channels and central venous catheters. Recognizing the current lack of effective eradication of��in vivo��microbial biofilm, implicated in diabetic foot ulcers and chronic obstructive pulmonary disease, investigators at the Center developed a PAS-based wound dressing therapy and demonstrated its efficacy and safety in disrupting MRSA wound biofilms, the culprit responsible for life-threatening bacteremia. Further, these discoveries are being expanded to meet the challenge of disinfection beyond medicine, for example PEF-reduced biofouling control of liquid food (e.g., orange juice), PAS-enhanced animal food safety, and PAS-based control of COVID spread by inactivating SARS-CoV-2 binding with human cells.

Of diverse reactive species and ionic species produced by cold plasma, hydrogen peroxide and nitric oxide are known to promote cell proliferation and angiogenesis, respectively. These contribute to the basis of multiple successful clinical studies of cold plasma for wound healing. Interestingly, cold plasma activates, in a dose-dependent fashion, major signaling pathways important in regenerative medicine, for example Nrf2 for abatement of excessive oxidative stress common in injured tissues, Wnt for migration of stem cells, and HIF-1 alpha for angiogenesis. These studies suggest that cold plasma may be applicable beyond skin wounds. As an example, investigators at the Center for Bioelectrics discovered that cold plasma elicits neuroprotection against glutamate excitotoxicity by elevating cellular antioxidant capacity, a desirable function for treatment of stroke and spinal cord injury. In the case of vitiligo in which dysregulated T cells attack melanocytes to cause skin depigmentation and for which there is no cure, investigators innovated a gel prepared with cold plasma that disrupts T cell attack on melanocytes, arrests excessive oxidative stress, and promotes re-pigmentation of vitiligo lesion in animal models. This��in vivo��efficacy is successfully demonstrated in a controlled and randomized clinical trial. Current focus is to improve mechanistic insights and translational readiness.

Acute Lung Injury (ALI), acute respiratory distress syndrome (ARDS) and Pulmonary Fibrosis (PF) are major determinates of morbidity and mortality. FDA-approved therapeutic interventions are limited, and new drugs are thus needed. Heat Shock Proteins (HSP) are chaperones that assist a high number of��client��proteins during their folding, stabilization and/or degradation. HSP90, the most ubiquitous protein of the family, plays a major role during lung injury and inflammation. Indeed, HSP90 is a critical regulator of pulmonary endothelial permeability and modulates key proteins, including RhoA, ROCK1, cofilin and VE-cadherin, thus participating in the development of alveolar edema. Consequently, HSP90 inhibitors control at multiple levels both inflammation and lung injury. Among the >400 client proteins, HSP90 stabilizes Transforming Growth Factor-β (TGF-β), its receptor and Raf, ERK and Smads signaling, that are directly involved in the development of chronic lung injury. HSP90 inhibitors reduce mortality in several animal models of ALI and prevent the development of Pulmonary Fibrosis. Further investigations are required to establish optimal dose strategy, potency, and therapeutic schemes of the various new HSP90 inhibitors.

The Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) Pandemic has infected more than 300 million people and caused the death of over 5 million globally. The spike protein on the surface of the virus is capable of eliciting a strong inflammatory reaction, provoking vasculitis, thrombotic disease and white blood cells infiltration, major determinants of death in patients with COVID-19.��In vivo��and��in vitro��studies suggest that a single exposure to the spike protein subunit 1 (S1SP) provokes acute lung injury and damages endothelial barrier function leading to increase permeability. Protein Tyrosine Phosphatase 4A3 removes phosphate groups from target proteins, thus regulating a large number of cellular processes. PTP4A3 is a critical regulator of endothelial function and a strong anti-inflammatory agent, as it inhibits STAT3 and NF-kB. We are investigating the first specific PTP4A3 inhibitor, KVX-053 (developed by KeViRX) as a candidate to block Spike protein-induced endothelial permeability, modulate the cytokine storm and prevent the development of acute lung injury