Reversible electroporation of vegetable tissues - metabolic consequences and applications

This review explores recent findings on gross metabolic responses of vegetable tissue induced by the application of pulsed electric field as well as discusses potential industrial applications of reversible electroporation.Este artículo explora recientes investigaciones sobre respuestas metabólicas de tejidos vegetales inducidas por la aplicación de pulsos eléctricos. También se discuten potenciales aplicaciones industriales de la electroporación reversible

Pulsed electric field in combination with vacuum impregnation with trehalose improves the freezing tolerance of spinach leaves

Pulsed electric fields in combination with vacuum infusion have been utilized to impregnate cells with trehalose, aiming at substantially improving the freezing tolerance of spinach leaves. Spinach samples were first treated with ten trains of bi-polar, rectangular electric field pulses with a nominal electric field strength of 580 V/cm and immediately immersed in a 40% (w/w) solution of trehalose under vacuum for 20 min. The samples were kept in the trehalose solution for 2.5 h at atmospheric pressure, immersed in deionised water at 4 ºC overnight, frozen in liquid nitrogen and thawed in water at room temperature. The leaves were evaluated for cell damage with microscopic observations and wilting tests. The results provided evidence that the impregnation with trehalose by the combined actions of electric fields and vacuum impregnation drastically improved the freezing tolerance of the spinach leaves

Pulsed electric field-induced cell permeabilisation of potato tissue lead to sustained metabolic changes

Metabolite profiling was used to characterize stress responses of potato tissue subjected to reversible electroporation, providing insights on how potato tissue responds to a physical stimulus such as pulsed electric fields (PEF), which is an artificial stress. Wounded potato tissue was subjected to field strengths ranging from 200 to 400 V/cm, with a single rectangular pulse of 1 ms. Electroporation was demonstrated by propidium iodide staining of the cells nucleae. Metabolic profiling of data obtained through GC/TOF-MS complemented with orthogonal projections to latent structures (OPLS) clustering analysis showed that 24 h after the application of PEF, potato metabolism shows PEF-specific responses characterized by the changes in the hexose pool that may involve starch and ascorbic acid degradation

Metabolomic evaluation of pulsed electric field-induced stress on potato tissue

Metabolite profiling was used to characterize stress responses of potato tissue subjected to reversible electroporation, providing insights on how potato tissue responds to a physical stimulus such as pulsed electric fields (PEF), which is an artificial stress. Wounded potato tissue was subjected to field strengths ranging from 200 to 400 V/cm, with a single rectangular pulse of 1 ms. Electroporation was demonstrated by propidium iodide staining of the cell nucleae. Metabolic profiling of data obtained through GC/TOF-MS and UPLC/TOF-MS complemented with orthogonal projections to latent structures clustering analysis showed that 24 h after the application of PEF, potato metabolism shows PEF-specific responses characterized by the changes in the hexose pool that may involve starch and ascorbic acid degradation.The Royal Physiographic Society in Lund, SwedenPortuguese Foundation of Science (FCT), PortugalDepartment of Cell and Organism Biology, Lund Universit

X-ray microtomography provides new insights into vacuum impregnation of spinach leaves

Vacuum impregnation is used in the food industry to facilitate the impregnation of porous products with, e.g. firming, antioxidant, antimicrobial or cryoprotective agents. X-ray micro-tomography (CT) was used to study the process of vacuum impregnation in spinach leaves. Low (300 mbar absolute pressure) and mild vacuum (150 mbar absolute pressure) impregnation protocols were used to impregnate an isotonic solution of trehalose in the leaves and CT was used to make observations of the cross section of the impregnated samples and quantify their porosity. Results revealed that the free volume in the spongy mesophyll is easier to impregnate than the spaces around the palisade mesophyll. The low vacuum impregnation protocol provoked less impregnation close to the edge of the leaf than in its centre, probably accounting for an influence of the tissue structure on impregnation. The vacuum impregnation protocols tested in this investigation drastically decreased the proportion of large pores (>100 m) and increased the proportion of small pores (<50 m). The mild vacuum impregnation protocol, which was designed on the basis of measured apparent porosity, did not achieve full impregnation of the tissue.V. Panarese acknowledges the financial support from the Portuguese Foundation of Science (FCT). F. Gomez Galindo acknowledges the financial support from European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement no. 245280, also known under the acronym PRESERF. Financial support of FWO Vlaanderen (project G.0645.13), the Flemish government agency for Innovation by Science and Technology (project IWT SBO120033 TomFood) and the University of Leuven (project OT 12/055) is gratefully acknowledged. Dennis Cantre is an IRO scholar of KU Leuven. We also acknowledge the Hercules foundation for supporting the X-ray CT facility (AKUL001(HER/09/016))

Modeling electroporation of the non-treated and vacuum impregnated heterogeneous tissue of spinach leaves

Uniform electroporation of the heterogeneous structure of spinach leaf cross section is a technological challenge that is addressed in this investigation. Three dimensional models were created with cells arranged in specific tissue types, considering a leaf with its air fraction and a leaf where the air fraction was replaced by a solution of known properties using vacuum impregnation. The models were validated before electroporation, in the frequency domain, where alternating voltage and current signal at frequencies from 20 Hz to 1 MHz were used to measure conductivity of the tissue. They were also validated through measurements of current during electroporation when a single 250 μs rectangular pulse with amplitudes ranging from 50 to 500 V was applied. Model validations show that both the frequency dependent conductivity and electroporation are well predicted. The importance of the wax layer and stomata in the model is thoroughly discussed. Industrial relevance: Our aim was to investigate electroporation of the spinach leaf by developing a model which would enable us to meet the technological challenge of achieving uniform electroporation in a highly heterogeneous structure in the context of a process aimed at improving freezing stability of plant foods. Pulsed electric field treatment may be used to introduce the cryoprotectant molecules into the cells, and hence improve the structure and properties of frozen food plants

Effects of pulsed electric field on the viscoelastic properties of potato tissue

We have investigated whether transient permeabilization caused by the application of pulsed electric field would give rise to transient changes in the potato tissue viscoelastic properties. Potato tissue was subjected to nominal field strengths (E) ranging from 30 to 500 V/cm, with a single rectangular pulse of 10−5, 10−4, or 10−3 s. The changes on the viscoelastic properties of potato tissue during pulsed electric fields (PEF) were monitored through small amplitude oscillatory dynamic rheological measurements. The elastic (G′) and viscous moduli (G″) were measured every 30 s after the delivery of the pulse and the loss tangent change (tan-δ) was calculated. The results were correlated with measurements of changes on electrical resistance during the delivery of the pulse. Results show a drastic increase of tan-δ in the first 30 s after the application of the pulse, followed by a decrease 1 min after pulsation. This response is strongly influenced by pulsing conditions and is independent of the total permeabilization achieved by the pulse. Our results, supported by similar measurements on osmotically dehydrated control samples, clearly show that PEF causes a rapid change of the viscoelastic properties of the tissue that could be attributed to a partial loss in turgor pressure. This would be an expected consequence of electroporation. The recovery of tan-δ to values similar to those before pulsation strongly suggests recovery of cell membrane properties and turgor, pointing at reversible permeabilization of the cells. A slight increase of stiffness traduced by a negative change of tan-δ after application of certain PEF conditions may also give an indication of events occurring on cell wall structure due to stress responses. This study set the basis for further investigations on the complex cell stress physiology involving both cell membrane functional properties and cell wall structure that would influence tissue physical properties upon PEF application.Fundação para a Ciência e a Tecnologia (FCT

Exploring metabolic responses of potato tissue induced by electric pulses

In this study, we investigated the metabolic responses of potato tissue induced by pulsed electric field (PEF). Potato tissue was subjected to field strengths ranging from 30 to 500 V/cm, with a single rectangular pulse of 10 μs, 100 μs, or 1 ms. Metabolic responses were monitored using isothermal calorimetry, changes on electrical resistance during the delivery of the pulse, as well as impedance measurements. Our results show that the metabolic response involves oxygen consuming pathways as well as other unidentified events that are shown to be insensitive to metabolic inhibitors such as KCN and sodium azide. The metabolic response is strongly dependent on pulsing conditions and is independent of the total permeabilization achieved by the pulse. Evidence shows that calorimetry is a simple and powerful method for exploring conditions for metabolic stimulation, providing information on metabolic responses that can not be obtained from electrical measurements. This study set the basis for further investigations on defense-related consequences of PEF-induced stress.Sparbanksstiftelsen Färs & Frosta (Sweden).Fundação para a Ciência e a Tecnologia (FCT).Lund University (Sweden).Department of Cell and Organism Biology; Department of Plant Biochemistry

Intermediate Molecular Phenotypes to Identify Genetic Markers of Anthracycline-Induced Cardiotoxicity Risk.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice (n = 165) generated by backcrossing were treated with doxorubicin and docetaxel. We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients (n = 71) and women with breast cancer (n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.J.P.L.’s lab is sponsored by Grant PID2020-118527RB-I00 funded by MCIN/AEI/10.13039/ 501100011039; Grant PDC2021-121735-I00 funded by MCIN/AEI/10.13039/501100011039 and by the “European Union Next Generation EU/PRTR”, the Regional Government of Castile and León (CSI144P20). J.P.L. and P.L.S. are supported by the Carlos III Health Institute (PIE14/00066). AGN laboratory and human patients’ studies are supported by an ISCIII project grant (PI18/01242). The Human Genotyping unit is a member of CeGen, PRB3, and is supported by grant PT17/0019 of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. SCLl is supported by MINECO/FEDER research grants (RTI2018-094130-B-100). CH was supported by the Department of Defense (DoD) BCRP, No. BC190820; and the National Cancer Institute (NCI) at the National Institutes of Health (NIH), No. R01CA184476. Lawrence Berkeley National Laboratory (LBNL) is a multi-program national laboratory operated by the University of California for the DOE under contract DE AC02-05CH11231. The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023 of the PE I + D +i, 2017–2020, funded by ISCIII and FEDER. RCC is funded by fellowships from the Spanish Regional Government of Castile and León. NGS is a recipient of an FPU fellowship (MINECO/FEDER). hiPSC-CM studies were funded in part by the “la Caixa” Banking Foundation under the project code HR18-00304 and a Severo Ochoa CNIC Intramural Project (Exp. 12-2016 IGP) to J.J.S