Identification of Proteins Involved in Cell Membrane Permeabilization by Nanosecond Electric Pulses (nsEP)

The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R \u3e 0.9, p \u3c 0.02) with their LD50 for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP

Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD

How to Alleviate Cardiac Injury From Electric Shocks at the Cellular Level

Electric shocks, the only effective therapy for ventricular fibrillation, also electroporate cardiac cells and contribute to the high-mortality post-cardiac arrest syndrome. Copolymers such as Poloxamer 188 (P188) are known to preserve the membrane integrity and viability of electroporated cells, but their utility against cardiac injury from cardiopulmonary resuscitation (CPR) remains to be established. We studied the time course of cell killing, mechanisms of cell death, and protection with P188 in AC16 human cardiomyocytes exposed to micro- or nanosecond pulsed electric field (μsPEF and nsPEF) shocks. A 3D printer was customized with an electrode holder to precisely position electrodes orthogonal to a cell monolayer in a nanofiber multiwell plate. Trains of nsPEF shocks (200, 300-ns pulses at 1.74 kV) or μsPEF shocks (20, 100-μs pulses at 300 V) produced a non-uniform electric field enabling efficient measurements of the lethal effect in a wide range of the electric field strength. Cell viability and caspase 3/7 expression were measured by fluorescent microscopy 2–24 h after the treatment. nsPEF shocks caused little or no caspase 3/7 activation; most of the lethally injured cells were permeable to propidium dye already at 2 h after the exposure. In contrast, μsPEF shocks caused strong activation of caspase 3/7 at 2 h and the number of dead cells grew up to 24 h, indicating the prevalence of the apoptotic death pathway. P188 at 0.2–1% reduced cell death, suggesting its potential utility in vivo to alleviate electric injury from defibrillation

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca\u3csup\u3e2+\u3c/sup\u3e and Ba\u3csup\u3e2+\u3c/sup\u3e Ions

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within \u3c1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than \u3e7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns–10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15–20% higher LD50, p \u3c 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30–40% lower LD50 (p \u3c 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation

Analysis of fractional order inductive transducers

Inductive sensors have been widely used for precise measurement of position, displacement, temperature and pressure as they are extremely reliable in the harsh industrial atmosphere. In this paper, fractional order models of inductive transducers namely fractional order variable reluctance inductive transducer and fractional order resonant inductive transducer are developed and its characteristics are analysed. The impact of fractional order parameter on the characteristics like sensitivity and nonlinearity of the fractional order transducer are studied. The results are enriched when a fractional order inductor is connected in parallel to the fractional order resonant inductive transducer

Bossed diaphragm coupled fixed guided beam structure for MEMS based piezoresistive pressure sensor

Purpose Structures play a very important role in developing pressure sensors with good sensitivity and linearity, as they undergo deformation to the input pressure and function as the primary sensing element of the sensor. To achieve high sensitivity, thinner diaphragms are required; however, excessively thin diaphragms may induce large deflection and instability, leading to the unfavorable performances of a sensor in terms of linearity and repeatability. Thereby, importance is given to the development of innovative structures that offer good linearity and sensitivity. This paper aims to investigate the sensitivity of a bossed diaphragm coupled fixed guided beam three-dimensional (3D) structure for pressure sensor applications. Design/methodology/approach The proposed sensor comprises of mainly two sensing elements: the first being the 3D mechanical structure made of bulk silicon consisting of boss square diaphragm along with a fixed guided beam landing on to its center, forming the primary sensing element, and the diffused piezoresistors, which form the secondary sensing element, are embedded in the tensile and compression regions of the fixed guided beam. This micro mechanical 3 D structure is packaged for applying input pressure to the bottom of boss diaphragm. The sensor without pressure load has no deflection of the diaphragm; hence, no strain is observed on the fixed guided beam and also there is no change in the output voltage. When an input pressure P is applied through the pressure port, there is a deformation in the diaphragm causing a deflection, which displaces the mass and the fixed guided beam vertically, causing strain on the fixed guided beam, with tensile strain toward the guided end and compressive strain toward the fixed end of the close magnitudes. The geometrical dimensions of the structure, such as the diaphragm, boss and fixed guided beam, are optimized for linearity and maximum strain for an applied input pressure range of 0 to 10 bar. The structure is also analyzed analytically, numerically and experimentally, and the results are compared. Findings The structure offers equal magnitudes of tensile and compressive stresses on the surface of the fixed guided beam. It also offers good linearity and sensitivity. The analytical, simulation and experimental studies of this sensor are introduced and the results correlate with each other. Customized process steps are followed wherein two silicon-on-insulator (SOI) wafers are fusion bonded together, with SOI-1 wafer used to realize the diaphragm along with the boss and SOI-2 wafer to realize the fixed guided beam, leading to formation of a 3D structure. The geometrical dimensions of the structure, such as the diaphragm, boss and fixed guided beam, are optimized for linearity and maximum strain for an applied input pressure range of 0 to10 bar. Originality/value This paper presents a unique and compact 3D micro-mechanical structure pressure sensor with a rigid center square diaphragm (boss diaphragm) and a fixed guided beam landing at its center, with diffused piezoresistors embedded in the tensile and compression regions of the fixed guided beam. A total of six masks were involved to realize and fabricate the 3D structure and the sensor, which is presumed to be the first of its kind in the fabrication of MEMS-based piezoresistive pressure sensor

A Novel Piezoelectric Energy Harvester Using a Multi-Stepped Beam with Rectangular Cavities

In vibration-based piezoelectric energy harvesters, one of the major critical issues is increasing the bandwidth and output voltage simultaneously. This manuscript explores a new technique for broadening the operating frequency range and enhancing the output voltage of the piezoelectric material-based energy harvester by appropriate structural tailoring. The wide bandwidth and the improvement in harvested output are accomplished by means of a multi-stepped cantilever beam shaped with rectangular cavities. The harvester is mathematically modeled and analyzed for modal characteristics. It was demonstrated from the outcome that the first two consecutive mode frequencies could be brought closer and the output power was large at both the resonant frequencies compared to the regular cantilever beam energy harvester. The results obtained from experimentation were in agreement with analytical results