Optimization of Pulsed Electric Field Treatment for the Extraction of Bioactive Compounds from Blackcurrant

The objective of this work was to enhance the bioactive compounds extraction of blackcurrant using pulsed electric fields (PEF) technology. An experimental design was performed to find the best PEF conditions using the desirability approach and response surface methodology. The effect of the electric field strength and the treatment time over the total polyphenolic content (TPC) and the antioxidant activity (AA) was analyzed. The optimum treating conditions were found to be 1318 V/cm and 315 pulses, and resulted in increments of 19%, 45%, and 6%, for TPC, AA, and total monomeric anthocyanins, respectively. Two initial temperatures were studied (10 and 22 °C) during electroporation. A significant effect of temperature over PEF treatment was observed. The PEF treatment was appropriate for increasing the extraction of bioactive compounds, leading to improved blackcurrant juices that could be used as ingredients for functional foods.Fil: Gagneten, Maite. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias. Instituto de Tecnología de Alimentos y Procesos Químicos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Tecnología de Alimentos y Procesos Químicos; ArgentinaFil: Leiva, Graciela Edith. Universidad de Buenos Aires; ArgentinaFil: Salvatori, Daniela Marisol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; ArgentinaFil: Schebor, Carolina Claudia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias. Instituto de Tecnología de Alimentos y Procesos Químicos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Tecnología de Alimentos y Procesos Químicos; ArgentinaFil: Olaiz, Nahuel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentin

Canine Oral Eosinophilic Granuloma Treated with Electrochemotherapy

A case of a canine oral eosinophilic granuloma in a 14-year-old female crossbred is described. The dog was presented with a history of ptyalism, halitosis, local pain, decreased appetite, and blood staining noted on food and water bowls. Clinical, hematologic, and biochemical examinations, abdominal ultrasonography, and 3-view chest radiographs were performed, and no metastases were found. Histopathologic examination of two 6 mm punch biopsies from the oral lesion revealed the presence of eosinophilic granulomatous lesions in the submucosa. After treatment with corticosteroids and wide spectrum antibiotics no significant changes in clinical signs and lesion size were observed. Electrochemotherapy (ECT), a novel tumor treatment routinely used for cutaneous and subcutaneous tumors in human patients in the European Union since 2006, was used to treat the eosinophilic granuloma. The procedure was performed under general anesthesia, followed by intravenous administration of bleomycin. Six weeks after treatment a complete response with disappearance of the mass and improvement of clinical signs were observed.Fil: Tellado, Matías Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Michinski, Sebastián Diego. Instituto Tecnológico de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olaiz, Nahuel Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maglietti, Felipe Horacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marshall, Guillermo Ricardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The Role of pH Fronts in Reversible Electroporation

We present experimental measurements and theoretical predictions of ion transport in agar gels during reversible electroporation (ECT) for conditions typical to many clinical studies found in the literature, revealing the presence of pH fronts emerging from both electrodes. These results suggest that pH fronts are immediate and substantial. Since they might give rise to tissue necrosis, an unwanted condition in clinical applications of ECT as well as in irreversible electroporation (IRE) and in electrogenetherapy (EGT), it is important to quantify their extent and evolution. Here, a tracking technique is used to follow the space-time evolution of these pH fronts. It is found that they scale in time as , characteristic of a predominantly diffusive process. Comparing ECT pH fronts with those arising in electrotherapy (EChT), another treatment applying constant electric fields whose main goal is tissue necrosis, a striking result is observed: anodic acidification is larger in ECT than in EChT, suggesting that tissue necrosis could also be greater. Ways to minimize these adverse effects in ECT are suggested

Bubble Formation in Pulsed Electric Field Technology May Pose Limitations

Currently, increasing amounts of pulsed electric fields (PEF) are employed to improve a person’s life quality. This technology is based on the application of the shortest high voltage electrical pulse, which generates an increment over the cell membrane permeability. When applying these pulses, an unwanted effect is electrolysis, which could alter the treatment. This work focused on the study of the local variations of the electric field and current density around the bubbles formed by the electrolysis of water by PEF technology and how these variations alter the electroporation protocol. The assays, in the present work, were carried out at 2 KV/cm, 1.2 KV/cm and 0.6 KV/cm in water, adjusting the conductivity with NaCl at 2365 μs/cm with a single pulse of 800 μs. The measurements of the bubble diameter variations due to electrolysis as a function of time allowed us to develop an experimental model of the behavior of the bubble diameter vs. time, which was used for simulation purposes. In the in silico model, we calculated that the electric field and observed an increment of current density around the bubble can be up to four times the base value due to the edge effect around it, while the thermal effects were undesirable due to the short duration of the pulses (variations of ±0.1 °C are undesirable). This research revealed that the rise of electric current is not just because of the shift in electrical conductivity due to chemical and thermal effects, but also varies with the bubble coverage over the electrode surface and variations in the local electric field by edge effect. All these variations can conduce to unwanted limitations over PEF treatment. In the future, we recommend tests on the variation of local current conductivity and electric fields

The role of additional pulses in electropermeabilization protocols.

Electropermeabilization (EP) based protocols such as those applied in medicine, food processing or environmental management, are well established and widely used. The applied voltage, as well as tissue electric conductivity, are of utmost importance for assessing final electropermeabilized area and thus EP effectiveness. Experimental results from literature report that, under certain EP protocols, consecutive pulses increase tissue electric conductivity and even the permeabilization amount. Here we introduce a theoretical model that takes into account this effect in the application of an EP-based protocol, and its validation with experimental measurements. The theoretical model describes the electric field distribution by a nonlinear Laplace equation with a variable conductivity coefficient depending on the electric field, the temperature and the quantity of pulses, and the Penne's Bioheat equation for temperature variations. In the experiments, a vegetable tissue model (potato slice) is used for measuring electric currents and tissue electropermeabilized area in different EP protocols. Experimental measurements show that, during sequential pulses and keeping constant the applied voltage, the electric current density and the blackened (electropermeabilized) area increase. This behavior can only be attributed to a rise in the electric conductivity due to a higher number of pulses. Accordingly, we present a theoretical modeling of an EP protocol that predicts correctly the increment in the electric current density observed experimentally during the addition of pulses. The model also demonstrates that the electric current increase is due to a rise in the electric conductivity, in turn induced by temperature and pulse number, with no significant changes in the electric field distribution. The EP model introduced, based on a novel formulation of the electric conductivity, leads to a more realistic description of the EP phenomenon, hopefully providing more accurate predictions of treatment outcomes

Combined local and systemic bleomycin administration in electrochemotherapy to reduce the number of treatment sessions

Electrochemotherapy (ECT), a medical treatment widely used in human patients for tumor treatment, increases bleomycin toxicity by 1000 fold in the treated area with an objective response rate of around 80%. Despite its high response rate, there are still 20% of cases in which the patients are not responding. This could be ascribed to the fact that bleomycin, when administered systemically, is not reaching the whole tumor mass properly because of the characteristics of tumor vascularization, in which case local administration could cover areas that are unreachable by systemic administration

Minimally invasive electrochemotherapy procedure for treating nasal duct tumors in dogs using a single needle electrode

Nasal cavity tumors are usually diagnosed late, when they already have infiltrated adjacent tissues thus requiring very aggressive treatments with serious side effects. Here we use electrochemotherapy (ECT), a well demonstrated treatment modality for superficial tumors

Towards an optimal dose-response relationship in gene electrotransfer protocols

In search of an optimal gene electrotransfer (GET) protocol, an electroporation-based (EP) tumor treatment with great potential as a non-viral gene-delivery system, the concept of the dose-response relationship is introduced. It is shown that a reliable dose parameter is the pulse dosage and reliable response parameters are the reversibly electroporated tissue area as well as the unwanted damaged tissue area and plasmid damage due to pH. The standard stationary EP model consists in computing the reversibly electroporated tissue area in the first pulse as the region of tissue subjected to an electric field distribution higher than an electric field threshold for EP, where the electric field threshold comes from an experimental measurement and the electric field distribution from the solution of the nonlinear stationary Laplace equation for the electrostatic potential. The extended standard EP model introduced here consists in replicating for n consecutive pulses the standard EP model, via the experimental measurement in time of the successive thresholds. Because experimental data of this threshold variation is lacking, an exponential time decay function is assumed based on experimental measurements. The damage induced by pH fronts is defined as the tissue area subjected to pH abrupt changes above a basic threshold or below an acid threshold, where these changes come from numerical solutions via the electrolytic ablation (EA) model for EP-based protocols and the basic and acid thresholds from experiments. An optimal dose-response relationship in a GET protocol, for the range of pulse intensities with fixed pulse length and frequency, tested here, is predicted as the critical pulse dosage yielding maximum reversibly electroporated tissue area with minimal tissue area damage induced by pH fronts. Moreover, since damage induced by pH changes is proportional to the Coulomb dosage, damage induced by pH fronts is negligible in typical EP-based tumor protocols such as in electrochemotherapy (ECT) and irreversible electroporation (IRE) but not in GET, due to the most often longer pulses applied/used, i.e. higher dosage applied.Fil: Luján, Emmanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Simulación Computacional para Aplicaciones Tecnológicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; ArgentinaFil: Marino, Matias Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; ArgentinaFil: Olaiz, Nahuel Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; ArgentinaFil: Marshall, Guillermo Ricardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentin

The role of pH fronts in tissue electroporation based treatments.

Treatments based on electroporation (EP) induce the formation of pores in cell membranes due to the application of pulsed electric fields. We present experimental evidence of the existence of pH fronts emerging from both electrodes during treatments based on tissue EP, for conditions found in many studies, and that these fronts are immediate and substantial. pH fronts are indirectly measured through the evanescence time (ET), defined as the time required for the tissue buffer to neutralize them. The ET was measured through a pH indicator imaged at a series of time intervals using a four-cluster hard fuzzy-c-means algorithm to segment pixels corresponding to the pH indicator at every frame. The ET was calculated as the time during which the number of pixels was 10% of those in the initial frame. While in EP-based treatments such as reversible (ECT) and irreversible electroporation (IRE) the ET is very short (though enough to cause minor injuries) due to electric pulse characteristics and biological buffers present in the tissue, in gene electrotransfer (GET), ET is much longer, enough to denaturate plasmids and produce cell damage. When any of the electric pulse parameters is doubled or tripled the ET grows and, remarkably, when any of the pulse parameters in GET is halved, the ET drops significantly. Reducing pH fronts has relevant implications for GET treatment efficiency, due to a substantial reduction of plasmid damage and cell loss