Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing

BACKGROUND Mesenchymal stem cells(MSCs)modulated by various exogenous signals have been applied extensively in regenerative medicine research.Notably,nanosecond pulsed electric fields(nsPEFs),characterized by short duration and high strength,significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways.Consequently,we used transcriptomics to study changes in messenger RNA(mRNA),long noncoding RNA(lncRNA),microRNA(miRNA),and circular RNA expression during nsPEFs application.AIM To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs.METHODS The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing.MSCs were pretreated with 5-pulse nsPEFs(100 ns at 10 kV/cm,1 Hz),followed by total RNA isolation.Each transcript was normalized by fragments per kilobase per million.Fold change and difference significance were applied to screen the differentially expressed genes(DEGs).Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions,complemented by quantitative polymerase chain reaction verification.RESULTS In total,263 DEGs were discovered,with 92 upregulated and 171 downregulated.DEGs were predominantly enriched in epithelial cell proliferation,osteoblast differentiation,mesenchymal cell differentiation,nuclear division,and wound healing.Regarding cellular components,DEGs are primarily involved in condensed chromosome,chromosomal region,actin cytoskeleton,and kinetochore.From aspect of molecular functions,DEGs are mainly involved in glycosaminoglycan binding,integrin binding,nuclear steroid receptor activity,cytoskeletal motor activity,and steroid binding.Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation.CONCLUSION Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs,2 miRNAs,and 65 lncRNAs.Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways,which are involved in vesicular transport,calcium ion transport,cytoskeleton,and cell differentiation.

Phyllanthuse emblica polyphenols:Optimization of high-voltage pulsed electric field assisted extraction,an antioxidant and anti-inflammatory effects in vitro

Background:The polyphenols extraction of Phyllanthus emblica is primarily carried out using organic solvents,and assisted by physical fields such as ultrasound and microwave for extraction.High voltage pulsed electric field technology(PEF)is a non-thermal processing technology that has high efficiency and minimal damage to thermosensitive substances.PEF has been applied to plant extraction in many studies,however,the extraction of polyphenols from Phyllanthus emblica using the PEF has still not been reported;Objective:This study explores the optimal extraction process of polyphenols from Phyllanthus emblica using the PEF,and investigates its relaxation and anti-wrinkle based on anti-oxidation and anti-inflammatory experiment,in order to develop a Phyllanthus emblica extract with substantial efficacy;Materials and Methods:The method of Phyllanthus emblica extract using PEF is established,and compared with a traditional extraction method.The experimental conditions,such as electric field intensity(0.5–6.0 kV/cm),pulse times(20−120),extraction time(0–60 min)and material concentration(0.5%∼3%),are investigated and optimized using orthogonal experiments;Results:the polyphenols in the Phyllanthus emblica extract were highest at the electric field intensity of 5 kV/cm,120 pulses,extraction time of 30 min,and 2%material concentration.The PEFcontained more polyphenols than the conventional water extraction and ultrasound-assisted extraction.The Phyllanthus emblica extract had substantial antioxidant and anti-inflammatory effects,with a good clearance effect on DPPH(IC50 of 1.82%)and ABTS(IC50 of 1.80%)radicals.At the Phyllanthus emblica extract concentration of 1.25%,inflammatory factors(TNF-α)were reduced by 47.08%;and Conclusion:The PEF is a leading-edge and promising method for preparing Phyllanthus emblica extracts.

Application of a Pulsed Electric Field to Modify the Free Energy of the Active Site of an Azoreductase

The Gibbs free energy is strongly related to the stability and catalytic function of an enzyme through the energetic changes that occur in the chemical reactions the enzyme catalyzes. In this in silico study, a pulsed electric field was applied to an azoreductase, and its effect on the Gibbs free energy of molecular docking with two dyes was measured. We propose that certain stimuli from a pulsed electric field favor the structural stability of the enzyme by promoting an arrangement in the active site, potentially leading to an enhancement of enzymatic activity overall.

Demulsification Behavior, Characteristics, and Performance of Surfactant Stabilized Oil-in-Water Emulsion under Bidirectional Pulsed Electric Field

As a novel electric demulsification method,bidirectional pulsed electric field(BPEF)was employed to demulsify the surfactant stabilized oil-in-water(SSO/W)emulsion for oil/water separation in this work.The demulsification behavior,characteristics,and stages under BPEF were explored.It was discovered that BPEF drove SSO/W emulsion to move and form vortexes,during which the oil droplets aggregated and accumulated to generate an oil droplet layer(ODL).ODL subsequently transformed into a continuous oil layer(COL)leading to the demulsification and separation of SSO/W emulsion.The conversion rate of ODL to COL was defined and used to evaluate the demulsification process and reflect the coalescence ability and transformation efficiency of dispersed oil droplets into COL.Furthermore,the effects of BPEF voltage,frequency,duty cycle,ratio of pulse output time,and surfactant type and content on the demulsification performance were examined.The optimal values of BPEF parameters for demulsification operation were 400 V,25 Hz,50%,and 4:1.O/W emulsion containing anionic surfactant was apt to be demulsified by BPEF,nonionic surfactant took the second place and cationic surfactant was the most difficult.A high surfactant content was not conducive to the BPEF demulsification.This work is anticipated to provide useful guidance for oil/water separation and oil recovery from actual emulsified oily wastewater by BPEF.

Recent progress in pulsed electric field ablation for liver cancer

The number of liver cancer patients is likely to continue to increase in the coming decades due to the aging of the population and changing risk factors.Traditional treatments cannot meet the needs of all patients.New treatment methods evolved from pulsed electric field ablation are expected to lead to breakthroughs in the treatment of liver cancer.This paper reviews the safety and efficacy of irreversible electroporation in clinical studies,the methods to detect and evaluate its ablation effect,the improvements in equipment and its antitumor effect,and animal and clinical trials on electrochemotherapy.We also summarize studies on the most novel nanosecond pulsed electric field ablation techniques in vitro and in vivo.These research results are certain to promote the progress of pulsed electric field in the treatment of liver cancer.

Removal of phenol by activated alumina bed in pulsed high-voltage electric field

A new process for removing the pollutants in aqueous solution-activated alumina bed in pulsed high-voltage electric field was investigated for the removal of phenol under different conditions. The experimental results indicated the increase in removal rate with increasing applied voltage, increasing pH value of the solution, aeration, and adding Fe^2＋. The removal rate of phenol could reach 72.1% when air aeration flow rate was 1200 ml/min, and 88.2% when 0.05 mmol/L Fe^2＋ was added into the solution under the conditions of applied voltage 25 kV, initial phenol concentration of 5 mg/L, and initial pH value 5.5. The addition of sodium carbonate reduced the phenol removal rate. In the pulsed high-voltage electric field, local discharge occurred at the surface of activated alumina, which promoted phenol degradation in the thin water film. At the same time, the space-time distribution of gas-liquid phases was more uniform and the contact areas of the activated species generated from the discharge and the pollutant molecules were much wider due to the effect of the activated alumina bed. The synthetical effects of the pulsed high-voltage electric field and the activated alumina particles accelerated phenol degradation.

Effects of High-voltage Pulse Electric Field Treatment on the Structure Stability of Konjac Glucomannan

Structures of KGM treated in two high-voltage pulse electric fields were characterized by infrared spectroscopy,Raman spectroscopy,X-ray diffraction and so on.The results showed that intermolecular hydrogen bonding interactions of KGM were reduced after being treated with high-voltage pulse electric field,but there was no significant effect on its fiber chain form and thermal characteristics.Results of the study can provide a useful reference for further study on the structure and property of KGM,and especially can provide theoretical basis for the effect of physical field on the foodstuff deep processing related to KGM.

Tumor ablation with nanosecond pulsed electric fields

Liver cancer is one of the most malignant cancers. It is reported that 600 000 patients died from liver cancer every year. [1,2] Hepatocellular carcinoma (HCC) is a particular problem because symptoms are not evident until the disease has progressed and hepatitis B, which is prominent specific regions of Asia, is a common precursor of the disease. There are many liver cancer

Influence of pulsed electric field on enzymes, bacteria and volatile flavor compounds of unpasteurized sake

A pulsed electric field(PEF) was applied to unpasteurized sake at constant temperatures, at which α-amylase was not inactivated. We adjusted the input energy to be identical for the temperatures by changing the number of PEF application, because the current significantly increased with the temperature, even the amplitude of the applied voltage was identical. As a result, the α-amylase was seemed to be inactivated by PEF application, not due to thermal effect.The glucoamylase was significantly inactivated by PEF. Moreover, the acid carboxypeptidase was inactivated by PEF at 4 °C but significantly activated at 25 °C. These results show that the sensitivity of enzyme to PEF application differs depending on the types of enzyme and treatment temperature. On the other hand, the colony number of bacteria was remarkably decreased, but the amount of the volatile flavor compounds was not decreased by PEF application.

Design and Experiments of the High Voltage Pulsed Electric Fields Sterilization System

This experiment designed the pulsed electric fields （PEF） of high intensity of 100 kV· cm^-1 sterilization system. Fluorescent pseudomonas as target cell was operated 180 s in the PEF. By observing the difference of the bacteria before and after the disposal by TEM, it is found that the cell walls of the treated bacteria were broken. Irreversible perforations were formed on the cell membrane. The cell inclusions and cell fragments were leaked. The cell died as a result. The results showed that the PEF sterilization system designed can be used for liquid food sterilization experiments.

Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

BACKGROUND： Electromagnetic radiation can influence dopamine （DA） synthesis in brain tissues or ceils, but electromagnetic frequencies, intensities, and radiation time can produce different effects. In addition, the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial. OBJECTIVE： To determine tyrosine hydroxylase （TH） expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field （LF-PEF） stimulation, and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors. DESIGN, TIME AND SETTING： A parallel, controlled, cell experiment was performed at the Laboratory of Cell Biology, School of Life Science, East China Normal University, between January and October 2006. MATERIALS： PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Nerve growth factor was purchased from PeproTech, USA. The protein kinase A inhibitor, H-89, and mitogen-activated protein kinase kinase inhibitor, U0126, were purchased from Sigma, USA. METHODS： （1） Following routine culture in Dulbecco＇s modified eagle medium, primary PC12 cells were stimulated under LF-PEF （pulse frequency 50.Hz, pulse width 20 μs, peak field strength 1 V/m） for 5, 10, 15, 20, and 30 minutes. （2） Inhibitors （H-89 or U0126, 1 μmol/L） were added 30 minutes before LF-PEF stimulation for 10 minutes. MAIN OUTCOME MEASURES： （1） TH expression was determined by Western blot in PC12 cells at 0.5, 1,2, 3, and 4 days after LF-PEF stimulation. Similarly, DA was measured by high-performance liquid chromatography in media at 2, 3, 4, or 5 days after LF-PEE （2） TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEE RESULTS： （1） Short-term LF-PEF stimulation （5 and 10 minutes） increased TH expression and media DA levels after short-term culture （2 days） （P 〈 0.01）, but both parameters decreased with longer culture （3 4 days） （P 〈 0.01）. Long-term LF-PEF stimulation （15, 20, or 30 minutes） decreased TH and DA synthesis, followed by a rapid increase （P 〈 0.01）. （2） H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes, while the inhibition rate of U0126 was 53.2%. CONCLUSION： Short-term LF-PEF first promotes then inhibits, while long-term LF-PEF first inhibits then promotes, TH and DA synthesis. LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.

Verification of Drainage Sterilization System Uses Low-Voltage Pulsed Electric Field in a Prawn Farm

It is necessary to take measures against infectious diseases in the Southeast Asian prawn farming industry. In giant tiger prawn （Penaeus monodon） aquaculture, diseases caused by viruses such as white spot syndrome virus （WSSV） and bacteria such as Vibrio have become a menace. Appropriate treatments of prawn culture pond＇s waters are advocated for preventing and controlling pathogens. The purpose of this study was to conduct an antimicrobial water treatment using a low-voltage pulsed electric field sterilization. Here we prepared a mechanical low-voltage pulsed electric field system with copper wire coiling around a titanium ring. The viability of WSSV in seawater was examined by prawn infectivity experiments. We inoculated healthy prawns （approximately 10-15 g） with the WSSV master sample that passed 0-3 times through the system. WSSV infection in prawns decreased according to the number of passes through the system. Healthy prawns survived for〉 10 days without feeding, where as prawns inoculated with the WSSV master sample showed symptoms of white spot and died in about 4 days. Two-thirds of the prawns inoculated with the WSSV master sample that was passed once through the system developed WSSV symptoms and were polymerase chain reaction （PCR） positive. However, no symptoms were observed and prawns were PCR negative when the WSSV master sample was passed three times through the system. Based on these results, we purpose that a low-voltage pulsed electric field system will serve as an efficient pond drainage sterilization system and will replace conventional treatments using chemicals such as sodium hypochlorite.

Study of the selective effect on cells induced by nanosecond pulsed electric field with the resistor-capacitor circuit model

A resistor-capacitor(RC)circuit model is proposed to study the effect of nanosecond pulsed electricfield on cells according to the structure and electrical parameters of cells.After a nanosecond step fieldhas been applied,the variation of voltages across cytomembrane and mitochondria membrane both in nor-mal and in malignant cells are studied with this model.The time for selectively targeting the mitochondriamembrane and malignant cell can be evaluated much easily with curves that show the variation of voltageacross each membrane with time.Ramp field is the typical field applied in electrobiology.The voltagesacross each membrane induced by ramp field are analyzed with this model.To selectively target the mito-chondria membrane,proper range of ramp slope is needed.It is relatively difficult to decide the range ofa slope to selectively affect the malignant cell.Under some conditions,such a range even does not exist.

Effects of Various Pulsed Electric Field Conditions on Cell Disintegration and Mass Transfer of Sugar Beet

Pulsed electric field is an innovative method for non-thermal food processing. The effects of pulsed electric field on cell disintegration and mass transfer in sugar beet were studied. Sugar beet slices were treated by various Field strengths （0.5-6 kV cm^-1）, capacity of capacitors （0.5, 8 and 32 μF） and pulse numbers （1-100 pulses）. The cell disintegration index and energy input for each treated sample was evaluated. The results showed that the cell membrane of PEF （Pulsed Electric Field） treated samples using 2 kV cm^-1, 8 μF after 20 pulses, and 3 kV cm1, 8 μF after 10 pulses were rapidly disintegrated in less than 1 min. Most important parameters during cell permeabilization were the total energy input followed by field strength. Energy efficiency index was defined as cell disintegration index per energy input unit. The maximum efficiency index was achieved using 2 kV cm^-1, 8 μF after 5 pulses. Comparison between PEF pretreatment and thermal method showed that large amount of sugar may be extracted after PEF pretreatment using field strength of 2 Kv cm^-1, 8μF in less than 10 min at ambient temperature. In.addition, the consumed energy for thermal treatment was approximately 20 to 50 times more than PEF pretreatment. Optimizing the field conditions in PEF treatment is an important factor to achieve high amount of sugar extraction from sugar beet.

Structure optimization of pulsed electric field processor and its sterilization effect

Pulsed electric field(PEF)technology is a highly promising non-thermal processing technique,which holds great potential for sterilizing liquid food.The effectiveness of this treatment depends on two crucial factors:pulse parameters and the structure of the processor.In this study,we focus on optimizing the shape of insulators within the PEF processor to improve the sterilization effect.This research provides a reliable technical reference for designing industrial equipment.The numerical simulation of multi-physics fields was employed to analyze the temperature,electric field,and fluid field distributions for different insulators.Ultimately,the geometric structure of the insulator was optimized by embedding a 0.7-mm arc.Sterilization experiments were then conducted on Escherichia coli and Staphylococcus aureus suspensions with conductivities similar to those of real grapefruit juice.The sterilization effect of the processor with the optimized structure and the maximum applied voltage was examined at the same driving frequency.The results indicate that compared to the original structure,there is an increase in withstanding voltage by 5–10 kV while maintaining significantly improved bactericidal effects at the same applied voltage.Furthermore,we preliminarily discussed the sterilization mechanism by combining electroporation theory with the electroporation threshold of S.aureus.

STUDY ON THE MECHANISM OF GRAPHITIZATION IN MOLTEN CAST IRON PROMOTED BY ELECTROPULSE DISCHARGE

From the points of both molten cast iron structure and the appearing ratio of electrons in outer-layer of different atoms, analysis on enhancement mechanism of graphitization ability after processing of the iron by pulse electric discharge has been made, and the theory has been proofed by corresponding experiments. The results show that when the molten cast iron is being processed by pulse electric discharge, both the size of crystal embryos that composed by Fe and C atoms as well as the number of clusters can be reduced, even be separated by such discharging; consequently results in the segre- gation of C atoms in the molten cast iron near the cathode of discharging. The nucleation of graphite in these areas of the iron has been promoted at the discharging temperature; even though such degree has not been reached, the most favorable nucleation conditions of graphite can be at least created. Under the preconditions of not breaking up the graphite crystal embryos, with proper adjustment of discharging frequency, the stronger of the electric field and the longer of the pulsation treatment time are, the more graphitization ability of the molten cast iron is. The theoretical analysis on the above rules consists well with experimental results.

The latest research progress on minimally invasive treatments for hepatocellular carcinoma

Background:Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide.Due to the high prevalence of hepatitis B virus(HBV)infection in China,the incidence of HCC in China is high,and liver cirrhosis caused by chronic hepatitis also brings great challenges to treatment.This paper reviewed the latest research progress on minimally invasive treatments for HCC,including percutaneous thermal ablation and new nonthermal ablation techniques,and introduced the principles,advantages,and clinical applications of various therapeutic methods in detail.Data sources:The data of treatments for HCC were systematically collected from the Pub Med,Science Direct,American Chemical Society and Web of Science databases published in English,using“minimally invasive”and“hepatocellular carcinoma”or“liver cancer”as the keywords.Results:Percutaneous thermal ablation is still a first-line strategy for the minimally invasive treatment of HCC.The effect of microwave ablation(MWA)on downgrading treatment before liver transplantation is better than that of radiofrequency ablation(RFA),while RFA is more widely used in the clinical practice.High-intensity focused ultrasound(HIFU)is mainly used for the palliative treatment of advanced liver cancer.Electrochemotherapy(ECT)delivers chemotherapeutic drugs to the target cells while reducing the blood supply around HCC.Irreversible electroporation(IRE)uses a microsecond-pulsed electric field that induces apoptosis and necrosis and triggers a systemic immune response.The nanosecond pulsed electric field(ns PEF)has achieved a good response in the ablation of mice with HCC,but it has not been reported in China for the treatment of human HCC.Conclusions:A variety of minimally invasive treatments provide a sufficient survival advantage for HCC patients.Nonthermal ablation will lead to a new wave with its unique advantage of antitumor recurrence and metastasis.

Changes of intracellular Na^+ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na+ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na+ concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na+ concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na , K -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell perme-abilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell mem-branes are discussed.

Effects of high intensity pulsed electric fields on yield and chemical composition of rose essential oil

High intensity pulsed electric fields(PEF)combined with distillation was used to extract essential oil from rose.Three main parameters,namely electric field intensity,pulse number and distillation times,which can affect the efficiency of extraction,were optimized.Then,in order to demonstrate the feasibility of the method,oil obtained under optimal conditions was compared quantitatively and qualitatively with that obtained by conventional hydro distillation.Considering the extraction yield,the optimal parameters combination was obtained with electric field intensity of 20 kV/cm,pulse number of 8 and distillation time of 2 h.PEF treatment had no significant influence on the quality of essential oils.However,there was an increase in methyl eugenol percentage,which is a disadvantage from qualitative point of view.

Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo

Mesenchymal stem cells(MSCs) are important cell sources in cartilage tissue development and homeostasis,and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration.Here we report the effects of combining nanosecond pulsed electric fields(ns PEFs) followed by treatment with ghrelin(a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs.ns PEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs,and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined,which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats.We further found that ns PEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway.Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway,and RNA-seq results indicated 243 genes were regulated,and JAK-STAT signaling pathway was involved.Interestingly,the sequential order of applying these two stimuli is critical,with ns PEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo,but not vice versa.This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.